Polyelectrolyte Adsorption Research Articles

Drug Release Properties of Diflunisal from Layer-By-Layer Self-Assembled κ-Carrageenan/Chitosan Nanocapsules: Effect of Deposited Layers.

Engineering of multifunctional drug nanocarriers combining stability and good release properties remains a great challenge. In this work, natural polymers κ-carrageenan (κ-CAR) and chitosan (CS) were deposited onto olive oil nanoemulsion droplets (NE) via layer-by-layer (LbL) self-assembly to study the release mechanisms of the anti-inflammatory diflunisal (DF) as a lipophilic drug model. The nano-systems were characterized by dynamic light scattering (DLS), zeta potential (ζ-potential) measurements, transmission electron microscopy (TEM), scanning electron microscopy (SEM), X-ray energy dispersive spectroscopy (XEDS) and Fourier transform infrared spectroscopy (FTIR) to confirm the NE-coating with polymer layers. In addition, kinetic release studies of DF were developed by the dialysis diffusion bag technique. Mathematical models were applied to investigate the release mechanisms. The results showed that stable and suitably sized nanocapsules (~300 nm) were formed. Also, the consecutive adsorption of polyelectrolytes by charge reversal was evidenced. More interestingly, the drug release mechanism varied depending on the number of layers deposited. The nanosized systems containing up to two layers showed anomalous transport and first order kinetics. Formulations with three and four layers exhibited Case II transport releasing diflunisal with zero order kinetics. Hence, our results suggest that these polyelectrolyte nanocapsules have great potential as a multifunctional nanocarrier for drug delivery applications.

Novel magneto-responsive nanoplatforms based on MnFe2O4 nanoparticles layer-by-layer functionalized with chitosan and sodium alginate for magnetic controlled release of curcumin

Remotely assisted drug delivery by means of magnetic biopolymeric nanoplatforms has been utilized as an important tool to improve the delivery/release of hydrophobic drugs and to address their low cargo capacity. In this work, MnFe2O4 magnetic nanoparticles (MNPs) were synthesized by thermal decomposition, coated with citrate and then functionalized with the layer-by-layer (LbL) assembly of polyelectrolyte multilayers, with chitosan as polycation and sodium alginate as polyanion. Simultaneous conductimetric and potentiometric titrations were employed to optimize the LbL deposition and to enhance the loading capacity of nanoplatforms for curcumin, a hydrophobic drug used in cancer treatment. ~200 nm sized biopolymer platforms with ~12 nm homogeneously embedded MNPs were obtained and characterized by means of XRD, HRTEM, DLS, TGA, FTIR, XPS and fluorescence spectroscopy techniques to access structural, morphological and surface properties, to probe biopolymer functionalization and to quantify drug-loading. Charge reversals (±30 mV) after each deposition confirmed polyelectrolyte adsorption and a stable LbL assembly. Magnetic interparticle interaction was reduced in the biopolymeric structure, hinting at an optimized performance in magnetic hyperthermia for magneto-assisted drug release applications. Curcumin was encapsulated, resulting in an enhanced payload (~100 μg/mg). Nanocytotoxicity assays showed that the biopolymer capping enhanced the biocompatibility of nanoplatforms, maintaining entrapped curcumin. Our results indicate the potential of synthesized nanoplatforms as an alternative way of remotely delivering/releasing curcumin for medical purposes, upon application of an alternating magnetic field, demonstrating improved efficiency and reduced toxicity.

Strong cationic polyelectrolyte adsorption on a water swollen cellulosic biomaterial and its relevance on microstructure and rheological properties

A multiscale approach was taken to study the swelling and viscosifying properties of a cellulose-rich biomaterial as a function of environmental conditions. First, the impact of pH and ionic strength on the surface properties of the fiber was investigated by means of potentiometry and cationic polyelectrolyte adsorption. Three adsorption regimes were identified: the first one occurred in acidic conditions and was related to the ionization of the carboxylic groups of pectin. A very sharp adsorption was then observed in the mild-acidic to neutral region which was most likely related to a conformation change in the plant cell wall architecture. The third adsorption regime happened in the alkaline region and was related to ionization effects. Based on this information, the impact of environmental conditions on the hydrated microstructure was studied in DSC by means of thermoporosimetry. It clearly demonstrated a structural collapse of the hydrated fiber when pH decreased or the amounts of ions increased. This phenomenon was in line with the surface charge measurements. Finally, the link between the wet microstructure and the macroscale behavior of the fiber was demonstrated by studying the flow properties of the reconstituted fiber, below and above its percolation threshold. In line with the surface properties and microstructure data, yield stresses increased when the pH was adjusted from acidic to alkaline conditions. Salt addition resulted in a loss of the texturizing properties.

Inorganic Crystallization Engineered by the Dynamic Adsorption of Linear and Particulate Polyelectrolytes

For most of natural hybrid materials, the shape of mineral crystals is controlled by natural polymers (e.g., proteins) to provide designated and unique physical properties and functions. Synthetic hybrid materials, which show comparable performance to the natural ones, require well-controlled crystal growth. In particular, the dynamic process including adsorption of the polymers on the crystal surface plays a significant role. We investigated CaSO4·0.5H2O crystallization in the presence of linear or particulate charged macromolecules, which can interact electrostatically with the crystals. Not only the static factors such as concentration and charge fraction but also the diffusion dynamics of the charged macromolecules were found crucial to involve the complete inhibition of growth and the subsequent shape transition of the crystals. This leads to the elucidation of semiquantitative relationships between dynamic factors such as the diffusion of macromolecules and the rate of crystal growth to the growth i...

Layer-by-layer hollow photosensitizer microcapsule design via a manganese carbonate hard template for photodynamic therapy in cells

Microcapsules fabricated using layer-by-layer self-assembly have unique properties, making them attractive for drug delivery applications. The technique has been improved, allowing the deposition of multiple layers of oppositely charged polyelectrolytes on spherical, colloidal templates. These templates can be decomposed by coating multiple layers, resulting in hollow shells. In this paper, we describe a novel drug delivery system for loading photosensitizer drugs into hollow multilayered microcapsules for photoprocess applications. Manganese carbonate particles were prepared by mixing NH4HCO3 and MnSO4 and performing consecutive polyelectrolyte adsorption processes onto these templates using poly-(sodium 4-styrene sulfonate) and poly-(allylamine hydrocholoride). A photosensitizer was also incorporated into the layers. Hollow spheres were fabricated by removing the cores in the acidic solution. The hollow, multilayered microcapsules were studied by scanning electron microscopy, steady-state, and time-resolved techniques. Their biological activity was evaluated in vitro with cancer cells using a conventional MTT assay. The synthesized CaCO3 microparticles were uniform, non-aggregated, and highly porous spheres. The phthalocyanine derivatives loaded in the microcapsules maintained their photophysical behaviour after encapsulation. The spectroscopic results presented here showed excellent photophysical behaviour of the studied drug. We observed a desirable increase in singlet oxygen production, which is favourable for the PDT protocol. Cell viability after treatment was determined and the proposed microcapsules caused 80% cell death compared to the control. The results demonstrate that photosensitizer adsorption into the CaCO3 microparticle voids together with the layer-by-layer assembly of biopolymers provide a method for the fabrication of biocompatible microcapsules for use as biomaterials.

Modified carbons for enhanced nucleophilic substitution reactions of adsorbed methyl bromide

Activated carbons are highly-effective adsorbents for the passive removal of organic pollutants from aqueous and gaseous phases. However, the pollutant remains chemically unchanged and poorly available to dissolved reagents. In this study, we developed quaternary ammonium (QA)-modified carbon adsorbent-catalysts that can catalyze direct reactions between adsorbed molecules and anionic reagents, thereby permitting a trap-and-degrade strategy. The concept was applied to base hydrolysis of methyl bromide (CH3Br, MeBr), a quarantine and pre-shipment fumigant of concern as an ozone-depleting gas. Modifications of the carbons included irreversible adsorption of the cationic polyelectrolyte, poly (diallyldimethylammonium chloride) (PDADMAC), and covalent grafting of QA groups via precursors (Quat188 and Quab360). In mixtures of the carbon in 1 M NaOH at 55 °C under conditions where hydrolysis in the aqueous phase was negligible, first-order rate constants were increased by up to a factor of 7.4 compared to the unmodified carbon. Combined PDADMAC/Quat188 modification was superior to the corresponding individual modification. Rate acceleration was due to attraction of hydroxide ions to the newly-created anion exchange sites. This is supported by effects of QA modification on anion exchange capacity, point of zero charge, and isoelectric point, as well as by the results of competition experiments with otherwise-inert monovalent anions. The modified catalysts are robust for at least five hydrolysis cycles. QA modification also greatly enhances hydrolysis at unadjusted pH (8.6–10.2) and nucleophilic reaction by thiosulfate. QA modification of carbons is a promising approach for in situ trapping and degrading MeBr or other hydrophobic compounds that are reactive toward anionic reagents.

Automated self-assembly and electrical characterization of nanostructured films

Abstract

Modulation of Polyelectrolyte Adsorption on Nanoparticles and Nanochannels by Surface Curvature

This paper presents theoretical results on the adsorption of polyelectrolyte chains on surfaces with opposite charge and nanoscale curvature. The theory predicts that increasing the surface curvature can either increase or decrease the amount of adsorbed polyelectrolyte, depending on the type of curvature (convex or concave) and whether the polyelectrolyte undercompensates or overcompensates the initial charge of the substrate. For small bulk salt concentration (10–4 M), increasing the curvature of the surface displaces the adsorption equilibrium of the polyelectrolyte in order to decrease the absolute value of the effective charge density for concave surfaces (nanochannels) or to increase it for convex surfaces (nanoparticles). This behavior is traced back to the dependence of the total free energy as a function of the curvature of the surface. For intermediate salt concentrations (0.01–0.1 M), the magnitude of the effect is larger than that for low salt concentrations, although the general picture becom...

Adsorption of Polyelectrolyte onto Nanosilica Synthesized from Rice Husk: Characteristics, Mechanisms, and Application for Antibiotic Removal.

Adsorption of the polyelectrolyte polydiallyldimethylammonium chloride (PDADMAC) onto nanosilica (SiO2) fabricated from rice husk was studied in this work. Nanosilica was characterized by X-ray diffraction, Fourier-transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). Adsorption of PDADMAC onto SiO2 increased with increasing pH because the negative charge of SiO2 is higher at high pH. Adsorption isotherms of PDADMAC onto silica at different KCl concentrations were fitted well by a two-step adsorption model. Adsorption mechanisms of PDADMAC onto SiO2 are discussed on the basis of surface charge change, evaluation by ζ potential, surface modification by FTIR measurements, and the adsorption isotherm. The application of PDADMAC adsorption onto SiO2 to remove amoxicillin antibiotic (AMX) was also studied. Experimental conditions such as contact time, pH, and adsorbent dosage for removal of AMX using SiO2 modified with PDADMAC were systematically optimized and found to be 180 min, pH 10, and 10 mg/mL, respectively. The removal efficiency of AMX using PDADMAC-modified SiO2 increased significantly from 19.1% to 92.3% under optimum adsorptive conditions. We indicate that PDADMAC-modified SiO2 rice husk is a novel adsorbent for removal of antibiotics from aqueous solution.

Study of the Liquid/Vapor Interfacial Properties of Concentrated Polyelectrolyte–Surfactant Mixtures Using Surface Tensiometry and Neutron Reflectometry: Equilibrium, Adsorption Kinetics, and Dilational Rheology

The adsorption of mixtures of poly(diallyldimethylammonium chloride) (PDADMAC) and sodium N-lauroyl-N-methyltaurate (SLMT) at the water / vapor interface has been studied using drop profile tensiometry and neutron reflectometry. This study sheds light on the mechanisms involved in the adsorption of polyelectrolyte – oppositely charged surfactants by the characterization of both equilibrium and dynamics features associated with the layer formation at the fluid interface. The results are discussed in terms of an adsorption – equilibration of the interfacial layers as a two-step process: the initial stages involve the adsorption of polyelectrolyte – surfactant complexes formed in the bulk, and a subsequent stage involves reorganization of the interface. This work contributes to the understanding of the physico-chemical features of systems that undergo complex bulk and interfacial interactions with importance in science and technology.

Controlling Polyelectrolyte Adsorption onto Carbon Nanotubes by Tuning Ion-Image Interactions.

Understanding and controlling polyelectrolyte adsorption onto carbon nanotubes is a fundamental challenge in nanotechnology. Polyelectrolytes have been shown to stabilize nanotube suspensions through adsorbing onto the nanotube surface, and polyelectrolyte-coated nanotubes are emerging as building blocks for complex and addressable self-assembly. Conventional wisdom suggests that polyelectrolyte adsorption onto nanotubes is driven by specific chemical or van der Waals interactions. We develop a simple mean-field model and show that ion-image attraction significantly effects adsorption onto conducting nanotubes at low salt concentrations. Our theory suggests a simple strategy to selectively and reversibly functionalize carbon nanotubes on the basis of their electronic structures, which in turn modify the ion-image attraction.

The Effect of Surface Charge on Adsorption of a Cationic Polyelectrolyte

The regularities of adsorption of a cationic polyelectrolyte, poly(diallyldimethylammonium chloride), on the surface of fused quartz are studied at different values of solution pH by capillary electrokinetics. It is shown that the polyelectrolyte adsorption on a negatively charged surface depends on the value of the surface charge and increases with its growth. At a low charge value (pH 3.8), the polyelectrolyte adsorption increases the quartz surface charge. The driving forces of the adsorption are both electrostatic interaction and forces of nonelectrostatic nature, probably hydrophobic interactions and a change in entropy due to the displacement of counterions from a double layer. The adsorption of poly(diallyldimethylammonium chloride) on quartz from alkaline and neutral solutions is irreversible, which indicates the key role of the electrostatic interaction. At low values of the surface charge, the nonelectrostatic interactions play the main role, thereby resulting in polyelectrolyte desorption.

Adsorption of charged macromolecules upon multicomponent responsive surfaces.

Adsorption of polyions onto charged surfaces has long been recognized as a crucial phenomenon in biological and technological applications. An intuitive model relating polyelectrolyte adsorption with the imposed features of polarizable surfaces of different compositions and charges is proposed based on Monte Carlo simulations using a coarse-grained approach. The excellent performance of the equation allows simultaneously describing a wide range of adsorption regimes and accounting for specific non-monotonic trends. For a constant surface charge density, the surface composition governs adsorption, promoting variations exceeding 100%. Adsorption increases with the number of attractive charges in the surface until reaching a maximum, decreasing thereafter due to the presence of polyanion-like charged particles. The presence of crowders hampers adsorption. These results can be used to efficiently predict and modulate the interaction between charged macromolecules and different substrates with direct implications in de novo designs of vehicles and biomedical devices.

Angular scanning and variable wavelength surface plasmon resonance allowing free sensor surface selection for optimum material- and bio-sensing

A variable-wavelength Kretschmann configuration surface plasmon resonance (SPR) apparatus with angle scanning is presented. The setup provides the possibility of selecting the optimum wavelength with respect to the properties of the metal layer of the sensorchip, sample matrix, and biomolecular interaction of interest. Monitoring SPR curves over a wide angular range (39°) permits simultaneous determination of the total internal reflection angle (TIR), the resonance angle, and the intensity and width of the SPR dip, which are essential parameters for measuring binding events and achieving optimum sensitivity. The new apparatus was evaluated by recording full SPR curves at different wavelengths ranging from 600 to 890 nm using sensor surfaces of silver, gold and gold with deposited silicon oxide, aluminum oxide, titanium oxide and indium tin oxide which were exposed to air and an aqueous solution of sodium chloride. Clear wavelength dependencies of sensor-material resonance angles and SPR-dip widths were demonstrated. In order to investigate the capability of the system to probe molecular binding to different sensor surfaces, the layer-by-layer adsorption of charged polyelectrolytes was monitored in angular scanning mode at 600, 670, 785, and 890 nm. Although at longer wavelengths lower angular shifts were observed as result of layer deposition, the sharper dip, wider detection window and better signal-to-noise ratios at these wavelengths can be beneficial for binding studies. The applicability for biosensing was tested by immobilizing human serum albumin (HSA) on an aluminum-oxide-coated gold sensor using a new procedure and measuring the binding of anti-HSA antibodies at the optimal wavelength (890 nm) in angular-scanning and fixed-angle mode. The HSA biosensor showed negligible non-specific interaction and yielded almost ten times better sensitivity than obtained with a conventional gold-dextran-based sensor operated at 670/785 nm. Analysis of anti-HSA samples pre-incubated with different concentrations of HSA allowed measurement of the IC50 value. The reported data demonstrate the usefulness of the presented variable-wavelength angle-scanning SPR instrument, permitting continuous recording of full SPR curves in time at any selected wavelength in the 600–890 nm range using a sensor material of choice.

Effect of nanoparticles size and polyelectrolyte on nanoparticles aggregation in a cellulose fibrous matrix

Controlling nanoparticles (NPs) aggregation in cellulose/NPs composites allows to optimise NPs driven properties and their applications. Polyelectrolytes are used to control NPs aggregation and their retention within the fibrous matrix. Here, we aim at evaluating how a polyelectrolyte (Cationic Polyacrylamide; CPAM, molecular weight: 13MDa, charge: 50%, Radius of gyration: 30–36nm) adsorbs and re-conforms onto the surface of silica(SiO2) NPs differing in diameter (8, 22 and 74nm) and to investigate the respective NPs aggregation in cellulose matrices.SEM shows the local area distribution of NPs in composites. Ultra-SAXS (USAXS) allows to evaluate the average NPs size distribution and the inter-particle interactions at length scale ranging from 1 to 1000nm.USAXS data analysis reveals that CPAM covers multiple NPs of the smaller diameter (8nm), presumably with a single chain to form large size NPs aggregates. As the NPs diameter is increased to 22nm, CPAM re-conforms over NP surface forming a large shell of thickness 5.5nm. For the composites with NPs of diameter 74nm, the CPAM chain re-conforms further onto NP surface and the surrounding shell thickness decreases to 2.2nm. Structure factor analysis reveals higher structural ordering for NPs as increases their diameter, which is caused by different conformations adopted by CPAM onto NPs surface.

Modeling of Polyelectrolyte Adsorption from Micellar Solutions onto Biomimetic Substrates.

Depositing cationic polyelectrolytes (PEs) from micellar solutions that include surfactants (SU) onto surfaces is a rich, complex, highly relevant, and challenging topic that covers a broad field of practical applications (e.g., from industrial to personal care). The role of the molecular architecture of the constituents of the PEs are often overruled, or at least and either, underestimated in regard to the surface properties. In this work, we aim to evaluate the effect of a model biomimetic surface that shares the key characteristics of the extreme surface of hair and its concomitant chemo- and physisorbed properties onto the deposition of a complex PEs:SU system. To tackle out the effect of the molecular architecture of the PEs, we consider (i) a purely linear and hydrophilic PE (P100) and (ii) a PE with lateral amphiphilic chains (PegPE). Using numerical self-consistent field calculations, we show that the architecture of the constituents interfere with the surface properties in a nonintuitive way such that, depending on the amphiphilicity and hydrophilicity of the PEs and the hydrophobicity of the surface, a re-entrant adsorbing transition can be observed, the lipid coverage of the model hair surface being the unique control parameter. Such a behavior is rationalized by the anticooperative associative properties of the coacervate micelles in solution, which is also controlled by the architecture of the PEs and SU. We now expect that PEs adsorption, as a rule, is governed by the molecular details of the species in solution as well as the surface specificities. We emphasize that molecular realistic modeling is essential to rationalize and optimize the adsorption process of, for example, polymer conditioning agents in water-rinsed cosmetic or textile applications.

Layered double hydroxide nanoparticles customization by polyelectrolyte adsorption: mechanism and effect on particle aggregation

Adsorption of polyelectrolytes (PEs) is a common strategy for stabilizing nanoparticles and customizing their properties. Although scarcely explored for layered double hydroxide nanoparticles (LDH-NPs), it can be used to increase their functionality in pharmaceutical and biomedical applications, among others. In this work, LDH-NPs intercalated with chloride, ibuprofen and ketoprofen were synthesized and modified with carboxylate containing PEs with different structures and physicochemical properties. The PEs adsorption mechanism on LDH-NPs and its effect on the aggregation of the obtained hybrids were studied by zeta potential and hydrodynamic diameter determinations on chloride intercalated LDH-NPs dispersions in the presence of polyacrylate (PA) at different concentrations. PA adsorption behavior was compared to that of citrate anion to contrast the adsorption mechanism of PEs and anions. Afterwards, the study was extended to other PEs (alginate and carbomer) as well as to drug intercalated LDH-NPs. PA adsorbed at the surface of LDH-NPs with high affinity: at low concentrations, PA was irreversibly adsorbed due to the positive structural charge of LDH-NPs and the flexible and negatively charged structure of PA. This high affinity caused a particle charge inversion, which results in negatively charged LDH-NPs stabilized by both electrostatic interactions and steric hindrance. Due to their structure, alginate and carbomer showed a lower affinity and, consequently, a lower capacity to produce LDH-NPs disaggregation. Finally, PEs adsorption allowed enhancing the disaggregation and modifying the interfacial properties of both ibuprofen and ketoprofen intercalated LDH-NPs producing only a small release of the drug loading.

Influence of Protamine Functionalization on the Colloidal Stability of 1D and 2D Titanium Oxide Nanostructures.

The colloidal stability of titanium oxide nanosheets (TNS) and nanowires (TiONW) was studied in the presence of protamine (natural polyelectrolyte) in aqueous dispersions, where the nanostructures possessed negative net charge, and the protamine was positively charged. Regardless of their shape, similar charging and aggregation behaviors were observed for both TNS and TiONW. Electrophoretic experiments performed at different protamine loadings revealed that the adsorption of protamine led to charge neutralization and charge inversion depending on the polyelectrolyte dose applied. Light scattering measurements indicated unstable dispersions once the surface charge was close to zero or slow aggregation below and above the charge neutralization point with negatively or positively charged nanostructures, respectively. These stability regimes were confirmed by the electron microscopy images taken at different polyelectrolyte loadings. The protamine dose and salt-dependent colloidal stability confirmed the presence of DLVO-type interparticle forces, and no experimental evidence was found for additional interactions (e.g., patch-charge, hydrophobic, or steric forces), which are usually present in similar polyelectrolyte-particle systems. These findings indicate that the polyelectrolyte adsorbs on the TNS and TiONW surfaces in a flat and extended conformation giving rise to the absence of surface heterogeneities. Therefore, protamine is an excellent biocompatible candidate to form smooth surfaces, for instance in multilayers composed of polyelectrolytes and particles to be used in biomedical applications.

Reconciling DLVO and non-DLVO Forces and Their Implications for Ion Rejection by a Polyamide Membrane.

Recognizing the significance of surface interactions for ion rejection and membrane fouling in nanofiltration, we revise the theories of DLVO (named after Derjaguin, Landau, Verwey, and Overbeek) and non-DLVO forces in the context of polyamide active layers. Using an atomic force microscope, surface forces between polyamide active layers and a micrometer-large and smooth silica colloid were measured in electrolyte solutions of representative monovalent and divalent ions. While the analysis of DLVO forces, accounting for surface roughness, provides how surface charge of the active layer changes with electrolyte concentration, scrutiny of non-DLVO hydration forces gives molecular insight into the composition of the membrane-solution interface. Importantly, we report an expansion of the diffuse layer at high ionic strength, consistent with the recent development of the electrical double layer theory, but in contrast to the widely accepted phenomenon of aggregation in the secondary minimum. Further, the enhanced repulsion acting on modified membranes via polyelectrolyte adsorption can be quantitatively predicted by DLVO and non-DLVO forces. This work serves to solve past misunderstandings about the interaction forces acting on nanofiltration membranes, and it provides guidance for future work on the relation between surface properties and rejection mechanisms and fouling.

The interaction of clozapine loaded nanocapsules with the hCMEC/D3 cells – In vitro model of blood brain barrier

Despite progress in the development of novel pharmacological compounds, their efficacy in the treatment of neuropathologies is not satisfactory. One strategy to achieve safe and efficient brain targeting therapy is to design nanocarriers capable of transporting antipsychotic drugs through the BBB (without affecting the normal functions of the barrier) in a defined part of the brain. Here we investigate the interaction of clozapine-loaded polymeric Nano capsules (CLO-NCs) with hCMEC/D3 (human cerebral microvascular endothelial cells, D3 clone) cells that constitutes an in vitro model of the blood brain barrier (BBB). CLO-NCs (average size of 100nm) were constructed by the technique of sequential adsorption of polyelectrolytes (LbL), using biocompatible polyanion PGA (Poly-l-glutamic acid sodium salt) and polycation PLL (poly L-lysine) on clozapine-loaded nano-emulsion cores. Pegylated external layers were prepared using PGA-g(39)-PEG (PGA grafted by PEG poly(ethylene glycol)). The influence of the physicochemical properties of the CLO-NCs (charge, size, surface modification) on cell viability was determined. Advanced studies of CLO-NCs internalization (including endocytosis and transcytosis experiments) using confocal microscopy, flow cytometry and fluorescence spectroscopy are presented. Our results indicate that among the studied NCs, the pegylated clozapine-loaded NCs were the most protected from their uptake by macrophages, and they were the least toxic to hCMEC/D3 cells. They were also the most efficient in the transcytosis experiment, which serves as an indicator of their ability to cross a model BBB.