PDADMAC/Alginate-Coated Gold Nanorod For Eradication of Staphylococcus Aureus Biofilms.

Introduction: Over 75% of clinical microbiological infections are caused by bacterial biofilms that grow on wounds or implantable medical devices. This work describes the development of a new poly(diallyldimethylammonium chloride) (PDADMAC)/alginate-coated gold nanorod (GNR/Alg/PDADMAC) that effectively disintegrates the biofilms of Staphylococcus aureus (S. aureus), a prominent pathogen responsible for hospital-acquired infections. Methods: GNR was synthesised via seed-mediated growth method, and the resulting nanoparticles were coated first with Alg and then PDADMAC. FTIR, zeta potential, transmission electron microscopy, and UV-Vis spectrophotometry analysis were performed to characterise the nanoparticles. The efficacy and speed of the non-coated GNR and GNR/Alg/PDADMAC in disintegrating S. aureus-preformed biofilms, as well as their in vitro biocompatibility (L929 murine fibroblast) were then studied. Results: The synthesised GNR/Alg/PDADMAC (mean length: 55.71 ± 1.15 nm, mean width: 23.70 ± 1.13 nm, aspect ratio: 2.35) was biocompatible and potent in eradicating preformed biofilms of methicillin-resistant (MRSA) and methicillin-susceptible S. aureus (MSSA) when compared to triclosan, an antiseptic used for disinfecting S. aureus colonisation on abiotic surfaces in the hospital. The minimum biofilm eradication concentrations of GNR/Alg/PDADMAC (MBEC50 for MRSA biofilm = 0.029 nM; MBEC50 for MSSA biofilm = 0.032 nM) were significantly lower than those of triclosan (MBEC50 for MRSA biofilm = 10,784 nM; MBEC50 for MRSA biofilm 5967 nM). Moreover, GNR/Alg/PDADMAC was effective in eradicating 50% of MRSA and MSSA biofilms within 17 min when used at a low concentration (0.15 nM), similar to triclosan at a much higher concentration (50 µM). Disintegration of MRSA and MSSA biofilms was confirmed by field emission scanning electron microscopy and confocal laser scanning microscopy. Conclusion: These findings support the potential application of GNR/Alg/PDADMAC as an alternative agent to conventional antiseptics and antibiotics for the eradication of medically important MRSA and MSSA biofilms.

Conjugation of Peptides to Gold Nanoparticles.

Peptides and proteins have played an important role in many biological processes, functioning as enzymes, hormones, ligands, receptors, cell mediators, and structural components of cells. Being intrinsic molecules in signaling pathways, peptides allow for therapeutic intervention that closely mimic natural signaling cascades. However, the short chain of amino acids in free peptides is susceptible to proteolysis in vivo. Conjugation of peptides onto nanoparticles has been used as a strategy to extend peptide half-life through conferring steric hindrance and a high packing density that prevents proteolytic enzymes to degrade them. Here, we describe a method to conjugate the anticancer p53 peptides as our model peptide onto 12 nm gold nanoparticles (AuNPs) to form the AuNP-p53 peptide conjugate. Conjugation of the p53 short-chain peptide of 25 amino acids occurs through a combination of electrostatic interactions and covalent bonds between cysteine residues at the N-terminal of the peptide and the surface of the AuNPs. The AuNPs and AuNP-p53 are characterized by UV-Vis spectroscopy for its optical absorbance and zetasizer for their hydrodynamic diameter and zeta potential. The semiquantitative analysis of the amount of conjugated peptides on the AuNPs and peptide stability under trypsin treatment is performed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Stealthiness and Hematocompatibility of Gold Nanoparticles with Pre-Formed Protein Corona.

Studies have established that a serum protein corona pre-formed around gold nanorods (NRs) could be exploited for loading photosensitizers and chemotherapeutics to result in efficient cell kill in vitro with an extremely low dose. In this study, we further demonstrated that pre-forming a serum protein corona (PC) around citrate-capped NRs (NR-Cit) to form NR-PC conferred them stealth property and high hematocompatibility similar to the common strategy of PEGylating NRs, which would otherwise not be able to evade the immune system. Specifically, the NR-PC caused minimal complement activation with significantly lower formation of the terminal complement complex SC5b-9 measured in human serum containing NR-PC, and this resulted in low uptake by phagocytic U937 monocytes of 5.9% of the initial gold dose compared to 55.8% of NR-Cit. In addition, NR-PC exhibited very low hemolytic activity of less than 0.2% hemolysis with no observable effect on RBC morphology as opposed to 0.6% for NR-Cit at the same concentration of 1 nM NRs. Furthermore, we showed that the high hematocompatibility and stealth property of NR-PC were maintained even after the loading of small molecules, photosensitizer Chlorine e6 (Ce6), into the protein corona, thus further establishing the potential clinical relevance of exploiting the inevitably formed serum protein corona on nanoparticles as an effective delivery vector for small molecular therapeutics.

In situ measurements of intracellular thermal conductivity using heater-thermometer hybrid diamond nanosensors.

Understanding heat dissipation processes at nanoscale during cellular thermogenesis is essential to clarify the relationships between the heat and biological processes in cells and organisms. A key parameter determining the heat flux inside a cell is the local thermal conductivity, a factor poorly investigated both experimentally and theoretically. Here, using a nanoheater/nanothermometer hybrid made of a polydopamine encapsulating a fluorescent nanodiamond, we measured the intracellular thermal conductivities of HeLa and MCF-7 cells with a spatial resolution of about 200 nm. The mean values determined in these two cell lines are both 0.11 ± 0.04 W m-1 K-1, which is significantly smaller than that of water. Bayesian analysis of the data suggests there is a variation of the thermal conductivity within a cell. These results make the biological impact of transient temperature spikes in a cell much more feasible, and suggest that cells may use heat flux for short-distance thermal signaling.

Dynamics of Human Serum Albumin Corona Formation on Gold Nanorods with Different Surface Ligands In Silico.

The interaction between human serum albumin (HSA) and nanoparticles (NPs) to form HSA corona has widely been studied since endogenous functions of albumin are highly attractive for drug delivery. However, a full understanding of the molecular dynamics and factors behind the formation of HSA corona, including interactions between HSA and different surface ligands and between neighboring HSA molecules, resulting in conformational change of HSA is presently lacking. Here, we assembled 14 HSA molecules around gold nanorods (AuNRs) with different surface chemistries (bare gold surface, cetyltrimethylammonium bromide (CTAB), polystyrene sulfonate (PSS), and polydiallyldimethylammonium chloride (PDADMAC)) in silico and examined the dynamics of HSA corona formation using coarse-grained molecular dynamics for 300 ns of simulation. We observed that PDADMAC, being more flexible than PSS, resulted in all HSA molecules moving toward AuNR-PDADMAC, while the instability of CTAB on AuNR resulted in fewer HSA molecules moving toward AuNR-CTAB compared to AuNR-PSS. HSA molecules around AuNR-PDADMAC also exhibited the largest conformational change in terms of their radius of gyration (Rg) and root mean square deviation (RMSD). In the absence of surface ligands, HSA molecules around the bare AuNR were susceptible to steric hindrance with conformational change observed in terms of their RMSD but not their Rg unlike that of HSA molecules around AuNR-PDADMAC. The insights gained from the inclusion of neighboring HSA molecules in the simulation of corona formation could be more representative than examining a single adsorbed HSA molecule on AuNRs with different surface passivations.

Conjugation with gold nanoparticles improves the stability of the KT2 peptide and maintains its anticancer properties.

One of the major weaknesses of therapeutic peptides is their sensitivity to degradation by proteolytic enzymes in vivo. Gold nanoparticles (GNPs) are a good carrier for therapeutic peptides to improve their stability and cellular uptake in vitro and in vivo. We conjugated the anticancer KT2 peptide as an anticancer peptide model to PEGylated GNPs (GNPs-PEG) and investigated the peptide stability, cellular uptake and ability of the GNPs-KT2-PEG conjugates to induce MDA-MB-231 human breast cancer cell death. We found that 11 nm GNPs protected the conjugated KT2 peptide from trypsin proteolysis, keeping it stable up to 0.128% trypsin, which is higher than the serum trypsin concentration (range 0.0000285 ± 0.0000125%) reported by Lake-Bakaar, G. et al., 1979. GNPs significantly enhanced the cellular uptake of KT2 peptides after conjugation. Free KT2 peptides pretreated with trypsin were not able to kill MDA-MB-231 cells due to proteolysis, while GNPs-KT2-PEG was still able to exert effective cancer cell killing after trypsin treatment at levels comparable to GNPs-KT2-PEG without enzyme pretreatment. The outcome of this study highlights the utility of conjugated anticancer peptides on nanoparticles to improve peptide stability and retain anticancer ability.

Innate immune activation by conditioned medium of cancer cells following combined phototherapy with photosensitizer-loaded gold nanorods.

Nanoparticle-based phototherapy has evolved to include immunotherapy as an effective treatment combination for cancers through inducing anti-cancer immune activation leading to downstream adaptive responses and immune protection. However, most cancer phototherapy studies that claimed anti-cancer immunogenic effects often included exogenous immunostimulants to potentiate immune responses and did not clearly establish their effects on immune cells. In this study, we showed that combined photodynamic (PDT) and photothermal therapy (PTT) using gold nanorods (NRs) loaded with the photosensitizer chlorin e6 (Ce6) on endogenously formed mouse serum (MS) protein coronas (i.e., NR-MS-Ce6) on EMT6 murine mammary carcinoma cells could potentiate the activation of both J774A.1 macrophages and DC2.4 dendritic cells. The activation of these innate immune cells by the conditioned media from cancer cells treated with combined PDT + PTT was cell-type and number dependent. While treated B16-OVA murine melanoma cells induced lower activation levels for both immune cell types compared to EMT6, they caused higher pro-inflammatory cytokine secretion levels. Our study suggests the importance of immunological investigations to complement any nanoparticle-based therapeutic interventions to better evaluate their efficacy. This could be achieved through a simple approach to screen for the first line of immune responses arising from these therapies prior to in vivo studies.

Polyelectrolyte stiffness on gold nanorods mediates cell membrane damage.

Charge and surface chemistry of gold nanorods (AuNRs) are often considered the predictive factors for cell membrane damage. Unfortunately, extensive research on AuNR passivated with polyelectrolyte (PE) ligand shell (AuNR-PE) has hitherto left a vital knowledge gap between the mechanical stability of the ligand shell and the cytotoxicity of AuNR-PEs. Here, the agreement between unbiased coarse-grained molecular dynamics (CGMD) simulation and empirical outcomes on hemolysis of red blood cells by AuNR-PEs demonstrates for the first time, a direct impact of the mechanical stability of the PE shell passivating the AuNRs on the lipid membrane rupture. Such mechanical stability is ultimately modulated by the rigidity of the PE components. The CGMD simulation results also reveal the mechanism where the PE chain adsorbs near the surface of the lipid bilayer without penetrating the hydrophobic core of the bilayer, which allows the hydrophobic AuNR core to be in direct contact with the hydrophobic interior of the lipid bilayer, thereby perforating the lipid membrane to induce membrane damage.

Rapid Detection of Carbapenemase-Producing Enterobacteriacae Based on Surface-Enhanced Raman Spectroscopy with Gold Nanostars.

The emergence and rapid spread of antibiotic resistance poses a serious threat to healthcare systems across the globe. The existence of carbapenemase-producing Enterobacteriaceae (CPE) such as Klebsiella pneumoniae renders the use of carbapenems, the last-resort class of ß-lactam antibiotics, ineffective against bacterial infections, often leading to CPE-associated mortalities. Current methods of detection such as the Carba NP test and modified Hodge's test require hours to days to detect, which delays the response to isolate patients for rapid intervention. Here, we developed a surface-enhanced Raman scattering (SERS)-based detection scheme which utilizes gold nanostars conjugated to a ß-lactam antibiotic ceftriaxone (CRO) as a beacon for rapid detection of bacterial ß-lactamase secreted by Delhi metalloproteinase (NDM)-producing Escherichia coli as our CPE model with carbapenemase activity. The cleavage of ß-lactam ring in CRO by NDM (Class B ß-lactamase) caused a detectable reduction in SERS intensities at 722, 1358, and 1495 cm-1 within 25 min. Ratiometric analysis of the SERS peaks at 722, 1358, and 1495 cm-1 normalized against the Raman peak of polystyrene cuvette at 620 cm-1 showed the peak at 1358 cm-1 having the most significant change in intensity upon CPE detection. This reduced detection time has not been reported to date for CPE detection, and our novel approach using SERS could be extended to detect the activity of other classes of ß-lactamases to broaden its clinical utility.

Mannitol-induced gold nanoparticle aggregation for the ligand-free detection of viral particles.

Traditional virus detection methods require ligands that bind to either viral capsid proteins or viral nucleic acids. Ligands are typically antibodies or oligonucleotides and they are expensive, have limited chemical stability, and can only detect one specific type of virus at a time. Here, the biochemical surface properties of viruses are exploited for ligand-free, nonspecific virus detection. It has been found that the osmolyte mannitol can preferentially aggregate virus, while leaving proteins in solution. This led to the development of a ligand-free detection of virus using gold nanoparticle (AuNP) aggregation. Porcine parvovirus (PPV) was incubated with AuNPs and aggregation of the PPV-AuNP complex with mannitol was detected by dynamic light scattering (DLS). The lowest detectable concentration of PPV was estimated to be 106 MTT50 per mL, which is lower than standard antibody assays. PPV was also detected when swabbed from a dry surface and in the presence of a protein solution matrix. The enveloped bovine viral diarrhea virus (BVDV) was also detected using mannitol-induced aggregation of BVDV-coated AuNPs. The lowest detectable concentration of BVDV was estimated to be 104 MTT50 per mL. This demonstrates that gold nanoparticle aggregation can detect virus without the use of specific ligands.

Mucopenetration and biocompatibility of polydopamine surfaces for delivery in an Ex Vivo porcine bladder.

Urine voiding and the presence of a mucus layer on the apical surface of the urothelium are two major challenges towards an effective intravesical drug delivery for bladder malignancies. Improved bioavailability to the underlying bladder tissue could be achieved with delivery vectors that diffuse efficiently through the bladder mucus. Pegylation of delivery vectors remains the existing "gold standard" to enhance mucosal delivery despite known poor cell uptake and reported PEG sensitivity. Here, we showed improved mucopenetration of carboxylated polystyrene (PS) nanoparticles (NPs) passivated with a polydopamine (PDA) surface, at similar level as PEG. While the diffusion of PS NPs in mucus was retarded by ~1000-fold, PS-PDA diffused only 6-fold slower in mucus than water. This enabled faster and deeper penetration of PS-PDA into porcine bladder tissue beneath the mucus layer. The same PDA surface also conferred biocompatibility and enabled photothermal therapy (PTT) with significant surface disruption on an ex vivo porcine bladder model upon localized laser irradiation, which was not possible with PEG. Our outcomes suggested the facile and versatile PDA surface passivation of nanoparticles as an enabler for dual purposes of enhancing mucopenetration and allowing photothermal therapy on bladder tissue, which has not been demonstrated to date.

Exploiting Protein Corona around Gold Nanoparticles Conjugated to p53 Activating Peptides To Increase the Level of Stable p53 Proteins in Cells.

Therapeutic peptides suffer from major drawbacks such as peptide degradation in vivo due to proteolysis. Gold nanoparticles (AuNPs) are an effective carrier for therapeutic peptides that improve their stability in vivo, while also enabling nonspecific adsorption of complementary proteins to enhance their effectiveness. Using p53 peptide as a model known to disrupt the intracellular MDM2-p53 protein-protein interaction which tags the endogenous p53 proteins for degradation, we conjugated p53 peptides to AuNPs (AuNP-p53) and examined the functionality of AuNP-p53 to release the endogenous p53 proteins from being tagged for degradation, thereby increasing the level of stable p53 proteins in acute myeloid leukemia 2 (AML2) cells. We found that AuNPs did not just protect conjugated p53 peptides from trypsin degradation, but also helped to recruit 56.5% and 26.4% of total MDM2 and p53 proteins in the cells to form a protein corona around AuNP-p53. The proximity of MDM2/p53 complexes and p53 peptide on the surface of AuNP-p53 facilitated the action of p53 peptides to cause a sustained elevation of the p53 level in AML2 cells up to 6 h, which was not possible with free p53 peptide alone at the same concentration. Even a 20-fold higher concentration of free p53 peptide caused only a short-lived elevated p53 level of 1 h. The outcome of this study highlights the utility of combining conjugated ligands and complementary protein adsorption on nanoparticles to improve the biological functionality of the therapeutic ligands.

Polydopamine Coating Enhances Mucopenetration and Cell Uptake of Nanoparticles.

Mucus is an endogenous viscoelastic biopolymer barrier that limits the entry of foreign pathogens and therapeutic carriers to the underlying mucosal cells. This could be overcome with a hydrophilic and nonpositively charged carrier surface that minimizes interactions with the mucin glycoprotein fibers. Although PEGylation remains an attractive surface strategy to enhance mucopenetration, cell uptake of PEGylated nanoparticles (NPs) often remains poor. Here, we demonstrated polydopamine (PDA) coating to enhance both mucopenetration and cell uptake of NPs. PDA was polymerized on carboxylated polystyrene (PS) NPs to form a PDA coating, and the resulting PS-PDA achieved a similar level of mucopenetration as our PEGylated PS (PS-PEG) positive control in three separate studies: NP-mucin interaction test, transwell assay, and multiple particle tracking. Compared to water, the diffusions of PS-PDA and PS-PEG in reconstituted mucus solution were only 3.5 and 2.4 times slower, respectively, whereas the diffusion of bare PS was slowed by up to 250 times. However, the uptake of PS-PDA (61.2 ± 6.1%) was almost three times higher than PS-PEG (24.6 ± 5.4%) in T24 cells, which were used as a model for underlying mucosal cells. Our results showed a novel unreported functionality of PDA coating in enhancing both mucopenetration and cell uptake of NPs for mucosal drug delivery applications, not possible with conventional PEGylation strategies.

Enhanced Secretion of Functional Insulin with DNA-Functionalized Gold Nanoparticles in Cells.

We have previously shown the use of gold nanoparticles (AuNPs) functionalized with DNA (AuNP-DNA) to increase insulin mRNA translation in a cell-free system. In this study, we translate the concept into a whole cell system to demonstrate functionality despite the additional complexity of intracellular delivery and mRNA translation inside living cells. We selected an insulin-secreting pancreatic islet cell line, RIN-5F, as our model and designed a DNA oligomer (insDNA) that is complementary to the 3'-untranslated region of insulin mRNA for conjugation to AuNPs (AuNP-insDNA). AuNP-insDNA was stable in the extracellular environment of RIN-5F cells for up to 24 h, without eliciting any cell toxicity. Upon cellular entry, AuNP-insDNA was able to sustain enhanced insulin secretion from 6 to 12 h post-incubation, peaking at 10 h with an enhancement factor of 1.69-fold. This enhancement was not observed when insDNA was removed or replaced with poly thymine or poly adenine DNAs. The enhanced insulin secreted was 100% functional and capable of binding to its insulin receptor. The outcome of this study demonstrated the feasibility of AuNP-DNA to enhance the synthesis of proteins in whole cells and could serve as a new direction of invoking a patient's own beta cells to increase insulin secretion for treatment of diabetes.

Protein Corona Formed from Different Blood Plasma Proteins Affects the Colloidal Stability of Nanoparticles Differently.

Significant progress in the characterization of protein corona has been made. However, insights on how the corona affects the aggregation of nanoparticles (NPs) and consequent biological identity are still lacking. Here, we examined how the corona formed from four major serum proteins, immunoglobulin G (IgG), fibrinogen (FBG), apolipoprotein A1 (ApoA1), and human serum albumin (HSA), over a range of concentrations affects the aggregation of gold NPs (AuNPs). We found that at physiological pH of 7.4, all four proteins aggregated the AuNPs at low concentrations but conferred colloidal stability at high concentrations due to the complete "corona coat" around individual AuNPs. Due to their immune-related functions, IgG and FBG aggregated the AuNPs to a greater extent compared to HSA and ApoA1 which were mostly involved in transport of small molecules. We then introduced the AuNP-corona formed from each protein into an acidic solution at pH 6.2 with high ionic concentration for up to 24 h as a model of the tumor microenvironment to examine for changes in their aggregation. We observed that protein corona formation sterically stabilized the AuNP-corona for all four proteins, resulting in a smaller increase in aggregation and size compared to citrate-capped AuNPs. This was especially true for corona formed at high protein:AuNP ratios. Our study therefore showed that the formation of a complete "corona coat" around NPs at sufficiently high protein:NP ratio was required for colloidal stability of designed NP systems in both physiological and cancer microenvironment to maintain efficiency and efficacy in cancer drug delivery.

Size-dependent neutralizing activity of gold nanoparticle-based subunit vaccine against dengue virus.

Dengue results in substantial human morbidity and significant socio-economic impacts, but a specific dengue therapeutic is not available. The currently available dengue vaccine has low efficacy and high rate of adverse effects, necessitating different strategies for the development of a safer and more efficient vaccine against dengue virus. We describe here a hybrid combination of different-sized gold nanoparticles (AuNPs) and domain III of envelope glycoprotein derived from serotype 2 of dengue virus (EDIII) as dengue subunit vaccine. The efficacy of the EDIII-functionalized AuNPs (AuNP-E) to induce neutralizing antibody in BALB/c mice is evaluated. Obtained results show that AuNP-E induced a high level of antibody which mediates serotype-specific neutralization of dengue virus. More importantly, the level of antibody is dependent on both the size of AuNPs and the concentration of AuNP-E, implicating the possibility to modulate it through adjusting these parameters. These results represent an important step towards the development of tetravalent AuNP-based subunit dengue vaccine. STATEMENT OF SIGNIFICANCE: This research presents a novel subunit vaccine against dengue virus using a hybrid comprising gold nanoparticles (AuNPs) and domain III of envelop protein (EDIII). We proved the neutralizing activity of anti-EDIII antibody induced in immunized mice on Dengue virus serotype 2 in an AuNP core size and concentration dependent manner. The hybrid concept behind this work could also be adopted for the development of a tetravalent vaccine against four serotypes of Dengue virus.

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.

Influence of protein corona and caveolae-mediated endocytosis on nanoparticle uptake and transcytosis.

Transcytosis of nanoparticles (NPs) is emerging as an attractive alternative to the paracellular route in cancer drug delivery with studies suggesting targeting caveolae-mediated endocytosis to maximize NP transcytosis. However, there are limited studies on transcytosis of NPs, especially for corona-coated NPs. Most studies focused on cellular uptake as an indirect measure of the NP's transcellular permeability (Pd). Here, we probed the effect of protein corona on the uptake and transcytosis of 20, 40, 100, and 200 nm polystyrene nanoparticles (pNP-PC) across HUVECs in a microfluidic channel that modelled the microvasculature. We observed increased cell uptake with size of pNP-PC although it was the smallest 20 nm pNP-PC that exhibited the highest transcellular Pd. In the absence of corona however, cell uptake decreased with size, and the largest 200 nm pNP-PEG exhibited the lowest transcellular Pd. By inhibiting caveolae-mediated endocytosis in HUVECs, smaller pNPs had a larger drop in cell uptake than larger pNPs, regardless of surface coating. However, only the smallest (20 nm) and largest (200 nm) pNP-PC had a decrease in Pd following inhibition with MßCD. Our findings showed that the protein corona affected the transcytosis of NPs, and their uptake by caveolae-mediated endocytosis did not necessarily lead to transcytosis.

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.

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.