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Radboud University, 10 februari 2020, Promotor : Vries, I.J.M. de Co-promotores : Srinivas, M., Koshkina, O., Tagit, O., Contains fulltext : 215256.pdf (publisher's version ) (Open Access)

Contains fulltext : 229164.pdf (Publisher’s version ) (Open Access), Perfluorocarbons hold great promise both as imaging agents, particularly for (19)F MRI, and in therapy, such as oxygen delivery. (19)F MRI is unique in its ability to unambiguously track and quantify a tracer while maintaining anatomic context, and without the use of ionizing radiation. This is particularly well-suited for inflammation imaging and quantitative cell tracking. However, perfluorocarbons, which are best suited for imaging - like perfluoro-15-crown-5 ether (PFCE) - tend to have extremely long biological retention. Here, we showed that the use of a multi-core PLGA nanoparticle entrapping PFCE allows for a 15-fold reduction of half-life in vivo compared to what is reported in literature. This unexpected rapid decrease in (19)F signal was observed in liver, spleen and within the infarcted region after myocardial infarction and was confirmed by whole body NMR spectroscopy. We demonstrate that the fast clearance is due to disassembly of the ~200 nm nanoparticle into ~30 nm domains that remain soluble and are cleared quickly. We show here that the nanoparticle ultrastructure has a direct impact on in vivo clearance of its cargo i.e. allowing fast release of PFCE, and therefore also bringing the possibility of multifunctional nanoparticle-based imaging to translational imaging, therapy and diagnostics.

Contains fulltext : 217381.pdf (publisher's version ) (Open Access)

Contains fulltext : 229134.pdf (publisher's version ) (Open Access), Perfluorocarbon-loaded nanoparticles are powerful theranostic agents, which are used in the therapy of cancer and stroke and as imaging agents for ultrasound and (19)F magnetic resonance imaging (MRI). Scaling up the production of perfluorocarbon-loaded nanoparticles is essential for clinical translation. However, it represents a major challenge as perfluorocarbons are hydrophobic and lipophobic. We developed a method for continuous-flow production of perfluorocarbon-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles using a modular microfluidic system, with sufficient yields for clinical use. We combined two slit interdigital micromixers with a sonication flow cell to achieve efficient mixing of three phases: liquid perfluorocarbon, PLGA in organic solvent, and aqueous surfactant solution. The production rate was at least 30 times higher than with the conventional formulation. The characteristics of nanoparticles can be adjusted by changing the flow rates and type of solvent, resulting in a high PFC loading of 20-60 wt % and radii below 200 nm. The nanoparticles are nontoxic, suitable for (19)F MRI and ultrasound imaging, and can dissolve oxygen. In vivo (19)F MRI with perfluoro-15-crown-5 ether-loaded nanoparticles showed similar biodistribution as nanoparticles made with the conventional method and a fast clearance from the organs. Overall, we developed a continuous, modular method for scaled-up production of perfluorocarbon-loaded nanoparticles that can be potentially adapted for the production of other multiphase systems. Thus, it will facilitate the clinical translation of theranostic agents in the future.

Contains fulltext : 204774.pdf (publisher's version ) (Open Access)

Item does not contain fulltext, Nano- and microparticles have increasingly widespread applications in nanomedicine, ranging from drug delivery to imaging. Poly(lactic-co-glycolic acid) (PLGA) particles are the most widely-applied type of particles due to their biocompatibility and biodegradability. Here, we discuss the preparation of PLGA particles, and various modifications to tailor particles for applications in biological systems. We highlight new preparation approaches, including microfluidics and PRINT method, and modifications of PLGA particles resulting in novel or responsive properties, such as Janus or upconversion particles. Finally, we describe how the preparation methods can- and should-be adapted to tailor the properties of particles for the desired biomedical application. Our aim is to enable researchers who work with PLGA particles to better appreciate the effects of the selected preparation procedure on the final properties of the particles and its biological implications. STATEMENT OF SIGNIFICANCE: Nanoparticles are increasingly important in the field of biomedicine. Particles made of polymers are in the spotlight, due to their biodegradability, biocompatibility, versatility. In this review, we aim to discuss the range of formulation techniques, manipulations, and applications of poly(lactic-co-glycolic acid) (PLGA) particles, to enable a researcher to effectively select or design the optimal particles for their application. We describe the various techniques of PLGA particle synthesis and their impact on possible applications. We focus on recent developments in the field of PLGA particles, and new synthesis techniques that have emerged over the past years. Overall, we show how the chemistry of PLGA particles can be adapted to solve pressing biological needs.

Contains fulltext : 183916.pdf (publisher's version ) (Open Access)

Contains fulltext : 193511.pdf (publisher's version ) (Open Access), Photoacoustic imaging (PAI) is an emerging biomedical imaging technique that is now coming to the clinic. It has a penetration depth of a few centimeters and generates useful endogenous contrast, particularly from melanin and oxy-/deoxyhemoglobin. Indocyanine green (ICG) is a Food and Drug Administration-approved contrast agents for human applications, which can be also used in PAI. It is a small molecule dye with limited applications due to its fast clearance, rapid protein binding, and bleaching effect. Methods: Here, we entrap ICG in a poly(lactic-co-glycolic acid) nanoparticles together with a perfluorocarbon (PFC) using single emulsion method. These nanoparticles and nanoparticle-loaded dendritic cells were imaged with PA, (19)F MR, and fluorescence imaging in vitro and in vivo. Results: We formulated particles with an average diameter of 200 nm. The encapsulation of ICG within nanoparticles decreased its photobleaching and increased the retention of the signal within cells, making it available for applications such as cell imaging. As little as 0.1x10(6) cells could be detected in vivo with PAI using automated spectral unmixing. Furthermore, we observed the accumulation of ICG signal in the lymph node after subcutaneous injection of nanoparticles. Conclusion: We show that we can label primary human dendritic cells with the nanoparticles and image them in vitro and in vivo, in a multimodal manner. This work demonstrates the potential of combining PAI and (19)F MRI for cell imaging and lymph node detection using nanoparticles that are currently produced at GMP-grade for clinical use.

Contains fulltext : 177175.pdf (Publisher’s version ) (Open Access), Calcium phosphate cement (CPC) is used in bone repair because of its biocompatibility. However, high similarity between CPC and the natural osseous phase results in poor image contrast in most of the available in vivo imaging techniques such as computed tomography (CT) and magnetic resonance imaging (MRI). For accurate identification and localization during and after implantation in vivo, a composition with enhanced image contrast is needed. In this study, we labeled CPC with perfluoro-15-crown-5-ether-loaded (PFCE) poly(latic-co-glycolic acid) nanoparticles (hydrodynamic radius 100 nm) and gold nanoparticles (diameter 40 nm), as 19F MRI and CT contrast agents, respectively. The resulting CPC/PFCE/gold composite is implanted in a rat model for in vivo longitudinal imaging. Our findings show that the incorporation of the two types of different nanoparticles did result in adequate handling properties of the cement. Qualitative and quantitative long-term assessment of CPC/PFCE/gold degradation was achieved in vivo and correlated to the new bone formation. Finally, no adverse biological effects on the bone tissue are observed via histology. In conclusion, an easy and efficient strategy for following CPC implantation and degradation in vivo is developed. As all materials used are biocompatible, this CPC/PFCE/gold composite is clinically applicable.

Item does not contain fulltext, Due to the adsorption of biomolecules, the control of the biodistribution of nanoparticles is still one of the major challenges of nanomedicine. Poly(2-ethyl-2-oxazoline) (PEtOx) for surface modification of nanoparticles is applied and both protein adsorption and cellular uptake of PEtOxylated nanoparticles versus nanoparticles coated with poly(ethylene glycol) (PEG) and non-coated positively and negatively charged nanoparticles are compared. Therefore, fluorescent poly(organosiloxane) nanoparticles of 15 nm radius are synthesized, which are used as a scaffold for surface modification in a grafting onto approach. With multi-angle dynamic light scattering, asymmetrical flow field-flow fractionation, gel electrophoresis, and liquid chromatography-mass spectrometry, it is demonstrated that protein adsorption on PEtOxylated nanoparticles is extremely low, similar as on PEGylated nanoparticles. Moreover, quantitative microscopy reveals that PEtOxylation significantly reduces the non-specific cellular uptake, particularly by macrophage-like cells. Collectively, studies demonstrate that PEtOx is a very effective alternative to PEG for stealth modification of the surface of nanoparticles.

Item does not contain fulltext, The protein corona, which forms on the nanoparticle's surface in most biological media, determines the nanoparticle's physicochemical characteristics. The formation of the protein corona has a significant impact on the biodistribution and clearance of nanoparticles in vivo. Therefore, the ability to influence the formation of the protein corona is essential to most biomedical applications, including drug delivery and imaging. In this study, we investigate the protein adsorption on nanoparticles with a hydrodynamic radius of 30 nm and a coating of thermoresponsive poly(2-isopropyl-2-oxazoline) in serum. Using multiangle dynamic light scattering (DLS) we demonstrate that heating of the nanoparticles above their phase separation temperature induces the formation of agglomerates, with a hydrodynamic radius of 1 mum. In serum, noticeably stronger agglomeration occurs at lower temperatures compared to serum-free conditions. Cryogenic transmission electron microscopy (cryo-TEM) revealed a high packing density of agglomerates when serum was not present. In contrast, in the presence of serum, agglomerated nanoparticles were loosely packed, indicating that proteins are intercalated between them. Moreover, an increase in protein content is observed upon heating, confirming that protein adsorption is induced by the alteration of the surface during phase separation. After cooling and switching the surface back, most of the agglomerates were dissolved and the main fraction returned to the original size of approximately 30 nm as shown by asymmetrical flow-field flow fractionation (AF-FFF) and DLS. Furthermore, the amounts of adsorbed proteins are similar before and after heating the nanoparticles to above their phase-separation temperature. Overall, our results demonstrate that the thermoresponsivity of the polymer coating enables turning the corona formation on nanoparticles on and off in situ. As the local heating of body areas can be easily done in vivo, the thermoresponsive co

Contains fulltext : 139274.pdf (publisher's version ) (Open Access), Due to the recent widespread application of nanomaterials to biological systems, a careful consideration of their physiological impact is required. This demands an understanding of the complex processes at the bio-nano interface. Therefore, a comprehensive and accurate characterization of the material under physiological conditions is crucial to correlate the observed biological impact with defined colloidal properties. As promising candidates for biomedical applications, two SiO2-based nanomaterial systems were chosen for extensive size characterization to investigate the agglomeration behavior under physiological conditions. To combine the benefits of different characterization techniques and to compensate for their respective drawbacks, transmission electron microscopy, dynamic light scattering and asymmetric flow field-flow fractionation were applied. The investigated particle systems were (i) negatively charged silica particles and (ii) poly(organosiloxane) particles offering variable surface modification opportunities (positively charged, polymer coated). It is shown that the surface properties primarily determine the agglomeration state of the particles and therefore their effective size, especially under physiological conditions. Thus, the biological identity of a nanomaterial is clearly influenced by differentiating surface properties.

Item does not contain fulltext, The alveolar lung surfactant (LS) is a complex lipid protein mixture that forms an interfacial monolayer reducing the surface tension to near zero values and thus preventing the lungs from collapse. Due to the expanding field of nanotechnology and the corresponding unavoidable exposure of human beings from the air, it is crucial to study the potential effects of nanoparticles (NPs) on the structural organization of the lung surfactant system. In the present study, we investigated both, the domain structure in pure DPPC monolayers as well as in lung surfactant model systems. In the pure lipid system we found that two different sized hydrophobic polymeric nanoparticles with diameter of ~12 nm and ~136 nm have contrasting effect on the functional and structural behavior. The small nanoparticles inserted into fluid domains at the LE-LC phase transition are not visibly disturbing the phase transition but disrupting the domain morphology of the LE phase. The large nanoparticles led to an expanded isotherm and to a significant decrease in the line tension and thus to a drastic disruption of the domain structures at a much lower number of nanoparticles with respect to the lipid. The surface activity of the model LS films again showed drastic variations due to presence of different sized NPs illustrated by the film balance isotherms and the atomic force microscopy. AFM revealed laterally profuse multilayer protrusion formation on compression but only in the presence of 136 nm sized nanoparticles. Moreover we investigated the vesicle insertion process into a preformed monolayer. A severe inhibition was observed only in the presence of ~136 nm NPs compared to minor effects in the presence of ~12 nm NPs. Our study clearly shows that the size of the nanoparticles made of the same material determines the interaction with biological membranes.