Probing the in vitro binding mechanism between human serum albumin and La2 O2 CO3 nanoparticles

The interaction of La₂ O₂ CO₃ nanoparticles (La NPs) with human serum albumin (HSA) has been studied. Analysis of the fluorescence quenching data of HSA using Stern–Volmer method showed that La NPs quenched HSA fluorescence in static quenching mode. Thermodynamic analysis indicated that hydrogen bonds and Van der Waals interactions play a major role for HSA–La NPs associations. Fluorescent displacement measurements confirmed that the primary binding site of La NPs was mainly located within site I (subdomain IIA) of HSA. The binding distance was calculated by using Forster resonance energy transfer theory. Also, the results of Fourier‐transform infrared spectroscopy, circular dichroism, three‐dimensional fluorescence and UV–visible measurements indicated that the binding of above La NPs to HSA may induce conformational and micro‐environmental changes of protein. This study suggested that the conformational change of HSA was at secondary structure of it and the biological activity of this protein was changed in the present of La NPs.Inspec keywords: proteins, molecular biophysics, molecular configurations, nanoparticles, lanthanum compounds, fluorescence, radiation quenching, thermal analysis, biothermics, biochemistry, hydrogen bonds, van der Waals forces, Fourier transform infrared spectra, circular dichroism, visible spectra, ultraviolet spectra, nanobiotechnologyOther keywords: in vitro binding mechanism, human serum albumin, La₂ O₂ CO₃ nanoparticles, Stern‐Volmer method, La NPs quenched HSA fluorescence, static quenching mode, thermodynamic analysis, hydrogen bonds, Van der Waals interactions, HSA‐La NPs associations, fluorescent displacement measurements, primary binding site, subdomain IIA, binding distance, Forster resonance energy transfer theory, Fourier‐transform infrared spectroscopy, circular dichroism, three‐dimensional fluorescence, UV‐visible measurements, conformational changes, microenvironmental changes, protein, secondary structure, La₂ O₂ CO₃      

Intracellular delivery of etoposide loaded biodegradable nanoparticles: cytotoxicity and cellular uptake studies

The preferred delivery systems for anticancer drugs would be the one which would have selective and effective destruction of cancer cells. In the present study etoposide (ETO) loaded nanoparticles (NP) were prepared using PLGA (ETO-PLGA NP), PLGA-MPEG block copolymer (ETO-PLGA-MPEG NP) and PLGA-Pluronic copolymer (ETO-PLGA-PLU NP) and they were evaluated for cytotoxicity and cellular uptake studies using two cancer cell lines, L1210 and DU145. The IC50 values for L1210 cells were 18.0, 6.2, 4.8 and 5.4 microM and for DU145 cells the IC50 values were 98.4, 75.1, 60.1 and 71.3 microM for ETO, ETO-PLGA NP, ETO-PLGA-MPEG NP and ETO-PLGA-PLU NP respectively. The increased cytotoxicities were attributed to increased uptake of the NPs by the cells. Moreover the ETO loaded PLGA-MPEG NP and PLGA-Pluronic NP showed a sustained cytotoxic effect till 5 days on both the cell lines. Results of the long term cytotoxicity study concluded that the drug loaded PLGA nanoparticulate formulations were efficient in decreasing the viability of the L1210 cells over a period of three days, whereas the pure drug exerted its maximum efficiency on the day one itself. Z-stack confocal images of NPs showed fluorescence activity in each section of DU 145 and L1210 cells indicating that the nanoparticles were internalized by the cells. The study concluded that ETO loaded PLGA NPs had higher cytotoxicity compared with that of the free drug and ETO-PLGA-MPEG NP and ETO-PLGA-PLU NP had higher cell uptake efficiency compared with that of ETO-PLGA NP. The developed PLGA based NPs shows promise to be used for cancer therapy.     

Targeting and penetration of virus receptor bearing cells by nanoparticles coated with envelope proteins of Moloney murine leukemia virus

One of the most important steps in a productive viral infection is when the virus fuses to a cell membrane and delivers its genome into the cell cytosol. This dynamic event is mediated by interactions between specific virus envelope proteins with their cell-bound receptors. This process is exemplified by Moloney murine leukemia virus (Mo-MLV) where envelope protein interaction with its receptor, mCAT-1, leads to virus-cell membrane fusion and infection of cells. Here, fluorescent nanoparticles (NPs) were coated with Mo-MLV derived membranes (Mo-NPs) by extrusion. Electron microscopy and biochemical analysis showed tight association of the virus membranes and NPs. The coated NPs mimic native virus by binding and entering only cells expressing the virus receptor. Confocal microscopy revealed that the coated NPs were taken up into endocytic compartments containing receptor and were also seen associated with caveolin, a marker of caveolae. To demonstrate that the Mo-NPs could escape endosomes and deliver a protein cargo into the cell cytosol, beta-lactamase (betalac) was covalently coupled to the Mo-NP cores and incubated with cells. betalac activity was only detected in the cytosol of mCAT-1-expressing cells. This is the first time that virus proteins have been used to specifically target NPs to receptor-bearing cells as well as penetration into the cell cytosol. Extrusion provides a rapid, detergent-free method to couple virus membranes to NPs and should be readily applicable for many other virus and NP types.     

Direct optical detection of viral nucleoprotein binding to an anti-influenza aptamer

We have demonstrated label-free optical detection of viral nucleoprotein binding to a polyvalent anti-influenza aptamer by monitoring the surface-enhanced Raman (SERS) spectra of the aptamer-nucleoprotein complex. The SERS spectra demonstrated that selective binding of the aptamer-nucleoprotein complex could be differentiated from that of the aptamer alone based solely on the direct spectral signature for the aptamer-nucleoprotein complex. Multivariate statistical methods, including principal components analysis, hierarchical clustering, and partial least squares, were used to confirm statistically significant differences between the spectra of the aptamer-nucleoprotein complex and the spectra of the unbound aptamer. Two separate negative controls were used to evaluate the specificity of binding of the viral nucleoproteins to this aptamer. In both cases, no spectral changes were observed that showed protein binding to the control surfaces, indicating a high degree of specificity for the binding of influenza viral nucleoproteins only to the influenza-specific aptamer. Statistical analysis of the spectra supports this interpretation. AFM images demonstrate morphological changes consistent with formation of the influenza aptamer-nucleoprotein complex. These results provide the first evidence for the use of aptamer-modified SERS substrates as diagnostic tools for influenza virus detection in a complex biological matrix.     

Multiscale modeling and uncertainty quantification in nanoparticle-mediated drug/gene delivery

Nanoparticle (NP)-mediated drug/gene delivery involves phenomena at broad range spatial and temporal scales. The interplay between these phenomena makes the NP-mediated drug/gene delivery process very complex. In this paper, we have identified four key steps in the NP-mediated drug/gene delivery: (i) design and synthesis of delivery vehicle/platform; (ii) microcirculation of drug carriers (NPs) in the blood flow; (iii) adhesion of NPs to vessel wall during the microcirculation and (iv) endocytosis and exocytosis of NPs. To elucidate the underlying physical mechanisms behind these four key steps, we have developed a multiscale computational framework, by combining all-atomistic simulation, coarse-grained molecular dynamics and the immersed molecular electrokinetic finite element method (IMEFEM). The multiscale computational framework has been demonstrated to successfully capture the binding between nanodiamond, polyethylenimine and small inference RNA, margination of NP in the microcirculation, adhesion of NP to vessel wall under shear flow, as well as the receptor-mediated endocytosis of NPs. Moreover, the uncertainties in the microcirculation of NPs has also been quantified through IMEFEM with a Bayesian updating algorithm. The paper ends with a critical discussion of future opportunities and key challenges in the multiscale modeling of NP-mediated drug/gene delivery. The present multiscale modeling framework can help us to optimize and design more efficient drug carriers in the future.     

Surface Functionality of Nanoparticles Determines Cellular Uptake Mechanisms in Mammalian Cells

Nanoparticles (NPs) are versatile scaffolds for numerous biomedical applications including drug delivery and bioimaging. The surface functionality of NPs essentially dictates intracellular NP uptake and controls their therapeutic action. Using several pharmacological inhibitors, it is demonstrated that the cellular uptake mechanisms of cationic gold NPs in both cancer (HeLa) and normal cells (MCF10A) strongly depend on the NP surface monolayer, and mostly involve caveolae and dynamin‐dependent pathways as well as specific cell surface receptors (scavenger receptors). Moreover, these NPs show different uptake mechanisms in cancer and normal cells, providing an opportunity to develop NPs with improved selectivity for delivery applications.     

Shuttle‐Mediated Nanoparticle Delivery to the Blood–Brain Barrier

Many therapeutic drugs are excluded from entering the brain due to their lack of transport through the blood–brain barrier (BBB). The development of new strategies for enhancing drug delivery to the brain is of great importance in diagnostics and therapeutics of central nervous diseases. To overcome this problem, a viral fusion peptide (gH625) derived from the glycoprotein gH of Herpes simplex virus type 1 is developed, which possesses several advantages including high cell translocation potency, absence of toxicity of the peptide itself, and the feasibility as an efficient carrier for delivering therapeutics. Therefore, it is hypothesized that brain delivery of nanoparticles conjugated with gH625 should be efficiently enhanced. The surface of fluorescent aminated polystyrene nanoparticles (NPs) is functionalized with gH625 via a covalent binding procedure, and the NP uptake mechanism and permeation across in vitro BBB models are studied. At early incubation times, the uptake of NPs with gH625 by brain endothelial cells is greater than that of the NPs without the peptide, and their intracellular motion is mainly characterized by a random walk behavior. Most importantly, gH625 peptide decreases NP intracellular accumulation as large aggregates and enhances the NP BBB crossing. In summary, these results establish that surface functionalization with gH625 may change NP fate by providing a good strategy for the design of promising carriers to deliver drugs across the BBB for the treatment of brain diseases.     

Effect of tin oxide nanoparticle binding on the structure and activity of α-amylase from Bacillus amyloliquefaciens

Proteins adsorbed on nanoparticles (NPs) are being used in biotechnology, biosensors and drug delivery. However, understanding the effect of NPs on the structure of proteins is still in a nascent state. In the present paper tin oxide (SnO2) NPs were synthesized by the reaction of SnCl4·5H2O in methanol via the sol-gel method and characterized by x-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR) and transmission electron microscopy (TEM). The binding of these SnO2-NPs with α-amylase was investigated by using UV-vis, fluorescence and circular dichroism (CD) spectroscopic techniques. A strong quenching of tryptophan fluorescence intensity in α-amylase was observed due to formation of a ground state complex with SnO2-NPs. Far-UV CD spectra showed that the secondary structure of α-amylase was changed in the presence of NPs. The Michaelis-Menten constant (K(m)), was found to be 26.96 and 28.45 mg ml(-1), while V(max) was 4.173 and 3.116 mg ml(-1) min(-1) for free and NP-bound enzyme, respectively.     

Three‐Dimensional Gold Nanoparticle Clusters with Tunable Cores Templated by a Viral Protein Scaffold

Assembling nanoparticles (NPs) into ordered architectures remains a challenge in the field of nanotechnology. Templated strategies have been widely utilized for NP assembly. As typical biological nanostructures, virus‐based NPs (VNPs) have shown great promise in templating NP assembly. Here it is illustrated that the VNP of simian virus 40 (SV40) is a powerful scaffold in directing the assembly of 3D hybrid nanoarchitectures with one NP encapsulated inside as a core and a cluster of gold NPs (AuNPs) on the outer surface of the SV40 VNP as a shell, in which the core NPs can be CdSe/ZnS quantum dots (QDs), Ag₂S QDs, or AuNPs. The assembling of AuNPs onto the SV40 VNP surface is determined by the interactions between the AuNPs and the amine groups on the outer surface of SV40 VNPs. It is expected that the VNP guided 3D hybrid nanoarchitectures provide ideal models for NP interaction studies and open new opportunities for integrating various functionalities in NP assemblies.     

Spectroscopic and electrochemical studies on the molecular interaction between copper sulphide nanoparticles and bovine serum albumin

The interaction of covellite hexagonal phase of copper sulphide nanoparticles (CuS NPs) with bovine serum albumin (BSA) was examined systematically by using fluorescence, UV–visible, circular dichroism (CD), Fourier transform infrared (FTIR), dynamic light scattering (DLS) and molecular modelling techniques. Electrochemical method was studied to further confirm the interaction of BSA with CuS NPs. The results of fluorescence studies demonstrated that fluorescence of BSA was quenched by CuS NPs via a static quenching mechanism. The negative values of thermodynamic parameters (ΔG, ΔH and ΔS) indicated that the binding process is spontaneous, exothermic and van der Waals force or hydrogen bonding plays major roles in the interaction of CuS NPs with BSA. The interaction of CuS NPs with Trp residue was established by synchronous studies, and competitive binding studies revealed that Trp-212 of subdomain IIA was involved in the interaction with these nanoparticles. Further, the efficiency of energy transferred and the distance between fluorophore (BSA) and acceptor (CuS NPs) were calculated using Forster’s resonance energy transfer theory. The results of UV–visible, CD, FTIR and DLS revealed that the CuS NPs interact with BSA by inducing the conformational changes in secondary structure and reducing the α-helix content of BSA. Molecular modelling studies suggested that CuS NPs bind to site I of sub domain IIA of BSA. The results of spectroscopic and molecular docking studies were complimented by the electrochemical techniques.     

Formation of a Monolayer Protein Corona around Polystyrene Nanoparticles and Implications for Nanoparticle Agglomeration

Nanoparticle (NP) interactions with cells and organisms are mediated by a biomolecular adsorption layer, the so‐called “protein corona.” An in‐depth understanding of the corona is a prerequisite to successful and safe application of NPs in biology and medicine. In this work, earlier in situ investigations on small NPs are extended to large polystyrene (PS) NPs of up to 100 nm diameter, using human transferrin (Tf) and human serum albumin (HSA) as model proteins. Direct NP sizing experiments reveal a reversibly bound monolayer protein shell (under saturating conditions) on hydrophilic, carboxyl‐functionalized (PS‐COOH) NPs, as was earlier observed for much smaller NPs. In contrast, protein binding on hydrophobic, sulfated (PS‐OSO₃H) NPs in solvent of low ionic strength is completely irreversible; nevertheless, the thickness of the observed protein corona again corresponds to a protein monolayer. Under conditions of reduced charge repulsion (higher ionic strength), the NPs are colloidally unstable and form large clusters below a certain protein–NP stoichiometric ratio, indicating that the adsorbed proteins induce NP agglomeration. This comprehensive characterization of the persistent protein corona on PS‐OSO₃H NPs by nanoparticle sizing and quantitative fluorescence microscopy/nanoscopy reveals mechanistic aspects of molecular interactions occurring during exposure of NPs to biofluids.     

Immune Cell‐Mediated Biodegradable Theranostic Nanoparticles for Melanoma Targeting and Drug Delivery

Although tremendous efforts have been made on targeted drug delivery systems, current therapy outcomes still suffer from low circulating time and limited targeting efficiency. The integration of cell‐mediated drug delivery and theranostic nanomedicine can potentially improve cancer management in both therapeutic and diagnostic applications. By taking advantage of innate immune cell's ability to target tumor cells, the authors develop a novel drug delivery system by using macrophages as both nanoparticle (NP) carriers and navigators to achieve cancer‐specific drug delivery. Theranostic NPs are fabricated from a unique polymer, biodegradable photoluminescent poly (lactic acid) (BPLP‐PLA), which possesses strong fluorescence, biodegradability, and cytocompatibility. In order to minimize the toxicity of cancer drugs to immune cells and other healthy cells, an anti‐BRAF V600E mutant melanoma specific drug (PLX4032) is loaded into BPLP‐PLA nanoparticles. Muramyl tripeptide is also conjugated onto the nanoparticles to improve the nanoparticle loading efficiency. The resulting nanoparticles are internalized within macrophages, which are tracked via the intrinsic fluorescence of BPLP‐PLA. Macrophages carrying nanoparticles deliver drugs to melanoma cells via cell–cell binding. Pharmacological studies also indicate that the PLX4032 loaded nanoparticles effectively kill melanoma cells. The “self‐powered” immune cell‐mediated drug delivery system demonstrates a potentially significant advancement in targeted theranostic cancer nanotechnologies.     

Preparation and in vitro evaluation of meloxicam-loaded PLGA nanoparticles on HT-29 human colon adenocarcinoma cells

Context: The inhibitors of cyclooxygenase (COX)-2 play an important role in cancer chemoprevention. Certain COX-2 inhibitors exert antiproliferative and pro-apoptotic effects on cancer cells.

Objective: In this study, meloxicam, which is an enolic acid-type preferential COX-2 inhibitor, was encapsulated in poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs) to maintain local high concentration, and its efficacy was determined.

Methods: NPs were prepared by using salting-out and emulsion-evaporation steps. Meloxicam-loaded NP formulations were evaluated with respect to the drug loading, particle size, polydispersity index, zeta potential, drug release rate, and residual poly(vinyl alcohol) (PVA) percentage. The effects of PLGA and PVA molecular weight variations on the physicochemical properties of NPs were investigated. Stability of meloxicam in NPs was assessed over 3 months. COX-2 expressing human colon adenocarcinoma cell line HT-29 was used in cellular uptake and viability assays.

Results: NPs had a spherical shape and a negative zeta potential, and their size ranged between 170–231?nm with a lower polydispersity index. NPs prepared with high molecular weight PLGA were shown to be physically stable over three months at 4°C. The increase in molecular weight of the polymer and emulsifier reduced the in vitro release rate of meloxicam from NPs. Meloxicam-loaded NPs showed cytotoxic effects on HT-29 cells markedly at 800 µM. Cancer cells had high uptake of coumarin-6-loaded NPs.

Conclusion: The PLGA NPs developed in this study can be a potentially effective drug delivery system of meloxicam for the treatment of colon cancer.     

A spectroscopic study on the interaction between gold nanoparticles and hemoglobin

The interaction between horse hemoglobin and gold nanoparticles was studied using optical spectroscopy. UV-vis and fluorescence spectra show that a spontaneous binding process occurred between hemoglobin and gold nanoparticles. The Soret band of hemoglobin in the presence of gold nanoparticles does not show significant changes, which proves that the protein retained its biological function. A shift to longer wavelengths appears in the plasmonic band of gold nanoparticles upon the attachment of hemoglobin molecules. Gold nanoparticles quench the fluorescence emission of tryptophan residues in the structure of hemoglobin. The Stern-Volmer quenching constant, the binding constant and the number of binding sites were also calculated. Thermodynamic parameters indicate that the binding was mainly due to hydrophobic interactions.     

Tuning Cellular Response to Nanoparticles via Surface Chemistry and Aggregation

The aggregation of gold nanoparticles (Au NPs) in cell media is a common phenomenon that can influence NP‐cell interactions. Here, we control Au NP aggregation in cell media and study the impact of Au NP aggregation on human dermal fibroblast (HDF) cells. By first adding Au NPs to fetal bovine serum (FBS) and then subsequently to a buffer, aggregation can be avoided. Aggregation of Au NPs also can be avoided by coating Au NPs with other biomolecules such as lipids. The aggregation state of the Au NPs influences cellular toxicity and Au NP uptake: non‐aggregated cationic Au NPs are four‐fold less toxic to HDF cells than aggregated cationic Au NPs, and the uptake of non‐aggregated anionic citrate Au NPs is three orders of magnitude less than that of aggregated citrate Au NPs. Upon uptake of Au NPs, cellular F‐actin fiber formation is disrupted and actin dots are predominant. When lipid‐coated Au NPs are doped with a fluorescent lipid (F‐lipid) and incubated with HDF cells, the fluorescence from the F‐lipid was found throughout the cell, showing that lipids can dissociate from the Au NP surface upon entering the cell.     

Nanoparticle technology for drug delivery across the blood-brain barrier

Nanoparticles (NP) are solid colloidal particles ranging in size from 1 to 1000 nm that are utilized as drug delivery agents. The use of NPs to deliver drugs to the brain across the blood-brain barrier (BBB) may provide a significant advantage to current strategies. The primary advantage of NP carrier technology is that NPs mask the blood-brain barrier limiting characteristics of the therapeutic drug molecule. Furthermore, this system may slow drug release in the brain, decreasing peripheral toxicity. This review evaluates previous strategies of brain drug delivery, discusses NP transport across the BBB, and describes primary methods of NP preparation and characterization. Further, influencing manufacturing factors (type of polymers and surfactants, NP size, and the drug molecule) are detailed in relation to movement of the drug delivery agent across the BBB. Currently, reports evaluating NPs for brain delivery have studied anesthetic and chemotherapeutic agents. These studies are reviewed for efficacy and mechanisms of transport. Physiological factors such as phagocytic activity of the reticuloendothelial system and protein opsonization may limit the amount of brain delivered drug and methods to avoid these issues are also discussed. NP technology appears to have significant promise in delivering therapeutic molecules across the BBB.     

Fluorescent polymeric nanoparticles: aggregation and phase behavior of pyrene and amphotericin B molecules in nanoparticle cores

The state of aggregation of compounds, especially drugs, in the cores of nanoparticles (NPs) formed by rapid precipitation is a significant unresolved issue. The state can control the dissolution kinetics from the NP, bioavailability, and chemical stability of the compound. A block-copolymer-directed rapid precipitation process is used to form ≈100 nm NPs comprising mixtures of hydrophobic species including fluorescent probe molecules. Fluorescence measurements are used to probe the state of aggregation and dynamics of rearrangement of pyrene (Py), Hostasol Yellow (HosY), and amphotericin B (AmpB) in NP cores. The Flory-Huggins theory of mixing is used to predict the miscibility or phase separation of the fluorophores from the host NP core material (polystyrene, cholesterol, or polycaprolactone). For Py, excimer fluorescence shows an initial microphase separation in the polystyrene core. Over time the Py redistributes more uniformly with a decrease in excimer and increase in monomer fluorescence. The Flory-Huggins theory predicts the miscibility. For HosY, the fluorescence quenching is not time-dependent, thus indicating stability of the microphase-separated fluorophores, which is consistent with the Flory-Huggins theory calculations. For the drug compound AmpB, the amphiphilic character of the molecule creates unusual "anti-Ostwald" ripening behavior in which the size distribution decreases and narrows over time, and the fluorescence demonstrates an increased ordering in the NP core over time--opposite to the behavior observed for Py.     

Fluorometric detection of ca(2+) based on an induced change in the conformation of sol-gel entrapped parvalbumin

We report the development of a fluorometric detection strategy for Ca(2+) based on induced changes in the conformation of cod III parvalbumin entrapped within a sol-gel processed glass. The detection scheme utilizes a fluorescent allosteric signal transduction (FAST) strategy wherein conformational changes induced by Ca(2+) binding result in alterations in the intrinsic fluorescence from the single tryptophan residue at position 102. Intrinsic fluorescence was also used to examine chemically induced changes in protein structure to ascertain the effects of entrapment on the conformational motions and stability of the protein. Fluorescence analysis indicated that the behavior of the protein depended on the entrapment protocols used. The entrapped protein retained conformational flexibility similar to that observed in solution and remained accessible to analytes such as Ca(2+). Entrapment also caused improvements in protein stability against chemical denaturants. However, entrapment caused the apparent affinity constant for binding of Ca(2+) to decrease substantially with aging time. Even so, in optimum cases, fluorometric detection of Ca(2+) could be done over a 600 μM range with a limit of detection of 3 μM and with no interference from divalent ions such as Mg(2+), Sr(2+), or Cd(2+), indicating the viability of using sol-gel entrapped FAST proteins for the detection of Ca(2+).     

Exploring Protein–Nanoparticle Interactions with Coarse‐Grained Protein Folding Models

Understanding the fundamental biophysics behind protein–nanoparticle (NP) interactions is essential for the design and engineering bio‐NP systems. The authors describe the development of a coarse‐grained protein–NP model that utilizes a structure centric protein model. A key feature of the protein–NP model is the quantitative inclusion of the hydrophobic character of residues in the protein and their interactions with the NP surface. In addition, the curvature of the NP is taken into account, capturing the protein behavior on NPs of different size. The authors evaluate this model by comparison with experimental results for structure and adsorption of a model protein interacting with an NP. It is demonstrated that the simulation results recapitulate the structure of the small α/β protein GB1 on the NP for data from circular dichroism and fluorescence spectroscopy. In addition, the calculated protein adsorption free energy agrees well with the experimental value. The authors predict the dependence of protein folding on the NP size, surface chemistry, and temperature. The model has the potential to guide NP design efforts by predicting protein behavior on NP surfaces with various chemical properties and curvatures.