Thermally Switchable Nanogate Based on Polymer Phase Transition.

Mimicking and extending the gating properties of biological pores is of paramount interest for the fabrication of membranes that could be used in filtration or drug processing. Here, we build a selective and switchable nanopore for macromolecular cargo transport. Our approach exploits polymer graftings within artificial nanopores to control the translocation of biomolecules. To measure transport at the scale of individual biomolecules, we use fluorescence microscopy with a zero-mode waveguide set up. We show that grafting polymers that exhibit a lower critical solution temperature creates a toggle switch between an open and closed state of the nanopore depending on the temperature. We demonstrate tight control over the transport of DNA and viral capsids with a sharp transition (∼1 °C) and present a simple physical model that predicts key features of this transition. Our approach provides the potential for controllable and responsive nanopores in a range of applications.

Optical Quantification by Nanopores of Viruses, Extracellular Vesicles, and Nanoparticles.

Nanopores combined with optical approaches can be used to detect viral particles. In this work, we demonstrate the ability of hydrodynamical driving and optical sensing to identify and quantify viral particles in a biological sample. We have developed a simple and rapid method which requires only fluorescent labeling of the particles and can therefore be applied to a wide range of virus type. The system operates in real time and at the single particle level while providing a low error on concentration (4%) and a low limit of detection of 105 particles/mL for an acquisition time of 60 s with the ability to increase the acquisition time to achieve a lower limit.

Gene transfer to skeletal muscle using hydrodynamic limb vein injection: current applications, hurdles and possible optimizations.

Hydrodynamic limb vein injection is an in vivo locoregional gene delivery method. It consists of administrating a large volume of solution containing nucleic acid constructs in a limb with both blood inflow and outflow temporarily blocked using a tourniquet. The fast, high pressure delivery allows the musculature of the whole limb to be reached. The skeletal muscle is a tissue of choice for a variety of gene transfer applications, including gene therapy for Duchenne muscular dystrophy or other myopathies, as well as for the production of antibodies or other proteins with broad therapeutic effects. Hydrodynamic limb vein delivery has been evaluated with success in a large range of animal models. It has also proven to be safe and well-tolerated in muscular dystrophy patients, thus supporting its translation to the clinic. However, some possible limitations may occur at different steps of the delivery process. Here, we have highlighted the interests, bottlenecks and potential improvements that could further optimize non-viral gene transfer following hydrodynamic limb vein injection.

Conformation-dependent membrane permeabilization by neurotoxic PrP oligomers: The role of the H2H3 oligomerization domain.

The relationship between prion propagation and the generation of neurotoxic species and clinical onset remains unclear. Several converging lines of evidence suggest that interactions with lipids promote various precursors to form aggregation-prone states that are involved in amyloid fibrils. Here, we compared the cytotoxicities of different soluble isolated oligomeric constructs from murine full-length PrP and from the restricted helical H2H3 domain with their effects on lipid vesicles. The helical H2H3 domain is suggested to be the minimal region of PrP involved in the oligomerization process. The discrete PrP oligomers of both the full-length sequence and the H2H3 domain have de novo ß-sheeted structure when interacting with the membrane. They were shown to permeabilize synthetic negatively charged vesicles in a dose-dependent manner. Restricting the polymerization domain of the full-length PrP to the H2H3 helices strongly diminished the ability of the corresponding oligomers to associate with the lipid vesicles. Furthermore, the membrane impairment mechanism occurs differently for the full-length PrP oligomers and the H2H3 helices, as shown by dye-release and black lipid membrane experiments. The membrane damage caused by the full-length PrP oligomers is correlated to their neuronal toxicity at submicromolar concentrations, as shown by cell culture assays. Although oligomers of synthetic H2H3 could compromise in vitro cell homeostasis, they followed a membrane-disruptive pattern that was different from the full-length oligomers, as revealed by the role of PrPC in cell viability assays.

Polyglycidol-Stabilized Nanoparticles as a Promising Alternative to Nanoparticle PEGylation: Polymer Synthesis and Protein Fouling Considerations.

We herein demonstrate the outstanding protein-repelling characteristic of star-like micelles and polymersomes manufactured from amphiphilic block copolymers made by poly(butylene oxide) (PBO) hydrophobic segments and polyglycidol (PGL) hydrophilic outer shells. Although positively charged proteins (herein modeled by lysozyme) may adsorb onto the surface of micelles and polymersomes where the assemblies are stabilized by short PGL chains (degree of polymerization smaller than 15), the protein adsorption vanishes when the degree of polymerization of the hydrophilic segment (PGL) is higher than ∼20, regardless the morphology. This has been probed by using three different model proteins which are remarkably different concerning molecular weight, size, and zeta potential (bovine serum albumin (BSA), lysozyme, and immunoglobulin G (IgG)). Indeed, the adsorption of the most abundant plasma protein (herein modeled as BSA) is circumvented even by using very short PGL shells due to the highly negative zeta potential of the produced assemblies which presumably promote protein-nanoparticle electrostatic repulsion. The negative zeta potential, on the other hand, enables lysozyme adsorption, and the phenomenon is governed by electrostatic forces as evidenced by isothermal titration calorimetry. Nevertheless, the protein coating can be circumvented by slightly increasing the degree of polymerization of the hydrophilic segment. Notably, the PGL length required to circumvent protein fouling is significantly smaller than the one required for PEO. This feature and the safety concerns regarding the synthetic procedures on the preparation of poly(ethylene oxide)-based amphiphilic copolymers might make polyglycidol a promising alternative toward the production of nonfouling spherical particles.

Polynucleotide transport through lipid membrane in the presence of starburst cyclodextrin-based poly(ethylene glycol)s.

Symmetrical cyclodextrin-based 14-arm star polymers with poly(ethylene glycol) PEG branches were synthesized and characterized. Interactions of the star polymers with lipid bilayers were studied by the "black lipid membrane" technique in order to demonstrate the formation of monomolecular artificial channels. The conditions for the insertion are mainly based on dimensions and amphiphilic properties of the star polymers, in particular the molar mass of the water-soluble polymer branches. Translocation of single-strand DNA (ssDNA) through those synthetic nanopores was investigated, and the close dimension between the cross-section of ssDNA and the cyclodextrin cavity led to an energy barrier that slowed down the translocation process.

Use of Primary and Secondary Polyvinylamines for Efficient Gene Transfection.

Gene transfection with polymeric carrier remains a challenge; particularly, high transfection levels combined with low toxicity are hard to achieve. We herein revisit polyvinylamines, an old and neglected family of cationic polymers. They can be readily obtained by controlled hydrolysis of polyvinylamides prepared through (controlled) radical polymerization. A series of tailor-made and well-defined polyvinylamines bearing primary amino groups, and poly(N-methylvinylamine) bearing secondary amines, were evaluated for the transfection of cells with pDNA as a function of their molar mass, molar mass distribution, and degree of deacetylation. Unexpected high transfection levels, in combination with low cytotoxicity were recorded for both series. Surprisingly, a great impact of the molar mass was observed for the primary amine polyvinylamine series, whereas the results were mostly independent of molar mass or dispersity for the polymer bearing secondary amine. It was further established that a certain percentage of acetamide groups increased the transfection level, while maintaining low cytotoxicity. These results highlight for the first time the real potential of polyvinylamines as gene carriers, and make these polymers very attractive for further development in gene therapy.

Generation of Silicone Poly-HIPEs with Controlled Pore Sizes via Reactive Emulsion Stabilization.

Macrocellular silicone polymers are obtained after solidification of the continuous phase of a poly(dimethylsiloxane) emulsion, which contains poly(ethylene glycol) drops of sub-millimetric dimensions. Coalescence of the liquid template emulsion is prohibited by a reactive blending approach. The relationship is investigated in detail between the interfacial properties and the emulsion stability, and micro- and millifluidic techniques are used to generate macrocellular polymers with controlled structural properties over a wider range of cell sizes (0.2-2 mm) and volume fractions of the continuous phase (0.1%-40%). This approach could easily be transferred to a wide range of polymeric systems.

Poly(2-methyl-2-oxazoline)-b-poly(tetrahydrofuran)-b-poly(2-methyl-2-oxazoline) amphiphilic triblock copolymers: synthesis, physicochemical characterizations, and hydrosolubilizing properties.

Block copolymers assembled into micelles have gained a lot of attention to improve drug delivery. The recent drawbacks of the poly(ethylene oxide) blocks (PEO) contained in amphiphilic pluronics derivatives made of a central poly(propylene oxide) block surrounded by two PEO blocks were recently revealed, opening the way to the design of new amphiphilic block copolymers able to self-assemble in water and to entrap molecules of interest. Here, a family of p(methyloxazoline)-b-p(tetrahydrofuran)-b-p(methyloxazoline) triblock copolymers (called TBCP) is synthesized using cationic ring opening polymerization. Studies of micelle formation using dynamic light scattering, isothermal titration calorimetry (ITC), NMR diffusion-ordered spectroscopy (DOSY), and fluorescence experiments lead us to draw a relationship between copolymer structure and the physicochemical properties of the block copolymers (critical micellar concentration (CMC), Nagg, core diameter, shell thickness, etc.). The packing parameter of the block copolymers indicates the formation of a core-corona structure. Hydrosolubilizing properties of TBCPs were exemplified with curcumin selected as a highly insoluble drug model. Curcumin, a natural polyphenolic compound, has shown a large spectrum of biological and pharmacological activity, including anti-inflammatory, antimicrobial, antioxidant, and anticarcinogenic activities. An optimized formulation process reveals that the aggregation number is the parameter affecting drug encapsulation. Patch clamp experiments carried out to study the interaction of TBCP with the cell membrane demonstrate their permeation property suitable to promote the cellular internalization of curcumin.

Evaluation of polymersome permeability as a fundamental aspect towards the development of artificial cells and nanofactories.

Polymersomes are synthetic vesicles with potential use in healthcare, chemical transformations in confined environment (nanofactories), and in the construction of artificial cells and organelles. In this framework, one of the most important features of such supramolecular structures is the permeability behavior allowing for selective control of mass exchange between the inner and outer compartments. The use of biological and synthetic nanopores in this regard is the most common strategy to impart permeability nevertheless, this typically requires fairly complex strategies to enable porosity. Yet, investigations concerning the permeability of polymer vesicles to different analytes still requires further exploration and, taking these considerations into account, we have detailed investigated the permeability behavior of a variety of polymersomes with regard to different analytes (water, protons, and rhodamine B) which were selected as models for solvents, ions, and small molecules. Polymersomes based on hydrophilic blocks of poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) or PEO (poly(ethylene oxide)) linked to the non-responsive blocks poly[N-(4-isopropylphenylacetamide)ethyl methacrylate] (PPPhA) or poly(methyl methacrylate) (PMMA), or to the stimuli pH-responsive block poly[2-(diisopropylamino)ethyl methacrylate] (PDPA) have been investigated. Interestingly, the produced PEO-based vesicles are notably larger than the ones produced using PHPMA-containing block copolymers. The experimental results reveal that all the vesicles are inherently permeable to some extent with permeability behavior following exponential profiles. Nevertheless, polymersomes based on PMMA as the hydrophobic component were demonstrated to be the least permeable to the small molecule rhodamine B as well as to water. The synthetic vesicles based on the pH-responsive PDPA block exhibited restrictive and notably slow proton permeability as attributed to partial chain protonation upon acidification of the medium. The dye permeability was evidenced to be much slower than ion or solvent diffusion, and in the case of pH-responsive assemblies, it was demonstrated to also depend on the ionic strength of the environment. These findings are understood to be highly relevant towards polymer selection for the production of synthetic vesicles with selective and time-dependent permeability, and it may thus contribute in advancing biomimicry and nanomedicine.

Neutral Block Copolymer Assisted Gene Delivery using Hydrodynamic Limb Vein Injection.

Three different amphiphilic block copolymer families are synthesized to investigate new opportunities to enhance gene delivery via Hydrodynamic Limb Vein (HLV) injections. First a polyoxazoline-based family containing mostly one poly(2-methyl-2-oxazoline) (PMeOx) block and a second block POx with an ethyl (EtOx), isopropyl (iPrOx) or phenyl substituent (PhOx) is synthesized. Then an ABC poly(2-ethyl-2-oxazoline)-b-poly(2-n-propyl-2-oxazoline)-b-poly(2-methyl-2-oxazoline) triblock copolymer is synthesized, with a thermosensitive middle block. Finally, polyglycidol-b-polybutylenoxide-b-polyglycidol copolymers with various molar masses and amphiphilic balance are produced. The simple architecture of neutral amphiphilic triblock copolymer is not sufficient to obtain enhanced in vivo gene transfection. Double or triple amphiphilic neutral block copolymers are improving the in vivo transfection performances through HLV administration as far as a block having an lower critical solution temperature is incorporated in the vector. The molar mass of the copolymer does not seem to affect the vector performances in a significant manner.

Soft jamming of viral particles in nanopores.

Viruses have remarkable physical properties and complex interactions with their environment. However, their aggregation in confined spaces remains unexplored, although this phenomenon is of paramount importance for understanding viral infectivity. Using hydrodynamical driving and optical detection, we developed a method to detect the transport of single virus in real time through synthetic nanopores. We unveiled a jamming phenomenon specifically associated with virus confinement under flow. We showed that the interactions of viral particles with themselves and with the pore surface were critical for clog formation. Based on the detailed screening of the physical and chemical determinants, we proposed a simple dynamical model that recapitulated all the experimental observations. Our results pave the way for the study of jamming phenomena in the presence of more complex interactions.

Engineering of ion permeable planar membranes and polymersomes based on ß-cyclodextrin-cored star copolymers.

For polymersome-based nanoreactor purposes, we herein present the synthesis and characterization of well-defined star amphiphilic copolymers composed of a beta-cyclodextrin (ßCD) core and seven poly(butylene oxide)-block-polyglycidol (PBO-PGL) arms per side (ßCD-(PBO-PGL)14). The self-assembly behavior of 14-armed ßCD-(PBO-PGL)14 and PGL-PBO-PGL (linear analogues without the ßCD segment) was investigated using scattering techniques for comparison. The morphologies, including vesicles and micelles, are governed by the hydrophobic-to-hydrophilic (weight) ratio, regardless of the polymer architecture (linear or star). Interestingly, despite notable differences in polymer conformation, the produced supramolecular structures were evidenced to be fairly similar on the structural point of view. We subsequently investigated the ion permeability of the membranes of the self-assemblies focusing on the impact of the presence of ßCD. The results demonstrated that the ßCD-containing vesicular membranes are less permeable to H+, compared with ßCD-free vesicular membranes. The presence of ßCD in planar membranes also influences the K+Cl- permeability to some extent. Thus, ßCD-containing membranes can be considered as potential candidates in designing nano-containers towards applications where precise changes in environmental pH are required.

Synthesis of Double Hydrophilic Block Copolymers Poly(2-isopropyl-2-oxazoline-b-ethylenimine) and their DNA Transfection Efficiency.

Gene delivery is now a part of the therapeutic arsenal for vaccination and treatments of inherited or acquired diseases. Polymers represent an opportunity to develop new synthetic vectors for gene transfer, with a prerequisite of improved delivery and reduced toxicity compared to existing polymers. Here, the synthesis in a two-step's procedure of linear poly(ethylenimine-b-2-isopropyl-2-oxazoline) block copolymers with the linear polyethylenimine (lPEI) block of various molar masses is reported; the molar mass of the poly(2-isopropyl-2-oxazoline) (PiPrOx) block has been set to 7 kg mol-1 . Plasmid DNA condensation is successfully achieved, and in vitro transfection efficiency of the copolymers is at least comparable to that obtained with the lPEI of same molar mass. lPEI-b-PiPrOx block copolymers are however less cytotoxic than their linear counterparts. PiPrOx can be a good alternative to PEG which is often used in drug delivery systems. The grafting of histidine moieties on the lPEI block of lPEI-b-PiPrOx does not provide any real improvement of the transfection efficiency. A weak DNA condensation is observed, due to increased steric hindrance along the lPEI backbone. The low cytotoxicity of lPEI-b-PiPrOx makes this family a good candidate for future gene delivery developments.

Evidence of DNA transfer across a model membrane by a neutral amphiphilic block copolymer.

BACKGROUND: Neutral amphiphilic triblock copolymers have been shown to be efficient for gene transfection in vivo, especially by direct injection into the muscle. To contribute to a better understanding of the underlying mechanisms, in the present study, we investigated the properties of a poly(ethylene oxide-b-4-vinylpyridine) diblock copolymer as vector for nucleic acid transfer, with the particular aim of shedding some light on a possible mechanism explaining the internalization of DNA by the transfected cells. METHODS: Complexation of plasmid DNA with the PEO-b-P4VP diblock copolymer was investigated by ethidium bromide exclusion and gel electrophoresis assays. Interaction of the copolymer with a lipid model membrane was evaluated by electrophysiological assays and quantification of plasmid DNA was performed by quantitative polymerase chain reaction. In vivo luciferase transfection assays were finally performed. RESULTS: The diblock copolymer was found to poorly interact with DNA up to a mass ratio (copolymer/DNA) as high as 150. At a concentration of 36 µg/ml, it induced the formation of mainly transient (but sometimes permanent) pores and the formation of those pores allowed the translocation of plasmid DNA across the model membrane. However, only low transgene expression was obtained; the luciferase levels observed with the diblock being of the same order of magnitude as those observed with the corresponding PEO and P4VP homopolymers. CONCLUSIONS: These results strongly suggest that gene transfection by neutral block copolymers may involve the formation of cellular pores; in addition, they also highlight that in vivo gene transfection requires the use of adequately soluble block copolymers.

Supramolecular assemblies of histidinylated α-cyclodextrin in the presence of DNA scaffold during CDplexes formation.

α-Cyclodextrin was transformed in a cationic unit after per substitution with histidine (His-α-CD) and lysine (Lys-α-CD) molecules on the primary face. His-α-CD and Lys-α-CD were used to form electrostatic complexes (CDplexes) with a plasmid DNA encoding luciferase gene, and the ability of CDplexes to transfect mammalian cells was examined using HEK293-T7 cells. The luciferase activity in cells transfected with His-α-CDplexes was 8-fold higher than that obtained Lys-α-CDplexes. When the transfection was carried out in the presence of chloroquine, the luciferase activity with His-α-CDplexes and Lys-α-CDplexes increased 6 and 25 times, respectively. The lower enhancement with His-α-CDplexes confirmed that histidine induced a proton sponge effect inside endosomes upon imidazole protonation, favoring DNA delivery in the cytosol. At the same time, we found that the condensation of DNA with His-α-CD was unexpectedly stronger than that obtained with the lysyl-α-CD counterpart. Moreover, it was as strong as that observed with high molecular weight polylysine. NMR (ROESY and DOSY) investigations in the absence of DNA showed that an inclusion complex is formed between the imidazole ring of histidine and the hydrophobic cavity of CD but no His-α-CD polymers can be formed by intermolecular interactions. These results suggest that intermolecular interactions between imidazole and His-α-CD cavity could be involved to form supramolecular assemblies in the presence of a DNA scaffold leading to DNA condensation into low diameter particles.

Current Designs of Polymeric Platforms Towards the Delivery of Nucleic Acids Inside the Cells with Focus on Polyethylenimine.

BACKGROUND: Gene delivery is a promising technology for treating diseases linked to abnormal gene expression. Since nucleic acids are the therapeutic entities in such approach, a transfecting vector is required because the macromolecules are not able to efficiently enter the cells by themselves. Viral vectors have been evidenced to be highly effective in this context; however, they suffer from fundamental drawbacks, such as the ability to stimulate immune responses. The development of synthetic vectors has accordingly emerged as an alternative. OBJECTIVES: Gene delivery by using non-viral vectors is a multi-step process that poses many challenges, either regarding the extracellular or intracellular media. We explore the delivery pathway and afterwards, we review the main classes of non-viral gene delivery vectors. We further focus on the progresses concerning polyethylenimine-based polymer-nucleic acid polyplexes, which have emerged as one of the most efficient systems for delivering genetic material inside the cells. DISCUSSION: The complexity of the whole transfection pathway, along with a lack of fundamental understanding, particularly regarding the intracellular trafficking of nucleic acids complexed to non-viral vectors, probably justifies the current (beginning of 2021) limited number of formulations that have progressed to clinical trials. Truly, successful medical developments still require a lot of basic research. CONCLUSION: Advances in macromolecular chemistry and high-resolution imaging techniques will be useful to understand fundamental aspects towards further optimizations and future applications. More investigations concerning the dynamics, thermodynamics and structural parameters of polyplexes would be valuable since they can be connected to the different levels of transfection efficiency hitherto evidenced.

DNA nuclear targeting sequences for enhanced non-viral gene transfer: An in vitro and in vivo study.

An important bottleneck for non-viral gene transfer commonly relates to translocation of nucleic acids into the nuclear compartment of target cells. So-called 3NFs are optimized short nucleotide sequences able to interact with the transcription factor nuclear factor κB (NF-κB), which can enhance the nuclear import of plasmid DNA (pDNA) carrying such motifs. In this work, we first designed a consistent set of six pDNAs featuring a common backbone and only varying in their 3NF sequences. These constructions were then transfected under various experimental settings. In vitro, cationic polymer-assisted pDNA delivery in five human-derived cell lines showed the potential advantage of 3NF carrying pDNA in diverse cellular contexts. In vivo, naked pDNAs were hydrodynamically delivered to muscle hindlimbs in healthy mice; this direct accurate comparative (in the absence of any gene carrier) revealed modest but consistent trends in favor of the pDNAs equipped with 3NF. In summary, the results reported emphasize the implications of various parameters on NF-κB-mediated pDNA nuclear import; under specific conditions, 3NF can provide modest to substantial advantages for pDNA gene transfer, in vitro as well as in vivo. This study thus further underscores the potential of optimized nuclear import for more efficient non-viral gene transfer applications.

Helically shaped cation receptor: design, synthesis, characterisation and first application to ion transport.

The methyl ester of 8-oxo-8H-indeno[2',1':7,8]naphtho[1,2-b]thiophene-2-carboxylic acid (1) and its corresponding PEGylated ester were synthesised and fully characterised. X-ray diffraction studies on (1) confirmed the helical structure of the receptor and that it is self-assembled into layers by π-π interactions. An in-depth study by DFT calculations and MS experiments (ESI-MS, MS/MS, IMRPD and ESI-IMS-MS) was carried out between (1) and the physiological cation K+. The formation of supramolecular complexes between (1) and K+ with different stoichiometries was demonstrated and the cation K+ preferentially interacts with the oxygen atoms of the carbonyl bond of the ketone and ester groups and the sulphur atom of the heterocycle. The ability of the two synthesized aromatic architectures to transport ions across a model lipid membrane has been studied by electrophysiology experiments. The formation of pores was observed, even at nanomolar concentrations. Since the PEGylated molecule showed more regular pore definitions than the hydrophobic molecule, the introduction of a polar hydrophilic chain made it possible to control the orientation of the aromatic architectures within the membrane.

Temperature-Sensitive Amphiphilic Non-Ionic Triblock Copolymers for Enhanced In Vivo Skeletal Muscle Transfection.

It is reported that low concentration of amphiphilic triblock copolymers of pMeOx-b-pTHF-b-pMeOx structure (TBCPs) improves gene expression in skeletal muscle upon intramuscular co-injection with plasmid DNA. Physicochemical studies carried out to understand the involved mechanism show that a phase transition of TBCPs under their unimer state is induced when the temperature is elevated from 25 to 37 °C, the body temperature. Several lines of evidences suggest that TBCP insertion in a lipid bilayer causes enough lipid bilayer destabilization and even pore formation, a phenomenon heightened during the phase transition of TBCPs. Interestingly, this property allows DNA translocation across the lipid bilayer model. Overall, the results indicate that TBCPs exhibiting a phase transition at the body temperature is promising to favor in vivo pDNA translocation in skeletal muscle cells for gene therapy applications.