In Vitro and In Vivo Studies on HPMA-Based Polymeric Micelles Loaded with Curcumin.

Curcumin-loaded polymeric micelles composed of poly(ethylene glycol)-b-poly(N-2-benzoyloxypropyl methacrylamide) (mPEG-b-p(HPMA-Bz)) were prepared to solubilize and improve the pharmacokinetics of curcumin. Curcumin-loaded micelles were prepared by a nanoprecipitation method using mPEG5kDa-b-p(HPMA-Bz) copolymers with varying molecular weight of the hydrophobic block (5.2, 10.0, and 17.1 kDa). At equal curcumin loading, micelles composed of mPEG5kDa-b-p(HPMA-Bz)17.1kDa showed better curcumin retention in both phosphate-buffered saline (PBS) and plasma at 37 °C than micelles based on block copolymers with smaller hydrophobic blocks. No change in micelle size was observed during 24 h incubation in plasma using asymmetrical flow field-flow fractionation (AF4), attesting to particle stability. However, 22-49% of the curcumin loading was released from the micelles during 24 h from formulations with the highest to the lowest molecular weight p(HPMA-Bz), respectively, in plasma. AF4 analysis further showed that the released curcumin was subsequently solubilized by albumin. In vitro analyses revealed that the curcumin-loaded mPEG5kDa-b-p(HPMA-Bz)17.1kDa micelles were internalized by different types of cancer cells, resulting in curcumin-induced cell death. Intravenously administered curcumin-loaded, Cy7-labeled mPEG5kDa-b-p(HPMA-Bz)17.1kDa micelles in mice at 50 mg curcumin/kg showed a long circulation half-life for the micelles (t1/2 = 42 h), in line with the AF4 results. In contrast, the circulation time of curcumin was considerably shorter than that of the micelles (t1/2α = 0.11, t1/2ß = 2.5 h) but ∼5 times longer than has been reported for free curcumin (t1/2α = 0.02 h). The faster clearance of curcumin in vivo compared to in vitro studies can be attributed to the interaction of curcumin with blood cells. Despite the excellent solubilizing effect of these micelles, no cytostatic effect was achieved in neuroblastoma-bearing mice, possibly because of the low sensitivity of the Neuro2A cells to curcumin.

Quantification of the cellular dose and characterization of nanoparticle transport during in vitro testing.

BACKGROUND: The constant increase of the use of nanomaterials in consumer products is making increasingly urgent that standardized and reliable in vitro test methods for toxicity screening be made available to the scientific community. For this purpose, the determination of the cellular dose, i.e. the amount of nanomaterials effectively in contact with the cells is fundamental for a trustworthy determination of nanomaterial dose responses. This has often been overlooked in the literature making it difficult to undertake a comparison of datasets from different studies. Characterization of the mechanisms involved in nanomaterial transport and the determination of the cellular dose is essential for the development of predictive numerical models and reliable in vitro screening methods. RESULTS: This work aims to relate key physico-chemical properties of gold nanoparticles (NPs) to the kinetics of their deposition on the cellular monolayer. Firstly, an extensive characterization of NPs in complete culture cell medium was performed to determine the diameter and the apparent mass density of the formed NP-serum protein complexes. Subsequently, the kinetics of deposition were studied by UV-vis absorbance measurements in the presence or absence of cells. The fraction of NPs deposited on the cellular layer was found to be highly dependent on NP size and apparent density because these two parameters influence the NP transport. The NP deposition occurred in two phases: phase 1, which consists of cellular uptake driven by the NP-cell affinity, and phase 2 consisting mainly of NP deposition onto the cellular membrane. CONCLUSION: The fraction of deposited NPs is very different from the initial concentration applied in the in vitro assay, and is highly dependent of the size and density of the NPs, on the associated transport rate and on the exposure duration. This study shows that an accurate characterization is needed and suitable experimental conditions such as initial concentration of NPs and liquid height in the wells has to be considered since they strongly influence the cellular dose and the nature of interactions of NPs with the cells.

Proteolytic control of TGF-ß co-receptor activity by BMP-1/tolloid-like proteases revealed by quantitative iTRAQ proteomics.

The metalloproteinase BMP-1 (bone morphogenetic protein-1) plays a major role in the control of extracellular matrix (ECM) assembly and growth factor activation. Most of the growth factors activated by BMP-1 are members of the TGF-ß superfamily known to regulate multiple biological processes including embryonic development, wound healing, inflammation and tumor progression. In this study, we used an iTRAQ (isobaric tags for relative and absolute quantification)-based quantitative proteomic approach to reveal the release of proteolytic fragments from the cell surface or the ECM by BMP-1. Thirty-eight extracellular proteins were found in significantly higher or lower amounts in the conditioned medium of HT1080 cells overexpressing BMP-1 and thus, could be considered as candidate substrates. Strikingly, three of these new candidates (betaglycan, CD109 and neuropilin-1) were TGF-ß co-receptors, also acting as antagonists when released from the cell surface, and were chosen for further substrate validation. Betaglycan and CD109 proved to be directly cleaved by BMP-1 and the corresponding cleavage sites were extensively characterized using a new mass spectrometry approach. Furthermore, we could show that the ability of betaglycan and CD109 to interact with TGF-ß was altered after cleavage by BMP-1, leading to increased and prolonged SMAD2 phosphorylation in BMP-1-overexpressing cells. Betaglycan processing was also observed in primary corneal keratocytes, indicating a general and novel mechanism by which BMP-1 directly affects signaling by controlling TGF-ß co-receptor activity. The proteomic data have been submitted to ProteomeXchange with the identifier PXD000786 and doi: 10.6019/PXD000786 .

Correlation between in vitro stability and pharmacokinetics of poly(ε-caprolactone)-based micelles loaded with a photosensitizer.

Polymeric micelles are extensively investigated as drug delivery systems for hydrophobic drugs including photosensitizers (PSs). In order to benefit from micelles as targeted delivery systems for PS, rather than only solubilizers, the stability and cargo retention of the (PS-loaded) micelles should be properly assessed in biologically relevant media to get insight into the essential parameters predicting their in vivo performance (i.e., pharmacokinetics). In the present study, asymmetric flow field-flow fractionation (AF4) was used to investigate the in vitro stability in human plasma of empty and meta-tetra(hydroxyphenyl)chlorin (mTHPC)-loaded dithiolane-crosslinked micelles based on poly(ɛ-caprolactone)-co-poly(1,2-dithiolane­carbonate)-b-poly(ethylene glycol) (p(CL-co-DTC)-PEG) and non (covalently)-crosslinked micelles composed of poly(ε-caprolactone)-b-poly(ethylene glycol) (pCL-PEG). AF4 allows separation of the micelles from plasma proteins, which showed that small non (covalently)-crosslinked pCL9-PEG (17 nm) and pCL15-PEG (22 nm) micelles had lower stability in plasma than pCL23-PEG micelles with larger size (43 nm) and higher degree of crystallinity of pCL, and had also lower stability than covalently crosslinked p(CL9-DTC3.9)-PEG and p(CL18-DTC7.5)-PEG micelles with similar small sizes (~20 nm). In addition, PS (re)distribution to specific plasma proteins was observed by AF4, giving strong indications for the (in)stability of PS-loaded micelles in plasma. Nevertheless, fluorescence spectroscopy in human plasma showed that the retention of mTHPC in non (covalently)-crosslinked but semi-crystalline pCL23-PEG micelles (>8 h) was much longer than that in covalently crosslinked p(CL18-DTC7.5)-PEG micelles (~4 h). In line with this, in vivo circulation kinetics showed that pCL23-PEG micelles loaded with mTHPC had significantly longer half-life values (t½-ß of micelles and mTHPC was 14 and 18 h, respectively) than covalently crosslinked p(CL18-DTC7.5)-PEG micelles (t½-ß of both micelles and mTHPC was ~2 h). As a consequence, long circulating pCL23-PEG micelles resulted in significantly higher tumor accumulation of both the micelles and loaded mTHPC as compared to short circulating p(CL18-DTC7.5)-PEG micelles. These in vivo data were in good agreement with the in vitro stability studies. In conclusion, the present study points out that AF4 and fluorescence spectroscopy are excellent tools to evaluate the (in)stability of nanoparticles in biological media and thus predict the (in)stability of drug loaded nanoparticles after i.v. administration, which is favorable to screen promising delivery systems with reduced experimental time and costs and without excessive use of animals.

Rational design of multi-functional gold nanoparticles with controlled biomolecule adsorption: a multi-method approach for in-depth characterization.

Multi-functionalized nanoparticles are of great interest in biotechnology and biomedicine, especially for diagnostic and therapeutic purposes. However, at the moment the characterization of complex, multi-functional nanoparticles is still challenging and this hampers the development of advanced nanomaterials for biological applications. In this work, we have designed a model system consisting of gold nanoparticles functionalized with two differentially-terminated poly(ethylene oxide) ligands, providing both "stealth" properties and protein-binding capabilities to the nanoparticles. We use a combination of techniques (Centrifugal Liquid Sedimentation, Dynamic Light Scattering, Flow Field Flow Fractionation, Transmission Electron Microscopy, and Circular Dichroism) to: (i) monitor and quantify the ratios of ligand molecules per nanoparticle; (ii) determine the effect of coating density on non-specific protein adsorption; (iii) to assess the number and structure of the covalently-bound proteins. This article aims at comparing the complementary outcomes from typical and orthogonal techniques used in nanoparticle characterization by employing a versatile nanoparticle-ligands-biomolecule model system.

Gold nanoparticles increases UV and thermal stability of human serum albumin.

Ultraviolet (UV) radiation, temperature, and time can degrade proteins. Here, the authors show that gold nanoparticles significantly protect human serum albumin from denaturation when exposed to "stressing" conditions such as UV irradiation and sustained exposure in suboptimal conditions. In particular, the authors show that gold nanoparticles significantly reduce the decrease in secondary structure induced by UV irradiation or extended exposure to ambient temperature.

Highly Flexible Platform for Tuning Surface Properties of Silica Nanoparticles and Monitoring Their Biological Interaction.

The following work presents a simple, reliable and scalable seeding-growth methodology to prepare silica nanoparticles (SiO2 NPs) (20, 30, 50 and 80 nm) directly in aqueous phase, both as plain- as well as fluorescent-labeled silica. The amount of fluorescent label per particle remained constant regardless of size, which facilitates measurements in terms of number-based concentrations. SiO2 NPs in dispersion were functionalized with an epoxysilane, thus providing a flexible platform for the covalent linkage of wide variety of molecules under mild experimental conditions. This approach was validated with ethylenediamine, two different amino acids and three akylamines to generate a variety of surface modifications. Accurate characterization of particle size, size distributions, morphology and surface chemistry is provided, both for as-synthesized particles and after incubation in cell culture medium. The impact of physicochemical properties of SiO2 NPs was investigated with human alveolar basal epithelial cells (A549) such as the effect in cytotoxicity, cell internalization and membrane interaction.

Determination of the structure and morphology of gold nanoparticle-HSA protein complexes.

We propose a simple method to determine the structure and morphology of nanoparticle protein complexes. By combining a separation method with online size measurements, density measurements and circular dichroism, we could identify the number of proteins bound to each nanoparticle and their secondary structure changes in the complex. This method provides much-needed experimental information on the interaction of proteins with nanoparticles and on the behavior of nanoparticles in biological systems.

Singlet oxygen plays a key role in the toxicity and DNA damage caused by nanometric TiO2 in human keratinocytes.

Nanometric TiO2 has been reported to be cytotoxic and genotoxic in different in vitro models when activated by UV light. However, a clear picture of the species mediating the observed toxic effects is still missing. Here, a nanometric TiO2 powder has been modified at the surface to completely inhibit its photo-catalytic activity and to inhibit the generation of all reactive species except for singlet oxygen. The prepared powders have been tested for their ability to induce strand breaks in plasmid DNA and for their cytotoxicity and genotoxicity toward human keratinocyte (HaCaT) cells (100-500 µg mL(-1), 15 min UVA/B exposure at 216-36 mJ m(-2) respectively). The data reported herein indicate that the photo-toxicity of TiO2 is mainly triggered by particle-derived singlet oxygen. The data presented herein contribute to the knowledge of structure-activity relationships which are needed for the design of safe nanomaterials.