Non-specific adsorption of complement proteins affects complement activation pathways of gold nanomaterials

<p>The complement system is a key humoral component of innate immunity, serving as the first line of defense against intruders, including foreign synthetic nanomaterials. Although gold nanomaterials (AuNMs) are widely used in nanomedicine, their immunological response is not well understood. Using AuNMs of three shapes commonly used in biomedical applications: spherical gold nanoparticles, gold nanostars and gold nanorods, we demonstrated that AuNMs activated whole complement system, leading to the formation of SC5b-9 complex. All three complement pathways were simultaneously activated by all the AuNMs. Recognition molecules of the complement system interacted with all AuNMs <i>in vitro</i>, except for l-ficolin, but the correlation between these interactions and corresponding complement pathway activation was only observed in the classical and alternative pathways. We also observed the mediating role of complement activation in cellular uptake of all AuNMs by human U937 promonocytic cells, which expresses complement receptors. Taken together, our results highlighted the potential immunological challenges for clinical applications of AuNMs that were often overlooked.</p

Exploiting the Protein Corona around Gold Nanorods for Loading and Triggered Release

We form coronas of serum proteins on gold nanorods (NRs) coated with cetyltrimethylammonium bromide (CTAB). These coronas can be exploited for their ability to hold small molecular therapeutics at a capacity much higher (∼5–10×) than what covalent conjugation strategies can achieve. Coronas are loaded with DNA oligonucleotides and Doxorubicin, showing that they can hold species of either negative or positive charge. Payload capacity varies with assembly strategy, ionic strength, and loading concentration. Payload release can be achieved by increasing the temperature or by ultrafast laser excitation of the NRs at their longitudinal surface plasmon resonance. DNA leakage from the corona is minimal within the first 3 days of preparation, although Dox leakage was more significant. The coronas also stabilize the NRs in buffer and biological media. This study demonstrates the biological utility of the protein corona around nanomaterials, contrasting the common view of the corona as an undesirable biological response

Complement Activation by PEGylated Gold Nanoparticles

Gold nanoparticles (AuNPs) are widely used in biomedical applications, but much less is known about their immunological properties, particularly their interaction with the complement system, a key component of innate immunity serving as an indicator of their biocompatibility. Using a library of different-sized AuNPs (10, 20, 40, and 80 nm) passivated with polyethylene glycol (PEG) of different molecular weight (<i>M</i>_w = 1, 2, 5, and 10 kDa), we demonstrated that citrate-capped AuNPs activated the whole complement system in a size-dependent manner, characterized by the formation of the end-point activation product, SC5b-9, in human serum. Although PEGylation of AuNPs mitigated, but did not abolish, the activation level, complement activation by PEGylated AuNPs was independent of both the core size of AuNPs and the molecular weight of PEG. The cellular uptake of both citrate-capped and PEGylated AuNPs by human U937 promonocytic cells which expresses complement receptors were highly correlated to the level of complement activation. Taken together, our results provided new insights on the innate complement activation by PEGylated AuNPs that are widely considered to be inert biocompatible nanomaterials

Optimizing the Properties of the Protein Corona Surrounding Nanoparticles for Tuning Payload Release

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

Protein Coronas on Gold Nanorods Passivated with Amphiphilic Ligands Affect Cytotoxicity and Cellular Response to Penicillin/Streptomycin

We probe how amphiphilic ligands (ALs) of four different types affect the formation of protein coronas on gold nanorods (NRs) and their impact on cellular response. NRs coated with cetyltrimethylammonium bromide were ligand exchanged with polyoxyethylene[10]cetyl ether, oligofectamine, and phosphatidylserine (PS). Protein coronas from equine serum (ES) were formed on these NR-ALs, and their colloidal stability, as well as cell uptake, proliferation, oxidative stress, and gene expression, were examined. We find that the protein corona that forms and its colloidal stability are affected by AL type and that the cellular response to these NR-AL-coronas (NR-AL-ES) is both ligand and corona dependent. We also find that the presence of common cell culture supplement penicillin/streptomycin can impact the colloidal stability and cellular response of NR-AL and NR-AL-ES, showing that the cell response is not necessarily inert to pen/strep when in the presence of nanoparticles. Although the protein corona is what the cells see, the underlying surface ligands evidently play an important role in shaping and defining the physical characteristics of the corona, which ultimately impacts the cellular response. Further, the results of this study suggest that the cellular behavior toward NR-AL is mediated by not only the type of AL and the protein corona it forms but also its resulting colloidal stability and interaction with cell culture supplements

Quantifying Vascular Distribution and Adhesion of Nanoparticles with Protein Corona in Microflow

The protein corona has emerged as an important determinant of biological response in nanoparticle (NP) drug delivery. However, there is presently no reported study on how the protein corona affects the behavior of NPs in microflow and its subsequent interactions with the vascular endothelium, which could affect their delivery to the target tumor site regardless of its targeting mechanism. Furthermore, a consensus on the role of physical and surface characteristics of NPs in affecting the margination of NPs is lacking due to different methods of quantifying margination. In this study, we examine how the particle adhesion (PA) method and particle distribution (PD) method quantify the margination of 20, 40, 100, and 200 nm polystyrene NPs (pNPs) differently in fibronectin or pluronic F-127-coated microfluidic straight channels. We found that PA reduced with increasing pNP size, whereas the PD was similar across all pNP sizes regardless of channel coating. We then formed a protein corona on all pNPs (pNPs-PC) and found that the protein corona increased the adhesion of 40–200 nm pNPs in fibronectin-coated channels, with no size dependence between them except for 40 nm, which had significantly higher particle adhesion. The PA method was also dependent on channel coating, whereas the PD method was independent of channel coating. These results suggested that the PA method was more amenable to surface interactions between the pNPs and the channel wall while providing a measure of the amount of NPs that interacted with the channel walls, whereas the PD method provided a representation of their distribution across the channel due to margination. The two methods complement each other to elucidate a more holistic understanding of how different factors might affect a NP’s margination in future studies

Polydopamine Nanoparticles Enhance Drug Release for Combined Photodynamic and Photothermal Therapy

Our study shows a facile two-step method which does not require the use of core templates to load a hydrophobic photosensitizer drug chlorin e6 (Ce6) within polydopamine (PDA) nanoparticles (NPs) while maintaining the intrinsic surface properties of PDA NPs. This structure is significantly different from hollow nanocapsules which are less stiff as they do not possess a core. To our knowledge, there exist no similar studies in the literature on drug loading within the polymer matrix of PDA NPs. We characterized the drug loading and release behavior of the photosensitizer Ce6 and demonstrated the therapeutic efficacy of the combined photodynamic (PDT) and photothermal therapy (PTT) from Ce6 and PDA, respectively, under a single wavelength of 665 nm irradiation on bladder cancer cells. We obtained a saturated loading amount of 14.2 ± 0.85 μM Ce6 in 1 nM PDA NPs by incubating 1 mg/mL dopamine solution with 140 μM of Ce6 for 20 h. The PDA NPs maintained colloidal stability in biological media, whereas the pi–pi (π–π) interaction between PDA and Ce6 enabled a release profile of the photosensitizer until day 5. Interestingly, loading of Ce6 in the polymer matrix of PDA NPs significantly enhanced the cell uptake because of endocytosis. An increased cell kill was observed with the combined PDT + PTT from 1 nM PDA–Ce6 compared to that with PTT alone with 1 nM PDA and PDT alone with 15 μM equivalent concentration of free Ce6. PDA–Ce6 NPs could be a promising PDT/PTT therapeutic agent for cancer therapy

Exploiting the Protein Corona from Cell Lysate on DNA Functionalized Gold Nanoparticles for Enhanced mRNA Translation

This study describes the use of DNA functionalized gold nanoparticles (AuNPs) to enhance the synthesis of proteins in cell lysate and examines the mechanisms behind the enhanced mRNA translation. With an appropriate DNA oligomer sequence that hybridizes to the 3′-untranslated region of two mRNA of interest, insulin and green fluorescent protein (GFP), we found that these DNA conjugated AuNPs (AuNP-DNA) introduced into HeLa cell lysate enhanced the synthesis of insulin and GFP by up to 2.18 and 1.80-fold, respectively, over baseline production with just the mRNA present. The insulin synthesis was markedly reduced with non-DNA citrate-capped AuNP (1.25-fold) and AuNP-DNA with a nonspecific poly­(T) sequence (1.25-fold). We showed that both nonspecific adsorption of ribosomes and translation factors to form a lysate protein corona on AuNP-DNA and weak hybridization between DNA oligomers and mRNA of interest were important factors that brought translation factors, ribosomes, and mRNA into close proximity of each other. This could reduce the recycling time of ribosomes during mRNA translation, thereby increasing the efficiency of protein synthesis. The outcome of this work shows that with rational DNA design, it could be possible to modulate intracellular biological processes with AuNP-DNA and increase their production of proteins for various biomedical applications

Protein Corona around Gold Nanorods as a Drug Carrier for Multimodal Cancer Therapy

A single nanodevice based on gold nanorods (NRs) coloaded with a photosensitizer, Chlorin e6 (Ce6), and a chemotherapeutic, Doxorubicin (Dox), on its endogenously formed human serum (HS) protein corona, i.e., NR-HS-Ce6-Dox was developed with the aim of performing multimodal cancer therapy: photodynamic (PDT), photothermal (PTT) and chemotherapy (CTX) simultaneously upon irradiation with a single 665 nm laser. Here, the excitation of NRs and Ce6 resulted in photothermal ablation (PTT), and production of reactive oxygen species (ROS) to kill Cal 27 oral squamous cell carcinoma (OSCC) cells by oxidative stress (PDT) respectively, while the laser-triggered release of Dox intercalated into the DNA of cancer cells to result in DNA damage and cell death (CTX). High laser-triggered Dox release efficiency of 71.5% and strong plasmonic enhancement of ROS production by Ce6 (4.8-fold increase compared to free Ce6) was observed. Uptake of both Ce6 and Dox by Cal 27 cells was greatly enhanced, with 3.3 and 52 times higher intracellular Dox and Ce6 fluorescence observed, respectively, 6 h after dosing with NR-HS-Ce6-Dox compared to free drugs. The simultaneous trimodal therapy achieved a near complete eradication of cancer cells (98.7% cell death) with an extremely low dose of 15 pM NR-HS-Ce6-Dox loaded with just 1.26 nM Ce6 and 12.5 nM Dox due to strong synergistic enhancement in cancer cell kill compared to individual therapies performed separately. No dark toxicities were observed. These drug concentrations were far lower than any previously reported in vitro, thus eliminating any potential systemic toxicity of these agents

Quantitative and Label-Free Detection of Protein Kinase A Activity Based on Surface-Enhanced Raman Spectroscopy with Gold Nanostars

The activity of extracellular protein kinase A (PKA) is known to be a biomarker for cancer. However, conventional PKA assays based on colorimetric, radioactive, and fluorometric techniques suffer from intensive labeling-related preparations, background interference, photobleaching, and safety concerns. While surface-enhanced Raman spectroscopy (SERS)-based assays have been developed for various enzymes to address these limitations, their use in probing PKA activity is limited due to subtle changes in the Raman spectrum with phosphorylation. Here, we developed a robust colloidal SERS-based scheme for label-free quantitative measurement of PKA activity using gold nanostars (AuNS) as a SERS substrate functionalized with bovine serum albumin (BSA)–kemptide (Kem) bioconjugate (AuNS–BSA–Kem), where BSA conferred colloidal stability and Kem is a high-affinity peptide substrate for PKA. By performing principle component analysis (PCA) on the SERS spectrum, we identified two Raman peaks at 725 and 1395 cm^–1, whose ratiometric intensity change provided a quantitative measure of Kem phosphorylation by PKA in vitro and allowed us to distinguish MDA-MB-231 and MCF-7 breast cancer cells known to overexpress extracellular PKA catalytic subunits from noncancerous human umbilical vein endothelial cells (HUVEC) based on their PKA activity in cell culture supernatant. The outcome demonstrated potential application of AuNS–BSA–Kem as a SERS probe for cancer screening based on PKA activity