Preparation of Selenium-Based Drug-Modified Polymeric Ligand-Functionalised Fe3O4 Nanoparticles as Multimodal Drug Carrier and Magnetic Hyperthermia Inductor.

In recent years, much effort has been invested into developing multifunctional drug delivery systems to overcome the drawbacks of conventional carriers. Magnetic nanoparticles are not generally used as carriers but can be functionalised with several different biomolecules and their size can be tailored to present a hyperthermia response, allowing for the design of multifunctional systems which can be active in therapies. In this work, we have designed a drug carrier nanosystem based on Fe3O4 nanoparticles with large heating power and 4-amino-2-pentylselenoquinazoline as an attached drug that exhibits oxidative properties and high selectivity against a variety of cancer malignant cells. For this propose, two samples composed of homogeneous Fe3O4 nanoparticles (NPs) with different sizes, shapes, and magnetic properties have been synthesised and characterised. The surface modification of the prepared Fe3O4 nanoparticles has been developed using copolymers composed of poly(ethylene-alt-maleic anhydride), dodecylamine, polyethylene glycol and the drug 4-amino-2-pentylselenoquinazoline. The obtained nanosystems were properly characterised. Their in vitro efficacy in colon cancer cells and as magnetic hyperthermia inductors was analysed, thereby leaving the door open for their potential application as multimodal agents.

Design of disintegrable nanoassemblies to release multiple small-sized nanoparticles.

The therapeutic and diagnostic effects of nanoparticles highly depend on the efficiency of their delivery to targeted tissues, such as tumors. The size of nanoparticles, among other characteristics, plays a crucial role in determining their tissue penetration and retention. Small nanoparticles may penetrate deeper into tumor parenchyma but are poorly retained, whereas large ones are distributed around tumor blood vessels. Thus, compared to smaller individual nanoparticles, assemblies of such nanoparticles due to their larger size are favorable for prolonged blood circulation and enhanced tumor accumulation. Upon reaching the targeted tissues, nanoassemblies may dissociate at the target region and release the smaller nanoparticles, which is beneficial for their distribution at the target site and ultimate clearance. The recent emerging strategy that combines small nanoparticles into larger, biodegradable nanoassemblies has been demonstrated by several groups. This review summarizes a variety of chemical and structural designs for constructing stimuli-responsive disintegrable nanoassemblies as well as their different disassembly routes. These nanoassemblies have been applied as demonstrators in the fields of cancer therapy, antibacterial infection, ischemic stroke recovery, bioimaging, and diagnostics. Finally, we summarize stimuli-responsive mechanisms and their corresponding nanomedicine designing strategies, and discuss potential challenges and barriers towards clinical translation.

Quantitative Comparison of Gold Nanoparticle Delivery via the Enhanced Permeation and Retention (EPR) Effect and Mesenchymal Stem Cell (MSC)-Based Targeting.

There are still some gaps in existing knowledge in the field of cancer nanotheranostics, e.g., the efficiency of nanoparticle-loaded cells for targeted delivery. In the current study, gold nanoparticles (Au NPs) were delivered to tumors in both subcutaneous tumor and lung metastasis tumor models by intravenous injection of either free Au NPs or of human bone marrow mesenchymal stem cells (MSCs), which were loaded with endocytosed Au NPs. By making injections with the same dose of administrated Au NPs, it was possible to directly compare tumor targeting of both delivery modes. Hereby, the passive targeting of tumor by the plain Au NPs was facilitated by the enhanced permeation and retention (EPR) effect. Au NP retention by tumors, as well as tumor penetration, were found to be improved up to 2.4-to-9.3-fold when comparing the MSC-mediated delivery of Au NPs to the delivery of the plain Au NPs via EPR effect on day 7 post administration. While the absolute retention of Au NPs in the tumor remained low, our data show that, upon injection of the same amount of Au NPs, in fact MSC-mediated delivery is quantitatively higher than EPR-mediated delivery of NPs by half an order of magnitude.

Deducing the cellular mechanisms associated with the potential genotoxic impact of gold and silver engineered nanoparticles upon different lung epithelial cell lines in vitro.

Human ENP exposure is inevitable and the novel, size-dependent physicochemical properties that enable ENPs to be beneficial in innovative technologies are concomitantly causing heightened public concerns as to their potential adverse effects upon human health. This study aims to deduce the mechanisms associated with potential ENP mediated (geno)toxicity and impact upon telomere integrity, if any, of varying concentrations of both ∼16 nm (4.34 × 10-3 to 17.36 × 10-3 mg/mL) Gold (Au) and ∼14 nm (0.85 × 10-5 to 3.32 × 10-5 mg/mL) Silver (Ag) ENPs upon two commonly used lung epithelial cell lines, 16HBE14o- and A549. Following cytotoxicity analysis (via Trypan Blue and Lactate Dehydrogenase assay), two sub-lethal concentrations were selected for genotoxicity analysis using the cytokinesis-blocked micronucleus assay. Whilst both ENP types induced significant oxidative stress, Ag ENPs (1.66 × 10-5 mg/mL) did not display a significant genotoxic response in either epithelial cell lines, but Au ENPs (8.68 × 10-3 mg/mL) showed a highly significant 2.63-fold and 2.4-fold increase in micronucleus frequency in A549 and 16HBE14o- cells respectively. It is hypothesized that the DNA damage induced by acute 24-h Au ENP exposure resulted in a cell cycle stall indicated by the increased mononuclear cell fraction (>6.0-fold) and cytostasis level. Albeit insignificant, a small reduction in telomere length was observed following acute exposure to both ENPs which could indicate the potential for ENP mediated telomere attrition. Finally, from the data shown, both in vitro lung cell cultures (16HBE14o- and A549) are equally as suitable and reliable for the in vitro ENP hazard identification approach adopted in this study.

Influence of the Modulation of the Protein Corona on Gene Expression Using Polyethylenimine (PEI) Polyplexes as Delivery Vehicle.

The protein corona can significantly modulate the physicochemical properties and gene delivery of polyethylenimine (PEI)/DNA complexes (polyplexes). The effects of the protein corona on the transfection have been well studied in terms of averaged gene expression in a whole cell population. Such evaluation methods give excellent and reliable statistics, but they in general provide the final transfection efficiency without reflecting the dynamic process of gene expression. In this regard the influence of bovine serum albumin (BSA) on the gene expression of PEI polyplexes also on a single cell level via live imaging is analyzed. The results reveal that although the BSA corona causes difference in the overall gene expression and mRNA transcription, the gene expression behavior on the level of individual cell is similar, including the mitosis-dependent expression, distributions of onset time, expression pattern in two daughter cells, and expression kinetics in successfully transfected cells. Comparison of single cell and ensemble data on whole cell cultures indicate that the protein corona does not alter the transfection process after nuclear entry, including cell division, polyplex dissociation, and protein expression. Its influence on other steps of in vitro gene delivery before nuclear entry shall render the difference in the overall transfection.

X-ray Fluorescence Uptake Measurement of Functionalized Gold Nanoparticles in Tumor Cell Microsamples.

Quantitative cellular in vitro nanoparticle uptake measurements are possible with a large number of different techniques, however, all have their respective restrictions. Here, we demonstrate the application of synchrotron-based X-ray fluorescence imaging (XFI) on prostate tumor cells, which have internalized differently functionalized gold nanoparticles. Total nanoparticle uptake on the order of a few hundred picograms could be conveniently observed with microsamples consisting of only a few hundreds of cells. A comparison with mass spectroscopy quantification is provided, experimental results are both supported and sensitivity limits of this XFI approach extrapolated by Monte-Carlo simulations, yielding a minimum detectable nanoparticle mass of just 5 pg. This study demonstrates the high sensitivity level of XFI, allowing non-destructive uptake measurements with very small microsamples within just seconds of irradiation time.

Multimodal Imaging of Pancreatic Ductal Adenocarcinoma Using Multifunctional Nanoparticles as Contrast Agents.

Late diagnosis and refractory behavior toward current treatment protocols make pancreatic ductal adenocarcinoma (PDAC) one of the most difficult cancer forms to treat. The imaging-based approach plays an important role to identify potentially curable PDAC patients in high-risk groups at the early stage. In the present study, we developed a core-shell structured gold nanorod (AuNR) as a contrast agent for multimodal imaging and investigated its application for PDAC diagnosis. The composite nanoparticles composed of a AuNR core inside a layer of mesoporous silica that was then coated with a gadolinium oxide carbonate shell (AuNR-SiO2-Gd) are designed to be used in magnetic resonance imaging (MRI), X-ray computed tomography (CT), and photoacoustic imaging (PAI). A phantom study with the AuNR-SiO2-Gd NPs demonstrated higher MRI contrast compared to Gadovist and higher X-ray attenuation than Visipaque. A strong, stable, and broad wavelength range signal with a peak at 800 nm was observed in PAI. The AuNR-SiO2-Gd NPs showed significant contrast enhancement under PAI/MRI/CT in both the liver and spleen of control mice after intravenous administration. The utility in PDAC was studied in a genetically engineered mouse model carrying Kras and p53 mutations, which develops spontaneous tumors and keeps the desmoplasia and hypovascularity feature of PDAC in patients. The AuNR-SiO2-Gd NPs were highly accumulated in the surrounding soft tissues but were sparsely distributed throughout the tumor due to dense stroma infiltration and poor tumor vascularization. Hence, a negative contrast within the tumor area in CT/PAI and a positive contrast in MRI were observed. In conclusion, AuNR-SiO2-Gd NPs have good potential to be developed as a multimodal contrast agent for PDAC, which might improve early diagnosis and benefit the clinical outcome for PDAC patients.

Correction: Metabolic pathway for the universal fluorescent recognition of tumor cells.

[This corrects the article DOI: 10.18632/oncotarget.18551.].

Nonradioactive Cell Assay for the Evaluation of Modular Prostate-Specific Membrane Antigen Targeting Ligands via Inductively Coupled Plasma Mass Spectrometry.

The development of novel prostate-specific membrane antigen (PSMA)-targeted radioactive theranostic agents is currently limited to facilities capable of working with high-energy radioisotopes. Even preselection of lead structures in vitro relies mostly on radioactive assays with PSMA(+) LNCaP and PSMA(-) PC-3 cells. Assays utilizing radioisotopes are time consuming, costly, and limit discovery to a small group of scientists with special facilities. Nonradioactive alternatives are therefore needed in the field. In this paper, we describe an inductively coupled plasma mass spectrometry (ICP-MS)-based method for the evaluation of PSMA-targeting ligands conjugated to DOTA-chelates of Europium. This method is based on LNCaP and PC-3 cells and has been validated with the well-established targeting ligand PSMA-617.

Confining Iron Oxide Nanocubes inside Submicrometric Cavities as a Key Strategy To Preserve Magnetic Heat Losses in an Intracellular Environment.

The design of magnetic nanostructures whose magnetic heating efficiency remains unaffected at the tumor site is a fundamental requirement to further advance magnetic hyperthermia in the clinic. This work demonstrates that the confinement of magnetic nanoparticles (NPs) into a sub-micrometer cavity is a key strategy to enable a certain degree of nanoparticle motion and minimize aggregation effects, consequently preserving the magnetic heat loss of iron oxide nanocubes (IONCs) under different conditions, including intracellular environments. We fabricated magnetic layer-by-layer (LbL) self-assembled polyelectrolyte sub-micrometer capsules using three different approaches, and we studied their heating efficiency as obtained in aqueous dispersions and after internalization by tumor cells. First, IONCs were added to the hollow cavities of LbL submicrocapsules, allowing the IONCs to move to a certain extent in the capsule cavities. Second, IONCs were coencapsulated into solid calcium carbonate cores coated with LbL polymer shells. Third, IONCs were incorporated within the polymer layers of the LbL capsule walls. In aqueous solution, higher specific absorption rate (SAR) values were related to those of free IONCs, while lower SAR values were recorded for capsule/core assemblies. However, after uptake by cancer cell lines (SKOV-3 cells), the SAR values of the free IONCs were significantly lower than those observed for capsule/core assemblies, especially after prolonged incubation periods (24 and 48 h). These results show that IONCs packed into submicrocavities preserve the magnetic losses, as the SAR values remained almost invariable. Conversely, free IONCs without the protective capsule shell agglomerated and their magnetic losses were strongly reduced. Indeed, IONC-loaded capsules and free IONCs reside inside endosomal and lysosomal compartments after cellular uptake and show strongly reduced magnetic losses due to the immobilization and aggregation in centrosymmetrical structures in the intracellular vesicles. The confinement of IONCs into sub-micrometer cavities is a key strategy to provide a sustained and predictable heating dose inside biological matrices.

Biodegradable Alginate Polyelectrolyte Capsules As Plausible Biocompatible Delivery Carriers.

Polyelectrolyte capsules made of different biodegradable and nonbiodegradable polymers can be designed as systems for effective encapsulation and delivery of compounds. The objective of this work was to synthesize biocompatible and biodegradable capsules (<1 µm) by the layer-by-layer (LbL) approach using alginate (ALGI) and poly-l-arginine (PARG) polyelectrolytes with a pH-sensitive outer layer of EUDRAGIT L 100 (EuL) polymer. Those capsules were loaded with curcumin as a model therapeutic drug, which possesses antioxidant, anti-inflammatory, and anticancer activity. Encapsulation of drugs inside capsules protects its therapeutic activity and increases its bioavailability. We report the capsule stability, loading efficiency, drug release, as well as capsule degradation studies as a function of pH. Furthermore, in vitro biocompatibility studies of capsules including cell viability and uptake studies were performed using HeLa cells. The here synthesized capsules exhibited good reproducibility, spherical shape, and high monodispersibility. The capsules showed good loading efficiency and drug release profile dependent upon pH environment. The in vitro studies indicate that the capsules exhibited acceptable biocompatibility and are highly internalized by cells. Our study thus suggests that alginate LbL capsules could be used as an efficient drug carrier with effective encapsulation and successful in vitro release of cargo in the cell.

Adaptive metabolic pattern biomarker for disease monitoring and staging of lung cancer with liquid biopsy.

In this manuscript, we demonstrate the applicability of a metabolic liquid biopsy for the monitoring and staging of patients with lung cancer. This method provides an unbiased detection strategy to establish a more precise correlation between CTC quantification and the actual burden of disease, therefore improving the accuracy of staging based on current imaging techniques. Also, by applying statistical analysis techniques and probabilistic models to the metabolic status and distribution of peripheral blood mononuclear cell (PBMC) populations "perturbed" by the presence of CTCs, a new category of adaptive metabolic pattern biomarker (AMPB) is described and unambiguously correlated to the different clinical stages of the patients. In fact, this strategy allows for classification of different categories of disease within a single stage (stage IV) before computed tomography (CT) and positron emission tomography (PET) scans and with lower uncertainty.

Detailed investigation on how the protein corona modulates the physicochemical properties and gene delivery of polyethylenimine (PEI) polyplexes.

Given the various cationic polymers developed as non-viral gene delivery vectors, polyethylenimine (PEI) has been/is frequently used in in vitro transfection. However, the primary drawback limiting its in vivo applications is the sharp decrease in transfection efficiency in the presence of serum. Here, we investigated the influences of serum proteins or bovine serum albumin (BSA) on the physicochemical properties of PEI/DNA complexes (polyplexes), including hydrodynamic diameters and agglomeration behavior, zeta potentials, morphologies, and sensitivity to the presence of salt. Mechanism studies revealed that the protein corona determined the endocytic rates and pathways, intracellular transport, the rate of endo/lysosomal trafficking, vesicle escape efficiency, and thus the overall gene expression levels. This work offers mechanistic insights for the serum-induced suppression of transfection efficiency, which is the reduced endo/lysosomal escape of the protein-coated polyplexes, in contrast to the protein free polyplexes.

Investigation of the Viability of Cells upon Co-Exposure to Gold and Iron Oxide Nanoparticles.

Cell lines were exposed either to mixtures of gold and iron oxide nanoparticles, or to a hybrid nanoparticle with gold and iron oxide domain. In the case of simultaneous exposure to gold and iron oxide nanoparticles, enhanced toxicity as compared to the exposure to only one type of nanoparticles was observed. An indication was found that, at equivalent concentrations, the hybrid nanoparticles may slightly reduce cell viability more strongly than mixtures of both nanoparticle types. The results suggest that composite nanomaterials, in which different materials are present in particle form, need to be analyzed carefully, as not only the concentration of the respective materials but also their arrangement may influence their toxicity.

Study of Fluorinated Quantum Dots-Protein Interactions at the Oil/Water Interface by Interfacial Surface Tension Changes.

Understanding the interaction of nanoparticles with proteins and how this interaction modifies the nanoparticles’ surface is crucial before their use for biomedical applications. Since fluorinated materials are emerging as potential imaging probes and delivery vehicles, their interaction with proteins of biological interest must be studied in order to be able to predict their performance in real scenarios. It is known that fluorinated planar surfaces may repel the unspecific adsorption of proteins but little is known regarding the same process on fluorinated nanoparticles due to the scarce examples in the literature. In this context, the aim of this work is to propose a simple and fast methodology to study fluorinated nanoparticle-protein interactions based on interfacial surface tension (IFT) measurements. This technique is particularly interesting for fluorinated nanoparticles due to their increased hydrophobicity. Our study is based on the determination of IFT variations due to the interaction of quantum dots of ca. 5 nm inorganic core/shell diameter coated with fluorinated ligands (QD_F) with several proteins at the oil/water interface. Based on the results, we conclude that the presence of QD_F do not disrupt protein spontaneous film formation at the oil/water interface. Even if at very low concentrations of proteins the film formation in the presence of QD_F shows a slower rate, the final interfacial tension reached is similar to that obtained in the absence of QD_F. The differential behaviour of the studied proteins (bovine serum albumin, fibrinogen and apotransferrin) has been discussed on the basis of the adsorption affinity of each protein towards DCM/water interface and their different sizes. Additionally, it has been clearly demonstrated that the proposed methodology can serve as a complementary technique to other reported direct and indirect methods for the evaluation of nanoparticle-protein interactions at low protein concentrations.

Involvement of two uptake mechanisms of gold and iron oxide nanoparticles in a co-exposure scenario using mouse macrophages.

Little is known about the simultaneous uptake of different engineered nanoparticle types, as it can be expected in our daily life. In order to test such co-exposure effects, murine macrophages (J774A.1 cell line) were incubated with gold (AuNPs) and iron oxide nanoparticles (FeO x NPs) either alone or combined. Environmental scanning electron microscopy revealed that single NPs of both types bound within minutes on the cell surface but with a distinctive difference between FeO x NPs and AuNPs. Uptake analysis studies based on laser scanning microscopy, transmission electron microscopy, and inductively coupled plasma optical emission spectrometry revealed intracellular appearance of both NP types in all exposure scenarios and a time-dependent increase. This increase was higher for both AuNPs and FeO x NPs during co-exposure. Cells treated with endocytotic inhibitors recovered after co-exposure, which additionally hinted that two uptake mechanisms are involved. Cross-talk between uptake pathways is relevant for toxicological studies: Co-exposure acts as an uptake accelerant. If the goal is to maximize the cellular uptake, e.g., for the delivery of pharmaceutical agents, this can be beneficial. However, co-exposure should also be taken into account in the case of risk assessment of occupational settings. The demonstration of co-exposure-invoked pathway interactions reveals that synergetic nanoparticle effects, either positive or negative, must be considered for nanotechnology and nanomedicine in particular to develop to its full potential.

Metabolic pathway for the universal fluorescent recognition of tumor cells.

Quantification of circulating tumor cells (CTCs) in blood samples from cancer patients is a non-invasive approach to monitoring the status of the disease. Most of the methods proposed in the recent years are phenomenological and rely on the use of antibodies labelled with fluorophores, magnetic particles, or immobilized on surfaces to capture the CTCs. Herein, we designed and optimized a method that employs a glucose analogue labelled with a fluorophore which takes advantage of the different metabolic pathways of cancer cells to discern them from normal ones. Notably, we demonstrate that fluorescence signal in tumor cells can be greatly maximized by applying hyperoxia conditions without damaging the cells. These results are demonstrated by means of confocal fluorescence and flow-cytometry measurements in peripheral blood mononuclear cells (PBMC) extracted after Ficoll of human blood samples and spiked with a known concentration of MCF-7 tumor cells.

Maintenance of cellular respiration indicates drug resistance in acute myeloid leukemia.

Primary resistance to induction therapy is an unsolved clinical problem in acute myeloid leukemia (AML). Here we investigated drug resistance in AML at the level of cellular metabolism in order to identify early predictors of therapeutic response. Using extracellular flux analysis, we compared metabolic drug responses in AML cell lines sensitive or resistant to cytarabine or sorafenib after 24h of drug treatment to a small cell lung cancer (SCLC) cell line exposed to etoposide. Only drug-resistant AML cells maintained oxidative metabolism upon drug exposure while SCLC cells displayed an overall metabolic shift towards glycolysis, i.e. a Warburg effect to escape drug toxicity. Moreover, primary AML blasts displayed very low glycolytic activity, while oxygen consumption was readily detectable, indicating an essential role of oxidative pathways in the bioenergetics of AML blasts. In line with these observations, analysis of the mitochondrial membrane potential using tetramethylrhodamine ethyl ester staining and flow cytometry allowed for clear discrimination between drug sensitive and resistant AML cell line clones and primary blasts after 24h of treatment with cytarabine or sorafenib. Our data reveal a distinct metabolic phenotype of resistant AML cells and suggest that disrupting oxidative metabolism rather than glycolysis may enhance the cytotoxic effects of chemotherapy in AML.

Direct protein quantification in complex sample solutions by surface-engineered nanorod probes.

Detecting biomarkers from complex sample solutions is the key objective of molecular diagnostics. Being able to do so in a simple approach that does not require laborious sample preparation, sophisticated equipment and trained staff is vital for point-of-care applications. Here, we report on the specific detection of the breast cancer biomarker sHER2 directly from serum and saliva samples by a nanorod-based homogeneous biosensing approach, which is easy to operate as it only requires mixing of the samples with the nanorod probes. By careful nanorod surface engineering and homogeneous assay design, we demonstrate that the formation of a protein corona around the nanoparticles does not limit the applicability of our detection method, but on the contrary enables us to conduct in-situ reference measurements, thus further strengthening the point-of-care applicability of our method. Making use of sandwich assays on top of the nanorods, we obtain a limit of detection of 110 pM and 470 pM in 10-fold diluted spiked saliva and serum samples, respectively. In conclusion, our results open up numerous applications in direct protein biomarker quantification, specifically in point-of-care settings where resources are limited and ease-of-use is of essence.