Liposomal Enzyme Nanoreactors Based on Nanoconfinement for Efficient Antitumor Therapy.

Enzymatic reactions can consume endogenous nutrients of tumors and produce cytotoxic species and are therefore promising tools for treating malignant tumors. Inspired by nature where enzymes are compartmentalized in membranes to achieve high reaction efficiency and separate biological processes with the environment, we develop liposomal nanoreactors that can perform enzymatic cascade reactions in the aqueous nanoconfinement of liposomes. The nanoreactors effectively inhibited tumor growth in vivo by consuming tumor nutrients (glucose and oxygen) and producing highly cytotoxic hydroxyl radicals (⋅OH). Co-compartmentalization of glucose oxidase (GOx) and horseradish peroxidase (HRP) in liposomes could increase local concentration of the intermediate product hydrogen peroxide (H2 O2 ) as well as the acidity due to the generation of gluconic acid by GOx. Both H2 O2 and acidity accelerate the second-step reaction by HRP, hence improving the overall efficiency of the cascade reaction. The biomimetic compartmentalization of enzymatic tandem reactions in biocompatible liposomes provides a promising direction for developing catalytic nanomedicines in antitumor therapy.

Temperature-Responsive Nanoparticles Enable Specific Binding of Apolipoproteins from Human Plasma.

Apolipoproteins are an important class of proteins because they provide a so-called stealth effect to nanoparticles. The stealth effect on nanocarriers leads to a reduced unspecific uptake into immune cells and thereby to a prolonged blood circulation time. Herein, a novel strategy to bind apolipoproteins specifically on nanoparticles by adjusting the temperature during their incubation in human plasma is presented. This specific binding, in turn, allows a control of the stealth behavior of the nanoparticles. Nanoparticles with a well-defined poly(N-isopropylacrylamide) shell are prepared, displaying a reversible change of hydrophobicity at a temperature around 32 °C. It is shown by label-free quantitative liquid chromatography-mass spectrometry that the nanoparticles are largely enriched with apolipoprotein J (clusterin) at 25 °C while they are enriched with apolipoprotein A1 and apolipoprotein E at 37 °C. The temperature-dependent protein binding is found to significantly influence the uptake of the nanoparticles by RAW264.7 and HeLa cells. The findings imply that the functionalization of nanoparticles with temperature-responsive materials is a suitable method for imparting stealth properties to nanocarriers for drug-delivery.

Why do ants differ in acclimatory ability? Biophysical mechanisms behind cuticular hydrocarbon acclimation across species.

Maintaining water balance is vital for terrestrial organisms. Insects protect themselves against desiccation via cuticular hydrocarbons (CHCs). CHC layers are complex mixtures of solid and liquid hydrocarbons, with a surprisingly diverse composition across species. This variation may translate into differential phase behaviour, and hence varying waterproofing capacity. This is especially relevant when temperatures change, which requires acclimatory CHC changes to maintain waterproofing. Nevertheless, the physical consequences of CHC variation are still little understood. We studied acclimatory responses and their consequences for CHC composition, phase behaviour and drought survival in three congeneric ant species. Colony sub-groups were kept under cool, warm and fluctuating temperature regimes. Lasius niger and Lasius platythorax, both of which are rich in methyl-branched alkanes, showed largely predictable acclimatory changes of the CHC profile. In both species, warm acclimation increased drought resistance. Warm acclimation increased the proportion of solid compounds in L. niger but not in L. platythorax. In both species, the CHC layer formed a liquid matrix of constantly low viscosity, which contained highly viscous and solid parts. This phase heterogeneity may be adaptive, increasing robustness to temperature fluctuations. In Lasius brunneus, which is rich in unsaturated hydrocarbons, acclimatory CHC changes were less predictable, and warm acclimation did not enhance drought survival. The CHC layer was more homogeneous, but matrix viscosity changed with acclimation. We showed that ant species use different physical mechanisms to enhance waterproofing during acclimation. Hence, the ability to acclimate, and thus climatic niche breadth, may strongly depend on species-specific CHC profile.

Poly(ethylene glycol)-Based Surfactant Reduces the Conformational Change of Adsorbed Proteins on Nanoparticles.

When in contact with a biological medium, the surfaces of nanoparticles are usually covered by proteins. In this regard, it was found that poly(ethylene glycol) (PEG) promotes the "stealth effect". This implies a reduction of unspecific protein adsorption and cellular uptake. Although information about the PEG-protein interaction was reported, more accurate and sophisticated structure and dynamics analyses are needed to understand the interaction processes in detail. This work studies the PEG-protein interaction using model nanoparticles stabilized either by the PEG-based surfactant Lutensol AT50 or sodium dodecyl sulfate. The interaction with human serum albumin was studied using neutron scattering techniques. The parameters obtained by small-angle neutron scattering yielded information about the adsorbed protein layer thickness. Protein structure changes were detected via differential scanning fluorimetry and elastic neutron scattering. This combination gives a better insight into the PEG-protein interaction, contributing to the design of nanomaterials for medical applications.

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.

Dual Stimuli-Responsive Dynamic Covalent Peptide Tags: Toward Sequence-Controlled Release in Tumor-like Microenvironments.

Dynamic covalent chemistry (DCvC) has emerged as a versatile synthetic tool for devising stable, stimuli-responsive linkers or conjugates. The interplay of binding affinity, association and dissociation constants exhibits a strong influence on the selectivity of the reaction, the conversion rate, as well as the stability in aqueous solutions. Nevertheless, dynamic covalent interactions often exhibit fast binding and fast dissociation events or vice versa, affecting their conversion rates or stabilities. To overcome the limitation in linker design, we reported herein dual responsive dynamic covalent peptide tags combining a pH responsive boronate ester with fast association and dissociation rates, and a redox-active disulfide with slow formation and dissociation rate. Precoordination by boronic acid-catechol interaction improves self-sorting and selectivity in disulfide formation into heterodimers. The resulting bis-peptide conjugate exhibited improved complex stability in aqueous solution and acidic tumor-like extracellular microenvironment. Furthermore, the conjugate responds to pH changes within the physiological range as well as to redox conditions found inside cancer cells. Such tags hold great promise, through cooperative effects, for controlling the stability of bioconjugates under dilution in aqueous media, as well as designing intelligent pharmaceutics that react to distinct biological stimuli in cells.

From Hexaphenylbenzene to 1,2,3,4,5,6-Hexacyclohexylcyclohexane.

The hydrogenation of hexaphenylbenzene was studied, affording novel partially hydrogenated hexacyclohexylbenzene (HCB) as well as fully hydrogenated 1,2,3,4,5,6-hexacyclohexylcyclohexane (HCC) as an unprecedented "oligocyclohexyl" molecule. The reaction process was analyzed by mass spectrometry with atmospheric pressure chemical ionization and high-performance liquid chromatography. From a crude product mixture, two different crystals with flake- and block-shapes could be grown and analyzed by X-ray crystallography, revealing their structures as HCB and HCC. While a geared arrangement of cyclohexyl substitutes was found in HCB, two isomeric structures were identified in HCC crystal with chair and twist-boat conformations of the central cyclohexane.

The Influence of Nanoparticle Shape on Protein Corona Formation.

Nanoparticles have become an important utility in many areas of medical treatment such as targeted drug and treatment delivery as well as imaging and diagnostics. These advances require a complete understanding of nanoparticles' fate once placed in the body. Upon exposure to blood, proteins adsorb onto the nanoparticles surface and form a protein corona, which determines the particles' biological fate. This study reports on the protein corona formation from blood serum and plasma on spherical and rod-shaped nanoparticles. These two types of mesoporous silica nanoparticles have identical chemistry, porosity, surface potential, and size in the y-dimension, one being a sphere and the other a rod shape. The results show a significantly larger amount of protein attaching from both plasma and serum on the rod-like particles compared to the spheres. Interrogation of the protein corona by liquid chromatography-mass spectrometry reveals shape-dependent differences in the adsorption of immunoglobulins and albumin proteins from both plasma and serum. This study points to the need for taking nanoparticle shape into consideration because it can have a significant impact on the fate and therapeutic potential of nanoparticles when placed in the body.

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.

Communication versus waterproofing: the physics of insect cuticular hydrocarbons.

Understanding the evolution of complex traits is among the major challenges in biology. One such trait is the cuticular hydrocarbon (CHC) layer in insects. It protects against desiccation and provides communication signals, especially in social insects. CHC composition is highly diverse within and across species. To understand the adaptive value of this chemical diversity, we must understand how it affects biological functionality. So far, CHCs have received ample research attention, but their physical properties were little studied. We argue that these properties determine their biological functionality, and are vital to understanding how CHC composition affects their adaptive value. We investigated melting behaviour and viscosity of CHCs from 11 ant species using differential scanning calorimetry and a novel microrheological technique. CHCs began melting below -45°C, and often were entirely liquid only above 30°C. Thus, they formed a solid-liquid mixture under ambient conditions, which contrasts to previous assumptions of entirely solid layers in many species. This may be adaptive as only biphasic CHC layers ensure uniform coating of the insect body, which is necessary for waterproofing. CHC viscosity was mostly between 0.1 and 0.2 Pa s-1, thus similar to motor oils. Surprisingly, chemically different CHC profiles had similar viscosities, suggesting that a certain viscosity level is adaptive and ensures that communication signals can be perceived. With this study, we draw attention to the importance of studying the physics of CHC layers. Only by understanding how chemical and physical mechanisms enable CHC functionality can we understand the causes and consequences of CHC diversification.

Functionalization of Liposomes with Hydrophilic Polymers Results in Macrophage Uptake Independent of the Protein Corona.

Liposomes are established drug carriers that are employed to transport and deliver hydrophilic drugs in the body. To minimize unspecific cellular uptake, nanocarriers are commonly modified with poly(ethylene glycol) (PEG), which is known to minimize unspecific protein adsorption. However, to date, it has not been studied whether this is an intrinsic and specific property of PEG or if it can be transferred to hyperbranched polyglycerol (hbPG) as well. Additionally, it remains unclear if the reduction of unspecific cell uptake is independent of the "basic" carrier at which a surface functionalization with polymers is usually applied. Therefore, we studied the protein corona of differently functionalized liposomes (unfunctionalized vs PEG or hbPG-functionalized) using PEGylated and PGylated lipids. Their cellular uptake in macrophages was compared. For all three liposomal samples, rather similar protein corona compositions were found, and also-more importantly-the total amount of proteins adsorbed was very low compared to other nanoparticles. Interestingly, the cellular uptake was then significantly changed by the surface functionalization itself, despite the adsorption of a small amount of proteins: although the PEGylation of liposomes resulted in the abovementioned decreased cell uptake, functionalization with hbPG lead to enhanced macrophage interaction-both in the media with and without proteins. In comparison to other nanocarrier systems, this seems to be a liposome-specific effect related to the low amount of adsorbed proteins.

Possibilities and Limitations of Different Separation Techniques for the Analysis of the Protein Corona.

One of the biggest challenges in the field of nanomedicine is the adsorption of biomolecules on the nanomaterial upon contact with a biological medium. The interactions of the resulting protein corona are essential for their behavior in a biological system. Thus, it is now commonly accepted that understanding the formation and consequently understanding the influence of the protein corona on the biological response is crucial. However, the outcome of the protein corona characterization cannot easily be compared between different studies and techniques, since many different sample preparation procedures exist that are suitable for different materials or methods. Depending on the applied procedure, the nanomaterial-protein system will be altered in a certain way, so that it is necessary to consider the individual influence on the protein corona. Accordingly, the aim of this Minireview is to give an overview of the applied sample preparation methods for the analysis of the protein corona and to evaluate their influence on the outcome of the results especially with regard to the introduced terms "soft" and "hard protein corona". Special focus will be placed on the comparison of the most commonly used techniques such as centrifugation, magnetic, and chromatographic separation.

The Transferability from Animal Models to Humans: Challenges Regarding Aggregation and Protein Corona Formation of Nanoparticles.

Nanomaterials are interesting candidates for applications in medicine as drug delivery or diagnostic agents. For safe application, they have to be evaluated in in vitro and in vivo models to finally be translated to human clinical trials. However, often those transfer processes fail, and it is not completely understood whether in vitro models leading to these animal models can reliably be compared to the situation in humans. In particular, the interaction of nanomaterials with components from different blood plasma sources is difficult to compare, and the outcomes of those interactions with respect to body distribution and cell uptake are unclear. Therefore, we investigated the interactions of differently functionalized polymeric and inorganic nanoparticles with human, mouse, rabbit, and sheep plasma. The focus was put on the determination of aggregation events of the nanoparticles occurring in concentrated plasma and the correlation with the respectively formed protein coronas. Both the stability in plasma as well as the types of adsorbed proteins were found to strongly depend on the plasma source. Thus, we suggest evaluating the potential use of nanocarriers always in the plasma source of the chosen animal model for in vitro studies as well as in human plasma to pin down differences and eventually enable transfer into clinical trials in humans.

Denaturation via Surfactants Changes Composition of Protein Corona.

The use of nanocarriers as drug delivery vehicles brings them into contact with blood plasma proteins. Polymeric nanocarriers require some sort of surfactant to ensure colloidal stability. Formation of the protein corona is therefore determined not only by the intrinsic properties of the nanocarrier itself but also by the accompanying surfactant. Although it is well-known that surfactants have an impact on protein structure, only few studies were conducted on the specific effect of surfactants on the composition of protein corona of nanocarriers. Therefore, we analyzed the composition of the protein corona on "stealth" nanoparticles with additional surfactant (cetyltrimethylammonium chloride, CTMA-Cl) after plasma incubation. Additional CTMA-Cl led to an enrichment of apolipoprotein-A1 and vitronectin in the corona, while less clusterin could be found. Further, the structural stability of apolipoprotein-A1 and clusterin was monitored for a wide range of CTMA-Cl concentrations. Clusterin turned out to be more sensitive to CTMA-Cl, with denaturation occurring at lower concentrations.

Engineering Proteins at Interfaces: From Complementary Characterization to Material Surfaces with Designed Functions.

Once materials come into contact with a biological fluid containing proteins, proteins are generally-whether desired or not-attracted by the material's surface and adsorb onto it. The aim of this Review is to give an overview of the most commonly used characterization methods employed to gain a better understanding of the adsorption processes on either planar or curved surfaces. We continue to illustrate the benefit of combining different methods to different surface geometries of the material. The thus obtained insight ideally paves the way for engineering functional materials that interact with proteins in a predetermined manner.

Anti-PEG antibodies enriched in the protein corona of PEGylated nanocarriers impact the cell uptake.

Poly(ethylene glycol) (PEG) is the gold standard used to reduce unspecific protein adsorption and prolong nanocarrier circulation time. However, this stealth effect could be counteracted by the increasing prevalence of anti-PEG antibodies in the bloodstream. Up to now, the presence of anti-PEG antibodies in the protein corona and their effect on cell uptake has not been investigated yet. Our results showed a high concentration and prevalence of anti-PEG antibodies in the German population. PEGylated nanocarriers exhibited a higher level of anti-PEG antibodies in the protein corona compared to non-PEGylated, which lead to higher uptake in macrophages. Consequently, the anti-PEG antibodies in the protein corona could mitigate the stealth effect of PEG, leading to accelerated blood clearance and unwanted side effects.

A new methodology combining QCM-D and proteomic profiling enables characterization of protein adsorption on 2D surfaces.

One of the critical features of biomedical material design is controlling the plasma protein adsorption to modulate the material behavior in biological media. Protein adsorption is highly influenced by the material surfaces and the proteins present in the biological medium. Thus, it is necessary to study protein-surface interactions that eventually take place on nanomaterials introduced into the body by the use of human plasma. However, very little information is available about human plasma interaction with planar surfaces under physiological conditions. Due to the limitation of the current characterization techniques to investigate the complicated interaction between the complex milieu of plasma proteins and planar materials, most efforts have focused on single proteins. To face this challenge, we have developed a new methodology based on the combination of quartz crystal microbalance with dissipation monitoring (QCM-D) and liquid chromatography coupled with mass spectrometry (LC-MS) to obtain information about protein-surface interactions on planar surfaces. First, QCM-D allowed us to determine the adsorbed protein mass and layer thickness. After detaching the proteins by a surfactant treatment, LC-MS analysis revealed the proteomic profile. Here, we have investigated three base materials, polystyrene (PS), gold (Au), and silica (SiO2) with or without precoating and compared the protein profiles.

Systematic modulation of the lipid composition enables the tuning of liposome cellular uptake.

As liposomes have been widely explored as drug delivery carriers over the past decades, they are one of the most promising platforms due to their biocompatibility and versatility for surface functionalization. However, to improve the specific design of liposomes for future biomedical applications such as nanovaccines, it is necessary to understand how these systems interact with cell membranes, as most of their potential applications require them to be internalized by cells. Even though several investigations on the cellular uptake of liposomes were conducted, the effect of the liposome membrane properties on internalization in different cell lines remains unclear. Here, we demonstrate how the cellular uptake behavior of liposomes can be driven towards preferential interaction with dendritic cells (DC2.4) as compared to macrophages (RAW264.7) by tuning the lipid composition with varied molar ratios of the lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). Cellular internalization efficiency was analyzed by flow cytometry, as well as liposome-cell membrane co-localization by confocal laser scanning microscopy. The corresponding proteomic analysis of the protein corona was performed in order to unravel the possible effect on the internalization. The obtained results of this work reveal that it is possible to modulate the cellular uptake towards enhanced internalization by dendritic cells just by modifying the applied lipids and, thus, mainly the physico-chemical properties of the liposomes. STATEMENT OF SIGNIFICANCE: In the field of nanomedicine, it is of key importance to develop new specific and efficient drug carriers. In this sense, liposomes are one of the most widely known carrier types and used in clinics with good results. However, the exact interaction mechanisms of liposomes with cells remain unclear, which is of great importance for the design of new drug delivery platforms. Therefore, in this work we demonstrate that cellular uptake depends on the lipid composition. We are able to enhance the uptake in a specific cell type just by tuning the content of a lipid in the liposome membrane. This finding could be a step towards the selective design of liposomes to be internalized by specific cells with promising applications in biomedicine.

Antibacterial Nanostructured Surfaces Modulate Protein Adsorption, Inflammatory Responses, and Fibrous Capsule Formation.

The present study interrogates the interaction of highly efficient antibacterial surfaces containing sharp nanostructures with blood proteins and the subsequent immunological consequences, processes that are of key importance for the fate of every implantable biomaterial. Studies with human serum and plasma pointed to significant differences in the composition of the protein corona that formed on control and nanostructured surfaces. Quantitative analysis using liquid chromatography-mass spectrometry demonstrated that the nanostructured surface attracted more vitronectin and less complement proteins compared to the untreated control. In turn, the protein corona composition modulated the adhesion and cytokine expression by immune cells. Monocytes produced lower amounts of pro-inflammatory cytokines and expressed more anti-inflammatory factors on the nanostructured surface. Studies using an in vivo subcutaneous mouse model showed reduced fibrous capsule thickness which could be a consequence of the attenuated inflammatory response. The results from this work suggest that antibacterial surface modification with sharp spike-like nanostructures may not only lead to the reduction of inflammation but also more favorable foreign body response and enhanced healing, processes that are beneficial for most medical devices implanted in patients.