Controlling the Stealth Effect of Nanocarriers through Understanding the Protein Corona

The past decade has seen a significant increase in interest in the use of polymeric nanocarriers in medical applications. In particular, when used as drug vectors in targeted delivery, nanocarriers could overcome many obstacles for drug therapy. Nevertheless, their application is still impeded by the complex composition of the blood proteins covering the particle surface, termed the protein corona. The protein corona complicates any prediction of cell interactions, biodistribution, and toxicity. In particular, the unspecific uptake of nanocarriers is a major obstacle in clinical studies. This Minireview provides an overview of what we currently know about the characteristics of the protein corona of nanocarriers, with a focus on surface functionalization that reduces unspecific uptake (the stealth effect). The ongoing improvement of nanocarriers to allow them to meet all the requirements necessary for successful application, including targeted delivery and stealth, are further discussed.     

DNA‐Hybrid‐Gated Multifunctional Mesoporous Silica Nanocarriers for Dual‐Targeted and MicroRNA‐Responsive Controlled Drug Delivery

The design of an ideal drug delivery system with targeted recognition and zero premature release, especially controlled and specific release that is triggered by an exclusive endogenous stimulus, is a great challenge. A traceable and aptamer‐targeted drug nanocarrier has now been developed; the nanocarrier was obtained by capping mesoporous silica‐coated quantum dots with a programmable DNA hybrid, and the drug release was controlled by microRNA. Once the nanocarriers had been delivered into HeLa cells by aptamer‐mediated recognition and endocytosis, the overexpressed endogenous miR‐21 served as an exclusive key to unlock the nanocarriers by competitive hybridization with the DNA hybrid, which led to a sustained lethality of the HeLa cells. If microRNA that is exclusively expressed in specific pathological cell was screened, a combination of chemotherapy and gene therapy should pave the way for a targeted and personalized treatment of human diseases.     

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.     

Carbohydrate‐Based Nanocarriers Exhibiting Specific Cell Targeting with Minimum Influence from the Protein Corona

Whenever nanoparticles encounter biological fluids like blood, proteins adsorb on their surface and form a so‐called protein corona. Although its importance is widely accepted, information on the influence of surface functionalization of nanocarriers on the protein corona is still sparse, especially concerning how the functionalization of PEGylated nanocarriers with targeting agents will affect protein corona formation and how the protein corona may in turn influence the targeting effect. Herein, hydroxyethyl starch nanocarriers (HES‐NCs) were prepared, PEGylated, and modified on the outer PEG layer with mannose to target dendritic cells (DCs). Their interaction with human plasma was then studied. Low overall protein adsorption with a distinct protein pattern and high specific affinity for DC binding were observed, thus indicating an efficient combination of “stealth” and targeting behavior.     

Pay Attention to Biological Nanoparticles when Studying the Protein Corona on Nanomedicines

The protein corona of nanoparticles has in recent years received considerable attention, and even been postulated to be the missing link in the translation of nanomedicines from benchtop to bedside. We highlight the different types of biological nanoparticles present in blood that need to be considered in the protein corona research field. We map their size, density, and plasma concentrations, and use this information to stress potential challenges related to the isolation of nanomedicines—with a particular focus on liposomes—when using the traditional isolation methods that separate according to size and density.     

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 composition of the protein corona and the cell interaction. Upon a significant change in hydrophilicity, the overall amount of blood proteins adsorbed on the nanocarrier remained unchanged, while the protein composition varied. This result underlines the importance of the protein type for the protein corona and cellular uptake.     

Polyglycerol‐Based Copper Chelators for the Transport and Release of Copper Ions in Biological Environments

Here, the synthesis and characterization of three improved nanosystems is presented based on amino functionalized hyperbranched polyglycerol (hPG; M_w = 16.8 kDa) as potential copper(ii ) chelators. The ligands, N‐methyl‐N‐picolylglycine amide, 2,6‐pyridine dicarboxylic acid monoamide, and cyclam tetraacetic acid (TETA) monoamide, are covalently attached to the polymer with amide bonds. In this paper, the Cu(ii ) loading capacity, the stability of the Cu(ii )‐loaded carriers at different pHs, with competing ligands and in human serum, as well as the transport of Cu(ii ) in biological systems are investigated. For the first time, a different cytotoxicity of functionalized polymer nanoparticles with and without Cu(ii ) is observed. The cyclam‐based carrier combines the highest loading capacity (29 Cu ions/nanoparticle), best stability with respect to pH and EDTA (45% remaining Cu after 24 h), lowest cytotoxicity (IC₅₀ > 100 × 10^?6m (unloaded), 1500 × 10^?6m Cu(ii ); Cu:carrier 29:1), and the highest stability in human serum.

     

Gold Nanocarriers for Macrophage‐Targeted Therapy of Human Immunodeficiency Virus

The human immunodeficiency virus (HIV) continues to be a global pandemic and there is an urgent need for innovative treatment. Immune cells represent a major target of virus infection, but are also therapeutic targets. Currently, no antiretroviral therapy targets macrophages, which function as portal of entry and as major long‐term deposit of HIV. It has been shown before that human macrophages efficiently internalize gold nanoparticles, a fact which might be used to target them with drug‐nanoparticle conjugates. Here, the authors use gold nanocarriers to facilitate delivery of stavudine, a widely used antiretroviral drug, to primary human macrophages. Using an ease‐of‐use coupling method, a striking potentiation of stavudine intake by macrophages using gold nanocarriers is shown. Further, the carriers induce a specific subtype of proinflammatory activation indicative for antiviral activity of macrophages, which suggests promising novel treatment options for HIV.

     

Polyethyleneimine‐Coated Gold Nanoparticles: Straightforward Preparation of Efficient DNA Delivery Nanocarriers

A novel one‐pot method for the synthesis of polyethyleneimine (PEI)‐coated gold nanoparticles (AuPEI‐NPs) that combines the reductant–stabilizer properties of PEI with microwave irradiation starting from hydrogen tetrachloroaurate acid (HAuCl₄) and branched PEI 25 kDa (b25kPEI) was explored. The method was straightforward, green, and low costing, for which the Au/PEI ratio (1:1 to 1:128 w/w) was a key parameter to modulate their capabilities as DNA delivery nanocarriers. Transfection assays in CHO‐k1 cells demonstrated that AuPEI‐NPs with 1:16 and 1:32 w/w ratios behaved as effective DNA gene vectors with improved transfection efficiencies (twofold) and significantly lower toxicity than unmodified b25kPEI and Lipofectamine 2000. The transfection mediated by these AuPEI‐NP–DNA polyplexes preferentially used the caveolae‐mediated route for intracellular internalization, as shown by studies performed by using specific internalization inhibitors as well as colocalization with markers of clathrin‐ and caveolae‐dependent pathways. The AuPEI‐NP polyplexes preferentially used the more efficient caveolae internalization pathway to promote transfection, a fact that supports their higher transfection efficiency relative to that of Lipofectamine 2000. In addition, intracellular trafficking of the AuPEI‐NPs was studied by transmission electron microscopy.     

Biomedical and Pharmacological Uses of Fluorescein Isothiocyanate Chitosan‐Based Nanocarriers

Chitosan‐based nanocarriers (ChNCs) are considered suitable drug carriers due to their ability to encapsulate a variety of drugs and cross biological barriers to deliver the cargo to their target site. Fluorescein isothiocyanate‐labeled chitosan‐based NCs (FITC@ChNCs) are used extensively in biomedical and pharmacological applications. The main advantage of using FITC@ChNCs consists of the ability to track their fate both intra and extracellularly. This journey is strictly dependent on the physico‐chemical properties of the carrier and the cell types under investigation. Other applications make use of fluorescent ChNCs in cell labeling for the detection of disorders in vivo and controlling of living cells in situ. This review describes the use of FITC@ChNCs in the various applications with a focus on understanding their usefulness in labeled drug‐delivery systems.     

Protein Delivery System Containing a Nickel‐Immobilized Polymer for Multimerization of Affinity‐Purified His‐Tagged Proteins Enhances Cytosolic Transfer

Recombinant proteins with cytosolic or nuclear activities are emerging as tools for interfering with cellular functions. Because such tools rely on vehicles for crossing the plasma membrane we developed a protein delivery system consisting in the assembly of pyridylthiourea‐grafted polyethylenimine (πPEI) with affinity‐purified His‐tagged proteins pre‐organized onto a nickel‐immobilized polymeric guide. The guide was prepared by functionalization of an ornithine polymer with nitrilotriacetic acid groups and shown to bind several His‐tagged proteins. Superstructures were visualized by electron and atomic force microscopy using 2 nm His‐tagged gold nanoparticles as probes. The whole system efficiently carried the green fluorescent protein, single‐chain antibodies or caspase 3, into the cytosol of living cells. Transduction of the protease caspase 3 induced apoptosis in two cancer cell lines, demonstrating that this new protein delivery method could be used to interfere with cellular functions.     

Aggregation Behavior of Non‐ionic Twinned Amphiphiles and Their Application as Biomedical Nanocarriers

A new class of twinned amphiphiles was developed by conjugating a pair of hydrophilic head groups from mPEG chains (M _n: 350 or 1000) and a pair of hydrophobic segments from linear alkyl chains (C₁₁ or C₁₈) through a novel spacer synthesized from glycerol and p ‐hydroxybenzoic acid. The aggregation phenomena of the amphiphiles were proven by DLS and fluorescence experiments, whereas size and morphology of the aggregates were evaluated by cryo‐TEM. The measurements proved the formation of globular, thread‐like or rod‐like micelles as well as planar double‐layer assemblies, depending on the amphiphile's molecular structure. The applicability of these non‐ionic amphiphilic systems as nanocarriers for hydrophobic guest molecules was demonstrated by encapsulating a hydrophobic dye, Nile Red, and a hydrophobic drug, Nimodipine. The transport capacity results for both Nimodipine and Nile Red prove them as a promising candidate for drug delivery.     

Stimulus‐Induced Conformational Transformation of a Cyclic Peptide for Selective Cell‐Targeting On–Off Gatekeeper for Mesoporous Nanocarriers

α_vβ₃ Integrin is upregulated on many cancer cells. We designed a dual functional cyclic peptide gatekeeper with a capability of stimuli‐responsive conformational transformation which could serve as a selective cell‐targeting on–off gatekeeper for mesoporous nanocarriers. The advantage of employing the motif of stimuli‐induced conformational transformation of cyclic peptides is that they could be utilized not only as an on–off gatekeeper for the triggered release of cargo drugs but also as a targeting ligand of the carriers to desired cells with their respective binding receptors. The peptide gatekeepers on the surface of nanocarriers exhibited on–off gatekeeping via conformational transformation triggered by intracellular glutathione levels of the cancer cells. The cyclic RGD sequence of the peptide gatekeepers enhanced the intracellular uptake into tumor cells (A549) and the therapeutic efficacy of the nanocarrier.     

Tumor‐Acidity Activated Surface Charge‐Conversion of Polymeric Nanocarriers for Enhanced Cell Adhesion and Targeted Drug Release

The development of stimuli‐responsive polymeric nanocarriers could significantly enhance drug bioavailability due to improved pharmacokinetics and biodistribution. However, in the drug delivery process, the poor cell uptake of drug‐loaded carriers has greatly limited the therapeutic efficiency for anti‐cancer applications. Herein, 2,3‐dimethylmaleic anhydride (DMMA) is engineered into the well‐defined biodegradable amphiphilic block copolymer poly(D,L‐lactide)‐block‐poly(2‐aminoethyl methacrylate) (PLA‐b‐PAEMA) to construct a tumor‐acidity activated nanocarrier (PLA‐b‐PAEMA/DMMA) for potential tumor therapy. After the loading of positively charged DOX·HCl into the negatively charged corona structure through electrostatic attraction, this carrier is expected to prolong the blood circulation time and smartly convert surface charge from negative to positive for enhanced tumor cell uptake and targeted drug release. Furthermore, this carrier exhibits additional cytotoxicity for tumor cells after the tumor‐acidity activated surface charge‐conversion from negative to positive. Thus, this smart carrier is a feasible candidate for potential cancer therapy.

     

基于二氧化硅胶体晶体薄膜和反射干涉光谱的蛋白冠监测方法

生物分子与纳米材料作用形成的"蛋白冠"影响纳米材料的物理和化学性质,目前缺少有效的原位实时技术监测蛋白冠的形成过程.本研究基于二氧化硅胶体晶体薄膜和反射干涉光谱法,研究了三种代表性血液蛋白质在二氧化硅纳米粒子表面的蛋白冠形成过程,结果表明这三种蛋白具有不同的蛋白冠形成过程及参数;研究了人血清白蛋白在三种表面曲率的二氧化...     

Monodisperse Poly(lactide‐co‐glycolic acid)‐Based Nanocarriers for Gene Transfection

This contribution describes a simple, aerosol‐based method for fabricating monodisperse particles containing mixtures of poly(lactide‐co‐glycolic acid) [PLGA], protamine sulfate (Prot), and poly(l‐ lysine) [PLL] as nanocarriers for gene transfection. Aqueous solutions of PLGA, Prot, and PLL were collison‐atomized, and the resulting aerosolized droplets were dried “on the fly” to form solid particles, which then were electrostatically size‐classified into 50, 100, and 200 nm mobility diameter samples. Measurements of cell viability and transfection reveal that the fabricated nanocarriers have a lower cytotoxicity (>85% in cell viability) and a higher transfection efficiency [>8.7 × 10⁵ in relative light units (RLU) mg⁻¹] than does 25 kDa polyethyleneimine (≈50% and 6.8 × 10⁵ RLU mg⁻¹).     

Engineering the Protein Corona Structure on Gold Nanoclusters Enables Red-Shifted Emissions in the Second Near-infrared Window for Gastrointestinal Imaging

The application of NIR-II emitters for gastrointestinal (GI) tract imaging remains challenging due to fluorescence quenching in the digestive microenvironment. Herein, we report that red-shifting of the fluorescence emission of Au nanoclusters (AuNCs) into NIR-II region with improved quantum yields (QY) could be achieved by engineering a protein corona structure consisting of a ribonuclease-A (RNase-A) on the particle surfaces. RNase-A-encapsulated AuNCs (RNase-A@AuNCs) displayed emissions at 1050 nm with a 1.9 % QY. Compared to rare earth and silver-based NIR-II emitters, RNase-A@AuNCs had excellent biocompatibility, showing >50-fold higher sensitivity in GI tract, and migrated homogenously during gastrointestinal peristalsis to allow visualization of the detailed structures of the GI tract. RNase-A@AuNCs could successfully examine intestinal tumor mice from healthy mice, indicating a potential utility for early diagnosis of intestinal tumors.     

Design and Preparation of Single‐Chain Nanocarriers Mimicking Disordered Proteins for Combined Delivery of Dermal Bioactive Cargos

Inspired by the multifunctionality of vitamin D‐binding protein and the multiple transient‐binding behavior of some intrinsically disordered proteins (IDPs), a polymeric platform is designed, prepared, and characterized for combined delivery of dermal protective and anticancer bioactive cargos on the basis of artificial single‐chain nano‐objects mimicking IDPs. For the first time ever, simultaneous delivery of folic acid or vitamin B₉, and hinokitiol, a relevant natural bioactive compound that exhibits anticancer activity against human malignant melanoma cells, from these multidirectionally self‐assembled unimolecular nanocarriers is illustrated.

     

Interaction of Polystyrene Nanoparticles with Supported Lipid Bilayers: Impact of Nanoparticle Size and Protein Corona

Polystyrene is one of the most widely used plastics. This article reports on the interaction of 50 and 210 nm polystyrene nanoparticles (PSNPs) with human serum albumin (HSA) and transferrin (Tf), as well as their effect on supported lipid bilayers (SLBs), using experimental and theoretical approaches. Dynamic light scattering (DLS) and atomic force microscopy (AFM) measurements show that the increase in diameter for the PSNP-protein bioconjugates depends on nanoparticle size and type of proteins. The circular dichroism (CD) spectroscopy results demonstrate that the proteins preserve their structures when they interact with PSNPs at physiological temperatures. The quartz crystal microbalance (QCM) technique reveals that PSNPs and their bioconjugates show no strong interactions with SLBs. On the contrary, the molecular dynamics simulations (MDS) show that both proteins bind strongly to the lipid bilayer (SLBs) when compared to their binding to a polystyrene surface model. The interaction is strongly dependent on the protein and lipid bilayer composition. Both the PSNPs and their bioconjugates show no toxicity in human umbilical vein endothelial (HUVEC) cells; however, bare 210 nm PSNPs and 50 nm PSNP-Tf bioconjugates show an increase in reactive oxygen species production. This study may be relevant for assessing the impact of plastics on health.