Tuning the Cross-Linking Density and Cross-Linker in Core Cross-Linked Polymeric Micelles and Its Effects on the Particle Stability in Human Blood Plasma and Mice.

Core cross-linked polymeric micelles (CCPMs) are designed to improve the therapeutic profile of hydrophobic drugs, reduce or completely avoid protein corona formation, and offer prolonged circulation times, a prerequisite for passive or active targeting. In this study, we tuned the CCPM stability by using bifunctional or trifunctional cross-linkers and varying the cross-linkable polymer block length. For CCPMs, amphiphilic thiol-reactive polypept(o)ides of polysarcosine-block-poly(S-ethylsulfonyl-l-cysteine) [pSar-b-pCys(SO2Et)] were employed. While the pCys(SO2Et) chain lengths varied from Xn = 17 to 30, bivalent (derivatives of dihydrolipoic acid) and trivalent (sarcosine/cysteine pentapeptide) cross-linkers have been applied. Asymmetrical flow field-flow fraction (AF4) displayed the absence of aggregates in human plasma, yet for non-cross-linked PM and CCPMs cross-linked with dihydrolipoic acid at [pCys(SO2Et)]17, increasing the cross-linking density or the pCys(SO2Et) chain lengths led to stable CCPMs. Interestingly, circulation time and biodistribution in mice of non-cross-linked and bivalently cross-linked CCPMs are comparable, while the trivalent peptide cross-linkers enhance the circulation half-life from 11 to 19 h.

Squaric Ester-Based Nanogels Induce No Distinct Protein Corona but Entrap Plasma Proteins into their Porous Hydrogel Network.

After intravenous administration of nanocarriers, plasma proteins may rapidly adsorb onto their surfaces. This process hampers the prediction of the nanocarriers' pharmacokinetics as it determines their physiological identity in a complex biological environment. Toward clinical translation it is therefore an essential prerequisite to investigate the nanocarriers' interaction with plasma proteins. Here, this work evaluates a highly "PEGylated" squaric ester-based nanogel with inherent prolonged blood circulation properties. After incubation with human blood plasma, the nanogels are isolated by asymmetrical flow-field flow fractionation. Multiangle light scattering measurements confirm the absence of significant size increases as well as aggregation upon plasma incubation. However, proteomic analyses by gel electrophoresis find minor absolute amounts of proteins (3 wt%), whereas label-free liquid chromatography mass spectrometry identify 65 enriched proteins. Interestingly, the relative abundance of these proteins is almost similar to their proportion in pure native plasma. Due to the nanogels' hydrated and porous network morphology, it is concluded that the detected proteins rather result from passive diffusion into the nanogel network than from specific interactions at the plasma particle interface. Consequently, these results do not indicate a classical surface protein corona but rather reflect the highly outer and inner stealth-like behavior of the porous hydrogel network.

Nanoparticles in the Biological Context: Surface Morphology and Protein Corona Formation.

A recent paper demonstrated that the formation of a protein corona is not a general property of all types of nanosized objects. In fact, it varies between a massive aggregation of plasma proteins onto the nanoparticle down to traces (e.g., a few proteins per 10 nanoparticles), which can only be determined by mass spectrometry in comparison to appropriate negative controls and background subtraction. Here, differences between various types of nanosized objects are discussed in order to determine general structure-property-relations from a physico-chemical viewpoint. It is highlighted that "not all nanoparticles are alike" and shown that their internal morphology, especially the difference between a strongly hydrated/swollen shell versus a sharp "hard" surface and its accessibility, is most relevant for biomedical applications.

Polymeric Nanoparticles with Neglectable Protein Corona.

The current understanding of nanoparticle-protein interactions indicates that they rapidly adsorb proteins upon introduction into a living organism. The formed protein corona determines thereafter identity and fate of nanoparticles in the body. The present study evaluates the protein affinity of three core-crosslinked polymeric nanoparticles with long circulation times, differing in the hydrophilic polymer material forming the particle surface, namely poly(N-2-hydroxypropylmethacrylamide) (pHPMA), polysarcosine (pSar), and poly(ethylene glycol) (PEG). This includes the nanotherapeutic CPC634, which is currently in clinical phase II evaluation. To investigate possible protein corona formation, the nanoparticles are incubated in human blood plasma and separated by asymmetrical flow field-flow fractionation (AF4). Notably, light scattering shows no detectable differences in particle size or polydispersity upon incubation with plasma for all nanoparticles, while in gel electrophoresis, minor amounts of proteins can be detected in the particle fraction. Label-free quantitative proteomics is additionally applied to analyze and quantify the composition of the proteins. It proves that some proteins are enriched, but their concentration is significantly less than one protein per particle. Thus, most of the nanoparticles are not associated with any proteins. Therefore, this work underlines that polymeric nanoparticles can be synthesized, for which a protein corona formation does not take place.

HPMA-Based Nanoparticles for Fast, Bioorthogonal iEDDA Ligation.

Fast and bioorthogonally reacting nanoparticles are attractive tools for biomedical applications such as tumor pretargeting. In this study, we designed an amphiphilic block copolymer system based on HPMA using different strategies to introduce the highly reactive click units 1,2,4,5-tetrazines (Tz) either at the chain end (Tz-CTA) or statistical into the hydrophobic block. This reactive group undergoes a rapid, bioorthogonal inverse electron-demand Diels-Alder reaction (iEDDA) with trans-cyclooctenes (TCO). Subsequently, this polymer platform was used for the preparation of different Tz-covered nanoparticles, such as micelles and colloids. Thereby it was found that the reactivity of the polymeric micelles is comparable to that of the low molar mass tetrazines. The core-cross-linked micelles can be successfully conjugated at rather low concentrations to large biomacromolecules like antibodies, not only in physiological buffer, but also in human blood plasma, which was confirmed by fluorescence correlation spectroscopy (FCS).

Living Light-Induced Crystallization-Driven Self-Assembly for Rapid Preparation of Semiconducting Nanofibers.

Well-defined nanostructures composed of conjugated polymers have attracted significant attention due to their intriguing electronic and optical properties. However, precise control of the size and uniformity of these semiconducting nanostructures is still rare and challenging, despite recent advances in strategies to obtain self-assembled nanostructures with narrow dispersions. Herein, we demonstrate the preparation of fluorescent conjugated block copolymers by one-shot polymerization and rapid formation of nanofibers in a few minutes via light-induced crystallization-driven self-assembly, driven by facile cis-to- trans photoisomerization of its poly( p-phenylenevinylene) blocks. Furthermore, living self-assembly was possible, allowing not only nanofibers with excellent length control and narrow size distribution but also ABA triblock comicelles and gradient comicelles, to be produced by seeded growth. Lastly, the seeded growth could be activated and deactivated repeatedly by switching the light on and off, analogous to light-induced living radical polymerization.

Polymeric Selectin Ligands Mimicking Complex Carbohydrates: From Selectin Binders to Modifiers of Macrophage Migration.

Novel polymeric cell adhesion inhibitors were developed in which the selectin tetrasaccharide sialyl-LewisX (SLeX ) is multivalently presented on a biocompatible poly(2-hydroxypropyl)methacrylamide (PHPMA) backbone either alone (P1) or in combination with O-sulfated tyramine side chains (P2). For comparison, corresponding polymeric glycomimetics were prepared in which the crucial "single carbohydrate" substructures fucose, galactose, and sialic acid side chains were randomly linked to the PHPMA backbone (P3 or P4 (O-sulfated tyramine)). All polymers have an identical degree of polymerization, as they are derived from the same precursor polymer. Binding assays to selectins, to activated endothelial cells, and to macrophages show that polyHPMA with SLeX is an excellent binder to E-, L-, and P-selectins. However, mimetic P4 can also achieve close to comparable binding affinities in in vitro measurements and surprisingly, it also significantly inhibits the migration of macrophages; this provides new perspectives for the therapy of severe inflammatory diseases.

Block copolymers from ionic liquids for the preparation of thin carbonaceous shells.

This paper describes the controlled radical polymerization of an ionic-liquid monomer by RAFT polymerization. This allows the control over the molecular weight of ionic liquid blocks in the range of 8000 and 22000 and of the block-copolymer synthesis. In this work we focus on block copolymers with an anchor block. They can be used to control the formation of TiO2 nanoparticles, which are functionalized thereafter with a block of ionic-liquid polymer. Pyrolysis of these polymer functionalized inorganic nanoparticles leads to TiO2 nanoparticles coated with a thin carbonaceous shell. Such materials may, e.g., be interesting as battery materials.

Functionalization of Active Ester-Based Polymersomes for Enhanced Cell Uptake and Stimuli-Responsive Cargo Release.

Poly(2,3-dihydroxypropyl methacrylamide) (P(DHPMA))-based amphiphilic block copolymers have recently proven to form polymer vesicles (polymersomes). In this work, we further expand their potential by incorporating (i) units for pH-dependent disintegration into the hydrophobic membrane and (ii) mannose as targeting unit into the hydrophilic block. This last step relies on the use of an active ester prepolymer. We confirm the stability of the polymersomes against detergents like Triton X-100 and their low cytotoxicity. The incorporation of 2-(2,2-dimethyl-1,3-dioxolane-4-yl)ethyl methacrylate into the hydrophobic block (lauryl methacrylate) allows a pH-responsive disintegration for cargo release. Efficient decomposition of the polymersome structure is monitored by dynamic light scattering. It is thus possible to include an active enzyme (glucose oxidase), which gets only active (is set free) after vesicle disintegration. In addition, the introduction of mannose as targeting structure allows enhanced and selective targeting of dendritic cells.

Reductive Decationizable Block Copolymers for Stimuli-Responsive mRNA Delivery.

Messenger ribonucleic acids (mRNAs) are considered as promising alternatives for transient gene therapy, but to overcome their poor pharmacokinetic properties, smart carriers are required for cellular uptake and stimuli-responsive release. In this work, a synthetic concept toward reductive decationizable cationic block copolymers for mRNA complexation is introduced. By combination of RAFT block copolymerization with postpolymerization modification, cationic block copolymers are generated with disulfide-linked primary amines. They allow effective polyplex formation with negatively charged mRNA and subsequent release under reductive conditions of the cytoplasm. In first in vitro experiments with fibroblasts and macrophages, tailor-made block copolymers mediate cell-specific mRNA transfection, as quantified by polyplex uptake and mRNA-encoding gene expression. Furthermore, RAFT polymerization provides access to heterotelechelic polymers with orthogonally addressable endgroup functionalities utilized to ligate targeting units onto the polyplex-forming block copolymers. The results exemplify the broad versatility of this reductive decationizable mRNA carrier system, especially toward further advanced mRNA delivery applications.

Pentafluorophenyl Ester-based Polymersomes as Nanosized Drug-Delivery Vehicles.

In this work, activated ester chemistry is employed to synthesize biocompatible and readily functionalizable polymersomes. Via aminolysis of pentafluorophenyl methacrylate-based precursor polymers, an N-(2-hydroxypropyl) methacrylamide (HPMA)-analog hydrophilic block is obtained. The precursor polymers can be versatile functionalized by simple addition of suitable primary amines during aminolysis as demonstrated using a fluorescent dye. Vesicle formation is proven by cryoTEM and light scattering. High encapsulation efficiencies for hydrophilic cargo like siRNA are achieved using dual centrifugation and safe encapsulation is demonstrated by gel electrophoresis. In vitro studies reveal low cytotoxicity and no protein adsorption-induced aggregation in human blood serum occurs, making the vesicles interesting candidates as nanosized drug carriers.

New Techniques to Assess In Vitro Release of siRNA from Nanoscale Polyplexes.

PURPOSE: Release of siRNA from nanoscale polyplexes is a crucial yet little investigated process, important during all stages of therapeutic research. Here we develop new methods to characterize polyplex stability early on in the development of new materials. METHODS: We used double fluorescent labeled siRNA to compare binding and stability of a panel of chemically highly diverse nanoscale polyplexes, including peptides, lipids, nanohydrogels, poly-L-lysine brushes, HPMA block copolymers and manganese oxide particles. Conventional EMSA and heparin competition methods were contrasted with a newly developed microscale thermophoresis (MST) assay, a near-equilibrium method that allows free choice of buffer conditions. Integrity of FRET-labeled siRNA was monitored in the presence of nucleases, in cell culture medium and inside living cells. This approach characterizes all relevant steps from polyplex stability, over uptake to in vitro knockdown capability. RESULTS: Diverging polyplex binding properties revealed drawbacks of conventional EMSA and heparin competition assays, where MST and FRET-based siRNA integrity measurements offered a better discrimination of differential binding strength. Since cell culture medium left siRNA in all polyplexes essentially intact, the relevant degradation events could be pinpointed to occur inside cells. Differential binding strength of the variegated polyplexes correlated only partially with intracellular degradation. The most successful compounds in RNAi showed intermediate binding strength in our assays. CONCLUSIONS: We introduce new methods for the efficient and informative characterization of siRNA polyplexes with special attention to stability. Comparing FRET-labeled siRNA in different polyplexes associates successful knockdown with intermediate siRNA stability in various steps from formulation to intracellular persistence.

Facile synthesis of fluorescent polymer nanoparticles by covalent modification-nanoprecipitation of amine-reactive ester polymers.

Emission wavelength control in fluorescent nanoparticles (NPs) is crucial for their applications. In the case of inorganic quantum dots or dye-impregnated silica NPs, such a control is readily achieved by changing the size of the particles or choosing appropriate fluorescent dyes, respectively. A similar modular approach for controlling the emission wavelength of fluo-rescent polymer NPs, however, is difficult. This article reports on fluorescent polymer NPs, the synthesis of which provides a platform for a modular approach towards the preparation of fluorescent NPs of desired emission wavelength. Atom-transfer radical polymerization (ATRP) is employed to synthesize reactive ester polymers, which are then easily modified with a commercially available dye and subsequently subjected to nanoprecipitation. The resulting NPs, with low size polydispersity, show an enhanced emission quantum yield when compared with the same dye molecules in solution.

Photosensitive functionalized surface-modified quantum dots for polymeric structures via two-photon-initiated polymerization technique.

In this paper, the surface modification of CdSe- and CdZnS-based quantum dots (QDs) with a functional silica shell is reported. Functionalized silica shells are prepared by two routes: either by ligand exchange and a modified Stöber process or by a miniemulsion process with amphiphilic poly(oxyethylene) nonylphenylether also know as Igepal CO-520 (IG) as oligomeric amphiphile and modified silica precursors. The polymerizable groups on the functionalized silica shell allow covalent bonding to a polymer matrix and prevent demixing during polymerization and crosslinking. This allows the homogeneous incorporation of QDs in a crosslinked polymer matrix. This paper furthermore demonstrates that the resulting QDs, which are i) shielded with a proper silica shell and ii) functionalized with crosslinkable groups, can be used in two-photon-initiated polymerization processes in combination with different photoresists to obtain highly luminescent 3D structures. The resulting luminescent structures are attractive candidates for photonics and metamaterials research.

Precursor polymers for the carbon coating of Au@ZnO multipods for application as active material in lithium-ion batteries.

The synthesis of statistical and block copolymers based on polyacrylonitrile, as a source for carbonaceous materials, and thiol-containing repeating units as inorganic nanoparticle anchoring groups is reported. These polymers are used to coat Au@ZnO multipod heteroparticles with polymer brushes. IR spectroscopy and transmission electron microscopy prove the successful binding of the polymer onto the inorganic nanostructures. Thermogravimetric analysis is applied to compare the binding ability of the block and statistical copolymers. Subsequently, the polymer coating is transformed into a carbonaceous (partially graphitic) coating by pyrolysis. The obtained carbon coating is characterized by Raman spectroscopy and energy-dispersive X-ray (EDX) spectroscopy. The benefit of the conformal carbon coating of the Au@ZnO multipods regarding its application as lithium-ion anode material is revealed by performing galvanostatic cycling, showing a highly enhanced and stabilized electrochemical performance of the carbon-coated particles (still 831 mAh g(-1) after 150 cycles) with respect to the uncoated ones (only 353 mAh g(-1) after 10 cycles).

Morphology control in biphasic hybrid systems of semiconducting materials.

Simple blends of inorganic nanocrystals and organic (semiconducting) polymers usually lead to macroscopic segregation. Thus, such blends typically exhibit inferior properties than expected. To overcome the problem of segregation, polymer coated nanocrystals (nanocomposites) have been developed. Such nanocomposites are highly miscible within the polymer matrix. In this Review, a summary of synthetic approaches to achieve stable nanocomposites in a semiconducting polymer matrix is presented. Furthermore, a theoretical background as well as an overview concerning morphology control of inorganic NCs in polymer matrices are provided. In addition, the morphologic behavior of highly anisotropic nanoparticles (i.e. liquid crystalline phase formation of nanorod-composites) and branched nanoparticles (spatial orientation of tetrapods) is described. Finally, the morphology requirements for the application of inorganic/organic hybrid systems in light emitting diodes and solar cells are discussed, and potential solutions to achieve the required morphologies are provided.

High-performance TiO2 nanoparticle/DOPA-polymer composites.

Many natural materials are complex composites whose mechanical properties are often outstanding considering the weak constituents from which they are assembled. Nacre, made of inorganic (CaCO3 ) and organic constituents, is a textbook example because of its strength and toughness, which are related to its hierarchical structure and its well-defined organic-inorganic interface. Emulating the construction principles of nacre using simple inorganic materials and polymers is essential for understanding how chemical composition and structure determine biomaterial functions. A hard multilayered nanocomposite is assembled based on alternating layers of TiO2 nanoparticles and a 3-hydroxy-tyramine (DOPA) substituted polymer (DOPA-polymer), strongly cemented together by chelation through infiltration of the polymer into the TiO2 mesocrystal. With a Young's modulus of 17.5 ± 2.5 GPa and a hardness of 1.1 ± 0.3 GPa the resulting material exhibits high resistance against elastic as well as plastic deformation. A key feature leading to the high strength is the strong adhesion of the DOPA-polymer to the TiO2 nanoparticles.

A minimal hydrophobicity is needed to employ amphiphilic p(HPMA)-co-p(LMA) random copolymers in membrane research.

Because a polymer environment might be milder than a detergent micelle, amphiphilic polymers have attracted attention as alternatives to detergents in membrane biochemistry. The polymer poly[N-(2-hydroxypropyl)-methacrylamid] [p(HPMA)] has recently been modified with hydrophobic lauryl methacrylate (LMA) moieties, resulting in the synthesis of amphiphilic p(HPMA)-co-p(LMA) polymers. p(HPMA)-co-p(LMA) polymers with a LMA content of 5 or 15% have unstable hydrophobic cores. This, on one hand, promotes interactions of the hydrophobic LMA moieties with membranes, resulting in membrane rupture, but at the same time prevents formation of a hydrophobic, membrane mimetic environment that is sufficiently stable for the incorporation of transmembrane proteins. On the other hand, the p(HPMA)-co-p(LMA) polymer with a LMA content of 25% forms a stable hydrophobic core structure, which prevents hydrophobic interactions with membrane lipids but allows stable incorporation of membrane proteins. On the basis of our data, it becomes obvious that amphiphilic polymers have to have threshold hydrophobicities should an application in membrane protein research be anticipated.

Size-dependent knockdown potential of siRNA-loaded cationic nanohydrogel particles.

To overcome the poor pharmacokinetic conditions of short double-stranded RNA molecules in RNA interference therapies, cationic nanohydrogel particles can be considered as alternative safe and stable carriers for oligonucleotide delivery. For understanding key parameters during this process, two different types of well-defined cationic nanohydrogel particles were synthesized, which provided nearly identical physicochemical properties with regards to their material composition and resulting siRNA loading characteristics. Yet, according to the manufacturing process using amphiphilic reactive ester block copolymers of pentafluorophenyl methacrylate (PFPMA) and tri(ethylene glycol)methyl ether methacrylate (MEO3MA) with similar compositions but different molecular weights, the resulting nanohydrogel particles differed in size after cross-linking with spermine (average diameter 40 vs 100 nm). This affected their knockdown potential significantly. Only the 40 nm sized cationic nanogel particles were able to generate moderate gene knockdown levels, which lasted, however, up to 3 days. Interestingly, primary cell uptake and colocalization studies with lysosomal compartments revealed that only these small sized nanogels were able to avoid acidic compartments of endolysosomal uptake pathways, which may contribute to their knockdown ability exclusively. To that respect, this size-dependent intracellular distribution behavior may be considered as an essential key parameter for tuning the knockdown potential of siRNA nanohydrogel particles, which may further contribute to the development of advanced siRNA carrier systems with improved knockdown potential.