Protein Corona: Prevention of Dominant IgG Adsorption on Nanocarriers in IgG‐Enriched Blood Plasma by Clusterin Precoating (Adv. Sci. 10/2019)

The development of nanocarriers for drug delivery is challenged by individual blood composition fluctuations. In article number 1802199, Svenja Morsbach and co‐workers report the accumulation of immunoglobulins in the protein corona of nanocarriers in IgG‐enriched blood plasma resulting in increased cell uptake. This could be prevented by pre‐coating the nanocarriers with the “stealth” protein clusterin. Cover design by Stefan Schuhmacher.

Covalently Binding of Bovine Serum Albumin to Unsaturated Poly(Globalide-Co-ε-Caprolactone) Nanoparticles by Thiol-Ene Reactions.

When nanoparticles (NPs) are introduced to a biological fluid, different proteins (and other biomolecules) rapidly get adsorbed onto their surface, forming a protein corona capable of giving to the NPs a new "identity" and determine their biological fate. Protein-nanoparticle conjugation can be used in order to promote specific interactions between living systems and nanocarriers. Non-covalent conjugates are less stable and more susceptible to desorption in biological media, which makes the development of engineered nanoparticle surfaces by covalent attachment an interesting topic. In this work, the surface of poly(globalide-co-e-caprolactone) (PGlCL) nanoparticles containing double bonds i…

Nanoparticle Shape: The Influence of Nanoparticle Shape on Protein Corona Formation (Small 25/2020)

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 chlorid…

<p>Silica Nanocapsules with Different Sizes and Physicochemical Properties as Suitable Nanocarriers for Uptake in T-Cells</p>

Introduction Adoptive T-cell immunotherapy emerged as a powerful and promising cancer therapy, as the problem regarding the immuno-reaction between different donors and recipients can be avoided. However, this approach is challenging. After long cultivation and expansion under laboratory media conditions, T-cells are losing their viability and function due to immune checkpoint proteins, leading to decreased efficiency in killing cancer cells. Therefore, a new strategy to improve T-cell survival and function is needed. With the advantages of nanotechnology and the biocompatibility of silica-based material, silica nanocapsules (SiNCs) provide an ideal delivery system to transport therapeutic …

Highly Loaded Semipermeable Nanocapsules for Magnetic Resonance Imaging.

Magnetic resonance imaging has become an essential tool in medicine for the investigation of physiological processes. The key issues related to contrast agents, i.e., substances that are injected in the body for imaging, are the efficient enhancement of contrast, their low toxicity, and their defined biodistribution. Polyurea nanocapsules containing the gadolinium complex Gadobutrol as a contrast agent in high local concentration and high relaxivity up to 40 s-1 mmol-1 L are described. A high concentration of the contrast agent inside the nanocapsules can be ensured by increasing the crystallinity in the shell of the nanocapsules. Nanocapsules from aliphatic polyurea are found to display hi…

Water-dispersed semiconductor nanoplatelets with high fluorescence brightness, chemical and colloidal stability

Quasi-two dimensional semiconductor nanoplatelets (NPLs) exhibit high spectral brightness and large absorption cross sections, making them promising for various applications including bioimaging. However, the synthesis of NPLs takes place in organic solvents, therefore they require phase transfer in order to use them in aqueous environments. The phase transfer of NPLs has so far been challenging with few examples in literature. This is likely due to the facile agglomeration of materials with plate-like geometries during the coating procedure. Here we demonstrate how to overcome agglomeration and transfer NPLs, individually coated with amphiphilic polymer chains, to aqueous phase. Upon one a…

Achieving dendritic cell subset-specific targeting in vivo by site-directed conjugation of targeting antibodies to nanocarriers

AbstractThe major challenge of nanocarrier-based anti-cancer vaccination approaches is the targeted delivery of antigens and immunostimulatory agents to cells of interest, such as specific subtypes of dendritic cells (DCs), in order to induce robust antigen-specific anti-tumor responses. An undirected cell and body distribution of nanocarriers can lead to unwanted delivery to other immune cell types like macrophages reducing the vaccine efficacy. An often-used approach to overcome this issue is the surface functionalization of nanocarriers with targeting moieties, such as antibodies, mediating cell type-specific interaction. Numerous studies could successfully prove the targeting efficiency…

Fully degradable protein nanocarriers by orthogonal photoclick tetrazole-ene chemistry for the encapsulation and release

The encapsulation of sensitive drugs into nanocarriers retaining their bioactivity and achieving selective release is a challenging topic in current drug delivery design. Established protocols rely on metal-catalyzed or unspecific reactions to build the (mostly synthetic) vehicles which may inhibit the drug's function. Triggered by light, the mild tetrazole–ene cycloaddition enables us to prepare protein nanocarriers (PNCs) preserving at the same time the bioactivity of the sensitive antitumor and antiviral cargo Resiquimod (R848). This catalyst-free reaction was designed to take place at the interface of aqueous nanodroplets in miniemulsion to produce core–shell PNCs with over 90% encapsul…

Bio-orthogonal triazolinedione (TAD) crosslinked protein nanocapsules affect protein adsorption and cell interaction

Albumin-based protein nanocarriers have been widely exploited as drug delivery systems, since they show excellent degradability, low toxicity, but at the same time provide high loading capacity and relevant uptake into cells. For the formation of protein nanocapsules, bio-orthogonal reactions are important so that the material to be encapsulated is not affected by the shell formation. We show that protein nanocapsules with narrow size distributions and low protein adsorption upon contact with blood serum can be synthesized by inverse miniemulsion and interfacial crosslinking of the protein using triazolinediones (TADs) as powerful dienophiles and enophiles, which smoothly perform electrophi…

Noncovalent Targeting of Nanocarriers to Immune Cells with Polyphosphoester‐Based Surfactants in Human Blood Plasma

Abstract Dendritic cells (DCs) are part of the immune system and can internalize pathogens by carbohydrate receptors. The uptake induces maturation and migration of the DCs resulting in an adaptive immune response by presenting antigens to T‐cells. Thus, targeted delivery to DCs is a powerful tool for immunotherapy. However, in blood, specific targeting is challenging as blood proteins adsorb to the nanocarriers and mask the targeting molecules. Additionally, covalent coupling of targeting groups to nanocarriers requires new chemistry for each nanocarrier, while a general strategy is missing. A general protocol by noncovalent adsorption of mannosylated polyphosphoesters (PPEs) on the nanoca…

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 …

Encapsulation of polyprodrugs enables an efficient and controlled release of dexamethasone

Water-soluble low molecular weight drugs, such as the synthetic glucocorticoid dexamethasone (DXM), can easily leak out of nanocarriers after encapsulation due to their hydrophilic nature and small size. This can lead to a reduced therapeutic efficacy and therefore to unwanted adverse effects on healthy tissue. Targeting DXM to inflammatory cells of the liver like Kupffer cells or macrophages is a promising approach to minimize typical side effects. Therefore, a controlled transport to the cells of interest and selective on-site release is crucial. Aim of this study was the development of a DXM-phosphate-based polyprodrug and the encapsulation in silica nanocontainers (SiO2 NCs) for the red…

Hydrophilicity Regulates the Stealth Properties of Polyphosphoester‐Coated Nanocarriers

Increasing the plasma half-life is an important goal in the development of drug carriers, and can be effectively achieved through the attachment of polymers, in particular poly(ethylene glycol) (PEG). While the increased plasma half-life has been suggested to be a result of decreased overall protein adsorption on the hydrophilic surface in combination with the adsorption of specific proteins, the molecular reasons for the success of PEG and other hydrophilic polymers are still widely unknown. We prepared polyphosphoester-coated nanocarriers with defined hydrophilicity to control the stealth properties of the polymer shell. We found that the log P value of the copolymer controls the composit…

Amphiphilic Dendrimers Control Protein Binding and Corona Formation on Liposome Nanocarriers

Amphiphilic polyphenylene dendrimers (PPDs) with distinct lipophilic and positively or negatively charged surface groups were adsorbed onto liposomes and their impact on protein adsorption in blood plasma was studied. The PPD corona reduced binding of specific opsonins and increased the adsorption of proteins controlling cellular uptake based on their surface patches.

Preparation of the Protein Corona: How Washing Shapes the Proteome and Influences Cellular Uptake

A protein coat, termed the protein corona, assembles around the nanocarriers´ surface once it gets in contact with a biological environment. We show that the media used for washing of the protein corona can be crucial. This is true for downstream analysis as well as for precoating for in vitro or in vivo use. This has been widely overlooked so far. We focus on the choice of eight different washing media and how they influence the composition of the hard protein corona of several nanocarriers incubated with human blood plasma and serum. SDS-PAGE and LC-MS analysis showed major differences in protein corona profiles when using diverse washing media. While plasma and serum proteins already hav…

The Protein Corona as a Confounding Variable of Nanoparticle-Mediated Targeted Vaccine Delivery

Nanocarriers (NC) are very promising tools for cancer immunotherapy. Whereas conventional vaccines are based on the administration of an antigen and an adjuvant in an independent fashion, nanovaccines can facilitate cell-specific co-delivery of antigen and adjuvant. Furthermore, nanovaccines can be decorated on their surface with molecules that facilitate target-specific antigen delivery to certain antigen-presenting cell types or tumor cells. However, the target cell-specific uptake of nanovaccines is highly dependent on the modifications of the nanocarrier itself. One of these is the formation of a protein corona around NC after in vivo administration, which may potently affect cell-speci…

Timing of Heparin Addition to the Biomolecular Corona Influences the Cellular Uptake of Nanocarriers.

Few studies have considered the interaction of nanocarriers with drugs and the implications for their individual efficiency. Here, we demonstrate that heparin, a common anticoagulant, interacts with nanocarriers. Hence, nanocarriers, precoated with heparin and plasma in different conditions, were incubated with cancer cells, as well as primary cells from human blood. The relation between the timing of the heparin's addition to the nanocarrier and the cellular uptake extent was assessed by flow cytometry. Through proteomics the effect of heparin on the biomolecular corona composition was determined. We found that HeLa cells, monocytes and macrophages reacted differently to the presence of he…

Prevention of Dominant IgG Adsorption on Nanocarriers in IgG‐Enriched Blood Plasma by Clusterin Precoating

Abstract Nanocarriers for medical applications must work reliably within organisms, independent of the individual differences in the blood proteome. Variation in the blood proteome, such as immunoglobulin levels, is a result of environmental, nutrition, and constitution conditions. This variation, however, should not influence the behavior of nanocarriers in biological media. The composition of the protein corona is investigated to understand the influence varying immunoglobulin levels in the blood plasma have on the interactions with nanocarriers. Specifically, the composition of the nanocarriers' coronas is analyzed after incubation in plasma with normal or elevated immunoglobulin G (IgG)…

Preservation of the soft protein corona in distinct flow allows identification of weakly bound proteins.

Abstract Nanocarriers that are used for targeted drug delivery come in contact with biological liquids and subsequently proteins will adsorb to the nanocarriers’ surface to form the so called ‘protein corona’. The protein corona defines the biological identity and determines the biological response towards the nanocarriers in the body. To make nanomedicine safe and reliable it is required to get a better insight into this protein corona and, therefore, the adsorbed proteins have to be characterized. Currently, centrifugation is the common method to isolate the protein corona for further investigations. However, with this method it is only possible to investigate the strongly bound proteins,…

Hydrophilie als bestimmender Faktor des Stealth-Effekts von Polyphosphoester-funktionalisierten Nanoträgern

Brush Conformation of Polyethylene Glycol Determines the Stealth Effect of Nanocarriers in the Low Protein Adsorption Regime

For nanocarriers with low protein affinity, we show that the interaction of nanocarriers with cells is mainly affected by the density, the molecular weight, and the conformation of polyethylene glycol (PEG) chains bound to the nanocarrier surface. We achieve a reduction of nonspecific uptake of ovalbumin nanocarriers by dendritic cells using densely packed PEG chains with a "brush" conformation instead of the collapsed "mushroom" conformation. We also control to a minor extent the dysopsonin adsorption by tailoring the conformation of attached PEG on the nanocarriers. The brush conformation of PEG leads to a stealth behavior of the nanocarriers with inhibited uptake by phagocytic cells, whi…

The conjugation strategy affects antibody orientation and targeting properties of nanocarriers.

Antibody-modified drug delivery systems in the nano-range have the ability to overcome current challenges for treating diseases due to their high specificity towards the targeted body region. However, no antibody-bound nanocarrier has been clinically approved to date. This missing clinical approval may be a result of the conjugation strategy that influences the spatial orientation of the attached antibody on the nanocarriers' surface. What is not missing, however, is a diverse selection of antibody to nanocarrier conjugation strategies that determine the success of an antibody functionalized drug delivery system. In this paper, two antibody conjugation strategies were compared by conjugatin…

Amphiphilic Polyphenylene Dendron Conjugates for Surface Remodeling of Adenovirus 5

Abstract Amphiphilic surface groups play an important role in many biological processes. The synthesis of amphiphilic polyphenylene dendrimer branches (dendrons), providing alternating hydrophilic and lipophilic surface groups and one reactive ethynyl group at the core is reported. The amphiphilic surface groups serve as biorecognition units that bind to the surface of adenovirus 5 (Ad5), which is a common vector in gene therapy. The Ad5/dendron complexes showed high gene transduction efficiencies in coxsackie‐adenovirus receptor (CAR)‐negative cells. Moreover, the dendrons offer incorporation of new functions at the dendron core by in situ post‐modifications, even when bound to the Ad5 sur…

Ultra-small gold nanoclusters assembled on plasma polymer-modified zeolites: a multifunctional nanohybrid with anti-haemorrhagic and anti-inflammatory properties

Refereed/Peer-reviewed Hemostatic agents are pivotal for managing clinical and traumatic bleeding during emergency and domestic circumstances. Herein, a novel functional hybrid nanocomposite material consisting of plasma polymer-modified zeolite 13X and ultra-small gold nanoclusters (AuNCs) was fabricated as an efficient hemostatic agent. The surface of zeolite 13X was functionalised with amine groups which served as binding sites for carboxylate terminated AuNCs. Protein corona studies revealed the enhanced adsorption of two proteins, namely, coagulation factors and plasminogen as a result of AuNCs immobilization on the zeolite surface. The immune response studies showed that the hybrid na…

A bio-orthogonal functionalization strategy for site-specific coupling of antibodies on vesicle surfaces after self-assembly

Attaching targeting ligands on the surface of self-assembled drug delivery systems is the key request for a controlled transport of the drug to a desired location. Most commonly, the amphiphilic molecules (blockcopolymers, lipids etc.) are therefore pre-functionalized before the self-assembly takes place. However, this strategy cannot be applied, if it interferes with the self-assembly process, if the introduced functional groups react with loaded cargo or if natural carriers like extracellular vesicles should be functionalized. Here, we present the site-specific coupling of antibodies to the surface of amino group-terminated liposomes via bio-orthogonal copper-free click chemistry after li…

The Role of the Protein Corona in the Uptake Process of Nanoparticles

Protein denaturation caused by heat inactivation detrimentally affects biomolecular corona formation and cellular uptake

Adsorption of blood proteins to the surface of nanocarriers is known to be the critical factor influencing cellular interactions and eventually determining the successful application of nanocarriers as drug carriers in vivo. There is an increasing number of reports summarizing large data sets of all identified corona proteins. However, to date our knowledge about the multiple mechanisms mediating interactions between proteins and nanocarriers is still limited. In this study, we investigate the influence of protein structure on the adsorption process and focus on the effect of heat inactivation of serum and plasma, which is a common cell culture procedure used to inactivate the complement sy…

Controlling protein interactions in blood for effective liver immunosuppressive therapy by silica nanocapsules

Immunosuppression with glucocorticoids is a common treatment for autoimmune liver diseases and after liver transplant, which is however associated with severe side-effects. Targeted delivery of glucocorticoids to inflammatory cells, e.g. liver macrophages and Kupffer cells, is a promising approach for minimizing side effects. Herein, we prepare core–shell silica nanocapsules (SiO2 NCs) via a sol–gel process confined in nanodroplets for targeted delivery of dexamethasone (DXM) for liver immunosuppressive therapy. DXM with concentrations up to 100 mg mL−1 in olive oil are encapsulated while encapsulation efficiency remains over 95% after 15 days. Internalization of NCs by non-parenchymal muri…

Protein Corona Mediated Stealth Properties of Biocompatible Carbohydrate‐based Nanocarriers

Unraveling the In Vivo Protein Corona

Understanding the behavior of nanoparticles upon contact with a physiological environment is of urgent need in order to improve their properties for a successful therapeutic application. Most commonly, the interaction of nanoparticles with plasma proteins are studied under in vitro conditions. However, this has been shown to not reflect the complex situation after in vivo administration. Therefore, here we focused on the investigation of magnetic nanoparticles with blood proteins under in vivo conditions. Importantly, we observed a radically different proteome in vivo in comparison to the in vitro situation underlining the significance of in vivo protein corona studies. Next to this, we fou…

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 fun…

Polyphosphoester surfactants as general stealth coatings for polymeric nanocarriers

Opsonization of nanocarriers is one of the most important biological barriers for controlled drug delivery. The typical way to prevent such unspecific protein adsorption and thus fast clearance by the immune system is the covalent modification of drug delivery vehicles with poly(ethylene glycol) (PEG), so-called PEGylation. Recently, polyphosphoesters (PPEs) were identified as adequate PEG substitutes, however with the benefits of controllable hydrophilicity, additional chemical functionality, or biodegradability. Here, we present a general strategy by non-covalent adsorption of different nonionic PPE-surfactants to nanocarriers with stealth properties. Polyphosphoester surfactants with dif…

How Low Can You Go? Low Densities of Poly(ethylene glycol) Surfactants Attract Stealth Proteins.

It is now well-established that the surface chemistry and “stealth” surface functionalities such as poly(ethylene glycol) (PEG) chains of nanocarriers play an important role to decrease unspecific protein adsorption of opsonizing proteins, to increase the enrichment of specific stealth proteins, and to prolong the circulation times of the nanocarriers. At the same time, PEG chains are used to provide colloidal stability for the nanoparticles. However, it is not clear how the chain length and density influence the unspecific and specific protein adsorption keeping at the same time the stability of the nanoparticles in a biological environment. Therefore, this study aims at characterizing the…