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Organisationseinheit der BAM
Characterization of polymer nanoparticles by asymmetrical flow field flow fractionation (AF-FFF)
(2010)
The role of cyclization in polycondensations is discussed for two different scenarios: thermodynamically-controlled polycondensation (TCPs) on the one hand and kinetically-controlled polycondensations (KCPs) on the other. The classical CarothersFlory theory of step-growth polymerization does not include cyclization reactions. However, TCPs involve the formation of cycles via back-biting degradation, and when the ringchain equilibrium is on the side of the cycles the main reaction products of the TCP will be cyclic oligomers. Two groups of examples are discussed: polycondensations of salicyclic acid derivatives (e.g. aspirin) and polycondensations of dibutyltin derivatives with long {alpha}-, {omega}-diols or dicarboxylic acids. Furthermore, various kinetically-controlled syntheses of polyesters and polyamides were studied and carefully optimized in the direction of high molecular weights. High fractions of cyclic oligomers and polymers were found by MALDI-TOF mass spectrometry, and their fractions increased with optimization of the process for molecular weight. These results disagree with the CarothersFlory theory but agree with the theoretical background of the RuggliZiegler dilution method (RZDM). When poly(ether-sulfone)s were prepared from 4,4'-difluorodiphenylsulfone and silylated bisphenol-A two different scenarios were found. With CsF as catalyst at a temperature of more than 145°C cyclic oligoethers were formed under thermodynamic control. When the polycondensation was promoted with K2CO3 in N-methylpyrolidone at ?145°C the formation of cyclic oligoethers and polyethers occurred under kinetic control. A new mathematical formula is presented correlating the average degree of polymerization with the conversion and taking into account the competition between cyclization and propagation.
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.
The protein corona, which forms on the nanoparticle's surface in most biological media, determines the nanoparticles 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 µm. 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 coating could potentially be used to induce the agglomeration of nanoparticles and proteins and the accumulation of nanoparticles in a targeted body region.
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.
Micromixer technology was used to prepare polymeric vesicles (Pluronic® L-121) dual loaded with the anti-cancer drug camptothecin and magnetic nanoparticles. Successful incorporation of the magnetic nanoparticles was confirmed by transmission electron microscopy. Dynamic light scattering measurements showed a relatively narrow size distribution of the hybrid polymersomes. Camptothecin polymersomes reduced the cell viability of prostate cancer cells (PC-3) measured after 72 h significantly, while drug-free polymersomes showed no cytotoxic effects. Covalent attachment of a cancer targeting peptide (bombesin) as well as a fluorescent label (Alexa Fluor® 647) to the hybrid polymersomes was performed and specific cell binding and internalization were shown by flow cytometry and confocal microscopy. Relaxometry measurements clearly demonstrated the capacity of magnetic polymersomes to generate significant T2-weighted MRI contrast and potentially allow for direct monitoring of the biodistribution of the polymersomes. Micromixer technology as an easy, fast and efficient way to manufacture hybrid polymersomes as theranostic drug delivery devices is a further step from basic research to personalized medicine.
The need for a better understanding of nanoparticleprotein interactions and the mechanisms governing the resulting colloidal stability has been emphasised in recent years. In the present contribution, the short and long term colloidal stability of silica nanoparticles (SNPs) and silica–poly(ethylene glycol) nanohybrids (Sil–PEG) have been scrutinised in a protein model system. Well-defined silica nanoparticles are rapidly covered by bovine serum albumin (BSA) and form small clusters after 20 min while large agglomerates are detected after 10 h depending on both particle size and nanoparticle–protein ratio. Oppositely, Sil–PEG hybrids present suppressive protein adsorption and enhanced short and long term colloidal stability in protein solution. No critical agglomeration was found for either system in the absence of protein, proving that instability found for SNPs must arise as a consequence of protein adsorption and not to high ionic environment. Analysis of the small angle X-ray scattering (SAXS) structure factor indicates a short-range attractive potential between particles in the silica-BSA system, which is in good agreement with a protein bridging agglomeration mechanism. The results presented here point out the importance of the nanoparticle surface properties on the ability to adsorb proteins and how the induced or depressed adsorption may potentially drive the resulting colloidal stability.
Polymeric vesicles (Pluronic® L-121) loaded with magnetic nanoparticles (MNP) and an anti-cancer drug (camptothecin) were prepared continuously in a micro mixing device. Characterization by TEM confirmed the successful incorporation of the MNP and DLS measurements showed a relatively narrow size distribution of the hybrid polymersomes. A very high drug loading of camptothecin (100 µg/ml in the polymersome formulation) was reached and a drug release study of loaded magnetic polymersomes has shown a sustained camptothecin release over several days. Carboxylation of Pluronic® L-121 was performed and enabled a further surface functionalization with bombesin, a 14 amino acid peptide, which binds specifically to the GRPR (gastrin releasing peptide receptor). This receptor is often overexpressed in tumor cells (e.g., human prostate cancer cells) and therefore a suitable target for cancer treatment. An additional fluorescence label with Alexa Fluor® 647 allow tracking of the polymersomes e.g., in cell experiments. Relaxivity measurements to evaluate the potential of magnetic polymersomes as MR contrast agent for in vivo imaging are in progress.
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.
The separation of colloidal nanocrystals from their original synthesis medium is an essential process step towards their application, however, the costs on a preparative scale are still a constraint. A new combination of approaches for the purification of hydrophobic Quantum Dots is presented, resulting in an efficient scalable process in regard to time and solvent consumption, using common laboratory equipment and low-cost materials. The procedure is based on a combination of solvent-induced adhesion and solid phase extraction. The platform allows the transition from manual handling towards automation, yielding an overall purification performance similar to one conventional batch precipitation/centrifugation step, which was investigated by thermogravimetry and gas chromatography. The distinct miscibility gaps between surfactants used as nanoparticle capping agents, original and extraction medium are clarified by their phase diagrams, which confirmed the outcome of the flow chemistry process. Furthermore, the solubility behavior of the Quantum Dots is put into context with the Hansen solubility parameters framework to reasonably decide upon appropriate solvent types.