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Photothermal effects on protein adsorption dynamics of PEGylated gold nanorods
Abstract
The effect of thermoplasmonic heating on the dynamics of the protein corona around polyethylene glycol (PEG) coated gold nanorods (GNRs) is reported. GNRs were surface-modified with densely packed shells composed of thiolated PEGs bearing carboxyl and/or amine end-terminal groups, which led to negatively, positively or neutral charged GNRs. The three types of PEGylated GNRs, although with subtle differences among them, present weak interactions (binding dissociation constants in the μM range) with the model protein bovine serum albumin (BSA), as determined by different methods including gel electrophoresis, laser Doppler anemometry and isothermal titration calorimetry (ITC). In general, plasmonic photothermal heating significantly changed the composition of the protein corona around GRNs, particularly, on the positively charged ones. Moreover, a pre-formed BSA corona on the PEGylated GNRs further decreased their already reduced uptake by cells. However, photothermal treatment of BSA-coated GNRs only produced a significant decreased internalization in the case of non-PEGylated control samples. Overall, our results indicate that differently surface charged PEGylations on GNRs affects the dynamics of protein corona formation, which is particularly relevant after photothermal treatment. Such effects are nonetheless non-significant concerning cellular uptake.
... GNRs can be functionalized to localize specifically within and around a tumor volume [21,29]. A recent study by Polo et al. demonstrated that plasmonic heating significantly changes the composition of protein corona around the GNRs which can be manipulated through surface charging of PEGylated GNRs [30]. However, the clinical application of PPTT for pancreatic cancer treatment will require precise control of GNR localization and thermal ablation, requiring further research to realize. ...
... A fiberoptic microneedle device (FMD) was developed by our group through leveraging an innovative light fluid coupling technique for co-delivering high power laser light (both visible and NIR) and liquid solutions (e.g., GNR solution) to a targeted tissue volume [30][31][32][33]. The sharp FMD tip is capable of penetrating soft tissue to infuse fluids and deliver light either peri-or intra-tumorally, reducing the chance of damaging healthy tissue in the optical path. ...
... The experimental setup was similar to the previous experiments except for the addition of the GNR solution transfused through the FMD by a syringe pump (Figure 3). The FMD was also coupled by a free coupler to the 808 or 1064 nm laser, which was delivered at the same irradiances as before (30,40, and 50 mW·mm −2 ). GNR concentrations infused included 0.1, 0.25, 0.5, 0.75, and 1 nM delivered at 1 mL/min for 60 s. ...
Plasmonic photothermal therapy (PPTT) has potential as a superior treatment method for pancreatic cancer, a disease with high mortality partially attributable to the currently non-selective treatment options. PPTT utilizes gold nanoparticles infused into a targeted tissue volume and exposed to a specific light wavelength to induce selective hyperthermia. The current study focuses on developing this approach within an ex vivo porcine pancreas model via an innovative fiberoptic microneedle device (FMD) for co-delivering light and gold nanoparticles. The effects of laser wavelengths (808 vs. 1064 nm), irradiances (20–50 mW·mm−2), and gold nanorod (GNR) concentrations (0.1–3 nM) on tissue temperature profiles were evaluated to assess and control hyperthermic generation. The GNRs had a peak absorbance at ~800 nm. Results showed that, at 808 nm, photon absorption and subsequent heat generation within tissue without GNRs was 65% less than 1064 nm. The combination of GNRs and 808 nm resulted in a 200% higher temperature rise than the 1064 nm under similar conditions. A computational model was developed to predict the temperature shift and was validated against experimental results with a deviation of <5%. These results show promise for both a predictive model and spatially selective, tunable treatment modality for pancreatic cancer.
... With the same number of free surface thiol groups as K19C GB3, the size effect of protein adsorption on PEGylated AuNP can be examined. Previous experiments using fluorescence spectroscopy found that BSA was buried inside the 10K PEG layer on an AuNP [14,26]. Our studies indicate that, due to the size of BSA, only a small concentration of BSA is adsorbed by all three types of PEGylated AuNP surfaces. ...
... Molecules 2021,26, 5788 ...
... Molecules 2021, 26, 5788 ...
Polyethylene glycol (PEG) surface conjugations are widely employed to render passivating properties to nanoparticles in biological applications. The benefits of surface passivation by PEG are reduced protein adsorption, diminished non-specific interactions, and improvement in pharmacokinetics. However, the limitations of PEG passivation remain an active area of research, and recent examples from the literature demonstrate how PEG passivation can fail. Here, we study the adsorption amount of biomolecules to PEGylated gold nanoparticles (AuNPs), focusing on how different protein properties influence binding. The AuNPs are PEGylated with three different sizes of conjugated PEG chains, and we examine interactions with proteins of different sizes, charges, and surface cysteine content. The experiments are carried out in vitro at physiologically relevant timescales to obtain the adsorption amounts and rates of each biomolecule on AuNP-PEGs of varying compositions. Our findings are relevant in understanding how protein size and the surface cysteine content affect binding, and our work reveals that cysteine residues can dramatically increase adsorption rates on PEGylated AuNPs. Moreover, shorter chain PEG molecules passivate the AuNP surface more effectively against all protein types.
... The short length of the thiol ligand was selected to allow high-density coating, potentially providing better resistance of the nanostructure (and therefore of the optical properties) to thermal annealing and laser irradiation [41]. Furthermore, compared to the bare NPs, the negatively charged PEG ligand coating helps reduce the strength of protein interactions with the surface [42,43], while a positive charge can lead to stronger interactions with proteins [44]. ...
... The interaction with proteins may affect the photophysical properties of photosensitizers, [56][57][58][59][60][61] so we evaluated the BGNP performances in a physiological-like conditions, investigating the possible effects of the biomolecular corona during laser the irradiation/heating process [44]. We repeated the irradiation experiment for the FBS-dispersed BGNPs, using the highest concentration of BGNPs (0.25 mM Au 0 BGNPs). ...
... Other nanoparticles, like gold nanorods, are known to change their structure with laser absorption [63,64]. Thus, it is important to determine the photothermal stability of BGNPs to exclude heat-induced morphological changes and consequent LSPR shifts, which could prevent further cell death during in vitro laser treat- The interaction with proteins may affect the photophysical properties of photosensitizers, [56][57][58][59][60][61] so we evaluated the BGNP performances in a physiological-like conditions, investigating the possible effects of the biomolecular corona during laser the irradiation/heating process [44]. We repeated the irradiation experiment for the FBS-dispersed BGNPs, using the highest concentration of BGNPs (0.25 mM Au 0 BGNPs). ...
Colorectal cancer (CRC) is a widespread and lethal disease. Relapses of the disease and metastasis are very common in instances of CRC, so adjuvant therapies have a crucial role in its treatment. Systemic toxic effects and the development of resistance during therapy limit the long-term efficacy of existing adjuvant therapeutic approaches. Consequently, the search for alternative strategies is necessary. Photothermal therapy (PTT) represents an innovative treatment for cancer with great potential. Here, we synthesize branched gold nanoparticles (BGNPs) as attractive agents for the photothermal eradication of colon cancer cells. By controlling the NP growth process, large absorption in the first NIR biological window was obtained. The FBS dispersed BGNPs are stable in physiological-like environments and show an extremely efficient light-to-heat conversion capability when irradiated with an 808-nm laser. Sequential cycles of heating and cooling do not affect the BGNP stability. The uptake of BGNPs in colon cancer cells was confirmed using flow cytometry and confocal microscopy, exploiting their intrinsic optical properties. In dark conditions, BGNPs are fully biocompatible and do not compromise cell viability, while an almost complete eradication of colon cancer cells was observed upon incubation with BGNPs and irradiation with an 808-nm laser source. The PTT treatment is characterized by an extremely rapid onset of action that leads to cell membrane rupture by induced hyperthermia, which is the trigger that promotes cancer cell death.
... Both in vitro and in vivo outcomes reveal that magnetothermal modulation can result in fewer opsonins, increase the pharmacokinetics of NPs, and improve cancer nanotherapy (Fig. 4B) [134]. Another earlier study has shown that the uptake of PEGylated AuNR (GNR)@BSA platforms by HeLa cells is independent of the surface charge ( Fig. 4C(i)) because of the stealth characteristics of the PEG modification [135]. However, the BSA pre-coating mitigated the cellular uptake of NPs relative to PEGylated counterparts without the BSA; this effect was charge-dependent ( Fig. 4C (ii)). ...
... Then, the analysis of the photothermal effect induced by laser irradiation (λ = 805 nm, 500 mW, 4 W/cm 2 , 3 min) on the GNR surface revealed a notable reduction in the amount of BSA attachment on the PEGylated GNR with a positive charge, both in completed and non-completed cell media (Fig. 4C (iii)). Nevertheless, no-apparent difference in the cellular internalization of the GNR@BSA platforms with and without laser irradiation was detected except for CTAB-modified GNRs (Fig. 4C (iv)) [135]. In another study, the effect of intravenous injection and the subsequent shear flow was investigated on the PC properties [136]. ...
Nanoparticles (NPs) have been used in numerous applications as anticancer, antibacterial and antioxidant agents. Artificial engineering of protein interactions with NPs in biological systems is crucial to develop potential NPs for drug delivery and cancer nanotherapy. The protein corona (PC) on the NP surface, displays an interface between biomacromolecules and NPs, governing their pharmacokinetics and pharmacodynamics. Upon interaction of proteins with the NPs, their surface features are modified and they can easily be removed from the circulation by the mononuclear phagocytic system (MPS). PC properties heavily depend on the biological microenvironment and NP physicochemical parameters. Based on this context, we have surveyed different approaches that have been used for artificial engineering of the PC composition on NP surfaces. We discussed the effects of NP size, shape, surface modifications (PEGylation, self-peptide, other polymers), and protein pre-coating on the PC properties. Additionally, other factors including protein source and structure, intravenous injection and the subsequent shear flow, plasma protein gradients, temperature and local heat transfer, and washing media were considered in the context of their effects on the PC properties and overall target cellular effects. Moreover, the effects of NP-PC complexes on cancer cells based on cellular interactions, organization of intracellular PC (IPC), targeted drug delivery (TDD) and regulation of burst drug release profile of nanoplat-forms, enhanced biocompatibility, and clinical applications were discussed followed by challenges and future perspective of the field. In conclusion, this paper can provide useful information to manipulate PC properties on the NP surface, thus trying to provide a literature survey to shorten their shipping from preclinical to clinical trials and to lay the basis for a personalized PC.
... For example, in a recent study, gold nanorods were covered with 5 kDa PEG chains carrying a -COOH terminal, -NH2 terminal, or equal combination of PEGs carrying both terminal groups ( Figure 3). The overall charges for these systems were negative, positive, or neutral (respectively), leading to nanorods with different binding affinities towards Bovine Serum Albumin (BSA), which exhibited the following trend: positively charged PEGylated-nanorods > negatively charged PEGylated-nanorods > neutral charged PEGylated-nanorods [41]. Likewise, a similar effect was observed when CdSe quantum dots (QDs) were coated with 0.6-1 kDa PEG chains containing -NH2, -COOH, or -OCH3 terminal groups, and their interaction with BSA protein was studied using agarose gel electrophoresis [42]. ...
... Nanorods covered with 5 kDa PEG chains carrying a -COOH terminal, -NH2 terminal, or an equal combination of PEGs carrying both terminal groups show different binding affinities towards Bovine Serum Albumin (BSA). Reprinted with permission from reference[41]. Copyright (2019) Elsevier. ...
Biofouling is a major issue in the field of nanomedicine and consists of the spontaneous and unwanted adsorption of biomolecules on engineered surfaces. In a biological context and referring to nanoparticles (NPs) acting as nanomedicines, the adsorption of biomolecules found in blood (mostly proteins) is known as protein corona. On the one hand, the protein corona, as it covers the NPs’ surface, can be considered the biological identity of engineered NPs, because the corona is what cells will “see” instead of the underlying NPs. As such, the protein corona will influence the fate, integrity, and performance of NPs in vivo. On the other hand, the physicochemical properties of the engineered NPs, such as their size, shape, charge, or hydrophobicity, will influence the identity of the proteins attracted to their surface. In this context, the design of coatings for NPs and surfaces that avoid biofouling is an active field of research. The gold standard in the field is the use of polyethylene glycol (PEG) molecules, although zwitterions have also proved to be efficient in preventing protein adhesion and fluorinated molecules are emerging as coatings with interesting properties. Hence, in this review, we will focus on recent examples of anti-biofouling coatings in three main areas, that is, PEGylated, zwitterionic, and fluorinated coatings.
... Magnetothermal regulation of protein corona composition presents another captivating area of research with the potential to yield specific biomarker collections [134]. Magnetic nanoparticles can convert an external alternating magnetic field into heat through hysteresis losses, forming the cornerstone for various applications in stimuli-responsive drug deliveries and imaging [135]. In the context of the protein corona, two key parameters affect the outcome: particle concentration and time of exposure. ...
Polymeric nanomaterials, nanogels, and solid nanoparticles can be fabricated using single or double emulsion methods. These materials hold great promise for various biomedical applications due to their biocompatibility, biodegradability, and their ability to control interactions with body fluids and cells. Despite the increasing use of nanoparticles in biomedicine and the plethora of publications on the topic, the biological behavior and efficacy of polymeric nanoparticles (PNPs) have not been as extensively studied as those of other nanoparticles. The gap between the potential of PNPs and their applications can mainly be attributed to the incomplete understanding of their biological identity. Under physiological conditions, such as specific temperatures and adequate protein concentrations, PNPs become coated with a “protein corona” (PC), rendering them potent tools for proteomics studies. In this review, we initially investigate the synthesis routes and chemical composition of conventional PNPs to better comprehend how they interact with proteins. Subsequently, we comprehensively explore the effects of material and biological parameters on the interactions between nanoparticles and proteins, encompassing reactions such as hydrophobic bonding and electrostatic interactions. Moreover, we delve into recent advances in PNP-based models that can be applied to nanoproteomics, discussing the new opportunities they offer for the clinical translation of nanoparticles and early prediction of diseases. By addressing these essential aspects, we aim to shed light on the potential of polymeric nanoparticles for biomedical applications and foster further research in this critical area.
... [204] Interestingly, the modified PC was found to alter the biological behavior of NPs, as cell uptake, cytotoxicity, and ROS production were different between laser-irradiated and non-irradiated PC-coated NPs. [203][204][205] Different observations were reported for gold nanoprisms and BSA. [206] The photothermal effect did not induce significant illumination. ...
Multiple inorganic nanoparticles (NPs) are currently being developed for nanomedicine. Various core materials and shapes are explored, but they all display a common hybrid structure, with organic ligands on their surface. These ligands play a key role in the NP colloidal stability and surface properties, and therefore strongly impact the biological fate of the NPs. However, ligands may be subject to reorganization, degradation, desorption, and exchange, both during their shelf-life and upon exposure to a biological environment. The question of ligand (in)stability at the surface of inorganic NPs has been little addressed in the literature. The goal of this review is to provide a portrait of this critical phenomenon. We identify and review here the different mechanisms likely to promote ligand instability and discuss the resulting biological fate of ligands. This review is aimed to provide a better understanding of these phenomena and to help researchers to design NP-based medicines with better clinical efficacy and translation ability.
... GNP synthesis is well established and reproducible, and their surface can be functionalised with a wide range of ligands, polymers or even biomolecules. [3][4][5] Despite the clinical potentials, nanomedicine suffers from several drawbacks, including the translation from laboratory to clinical products proceeding at a pace significantly lower than expected. [6] A well-known phenomenon occurs after NPs are exposed to a biological fluid, such as serum or human plasma (HP), where their surface is immediately covered by biomolecules that have a high affinity towards the surface of the NPs, forming the so-called biomolecular corona. ...
Hypothesis
Following blood administration, the pristine surface of nanoparticles (NPs) associates with biomolecules from the surrounding environment forming the so-called “biomolecular corona”. It is well accepted that the biomolecular corona dramatically affects the NP fate in the biological medium while the pristine surface is no longer available for binding. Recent studies have shown that the glycans associated with the proteins forming the corona have a role in the NP interaction with macrophages, but the glycan identities remain unknown. We aim here to identify the glycan composition of the biomolecular corona and to assess the role of these glycans in the interaction of the proteins from the corona with glycan binding biomolecules, such as lectins.
Experiments
In this study, we have characterized the biomolecular corona of citrate stabilised gold NPs after exposure of the NPs to blood plasma at two different plasma concentrations, mimicking the in vitro and in vivo conditions. We have extensively characterized the biomolecular corona using HILIC chromatography and shotgun proteomics. Following this, a lectin binding assay was carried out using Dynamic Light Scattering (DLS) and Fluorescence Correlation Spectroscopy (FCS) to assess whether proteins with known affinity towards specific glycans would bind to the corona.
Findings
Our findings highlighted that the protein corona composition is dependent on the exposing conditions. However, under both plasma concentrations, the biantennary sialylated glycans (A2G2S2) are enriched. DLS and FCS confirmed that the glycans are accessible for binding as the corona interacts with lectins with known affinity towards terminal sialic acids and the enzymatic removal of the glycans leads to a decrease in lectin affinity. This study shows for the first time that the glycans are present in the corona and that they could potentially be responsible for the modulation of NP biological processes as they can directly engage with glycan binding receptors that are highly expressed in an organism.
... The use of the photothermal properties of plasmonic NPs for photoinduced local heating is well-documented. 18 These properties are particularly useful for biological applications because the heating can be triggered by NIR light sources, 19 which excite the corresponding plasmon band of anisotropic metal NPs, usually gold NPs 20,21 such as nanorods, 22 nanostars, 23 and nanoprisms. 24 The thermoplasmonic approach has also been used in photothermal therapy (analogous to magnetic-induced hyperthermia for treatment of solid tumors) and NIR-gated delivery of payloads in tissues and cells. ...
We describe a microporous plasmonic nanoreactor to carry out designed near-infrared (NIR)-driven photothermal cyclizations inside living cells. As a proof of concept, we chose an intramolecular cyclization that is based on the nucleophilic attack of a pyridine onto an electrophilic carbon, a process that requires high activation energies and is typically achieved in bulk solution by heating at ∼90 °C. The core–shell nanoreactor (NR) has been designed to include a gold nanostar core, which is embedded within a metal–organic framework (MOF) based on a polymer-stabilized zeolitic imidazole framework-8 (ZIF-8). Once accumulated inside living cells, the MOF-based cloak of NRs allows an efficient diffusion of reactants into the plasmonic chamber, where they undergo the transformation upon near-IR illumination. The photothermal-driven reaction enables the intracellular generation of cyclic fluorescent products that can be tracked using fluorescence microscopy. The strategy may find different type of applications, such as for the spatio-temporal activation of prodrugs.
... 71 Only few techniques are able to study the in situ corona without a previous purification step, such as fluorescence correlation spectroscopy (FCS), 71,77 flow cytometry, 78 diffusionordered nuclear magnetic resonance (NMR) spectroscopy, 79 nanoparticle tracking analysis (NTA), 80 and isothermal titration calorimetry. 27,72 However, such studies are still rare. Importantly, to extract quantifiable data (e.g., dissociation constants, d h ) from these techniques, studies are usually restricted to model proteins (e.g., albumin, transferrin, fibrinogen, immunoglobulins, low-density lipoproteins). ...
... The choice of the protein model was based on the fact that serum albumin is the most abundant protein in the serum/plasma, and multiple mass spectrometry based studies have demonstrated that, usually, for metallic and polymeric nanoparticles, it is the protein that contributes the most to the protein corona formation [20,21]. BSA was labeled with FITC using the protocol described by Polo et al. [22]. FITC was dissolved in dimethyl sulfoxide (DMSO) (5 mg/mL) and was added to 1 mL of BSA 10 mg/mL dissolved in carbonate buffer (50 mM, pH = 9.2) at a ratio of 25 dye molecules per 1 BSA molecule. ...
The interactions of nanoparticles with living organisms are driven by an interface called the protein corona. This interface is formed when nanoparticles are introduced in biological milieu and proteins are adsorbed at nanoparticles’ surfaces. Understanding the factors that are responsible for the formation and the composition of the protein corona could reveal mechanistic insights that are involved in the interaction of nanoparticles with biological structures. Multiple studies have qualitatively described the protein corona, but just a few have proposed quantification methods, especially for gold nanoparticles. Using bovine serum albumin conjugated with fluorescein-5-isothiocyanate as a model protein, we developed a fluorescent-based quantification method for gold nanoparticles’ protein coronas. The impact of nanoparticle size and surface chemistry was studied, and our research emphasizes that size and surface chemistry are determinant factors: Bigger nanoparticles and amino-modified surface chemistry are responsible for higher protein adsorption compared to smaller ones and carboxyl- or methoxy-modified surface chemistry. The proposed method can be used to complete the full picture of the interactions of nanoparticles with biological milieu and to describe the parameters which govern these interactions for the better development of nanomedicines.
Gold nanoparticles (GNPs) are largely employed in diagnostics/biosensors and are among the most investigated nanomaterials in biology/medicine. However, few GNP‐based nanoformulations have received FDA approval to date, and promising in vitro studies have failed to translate to in vivo efficacy. One key factor is that biological fluids contain high concentrations of proteins, lipids, sugars, and metabolites, which can adsorb/interact with the GNP's surface, forming a layer called biomolecular corona (BMC). The BMC can mask prepared functionalities and target moieties, creating new surface chemistry and determining GNPs’ biological fate. Here, the current knowledge is summarized on GNP‐BMCs, analyzing the factors driving these interactions and the biological consequences. A partial fingerprint of GNP‐BMC analyzing common patterns of composition in the literature is extrapolated. However, a red flag is also risen concerning the current lack of data availability and regulated form of knowledge on BMC. Nanomedicine is still in its infancy, and relying on recently developed analytical and informatic tools offers an unprecedented opportunity to make a leap forward. However, a restart through robust shared protocols and data sharing is necessary to obtain “stronger roots”. This will create a path to exploiting BMC for human benefit, promoting the clinical translation of biomedical nanotools.
Hierarchical plasmonic nanostructures of a gold nanorod-covered microball by a syringe-injection reduction have a good potential for a selected single-cell calcium ionophore delivery and apoptosis induction in heterogenous cancer cells.
Starch nanoparticles (SNPs) were synthesized and characterized in vivo for their subacute biotoxicity. Animal experiments revealed that SNPs would not induce changes in inflammatory cytokines and gut microbiota, avoiding damage to mice's organs. Moreover, SNPs were rapidly excreted from the body after 6 h without any accumulation, demonstrating their biosafety. Based on these findings, SNPs were used for β-lactoglobulin (βLg) desensitization by formation of a protein corona. The thermodynamics and time evolution of βLg's secondary structure were investigated to address the desensitization mechanism. The results showed ∼1600 βLg molecules onto a single SNP coupled with significant changes in secondary structure formed a stable SNPs-βLg corona with binding affinity (Ka) of 8.4 ± 0.5 × 10⁵ M⁻¹. Functionally, the decrease of β-sheet destroyed the conformation of immunoglobulin E (IgE) epitopes and inhibited IgE combining capacity, achieving SNPs' desensitization to βLg. Additionally, it takes ∼2 h to complete changes in βLg's surface hydrophobicity and immunoglobulin E (IgE) combining capacity after incubation with SNPs, consistent with the time evolution of structure changes, indicating protein corona is response for the desensitization.
Nanoparticles interacting with proteins to form protein corona represent one of the most fundamental problems in the rapid development of nanotechnology. In the past decade, thousands of studies have pointed out this issue. Within multi-protein systems, the formation of protein corona is a homeostasis process in which proteins compete for the limited surface sites of nanoparticles. Besides, the formation of protein corona generally shows a tendency of evolving with time and involves many different driving forces controlled by properties of nanoparticles, proteins and environment. Therefore, recent research on the dynamic process and mechanisms of protein corona formation in both animals and plants are summarized in this review. The factors that affect the formation and the techniques that commonly used for protein corona analysis are proposed. Furthermore, in order to provide reference for the future research, the limitations and challenges in protein corona studies are assessed and the future perspectives are proposed.
Smart drug delivery systems represent state-of-the-art approaches for targeted therapy of life-threatening diseases such as cancer and cardiovascular diseases. Stimuli-responsive on-demand release of therapeutic agents at the diseased site can significantly limit serious adverse effects. In this study, we engineered a near-infrared (NIR) light-responsive liposomal gold nanorod-containing platform for on-demand delivery of proteins using a hybrid formulation of ultrasmall gold nanorods (AuNRs), thermosensitive phospholipid (DPPC) and non-ionic surfactant (Brij™ 58). In light-triggered release optimization studies, 55.6% (±4.8) of a FITC-labelled model protein, ovalbumin (MW 45 kDa) was released in 15 min upon NIR irradiation (785 nm, 1.35 W/cm² for 5 min). This platform was then utilized to test on-demand delivery of urokinase-plasminogen activator (uPA) for bleeding-free photothermally-assisted thrombolysis, where the photothermal effect of AuNRs would synergize with the released uPA in clot lysis. Urokinase light-responsive liposomes showed 80.7% (± 4.5) lysis of an in vitro halo-clot model in 30 min following NIR irradiation (785 nm, 1.35 W/cm² for 5 min) compared to 36.3% (± 4.4) and 15.5% (± 5.5) clot lysis from equivalent free uPA and non-irradiated liposomes respectively. These results show the potential of low-dose, site-specific thrombolysis via the combination of light-triggered delivery/release of uPA from liposomes combined with photothermal thrombolytic effects from gold nanorods. In conclusion, newly engineered, gold nanorod-based, NIR light-responsive liposomes represent a promising drug delivery system for site-directed, photothermally-stimulated therapeutic protein release.
Engineering the protein corona (PC) on nanodrugs is emerging as an effective approach to improve their pharmacokinetics and therapeutic efficacy, but conventional in vitro pre-programmed methods have shown great limitation for regulation of the PC in the complex and dynamic in vivo physiological environment. Here, we demonstrate an magnetothermal regulation approach that allows us to in situ modulate the in vivo PC composition on iron oxide nanoparticles for improved cancer nanotherapy. Experimental results revealed that the relative levels of major opsonins and dysopsonins in the PC can be tuned quantitatively by means of heat induction mediated by the nanoparticles under an alternating magnetic field. When the PC was magnetically optimized in vivo, the nanoparticles exhibited prolonged circulation and enhanced tumor delivery efficiency in mice, 2.53-fold and 2.02-fold higher respectively than the control. This led to a superior thermotherapeutic efficacy of systemically delivered nanoparticles. In vivo magnetothermal regulation of the PC on nanodrugs will find wide applications in biomedicine.
The last two decades have seen great advancements in fundamental understanding and applications of metallic nanoparticles stabilized by mixed-ligand monolayers. Identifying and controlling the organization of multiple ligands in the nanoparticle monolayer has been studied, and its effect on particle properties has been examined. Mixed-ligand protected particles have shown advantages over monoligand protected particles in fields such as catalysis, self-assembly, imaging, and drug delivery. In this Review, the use of mixed-ligand monolayer protected nanoparticles for sensing applications will be examined. This is the first time this subject is examined as a whole. Mixed-ligand nanoparticle-based sensors are revealed to be divided into four groups, each of which will be discussed. The first group consists of ligands that work cooperatively to improve the sensors' properties. In the second group, multiple ligands are utilized for sensing multiple analytes. The third group combines ligands used for analyte recognition and signal production. In the final group, a sensitive, but unstable, functional ligand is combined with a stabilizing ligand. The Review will conclude by discussing future challenges and potential research directions for this promising subject.
Translating the potential of thermoplasmonics to cell‐derived nanomaterials offers exciting opportunities to fabricate beyond state‐of‐art artificial biomimetic nanocomposites that upon illumination perform active tasks such as delivery of cargo in complex, dynamic media such as the cytosol of cells. Cell‐derived nanoparticles have shown stunning potential to implement cell‐specific functions, such as long blood circulation or targeting capabilities, into advanced drug delivery nanosystems. The biomimicry nanotechnology has now advanced to offer new and exciting opportunities to improve the commonly poor in vivo performance of most current nanomedicines, including evading the immune system and targeting specific tissues such as tumors, the latest remaining among the most wanted breakthroughs in nanomedicine. However, the use of cell‐derived nanocomposites as stimulus‐controlled drug delivery agents remains virtually unexplored. This study reports the fabrication of a plasmonic cell‐derived nanocomposite by integrating near‐infrared active gold nanorods in its structure. As a proof of concept, the plasmonic nanomembranes are loaded with cell non‐permeant antibodies, which upon near‐infrared stimulation can be released from the plasmonic nanomembranes into the cytosol of living cells, without impairing cell viability or the antibodies' function. These results set the stage for the development of photoactive cell‐derived nanocarriers, which in addition to cell‐specific functions promise straightforward access to spatiotemporal‐controlled intracellular delivery of antibodies.
We introduce an approach to synthesize macrocyclic poly(ethylene oxide)s containing a pendant protected thiol group (pSH-CPEO), which is demonstrated to be able to attach to gold surfaces without prior deprotection. Our strategy is based on a bimolecular approach by which a di-alkyne molecule derived from thiol-protected 3-mercapto-1,2-propanediol and a series of PEO bis(azides) of Mn = 2, 6, and 11 kg mol⁻¹ are coupled via copper-catalyzed azide–alkyne cycloaddition. The cyclization reaction was verified by size exclusion chromatography and matrix-assisted laser desorption ionization time of flight mass spectrometry. In addition, we used fast scanning calorimetry to evaluate the glass transition temperature (Tg) of the synthesized pSH-CPEOs. Thanks to the extremely rapid cooling power of this technique, PEO crystallization can be circumvented and, therefore, its fully amorphous state can be investigated. The results confirmed higher Tg values for macrocycles compared to their chemically equivalent linear precursors. This result highlights the importance of the chain end in affecting the Tg of polymers. Finally, to demonstrate the ability of pSH-CPEO to covalently attach to gold surfaces, pSH-CPEO samples were allowed to react with gold-coated glass slides and the surface properties were compared with those of the samples obtained by the reaction of linear α-thiol, ω-methoxy PEO (with and without protection of the thiol group) and gold-coated glass slides. X-ray photoelectron spectroscopy data confirmed the formation of Au–S linkages as well as the removal of the thiol protector group through the quantitative analysis of the chemical composition at the surface. The contact angle data of pSH-CPEO/gold exhibited increased hydrophilicity compared to bare gold and topological effects at the interface. A reaction mechanism between the 2,4-dinitrobenzene-protected thiol group and the gold surface is also proposed.
The physicochemical properties of a set of 21 different gold nanoparticles (spherical and rod-shaped nanoparticles (NPs) of different diameters with 3 different surface coatings) were studied. Protein corona formation, in vitro uptake, effect on cell viability and proliferation, and in vivo biodistribution of these NPs were determined. The relation of the results of the different NPs was analyzed by hierarchical cluster analysis (HCA), which will tell which NPs have most similar physicochemical properties and biological effects, without having to specify individual physicochemical parameters. The results show that the physicochemical properties of gold nanoparticles (Au NPs) are mainly accounted for by their hydrodynamic diameter and their zeta-potential. The formation of the protein corona is determined by the pH-dependence of their zeta-potential. While several reports found that in vitro uptake and in vivo biodistribution of NPs are correlated to individual physicochemical parameters, e.g. size, shape, or surface chemistry, such direct dependence in the investigated multidimensional set of NPs was not found in our study. This most likely is due to entanglement of the different parameters, which complicates the prediction of the biological effect of NPs in case multiple physicochemical properties are simultaneously varied. The in vitro uptake and in vivo biodistribution of NPs seem to be not directly driven by the protein corona, but the physicochemical properties determine as well the corona as they influence in vitro/in vivo behaviors, and thus the effect of the protein corona would be rather indirect.
Core-shell nanoparticles receive much attention for their current and potential applications in life sciences. Commonly, a dense shell of hydrated polymer, a polymer brush, is grafted to improve colloidal stability of functional nanoparticles and to prevent protein adsorption, aggregation, cell recognition and uptake. Until recently it was widely assumed that a polymer brush shell indeed prevents strong association of proteins, and that this leads to their superior “stealth” properties in vitro and in vivo. We show using T-dependent isothermal titration calorimetry on well-characterized monodisperse superparamagnetic iron oxide nanoparticles with controlled dense stealth polymer brush shells that “stealth” core-shell nanoparticles display significant attractive exothermic and enthalpic interactions with serum proteins, despite having excellent colloidal stability and negligible non-specific cell uptake. This observation is at room temperature shown to only weakly depend on variation of iron oxide core diameter and type of grafted stealth polymer: poly(ethylene glycol), poly(ethyl oxazoline), poly(isopropyl oxazoline) and poly(N-isopropyl acrylamide). Polymer brush shells with a critical solution temperature close to body temperature showed a strong temperature dependence in their interactions with proteins with a significant increase in protein binding energy with increased temperature. The stoichiometry of interaction is estimated to be near 1 : 1 for PEGylated nanoparticles and up to 10 : 1 for larger thermoresponsive nanoparticles, while the average free energy of interaction is enthalpically driven and comparable to a weak hydrogen bond.
Colloidal nanoparticles (NPs) are a versatile potential platform for in vivo nanomedicine. Inside blood circulation, NPs may undergo drastic changes, such as by formation of a protein corona. The in vivo corona cannot be completely emulated by the corona formed in blood. Thus, in situ detection in complex media, and ultimately in vivo, is required. Here we present a methodology for determining protein corona formation in complex media. NPs are labeled with ¹⁹F and their diffusion coefficient measured using ¹⁹F diffusion-ordered nuclear magnetic resonance (NMR) spectroscopy. ¹⁹F diffusion NMR measurements of hydrodynamic radii allow for in situ characterization of NPs in complex environments by quantification of protein adsorption to the surface of NPs, as determined by increase in hydrodynamic radius. The methodology is not optics based, and thus can be used in turbid environments, as in the presence of cells.
Photothermal desorption of molecules from plasmonic nanoparticles is an example of a light-triggered molecular release due to heating of the system. However, this phenomenon ought to work only if the molecule–nanoparticle interaction is exothermic in nature. In this study, we compare protein adsorption behavior onto gold nanoparticles for both endothermic and exothermic complexation reactions, and demonstrate that Le Chatelier’s principle can be applied to predict protein adsorption or desorption on nanomaterial surfaces. Polyelectrolyte-wrapped gold nanorods were used as adsorption platforms for two different proteins, which we were able to adsorb/desorb from the nanorod surface depending on the thermodynamics of their interactions. Furthermore, we show that the behaviors hold up under more complex biological environments such as fetal bovine serum.
Ultrasmall nanoparticles (USNPs), usually defined as NPs with core in the size range 1–3 nm, are a class of nanomaterials which show unique physicochemical properties, often different from larger NPs of the same material. Moreover, there are also indications that USNPs might have distinct properties in their biological interactions. For example, recent in vivo experiments suggest that some USNPs escape the liver, spleen, and kidney, in contrast to larger NPs that are strongly accumulated in the liver. Here, we present a simple approach to study the biomolecular interactions at the USNPs bio-nanointerface, opening up the possibility to systematically link these observations to microscopic molecular principles.
Ultrasmall nanoparticles (USNPs), usually defined as NPs with core in the size range 1-3 nm, are a class of nanomaterials which show unique physicochemical properties, often different from larger NPs of the same material. Moreover, there are also indications that USNPs might have distinct properties in their biological interactions. For example, recent in vivo experiments suggest that some USNPs escape the liver, spleen, and kidney, in contrast to larger NPs that are strongly accumulated in the liver. Here, we present a simple approach to study the biomolecular interactions at the USNPs bio-nanointerface, opening up the possibility to systematically link these observations to microscopic molecular principles.
The design and use of materials in the nanoscale size range for addressing medical and health-related issues continues to receive increasing interest. Research in nanomedicine spans a multitude of areas, including drug delivery, vaccine development, diagnosis and imaging tools, wearable devices, high-throughput screening platforms, etc. using biological, nonbiological, or hybrid materials. Many of these developments are starting to be translated into viable clinical products. Here, we provide an overview of recent developments in nanomedicine and highlight the current challenges and upcoming opportunities for the field and translation to the clinic.
A homologous nanoparticle library was synthesized in which gold nanoparticles were coated with polyethylene glycol, whereby the diameter of the gold cores, as well as the thickness of the shell of polyethylene glycol, was varied. Basic physicochemical parameters of this two-dimensional nanoparticle library, such as size, ζ-potential, hydrophilicity, elasticity, and catalytic activity ,were determined. Cell uptake of selected nanoparticles with equal size yet varying thickness of the polymer shell and their effect on basic structural and functional cell parameters was determined. Data indicates that thinner, more hydrophilic coatings, combined with the partial functionalization with quaternary ammonium cations, result in a more efficient uptake, which relates to significant effects on structural and functional cell parameters.
Cell-penetrating peptides (CPPs) have been widely used to deliver nanomaterials and other types of macromolecules into mammalian cells for therapeutic and diagnostic use. Cationic CPPs that bind to heparan sulfate (HS) proteoglycans on the cell surface induce potent endocytosis; however, the role of other surface receptors in this process is unclear. We describe the convergence of an HS-dependent pathway with the C-end rule (CendR) mechanism that enables peptide ligation with neuropilin-1 (NRP1), a cell surface receptor known to be involved in angiogenesis and vascular permeability. NRP1 binds peptides carrying a positive residue at the carboxyl terminus, a feature that is compatible with cationic CPPs, either intact or after proteolytic processing. We used CPP and CendR peptides, as well as HS- and NRP1-binding motifs from semaphorins, to explore the commonalities and differences of the HS and NRP1 pathways. We show that the CendR-NRP1 interaction determines the ability of CPPs to induce vascular permeability. We also show at the ultrastructural level, using a novel cell entry synchronization method, that both the HS and NRP1 pathways can initiate a macropinocytosis-like process and visualize these CPP-cargo complexes going through various endosomal compartments. Our results provide new insights into how CPPs exploit multiple surface receptor pathways for intracellular delivery.
Using quantitative models to predict the biological interactions of nanoparticles will accelerate the translation of nanotechnology. Here, we characterized the serum protein corona ‘fingerprint’ formed around a library of 105 surface-modified gold nanoparticles. Applying a bioinformatics-inspired approach, we developed a multivariate model that uses the protein corona fingerprint to predict cell association 50% more accurately than a model that uses parameters describing nanoparticle size, aggregation state, and surface charge. Our model implicates a set of hyaluronan-binding proteins as mediators of nanoparticle–cell interactions. This study establishes a framework for developing a comprehensive database of protein corona fingerprints and biological responses for multiple nanoparticle types. Such a database can be used to develop quantitative relationships that predict the biological responses to nanoparticles and will aid in uncovering the fundamental mechanisms of nano–bio interactions.
The protein adsorption layer (a.k.a. the "protein corona") that forms on the surface of colloidal nanoparticles plays an important role in their interaction with living matter. Thus, characterization of the protein corona is of utmost importance for understanding how exposure to nanoparticles affects the biological responses of cells and organisms. Although a lot of experimental studies have been reported in this direction, a comprehensive picture is still missing, in particular due to the multitude of different scenarios under which experiments have been performed. In this review an analysis of existing experimental data about the protein corona, and an outline for required future work will be given. In particular we review how existing simple analytical models such as the adopted Hill model may help to extract quantitative data from such experiments such as equilibrium dissociation and kinetic coefficients. Careful quantitative assessment of equilibrium and kinetic properties would allow for a comparison of protein binding data from the vast array of engineered nanoparticles, so that basic principles could be revealed. This review outlines that the field is in dire need of more quantitative studies to further our understanding of protein corona formation and its biological consequences.
It is now clearly emerging that besides size and shape, the other primary defining element of nanoscale objects in biological media is their long-lived protein ("hard") corona. This corona may be expressed as a durable, stabilizing coating of the bare surface of nanoparticle (NP) monomers, or it may be reflected in different subpopulations of particle assemblies, each presenting a durable protein coating. Using the approach and concepts of physical chemistry, we relate studies on the composition of the protein corona at different plasma concentrations with structural data on the complexes both in situ and free from excess plasma. This enables a high degree of confidence in the meaning of the hard protein corona in a biological context. Here, we present the protein adsorption for two compositionally different NPs, namely sulfonated polystyrene and silica NPs. NP-protein complexes are characterized by differential centrifugal sedimentation, dynamic light scattering, and zeta-potential both in situ and once isolated from plasma as a function of the protein/NP surface area ratio. We then introduce a semiquantitative determination of their hard corona composition using one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrospray liquid chromatography mass spectrometry, which allows us to follow the total binding isotherms for the particles, identifying simultaneously the nature and amount of the most relevant proteins as a function of the plasma concentration. We find that the hard corona can evolve quite significantly as one passes from protein concentrations appropriate to in vitro cell studies to those present in in vivo studies, which has deep implications for in vitro-in vivo extrapolations and will require some consideration in the future.
The chemical composition, size, shape and surface characteristics of nanoparticles affect the way proteins bind to these particles, and this in turn influences the way in which nanoparticles interact with cells and tissues. Nanomaterials bound with proteins can result in physiological and pathological changes, including macrophage uptake, blood coagulation, protein aggregation and complement activation, but the mechanisms that lead to these changes remain poorly understood. Here, we show that negatively charged poly(acrylic acid)-conjugated gold nanoparticles bind to and induce unfolding of fibrinogen, which promotes interaction with the integrin receptor, Mac-1. Activation of this receptor increases the NF-κB signalling pathway, resulting in the release of inflammatory cytokines. However, not all nanoparticles that bind to fibrinogen demonstrated this effect. Our results show that the binding of certain nanoparticles to fibrinogen in plasma offers an alternative mechanism to the more commonly described role of oxidative stress in the inflammatory response to nanomaterials.
Nanoparticles in a biological fluid (plasma, or otherwise) associate with a range of biopolymers, especially proteins, organized into the “protein corona” that is associated with the nanoparticle and continuously exchanging with the proteins in the environment. Methodologies to determine the corona and to understand its dependence on nanomaterial properties are likely to become important in bionanoscience. Here, we study the long-lived (“hard”) protein corona formed from human plasma for a range of nanoparticles that differ in surface properties and size. Six different polystyrene nanoparticles were studied: three different surface chemistries (plain PS, carboxyl-modified, and amine-modified) and two sizes of each (50 and 100 nm), enabling us to perform systematic studies of the effect of surface properties and size on the detailed protein coronas. Proteins in the corona that are conserved and unique across the nanoparticle types were identified and classified according to the protein functional properties. Remarkably, both size and surface properties were found to play a very significant role in determining the nanoparticle coronas on the different particles of identical materials. We comment on the future need for scientific understanding, characterization, and possibly some additional emphasis on standards for the surfaces of nanoparticles.
• bionanoscience
• mass spectrometry
• interactions
• proteomics
• human plasma
Owing to the complex and still not fully understood physiological environment, the development of traditional nano-sized drug delivery systems is very challenging for precision cancer therapy. It is very difficult to control the in vivo distribution of nanoparticles after intravenous injection. The ideal drug nanocarriers should not only have stealth surface for prolonged circulation time, but also possess enhanced cellular internalization in tumor sites. Unfortunately, the stealth surface and enhanced cellular uptake seem contradictory to each other. How to integrate the two opposite aspects into one system is a very herculean but meaningful task. As an alternative drug delivery strategy, chameleon-like drug delivery systems were developed to achieve long circulation time while maintaining enhanced cancer cell uptake. Such drug dnanocarriers can “turn off” their internalization ability during circulation. However, the enhanced cellular uptake can be readily activated upon arriving in tumor tissues. In this way, stealth surface and enhanced uptake are of dialectical unity in drug delivery. In this review, we focused on the surface engineering of drug nanocarriers to obtain simultaneous stealth surface in circulation and enhanced uptake in tumor. The current strategies and ongoing developments, including programmed tumor targeting strategies and some specific zwitterionic surfaces will be discussed in detail.
Recent years have witnessed the rapid development of plasmonic nanomaterials for applications in biology and medicine generating a new field, the so-called bioplasmonics. At present, plasmonic nanoparticles can be found in many applications serving as therapeutics, diagnostic or theranostic agents. The rapid advancement of bioplasmonics has gone hand in hand with the multiplication of the synthetic methods to produce finely-tailored plasmonic nanomaterials and the development of their surface functionalization tools, which is key to provide them with colloidal stability and/or specificity in complex biological media. This review aims to highlight basic physicochemical properties in the bioplasmonics context, as well as the most important bioapplications based on the properties of plasmonic colloids. Pioneering works that stimulated the rise of the bioplasmonics field, as well as most recent advances and future directions are discussed.
Synthesis, characterization, and applications of colloidal nanoparticles have been a prominent topic of current research interests within the last two decades. Available reports in the literature that describe the synthesis of colloidal nanoparticles are abundant with various degrees of reproducibility and simplicity. Moreover, different methods for the characterization of colloidal nanoparticles' basic properties are employed, resulting in conflicting results in many cases. Herein, we describe “in detail” selected standard protocols for the synthesis, purification, and characterization of various types of colloidal inorganic nanoparticles including gold nanoparticles, silver nanoparticles, iron oxide nanoparticles, and quantum dots. This report consists of five main parts: The first and the second part are dedicated to describing the synthesis of various types of hydrophobic and hydrophilic nanoparticles in organic solvents and in aqueous solutions, respectively. The third part describes surface modification of nanoparticles with focus on ligand exchange reactions, to allow phase transfer of nanoparticles from aqueous to organic solvents and vice versa. The fourth and the fifth part describe various general purification and characterization techniques used to purify and characterize nanoparticles, respectively. Collectively, this contribution does not aim to cover all available protocols in the literature to prepare inorganic nanoparticles, but rather provides detailed synthetic procedures to important inorganic nanocrystals with full description of their purification and characterization process.
Surface chemistry plays a deciding role in nanoparticle biodistribution, yet very little is known about how surface chemistry influences the sub-organ distributions of nanomaterials. Here, using quantitative imaging based on laser ablation inductively-coupled plasma mass spectrometry (LA-ICP-MS), we demonstrate that surface charge dictates the sub-organ distributions of nanoparticles in the kidney, liver, and spleen of mice intravenously injected with functionalized gold nanoparticles. Images of the kidney show that positively charged nanoparticles accumulate extensively in the glomeruli, the initial stage in filtering for the nephron, suggesting that these nanoparticles may be filtered by the kidney at a different rate than the neutral or negatively charged nanoparticles. We find that positively and negatively-charged nanoparticles accumulate extensively in the red pulp of the spleen. In contrast, uncharged nanoparticles accumulate in the white pulp and marginal zone of the spleen to a greater extent than the positively or negatively-charged nanoparticles. Moreover, these uncharged nanoparticles are also more likely to be found associated with Kupffer cells in the liver. Positively-charged nanoparticles accumulate extensively in liver hepatocytes, whereas negatively-charged nanoparticles show a broader distribution in the liver. Together these observations suggest that neutral nanoparticles having 2 nm cores may interact with the immune system to a greater extent than charged nanoparticles, highlighting the value of determining the sub-organ distributions of nanomaterials for delivery and imaging applications.
Using a family of cationic gold nanoparticles (NPs) with similar size and charge, we demonstrate that proper surface engineering can control the nature and identity of protein corona in physiological serum conditions. The protein coronas were highly dependent on the hydrophobicity and arrangement of chemical motifs on NP surface. The NPs were uptaken in macrophages in a corona-dependent manner, predominantly through recognition of specific complement proteins in the NP corona. Taken together, this study shows that surface functionality can be used to tune the protein corona formed on NP surface, dictating the interaction of NPs with macrophages.
The current gold standard to reduce non-specific cellular uptake of drug delivery vehicles is by covalent attachment of poly(ethylene glycol) (PEG). It is thought that PEG can reduce protein adsorption and thereby confer a stealth effect. Here, we show that polystyrene nanocarriers that have been modified with PEG or poly(ethyl ethylene phosphate) (PEEP) and exposed to plasma proteins exhibit a low cellular uptake, whereas those not exposed to plasma proteins show high non-specific uptake. Mass spectrometric analysis revealed that exposed nanocarriers formed a protein corona that contains an abundance of clusterin proteins (also known as apolipoprotein J). When the polymer-modified nanocarriers were incubated with clusterin, non-specific cellular uptake could be reduced. Our results show that in addition to reducing protein adsorption, PEG, and now PEEPs, can affect the composition of the protein corona that forms around nanocarriers, and the presence of distinct proteins is necessary to prevent non-specific cellular uptake.
Here we have investigated the effect of enshrouding polymer-coated nanoparticles (NPs) with polyethylene glycol (PEG) on the adsorption of proteins and uptake by cultured cells. PEG was covalently linked to the polymer surface to the maximal grafting density achievable under our experimental conditions. Changes in the effective hydrodynamic radius of the NPs upon adsorption of human serum albumin (HSA) and fibrinogen (FIB) were measured in situ by using fluorescence correlation spectroscopy (FCS). For NPs without a PEG shell, a thickness increase of around 3 nm, corresponding to HSA monolayer adsorption, was measured at high HSA concentration. Only 50% of this value was found for NPs with PEGylated surfaces. While the size increase clearly reveals formation of a protein corona also for PEGylated NPs, fluorescence lifetime measurements and quenching experiments suggest that the adsorbed HSA molecules are buried within the PEG shell. For FIB adsorption onto PEGylated NPs, even less change in NP diameter was observed. In vitro uptake of the NPs by 3T3 fibroblasts was reduced to around 10% upon PEGylation with PEG chains of 10 kDa. Thus, even though the PEG coatings did not completely prevent protein adsorption, the PEGylated NPs still displayed a pronounced reduction of cellular uptake with respect to bare NPs, which is to be expected if the adsorbed proteins are not exposed on the NP surface.
The physico-chemical properties of colloidal particles determine their uptake into cells. For a series of microparticles only one parameter, the mechanical stiffness, was varied, whereas other parameters such as size, shape, and charge were kept constant. The uptake was monitored in situ by analyzing individual particle trajectories including the progress of endocytosis, derived from local pH measurements around each particle. Evidence is presented that soft particles with low stiffness are transported faster to lysosomes than stiffer ones.
© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
In medicine, nanotechnology has sparked a rapidly growing interest as it promises to solve a number of issues associated with conventional therapeutic agents, including their poor water solubility (at least, for most anticancer drugs), lack of targeting capability, nonspecific distribution, systemic toxicity, and low therapeutic index. Over the past several decades, remarkable progress has been made in the development and application of engineered nanoparticles to treat cancer more effectively. For example, therapeutic agents have been integrated with nanoparticles engineered with optimal sizes, shapes, and surface properties to increase their solubility, prolong their circulation half-life, improve their biodistribution, and reduce their immunogenicity. Nanoparticles and their payloads have also been favorably delivered into tumors by taking advantage of the pathophysiological conditions, such as the enhanced permeability and retention effect, and the spatial variations in the pH value. Additionally, targeting ligands (e.g., small organic molecules, peptides, antibodies, and nucleic acids) have been added to the surface of nanoparticles to specifically target cancerous cells through selective binding to the receptors overexpressed on their surface. Furthermore, it has been demonstrated that multiple types of therapeutic drugs and/or diagnostic agents (e.g., contrast agents) could be delivered through the same carrier to enable combination therapy with a potential to overcome multidrug resistance, and real-time readout on the treatment efficacy. It is anticipated that precisely engineered nanoparticles will emerge as the next-generation platform for cancer therapy and many other biomedical applications.
In protein-rich environments such as the blood, the formation of a protein corona on receptor-targeting nanoparticles prevents target recognition. As a result, the ability of targeted nanoparticles to selectively bind to diseased cells is drastically inhibited. Backfilling the surface of a targeted nanoparticle with polyethylene glycol (PEG) molecules is demonstrated to reduce the formation of the protein corona and re-establishes specific binding. The length of the backfilled PEG molecules must be less than the length of the ligand linker; otherwise, PEG interferes with the binding of the targeting ligand to its corresponding cellular receptor.
It is well recognized that the primary interaction of most biological environments with nanoparticles (NPs) is strongly influenced by a long-lived ("hard") protein corona that surrounds the NP and remains strongly adsorbed to its surface. The amount and composition of associated proteins in the corona adsorbed onto the NPs is related to several important factors, including the physicochemical properties of the NPs and the composition of the protein solution. Here, for the first time, it is shown that plasmonic heat induction (by laser activation) leads to significant changes in the composition of the hard protein corona adsorbed on low aspect ratio gold nanorods. Using mass spectrometry, several proteins in the corona were identified whose concentrations change most substantially as a result of photoinduced (plasmonic) heating versus simple thermal heating. Molecular modeling suggests that the origin of these changes in protein adsorption may be the result of protein conformational changes in response to much higher local temperatures that occur near the gold nanorods during photoinduced, plasmonic heating. These results may define new applications in vivo for NPs with hyperthermia capability and better define the likely interactions of cells with NPs after plasmonic heating. Potential changes in the protein corona following hyperthermia treatment may influence the final biological fate of plasmonic NPs in clinical applications and help elucidate safety considerations for hyperthermia applications.
We manipulate the passive release rates of DNA payloads on protein coronas formed around nanoparticles (NPs) by varying the corona composition. The coronas are prepared using a mixture of hard and soft corona proteins. We form coronas around gold nanorods (NRs), nanobones (NBs), and carbon nanotubes (CNTs) from human serum (HS) and find that tuning the amount of human serum albumin (HSA) in the NR-coronas (NR-HS-DNA) changes the payload release profile. The effect of buffer strength, HS concentration, and concentration of the cetyltrimethylammonium bromide (CTAB) passivating the NP surfaces on passive release is explored. We find that corona properties play an important role in passive release, and concentrations of CTAB, HS, and phosphate buffer used in corona formation can tune payload release profiles. These advances in understanding protein corona properties bring us closer toward developing a set of basic design rules that enable their manipulation and optimization for particular biological applications.
Too hot to handle: The surroundings of magnetic nanoparticles can be heated by applying a magnetic field. Polymer-coated magnetic nanoparticles were functionalized with single-stranded DNA molecules and further hybridized with DNA modified with different fluorophores. By correlating the denaturation profiles of the DNA with the local temperature, temperature gradients for the vicinity of the excited nanoparticles were determined.
In biological fluids, proteins bind to the surface of nanoparticles to form a coating known as the protein corona, which can critically affect the interaction of the nanoparticles with living systems. As physiological systems are highly dynamic, it is important to obtain a time-resolved knowledge of protein-corona formation, development and biological relevancy. Here we show that label-free snapshot proteomics can be used to obtain quantitative time-resolved profiles of human plasma coronas formed on silica and polystyrene nanoparticles of various size and surface functionalization. Complex time- and nanoparticle-specific coronas, which comprise almost 300 different proteins, were found to form rapidly (<0.5 minutes) and, over time, to change significantly in terms of the amount of bound protein, but not in composition. Rapid corona formation is found to affect haemolysis, thrombocyte activation, nanoparticle uptake and endothelial cell death at an early exposure time.
Upon incorporation of nanoparticles (NPs) into the body, they are exposed to biological fluids, and their interaction with the dissolved biomolecules leads to the formation of the so-called protein corona on the surface of the NPs. The composition of the corona plays a crucial role in the biological fate of the NPs. While the effects of various physico-chemical parameters on the composition of the corona have been explored in depth, the role of temperature upon its formation has received much less attention. In this work, we have probed the effect of temperature on the protein composition on the surface of a set of NPs with various surface chemistries and electric charges. Our results indicate that the degree of protein coverage and the composition of the adsorbed proteins on the NPs surface depend on the temperature at which the protein corona is formed. Also, the uptake of NPs is affected by the temperature. Temperature is, thus, an important parameter that needs to be carefully controlled in quantitative studies of bio-nano interactions.
Local heating can be produced by iron oxide nanoparticles (IONPs) when exposed to an alternating magnetic field (AMF). To measure the temperature profile at the nanoparticle surface with a subnanometer resolution, here we present a molecular temperature probe based on the thermal decomposition of a thermo-sensitive molecule, namely, azobis[N-(2-carboxyethyl)-2-methylpropionamidine]. Fluoresceineamine (FA) was bound to the azo molecule at the IONP surface functionalized with poly(ethylene glycol) (PEG) spacers of different molecular weights. Significant local heating, with a temperature increase up to 45 °C, was found at distances below 0.5 nm from the surface of the nanoparticle, which decays exponentially with increasing distance. Furthermore, the temperature increase was found to scale linearly with the applied field at all distances. We implemented these findings in an AMF-triggered drug release system in which doxorubicin was covalently linked at different distances from the IONP surface bearing the same thermo-labile azo molecule. We demonstrated the AMF triggered distance-dependent release of the drug in a cytotoxicity assay on KB cancer cells.
Surface functionalization: Complete detoxification of gold nanorods was achieved by manipulating the position in the stability map between surface-adsorbing polyethylene glycol (PEG) and destabilizing ethanol. This robust procedure complements studies related to the effects of shape when contemplating the nanoparticle-cell interaction.
The search for understanding the interactions of nanosized materials with living organisms is leading to the rapid development of key applications, including improved drug delivery by targeting nanoparticles, and resolution of the potential threat of nanotechnological devices to organisms and the environment. Unless they are specifically designed to avoid it, nanoparticles in contact with biological fluids are rapidly covered by a selected group of biomolecules to form a corona that interacts with biological systems. Here we review the basic concept of the nanoparticle corona and its structure and composition, and highlight how the properties of the corona may be linked to its biological impacts. We conclude with a critical assessment of the key problems that need to be resolved in the near future.
Technological limitations have prevented the interrogation and manipulation of many signaling pathways in model and living systems required for the development of diagnostic and treatment modalities for diseases, such as cancer. In this work, we demonstrate that gold-coated liposomes are capable of encapsulation and on-demand release of signaling molecules with a spatial and temporal resolution leading to activation of signaling pathways in individual cells. We used HEK293 cells modified to overexpress the CCK2 receptor and achieved activation of this GPCR in a single cell via the localized release of an agonist of this receptor. This content release was triggered by illumination of the liposomes at wavelengths matching the plasmon resonance spectrum of the gold coating. The use of plasmon resonant liposomes may enable on-demand release of a broad range of molecules, without molecule chemical modification and using biologically safe near infrared light. In combination with the spectral tunability of plasmon resonant coating, this technology may allow for multiplexed interrogation of complex and diverse signaling pathways in model or living tissues with unprecedented spatial and temporal control.
Serum albumin (SA) is the most abundant plasma protein in mammals. SA is a multifunctional protein with extraordinary ligand binding capacity, making it a transporter molecule for a diverse range of metabolites, drugs, nutrients, metals and other molecules. Due to its ligand binding properties, albumins have wide clinical, pharmaceutical, and biochemical applications. Albumins are also allergenic, and exhibit a high degree of cross-reactivity due to significant sequence and structure similarity of SAs from different organisms. Here we present crystal structures of albumins from cattle (BSA), horse (ESA) and rabbit (RSA) sera. The structural data are correlated with the results of immunological studies of SAs. We also analyze the conservation or divergence of structures and sequences of SAs in the context of their potential allergenicity and cross-reactivity. In addition, we identified a previously uncharacterized ligand binding site in the structure of RSA, and calcium binding sites in the structure of BSA, which is the first serum albumin structure to contain metal ions.
Polyelectrolyte capsules with metal nanoparticles in their walls and fluorescently labeled polymers as cargo inside their cavity were prepared. Capsules were ingested by living cells with no uncontrolled release of the cargo upon the incorporation process. Photoinduced heating of the metal nanoparticles in the capsule walls lead to rupture of the capsule walls, and the polymeric cargo was released to the whole cytosol. Viability tests demonstrate that opening of capsules at moderate light intensities does not impair the cellular metabolism, whereas capsule opening at high light intensities ultimately leads to cell death.
Gold nanoparticles (Au NPs) from 5 to 100 nm in size synthesized with HAuCl(4) and sodium citrate were complexed with the plasma protein human serum albumin (HSA). Size, surface charge, and surface plasmon bands of the Au NPs are largely modified by the formation of a protein corona via electrostatic interactions and hydrogen bonding as revealed by thermodynamic data. Negative values of the entropy of binding suggested a restriction in the biomolecule mobility upon adsorption. The structure of the adsorbed protein molecules is slightly affected by the interaction with the metal surface, but this effect is enhanced as the NP curvature decreases. Also, it is observed that the protein molecules adsorbed onto the NP surface are more resistant to complete thermal denaturation than free protein ones as deduced from the increases in the melting temperature of the adsorbed protein. Differences in the conformations of the adsorbed protein molecules onto small (<40 nm) and large NPs were observed on the basis of ζ-potential data and FTIR spectroscopy, also suggesting a better resistance of adsorbed protein molecules to thermal denaturing conditions. We think this enhanced protein stability is responsible for a reduced formation of HSA amyloid-like fibrils in the presence of small Au NPs under HSA fibrillation conditions.
In this work, we explore the formation of the protein corona after exposure of metallic Au nanoparticles (NPs), with sizes ranging from 4 to 40 nm, to cell culture media containing 10% of fetal bovine serum. Under in vitro cell culture conditions, zeta potential measurements, UV-vis spectroscopy, dynamic light scattering and transmission electron microscope analysis were used to monitor the time evolution of the inorganic NP-protein corona formation and to characterize the stability of the NPs and their surface state at every stage of the experiment. As expected, the red-shift of the surface plasmon resonance peak, as well as the drop of surface charge and the increase of the hydrodynamic diameter indicated the conjugation of proteins to NPs. Remarkably, an evolution from a loosely attached toward an irreversible attached protein corona over time was observed. Mass spectrometry of the digested protein corona revealed albumin as the most abundant component which suggests an improved biocompatibility.
Short gold nanorods of average lengths ranging between 20 and 100 nm (with corresponding aspect ratios of 2 and 4) were synthesized in excellent yield (approximately 97%). These nanorods were characterized by dark-field microscopy, UV-visible spectrophotometry, and transmission electron microscopy. Temporal evolution of rod shape had also been followed by UV-visible spectrophotometry and transmission electron microscopy and indicates that the nanorods briefly increase in length, then increase slightly in width, as they grow. The effect of the synthetic parameters on the rod dimension and yield was explored to find out suitable conditions to produce short nanorods; short nanorods have both plasmon bands in the visible region of the spectrum, which is a valuable property for sensor applications.
- E Polo
- M F Navarro Poupard
- L Guerrini
- P Taboada
- B Pelaz
E. Polo, M.F. Navarro Poupard, L. Guerrini, P. Taboada, B. Pelaz, et al., Colloidal
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Phenotype determines nanoparticle uptake by human macrophages from liver and blood
- S A Macparland
- K M Tsoi
- B Ouyang
- X.-Z Ma
- J Manuel
S.A. MacParland, K.M. Tsoi, B. Ouyang, X.-Z. Ma, J. Manuel, et al., Phenotype
determines nanoparticle uptake by human macrophages from liver and
blood, ACS Nano 11 (2017) 2428-2443.
- M Carril
- D Padro
- P Pino
- C Carrillo-Carrion
- M Gallego
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Phenotype determines nanoparticle uptake by human macrophages from liver and blood
- MacParland