The AF4 elugrams for the three polymericnanoparticles are shown in Figure 3a–c. A run of pure plasma is pictured in red resulting in an intense peak between 6.5 and 10 min at the beginning of the measurements, which consists of the main fraction of small proteins (larger objects like lipoproteins and particles aggregates appear—at the end of each measure- ment—in the rinse peak). The elugrams of nanoparticles, which were incubated with phosphate buffered saline (PBS) instead of plasma, are presented in green. Since the nanopar- ticles are larger than most of the proteins they are eluting later and depending on the crossflow at different retention times (see experimental part). The elugrams of the mixtures between nanoparticles and pure plasma after incubation are shown in blue. During the measurements we observed—for all three cases—no sign of any significant loss of particles or proteins by unspecific adsorption in the device (loss of UV-intensity or adsorption onto the membrane, see Figure S6 in the Sup- porting Information experiments to plasma loss) or aggrega- tion in the AF4 channel. Furthermore, the nanoparticle peaks did not undergo any shift to later retention times after incu- bation (UV and MALS detector, for MALS detector see Figure S7 in the Supporting Information), which is a first indication that the size of the particles is not significantly altered during incubation with plasma. Nanoparticles, which aggregate with proteins and thus increase in size, would elute at later reten- tion times or even during the rinsing due to the smaller dif- fusion coefficient of the aggregate (see examples for PS NPs and different micelles in Figure S8 in the Supporting Informa- tion). This fact was confirmed by multiangle-light scattering of isolated particles, which were incubated with plasma or PBS before purification by AF4 (Figure 3d–f). For all three nanopar- ticles the hydrodynamic radius remains the same, whether they were incubated in PBS or plasma. These results confirm our starting hypothesis that the chosen materials remain intact and are not subjected to any dynamics after exposure to plasma. Moreover, these results further indicate that there is no major adsorption of proteins on the nanoparticles leading to a signifi- cant size increase of the complexes, which is in accordance to findings on liposomes as determined by AF4.  On the other
Having demonstrated the successful development of photo-sensitive microgels swellable and degradable in organic solvents, transferring this approach to aqueous media renders them highly interesting for a broad variety of applications such as sensors, 81 optics, 82 colloidal crystals, 83, 272 and biomedical fields. 2, 85, 273 In the latter, the controlled delivery of pharmaceutically active substances holds promise to be a key concept for future treatment of diseases. Regarding drug delivery applications, various types of polymeric carrier systems have been developed over the past years. Compared to macromolecular approaches such as polymer-drug conjugates, 274-275 particulate drug delivery systems are characterized by an enhanced protection of embedded active compounds against the body’s defense mechanisms, therefore enabling the delivery of drugs to diseased sites in high doses with minimal damage to healthy tissue. 276-277 In this context, micellar aggregates of amphiphilic block copolymers have been well established but are mainly applied for the incorporation of hydrophobic drugs into the hydrophobic micellar cores. 278 In contrast, microgels as intermolecularly crosslinked hydrophilic polymericnanoparticles allow the incorporation of water-soluble drugs, including proteins and nucleic acids, in the network. Furthermore, the utilization of biocompatible and nonantiogenic materials enhances the protection of the payload from hostile enzymes until the delivery to targeted tissues. 88 Additional advantages include the ease of preparation, high stability, and the good dispersibility in water. 88, 108, 279
Nanobiotechnology is a rapidly growing field with a large number of newly synthesized materials that have already been implemented in our daily life. The omnipresence of these nanomaterials raises several issues about nanosafety and the likelihood of interactions with biological systems (1). Especially, their administration in biomedical applications places high demands on the quality and safety of such nanomaterials. Fortunately, there is a way to achieve these high requirements. The improvement of nanoparticles mainly profits from the huge variety of possible synthesis protocols that can be applied to properly adapt nanomaterials for the desired applications. The tremendous number of synthesis routes provides a huge freedom to vary fundamental features such as drug release efficiency, blood circulation half-life or the size of the nanomaterial (1). However, the manifoldness of the diverse materials challenges life scientists to obtain a detailed view about nano-bio-interactions (1). To guarantee safe nanoproducts in the future, it is therefore absolutely necessary to gain knowledge about nano-bio-interactions. These include i. a. nanoparticulate cytotoxicity, cellular uptake mechanisms, their environmental disposition and nanoparticle-membrane interactions.
A convenient synthetic route based on the concept of nanoreactors (individual nanosized homogeneous entities) using the miniemulsion technique to overcome the aforementioned problems of other available techniques in producing uniformly crosslinked gelatin nanoparticles also with high crosslinking degrees is presented in this work. The miniemulsion technique is a straight forward approach, which does not rely on phase separation and offers the flexibility of conveniently varying the gelatin content and the degree of crosslinking using small amounts of surfactant. Glutardialdehyde, which is a known protein fixative, is used as the crosslinker. It is understood that the ε-amino groups of the lysine residues and the N-terminal amino groups of the protein are involved in the crosslinking with glutaraldehyde. Gelatin nanoparticles are prepared using inverse miniemulsion process, where p-xylene is used as the continuous phase. As gelatin itself is practically insoluble in p-xylene, it serves the purpose of hydrophilic agent (osmotic control agent) by itself in order to suppress Ostwald ripening. Different types of gelatin have been used without purification or fractionation and the amount of the crosslinker has been optimized. The stability of the dispersion, particle size, and the efficiency of crosslinking by analyzing the uncrosslinked free chains have been studied.  The optimized gelatin particles have been used as templates for hydroxyapatite crystals in this work.
Previous studies have shown that 10–20 mol% ligand (i.e., biotin and folate) density on the surface of polymericnanoparticles was op- timal for efficient cellular uptake [ 83 , 84 ]. We therefore fixed the biotin density at 10 mol% at the surface of the micelles. A549 (biotin receptor- positive) and HEK293 (biotin receptor-negative) cells were incubated with Cy3-labeled p(HPMAm)-b-p(HPMAm-Bz) micelles with or without biotin decoration for 1, 4, 8, and 24 h and stained with Hoechst 33430 before imaging. In non-cancerous HEK293 cells which do not express the biotin receptor, very low internalization of both biotinylated and nonbiotinylated micelles was observed ( Fig. 5 B & C). However, it is clear that the fluorescence intensity of biotinylated p(HPMAm)-b-p (HPMAm-Bz) micelles was significantly higher as compared to the nonbiotinylated micelles in biotin receptor-expressing A549 cells ( Fig. 5 A & C), confirming the enhanced internalization of biotinylated p (HPMAm)-b-p(HPMAm-Bz) micelles. Besides, the uptake of p (HPMAm)-b-p(HPMAm-Bz) micelles in these cells was time-dependent. In the presence of free biotin, biotinylated micelles showed significantly lower uptake by A549 cells whereas the uptake of nonbiotinylated micelles was not affected (Fig. S10). These observations validate that the biotinylated micelles are taken up via biotin/SMVT receptor- mediated endocytosis.
As stated in Section 2.3 and 2.4, nature uses a plethora of ways and motifs to stabilize its formed architectures that fold dynamically. There also exists a wide variety of studies presenting different groups and functionalities that induce single-chain nanoparticles (SCNPs), whereas only a few examples are reported for the synthesis of SCNPs that fuse several functionalities to prepare dual- and dynamically folded architectures. [108,110,141,250] Within the current section, the non-covalent interaction of hydrogen bonds of the Hamilton Wedge (HW)/cyanuric acid (CA) moiety is fused with a host-guest system consisting of benzo-21-crown-7 (host) and a secondary ammonium salt (guest). The methodologies are combined to prepare a dual-folded system that is unfolded dynamically through the addition of different chemical trigger signals. The orthogonal interaction during the folding and unfolding represents a step towards finely controlling the folding state. Further, the relatively new area of light induced collapse in the field SCNPs is addressed by the preparation of a self-reporting refoldable system. The current status (folded vs unfolded) of the system can be read out by fluorescence. The SCNPs show fluorescence, while the open linear chain does not fluoresce. Compared to the dynamic SCNP structures presented in Section 3.1, the single-chain polymericnanoparticles synthesized employing the repeat unit approach are less well-defined due to the statistical interaction of the folding motifs. However, one of the advantage of the repeat unit approach is that it is synthetically less demanding and hence dominant in literature. The current chapter discusses the methodologies pioneered within this thesis for the generation of non-covalent and dynamic covalent crosslinks into dynamically switchable SCNP systems.
knowledge, this is the first report on using the flow cytometer to differentiate between spherical and non-spherical micro and nanoparticles. The flow cytometer is a single particle measurement method, which can analyze large samples of 0.5–100 μm in very short time frames (up to 5000 particles/s) . By examining flow cytometry dot plots for forward and side scatter, spherical particles are found as small homogenous populations with relatively low scatter. This homogeneity is probably due to the isotropicity of the spherical particles, leading to a nearly identical scattering pattern for all the particles. In contrast, the non-spherical particles are anisotropic, leading to different scattering pattern for each particle depending on the orientation of the particle with respect to the incident light beam. Accordingly, non-spherical particles appear as a “cloud” rather than a homogeneous “spot” on the dot plot, with a shift towards higher forward and side scatter (see Figure II-9 and Figure II-10). These results allowed the differentiation between the two particle populations, as well as the determination of the two populations in preformed mixtures, and the levels of spherical contaminants (Figure II-10).
Interspaced Short Palindromic Repeats) technology. Delivery of the CRISPR-associated protein-9 nuclease (Cas9) complexed with a synthetic guide RNA (gRNA) into a cell enables genome cutting at a desired location, allowing existing genes to be silenced, removed or added. In chronic myeloid leukemia (CML) reciprocal translocation of chromosome 9 and 22t results in a breakpoint cluster region-abelson (BCR-ABL) fusion oncogene, which translates a relating BCR-ABL fusion protein. In a study by Liu and colleagues, this BCR-ABL fusion protein was knocked out via the delivery of a CRISPR/Cas9 plasmid (pCas9/gBCR- ABL), which was encapsulated in a PEG-PLGA-based cationic lipid-assisted polymeric NP. Successful knockout of the gene of interest was confirmed in vitro as well as in vivo in a CML mouse model. Mice that were i.v. injected with the nanocarrier showed a reduced number of myelogenous leukemia cells (K562) in blood and bone marrow and a significant prolonged survival rate after the treatment compared to naive and vehicle controls ( Liu et al., 2018 ).
With regard to lyophilization of siRNA nanoparticles, only limited information is available, as well. Wu et al.  reported that siRNA-loaded lipid particles could be prepared by hydration of a stable freeze-dried matrix, containing siRNA and lipids. But here, only this preparation method was compared to an established direct preparation procedure and the influence of the lyophilization process itself on particle size and biological activity was not studied. Kundu et al.  evaluated the storage stability of lyophilized lipid-based siRNA nanosome formulations, but also did not investigate the influence of the lyophilization process. Moreover, Andersen et al.  described lyophilized chitosan or lipid siRNA formulations that are coated on cell culture plates as high-throughput screening tool and reported that 10% sucrose was required to sufficiently preserve particle size and gene silencing activity. Additionally, Yadava et al.  reported that siRNA lipoplexes could only be lyophilized with maintenance of transfection efficacy and with only a slight increase in particle size in the presence of stabilizers, such as glucose or sucrose. Similar results were published by Werth et al. , who showed that 5% glucose was required to preserve the transfection efficacy of low molecular weight polyethylenimine based siRNA polyplexes, but atomic force microscopy demonstrated a broadening of the polyplex particle size.
L -lactate is converted into CO 2 , which is excreted through the lungs and it is converted to pyruvate, which enters the Krebs cycle. Glycolate on the other hand is either directly excreted through the renal system or it can be oxidized to glyoxylate, which is afterward further converted into glycine, serine, and pyruvate. The latter can again enter the Krebs cycle and is metabolized into CO 2 and H 2 O. ( Danhier et al., 2012 ; Silva et al., 2015 ). Typically, PLGA is produced by a catalyzed ring-opening copolymerization of LA and GA ( Dechy-Cabaret et al., 2004 ). PGA is a crystalline hydrophilic polymer with low water solubility and fast degradation rate under physiological conditions. On the contrary, PLA is a stiff and hydrophobic polymer with low mechanical strength. As a copolymer of both, PLGA inherits the intrinsic properties of its constitutional monomers where the polymeric content, based on LA/GA ratio and Mw, strongly affect its degradation rate. For example, with an increase in the LA/GA ratio, the overall PLGA hydrophobicity increases, which leads to lower degradation and thus slower drug release rate ( Engineer et al., 2011 ). Furthermore, the final Mw of the polymer also influences the degradation and drug release kinetics of the resulting formulations; i.e., with a decrease in the Mw, degradation as well as drug release rates both increase ( Xu et al., 2017 ). Next, degradation, release kinetics, and the Mw also correlate with the size of the resulting NPs formulate. These are crucial factors for the therapeutic performance of PLGA NPs. Despite the higher drug loading potential of larger sized formulations, achieving a lower nano-size range is essentially important for the ability of the NPs to overcome biological barriers and to reach the disease site. In this context, a study pointed to the impact of the Mw of four 1:1 (LA:GA) PLGA copolymers with different Mw of 14.5, 45, 85, and 213 kDa on polymeric degradation and release rate ( Mittal et al., 2007 ). With increasing Mw, the PLGA NPs degradation as well as its drug release decreased with a payload release under physiological conditions on day 18 of 95, 66, 50, and 23%, respectively. In addition it has been observed that the is higher the Mw of PLGA (6, 14.5, 63.6 kDa), the bigger is the size of NPs loaded with paclitaxel (PTX) (122 ± 3, 133 ± 2, 160 ± 2 nm) and also of NPs without PTX (117 ± 2, 132 ± 2, 159 ± 3 nm) ( Fonseca et al., 2002 ; Song et al., 2008 ). LA/GA ratio is an effective parameter in tailoring degradation time and drug release rate. The higher the GA content, the faster the resulting degradation rate ( Xu et al., 2017 ). Vice versa the drug release is prolonged with an increase in LA content (LA/GA: 50/50, 75/25 showed faster release kinetics then 100/0) ( Horisawa et al., 2002 ). Hence, these polymeric characteristics, as well as their size, are important to tailor hydrophobicity, drug loading efficacy, and the pharmacokinetic profile of PLGA formulations.
monomer present in the dispersed oil phase and a monomer present in the aqueous continuous phase. Breitenkamp and Emrick developed a method for the synthesis of larger capsules (~ 40 μm) made by cross-metathesis at the oil in water droplets interface [Bre_2003]. Additionally, a completely different approach was recently reported by Im et al. [ImJ_2005]. First, polystyrene nanospheres were swollen with toluene to increase their volumes followed by freezing with liquid nitrogen. The freezing involves shrinkage of the particles and hence the creation of a void inside the particles due to liquid to solid phase transition of toluene. The toluene is then evaporated below 0 °C, leaving a hole at the particle surface because of the flux of toluene. The final materials are hollow polystyrene nanocapsules. Upt to know, the techniques discussed above do not allow the encapsulation of hydrophilic compounds in a hollow capsule structure. Thus, no report exists about the synthesis of capsules by interfacial polycondensation in an inverse miniemulsion system (w/o). We present the synthesis of hollow nanocapsules made of polyurea, polythiourea or polyurethane. These nanoreactors are used for the reduction of silver nitrate to silver nanoparticles as model reactions. This is to our knowledge the first time that inorganic nanoparticles are encapsulated in a hollow structure without the use of a sacrificial core.
Healthcare and life science applications have drawn a great deal of interest for nanotechnology based systems and solutions. Nanostructured drugs and targeted drug delivery, sensing of target molecules, and imaging of cell and tissues are just a few examples for applications in medicine. However a wide variety of materials are used in medicine, the magnetic nanoparticles (MNPs) seem to hold one of the fastest moving and most exciting research areas. MNPs have a diverse range of applications from engineering to biomedical aspects. In fact, the application of MNPs to target tumor cells inside the human body was first conceived in the late 1970’s. [1,2] The key point was to inject the attached anticancer drugs to small magnetic spheres inside the body and concentrate the drug-loaded particles inside the tumor tissue under applying external magnetic fields (AMF), in order to reduce drug payload, and thereby reduce the side effects associated with chemotherapeutic agents.
Interfacial polymerization (see fig.12) is conventionally used for encapsulation in polymeric shells. The reaction of polymerization happens on the boundary between two immiscible (usually) liquid phases. One reactive monomer is dissolved in one phase, another reactive monomer - in another. When monomers meet on the interface the polymerization reaction takes place. The whole process is divided into two steps. On the first step an emulsion is formed. The first reactive monomer is dissolved in the dispersed phase, while the continuous phase usually contains emulsion stabilizer. On the second step the second reactive monomer is added to the continuous phase, leading to the polymeric shell formation on the surface of the emulsion droplets. The liquid content of the dispersed phase turns to be entrapped inside the polymer after the polymerization reaction is finished. The size and the shape of the final capsules are expected 94 to be equal to the ones of the original emulsion droplets, which serve as liquid templates for the capsules. Different kinds of emulsions can be used for interfacial polymerization. The most common way is to use conventional oil-in-water emulsions 95-125 , while inverse water-in-oil 103,126-128 and oil-in-oil 129-134 emulsions also have been used.
Up to now, much work has been devoted to the synthesis of nanomaterials and to their interaction with single protein types. However, biological media are multicomponent mixtures i.e. they usually contain more than one type of protein. For example, in human serum the most abundant proteins, albu- min (70%), IgG (14%), transferrin (5.7%), fibrinogen (2.8%), and α-antitrypsin (0.7%), cover 93% of the whole protein mass. In addition to these other proteins which function as enzymes and hormones are present. In such a complex mixture competitive and /or cooperative adsorption necessarily need to be considered. For spherical nanoparticles the competitive and /or cooperative adsorptions of diﬀerent protein types will lead to the formation of a protein corona. The composition of the protein corona will determine the physical and chemical properties of the nanoparticle and in a biological medium will determine its biological fate in terms of cellular response, biodistribution, clearance and toxicity. 9,26–34 Figure 1.3 a shows schematically how a spherical nanoparticle encased with a protein corona interacts at a cellular level with a membrane. The formation of a protein corona is a dynamic process and the composition of proteins will evolve with time (see Figure 1.3b) in analogy to the Vroman eﬀect for flat surfaces (see section 1.2). Although at some point equilibrium might be reached, i.e. association and
The results in this work show that the synthesis of bimetallic silver-platinum nanoparticles in different compositions from Ag10Pt90 to Ag90Pt10 in 10 mol% steps is possible through variation of the reductants and the reaction conditions. A seeded-growth method was applied which uses the galvanic exchange reaction. The size of the silver seeds affected the resulting nanoparticle size. With trisodium citrate and tannic acid as reductants, particles with diameters of 35 nm were obtained. Using sodium borohydride, 10 nm sized nanoparticles were achieved. At a pH of 3 during the reaction, two species could be found in one sample of which one was the hollow alloyed particle and the other a core-shell nanoparticle species. The core-shell particles show a silver core and a platinum shell. The number of alloyed particles in the samples increases with the silver composition. The alloy character was confirmed by UV/vis spectroscopy, EDX maps on single particles, and powder X-ray diffraction. A physical mixture was excluded by measuring physical mixtures of silver and platinum nanoparticles in different ratios by UV/vis spectroscopy. Powder X-ray diffraction showed a reflex shift to smaller angles with increasing silver composition. The nanoparticles are polycrystalline which was shown by HAADF-STEM and PXRD. The lattice constants of the different compositions follow Vegard’s law, although the lattice parameters are slightly contracted. At pH 10, 8 nm to 12 nm sized nanoparticles were obtained. Platinum rich samples showed pure platinum nanoparticles together with core-shell particles, whereas the compositions with moderate platinum amounts exhibited aggregated core-shell particles or alloys with a gradient structure. Particles with low platinum molar fractions revealed core-shell particles with a silver core and a platinum shell.
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Ultrasound (US) imaging is frequently employed in clinical practice because of its non-invasive nature, easy handling, low cost, real-time feedback, and broad applicability. Microbubbles (MB) extend the range of US applications towards functional and molecular imaging of (patho-) physiological phenomena in cardiovascular diseases and cancer [1,2]. MB are 1–5 µm-sized gas-filled vesicles, which are shell-stabilized by lipids, proteins, or polymers. Advances in MB formulation and functionalization have enabled the incorporation of various targeting ligands, imaging agents, and drug molecules, expanding their applicability towards multimodality imaging and direct and indirect drug delivery [3,4]. The suitability of different MB formulations for imaging and drug delivery is largely based on their shell composition (lipid-based soft MB vs. polymer-based hard MB). Hard-shelled polymeric MB, including poly(n-butyl cyanoacrylate) (PBCA) MB, are generally considered to be favorable for multimodality imaging and drug delivery [3–5].
As a visible-light activated photo-release from spin-silenced constructs had never been reported before, two possible issues were identified that needed to be addressed, be- fore transferring them to a complex polymeric system. Firstly, an insufficient ratio of si- lencing/turn-on would have resulted in issues with analysis, compromising the effective- ness of the self-reporting system. Secondly, the photo-release propagates via a reaction in excited electronic states – which were intentionally going to be influenced by unpaired electrons. At the time no in-depth studies of the proposed construct had been performed, confirming whether or not quenching of the excited state occurs before ISC and the ensuing radical cascade could take place. As polymeric systems are inherently difficult to analyse with respect to exact composition and chemical environment of individual groups, a sim- plified version was to be investigated first. The small molecule analogue to test this hy- pothesis is shown in Scheme 4.3.
release into HUH7 cells. The novel assumption that uptake into tumor tissue is an active process mediated by endothelial cells  might be an explanation. Attachment to the negatively charged glycocalyx of endothelial cells  is a prerequisite that can be met by cationic nanoparticles more easily . Another hypothesis is the binding of cationic nanoformulations to anionic lymphocytes and subsequent transportation into tumor tissue [150-152]. Though anionic net charge is considered advantageous regarding parameters like circulation time and shows comparable transfection efficacy in vitro, the cationic formulation prevails in our experiments. This can result from increased tumor accumulation because of its cationic charge leading to increased trans-endothelial uptake into solid tumor tissue. Additionally, HA induces receptor-mediated uptake into CD44 positive HUH7 cells , which can only function for the 0.1 equiv. of HA, due to the fact it can be determined in relevant levels in tumor tissue, in contrast to the anionic formulation.