Rubber particle proteins, HbREF and HbSRPP, show different interactions with model membranes.

The biomembrane surrounding rubber particles from the hevea latex is well known for its content of numerous allergen proteins. HbREF (Hevb1) and HbSRPP (Hevb3) are major components, linked on rubber particles, and they have been shown to be involved in rubber synthesis or quality (mass regulation), but their exact function is still to be determined. In this study we highlighted the different modes of interactions of both recombinant proteins with various membrane models (lipid monolayers, liposomes or supported bilayers, and multilamellar vesicles) to mimic the latex particle membrane. We combined various biophysical methods (polarization-modulation-infrared reflection-adsorption spectroscopy (PM-IRRAS)/ellipsometry, attenuated-total reflectance Fourier-transform infrared (ATR-FTIR), solid-state nuclear magnetic resonance (NMR), plasmon waveguide resonance (PWR), fluorescence spectroscopy) to elucidate their interactions. Small rubber particle protein (SRPP) shows less affinity than rubber elongation factor (REF) for the membranes but displays a kind of "covering" effect on the lipid headgroups without disturbing the membrane integrity. Its structure is conserved in the presence of lipids. Contrarily, REF demonstrates higher membrane affinity with changes in its aggregation properties, the amyloid nature of REF, which we previously reported, is not favored in the presence of lipids. REF binds and inserts into membranes. The membrane integrity is highly perturbed, and we suspect that REF is even able to remove lipids from the membrane leading to the formation of mixed micelles. These two homologous proteins show affinity to all membrane models tested but neatly differ in their interacting features. This could imply differential roles on the surface of rubber particles.

Nano-encapsulation of plitidepsin: in vivo pharmacokinetics, biodistribution, and efficacy in a renal xenograft tumor model.

PURPOSE: Plitidepsin is an antineoplasic currently in clinical evaluation in a phase III trial in multiple myeloma (ADMYRE). Presently, the hydrophobic drug plitidepsin is formulated using Cremophor®, an adjuvant associated with unwanted hypersensitivity reactions. In search of alternatives, we developed and tested two nanoparticle-based formulations of plitidepsin, aiming to modify/improve drug biodistribution and efficacy. METHODS: Using nanoprecipitation, plitidepsin was loaded in polymer nanoparticles made of amphiphilic block copolymers (i.e. PEG-b-PBLG or PTMC-b-PGA). The pharmacokinetics, biodistribution and therapeutic efficacy was assessed using a xenograft renal cancer mouse model (MRI-H-121 xenograft) upon administration of the different plitidepsin formulations at maximum tolerated multiple doses (0.20 and 0.25 mg/kg for Cremophor® and copolymer formulations, respectively). RESULTS: High plitidepsin loading efficiencies were obtained for both copolymer formulations. Considering pharmacokinetics, PEG-b-PBLG formulation showed lower plasma clearance, associated with higher AUC and Cmax than Cremophor® or PTMC-b-PGA formulations. Additionally, the PEG-b-PBLG formulation presented lower liver and kidney accumulation compared with the other two formulations, associated with an equivalent tumor distribution. Regarding the anticancer activity, all formulations elicited similar efficacy profiles, as compared to the Cremophor® formulation, successfully reducing tumor growth rate. CONCLUSIONS: Although the nanoparticle formulations present equivalent anticancer activity, compared to the Cremophor® formulation, they show improved biodistribution profiles, presenting novel tools for future plitidepsin-based therapies.

Magnetic responsive polymer composite materials.

Magnetic responsive materials are the topic of intense research due to their potential breakthrough applications in the biomedical, coatings, microfluidics and microelectronics fields. By merging magnetic and polymer materials one can obtain composites with exceptional magnetic responsive features. Magnetic actuation provides unique capabilities as it can be spatially and temporally controlled, and can additionally be operated externally to the system, providing a non-invasive approach to remote control. We identified three classes of magnetic responsive composite materials, according to their activation mode and intended applications, which can be defined by the following aspects. (A) Their ability to be deformed (stretching, bending, rotation) upon exposure to a magnetic field. (B) The possibility of remotely dragging them to a targeted area, called magnetic guidance, which is particularly interesting for biomedical applications, including cell and biomolecule guidance and separation. (C) The opportunity to use magnetic induction for thermoresponsive polymer materials actuation, which has shown promising results for controlled drug release and shape memory devices. For each category, essential design parameters that allow fine-tuning of the properties of these magnetic responsive composites are presented using key examples.

Polymeric micelles and vesicles: biological behavior evaluation using radiolabeling techniques.

The application of combined diagnosis and therapy through nanotechnology applications is attracting increasing attention worldwide. Polymeric self-assembled nanoparticles (NPs) have been studied for this purpose. Micelles and vesicles with or without a magnetic core can efficiently carry diagnostic and/or therapeutic agents to a desired target. The biological behavior of these NPs has been evaluated in this study, after radiolabeling with (99m)Tc. In vitro stability, in media that mimic the environment of the living body, was better for vesicles than for micelles at 1 h and decreased for both as time passed. After administration to healthy animals, all NPs presented major uptake at liver and spleen as expected. Biodistribution and imaging studies confirmed the higher uptake in these organs for the hybrid NPs and at higher extent for the ones with larger size, indicating that the magnetic load and size play an important role on in vivo distribution.

Biologically active polymersomes from amphiphilic glycopeptides.

Polypeptide block copolymers with different block length ratios were obtained by sequential ring-opening polymerization of benzyl-L-glutamate and propargylglycine (PG) N-carboxyanhydrides. Glycosylation of the poly(PG) block was obtained by Huisgens cycloaddition "click" reaction using azide-functionalized galactose. All copolymers were self-assembled using the nanoprecipitation method to obtain spherical and wormlike micelles as well as polymersomes depending on the block length ratio and the nanoprecipitation conditions. These structures display bioactive galactose units in the polymersome shell, as proven by selective lectin binding experiments.

Single-molecule mechanical unfolding experiments reveal a critical length for the formation of α-helices in peptides.

α-Helix is the most predominant secondary structure in proteins and supports many functions in biological machineries. The conformation of the helix is dictated by many factors such as its primary sequence, intramolecular interactions, or the effect of the close environment. Several computational studies have proposed that there is a critical maximum length for the formation of intact compact helical structures, supporting the fact that most intact α-helices in proteins are constituted of a small number of amino acids. To obtain a detailed picture on the formation of α-helices in peptides and their mechanical stability, we have synthesized a long homopolypeptide of about 90 amino acids, poly(γ-benzyl-l-glutamate), and investigated its mechanical behaviour by AFM-based single-molecule force spectroscopy. The characteristic plateaus observed in the force-extension curves reveal the unfolding of a series of small helices (from 1 to 4) of about 20 amino acid residues connected to each other, rather than a long helix of 90 residues. Our results suggest the formation of a tertiary structure made of short helices with kinks, instead of an intact compact helical structure for sequences of more than 20 amino acid residues. To our knowledge, this is the first experimental evidence supporting the concept of a helical critical length previously proposed by several computational studies.

Effect of the polymer nature on the structural organization of lipid/polymer particle assemblies.

The nano-organized LipoParticle assemblies, consisting of polymer particles coated with lipid layers, are investigated with the aim of evidencing the impact of the particle chemical nature on their physicochemical behavior. To this end, these colloidal systems are elaborated from anionic submicrometer poly(styrene) (P(St)) or poly(lactic acid) (PLA) particles, and lipid mixtures composed of zwitterionic 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC) and cationic 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP). As revealed by various experimental techniques, such as quasielastic light scattering, zeta potential measurements, transmission electron microscopy, and 1H NMR spectroscopy, the features of both LipoParticle systems are similar when cationic lipid formulations (DPPC/DPTAP mixtures) are used. This result emphasizes the major role of electrostatic interactions as driving forces in the assembly elaboration process. Conversely, the assemblies prepared only with the zwitterionic DPPC lipid are strongly dependent on the particle chemical nature. The structural characteristics of the assemblies based on PLA particles are not controlled and correspond to aggregates, contrary to P(St) particles. To understand this specific phenomenon, and to consequently improve the final organization of these assemblies which are potentially of great interest in biotechnology and biomedicine, numerous investigations are carried out such as the studies of the impact of the ionic strength and the pH of the preparation media, as well as the presence of ethanol (involved in the PLA particle synthesis) or the mean size of the lipid vesicles. From the resulting data and according to the nature of spherical solid support, hydrophobic effects, hydrogen bonds, or dipole-dipole interactions would also appear to influence the LipoParticle elaboration in the case of zwitterionic lipid formulation.

Cellular Uptake and Cytotoxic Effect of Epidermal Growth Factor Receptor Targeted and Plitidepsin Loaded Co-Polymeric Polymersomes on Colorectal Cancer Cell Lines.

Encapsulating chemotherapy drugs in targeted nanodelivery systems is one of the most promising approaches to tackle cancer disease, avoiding side effects of common treatment. In the last decade, several nanocarriers with different nature have been tested, but polypeptide-based copolymers have attracted considerable attention for their biocompatibility, controlled and slow biodegradability as well as their low toxicity. In this work, we synthesized, characterized and evaluated poly(trimethylene carbonate)-bock-poly(L-glutamic acid) derived polymersomes, targeted to epidermal growth factor receptor (EGFR), loaded with plitidepsin and ultimately tested in HT29 and LS174T colorectal cancer cell lines for specificity and efficacy. Furthermore, morphology, physico-chemical properties and plitidepsin loading were carefully investigated. A thorough in vitro cytotoxicity analysis of the unloaded polymersomes was carried out for biocompatibility check, studying viability, cell membrane asymmetry and reactive oxygen species levels. Those cytotoxicity assays showed good biocompatibility for plitidepsin-unloaded polymersomes. Cellular uptake and cytotoxic effect of EGFR targeted and plitidepsin loaded polymersome indicated that colorectal cancer cell lines were.more sensitive to anti-EGFR-drug-loaded than untargeted drug-loaded polymersomes. Also, in both cell lines, the use of untargeted polymersomes greatly reduced plitidepsin cytotoxicity as well as the cellular uptake, indicating that the use of this targeted nanocarrier is a promising approach to tackle colorectal cancer disease and avoid the undesired effects of the usual treatment. Furthermore, in vivo assays support the in vitro conclusions that EGFR targeted polymersomes could be a good drug delivery system. This work provides a proof of concept for the use of encapsulated targeted drugs as future therapeutic treatments for cancer.

Amphiphilic PEO-b-PBLG diblock and PBLG-b-PEO-b-PBLG triblock copolymer based nanoparticles: doxorubicin loading and in vitro evaluation.

Huisgen's 1,3-dipolar cycloaddition ("Click Chemestry") has been used to prepare amphiphilic PEO-b-PBLG diblock and PBLG-b-PEO-b-PBLG triblock copolymers as potential carriers of anticancer drugs. Spherical and flower shaped micelles (D ≈ 100 nm) were obtained from diblock and triblock copolymers respectively. DOX was effectively encapsulated up to 18 wt.% and 50-60% of it was steadily released from the micelles over a period of 7 d. Flow cytometry and fluorescence microscopy confirmed the effective intracellular uptake as well as the sustained release of DOX from micelles. These results suggest that the diblock as well as triblock copolymers are promising carriers for intra-cellular drug delivery.

Nano-thermometers with thermo-sensitive polymer grafted USPIOs behaving as positive contrast agents in low-field MRI.

Two commercial statistical copolymers of ethylene oxide and propylene oxide, Jeffamine® M-2005 (PEO5-st-PPO37) and M-2070 (PEO46-st-PPO13), exhibiting lower critical solution temperature (LCST) in water, were grafted onto the surface of ultra-small superparamagnetic iron oxide nanoparticles (USPIOs) using silanization and amide-bond coupling reactions. The LCSTs of the polymers in solution were measured by dynamic light scattering (DLS) and nuclear magnetic resonance (NMR). In accordance with the compositions of EO vs. PO, the transition temperature was measured to be 22 ± 2 °C for M-2005 by both DLS and NMR, while the LCST was much higher, 52 ± 2 °C, for M-2070 (a second transition was also detected above 80 °C by NMR in that case, ascribed to the full dehydration of chains at the molecular level). The resulting polymer-grafted USPIOs exhibit a temperature-responsive colloidal behaviour, their surface reversibly changing from hydrophilic below LCST to hydrophobic above it. This phenomenon was utilised to design thermo-sensitive contrast agents for MRI. Transverse relaxivities (r2) of the USPIO@PEO5-st-PPO37 core-shell nanoparticles were measured at 8.25, 20, 60, and 300 MHz. Nuclear magnetic resonance dispersion (NMRD) profiles, giving longitudinal relaxivities (r1) between 0.01 and 60 MHz, were acquired at temperatures ranging from 15 to 50 °C. For all tested frequencies except 300 MHz, both r1 and r2 decrease with temperature and show an inflection point at 25 °C, near the LCST. To illustrate the interest of such polymer-coated USPIOs for MRI thermometry, sample tubes were imaged on both low-field (8.25 MHz/0.194 Tesla) and high-field (300 MHz/7.05 Tesla) MRI scanners with either T1- or T2*-weighted spin echo sequences. The positive contrast on low-field MR images and the perfect linearity of the signal with a T2*-weighted sequence over the entire temperature range 15-50 °C render these LCST polymer coated USPIOs interesting positive contrast agents, also working as "nano-thermometers".

Magnetic field triggered drug release from polymersomes for cancer therapeutics.

Local and temporal control of drug release has for long been a main focus in the development of novel drug carriers. Polymersomes, which can load both hydrophilic and hydrophobic species and, at the same time, be tailored to respond to a desired stimulus, have drawn much attention over the last decade. Here we describe polymersomes able to encapsulate up to 6% (w/w) of doxorubicin (DOX) together with 30% (w/w) of superparamagnetic iron oxide nanoparticles (USPIO; γ-Fe2O3). Upon internalization in HeLa cells and when a high frequency AC magnetic field (14mT at 750kHz) was applied, the developed delivery system elicited an 18% increase in cell toxicity, associated with augmented DOX release kinetics. In order to ensure that the observed cytotoxicity arose from the increased doxorubicin release and not from a pure magnetic hyperthermia effect, polymersomes loaded with magnetic nanoparticles alone were also tested. In this case, no increased toxicity was observed. We hypothesize that the magnetic field is inducing a very local hyperthermia effect at the level of the polymersome membrane, increasing drug release. This approach opens new perspectives in the development of smart delivery systems able to release drug upon demand and therefore, improving treatment control.

Antibody-functionalized magnetic polymersomes: in vivo targeting and imaging of bone metastases using high resolution MRI.

Multifunctional polymersomes loaded with maghemite nanoparticles and grafted with an antibody, directed against human endothelial receptor 2, are developed as novel MRI contrast agents for bone metastasis imaging. Upon administration in mice bearing bone tumor grown from human breast cancer cells, MR images show targeting and enhanced retention of antibody-labeled polymersomes at the tumor site.

Hybrid iron oxide-copolymer micelles and vesicles as contrast agents for MRI: impact of the nanostructure on the relaxometric properties.

Magnetic resonance imaging (MRI) is at the forefront of non-invasive medical imaging techniques. It provides good spatial and temporal resolution that can be further improved by the use of contrast agents (CAs), providing a valuable tool for diagnostic purposes. Ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles are attractive MRI contrast agents due to their negative (T2) contrast enhancement capability and biocompatibility. Clusters of USPIOs with polymer material are of particular interest since they can sustain additional functionalities like drug delivery and targeting. Aiming to establish a relationship between the morphology of the clusters and their efficacy as MRI contrast agents (relaxometric properties), we prepared - using three different maghemite (γ-Fe2O3) USPIO diameters - a series of hybrid copolymer/iron oxide CAs presenting two different geometries (micellar or vesicular). The NMR relaxometry profiles confirmed the nature of the physical mechanisms inducing the increase of nuclear relaxation rates at low (magnetic anisotropy) and high (Curie relaxation) magnetic fields. A heuristic model, first proposed by Roch, Muller, Gillis, and Brooks, allowed the fitting of the whole longitudinal relaxivity r1(ν) profile, for samples with different magnetic core sizes. We show that both types of clusters exhibit transverse relaxivity (r2) values comparable to or higher than those of common contrast agents, over the whole tested frequency range. Moreover, in-depth analysis revealed substantially a linear relationship between r2 and the number of encapsulated USPIOs divided by the diameter of the clusters (NUSPIO/DH), for each USPIO size. The cluster structure (i.e. micelle or vesicle) appeared to have a mild influence on the transverse relaxivity value. Indeed, the r2 value was mainly governed by the individual size of the USPIOs, correlated with both the cluster external diameter and the magnetic material volume fraction.

Smart polymersomes for therapy and diagnosis: fast progress toward multifunctional biomimetic nanomedicines.

Rising from the shortcomings of modern day therapeutics there is a need for a controlled approach in carrier-mediated drug delivery. Polymeric vesicles, also called polymersomes, are powerful tools to address issues of efficacy, specificity, and controlled release of drugs to diseased tissues. These recent, biomimetic structures are able to overcome the body's natural defences, remaining stable for extended time in circulation, have tuneable membrane properties, allowing the control of membrane permeability and therefore of drug release, and have the potential to be functionalized for active targeting of specific tissues, reducing undesirable side effects. Extensive work has been carried out in order to attain multifunctional polymeric vesicles that respond to precise triggers (e.g., temperature, pH, redox, magnetic field, etc) with a spatial and temporal monitoring what may enable unprecedented control of drug release in the body. These versatile structures can be loaded with different type of (bio)molecules and nanoparticles, from drugs to contrast agents for medical imaging, and are able to accommodate them in different subcompartments of the vesicle (i.e., hydrophobic membrane and hydrophilic core). Multimodal targeted delivery system could be obtained from this unique platform, with abilities in both drug delivery and medical imaging contrast enhancement, widening the perspectives toward theranostics. Polymersomes offer a promising route toward more effective treatments with fewer side effects and superior outcomes.

Doxorubicin loaded magnetic polymersomes: theranostic nanocarriers for MR imaging and magneto-chemotherapy.

Hydrophobically modified maghemite (γ-Fe(2)O(3)) nanoparticles were encapsulated within the membrane of poly(trimethylene carbonate)-b-poly(l-glutamic acid) (PTMC-b-PGA) block copolymer vesicles using a nanoprecipitation process. This formation method gives simple access to highly magnetic nanoparticles (MNPs) (loaded up to 70 wt %) together with good control over the vesicles size (100-400 nm). The simultaneous loading of maghemite nanoparticles and doxorubicin was also achieved by nanoprecipitation. The deformation of the vesicle membrane under an applied magnetic field has been evidenced by small angle neutron scattering. These superparamagnetic hybrid self-assemblies display enhanced contrast properties that open potential applications for magnetic resonance imaging. They can also be guided in a magnetic field gradient. The feasibility of controlled drug release by radio frequency magnetic hyperthermia was demonstrated in the case of encapsulated doxorubicin molecules, showing the viability of the concept of magneto-chemotherapy. These magnetic polymersomes can be used as efficient multifunctional nanocarriers for combined therapy and imaging.

A simple method to achieve high doxorubicin loading in biodegradable polymersomes.

Doxorubicin (Dox), an anthracycline anticancer drug, was successfully incorporated into block copolymer vesicles of poly(trimethylene carbonate)-b-poly(L-glutamic acid) (PTMC-b-PGA) by a solvent-displacement (nanoprecipitation) method. pH conditions were shown to have a strong influence on loading capacity and release profiles. Substantial drug loading (47% w/w) was achieved at pH 10.5. After pH neutralization, aqueous dispersions of drug-loaded vesicles were found stable for a prolonged period of time (at least 6months) without vesicle disruption or drug precipitation. Dox-loaded vesicles exhibited in vitro pH and temperature-dependent drug release profiles: release kinetics fastened in acid conditions or by increasing temperature. These features strongly support the interest of developing PTMC-b-PGA polymersomes as carriers for the controlled delivery of Dox.

Steric stabilization of lipid/polymer particle assemblies by poly(ethylene glycol)-lipids.

Biocompatible and biodegradable assemblies consisting of spherical particles coated with lipid layers were prepared from sub-micrometer poly(lactic acid) particles and lipid mixtures composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-3-trimethylammonium-propane. These original colloidal assemblies, named LipoParticles, are of a great interest in biotechnology and biomedicine. Nevertheless, a major limitation of their use is their poor colloidal stability toward ionic strength. Indeed, electrostatic repulsions failed to stabilize LipoParticles in aqueous solutions containing more than 10 mM NaCl. By analogy with the extensive use of poly(ethylene glycol) (PEG)-lipid conjugates to improve the circulation lifetime of liposomes in vivo, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] with various PEG chain lengths was added to the lipid formulation. Here, we show that LipoParticle stabilization was enhanced at least up to 150 mM NaCl (for more than 1 year at 4 degrees C). To determine the structure of PEG-modified LipoParticles as a function of the PEG chain length and the PEG-lipid fraction in the lipid formulation, a thorough physicochemical characterization was carried out by means of many techniques including quasi-elastic light scattering, zeta potential measurements, transmission electron microscopy, 1H NMR spectroscopy, and small-angle X-ray scattering. Finally, an attempt was made to link the resulting structural data to the colloidal behavior of PEG-modified LipoParticles.