Evaluation and Screening of Biopharmaceuticals using Multi-Angle Dynamic Light Scattering.

Biopharmaceuticals are large, complex and labile therapeutic molecules prone to instability due to various factors during manufacturing. To ensure their safety, quality and efficacy, a wide range of critical quality attributes (CQAs) such as product concentration, aggregation, particle size, purity and turbidity have to be met. Size exclusion chromatography (SEC) is the gold standard to measure protein aggregation and degradation. However, other techniques such as dynamic light scattering (DLS) are employed in tandem to measure the particle size distribution (PSD) and polydispersity of biopharmaceutical formulations. In this study, the application of multi-angle dynamic light scattering (MADLS) was evaluated for the determination of particle size, particle concentration and aggregation in 3 different protein modalities, namely bovine serum albumin (BSA) and two biopharmaceuticals including a monoclonal antibody (mAb) and an enzyme. The obtained calibration curve (R2 > 0.95) for the particle number concentration of the 3 proteins and the observed correlation between MADLS and SEC (R2 = 0.9938) for the analysis of aggregation in the enzyme can be employed as a 3-in-1 approach to assessing particle size, concentration and aggregation for the screening and development of products while also reducing the number of samples and experiments required for analysis prior to other orthogonal tests.

Characterisation of particles in solution - a perspective on light scattering and comparative technologies.

We present here a perspective detailing the current state-of-the-art technologies for the characterisation of nanoparticles (NPs) in liquid suspension. We detail the technologies involved and assess their applications in the determination of NP size and concentration. We also investigate the parameters that can influence the results and put forward a cause and effect analysis of the principle factors influencing the measurement of NP size and concentration by NP tracking analysis and dynamic light scattering, to identify areas where uncertainties in the measurement can arise. Also included are technologies capable of characterising NPs in solution, whose measurements are not based on light scattering. It is hoped that the manuscript, with its detailed description of the methodologies involved, will assist scientists in selecting the appropriate technology for characterising their materials and enabling them to comply with regulatory agencies' demands for accurate and reliable NP size and concentration data.

Cellular uptake and biocompatibility of bismuth ferrite harmonic advanced nanoparticles.

Bismuth Ferrite (BFO) nanoparticles (BFO-NP) display interesting optical (nonlinear response) and magnetic properties which make them amenable for bio-oriented diagnostic applications as intra- and extra membrane contrast agents. Due to the relatively recent availability of this material in well dispersed nanometric form, its biocompatibility was not known to date. In this study, we present a thorough assessment of the effects of in vitro exposure of human adenocarcinoma (A549), lung squamous carcinoma (NCI-H520), and acute monocytic leukemia (THP-1) cell lines to uncoated and poly(ethylene glycol)-coated BFO-NP in the form of cytotoxicity, haemolytic response and biocompatibility. Our results support the attractiveness of the functional-BFO towards biomedical applications focused on advanced diagnostic imaging. FROM THE CLINICAL EDITOR: Bismuth Ferrite nanoparticles (BFO-NP) have been recently successfully introduced as photodynamic tools and imaging probes. However, how these nanoparticles interact with various cells at the cellular level remains poorly understood. In this study, the authors performed in vitro experiments to assess the effects of uncoated and PEG-coated BFO-NP in the form of cytotoxicity, haemolytic response and biocompatibility.

Heparin conjugated quantum dots for in vitro imaging applications.

In this work heparin-gelatine multi-layered cadmium telluride quantum dots (QDgel/hep) were synthesised using a novel 'one-pot' method. The QDs produced were characterised using various spectroscopic and physiochemical techniques. Suitable QDs were then selected and compared to thioglycolic acid stabilised quantum dots (QDTGA) and gelatine coated quantum dots (QDgel) for utilisation in in vitro imaging experiments on live and fixed permeabilised THP-1, A549 and Caco-2 cell lines. Exposure of live THP-1 cells to QDgel/hep resulted in localisation of the QDs to the nucleus of the cells. QDgel/hep show affinity for the nuclear compartment of fixed permeabilised THP-1 and A549 cells but remain confined to cytoplasm of fixed permeabilised Caco-2 cells. It is postulated that heparin binding to the CD11b receptor facilitates the internalisation of the QDs into the nucleus of THP-1 cells. In addition, the heparin layer may reduce the unfavourable thrombogenic nature of quantum dots observed in vivo. FROM THE CLINICAL EDITOR: In this study, heparin conjugated quantum dots were found to have superior imaging properties compared to its native counterparts. The authors postulate that heparin binding to the CD11b receptor facilitates QD internalization to the nucleus, and the heparin layer may reduce the in vivo thrombogenic properties of quantum dots.

A comparative analysis of extracellular vesicles (EVs) from human and feline plasma.

Extracellular vesicles (EVs) are nanoparticles found in all biological fluids, capable of transporting biological material around the body. Extensive research into the physiological role of EVs has led to the development of the Minimal Information for Studies of Extracellular Vesicles (MISEV) framework in 2018. This framework guides the standardisation of protocols in the EV field. To date, the focus has been on EVs of human origin. As comparative medicine progresses, there has been a drive to study similarities between diseases in humans and animals. To successfully research EVs in felines, we must validate the application of the MISEV guidelines in this group. EVs were isolated from the plasma of healthy humans and felines. EV characterisation was carried out according to the MISEV guidelines. Human and feline plasma showed a similar concentration of EVs, comparable expression of known EV markers and analogous particle to protein ratios. Mass spectrometry analyses showed that the proteomic signature of EVs from humans and felines were similar. Asymmetrical flow field flow fractionation, showed two distinct subpopulations of EVs isolated from human plasma, whereas only one subpopulation was isolated from feline plasma. Metabolomic profiling showed similar profiles for humans and felines. In conclusion, isolation, and characterisation of EVs from humans and felines show that MISEV2018 guidelines may also be applied to felines. Potential comparative medicine studies of EVs may provide a model for studying naturally occurring diseases in both humans and felines.

Folic acid modified gelatine coated quantum dots as potential reagents for in vitro cancer diagnostics.

BACKGROUND: Gelatine coating was previously shown to effectively reduce the cytotoxicity of CdTe Quantum Dots (QDs) which was a first step towards utilising them for biomedical applications. To be useful they also need to be target-specific which can be achieved by conjugating them with Folic Acid (FA). RESULTS: The modification of QDs with FA via an original "one-pot" synthetic route was proved successful by a range of characterisation techniques including UV-visible absorption spectroscopy, Photoluminescence (PL) emission spectroscopy, fluorescence life-time measurements, Transmission Electron Microscopy (TEM) and Dynamic Light Scattering (DLS). The resulting nanocomposites were tested in Caco-2 cell cultures which over-express FA receptors. The presence of FA on the surface of QDs significantly improved the uptake by targeted cells. CONCLUSIONS: The modification with folic acid enabled to achieve a significant cellular uptake and cytotoxicity towards a selected cancer cell lines (Caco-2) of gelatine-coated TGA-CdTe quantum dots, which demonstrated good potential for in vitro cancer diagnostics.

Investigating the Potential and Pitfalls of EV-Encapsulated MicroRNAs as Circulating Biomarkers of Breast Cancer.

Extracellular vesicles (EVs) shuttle microRNA (miRNA) throughout the circulation and are believed to represent a fingerprint of the releasing cell. We isolated and characterized serum EVs of breast tumour-bearing animals, breast cancer (BC) patients, and healthy controls. EVs were characterized using transmission electron microscopy (TEM), protein quantification, western blotting, and nanoparticle tracking analysis (NTA). Absolute quantitative (AQ)-PCR was employed to analyse EV-miR-451a expression. Isolated EVs had the appropriate morphology and size. Patient sera contained significantly more EVs than did healthy controls. In tumour-bearing animals, a correlation between serum EV number and tumour burden was observed. There was no significant relationship between EV protein yield and EV quantity determined by NTA, highlighting the requirement for direct quantification. Using AQ-PCR to relate miRNA copy number to EV yield, a significant increase in miRNA-451a copies/EV was detected in BC patient sera, suggesting potential as a novel biomarker of breast cancer.

The use of Raman microscopy to determine and localize vitamin E in biological samples.

Alpha-tocopherol (aT), the predominant form of vitamin E in mammals, is thought to prevent oxidation of polyunsaturated fatty acids. In the lung, aT is perceived to be accumulated in alveolar type II cells and secreted together with surfactant into the epithelial lining fluid. Conventionally, determination of aT and related compounds requires extraction with organic solvents. This study describes a new method to determine and image the distribution of aT and related compounds within cells and tissue sections using the light-scattering technique of Raman microscopy to enable high spatial as well as spectral resolution. This study compared the nondestructive analysis by Raman microscopy of vitamin E, in particular aT, in biological samples with data obtained using conventional HPLC analysis. Raman spectra were acquired at spatial resolutions of 2-0.8 microm. Multivariate analysis techniques were used for analyses and construction of corresponding maps showing the distribution of aT, alpha-tocopherol quinone (aTQ), and other constituents (hemes, proteins, DNA, and surfactant lipids). A combination of images enabled identification of colocalized constituents (heme/aTQ and aT/surfactant lipids). Our data demonstrate the ability of Raman microscopy to discriminate between different tocopherols and oxidation products in biological specimens without sample destruction. By enabling the visualization of lipid-protein interactions, Raman microscopy offers a novel method of investigating biological characterization of lipid-soluble compounds, including those that may be embedded in biological membranes such as aT.

Towards the Identification of an In Vitro Tool for Assessing the Biological Behavior of Aerosol Supplied Nanomaterials.

Nanoparticles (NP)-based inhalation systems for drug delivery can be administered in liquid form, by nebulization or using pressurized metered dose inhalers, and in solid form by means of dry powder inhalers. However, NP delivery to the lungs has many challenges including the formulation instability due to particle-particle interactions and subsequent aggregation, causing poor deposition in the small distal airways and subsequent alveolar macrophages activity, which could lead to inflammation. This work aims at providing an in vitro experimental design for investigating the correlation between the physico-chemical properties of NP, and their biological behavior, when they are used as NP-based inhalation treatments, comparing two different exposure systems. By means of an aerosol drug delivery nebulizer, human lung cells cultured at air-liquid interface (ALI) were exposed to two titanium dioxide NP (NM-100 and NM-101), obtained from the JRC repository. In parallel, ALI cultures were exposed to NP suspension by direct inoculation, i.e., by adding the NP suspensions on the apical side of the cell cultures with a pipette. The formulation stability of NP, measured as hydrodynamic size distributions, the cell viability, cell monolayer integrity, cell morphology and pro-inflammatory cytokines secretion were investigated. Our results demonstrated that the formulation stability of NM-100 and NM-101 was strongly dependent on the aggregation phenomena that occur in the conditions adopted for the biological experiments. Interestingly, comparable biological data between the two exposure methods used were observed, suggesting that the conventional exposure coupled to ALI culturing conditions offers a relevant in vitro tool for assessing the correlation between the physico-chemical properties of NP and their biological behavior, when NP are used as drug delivery systems.

Are existing standard methods suitable for the evaluation of nanomedicines: some case studies.

The use of nanotechnology in medical products has been demonstrated at laboratory scale, and many resulting nanomedicines are in the translational phase toward clinical applications, with global market trends indicating strong growth of the sector in the coming years. The translation of nanomedicines toward the clinic and subsequent commercialization may require the development of new or adaptation of existing standards to ensure the quality, safety and efficacy of such products. This work addresses some identified needs, and illustrates the shortcomings of currently used standardized methods when applied to medical-nanoparticles to assess particle size, drug loading, drug release and in vitro safety. Alternative physicochemical, and in vitro toxicology methods, with the potential to qualify as future standards supporting the evaluation of nanomedicine are provided.

The anticoagulant properties of cadmium telluride quantum dots.

The size-dependent optical properties of quantum dots (QDs) are frequently exploited for use in medical imaging and labelling applications. Similarly, presented here, they also elicit profound size-dependent anticoagulant properties. Cadmium telluride quantum dot (QDs) (3.2 nm) were shown to have a dramatic anticoagulant effect centred on around the intrinsic coagulation pathway, compared to their 3.6 nm counterparts. Several clinically relevant diagnostic tests were carried out over a concentration range of the QDs and demonstrated that the 3.2 nm QDs elicited their response on the intrinsic pathway as a whole, yet the activity of the individual intrinsic coagulation factors was not affected. The mechanism appears also to be strongly influenced by the concentration of calcium ions and not cadmium ions leached from the QDs. Static and shear-based primary haemostasis assays were also carried out, demonstrating a profound anticoagulant effect which was independent of platelets and phospholipids. The data presented here suggest that the physical-chemical properties of the QDs may have a role in the modulation of haemostasis and the coagulation cascade, in a yet not fully understood mechanism. This study has implications for the use of similar QDs as diagnostic or therapeutic tools in vivo, and for the occupational health and safety of those working with such materials.

Urinary nanovesicles captured by lectins or antibodies demonstrate variations in size and surface glycosylation profile.

AIM: The use of carbohydrate-binding proteins (lectins) to isolate urinary extracellular vesicles (uEVs) was investigated and the captured subpopulations were characterized. METHODS: Pooled uEVs from multiple healthy donors were exposed to lectin-conjugated or antibody-conjugated beads. Recovered uEVs were evaluated by protein estimation, transmission electron microscopy, nanoparticle tracking analysis and lectin microarray profiling. RESULTS: uEVs isolated by lectin- and antibody-based affinity capture exhibited distinct variations in size and surface content. Transmission electron microscopy confirmed similar EV diameters to those established by nanoparticle tracking analysis, but total particle counts did not correlate closely with protein-based quantification. Lectin microarray profiling demonstrated capture-dependent differences in surface glycosylation. CONCLUSION: Selective, carbohydrate-mediated EV isolation by lectin affinity approaches may prove immediately useful for research and find eventual use in clinical applications.

Targeted polyethylene glycol gold nanoparticles for the treatment of pancreatic cancer: from synthesis to proof-of-concept in vitro studies.

The main objective of this study was to optimize and characterize a drug delivery carrier for doxorubicin, intended to be intravenously administered, capable of improving the therapeutic index of the chemotherapeutic agent itself, and aimed at the treatment of pancreatic cancer. In light of this goal, we report a robust one-step method for the synthesis of dicarboxylic acid-terminated polyethylene glycol (PEG)-gold nanoparticles (AuNPs) and doxorubicin-loaded PEG-AuNPs, and their further antibody targeting (anti-Kv11.1 polyclonal antibody [pAb]). In in vitro proof-of-concept studies, we evaluated the influence of the nanocarrier and of the active targeting functionality on the anti-tumor efficacy of doxorubicin, with respect to its half-maximal effective concentration (EC50) and drug-triggered changes in the cell cycle. Our results demonstrated that the therapeutic efficacy of doxorubicin was positively influenced not only by the active targeting exploited through anti-Kv11.1-pAb but also by the drug coupling with a nanometer-sized delivery system, which indeed resulted in a 30-fold decrease of doxorubicin EC50, cell cycle blockage, and drug localization in the cell nuclei. The cell internalization pathway was strongly influenced by the active targeting of the Kv11.1 subunit of the human Ether-à-go-go related gene 1 (hERG1) channel aberrantly expressed on the membrane of pancreatic cancer cells. Targeted PEG-AuNPs were translocated into the lysosomes and were associated to an increased lysosomal function in PANC-1 cells. Additionally, doxorubicin release into an aqueous environment was almost negligible after 7 days, suggesting that drug release from PEG-AuNPs was triggered by enzymatic activity. Although preliminary, data gathered from this study have considerable potential in the application of safe-by-design nano-enabled drug-delivery systems (ie, nanomedicines) for the treatment of pancreatic cancer, a disease with a poor prognosis and one of the main current burdens of today's health care bill of industrialized countries.

A safe-by-design approach to the development of gold nanoboxes as carriers for internalization into cancer cells.

Gold nanomaterials are currently raising a significant interest for human welfare in the field of clinical diagnosis, therapeutics for chronic pathologies, as well as of many other biomedical applications. In particular, gold nanomaterials are becoming a promising technology for developing novel approaches and treatments against widespread societal diseases such as cancer. In this study, we investigated the potential of proprietary gold nanoboxes (AuNBs) as carriers for their perspective translation into multifunctional, pre-clinical nano-enabled systems for personalized medicine approaches against lung cancer. A safe-by-design, tiered approach, with systematic tests conducted in the early phases on uncoated AuNBs and more focused testing on the coated, drug-loaded nanomaterial toward the end, was adopted. Our results showed that uncoated AuNBs could effectively penetrate into human lung adenocarcinoma (A549) cells when in simple (mono-cultures) or complex (co- and three-dimensional-cultures) in vitro microenvironments mimicking the alveolar region of human lungs. Uncoated AuNBs were biologically inert in A549 cells and demonstrated signs of biodegradability. Concurrently, preliminary data revealed that coated, drug-loaded AuNBs could efficiently deliver a chemotherapeutic agent to A549 cells, corroborating the hypothesis that AuNBs could be used in the future for the development of personalized nano-enabled systems for lung cancer treatment.

Interlaboratory comparison of size measurements on nanoparticles using nanoparticle tracking analysis (NTA).

One of the key challenges in the field of nanoparticle (NP) analysis is in producing reliable and reproducible characterisation data for nanomaterials. This study looks at the reproducibility using a relatively new, but rapidly adopted, technique, Nanoparticle Tracking Analysis (NTA) on a range of particle sizes and materials in several different media. It describes the protocol development and presents both the data and analysis of results obtained from 12 laboratories, mostly based in Europe, who are primarily QualityNano members. QualityNano is an EU FP7 funded Research Infrastructure that integrates 28 European analytical and experimental facilities in nanotechnology, medicine and natural sciences with the goal of developing and implementing best practice and quality in all aspects of nanosafety assessment. This study looks at both the development of the protocol and how this leads to highly reproducible results amongst participants. In this study, the parameter being measured is the modal particle size.