Morphology and Mechanics of Star Copolymer Ultrathin Films Probed by Atomic Force Microscopy in the Air and in Liquid.

Star copolymer films were produced by using spin-coating, drop-casting, and casting deposition techniques, thus obtaining ultrathin and thick films, respectively. The morphology is generally flat, but it becomes substrate-dependent for ultrathin films where the planarization effect of films is not efficient. The indentation hardness of films was investigated by Force Volume Maps in both the air and liquid. In the air, ultrathin films are in the substrate-dominated zone and, thus, the elastic modulus E is overestimated, while E reaches its bulk value for drop-casted ultrathin and thick films. In liquid (water), E follows an exponential decay for all films with a minimum soaked time t0 of 0.37 and 2.65 h for ultrathin and drop-casted ultrathin and thick films, respectively. After this time, E saturates to a value on average 92% smaller than that measured in the air due to film swelling. Such results support the role of film morphology in the antimicrobial activity envisaged in the literature, suggesting also an additional role of film hardness.

Inherently Antimicrobial P(MMA-ran-DMAEMA) Copolymers Sensitive to Photodynamic Therapy: A Double Bactericidal Effect for Active Wound Dressing.

In this work, two compounds belonging to the BODIPY family, and previously investigated for their photosensitizing properties, have been bound to the amino-pendant groups of three random copolymers, with different amounts of methyl methacrylate (MMA) and 2-(dimethylamino)ethyl methacrylate (DMAEMA) in the backbone. The P(MMA-ran-DMAEMA) copolymers have inherently bactericidal activity, due to the amino groups of DMAEMA and to the quaternized nitrogens bounded to BODIPY. Systems consisting of filter paper discs coated with copolymers conjugated to BODIPY were tested on two model microorganisms, Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). On solid medium, irradiation with green light induced an antimicrobial effect, visible as a clear inhibition area around the coated disks. The system based on the copolymer with 43% DMAEMA and circa 0.70 wt/wt% of BODIPY was the most efficient in both bacterial species, and a selectivity for the Gram-positive model was observed, independently of the conjugated BODIPY. A residual antimicrobial activity was also observed after dark incubation, attributed to the inherently bactericidal properties of copolymers.

Hirudo verbana as a freshwater invertebrate model to assess the effects of polypropylene micro and nanoplastics dispersion in freshwater.

Plastics are a heterogeneous class of synthetic compounds that, due to their unique characteristics find numerous applications both in industrial and civil fields. However, despite the great advantages that these materials brought in everyday life, the plastic wastes resulting from their massive use represent one of the main environmental problems at the global level. Once released, plastics persist for a long time and are subjected both to biotic and abiotic processes leading to the formation of small particles, known as micro and to nanoplastics, that interact with organisms, accumulating inside tissues and risking to enter in the trophic chain. Among the different types of plastic, polypropylene (PP) is one of the diffused, widely exploited in food and textile industries for disposable packaging and to produce surgical masks. Owing to the huge distribution and the resultant abundant presence of PP waste products, it results necessary investigate the possible toxicity on living organisms. For these reasons, here we analyzed the effects of PP micro and nanoplastics dispersed in freshwater, using the medicinal leech Hirudo verbana as invertebrate model. To better follow the plastics fate, fluorescent particles, labeled with a fluorophore, have been used. Animals were examined at various timings after plastics exposure and results were analyzed by means of microscopy, immunofluorescent and molecular biology analyses. After assessing the entrance of PP fragments into leech tissues, the activation of the innate immune response was evaluated. The results show that the presence of micro and nanoplastics induces an initial physical protection that consists in the secretion of mucus, followed by an increase of blood vessels and the recruitment of immune cells, in particular macrophages. Moreover, macrophages were directly involved in both phagocytic and encapsulation processes, as demonstrated by acid phosphatase (ACP) histoenzymatic and Thioflavin-T assays, expressing specific pro-inflammatory factors, such as HvRNASET2 and HmAIF-1, as demonstrated by immunolocalization and qPCR experiments. Finally, the expression levels of genes related to oxidative stress-induced enzymes have been investigated, in order to evaluate the possible increase in reactive oxygen species (ROS), due to the entry into the leech tissues of PP micro and nanoplastics. This work allows deepening the current knowledge of the possible harmful effects on human health deriving from micro and nanoplastics dispersion, leading new insight about freshwater ecosystems that often represent the first environments interested in plastic pollution.

Interaction between surfaces decorated with like-charged pendants: Unravelling the interplay between energy and entropy leading to attraction.

HYPOTHESIS: The stronger motional coupling between monovalent counterions neutralizing homogeneously like-charged surfaces induced by an increase in charge density is known to foster inter-surface attraction. Compared to a uniformly distributed charge, point-like charges generate locally more intense fields, so that the correlation induced between counterions may be even stronger despite an identical total charge. It should thus be possible to induce surface attraction at lower charge densities than commonly expected. EXPERIMENTS: Monte Carlo simulations on primitive electrolyte models have been exploited to compute potential of mean force profiles and mobile ion densities for systems composed of two parallel surfaces bearing surface-tethered monovalent like-charged pendants as a function of the surface distance and pendant densities. FINDINGS: Surfaces bearing like-charged pendants are found to attract each other over a wide range of distances despite the presence of very low charge densities. Notwithstanding the attractive contribution to the inter-surface forces provided by electrostatic interactions, the entropic component of the system Helmholtz energy is found to play the key role in defining the overall magnitude. The latter finding appears justified by an increase in the relative delocalization of counterions upon decreasing the surface distance.

Inducing pH control over the critical micelle concentration of zwitterionic surfactants via polyacids adsorption: Effect of chain length and structure.

HYPOTHESIS: The critical concentration above which micelles form from zwitterionic surfactant solutions and their thermodynamic stability is affected by the interaction with weak Brønsted polyacid chains (An) via the formation of charged hydrogen bonds between the latter and anionic moieties. EXPERIMENTS: The interaction between zwitterionic micelles and polyacids capable of forming hydrogen bonds, and its dependence on the environmental pH and polymer structure, has been studied with constant-pH simulations and a restricted primitive model for all electrolytes. FINDINGS: At low pH, the formation of polyacid/micelle complexes is witnessed independently of the polymer size or structure, so that the concentration above which micelles form is substantially decreased compared to polyacid-free cases. Upon rising pH, polymer desorption takes place within a narrow range of pH values, its location markedly depending on the size and structure of polyacids, and on the relative disposition between headgroup charged moieties. Thus, the desorption onset for long linear polyacids (A60) interacting with sulphobetainic headgroups is roughly two pH units higher than for six decameric chains (6A10) adsorbed onto micelles bearing phosphorylcholinic headgroups. This effect, together with the preferential desorption of chain ends at intermediate pH, may be exploited for drug delivery purposes or building advanced metamaterials.

Synthesis of Di-Block Copolymers Poly (propylene ox-ide)-block-Poly (9-(2,3-epoxypropyl) Carbazole) via Sequential Addition of Monomers in One Pot.

Polypropylene oxide (PPO) and poly(9-(2,3-epoxypropyl) carbazole) (PEPK) di-block copolymers are prepared in one pot via sequential monomer addition by using i-PrONa/i-Bu3Al as an anionic catalytic system. An almost 100% monomer conversion is obtained, and the length of each block is controlled through the monomer/catalyst ratio used. Copolymer molecular weights are quite close to theoretical values calculated assuming the formation of one polymer chain per catalyst; therefore, it is hypothesized that the polymerization reaction proceeds with a living character. The synthesis appears to be particularly efficient and versatile. The calorimetric properties of copolymers obtained in this work are remarkable, since they show two distinct Tg values, corresponding to the PPO and PEPK blocks. The optical measurements of di-block copolymers show more analogous features than those of PEPK homopolymer. Copolymer solution emission spectra just exhibit isolated carbazole fluorescence, whereas in the solid state, film spectra show excimer fluorescence.

Hybrid giant lipid vesicles incorporating a PMMA-based copolymer.

BACKGROUND: In recent years, there has been a growing interest in the formation of copolymer-lipid hybrid self-assemblies, which allow combining and improving the main features of pure lipid-based and copolymer-based systems known for their potential applications in the biomedical field. As the most common method used to obtain giant vesicles is electroformation, most systems so far used low Tg polymers for their flexibility at room temperature. METHODS: Copolymers used in the hybrid vesicles have been synthesized by a modified version of the ATRP, namely the Activators ReGenerated by Electron Transfer ATRP and characterized by NMR and DSC. Giant hybrid vesicles have been obtained using electroformation and droplet transfer method. Confocal fluorescence microscopy was used to image the vesicles. RESULTS: Electroformation enabled to obtain hybrid vesicles in a narrow range of compositions (15 mol% was the maximum copolymer content). This range could be extended by the use of a droplet transfer method, which enabled obtaining hybrid vesicles incorporating a methacrylate-based polymer in a wide range of compositions. Proof of the hybrid composition was obtained by fluorescence microscopy using labeled lipids and copolymers. CONCLUSIONS: This work describes for the first time, to the best of our knowledge, the formation of giant hybrid polymer/lipid vesicles formed with such a content of a polymethylmethacrylate copolymer, the glass temperature of which is above room temperature. GENERAL SIGNIFICANCE: This work shows that polymer structures, more complex than the ones mostly employed, can be possibly included in giant hybrid vesicles by using the droplet transfer method. This will give easier access to functionalized and stimuli-responsive giant vesicles and to systems exhibiting a tunable permeability, these systems being relevant for biological and technological applications.

On the distribution of hydrophilic polyelectrolytes and their counterions around zwitterionic micelles: the possible impact on the charge density in solution.

Despite their charge neutrality, micelles composed of surfactants with zwitterionic headgroups selectively accumulate anions at their hydrophobic core/solution interphase due to electrostatic interactions if headgroup positive moieties are the innermost. This tendency may be markedly enhanced if polyions substitute simple ions. To investigate this possibility, solutions composed of zwitterionic micelles and hydrophilic polyanions have been investigated with Monte Carlo simulations representing the studied systems via primitive electrolyte models. Structural and energetic properties are obtained to highlight the impact of connecting simple ions into polyions on the interactions between electrolytes and micelles. Despite the latter, polyanions conserve their conformational properties. A marked increase in the concentration of charged species inside the micellar corona is, instead, found when polyions are present independently of their charge sign or the headgroup structure. Thus, polyelectrolytes act as "shuttle" for all charged species, with the potential of increasing reactions rates involving the latter due to mass effects. Besides, results for the polyions/micelles mixing free energy and Helmholtz energy profiles indicate that the critical micelle concentration is impacted minimally by hydrophilic polyelectrolytes, an outcome agreeing with experiments. This finding is entirely due to weak enthalpic effects while mixing hydrophilic polyions and micelles. A strong reduction in the screening of the micelle negative charge, acquired following the adsorption of anions in the corona and due to counterions layering just outside it (the so called "chameleon effect"), is forecasted when polyanions substitute monovalent anions.

Interface Counterion Localization Induces a Switch between Tight and Loose Configurations of Knotted Weak Polyacid Rings despite Intermonomer Coulomb Repulsions.

Stochastic simulations have been used to investigate the conformational behavior of knotted weak polyacid rings as a function of pH. Different from the commonly expected ionization-repulsion-expansion scheme upon increasing pH, theoretical results suggest a nonmonotonic behavior of the gyration radius Rg2. Polyelectrolyte recontraction at high ionization is induced by the weakening of Coulomb repulsion due to counterions (CIs) localizing at the interphase between the polymer and solvent, and the more marked it appears, the more complex is the knot topology. Compared with strong polyelectrolytic species of identical ionization, weak polyacids present tighter knots due to their ability to localize neutral monomers inside the tangled part. Increasing the solvent Bjerrum length enhances CIs localization, lowering the pH at which polyacids start decreasing their average size. A similar effect is also obtained by increasing the amount of "localizable" cations by adding salts.

Monte Carlo study of the effects of macroion charge distribution on the ionization and adsorption of weak polyelectrolytes and concurrent counterion release.

HYPOTHESIS: Adsorption of weak polyelectrolytes onto charged nanoparticles, and concurrent effects such as spatial partitioning of ions may be influenced by details of the polyelectrolyte structure (linear or star-like) and size, by the mobility of the nanoparticle surface charge, or the valence of the nanoparticle counterions. EXPERIMENTS: Ionization and complexation of weak polyelectrolytes on spherical macroions with monovalent and divalent countrions has been studied with constant-pH Monte Carlo titrations and primitive electrolyte models for linear and star-like polymers capable, also, of forming charged hydrogen bonds. Nanoparticles surface charge has been represented either as a single colloid-centered total charge (CCTC) or as surface-tethered mobile monovalent spherical charges (SMMSC). FINDINGS: Differences in the average number of adsorbed polyelectrolyte arms and their average charge, and in the relative amount of macroion counterions (m-CI's) released upon polymer adsorption are found between CCTC and SMMSC nanoparticles. The amount of the counterions released also depends on the polymer structure. As CCTC adsorbs a lower number of star-like species arms, the degree of condensation of polymer counterions (p-CI's) onto the polyelectrolyte is also substantially higher for the CCTC colloid, with a concurrent decrease of the osmotic coefficient values.

Impact of Charge Correlation, Chain Rigidity, and Chemical Specific Interactions on the Behavior of Weak Polyelectrolytes in Solution.

In this work, we performed titration simulations of weak linear polyelectrolytes via the Monte Carlo method and the constant pH ensemble aiming to understand how polyelectrolyte concentration, chain rigidity, and the formation of intra- and inter-chain charged hydrogen bonds (c-H-bonds) impact on ionization and conformations of polyacidic species, counterions (CIs) distribution, and system Helmholtz energy. Increasing polyelectrolyte concentration resulted in enhanced acidity for all cases investigated due to the increased screening of chain charges by CIs and, when possible, the formation of interchain c-H-bonds. Our simulations also evidenced that polyelectrolytes able to form c-H-bonds can populate simultaneously two conformational states (aggregated and unfolded) in a range of pH, the transition between the two appearing first order-like. To better understand how properties of two polyelectrolytic chains are modified by their relative distance, we performed window sampling (WS) simulations, which highlighted nontrivial features in the ionization and conformational behaviors. As byproducts of WS simulations, we obtained also the potential of mean force between two chains; from this, it emerges that the reversible work needed to reach a specific interchain distance does not always increase with the pH, especially for c-H-bonds forming semirigid chains brought at short distances.

Escherichia coli as a Model for the Description of the Antimicrobial Mechanism of a Cationic Polymer Surface: Cellular Target and Bacterial Contrast Response.

In this study, we use Escherichia coli as a model to investigate the antimicrobial mechanism of a film made of a copolymer based on monomethylether poly(ethylene glycol), methyl methacrylate, and 2-dimethyl(aminoethyl) methacrylate, whose surface is active towards Gram-negative and Gram-positive bacteria. The polymer contains not quaternized amino groups that can generate a charged surface by protonation when in contact with water. For this purpose, we adopted a dual strategy based on the analysis of cell damage caused by contact with the polymer surface and on the evaluation of the cell response to the surface toxic action. The lithic effect on the protoplasts of E. coli showed that the polymer surface can affect the structure of cytoplasmic membranes, while assays of calcein leakage from large unilamellar vesicles at different phospholipid compositions indicated that action on membranes does not need a functionally active cell. On the other hand, the significant increase in sensitivity to actinomycin D demonstrates that the polymer interferes also with the structure of the outer membrane, modifying its permeability. The study on gene expression, based on the analysis of the transcripts in a temporal window where the contact with the polymer is not lethal and the damage is reversible, showed that some key genes of the synthesis and maintenance of the outer membrane structure ( fabR, fadR, fabA, waaA, waaC, kdsA, pldA, and pagP), as well as regulators of cellular response to oxidative stress ( soxS), are more expressed when bacteria are exposed to the polymer surface. All together these results identified the outer membrane as the main cellular target of the antimicrobial surface and indicated a specific cellular response to damage, providing more information on the antimicrobial mechanism. In this perspective, data reported here could play a pivotal role in a microbial growth control strategy based not only on the structural improvements of the materials but also on the possibility of intervening on the cellular pathways involved in the contrast reaction to these and other polymers with similar mechanisms.

The effect of bicephalic stimulation of the dorsolateral prefrontal cortex on the attentional bias for threat: A transcranial direct current stimulation study.

Previous stimulation studies demonstrated that the dorsolateral prefrontal cortex (DLPFC) is involved in threat processing. According to a model of emotional processing, an unbalance between the two DLPFCs, with a hyperactivation of right frontal areas, is involved in the processing of negative emotions and genesis of anxiety. In the present study, we investigated the role of the right and left DLPFC in threat processing in healthy women who also completed the State-Trait Anxiety Inventory (STAI). We simultaneously modulated the activity of the right and left dorsolateral prefrontal cortex by applying bicephalic transcranial direct current stimulation (tDCS) before participants completed a modified version of the classic Posner task using threatening and nonthreatening stimuli as spatial cues. Anodal stimulation on the right DLPFC with a simultaneous cathodal stimulation over the left side induced a disengagement bias in individuals with low STAI scores and a facilitation bias in individuals with high STAI scores. Anodal stimulation on the left DLPFC with the simultaneous cathodal stimulation over the right side did not affect threat processing. The findings of the present study provided specific support to the hypothesis that unbalanced activation between left and right hemispheres with enhanced activation of the right DLPFC is critical in early top-down threat processing in healthy individuals.

pH-sensitive polymersomes: controlling swelling via copolymer structure and chemical composition.

pH-sensitive vesicles used as drug delivery systems (DDSs) are generally composed of protonable copolymers. The disaggregation of these nanoparticles (NPs) during drug release implies the dispersion of positively charged cytotoxic polyelectrolytes in the human body. To alleviate such issue, we synthesised A(BC)n amphiphilic block copolymers with linear (n = 1) and branched (n = 2) architectures to obtain pH-sensitive vesicles capable of releasing drugs in acidic conditions via controlled swelling instead of disaggregation. We obtained this feature by fine-tuning the relative amount of pH-sensitive and hydrophobic monomers. We studied pH-driven swelling by measuring NPs size in neutral and acidic conditions, the latter typical of tumours or inflamed tissues (pH∼6) and lysosomes (pH∼4.5). Dynamic light scattering (DLS) and zeta potential data provided useful indications about the influence of architecture and chemical composition on NPs swelling, stability and polycation release. Results demonstrated that vesicles made of linear copolymers with ∼22-28% in mol of protonable monomers in the 'BC' block swelled more than other species following a pH change from pH 7.4 to pH 4.5. We finally evaluated the cytotoxicity of vesicles composed of linear species, and paclitaxel (PTX) release from the latter in both cancer and normal cells.

Out of Equilibrium Self-Assembly of Janus Nanoparticles: Steering It from Disordered Amorphous to 2D Patterned Aggregates.

Solvent evaporation driven self-assembly of Janus nanoparticles (J-NPs) has been simulated employing lattice-gas models to investigate the possible emergence of new superlattices. Depending on the chemical nature of NP faces (hence solvophilicity and relative interaction strength), zebra-like or check-like patterns and micellar agglomerates can be obtained. Vesicle-like aggregates can be produced by micelle-based corrals during heterogeneous evaporation. Patterns formed during aggregation appear to be robust against changes in evaporation modality (i.e., spinodal or heterogeneous) or interaction strengths, and they are due to a strictly nanoscopic orientation of single J-NPs in all cases. Due to the latter feature, the aggregate size growth law N(t) ∝ ta has its exponent a markedly depending on the chemical nature of the J-NPs involved in spite of the unvaried growth mechanism. We interpret such a finding as connected to the increasingly stricter orientation pre-requirements for successful (binding) NP landing upon going from isotropic (a ≃ 0.50), to "zebra" (a ≃ 0.38), to "check" (a ≃ 0.23), and finally to "micelle" (a = 0.15-0.17) pattern forming NPs.

The Role of Charge Density and Hydrophobicity on the Biocidal Properties of Self-Protonable Polymeric Materials.

Intrinsic antimicrobial thermoplastic A(BC)n copolymers (n = 1, 2, 4), where A was poly(ethylene glycol) (PEG), BC was a random chain of methylmethacrylate (MMA), and alkyl-aminoethyl methacrylate (AAEMA), were synthesized and the antimicrobial activity and hemolyticity were evaluated on plaques obtained by casting as a function of the architecture, the N-substituent groups of the AAEMAs (methyl, ethyl, isopropyl, and tert-butyl groups) and the hydrophobic/charge density balance. Antimicrobial effectiveness and efficiency is controlled by the surface charge density and by the influence of N-alkyl groups on the surface morphology. Also interestingly, it is the absence of hemolitytic activity in all copolymers. In presence of Escherichia coli, the A(BC)2 copolymer with 40% of N-methyl groups is the most efficient, killing 91% of the bacteria already after 1.5 h.

Different insight into amphiphilic PEG-PLA copolymers: influence of macromolecular architecture on the micelle formation and cellular uptake.

One constrain in the use of micellar carriers as drug delivery systems (DDSs) is their low stability in aqueous solution. In this study "tree-shaped" copolymers of general formula mPEG-(PLA)n (n = 1, 2 or 4; mPEG = poly(ethylene glycol) monomethylether 2K or 5K Da; PLA = atactic or isotactic poly(lactide)) were synthesized to evaluate the architecture and chemical composition effect on the micelles formation and stability. Copolymers with mPEG/PLA ratio of about 1:1 wt/wt were obtained using a "core-first" synthetic route. Dynamic Light Scattering (DLS), Field Emission Scanning Electron Microscopy (FESEM), and Zeta Potential measurements showed that mPEG2K-(PD,LLA)2 copolymer, characterized by mPEG chain of 2000 Da and two blocks of atactic PLA, was able to form monodisperse and stable micelles. To analyze the interaction among micelles and tumor cells, FITC conjugated mPEG-(PLA)n were synthesized. The derived micelles were tested on two, histological different, tumor cell lines: HEK293t and HeLa cells. Fluorescence Activated Cells Sorter (FACS) analysis showed that the FITC conjugated mPEG2K-(PD,LLA)2 copolymer stain tumor cells with high efficiency. Our data demonstrate that both PEG size and PLA structure control the biological interaction between the micelles and biological systems. Moreover, using confocal microscopy analysis, the staining of tumor cells obtained after incubation with mPEG2K-(PD,LLA)2 was shown to be localized inside the tumor cells. Indeed, the mPEG2K-(PD,LLA)2 paclitaxel-loaded micelles mediate a potent antitumor cytotoxicity effect.

Modulating antimicrobial activity by synthesis: dendritic copolymers based on nonquaternized 2-(dimethylamino)ethyl methacrylate by Cu-mediated ATRP.

The synthesis of novel star-like heteroarms polymers A(BC)(n) containing m-PEG (block A), methylmethacrylate (MMA), and nonquaternized 2-(dimethylamino)ethyl methacrylate (DMAEMA) (blocks BC) is here reported. We demonstrated that copolymer films with comparable amounts of DMAEMA have antimicrobial properties strongly depending on the topological structure (i.e., the number of arms) of the composing copolymers. We interpret the highest antimicrobial activity of A(BC)(2) with respect to A(BC)(4) and linear copolymers (respectively, A(BC)(2) ≥ A(BC)(4) > A(BC)) as probably due to the formation of strong hydrogen bonds between close amino-ammonium groups in the A(BC)(2) film. Strong hydrogen bonds seem to be somewhat disfavored in the case of the linear species by the difference in both polymer architecture and film morphology compared with the A(BC)(2) and A(BC)(4) architectures.

Impact of polymer tacticity on the physico-chemical behaviour of polymers proposed as therapeutics.

Although water-soluble polymers are finding increasing use as polymer therapeutics, there has been little consideration of the effect of polymer stereochemistry on their physico-chemical and biological properties. The aim of this study was to investigate these properties using polymethacrylic acids (PMAs) of similar molecular weights with a difference in syndiotacticity of about 20% of rr triad content. Experiments to characterize the solution behaviour were conducted at pHs encountered during the transport, endocytic uptake and intracellular trafficking (7.4-3.0). These showed that with increasing rr triads, the polymer become less hydrophobic, a stronger acid, displayed a locally ordered solution conformation at pH 5.5, and interacted more strongly with dodecyl trimethylammonium bromide (DTAB) micelles. Preliminary cytotoxicity experiments using B16F10 melanoma cells showed lower toxicity in the concentration range of 1-100 µg/mL with increased rr triads. These observations indicate that the higher content of rr triads could drive a chain organization that minimize the influence of negative charges and so underline the importance of further, systematic studies to investigate the effect of tacticity on the behaviour of polymers in respect of their pharmacokinetics, toxicity and efficacy.

Establishment of subcellular fractionation techniques to monitor the intracellular fate of polymer therapeutics II. Identification of endosomal and lysosomal compartments in HepG2 cells combining single-step subcellular fractionation with fluorescent imaging.

As they are often designed for lysosomotropic, endosomotropic and/or transcellular delivery, an understanding of intracellular trafficking pathways is essential to enable optimised design of novel polymer therapeutics. Here, we describe a single-step density gradient subcellular fractionation method combined with fluorescent detection analysis that provides a new tool for characterisation of endocytic traffic of polymer therapeutics. Hepatoma (HepG2) cells were used as a model and cell breakage was optimised using a cell cracker to ensure assay of the whole cell population. After removal of unbroken cells and nuclei, the cell lysate as a post-nuclear supernatant (PNS) was layered onto an iodixanol (OptiPrep) density gradient optimised to 5-20%. Early endosomes, late endosomes and lysosomes were identified from gradient fractions by immunoblotting for marker proteins early endosome antigen 1 (EEA 1) and lysosomal associated membrane protein 1 (LAMP 1) using horseradish peroxidase or fluorescently-labelled secondary antibodies. Lysosomes were also detected using N-acetyl-beta-glucosamindase (Hex A) activity. In addition, cells were incubated with Texas-red labelled transferrin (TxR-Tf) for 5 min to specifically label early endosomes and this was directly detected from SDS-PAGE gels. Internalised macromolecules and colloidal particles can potentially alter vesicle buoyant density. To see if typical macromolecules of interest would alter vesicle density or perturb vesicle traffic, HepG2 cells were incubated with dextran or a polyethyleneglycol (PEG)-polyester dendron G4 (1 mg/ml for 24 h). The PEG-polyester dendron G4 caused a slight redistribution of endocytic structures to lower density fractions but immunofluorescence microscopy showed no obvious dendron effects. In conclusion, the combined subcellular fractionation with fluorescent imaging approach described here can be used as a tool for both fundamental cell biology research and/or the quantitative localisation of polymer therapeutics in the endocytic pathway.