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      

Nanoparticles Interacting with Proteins and Cells: A Systematic Study of Protein Surface Charge Effects

Despite intense research on biological and biomedical applications of nanoparticles, our understanding of their basic interactions with the biological environment is still incomplete. Systematic variation of the physicochemical properties of the nanoparticles is widely seen as a promising strategy to obtain further insights. In view of the key role of the protein adsorption layer forming on nanoparticles in contact with biofluids, we systematically varied the surface charge of proteins adsorbing onto nanoparticles by chemical modification so as to examine the effect of Coulomb forces in modulating nano‐bio interactions. We chose human serum albumin (HSA) as a model protein and ultra‐small, negatively charged fluorescent gold nanoclusters (AuNCs) as model nanoparticles. By using fluorescence and CD spectroscopies, we measured binding affinities and structural changes upon binding of the HSA variants. The strengths of the protein‐nanoparticle interactions were found to change substantially upon modifying the surface charge of HSA. Furthermore, by using inductively coupled plasma optical emission spectroscopy, confocal fluorescence microscopy, scanning transmission electron microscopy and cell viability assays, we observed that cellular interactions of the AuNCs, including their adherence to cell membranes, uptake efficiency and cytotoxicity, depended markedly on the different surface charges of the HSA variants adsorbed onto the nanoparticles. These results illustrate vividly that the cellular responses to nanoparticle exposure depend on the specific properties of the proteins that adsorb onto nanoparticles from biofluids.     

Protein Binding Characteristics of 2′-Benzoyloxycinnamaldehyde

The protein binding characteristic of 2′-Benzoyloxycinnamaldehyde (BCA) was investigated, which has demonstrated a potent antitumor effect against several human solid tumor cell lines and in human tumor xenograft nude mice. Protein binding of BCA in human serum was 86 ± 0.91% and the predominant binding protein of BCA was fatty-acid-free human serum albumin (HSA) (81 ± 0.91%). The binding of BCA to HSA was outlined by one class, and Ka and n of BCA were 1.65 × 10⁵ M^{? 1} and 0.374, respectively. Displacement studies with fluorescence probes suggested that BCA mainly binds to site I on HSA, and BCA-induced enhancement in site II binding. The limited drug–drug interaction experiments suggested that BCA influences both site I and site II drug–HSA bindings via different mechanisms; a competitive displacement and a probable allosteric conformational change in HSA, respectively.     

Chitosan based mucoadhesive nanoparticles of ketoconazole for bioavailability enhancement: formulation,optimization, in vitro and ex vivo evaluation

Purpose: The conventional dosage form of Ketoconazole (KZ) shows poor absorption due to rapid gastric emptying. Chitosan based mucoadhesive nanoparticles (NPs) of KZ were developed to efficiently release drug at its absorption window i.e. stomach and the site of action i.e. esophagus.

Method: The NPs were prepared by ionic gelation method. Concentration of polymer, cross-linking agent and ratio of drug/polymer as well as polymer/cross linking agent were optimized.

Results: NPs had 69.16?±?5.91% mucin binding efficiency, particle size of 382.6?±?2.384?nm, ζ potential of +48.1?mv and entrapment efficiency of 59.84 ± 1.088%. DSC thermogram indicated absence of any drug polymer interaction. The drug release was by controlled, non-fickian diffusion mechanism. Ex vivo diffusion studies were performed by emptying the stomach contents after 2?h to simulate in vivo gastric emptying. The results showed that drug diffusion from the solution across stomach mucosa stopped after emptying whereas that from the NPs continued upto 5?h. Hence we could conclude that the NPs must have adhered to the stomach mucosa and thereby would have been retained at this absorption site even after gastric emptying.

Conclusion: The orally delivered KZ loaded mucoadhesive NPs can be used as an efficient carrier for delivering drug at its absorption window i.e. the stomach and the site of action i.e. esophagus even after gastric emptying.     

Immobilization of albumin on magnetite nanoparticles

The magnetite (Fe₃O₄) nanoparticles were prepared by the co-precipitation of ferrous and ferric salts with NH₄OH, and then modified with 3-aminopropyltriethoxysilane (APTES) by silanization reaction and subsequent reaction with glutaraldehyde (GA) to obtain functional groups on their surface. The influence of different terminated groups on protein binding was studied with bare and modified magnetite nanoparticles. Amine terminated magnetite nanoparticles were shown the highest binding ability for immobilization process compared to Fe₃O₄ NPs and GA bonded NPs. This binding ability was shown by using sodium dodecyl polyacrylamide gel electrophoresis technique (SDS-PAGE). Albumin attached magnetite nanoparticles were also examined by Scanning Electron Microscopy (SEM).     

Protein binding characteristics of 2'-benzoyloxycinnamaldehyde

The protein binding characteristic of 2'-Benzoyloxycinnamaldehyde (BCA) was investigated, which has demonstrated a potent antitumor effect against several human solid tumor cell lines and in human tumor xenograft nude mice. Protein binding of BCA in human serum was 86 +/- 0.91% and the predominant binding protein of BCA was fatty-acid-free human serum albumin (HSA) (81 +/- 0.91%). The binding of BCA to HSA was outlined by one class, and Ka and n of BCA were 1.65 x 10(5) M(- 1) and 0.374, respectively. Displacement studies with fluorescence probes suggested that BCA mainly binds to site I on HSA, and BCA-induced enhancement in site II binding. The limited drug-drug interaction experiments suggested that BCA influences both site I and site II drug-HSA bindings via different mechanisms; a competitive displacement and a probable allosteric conformational change in HSA, respectively.     

Targeted biodegradable nanoparticles for drug delivery to smooth muscle cells

Targeted delivery of therapeutic agents to prevent smooth muscle cell (SMC) proliferation is important in averting restenosis (a narrowing of blood vessels). Since platelet derived growth factor (PDGF) receptors are over-expressed in proliferating SMCs after injury from cardiovascular interventions, such as angioplasty and stent implantation, our hypothesis is that conjugation of PDGF-BB (platelet-derived growth factor BB (homodimer)) peptides to biodegradable poly (D,L-lactic-co-glycolide) (PLGA) nanoparticles (NPs) would exhibit an increased uptake of these NPs by proliferating SMCs. In this study, poly (D,L-lactide-co-glycolide) (PLGA) nanoparticles containing dexamethasone were formulated and conjugated with PDGF-BB peptides. These NPs were stable, biocompatible, and exhibited a sustained drug release over 14 days. Various particle uptake studies using HASMCs (human aortic smooth muscle cells) demonstrated that PDGF-BB peptide-conjugated nanoparticles significantly increased cellular uptake and decreased proliferation of HASMCs compared to control nanoparticles (without conjugation of PDGF-BB peptides). These NPs were internalized primarily by clathrin-mediated endocytosis and macropinocytosis. Our in vitro results suggest that PDGF-BB peptide-conjugated NPs could represent as an effective targeted, sustained therapeutic delivery system to reduce restenosis and neointimal hyperplasia.     

In Situ Investigation on the Protein Corona Formation of Quantum Dots by Using Fluorescence Resonance Energy Transfer

A fundamental understanding of nanoparticle–protein corona and its interactions with biological systems is essential for future application of engineered nanomaterials. In this work, fluorescence resonance energy transfer (FRET) is employed for studying the protein adsorption behavior of nanoparticles. The adsorption of human serum albumin (HSA) onto the surface of InP@ZnS quantum dots (QDs) with different chirality (d ‐ and l ‐penicillamine) shows strong discernible differences in the binding behaviors including affinity and adsorption orientation that are obtained upon quantitative analysis of FRET data. Circular dichroism spectroscopy further confirms the differences in the conformational changes of HSA upon interaction with d ‐ and l ‐chiral QD surfaces. Consequently, the formed protein corona on chiral surfaces may affect their following biological interactions, such as possible protein exchange with serum proteins plasma as well as cellular interactions. These results vividly illustrate the potential of the FRET method as a simple yet versatile platform for quantitatively investigating biological interactions of nanoparticles.     

Folate encapsulation in PEG‐diamine grafted mesoporous Fe3 O4 nanoparticles for hyperthermia and in vitro assessment

Effective and targeted delivery of the antitumour drugs towards the specific cancer spot is the major motive of drug delivery. In this direction, suitably functionalised magnetic iron oxide nanoparticles (NPs) have been utilised as a theranostic agent for imaging, hyperthermia and drug delivery applications. Herein, the authors reported the preparation of multifunctional polyethyleneglycol‐diamine functionalised mesoporous superparamagnetic iron oxide NPs (SPION) prepared by a facile solvothermal method for biomedical applications. To endow targeting ability towards tumour site, folic acid (FA) is attached to the amine groups which are present on the NPs surface by 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide hydrochloride/N‐hydroxysuccinimide chemistry. FA attached SPION shows good colloidal stability and possesses high drug‐loading efficiency of ∼ 96% owing to its mesoporous nature and the electrostatic attachment of daunosamine (NH₃ ⁺) group of doxorubicin (DOX) towards the negative surface charge of carboxyl and hydroxyl group. The NPs possess superior magnetic properties in result endowed with high hyperthermic ability under alternating magnetic field reaching the hyperthermic temperature of 43°C within 223 s at NP''s concentration of 1 mg/ml. The functionalised NPs possess non‐appreciable toxicity in breast cancer cells (MCF‐7) which is triggered under DOX‐loaded SPION.Inspec keywords: nanoparticles, nanocomposites, mesoporous materials, colloids, biochemistry, nanomagnetics, molecular biophysics, tumours, superparamagnetism, drugs, toxicology, biomedical materials, nanofabrication, hyperthermia, cancer, magnetic particles, cellular biophysics, nanomedicine, iron compounds, drug delivery systems, filled polymers, biological organs, liquid phase depositionOther keywords: NP surface, colloidal stability, drug‐loading efficiency, hydroxyl group, magnetic properties, high hyperthermic ability, magnetic field, DOX‐loaded SPION, folate encapsulation, targeted delivery, antitumour drugs, specific cancer spot, magnetic iron oxide nanoparticles, theranostic agent, drug delivery applications, multifunctional polyethyleneglycol‐diamine, facile solvothermal method, biomedical applications, tumour site, amine groups, mesoporous superparamagnetic nanoparticles, PEG‐diamine grafted mesoporous nanoparticles, 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide hydrochloride‐N‐hydroxysuccinimide chemistry, daunosamine group, carboxyl group, breast cancer cells, temperature 43.0 degC, Fe₃ O₄      

Characterization of minor site probes for human serum albumin by high-performance affinity chromatography.

This study used high-performance affinity chromatography (HPAC) and immobilized human serum albumin (HSA) columns to examine the specificity and cross-reactivity of various compounds that have been proposed as markers for the minor binding sites of HSA. These agents included acetyldigitoxin and digitoxin as probes for the digitoxin site, phenol red as a probe for the bilirubin site, and cisor trans-clomiphene as markers for the tamoxifen site. None of these probes showed any significant binding at HSA's indole-benzodiazepine site. However, phenol red did bind at the warfarin-azapropazone site of HSA, and cis/trans-clomiphene gave positive allosteric effects caused by the binding of warfarin to HSA. Digitoxin and acetyldigitoxin were found to bind to a common, unique region on HSA; cis- and trans-clomiphene also appeared to interact at a unique site, although trans-clomiphene displayed additional direct competition with phenol red. From these results it was possible to develop a model that described the general relationship between these binding regions on HSA. This information should be useful in future studies that employ HPAC for characterizing the binding of HSA to other drugs or clinical agents.     

25th Anniversary Article: Interfacing Nanoparticles and Biology: New Strategies for Biomedicine

The exterior surface of nanoparticles (NPs) dictates the behavior of these systems with the outside world. Understanding the interactions of the NP surface functionality with biosystems enables the design and fabrication of effective platforms for therapeutics, diagnostics, and imaging agents. In this review, we highlight the role of chemistry in the engineering of nanomaterials, focusing on the fundamental role played by surface chemistry in controlling the interaction of NPs with proteins and cells.     

Development of Drug Delivery Systems from Vegetal Proteins: Legumin Nanoparticles

Legumin (storage protein from Pisum sativum L.) nanoparticles of about 250 nm were prepared by means of a pH-coacervation method and chemical cross-linking with glutaraldehyde. This preparative method enabled to avoid the use of organic solvents but only yielded about 27% of protein added as nanoparticles. No significant differences in size, percentage yield, and surface charge were obtained between legumin nanoparticles cross-linked with different glutaraldehyde concentrations. Legumin nanoparticles were quite stable in phosphate-buffered saline (PBS). They follow a zero-order degradation, and by increasing glutaraldehyde concentration, a longer half-life (t₅₀) was obtained. The amount of methylene blue (MB), used as a model of hydrophilic drug, loaded was about 6.2% of the initial dye. Its release from the nanoparticles consisted of a rapid initial phase followed by a slower second period. The rates in this second phase were inversely related to the degree of cross-linking.     

Fabrication of a nanocarrier system through self-assembly of plasma protein and its tumor targeting

Human serum albumin (HSA) nanoparticles hold great promise as a nanocarrier system for targeted drug delivery. The objective of this study was to explore the possibility of preparing size controllable albumin nanoparticles using the disulfide bond breaking reagent β-mercaptoethanol (β-ME). The results showed that the protein concentration and temperature had positive effects on the sizes of the albumin nanoparticles, while pH had a negative effect on the rate of nanoparticle formation. The addition of β-ME induced changes in HSA secondary structure and exposed the hydrophobic core of HSA, leading to the formation of nanoparticles. Human serum albumin nanoparticles could be internalized by MCF-7 cells and mainly accumulated in cytoplasm. After injection in tumor bearing mice, the HSA nanoparticles accumulated in tumor tissues, demonstrating the targeting ability of the nanoparticles. Therefore, human serum albumin can be fabricated into nanoparticles by breaking the disulfide bonds and these nanoparticles exhibit high tumor targeting ability. Human serum albumin nanoparticles could be ideal for the targeted delivery of pharmacologically active substances.     

Multifunctional Heterogeneous Carbon Nanotube Nanocomposites Assembled by DNA‐Binding Peptide Anchors

Although carbon nanotubes (CNTs) are remarkable materials with many exceptional characteristics, their poor chemical functionality limits their potential applications. Herein, a strategy is proposed for functionalizing CNTs, which can be achieved with any functional group (FG) without degrading their intrinsic structure by using a deoxyribonucleic acid (DNA)‐binding peptide (DBP) anchor. By employing a DBP tagged with a certain FG, such as thiol, biotin, and carboxyl acid, it is possible to introduce any FG with a controlled density on DNA‐wrapped CNTs. Additionally, different types of FGs can be introduced on CNTs simultaneously, using DBPs tagged with different FGs. This method can be used to prepare CNT nanocomposites containing different types of nanoparticles (NPs), such as Au NPs, magnetic NPs, and quantum dots. The CNT nanocomposites decorated with these NPs can be used as reusable catalase‐like nanocomposites with exceptional catalytic activities, owing to the synergistic effects of all the components. Additionally, the unique DBP–DNA interaction allows the on‐demand detachment of the NPs attached to the CNT surface; further, it facilitates a CNT chirality‐specific NP attachment and separation using the sequence‐specific programmable characteristics of oligonucleotides. The proposed method provides a novel chemistry platform for constructing new functional CNTs suitable for diverse applications.     

Enhanced cellular uptake of folic acid-conjugated PLGA-PEG nanoparticles loaded with vincristine sulfate in human breast cancer

The aim of this paper is to evaluate the cellular uptake of vincristine sulfate-loaded poly(lactic-co-glycolic acid)-polyethylene glycol (PLGA-PEG) nanoparticles with the folic acid modification (PLGA-PEG-folate NPs). PLGA-PEG-folate NPs were prepared using a water-oil-water emulsion solvent evaporation method. The particle size, surface morphology, drug encapsulation efficiency, and the drug release behavior were investigated. The NPs exhibited a biphasic drug release with a moderate initial burst followed by a sustained release profile. Internalization of the NPs labeled with coumarin- 6 by MCF-7 (Michigan Cancer Foundation-7) human breast cancer cells was quantitatively measured by microplate reader, and qualitatively analyzed by fluorescent microscopy and confocal laser scanning microscopy. The results showed PLGA-PEG-folate NPs achieved significantly higher cellular uptake in the folic acid receptor overexpressed MCF-7 cells, compared to PLGA-mPEG NPs without the folic acid modification. Due to the enhanced cellular uptake, PLGA-PEG-folate NPs displayed the highest cytotoxicity. Judged by IC(50) after 24 h culture, the therapeutic effects of the drug formulated in the NPs with surface modification could be 1.52 times, 3.91 times higher than that of PLGA-mPEG NPs and free vincristine sulfate, respectively.     

Controlled surface functionalization of silica nanospheres by covalent conjugation reactions and preparation of high density streptavidin nanoparticles

Silica nanoparticles with a diameter of 100 nm were covalently modified at their surface by adjustable amounts of amine and carboxyl functional groups. Bioconjugation studies of two proteins, streptavidin and streptactin, with the functional nanoparticles resulted in optimum binding of the proteins to a long-chain carboxyl-terminated linker. The surface functionalization of the nanoparticles was monitored by a variety of independent methods, including zeta-potential measurements, dynamic light scattering (DLS), scanning electron microscopy (SEM), particle charge detection titrations (PCD) and elemental analysis. At the surface of the nanoparticles, a functional surface group density of 1.8 amino groups per nm2 was realized. The amine functions were quantitatively transferred to carboxyl groups coupled with a linker elongation. Streptavidin was immobilized by covalent binding to the carboxyl linkers and resulted in a protein density at the surface of the nanoparticles that was three times higher than the highest binding densities at nanoparticles published to date. The binding capacity of the streptavidin-covered nanoparticles for ligand biotin was quantified by titration with biotin-4-fluorescein to 2.5 biotin binding sites per 100 nm2.     

Quinolino-triazole linked gold nanoparticles as sensitive 'turn-on' fluorescent Cd(2+) probes

Quinoline derivatives were brought into the surface of gold nanoparticles (Au NPs) through click chemistry. The fluorescence was quenched by Au NPs because of electron transfer between Au NPs and quinoline. However, upon addition of Cd(2+) to the quinoline-triazole Au NP solution, it exhibited an effective switch-on fluorescence response, owing to the coordination between quinoline and Cd(2+) which can efficiently block the electron transfer. What's more, the fluorescent sensor can effectively detect Cd(2+) in aqueous solution with a detection limit of 1.0 × 10(-5) M.     

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.     

Synthesis and characterization of gelatin nanoparticles using CDI/NHS as a non-toxic cross-linking system

Gelatin nanoparticles, cross-linked by a mixture of a water soluble carbodiimide (CDI) and N-hydroxysuccinimide (NHS) as a non-toxic cross-linking system, was prepared. The conventional two step desolvation method with acetone as the non-solvent was used. The mean size and size distribution as well as the morphology of the formed nanoparticles were evaluated and compared with those of nanoparticles cross-linked by glutaraldehyde (GA) as the most commonly used cross-linking agent. Furthermore, intrinsic viscosities of the nanoparticles cross-linked by CDI/NHS and GA were measured and compared under various conditions. The results showed the formation of smoother and more homogeneous nanoparticles with smaller size when CDI/NHS used as cross-linking agent under the same synthesis condition. Moreover, nanoparticles encapsulating paracetamol as a model drug were produced by the two different cross-linking agents and were characterized for drug entrapment and loading efficiencies and in vitro drug release. Both drug entrapment and loading efficiencies was higher in the CDI/NHS cross-linked nanoparticles; however, the release kinetics was comparable to that of nanoparticles cross-linked with GA. The differences in the characteristics of CDI/NHS and GA cross-linked nanoparticles were attributed to the different nature of network structures formed by the two cross-linking agents. On the whole, these results suggested that CDI/NHS cross-linked nanoparticles have high potential to be used for drug delivery application in preference to the nanoparticles synthesized by toxic cross-linking agents.     

A Self-Disguised Nanospy for Improving Drug Delivery Efficiency via Decreasing Drug Protonation

Nanoscale drug carriers play a crucial role in reducing side effects of chemotherapy drugs. However, the mononuclear phagocyte system (MPS) and the drug protonation after nanoparticles (NPs) burst release still limit the drug delivery efficiency. In this work, a self-disguised Nanospy is designed to overcome this problem. The Nanospy is composed of: i) poly (lactic-co-glycolic acid)-polyethylene glycol (PLGA-PEG) loading doxorubicin is the core structure of the Nanospy. ii) CD47 mimic peptides (CD47p) is linked to NPs which conveyed the “don't eat me” signal. iii) 4-(2-aminoethyl) benzenesulfonamide (AEBS) as the inhibitor of Carbonic anhydrase IX (CAIX) linked to NPs. Briefly, when the Nanospy circulates in the bloodstream, CD47p binds to the regulatory protein α (SIRPα) on the surface of macrophages, which causes the Nanospy escapes from phagocytosis. Subsequently, the Nanospy enriches in tumor and the AEBS reverses the acidic microenvironment of tumor. Due to above characteristics, the Nanospy reduces liver macrophage phagocytosis by 25% and increases tumor in situ DOX concentration by 56% compared to PLGA@DOX treatment. In addition, the Nanospy effectively inhibits tumor growth with a 63% volume reduction. This work presents a unique design to evade the capture of MPS and overcomes the influence of acidic tumor microenvironment (TME) on weakly alkaline drugs.