pH-Responsive Polyion Complex Vesicle with Polyphosphobetaine Shells.

When a bioactive molecule is taken into cells by endocytosis, it is sometimes unable to escape from the lysosomes, resulting in inefficient drug release. We prepared pH-responsive polyion complex (PIC) vesicles that collapse under acidic conditions such as those inside a lysosome. Furthermore, under acidic conditions, cationic polymer was released from the PIC vesicles to break the lysosome membranes. Diblock copolymers (P20M167 and P20A190) consisting of water-soluble zwitterionic poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) block and cationic or anionic blocks were synthesized via reversible addition-fragmentation chain transfer (RAFT) radical polymerization. Poly(3-(methacrylamidopropyl) trimethylammonium chloride) (PMAPTAC) and poly(sodium 6-acrylamidohexanoate) (PAaH) were used as the cationic and anionic blocks, respectively. The pendant hexanoate groups in the PAaH block are ionized in basic water and in phosphate buffered saline (PBS), while the hexanoate groups are protonated in acidic water. In basic water, PIC vesicles were formed from a charge neutralized mixture of oppositely charged diblock copolymers. At the interface of PIC vesicle and water exists biocompatible PMPC shells. Under acidic conditions, the PIC vesicles collapsed, because the charge balance shifted due to protonation of the PAaH block. After collapse of the PIC vesicles, P20A190 formed micelles composed of protonated PAaH core and PMPC shells, while P20M167 was released as unimers. PIC vesicles can encapsulate hydrophilic nonionic guest molecules into their hollow core. Under acidic conditions, the PIC vesicles can release the guest molecules and P20M167. The cationic P20M167 can break the lysosome membrane to efficiently release the guest molecules from the lysosomes to the cytoplasm.

Anticoagulant Properties of Poly(sodium 2-(acrylamido)-2-methylpropanesulfonate)-Based Di- and Triblock Polymers.

Di- and triblock copolymers with low dispersity of molecular weight were synthesized using radical addition-fragmentation chain transfer polymerization. The copolymers contained anionic poly(sodium 2-acrylamido-2-methylpropanesulfonate) (PAMPS) block as an anticoagulant component. The block added to lower the toxicity was either poly(ethylene glycol) (PEG) or poly(2-(methacryloyloxy)ethyl phosphorylcholine) (PMPC). The polymers prolonged clotting times both in vitro and in vivo. The influence of the polymer architecture and composition on the efficacy of anticoagulation and safety parameters was evaluated. The polymer with the optimal safety/efficacy profile was PEG47- b-PAMPS108, i.e., a block copolymer with the degrees of polymerization of PEG and PAMPS blocks equal to 47 and 108, respectively. The anticoagulant action of copolymers is probably mediated by antithrombin, but it differs from that of unfractionated heparin. PEG47- b-PAMPS108 also inhibited platelet aggregation in vitro and increased the prostacyclin production but had no antiplatelet properties in vivo. PEG47- b-PAMPS108 anticoagulant activity can be efficiently reversed with a copolymer of PEG and poly((3-(methacryloylamino)propyl)trimethylammonium chloride) (PMAPTAC) (PEG41- b-PMAPTAC53, HBC), which may be attributed to the formation of polyelectrolyte complexes with PEG shells without anticoagulant properties.

Complexes Covered with Phosphorylcholine Groups Prepared by Mixing Anionic Diblock Copolymers and Cationic Surfactants.

Anionic diblock copolymers (PmAn) composed of biocompatible polybetaine, poly(2-(methacryloyloxy)ethyl phosphorylcholine) (PMPC), and anionic poly(sodium 2-(acrylamido)-2-methylpropanesulfonate) (PAMPS) were synthesized via reversible addition-fragmentation chain transfer (RAFT) radical polymerization. Two types of diblock copolymers (P24A217 and P100A99) were prepared with different compositions. The PmAn/CTAB complexes were formed by a stoichiometrically charge-neutralized mixture of anionic PmAn and cationic cetyltrimethylammonium bromide (CTAB) micelles in water. The complexes prepared using P24A217 and P100A99 were vesicles and micelles, respectively, and were covered with hydrophilic PMPC shells. The complexes dissociated upon addition of NaCl because the complex was maintained through electrostatic interactions. The P24A217/CTAB vesicles could encapsulate uncharged hydrophilic guest molecules into the interior of the aqueous phase.

Preparation of upper critical solution temperature (UCST) responsive diblock copolymers bearing pendant ureido groups and their micelle formation behavior in water.

Poly(2-ureidoethyl methacrylate) (PUEM) was prepared via reversible addition-fragmentation chain transfer (RAFT) controlled radical polymerization and a post-modification reaction. PUEM shows upper critical solution temperature (UCST) behavior in aqueous solution. Although PUEM can dissolve in water above the UCST, it cannot dissolve in water below the UCST. Diblock copolymers (MmUn) composed of a biocompatible hydrophilic poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) block and a PUEM block with different compositions were prepared via RAFT radical polymerization and a post-modification reaction. "M" and "U" represent PMPC and PUEM blocks, respectively, and the subscripts represent the degree of polymerization of each block. M95U149 and M20U163 formed polymer micelles comprising a PUEM core and a PMPC shell below the critical aggregation temperature (Tc) in aqueous solution. Polymer micelles were formed from M20U163 below 32 °C, which can incorporate guest molecules into the core.

Preparation and characterization of polyion complex micelles with phosphobetaine shells.

A pair of oppositely charged diblock copolymers, poly(2-(methacryloyloxy)ethyl phosphorylcholine)-block-poly((3-(methacryloylamino)propyl)trimethylammonium chloride) (PMPC-b-PMAPTAC) and poly(2-(methacryloyloxy)ethyl phosphorylcholine)-block-poly(sodium 2-(acrylamido)-2-methylpropanesulfonate) (PMPC-b-PAMPS), was prepared via reversible addition-fragmentation chain transfer radical polymerization using a PMPC-based macro chain transfer agent. The pendant phosphorylcholine group in the hydrophilic PMPC block has anionic phosphate and cationic quaternary amino groups, which are neutralized within the pendant group. Therefore, the mixing of aqueous solutions of PMPC-b-PMAPTAC and PMPC-b-PAMPS leads to the spontaneous formation of simple core-shell spherical polyion complex (PIC) micelles comprising of a segregated PIC core and PMPC shells. The PIC micelles were characterized using (1)H NMR spin-spin (T2) and spin-lattice relaxation times (T1), diffusion-ordered NMR spectroscopy, static light scattering, dynamic light scattering (DLS), and transmission electron microscopy techniques. The hydrodynamic size of the PIC micelle depended on the mixing ratio of PMPC-b-PMAPTAC and PMPC-b-PAMPS; the maximum size occurred at the mixing ratio yielding stoichiometric charge neutralization. The PIC micelles disintegrated to become unimers with the addition of salts.

Photo Irradiation-Induced Core Crosslinked Poly(ethylene glycol)-block-poly(aspartic acid) Micelles: Optimization of Block Copolymer Synthesis and Characterization of Core Crosslinked Micelles.

We used photo irradiation to design core crosslinked polymeric micelles whose only significant physico-chemical change was in their physico-chemical stability, which helps elucidate poly(ethylene glycol) (PEG)-related immunogenicity. Synthetic routes and compositions of PEG-b-poly(aspartic acid) block copolymers were optimized with the control of n-alkyl chain length and photo-sensitive chalcone moieties. The conjugation ratio between n-alkyl chain and the chalcone moieties was controlled, and upon the mild photo irradiation of polymeric micelles, permanent crosslink proceeded in the micelle cores. In the optimized condition, the core crosslinked (CCL) micelles exhibited no dissociation while the non-CCL micelles exhibited dissociation. These results indicate that the photo-crosslinking reactions in the inner core were successful. A gel-permeation chromatography (GPC) measurement revealed a difference between the micellar-formation stability of CCL micelles and that of the non-CCL micelles. GPC experiments revealed that the CCL micelles were more stable than the non-CCL micelles. Our research also revealed that photo-crosslinking reactions did not change the core property for drug encapsulation. In conclusion, the prepared CCL micelles exhibited the same diameter, the same formula, and the same inner-core properties for drug encapsulation as did the non-CCL micelles. Moreover, the CCL micelles exhibited non-dissociable micelle formation, while the non-CCL micelles exhibited dissociation into single block copolymers.

Polyion Complex Vesicles with Solvated Phosphobetaine Shells Formed from Oppositely Charged Diblock Copolymers.

Diblock copolymers consisting of a hydrophilic poly(2-(methacryloyloxy)ethyl phosphorylcholine) (PMPC) block and either a cationic or anionic block were prepared from (3-(methacrylamido)propyl)trimethylammonium chloride (MAPTAC) or sodium 2-(acrylamido)-2-methylpropanesulfonate (AMPS). Polymers were synthesized via reversible addition-fragmentation chain transfer (RAFT) radical polymerization using a PMPC macro-chain transfer agent. The degree of polymerization for PMPC, cationic PMAPTAC, and anionic PAMPS blocks was 20, 190, and 196, respectively. Combining two solutions of oppositely charged diblock copolymers, PMPC-b-PMAPTAC and PMPC-b-PAMPS, led to the spontaneous formation of polyion complex vesicles (PICsomes). The PICsomes were characterized using ¹H NMR, static abd dynamic light scattering, transmittance electron microscopy (TEM), and atomic force microscopy. Maximum hydrodynamic radius (Rh) for the PICsome was observed at a neutral charge balance of the cationic and anionic diblock copolymers. The Rh value and aggregation number (Nagg) of PICsomes in 0.1 M NaCl was 78.0 nm and 7770, respectively. A spherical hollow vesicle structure was observed in TEM images. The hydrodynamic size of the PICsomes increased with concentration of the diblock copolymer solutions before mixing. Thus, the size of the PICsomes can be controlled by selecting an appropriate preparation method.

Polyion complex vesicles (PICsomes) from strong copolyelectrolytes. Stability and in vitro studies.

Polymer vesicles formed by a pair of oppositely charged diblock copolyelectrolytes (PICsomes) are considered as a good alternative to polymersomes formed by amphiphilic copolymers. Here, we report on inherent stability and in vitro biocompatibility of PICsomes prepared from a pair of oppositely charged zwitterionic-ionic copolymers, in which the ionic block is a strong polyelectrolyte. Our results demonstrated that the PICsomes are highly stable over a wide range of pH and temperatures. Direct microscopic observations revealed that the PICsomes retain their morphology in the presence of human serum. In vitro studies using human skin fibroblasts (HSFs) showed that the polymer vesicles are not cytotoxic and do not affect cell proliferation and adhesion. A model hydrophilic dye was effectively incorporated into the PICsomes by simple mixing. Using confocal microscopy observations, we demonstrated that the dye-loaded PICsomes are efficiently internalized by the cells and are located predominantly in endo/lysosomal compartments. Thus, the PICsomes have promising potential for use as nanocontainers for substances of biomedical interest.

Effect of Polycation Structure on Interaction with Lipid Membranes.

Interaction of polycations with lipid membranes is a very important issue in many biological and medical applications such as gene delivery or antibacterial usage. In this work, we address the influence of hydrophobic substitution of strong polycations containing quaternary ammonium groups on the polymer-zwitterionic membrane interactions. In particular, we focus on the polymer tendency to adsorb on or/and incorporate into the membrane. We used complementary experimental and computational methods to enhance our understanding of the mechanism of the polycation-membrane interactions. Polycation adsorption on liposomes was assessed using dynamic light scattering (DLS) and zeta potential measurements. The ability of the polymers to form hydrophilic pores in the membrane was evaluated using a calcein-release method. The polymer-membrane interaction at the molecular scale was explored by performing atomistic molecular dynamics (MD) simulations. Our results show that the length of the alkyl side groups plays an essential role in the polycation adhesion on the zwitterionic surface, while the degree of substitution affects the polycation ability to incorporate into the membrane. Both the experimental and computational results show that the membrane permeability can be dramatically affected by the amount of alkyl side groups attached to the polycation main chain.

Stable polymersomes based on ionic-zwitterionic block copolymers modified with superparamagnetic iron oxide nanoparticles for biomedical applications.

Stable polymersomes with semipermeable membranes were prepared by simple mixing of two oppositely charged diblock copolymers containing zwitterionic and cationic (PMPC20-b-PMAPTAC190) or anionic (PMPC20-b-PAMPS196) blocks. The formation of vesicular structures in the mixed solution of the block copolymers was confirmed by direct observation using the cryo-TEM technique. Superparamagnetic iron oxide nanoparticles coated with a cationic chitosan derivative (SPION/CCh) and decorated with a fluorescent probe molecule were next incorporated into the polymersome structure. The average diameter of SPION/CCh-polymersomes estimated using cryo-TEM was about 250 nm. Surface topography of the SPION/CCh-loaded vesicles was imaged using AFM and the magnetic properties of these objects were confirmed by MFM and MRI measurements. The ability of SPION/CCh-polymersomes to affect T2 relaxation time in MRI was evaluated based on the measurements of r2 relaxivity. The obtained value of r2 (573 ± 10 mM-1 s-1) was quite high. The cytotoxicity and intracellular uptake of the SPION/CCh-loaded vesicles into EA.hy926 cells were studied. The results indicate that the SPION/CCh-polymersomes seem to be internalized by vascular endothelium and are not cytotoxic to endothelial cells up to 1 µg Fe per mL. Therefore, it can be suggested that SPION/CCh-polymersomes could prove useful as T2 contrast agents in the MRI of endothelium.