Robust Polyion Complex Vesicles (PICsomes) Based on PEO-b-poly(amino acid) Copolymers Combining Electrostatic and Hydrophobic Interactions: Formation,siRNA Loading and Intracellular Delivery

Two pairs of oppositely charged PEO-b-poly(amino acid) copolymers with neutral poly(ethylene oxide) block and polypeptide block composed of the hydrophobic l -phenylalanine (Phe) amino acid mixed with either negative l -glutamic acid (Glu) or positive l -lysine (Lys) units are synthesized. N-carboxyanhydride (NCA) ring opening polymerization is performed with either PEO₄₆-NH₂ or PEO₁₁₄-NH₂ macroinitiators, leading respectively to PEO₄₆-b-P(Glu₁₀₀-co-Phe₆₅) and PEO₄₆-b-P(Lys₁₀₀-co-Phe₆₅), and PEO₁₁₄-b-P(Glu₆₀-co-Phe₄₀) and PEO₁₁₄-b-P(Lys₆₀-co-Phe₄₀). Polyion complexes (PIC) formed at near charge equilibrium led to vesicle formation (PICsomes), as shown by DLS, zetametry, and TEM. The good stability of PICsomes, even in high salinity media, is interpreted by π π stacking hydrophobic interactions between the Phe residues, playing the role of “physical cross-linking”. These PICsomes are successfully loaded with small interfering ribonucleic acid (siRNA) directed against firefly luciferase enzyme expression. They also exhibit minimal cell cytotoxicity while superior silencing efficacy is shown by cell bioluminescence assay as compared to free siRNA and a standard lipofectamine-siRNA complex. As such, self-assembly of oppositely charged PEO-b-poly(amino acids) block copolymers enables forming PICsomes of high stability thanks to π π interactions of the Phe co-monomer in the polypeptide block, with high potential as biocompatible nanocarriers for RNA interference.     

Synthesis and characterization of new ABA triblock copolymers with poly[3(S)-isobutylmorpholine-2,5-dione] and poly(ethylene oxide) blocks

ABA type block copolymers with poly[3(S)-isobutylmorpholine-2,5-dione] (PIBMD, A) and poly(ethylene oxide) (M_n = 6 000, PEO, B) blocks, PIBMD-b-PEO-b-PIBMD, were synthesized via ring-opening polymerization of 3(S)-isobutylmorpholine-2,5-dione in the presence of hydroxytelechelic poly-(ethylene oxide) with stannous octoate as a catalyst. M_n of the resulting copolymers increases with increasing 3(S)-isobutylmorpholine-2,5-dione content in the feed at constant mole ratio of monomer (M) to catalyst (C) (M/C = 125). No racemization of the leucine residue takes place during both homopolymerization of IBMD and polymerization of IBMD in the presence of PEO and Sn(Oct)₂. The melting temperature of the PIBMD segments in the block copolymers depends on the length of the PIBMD blocks. The melting temperature of the PEO blocks is lower than that of the homopolymer, and the crystallinity of the PEO block decreases with increasing length of the PIBMD blocks. The PIBMD block crystallizes first upon cooling from the melt. This leads to only imperfect crystallization or no crystallization of the PEO blocks.     

Regulation in free-radical polymerization of vinyl acetate and synthesis of end-group modified poly(vinyl alcohol)s

A kinetical investigation on the consumption of a chain transfer agent in the free-radical polymerization of vinyl acetate (VAc) was carried out. A new method to control the degree of polymerization of poly(vinyl acetate) (PVAc) or poly(vinyl alcohol) (PVA) as well as the end group content by continuous addition of a chain transfer agent, whose chain transfer constant is much larger than unity such as thiol compounds, during the course of VAc polymerization was proposed. The method using 2-mercaptoethanol was applied to the preparation of lowmolecular-weight PVAc and PVA with M?_w/M?_n ≈ 2.     

Morphology,crystallization and thermal behaviour of poly(ethylene oxide)/poly(vinyl acetate) blends

Blends of poly(ethylene oxide) (PEO) and poly(vinyl acetate) (PVAc) show a unique value of the glass transition temperature, intermediate between that of plain polymers. The addition of PVAc to PEO causes a depression in both the spherulite growth rate (G) and the overall kinetic rate constant (K_n). Such depression is larger at higher undercooling and, at a given crystallization temperature, it increases with the content of PVAc. The experimental G and K_n data were analyzed by means of latest kinetic theories in order to determine the influence of composition on the process of surface secondary nucleation. The melt behaviour of PEO/PVAc blends cannot be explained only in terms of diluent effects due to the compatibility of the components in the melt. Especially, at lower undercooling it is likely that annealing and morphological effects must also be taken into account. The morphology of thin films of blends, isothermally crystallized from melt, suggests that an amorphous mixed phase (PEO + PVAc) is formed in interlamellar regions. It was found that plain PEO crystals grow according to a regime I process of surface secondary nucleation while in the case of blends the crystals of PEO grow via regime II mechanism.     

Spherical Compound Micelles with Lamellar Stripes Self‐Assembled from Star Liquid Crystalline Diblock Copolymers in Solution

Detailed investigations on the self‐assembly of amphiphilic star block copolymers composed of three‐arm poly(ethylene oxide) (PEO) and poly(methacrylate) (PMAAz) with an azobenzene side chain (denoted as 3PEO‐b‐PMAAz) into stable spherical aggregates with clear lamellar stripes in solution are demonstrated. Four block copolymers, 3PEO₁₂‐b‐PMA(Az)₃₃, 3PEO₂₂‐b‐PMA(Az)₃₁, 3PEO₂₂‐b‐PMA(Az)₆₂, and linear PEO₆₈‐b‐PMA(Az)₃₁, are synthesized. The liquid crystalline properties of the block copolymers are studied by differential scanning calorimetry, polarized optical microscopy techniques, and wide‐angle X‐ray diffraction. The morphologies of the compound micelles self‐assembled in tetrahydrofuran (THF)/water mixtures are observed by means of transmission electron microscopy and scanning electron microscopy. The size of the spherical micelles is influenced by the self‐assembly conditions and the lengths of two blocks. The well‐defined three‐arm architecture of the hydrophilic blocks is a key structural element to the formation of stable spherical compound micelles. The micelle surface integrity is affected by the lengths of PEO blocks. The lamellar stripes are clearly observed on these micelles. This work provides a promising strategy to prepare functional stable spherical compound micelles self‐assembled by amphiphilic block copolymers in solution.     

Calcium Alcoholates of Hydroxytelechelic Poly(ethylene oxide)s as Initiators for the Ring‐Opening Polymerization of 3‐(S)‐Isopropylmorpholine‐2,5‐dione

Block copolymers with poly[3‐isopropylmorpholine‐2,5‐dione] (PIPMD) and poly(ethylene oxide) (M̄_n = 6 000, PEO) blocks, PIPMD‐b‐PEO‐b‐PIPMD, were synthesized via the ring‐opening polymerization of 3‐(S)‐isopropylmorpholine‐2,5‐dione (IPMD) in the presence of the calcium alcoholate of hydroxytelechelic poly(ethylene oxide) as an initiator at 140°C within 24 or 96 h. The number‐average molecular weight (M̄_n) of the resulting copolymers increases with increasing IPMD content in the feed and with reaction time. According to ¹H NMR spectroscopic analysis, about 40% of IPMD is racemized during polymerization within 96 h at 140°C, while about 12% is racemized within 24 h. The melting temperature of the PEO block upon first heating is lower than that of pure PEO homopolymer, and the melting endotherm decreases with increasing length of the PIPMD block upon second heating.     

Synthesis and Characterization of Amphiphilic Poly(ethylene oxide)‐block‐poly(hexyl methacrylate) Copolymers

We describe the preparation of amphiphilic diblock copolymers made of poly(ethylene oxide) (PEO) and poly(hexyl methacrylate) (PHMA) synthesized by anionic polymerization of ethylene oxide and subsequent atom transfer radical polymerization (ATRP) of hexyl methacrylate (HMA). The first block, PEO, is prepared by anionic polymerization of ethylene oxide in tetrahydrofuran. End capping is achieved by treatment of living PEO chain ends with 2‐bromoisobutyryl bromide to yield a macroinitiator for ATRP. The second block is added by polymerization of HMA, using the PEO macroinitiator in the presence of dibromobis(triphenylphosphine) nickel(II), NiBr₂(PPh₃)₂, as the catalyst. Kinetics studies reveal absence of termination consistent with controlled polymerization of HMA. GPC data show low polydispersities of the corresponding diblock copolymers. The microdomain structure of selected PEO‐block‐PHMA block copolymers is investigated by small angle X‐ray scattering experiments, revealing behavior expected from known diblock copolymer phase diagrams.

SAXS diffractograms of PEO‐block‐PHMA diblock copolymers with 16, 44, 68 wt.‐% PEO showing spherical (A), cylindrical (B), and lamellae (C) morphologies, respectively.     

Epoxy prepolymers cured with boron trifluoride-amine complexes, 2. Polymerization mechanisms

The kinetics of the polymerization of phenoxymethyloxirane ( 6 ) initiated by the BF₃ complex of 4-chloroaniline ( 7 ) solubilized in oligo(ethylene oxide) of molecular weight 300 (PEO) is characterized by an initial induction period, followed by a propagation step with a rate R_p = k [ 6 ] · [ 7 ]^0,32 · [PEO]^?0,55. The length of the induction period decreases with [ 7 ] and increases with [PEO]. However, R_p increases with [ 7 ] and decreases with [PEO]. The reaction can be explained by the activated monomer mechanism.     

Structure of polystyrene-block-poly(ethylene oxide) diblock copolymer micelles in water

Micellization in water of two homologous series of AB-type diblock copolymers, composed of polystyrene (PS) as the A block and poly(ethylene oxide) (PEO) as the B block, were investigated by small-angle X-ray scattering (SAXS) and dynamic light scattering (DLS). The copolymers have molecular weights M _n in the range 2 000—34 800, and have in a given series, the same number of repeating units of the PS block, (N_PS = 10 and 38), and a variable number of repeating units of the PEO block (N_PEO values in the range 23–704). In order to avoid secondary association of micelles, a dialysis technique was used to prepare the micellar systems, in the case of copolymers having high M _n values of the PS block. The experimental micelle properties such as the core radius R_c and the aggregation number N of non-equilibrium structures, so called “frozen micelles”, obtained by dialysis, were found to be independent of the copolymer characteristics. However, for equilibrium structures, obtained by direct solubilization of the copolymers (N_PS = 10) in water, R_c and N were found to decrease with increasing N_PEO for the homologous series.     

Functional poly[(ethylene oxide)-co-(β-benzyl-L-aspartate)] polymeric micelles: block copolymer synthesis and micelles formation

Well-defined α-methoxy-ω-amino and α-hydroxy-ω-amino poly(ethylene oxides) (PEOs), obtained by chemical modifications of α-hydroxy-ω-amino PEO, were studied for block copolymerization with β-benzyl-L -aspartate-N-carboxy anhydride (BLA-NCA); the block copolymers were obtained via polymerization of BLA-NCA with the primary amino end-groups of the PEOs as initiator in the mixture CHCl₃/N,N-dimethylformamide (DMF) (vol. ratio10/1). Gel-permeation chromatography (GPC) of both block copolymers showed the presence of BLA oligomers. α-Methoxy PEO/PBLA and α-hydroxy PEO/PBLA block copolymers were submitted to selective precipitation in 2-propanol; this method allowed total elimination of oligomers as shown by GPC of the purified block copolymers. Moreover, for each block copolymer, the number of BLA units determined by ¹H NMR spectroscopy (in CDCl₃) was in good agreement with the number calculated from the ratio BLA-NCA/amino end-groups of PEO. The polymeric micelles having hydroxy functions or methoxy groups on the outer-shell were prepared by dialysis against water of the corresponding solution of the pure block copolymers. These polymeric micelles were characterized by dynamic light scattering (diameter) and by fluorescence spectroscopy (critical micellar concentration, cmc) using pyrene as a fluorescence probe. Both polymeric micelles have a small diameter (<50nm) and a very low cmc (<20 mg/L in water).     

Effect of the number of crystallizable blocks on the lamellar crystalline structure of block copolymers from poly(4-tert-butylstyrene) and poly(ethylene oxide)

Linear block copolymers tBSEO with an amorphous block of poly(4-tert-butylstyrene) (PtBS) and a crystallizable block of poly(ethylene oxide) (PEO) as well as branched block copolymers tBS(EO)₂ with two PEO chains linked at the same end of a PtBS block exhibit a lamellar liquid-crystalline structure in the dry state as well as in the presence of a preferential solvent of the PtBS block. In this structure the crystallized chains are folded in two superposed layers. To investigate the effect of the number of the PEO blocks, a linear and a branched copolymer with identical relative molecular masses and copositions were studied by X-ray diffraction and differential scanning calorimetry. It was shown that the melting temperature, the degree of crystallinity, and the number of folds of the PEO chains are higher for the linear copolymer than for the branched one.     

Synthesis and characterization of poly(ethylene oxide) and poly(perfluorohexylethyl methacrylate) containing triblock copolymers

A new series of poly(perfluorohexylethyl methacrylate)‐block‐poly(ethylene oxide)‐block‐poly(perfluorohexylethyl methacrylate), PFMA‐b‐PEO‐b‐PFMA triblock copolymers has been synthesized by atom transfer radical polymerization using bifunctional PEO macroinitiators. The molecular structure of the block copolymers was confirmed by ¹H NMR spectroscopy and SEC. X‐ray scattering studies have been carried out to investigate their bulk properties. SAXS has shown cubic arrangement of spheres (bcc), hexagonally packed cylinders (hpc) and lamellar microdomain formation in the melt of triblock copolymers investigated, depending on composition. Crystallization was, however, found to destroy the ordered melt morphology and imposes a lamellar crystalline structure. WAXS, DSC and polarized light microscopy measurements confirmed the crystallization of PEO segments in block copolymers. Long PFMA blocks were found to have significant effect on PEO crystallization.

Synthesis of triblock copolymers of EO and FMA by ATRP.     

Well-defined graft copolymers containing a poly(methacrylate) backbone and functional or block poly(vinyl ether) side chains

The anionic random copolymerization of methyl methacrylate (MMA) and 2-(1-acetoxyethoxy)ethyl methacrylate (AEEMA) was carried out using 1,1-diphenylhexyllithium (DPHL) as initiator, in the presence of LiCl ([LiCl]/[DPHL]₀ = 2), in tetrahydrofuran (THF), at –60°C. The resulting polymer, poly-(MMA-co-AEEMA), has a controlled molecular weight and a narrow molecular weight distribution (M_w/M_n = 1.05 ˜ 1.09). Without quenching, toluene, EtAlCl₂ and a functional monomer [2-acetoxyethyl vinyl ether (AcVE), 2-chloroethyl vinyl ether (ClVE) or 2-vinyloxyethyl methacrylate (VEMA)] were introduced into the above THF solution of the copolymer at 20°C. Every side chain of AEEMA unit of poly(MMA-co-AEEMA) was activated by EtAlCl₂ to induce the cationic polymerization of the functional monomer. THF, which was used as solvent in the preparation of the copolymer of MMA and AEEMA, acted as a Lewis base in the latter cationic polymerization, thus stabilizing the propagating site. By using this procedure, a controlled cationic polymerization of a functional monomer was achieved and a well-defined graft copolymer with functional side chains was obtained. Instead of a single functional monomer, the simultaneous addition of isobutyl vinyl ether (IBVE) and AcVE, ClVE or VEMA during the second step cationic polymerization process generated a graft copolymer with random copolymer side chains. Furthermore, a graft copolymer with block side chains could also be prepared by performing a block copolymerization during the second cationic grafting step by adding sequentially AcVE (ClVE or VEMA) and IBVE or vice versa. Every graft copolymer thus obtained possessed a high purity, controlled graft number and molecular weight as well as a narrow molecular weight distribution (M_w/M_n = 1.12 ˜ 1.25).     

Synthesis of linear poly(4-vinylbenzaldehyde) by means of anionic living polymerization of 1,3-dimethyl-2-(4-vinylphenyl)imidazolidine and subsequent hydrolysis

Poly(4-vinylbenzaldehyde)s ( 4 ) of known chain lengths and of narrow molecular weight distributions (M?_w/M?_n ≈ 1,1) were synthesized by means of anionic living polymerization of 1,3-dimethyl-2-(4-vinylphenyl)imidazolidine ( 1 ) with oligo[α-methylstyryl]potassium ( 2 ) and subsequent acid hydrolysis to remove the imidazolidine protective group. The living polymer of 1 can initiate further polymerization of α-methylstyrene, resulting in the preparation of a triblock copolymer of the type poly[α-methylstyrene-b-(4-vinylbenzaldehyde)-b-α-methylstyrene] in quantitative yield after hydrolysis.     

Hypochromism of poly(N-vinylcarbazole)

The hypochromy of poly(N-vinylcarbazole) in relation to N-ethylcarbazole was estimated for samples of different origin (cationic and radical polymerization). The effect of solvent polarizability and the influence of temperature as well as of the addition of nonsolvent were studied. An increase in hypochromy of the ¹L_b transition and a decrease in that of the ¹L_a transition with increasing polarizability of the solvent were observed. No change in hypochromy connected with increasing temperature and addition of nonsolvent was found.     

Preparation and characterization of poly(methyl-3,3,3-trifluoropropylsiloxane-co-dimethylsiloxane)

Methyl-3,3,3-trifluoropropylsiloxane (F)-dimethylsiloxane (D) random and block copolymers were prepared. The random copolymers were prepared by equilibrium copolymerization starting from a mixture of cyclic F and D siloxanes with potassium silanolate as the catalyst. The F-D block copolymer was prepared by sequential anionic living polymerization of strained cyclic trisiloxanes using butyllithium as initiator, first polymerizing D₃ then adding F₃ after consumption of D₃. The copolymer microstructure was established by means of ²⁹Si NMR, differential scanning calorimetry (DSC), and gel-permeation chromatography (GPC). Characteristic glass transition temperature (T_g) shifts were observed depending on the F:D ratio of the random copolymers. It was demonstrated that the tensile strength of the poly(methyl-3,3,3-trifluoropropylsiloxane)-poly(dimethylsiloxane) (PTFPMS-PDMS) blend system was improved when either of the copolymers was added.     

Allyl End‐Functionalized Poly(ethylene oxide)‐block‐poly(methylidene malonate 2.1.2) Block Copolymers: Synthesis,Characterization, and Chemical Modification

Summary: Poly(ethylene oxide)‐block‐poly(methylidene malonate 2.1.2) block copolymer (PEO‐b‐PMM 2.1.2) bearing an allyl moiety at the poly(ethylene oxide) chain end was synthesized by sequential anionic polymerization of ethylene oxide (EO) and methylidene malonate 2.1.2 (MM 2.1.2). This allyl functional group was subsequently modified by reaction with thiol‐bearing functional groups to generate carboxyl and amino functionalized biodegradable block copolymers. These end‐group reactions, performed in good yields both in organic media and in aqueous micellar solutions, lead to functionalized PEO‐b‐PMM 2.1.2 copolymers which are of interest for cell targeting purposes.

Synthetic route to α‐allyl functionalized PEO‐b‐PMM 2.1.2 copolymers.     

Novel Olefin Block Copolymer,Polypropene‐block‐poly(methylene‐1,3‐cyclopentane‐co‐propene), Synthesized from Propene and 1,5‐Hexadiene by a Modified Stopped‐Flow Method

A block copolymer of propene and 1,5‐hexadiene, polypropene‐block‐poly(methylene‐1,3‐cyclopentane‐co‐propene) (PP‐b‐(PMCP‐co‐PP)), was synthesized using a modified stopped‐flow polymerization method with an MgCl₂‐supported Ziegler catalyst. Regarding the basic characteristics of the PP‐b‐(PMCP‐co‐PP), the block formation was investigated in detail. The obtained block copolymer showed a unimodal GPC curve without any peak in the low molecular weight region. It was also clear that the molecular weight of each part could be controlled by changing the polymerization time (from about 0.1 to 0.2 s). Furthermore, the elution pattern by temperature‐rising elution fractionation clearly showed that the block copolymer eluted in each temperature region between 20°C to 120°C was mainly composed of a unified component. Even after extraction with boiling heptane, the ¹³C NMR spectra of the block copolymer showed that the signals from PMCP‐co‐PP remained unchanged, but disappeared in the blend of polypropene and PMCP‐co‐PP. The differential scanning calorimetry results and optical microscopic observations indicated not only the formation of a block copolymer having a chemical linkage between polypropene and PMCP‐co‐PP, but also the regulation of the crystalline distribution in the block copolymer by changing the composition of each block part.     

Morphology of (methoxyethoxy/trifluoroethoxy)phosphazene copolymers

Block copolymers were synthesized by the anionically initiated copolymerization of (CH₃OCH₂CH₂O)(CF₃CH₂O)₂P? NSi(CH₃)₃, followed by the addition of (CF₃CH₂O)₃P? NSi(CH₃)₃. Random copolymers were made by simultaneous polymerization of these monomers. These copolymers exhibit a linear dependency on the mole fraction “m” of the repeating units bearing a methoxyethoxy pendant side group as well as on molecular weights. The thermal and morphological characteristics of the block copolymers are different from those of the random copolymers of analogous “m” and molecular weight. All copolymers undergo a mesophase T(1) transition for a range of temperatures depending upon “m” and molecular weights of the copolymers. Morphological and structural features essentially resemble those of the low molecular weight (trifluoroethoxy)phosphazene homopolymer. Upon heating and cooling the solution cast copolymer specimens through T(1), most of them transform into an orthorhombic form with the unit cell dimensions a = 2,060 nm, b = 0,940 nm and c = 0,486 nm from their initial monoclinic form with a = 1,003 nm, b = 0,937 nm, c = 0,486 nm and γ γ 91°. These unit cell dimensions agree completely with those of the low molecular weight PBFP. Complicated morphologies comprised of square and globular shapes that depend upon the copolymer composition were obtained from dilute tetrahydrofuran/p-xylene copolymer solutions. Electron microscopy directly reveals that chain extension occurs for the meltcrystallized copolymer specimen. The non-crystallizable minor component in the block copolymers is rejected from the crystal lattice. In the random copolymers, the methoxyethoxy pendant side group enters into the crystal lattice and influences their morphological and structural features.     

Effect of poly(dimethylsiloxane)-block-poly(oligo (ethylene glycol) methacrylate) amphiphilic block copolymers on dermal fibroblast viability and proliferation

Abstract

In this work, well-defined poly(dimethylsiloxane)-b-poly(oligo (ethylene glycol) methacrylate) (PDMS-b-POEGMA) amphiphilic block copolymers were synthesized and their effect on human dermal fibroblast were investigated. Anionic ring opening polymerization (ROP) and atom transfer radical polymerization (ATRP) were used to synthesis the block copolymers. The molecular weight of synthesized copolymers ranged from 1000 to 2300?Da by changing the number of both PDMS and POEGMA units. It was found that the copolymer having low molecular weight decreased the fibroblast viability and proliferation by inducing apoptosis. It was proved by flow cytometry and TUNEL assay that human dermal fibroblast experienced apoptosis after exposure to synthesized amphiphilic copolymers. The results of this work suggest the use of PDMS-b-POEGMA amphiphilic copolymers with low molecular weight for hypertrophic scars remediation.