Advanced characterisation methods for the analysis of nanoformulations and extracellular vesicles

Nanomedicine represents a challenging and highly multidisciplinary research field, concerned with the development and study of nanoformulations for diagnostic and/or therapeutic purposes. The nano-sized particles of interest are increasingly complex. Their translational potential has been hampered by difficulties in their thorough characterisation. On the nano scale, small variations in size and composition can have large implications for their pharmacodynamic and pharmacokinetic behaviour. More precise techniques are therefore required to address these challenges. This thesis describes novel, advanced characterisation methods designed for the detailed study of single nanoparticles and their interaction and uptake behaviour with cells. A platform technology for Single Particle Automated Raman Trapping Analysis – SPARTA - was developed, capable of non-destructive, label-free and automated comprehensive single particle analysis. With the SPARTA system, the composition, functionalisation, size and dynamic reactions on the surface can be investigated in detail, of a wide variety of nanoparticles, through their Raman spectra. A further improved, custom designed SPARTA 2.0 platform was built, optimised for the analysis of complex biological particles, such as EVs. EVs represent a high potential as biomarkers, studied here in the context of breast cancer. The SPARTA 2.0 platform was able to resolve compositional differences between non-cancerous and cancer cell-derived EVs with excellent sensitivity and specificity. This highlights the possibility for development of new minimally invasive diagnostic approaches. In addition, a new imaging strategy for investigation of the EV-cellular interaction is presented, based on 3D Focused Ion Beam – Scanning Electron Microscopy (FIB-SEM). FIB-SEM allows the generation of 3D models of the subcellular structure and visualisation of the cellular trafficking of nanoparticles. This represents a powerful new approach for investigating EV uptake. The methods developed in this thesis allow for the single particle-based analysis of a wide variety of nanoformulations and EVs, to aid in understanding their composition, applicability and cellular interactions.Open Acces

Coupling Lipid Nanoparticle Structure and Automated Single Particle Composition Analysis to Design Phospholipase Responsive Nanocarriers

Lipid nanoparticles (LNPs) are versatile structures with tunable physicochemical properties that are ideally suited as a platform for vaccine delivery and RNA therapeutics. A key barrier to LNP rational design is the inability to relate composition and structure to intracellular processing and function. Here we combine Single Particle Automated Raman Trapping Analysis (SPARTA®) with small angle scattering (SAXS / SANS) techniques to link LNP composition with internal structure and morphology and to monitor dynamic LNP - phospholipase D (PLD) interactions. Our analysis demonstrates that phospholipase D, a key intracellular trafficking mediator, can access the entire LNP lipid membrane to generate stable, anionic LNPs. PLD activity on vesicles with matched amounts of enzyme substrate was an order of magnitude lower, indicating that the LNP lipid membrane structure can be used to control enzyme interactions. This represents an opportunity to design enzyme-responsive LNP solutions for stimuli-responsive delivery and diseases where PLD is dysregulated

In Situ Gold Nanoparticle Gradient Formation in a 3D Meso- and Macroporous Polymer Matrix

Herein, the development and characterization of a 3D gradient structure of gold nanoparticles is described. The gradient of gold nanoparticles is made in situ in a macroporous nonionic block copolymer hydrogel matrix, through gold ion diffusion control. The polymer provides a matrix for diffusion of gold ions, acts as a template for controlling nanoparticle growth, and facilitates the in situ reduction of gold ions to gold nanoparticles. A clear gradient in gold nanoparticles is observed across the 3D space of the polymer matrix using scanning electron microscopy, fluorescence microscopy, atomic force microscopy, and thermogravimetric analysis. The particle gradient is further functionalized with both hydrophobic and hydrophilic groups via thiol-gold linkage to demonstrate the ability to form gradients with different chemical functionalities. Using additive manufacturing, the polymer can also be printed as a porous network with possible applications for 3D cell culturing in, e.g., biomaterials research

Shape-dependent antibacterial effects of non-cytotoxic gold nanoparticles

Gold nanoparticles (AuNPs) of various shapes (including spheres, stars and flowers), with similar dimensions, were synthesized and evaluated for their antibacterial effects toward Staphylococcus aureus, a bacterium responsible for numerous life-threatening infections worldwide. Optical growth curve measurements and Gompertz modeling showed significant AuNP shape- and concentration-dependent decreases in bacterial growth with increases in bacterial growth lag time. To evaluate prospective use in in vivo systems, the cytotoxicity of the same AuNPs was evaluated toward human dermal fibroblasts in vitro by 3-(4,5 dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tet razolium (MTS) viability assays and confocal microscopy. No indication of any mammalian cell toxicity or morphological effects was found. Additionally, it was observed that the AuNPs were readily internalized in fibroblasts after 4 days of incubation. Most importantly, the results of the present study showed that gold nanoflowers in particular possessed the most promising non-cytotoxic mammalian cell behavior with the greatest shape-dependent antibacterial activity-promising properties for their future investigation in a wide range of anti-infection applications

The use of Matti : a tangible user interface in physical rehabilitation to motivate children and older adults

Abstract: Highly motivated patients who enjoy their physical rehabilitation tend to attain better therapeutic outcomes. However, given the prolonged repetitive nature of postural control rehabilitation in older adults and children with developmental coordination disorder (DCD), motivation levels might drop quickly. Exergames and digital therapy tools could offer practical answers to this issue and allow therapists to provide patients objective outcome measurements. To incorporate digital innovation in clinical and rehabilitation practices, this study discusses Matti, an interactive system created as a customisable tangible user interface (TUI). Its usability in pediatric and geriatric physiotherapy contexts is explored and evaluated through user evaluation. The Matti device was found to be usable and enjoyable for exergaming rehabilitation. However, additional study on measuring capabilities is needed to enable the accurate and reliable objective outcome measurements through this TUI. Future research should analyse how this TUI and gamified postural control assessment affects patients' motivation and therapeutic outcomes

Seko : smart system for assisting home-based rehabilitation of knee arthroplasty patients

Knee arthroplasty is a surgical procedure that replaces severely arthritic or damaged knee joints. After undergoing this commonly performed procedure, there are still some patients who never fully recover. One of the reasons is the lack of patient compliance to the prescribed rehabilitation schedule. This paper describes the development of SEKO, a smart system which has the goal of improving the compliance of patients to the rehabilitation schedule after a knee arthroplasty. This is done by providing a system which connects a smart brace to a patient and his/her physiotherapist. The system exists out of three parts: the smart brace which uses multiple sensors that gather data of the movements and exercises performed by the patient, the mobile application which uses the gathered data to give feedback to the patient, and the web application which uses the data to keep the patient’s physiotherapist up to date on the rehabilitation performance and progress of the patient. The result is a proof of concept in the form of a fully functioning prototype, which was preliminary evaluated on the technical aspects and the usage scenario

In Vivo Biocompatibility and Immunogenicity of Metal-Phenolic Gelation

In vivo forming hydrogels are of interest for diverse biomedical applications due to their ease-of-use and minimal invasiveness and therefore high translational potential. Supramolecular hydrogels that can be assembled using metal–phenolic coordination of naturally occurring polyphenols and group IV metal ions (e.g. TiIV or ZrIV) provide a versatile and robust platform for engineering such materials. However, the in situ formation and in vivo response to this new class of materials has not yet been reported. Here, we demonstrate that metal–phenolic supramolecular gelation occurs successfully in vivo and we investigate the host response to the material over 14 weeks. The TiIV–tannic acid materials form stable gels that are well-tolerated following subcutaneous injection. Histology reveals a mild foreign body reaction, and titanium biodistribution studies show low accumulation in distal tissues. Compared to poloxamer-based hydrogels (commonly used for in vivo gelation), TiIV–tannic acid materials show substantially improved in vitro drug loading and release profile for the corticosteroid dexamethasone (from 10 days). These results provide essential in vivo characterization for this new class of metal–phenolic hydrogels, and highlight their potential suitability for biomedical applications in areas such as drug delivery and regenerative medicine.<br /

Surfactant Protein B promotes cytosolic SiRNA delivery by adopting a Virus-like mechanism of action

RNA therapeutics are poised to revolutionize medicine. To unlock the full potential of RNA drugs, safe and efficient (nano)formulations to deliver them inside target cells are required. Endosomal sequestration of nanocarriers represents a major bottleneck in nucleic acid delivery. Gaining more detailed information on the intracellular behavior of RNA nanocarriers is crucial to rationally develop delivery systems with improved therapeutic efficiency. Surfactant protein B (SPB) is a key component of pulmonary surfactant (PS), essential for mammalian breathing. In contrast to the general belief that PS should be regarded as a barrier for inhaled nanomedicines, we recently discovered the ability of SP-B to promote gene silencing by siRNA-loaded and lipid-coated nanogels. However, the mechanisms governing this process are poorly understood. The major objective of this work was to obtain mechanistic insights into the SP-B-mediated cellular delivery of siRNA. To this end, we combined siRNA knockdown experiments, confocal microscopy, and focused ion beam scanning electron microscopy imaging in an in vitro non-small-cell lung carcinoma model with lipid mixing assays on vesicles that mimic the composition of (intra)cellular membranes. Our work highlights a strong correlation between SP-B-mediated fusion with anionic endosomal membranes and cytosolic siRNA delivery, a mode of action resembling that of certain viruses and virus-derived cell-penetrating peptides. Building on these gained insights, we optimized the SP-B proteolipid composition, which dramatically improved delivery efficiency. Altogether, our work provides a mechanistic understanding of SP-B-induced perturbation of intracellular membranes, offering opportunities to fuel the rational design of SP-B-inspired RNA nanoformulations for inhalation therapy

Potent Virustatic Polymer-Lipid Nanomimics Block Viral Entry and Inhibit Malaria Parasites In Vivo

Infectious diseases continue to pose a substantial burden on global populations, requiring innovative broad-spectrum prophylactic and treatment alternatives. Here, we have designed modular synthetic polymer nanopartides that mimic functional components of host cell membranes, yielding multivalent nano-mimics that act by directly binding to varied pathogens. Nanomimic blood circulation time was prolonged by reformulating polymer-lipid hybrids. Femtomolar concentrations of the polymer nanomimics were sufficient to inhibit herpes simplex virus type 2 (HSV-2) entry into epithelial cells, while higher doses were needed against severe acute respiratory syndrome comnavirus 2 (SARS-CoV-2). Given their observed virustatic mode of action, the nanomimics were also tested with malaria parasite blood-stage merozoites, which lose their invasive capacity after a few minutes. Efficient inhibition of merozoite invasion of red blood cells was demonstrated both in vitro and in vivo using a preclinical rodent malaria model. We envision these nanomimics forming an adaptable platform for developing pathogen entry inhibitors and as immunomodulators, wherein nanomimic-inhibited pathogens can be secondarily targeted to sites of immune recognition