Ureido-derivatized polymers based on both poly(allylurea) and poly(L-citrulline) exhibit UCST-type phase transition behavior under physiologically relevant conditions.

There are few examples of polymers that exhibit upper critical solution temperature (UCST) behavior under physiological conditions of temperature, pH, and ionic strength. In this study, we demonstrated that polymers with ureido groups undergo UCST-type phase transitions under physiologically relevant conditions. Poly(allylurea) copolymers showed UCST behavior at pH 7.5 in 150 mM NaCl even at the low polymer concentration of 0.13 mg/mL. Their phase separation temperatures (T(p)) could be controlled up to 65 °C. Similar thermosensitivity was observed with copolypeptides consisting of L-citrulline having an ureido group. This is the first demonstration of a non-vinyl polymer that shows UCST behavior under physiologically relevant conditions. We suggest that the ureido modification will be useful for production of polymer materials with UCST behavior in aqueous media.

Thermosensitive transparent semi-interpenetrating polymer networks for wound dressing and cell adhesion control.

Thermosensitive, transparent, and flexible semi-interpenetrating polymer networks (semi-IPNs) composed of segmented polyurethane urea/poly(N-isopropylacrylamide) (SPUU/ PNiPAAm) were new class of materials, which holds promise for its potential use as wound dressings. A series of semi-IPNs, obtained via thermal initiated polymerization of NiPAAm, were characterized by infrared spectroscopy (IR), nuclear magnetic resonance (NMR), dynamic viscoelastic measurements, wide-angle X-ray diffraction (WAXD), and mechanical properties. The resulting semi-IPNs were also investigated for their dynamic water contact angles, thermodynamic interaction parameters, in vitro drug release, and cell adhesion and detachment. The semi-IPNs with differing compositions possess good mechanical properties in both dry and hydrated states. In addition, NIH3T3 fibroblasts can attach to and detach from these semi-IPN films with varying temperature. In addition, these film extracts do not show significant cytotoxicity. Therefore, these materials have great potential for the construction of a new generation of dressings and cell transplantation for wound healing.

Polyelectrolyte-assisted transconformation of a stem-loop DNA.

A cationic copolymer triggered dimerization of a self-complementary stem-loop DNA. The dimerization was faster than spontaneous dissociation of the dimer. Reversible transformation between stem-loop and dimer structures was driven by switching on/off copolymer activity.

Tumor delivery of Photofrin® by PLL-g-PEG for photodynamic therapy.

Photofrin® (porfimer sodium) is a photosensitive reagent used for photodynamic therapy (PDT) of tumors and dysplasias. Because only photo-irradiated sites are damaged, PDT is less invasive than systemic treatments. However, a photosensitive reaction is a major side effect of systemically delivered Photofrin. To enhance localization of Photofrin to tumors, we have formulated Photofrin with the tumor-localizing graft copolymer poly(ethylene glycol)-grafted poly(l-lysine), PLL-g-PEG. We demonstrate that Photofrin preferentially interacts with PLL-g-PEG through both ionic and hydrophobic interactions. The serum competitive study showed that the highly PEG-grafted PLL is better for preventing serum binding to the Photofrin/PLL-g-PEG complex. In tumor-bearing mice, formulation of Photofrin with PLL-g-PEG enhanced tumor localization of Photofrin as twice as Photofrin alone and concomitantly suppressed the photosensitivity reaction drastically.

Effective tumor treatment by VEGF siRNA complexed with hydrophobic poly(amino acid)-modified polyethylenimine.

Two copolymers are designed and synthesised for siRNA delivery based on polyethylenimine by grafting hydrophilic acrylamide segments and hydrophobic poly(γ-benzyl L-glutamate). The amphiphilic PEI-PBLG/siRNA complex demonstrates high gene silencing efficiency in the absence or presence of 10 vol% and 50 vol% sera in vitro. The anti-tumor effects in vivo are evaluated in luciferase-bearing mice expressing CT26 tumors. PEI-PBLG/siVEGF complex provides a higher and more sustained suppressive effect by reducing VEGF mRNA expression in the tumors, leading to higher tumor growth inhibition efficacy. Further studies on the potential use of the PEI-PBLG carrier system in mediating the silencing of genes other than VEGF or in other tumor models are recommended.

Dual crosslinked hydrogel nanoparticles by nanogel bottom-up method for sustained-release delivery.

Polysaccharide-PEG hybrid nanogels (CHPOA-PEGSH) crosslinked by both covalent ester bonds and physical interactions were prepared by the reaction of a thiol-modified poly(ethylene glycol) (PEGSH) with acryloyl-modified cholesterol-bearing pullulan (CHPOA). Experimental parameters, including CHPOA concentration, the degree of acryloyl substitution of CHPOA, and the initial amounts of CHPOA and PEGSH, were modified in order to assess their effect on the size of the nanogels (50-150 nm) and on their degradation kinetics, monitored by dynamic light scattering (DLS) and asymmetrical flow field-flow fractionation (AF4) chromatography. Rhodamine-labeled nanogels were injected intravenously into mice and their concentration in blood was determined by a fluorescence assay as a function of post-injection time. The elimination half-life (t(1/2)) of CHPOA-PEGSH nanoparticles was about 15-fold longer (18 h) than that of CHP nanogels (1.2 h). The half-life enhancement of CHPOA-PEGSH was attributed to the presence of the crosslinker PEG chains, which prevent non-specific protein adsorption, and to the slow hydrolysis kinetics of the crosslinking esters in the biological milieu. The hybrid CHPOA-PEGSH nanogels are expected to be useful as injectable nanocarriers for drugs and proteins, in view of their low surface fouling and slow hydrolysis rate.

The role of cationic comb-type copolymers in chaperoning DNA annealing.

G-rich oligonucleotides tend to fall into kinetically trapped unstable structures because of their conformational polymorphism. Nucleic acid chaperones accelerate association of nucleic acids assemblies into the thermodynamically most stable conformations by decreasing the energy barrier for breakage or re-assembly of base pairings. Here, we report that an artificial nucleic acid chaperone, a cationic comb-type copolymer, promotes tetramolecular quadruplex assembly from mixtures of two different G-rich sequences, 5'-TGGGGT-3' (TG(4)T) and 5'-TGGGGGT-3' (TG(5)T). A 1:1 mixture of TG(4)T and TG(5)T mainly gave [TG(4)T▪(TG(5)T)(3)], [(TG(4)T)(2)▪(TG(5)T)(2)] and [(TG(4)T)(3)▪TG(5)T] heteroquadruplexes when the mixture was annealed by cooling from 90 °C to 4 °C at 1.0 °C/min. At a cooling rate of 0.01 °C/min the mixture mostly assembled into [TG(4)T](4) and [TG(5)T](4) homoquadruplexes, indicating that homoquadruplexes were thermodynamically more stable than heteroquadruplexes. In the presence of the copolymer, mainly homoquadruplexes were obtained at cooling rate of 1 °C/min, suggesting that the copolymer promoted formation of the thermodynamically most stable structures. We also showed that the copolymer facilitated the recombination of heteroquadruplexes to homoquadruplexes even at 20-30 °C, implying that the copolymer can promote thermodynamically preferred quadruplex assembly from oligonucleotides trapped in metastable structures. These results show that the copolymer works as a DNA annealer that induces proper assembly of stable DNA structures from heterogeneous kinetically trapped mixtures of structures.

DNA assembly and re-assembly activated by cationic comb-type copolymer.

Guanine-rich oligonucleotides, such as TG(4)T and TG(5)T, assemble into a tetramolecular quadruplexes with layers of G-quartets stabilized by coordination to monovalent cations. Association rates of the quadruplexes are extremely slow, likely owing to electrostatic repulsion among the four strands. We have shown that comb-type copolymers with a polycation backbone and abundant hydrophilic graft chains form water-soluble polyelectrolyte complexes with DNA and promote DNA hybridization. Here, we report the effect of cationic comb-type copolymers on the kinetics of tetramolecular quadruplex formation. The copolymer significantly increased the association rate of tetramolecular quadruplexes without altering kinetic effects of metal cations in quadruplex formation. Dissociation rates of the quadruplexes were also accelerated by the copolymer suggesting that the copolymer has chaperone-like activity that reduces the energy barriers associated with dissociation and re-assembly of base pairs. This hypothesis was further supported by the observation that the copolymer activated the strand exchange reaction between the quadruplex and a constituting single-stranded.

Grafting of poly(ethylene glycol) to poly-lysine augments its lifetime in blood circulation and accumulation in tumors without loss of the ability to associate with siRNA.

Poly-lysine has been studied as a carrier for the delivery of drugs and nucleic acids for at least a decade. It is an especially attractive carrier for DNA and RNA, because of its condensed cationic charges. In our previous study, we showed that poly(ethylene glycol) (PEG) grafted to poly-L-lysine (PLL) remarkably increased the life time of a small interfering RNA (siRNA) in blood circulation. In this study, we prepared a new series of PEG-grafted PLL (PLL-g-PEG) with various lengths (PEG 2kDa, 5kDa, and 10kDa and PLL 28kDa and 40kDa), to evaluate masking effects of PEG on cationic charges of PLL in vivo and the structural implications for biodistribution and tumoral accumulation. The best in the series, 40K10P37 (40kDa of PLL, 10kDa of PEG, 37mol% grafting) with molecular weight of 10(6) as determined by Multi-Angle Laser Light Scattering (MALLS), accumulated in tumors at about 8% of the injected dose per gram of tissue. Interestingly, a PLL-g-PEG conjugate pre-mixed with murine sera prevented degradation of siRNA, suggesting that PLL-g-PEG preferentially associates with siRNA in sera. Our results indicate grafting of PEG to the side chains of PLL augments its lifetime in blood circulation and tumoral accumulation without loss of the ability to associate with siRNA and support further evaluation of these cationic delivery carriers.

Enhanced cell uptake via non-covalent decollation of a single-walled carbon nanotube-DNA hybrid with polyethylene glycol-grafted poly(l-lysine) labeled with an Alexa-dye and its efficient uptake in a cancer cell.

The use of single-walled carbon nanotubes (SWNTs) for biomedical applications is a promising approach due to their unique outer optical stimuli response properties, such as a photothermal response triggered by near-IR laser irradiation. The challenging task in order to realize such applications is to render the SWNTs biocompatible. For this purpose, the stable and homogeneous functionalization of the SWNTs with a molecule carrying a biocompatible group is very important. Here, we describe the design and synthesis of a polyanionic SWNT/DNA hybrid combined with a cationic poly(l-lysine) grafted by polyethylene glycol (PLL-g-PEG) to provide a supramolecular SWNT assembly. A titration experiment revealed that the assembly undergoes an approximately 1 : 1 reaction of the SWNT/DNA with PLL-g-PEG. We also found that SWNT/DNA is coated with PLL-g-PEG very homogeneously that avoids the non-specific binding of proteins on the SWNT surface. The experiment using the obtained supramolecular hybrid was carried out in vitro and a dramatic enhancement in the cell uptake efficiency compared to that of the SWNT/DNA hybrid without PLL-g-PEG was found.

Evaluation of polyanion-coated biodegradable polymeric micelles as drug delivery vehicles.

Polymeric micelles, as drug delivery vehicles, must achieve specific targeting and high stability in the body for efficient drug delivery. We recently reported the preparation of polyanion-coated biodegradable polymeric micelles by coating positively charged polymeric micelles consisting of poly(L-lysine)-block-poly(L-lactide) (PLys-b-PLLA) AB diblock copolymers with anionic hyaluronic acid (HA) by polyion complex (PIC) formation. The obtained HA-coated micelles showed significantly higher stability in aqueous solution. In this study, to evaluate the HA-coated polymeric micelles as a drug carrier, model drug release from the micelles and cytotoxicity of the micelles were investigated. The HA-coated micelles showed sustained release of model drugs and low cytotoxicity. It is known that there are receptors for HA on liver sinusoidal endothelial cells (LSEC). Specific interactions of HA-coated micelles with LSECs and Kupffer cells were investigated and compared with polymeric micelles coated with other polyanionic polysaccharides, i.e., heparin (Hep) and carboxymethyl-dextran (CMDex). Although Hep-coated micelles and CMDex-coated micelles were incorporated into both Kupffer cells and LSECs, HA-coated micelles were taken up only into LSECs. These results suggest HA-coated micelles have potential utility as drug delivery vehicles exhibiting specific accumulation into LSECs.

Synthesis, characterization and drug release of biocompatible/biodegradable non-toxic poly(urethane urea)s based on poly(epsilon-caprolactone)s and lysine-based diisocyanate.

Segmented poly(urethane urea)s (SPUUs) based on aliphatic diisocyanato (2,6-diisocyanato methyl caproate (lysine-based diisocyanate, LDI)), poly(epsilon-caprolactone diol)s (PCLs) with molecular weights 530, 1250 and 2000, and 1,4-butanediamine were synthesized in absence of catalyst. The resulting SPUUs, with different soft segment length, were characterized by suitable analytical techniques. The synthesized SPUUs had high molecular weights, low glass-transition temperatures (

Design of cationic graft copolymers as a potential inducer of B-Z transition.

Biological roles of transition from B form to Z form of DNA (B-Z transition) have recently received attention. The B-Z transition was also employed as driving machinery of a nano-mechanical DNA device. However, there are little reports of effective inducer of the B-Z transition. We previously reported that poly((L)-Lysine)-graft-dextran induces B-Z transition and grafted dextran plays an important role for the B-Z transition. In this report, we designed cationic graft copolymer as a potential inducer of B-Z transition. Series of the copolymers consisting of poly((L)-Lysine) backbone and abundant of dextran (Dex) or poly(ethylene glycol) (PEG) side chains were prepared. The B-Z transition of poly(dG-dC)/Poly(dG-dC) were observed in the presence of these copolymers. The copolymers having higher content of Dex or PEG effectively induced the B-Z transition compared to that having lower content. The result indicated that not only electrostatic interaction between DNA and the poly((L)-Lysine) backbone but also hydrophilic graft chains play a role for the B-Z transition. We speculated that Dex- or PEG-enriched environment is favorable for Z form.

Synthesis and characterization of semi-interpenetrating polymer networks based on polyurethane and N-isopropylacrylamide for wound dressing.

Thermosensitive semi-interpenetrating polymer networks (semi-IPNs) composed of crosslinked poly(N-isopropylacrylamide) (PNiPAAm) and linear segmented polyurethane urea (SPUU) were synthesized via thermal initiated free radical polymerization. Synthesized semi-IPNs of various compositions were characterized by Fourier transform infrared spectroscopy, water equilibrium swelling at different temperatures, drug lading, drug release, cell adhesion, and detachment. The semi-IPN films of all the compositions were transparent in dry state and negative thermosensitivity in their swelling ratio, that is, lower swelling levels with increasing temperature. The drug release study revealed that the rate of drug release is fast in case of pure SPUU compared to PNiPAAm and semi-IPN film. Drug release depended mainly on solubility of the drugs and physical networks between SPUU and PNiPAAm. Finally NIH3T3 cells were seeded on the semi-IPN films and found that cells were securely attached and proliferated to confluence. Upon cooling, cells were detached from the semi-IPN films. Therefore, the semi-IPN films may be good candidate materials for wound dressing applications.

A highly efficient siRNA carrier of PBLG modified hyperbranched PEI.

In search for effective non-viral gene vectors for the delivery of siRNA, a copolymer was designed and synthesized by grafting hydrophobic poly(gamma-benzyl L-glutamate) segment (PBLG) to hyperbranched polyethylenimine (PEI-PBLG). PEI-PBLG could efficiently deliver siRNA to cells to silence the targeted gene. Markedly, PEI-PBLG caused lower cytotoxicity in comparison to unmodified PEI. The siRNA complexed with PEI-PBLG showed a remarkable knockdown (75.23% relative to untreated cells, without changing the medium after 6 h of incubation) of the targeted luciferase gene in stable expressing luciferase CT26 cells while the Lipofectamine2000 and unmodified PEI could only achieve knockdown rates of 57.92% and 15.31%, respectively. The siRNA complexed with PEI-PBLG also demonstrated that it had greater gene silencing ability than unmodified PEI and Lipofectamin2000 in both 4T1 cells stably transfected with the luciferase gene and HeLa cells transiently transfected with the luciferase gene. The internalization efficiency of carrier/Alexa647-labeled siRNA was quantified using flow cytometry. PEI-PBLG/Alexa647-labeled siRNA showed internalization efficiency of 52.67% while PEI and Lipofectamine2000 demonstrated 27.23% and 37.91%, respectively. Confocal laser scanning microscopy (CLSM) assay also indicated that PEI-PBLG induced higher cell uptake efficiency than other commercial reagents. PEI-PBLG was shown to be a promising siRNA carrier with potential application in cancer therapy.

Polymer brush-stabilized polyplex for a siRNA carrier with long circulatory half-life.

Delivery systems of small interfering RNA (siRNA) are the key to siRNA therapeutic application. In this study, we prepared and evaluated a series of cationic comb-type copolymers (CCCs) possessing a polycationic backbone (less than 30 weight (wt) %) and abundant water-soluble side chains (more than 70 wt.%) as a siRNA carrier with prolonged blood circulation time. Markedly, the CCC with the higher side chain content (10 wt.% PLL and 90 wt.% PEG) showed stronger interaction with siRNA than that with the lower content (30 wt.% PLL and 70 wt.% PEG), suggesting that highly dense PEG brush reinforces interpolyelectrolyte complex between the PLL backbone and siRNA. The siRNA complexed with the CCC was resistant to nucleases in 90% plasma for 24 h in vitro. The CCC having the higher side chain content increased circulation time of siRNA in mouse bloodstream by 100-fold. Surprisingly, even when the CCC and siRNA were separately injected into mouse at 20 min interval, blood circulation of post-injected siRNA was significantly increased. These results imply that the CCC has higher selectivity in its ionic interaction with siRNA than other anionic substances in blood stream. To our knowledge, this is the first example of a polyplex carrier that prolongs blood circulation time of unmodified siRNA without resource-consuming preparation process.

Synthesis of a biocleavable polyrotaxane-plasmid DNA (pDNA) polyplex and its use for the rapid nonviral delivery of pDNA to cell nuclei.

This protocol provides a method for synthesizing a biocleavable polyrotaxane/plasmid DNA (pDNA) polyplex and for using it to deliver pDNA into cell nuclei. The biocleavable polyrotaxane is synthesized in four steps: (i) introduction of disulfide linkages at both terminals of PEG, (ii) preparation of an inclusion complex between disulfide-containing PEG and alpha-cyclodextrins (alpha-CDs), (iii) synthesis of polyrotaxane and (iv) modification of alpha-CDs in the polyrotaxane with dimethylethylenediamine. A polyplex of pDNA with the polyrotaxane is formed when the two compounds are dissolved together in a phosphate buffer. Subcellular localization of rhodamine-labeled pDNA in fluorescently labeled organelles is quantified by Z-series of confocal images captured by confocal laser scanning microscopy. Significant amounts of pDNA delivered to the nucleus can be expected as well as high transfection activity of the polyplex. This protocol can be completed in 23-32 d.

Biocleavable polyrotaxane-plasmid DNA polyplex for enhanced gene delivery.

A biocleavable polyrotaxane, having a necklace-like structure consisting of many cationic alpha-cyclodextrins (alpha-CDs) and a disulfide-introduced poly(ethylene glycol) (PEG), was synthesized and examined as a nonviral gene carrier. The polyrotaxane formed a stable polyplex having positively charged surface even at low charge ratio. This is likely to be due to structural factors of the polyrotaxane, such as the mobile motion of alpha-CDs in the necklace-like structure. Rapid endosomal escape was observed 90 min after transfection. The positively charged surface and the good buffering capacity are advantageous to show the proton sponge effect. The pDNA decondensation occurred through disulfide cleavage of the polyrotaxane and subsequent supramolecular dissociation of the noncovalent linkages between alpha-CDs and PEG. Transfection of the DMAE-SS-PRX polyplex is independent of the amount of free polycation. Those properties played a key role for delivery of pDNA clusters to the nucleus. Therefore, the polyplex nature and the supramolecular dissociation of the polyrotaxane contributed to the enhanced gene delivery.

Cationic comb-type copolymers as an artificial nucleic acid-chaperone: spectroscopic analyses for DNA/copolymer interaction.

We have reported previously that the cationic comb-type copolymer, PLL-g-Dex, accelerated DNA strand exchange reaction while stabilizing DNA duplexes. In this research, we showed the effects of cationic PLL-g-Dex copolymer on hybridization of self-complementary octamer oligonucleotides. NMR studies suggested that PLL-g-Dex copolymer preferentially interacted with a duplex DNA rather than a single-stranded DNA, and did not perturb local structures of DNA duplexes. CD studies also supported inert nature of the copolymer upon hybrid structure. From these results we concluded that the accelerating mechanisms of the copolymer did not involved partial perturbation of duplex structure.