Amphiphilic poly(L-lactide)-b-dendritic poly(L-lysine)s synthesized with a metal-free catalyst and new dendron initiators: chemical preparation and characterization

This study presents investigations on new approaches to novel biodegradable amphiphilic poly(L-lactide)-b-dendritic poly(L-lysine)s bearing well-defined structures. First, two new Boc-protected poly(L-lysine) dendron initiators G(2)OH 4 (generation = 2) and G(3)OH 6 (generation = 3) with hydroxyl end functional groups were efficiently derived from corresponding precursors 3 and 5 via methyl ester substitution with ethanolamine. Subsequently, two series of new diblock copolymers of poly(L-lactide)-b-dendritic Boc-protected poly(L-lysine)s (S1-S2, S3-S4) were prepared in chloroform through ring-opening copolymerization of poly(L-lactide)s with a metal-free catalyst of organic 4-(dimethylamino) pyridine (DMAP) in the presence of a corresponding new poly(L-lysine) dendron initiator. Further, molecular structures of the prepared new dendron initiators as well as those of poly(L-lactide)-b-dendritic Boc-protected poly(L-lysine)s bearing different dendron blocks and PLLA lengths were examined by means of nuclear magnetic resonance spectroscopy (NMR), gel permeation chromatography (GPC), mass spectrometry (ESI-MS, MALDI-FTMS), and thermal gravimetric analysis (TGA). The results demonstrated successful formation of the synthetic precursors, functional dendron initiators, and new diblock copolymers. In addition, the very narrow molecular weight distributions (PDI = 1.10-1.14) of these poly(L-lactide)-b-dendritic Boc-protected poly(L-lysine)s further indicated their well-defined molecular structures. After the efficient Boc-deprotection for the dendron amino groups with TFA/CH(2)Cl(2), new diblock poly(L-lactide)-b-dendritic poly(L-lysine)s bearing lipophilic PLLA and hydrophilic dendritic PLL were finally prepared. It was noteworthy that the MALDI-FTMS result showed that no appreciable intermolecular chain transesterification happened during the ROP of L-lactide catalyzed by the DMAP. Moreover, self-assembly of these new biodegradable amphiphilic copolymers in diverse solvents were also preliminarily studied.     

pH-sensitive cationic polymer gene delivery vehicle: N-Ac-poly(L-histidine)-graft-poly(L-lysine) comb shaped polymer

Advancing biotechnology spurs the development of new pharmaceutically engineered gene delivery vehicles. Poly(L-histidine) ?PLH? has been shown to induce membrane fusion at endosomal pH values, whereas PLL has a well documented efficacy in polyplex formation. Therefore, N-Ac-poly(L-histidine)-graft-poly(L-lysine) ?PLH-g-PLL? was synthesized by grafting poly(L-histidine) to poly(L-lysine) ?PLL?. PLH-g-PLL formed polyplex particles by electrostatic interactions with plasmid DNA ?pDNA?. The mean particle size of the polyplexes was in the range of 117 +/- 6 nm to 306 +/- 77 nm. PLH-g-PLL gene carrier demonstrated higher transfection efficacy in 293T cells than PLL at all equivalent weight ratios with pDNA. The inclusion of chloroquine as an endosomolytic agent enhanced transfection for both PLL and PLH-g-PLL gene carriers. PLH-g-PLL enhanced beta-galactosidase expression compared to PLL, but still increased in efficacy when chloroquine was included.     

A tetra(L-lysine)-grafted poly(organophosphazene) for gene delivery

In order to develop a new gene delivery vector, a novel cationic poly(organophosphazene) was synthesized by stepwise nucleophilic substitutions of poly(dichlorophosphazene) with a hydrophilic methoxy-poly(ethylene glycol) (MPEG) as a shielding group and a branched tetra(L-lysine), LysLys(LysEt)(2), as a cationic moiety. The cationic polymer has shown to form a polyplex by DNA condensation and very low in vitro cytotoxicity probably due to the shielding effect of MPEG, which provides a basis for improving the low gene transfection yield of cationic polyphosphazenes.     

Structural advantage of dendritic poly(L-lysine) for gene delivery into cells

This study aimed to investigate the relationships between structures of gene carrier molecules and their activities for gene delivery into cells. We compared 2 types of poly(L-lysine) as carriers, that is, dendritic poly(L-lysine) (KG6) and linear poly(L-lysine) (PLL). KG6 formed a neutral DNA complex, and its DNA compaction level was weaker than that of PLL. The amount of DNA binding and uptake into cells mediated by PLL was 4-fold higher than that with KG6. However, KG6-mediated gene expression was 100-fold higher than that by PLL. Since pK(a) values of terminal amines of KG6 were lowered even though small amounts of DNA were internalized into cells, sufficient DNA amounts for effective gene expression escaped to the cytosol due to the proton sponge effect in the endosome. In addition, weakly compacted DNA with KG6 was advantageous in accessing RNA polymerase in the cell nucleus. On the other hand, PLL did not show the proton sponge effect in the endosome and resulted in strong compaction of DNA. Even though large DNA amounts were internalized into cells, most of the DNA would not take part in gene expression systems in the nucleus. Amount of induced cytokine production after intravenous injection of DNA complexes with KG6 and PLL was low, and was similar to the case when DNA was injected alone. Therefore, no significant difference in effects on cytokine production was observed between KG6 and PLL.     

Effect of chain length and concentration of anionic surfactants on the conformational transitions of poly(L-ornithine) and poly(L-lysine) in aqueous solution

The conformation of poly(L-ornithine) (PLO) and poly(L-lysine) (PLL) in solutions of sodium alkyl sulfates, CH₃(CH₂)_nSO₄Na with n = 7, 9, 11, 13 and 15 was studied by circular dichroism. PLO adopts a helical conformation in all 5 homologs and PLL a β-form in only 4 of the homologs. With octyl sulfate PLL has a helical conformation instead. These conformations were observed in solution of surfactants both below and above the critical micelle concentration.     

Glycopolymer modification on physicochemical and biological properties of poly(L-lysine) for gene delivery

Poly(L-lysine) (PLL) has excellent plasmid DNA (pDNA) condensation capacity. However, the relatively high cytotoxicity and low transfection efficiency limit its application as gene delivery vectors. Here, well-defined glycopolymers are synthesized by reversible addition fragmentation transfer polymerization and grafted onto PLL to improve the gene delivery performance. After glycopolymer modification, PLL shows reduced cytotoxicity. By regulating the glycopolymer length and amino group substitution degree, the glycopolymer modified PLL can condense pDNA with proper strength, protect the condensed pDNA from degradation and release them in time. Transfection with NIH3T3 and HepG2 cells shows that the glycopolymer modified PLL has improved transfection efficiencies. The low cytotoxicity, effective pDNA protection and enhanced transfection efficiencies indicate that glycopolymer modification would be an effective strategy to improve the polycation properties for gene delivery.     

Coating of DNA/poly(L-lysine) complexes by covalent attachment of poly[N-(2-hydroxypropyl)methacrylamide

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymers (pHPMA) containing 4-nitrophenyl ester (ONp) or thiazolidine-2-thione (TT) reactive groups in side chains and telechelic/semitelechelic pHPMA with TT groups were designed as highly hydrophilic biocompatible polymers suitable for chemical coating of polyelectrolyte-based DNA-containing nanoparticles bearing amino groups on the surface. The course of the coating reaction carried out in aqueous solution was evaluated on model self-assembling polyelectrolyte DNA/poly(L-lysine) (DNA/PLL) complexes either by monitoring the amount of residual polymer reactive groups by UV spectroscopy or by monitoring changes in the weight-average molecular weight and hydrodynamic size of the complexes using light scattering methods. Physicochemical stability of the coated complexes in buffered saline solution was also investigated. Contrary to uncoated particles, the coated complexes showed remarkable stability to aggregate in 0.15 M NaCl. Coating with pHPMA had practically no effect on the size distribution of the most stable complexes prepared by complexation of DNA with high-molecular-weight PLL (M(w) = 134 000) as shown by dynamic light scattering. The coating reaction was faster and more efficient with multivalent HPMA copolymers containing TT reactive groups than that with HPMA copolymers containing ONp groups.     

Optimal poly(L-lysine) grafting density in hydrogels for promoting neural progenitor cell functions

Recently, we have developed a photopolymerizable poly(L-lysine) (PLL) that can be covalently incorporated into poly(ethylene glycol) diacrylate (PEGDA) hydrogels to improve their bioactivity by providing positive charges. To explore the potential of these PLL-grafted PEGDA hydrogels as a cell delivery vehicle and luminal filler in nerve guidance conduits for peripheral and central nerve regeneration, we varied the number of pendent PLL chains in the hydrogels by photo-cross-linking PEGDA with weight compositions of PLL (φ(PLL)) of 0, 1, 2, 3, and 5%. We further investigated the effect of PLL grafting density on E14 mouse neural progenitor cell (NPC) behavior including cell viability, attachment, proliferation, differentiation, and gene expression. The amount of actually grafted PLL and charge densities were characterized, showing a proportional increase with the feed composition φ(PLL). NPC viability in 3D hydrogels was significantly improved in a PLL grafting density-dependent manner at days 7 and 14 postencapsulation. Similarly, NPC attachment and proliferation were promoted on the PLL-grafted hydrogels with increasing φ(PLL) up to 2%. More intriguingly, NPC lineage commitment was dramatically altered by the amount of grafted PLL chains in the hydrogels. NPC differentiation demonstrated a parabolic or nonmonotonic dependence on φ(PLL), resulting in cells mostly differentiated toward mature neurons with extensive neurite formation and astrocytes rather than oligodendrocytes on the PLL-grafted hydrogels with φ(PLL) of 2%, whereas the neutral hydrogels and PLL-grafted hydrogels with higher φ(PLL) of 5% support NPC differentiation less. Gene expression of lineage markers further illustrated this trend, indicating that PLL-grafted hydrogels with an optimal φ(PLL) of 2% could be a promising cell carrier that promoted NPC functions for treatment of nerve injuries.     

Proliferative response of murine B-cell leukemia (BCL1) to poly(L-lysine)

Peripheral blood lymphocytes (PBL) isolated from BALB/c mice bearing a B-cell leukemia (BCL1) showed a marked proliferative response upon two days culturing with poly(L-lysine) (PLL) of various molecular weights. An inverse relationship was noted between the molecular weight of the PLL and the dose required for optimal proliferative response. PLL showed no proliferative activity when cultured with normal PBL or with lymphocytes isolated from the spleen or other lymphoid organs of BCL1-bearing mice. Double exposure to PLL and lipopolysaccharide (LPS) had a marked synergistic effect on BCL1 PBL stimulation but not on PBL isolated from normal mice. The data suggest that PLL, in contrast to LPS, may cause a selective proliferation of a subpopulation(s) of B-tumor cells at a particular stage(s) of differentiation.     

Poly(L-lysine)-graft-chitosan copolymers: synthesis, characterization, and gene transfection effect

Polypeptide/polysaccharide graft copolymers poly(L-lysine)-graft-chitosan (PLL-g-Chi) were prepared by ring-opening polymerization (ROP) of epsilon-benzoxycarbonyl L-lysine N-carboxyanhydrides (Z-L-lysine NCA) in the presence of 6-O-triphenylmethyl chitosan. The PLL-g-Chi copolymers were thoroughly characterized by 1H NMR, 13C NMR, Fourier transform infrared (FT-IR), and gel permeation chromatography (GPC). The number-average degree of polymerization of PLL grafted onto the chitosan backbone could be adjusted by controlling the feed ratio of NCA to 6-O-triphenylmethyl chitosan. The particle size of the complexes formed from the copolymer and calf thymus DNA was measured by dynamic light scattering (DLS). It was found in the range of 120 approximately 340 nm. The gel retardation electrophoresis showed that the PLL-g-Chi copolymers possessed better plasmid DNA-binding ability than chitosan. The gene transfection effect in HEK 293T cells of the copolymers was evaluated, and the results showed that the gene transfection ability of the copolymer was better than that of chitosan and was dependent on the PLL grafting ratio. The PLL-g-Chi copolymers could be used as effective gene delivery vectors.     

L-Phe end-capped poly(L-lactide) as macroinitiator for the synthesis of poly(L-lactide)-B-poly(L-lysine) block copolymer

A poly(L-lactide)-b-poly(Nepsilon-(Z)-L-lysine) (PLLA-b-PZLys) block copolymer was synthesized through the ring-opening polymerization of Nepsilon-(Z)-lysine-N-carboxyanhydride using L-Phe-terminated PLLA as a macroinitiator. The L-Phe-terminated PLLA was prepared through a novel three-step process. First, the hydroxyl-terminated PLLA was synthesized through the ring-opening polymerization of L-lactide initiated by n-butanol under the existence of tin(II) ethylhexanoate. Subsequently, the complete capping of the hydroxyl end group of PLLA with BOC-L-Phe was achieved by using a mixed anhydride of BOC-L-Phe under the catalysis of 4-(1-pyrrolidinyl) pyridine. Finally, the free amino end group was obtained by removal of the t-butoxycarbonyl group through trifluoroacetic acid treatment under anhydrous condition. All these treatments were conducted under mild conditions, thus avoiding the breakdown of the PLLA backbone. Poly(L-lactide)-b-poly(L-lysine) block copolymer was produced after deprotection treatment of PLLA-b-PZLys. The structure of the block copolymer was confirmed by 1H NMR, IR, and GPC. Adjustment of the ratio of the NCA monomer to the macroinitiator could control the chain length of the PLys block.     

Promoting nerve cell functions on hydrogels grafted with poly(L-lysine)

We present a novel photopolymerizable poly(L-lysine) (PLL) and use it to modify polyethylene glycol diacrylate (PEGDA) hydrogels for creating a better, permissive nerve cell niche. Compared with their neutral counterparts, these PLL-grafted hydrogels greatly enhance pheochromocytoma (PC12) cell survival in encapsulation, proliferation, and neurite growth and also promote neural progenitor cell proliferation and differentiation capacity, represented by percentages of both differentiated neurons and astrocytes. The role of efficiently controlled substrate stiffness in regulating nerve cell behavior is also investigated and a polymerizable cationic small molecule, [2-(methacryloyloxy)ethyl]-trimethylammonium chloride (MTAC), is used to compare with this newly developed PLL. The results indicate that these PLL-grafted hydrogels are promising biomaterials for nerve repair and regeneration.     

Chain length effects of oligo(L-lysine)-shelled dendrimers on interaction with DNA

The interaction of novel oligo(L-lysine)-shelled dendrimers (G3-PLL) with DNA was studied by means of circular dichroism spectroscopy, dynamic light scattering, and melting behavior of double-stranded DNA. G3-PLLs having various oligo(L-lysine) (PLL) segment (n = 5-40) were successfully synthesized by graft-polymerization of L-lysine NCA initiated with amino groups at the 3rd-generation poly(amidoamine) dendrimer surface. The ionization property of the newly prepared G3-PLLs were first examined. The evaluated pK(a) values (8.3-8.5) for G3-PLLs were found to be significantly lower than those (9.2-9.4) for the corresponding linear PLLs, probably due to alignment of PLL segments on the three-dimensional core surface. The binding experiments of G3-PLLs to DNA showed that double-stranded DNAs were fairly strongly bound to G3-PLLs primarily through electrostatic interactions. In addition, G3-PLLs served as a DNA cross-linker. A longer PLL-containing G3-PLL was found to interact with DNA more effectively than a shorter one.     

Mixtures of poly(triethylenetetramine/cystamine bisacrylamide) and poly(triethylenetetramine/cystamine bisacrylamide)-g-poly(ethylene glycol) for improved gene delivery

Branched disulfide-containing poly(amido ethyleneimines) (SS-PAEIs) are biodegradable polymeric gene carrier analogues of the well-studied, nondegradable, and often toxic branched polyethylenimines (bPEIs), but with distinct advantages for cellular transgene delivery. Clinical success of polycationic gene carriers is hampered by obscure design and formulation requirements. This present work reports synthetic and formulation properties for a graft copolymer of poly(ethylene glycol) (PEG) and a branched SS-PAEI, poly(triethylentetramine/cystaminebisacrylamide) (p(TETA/CBA)). Several laboratories have previously demonstrated the advantages of PEG conjugation to gene carriers, but have also shown that PEG conjugation may perturb plasmid DNA (pDNA) condensation, thereby interfering with nanoparticle formation. With this foundation, our studies sought to mix various amounts of p(TETA/CBA) and p(TETA/CBA)-g-PEG2k to alter the relative amount of PEG in each formulation used for polyplex formation. The influence of different PEG/polycation amounts in the formulations on polymer/nucleic acid nanoparticle (polyplex) size, surface charge, morphology, serum stability and transgene delivery was studied. Polyplex formulations were prepared using p(TETA/CBA)-g-PEG2k, p(TETA/CBA), and mixtures of the two species at 10/90 and 50/50 volumetric mixture ratios (wt/wt %), respectively. As expected, increasing the amount of PEG in the formulation adversely affects polyplex formation. However, optimal polymer mixtures could be identified using this facile approach to further clarify design and formulation requirements necessary to understand and optimize carrier stability and biological activity. This work demonstrates the feasibility to easily overcome typical problems observed when polycations are modified and thus avoids the need to synthesize multiple copolymers to identify optimal gene carrier candidates. This approach may be applied to other polycation-PEG preparations to alter polyplex characteristics for optimal stability and biological activity.     

Biological activity of oligonucleotide-poly(L-lysine) conjugates: mechanism of cell uptake

We have previously shown that antisense oligomers linked to poly(L-lysine) (PLL) exhibit antiviral properties against vesicular stomatitis virus (VSV) at concentrations lower than 1 microM. The conjugation to PLL provides an interesting alternative to natural or neutral oligomers to increase the biological effects of antisense oligomers. The internalization pathway of oligomer-PLL conjugates as compared to unconjugated oligomers has been studied in L929 cells. In parallel to their enhanced antiviral activity, PLL increases greatly the uptake of fluorescently tagged oligomers. This internalization follows a classical endocytic pathway and the oligomer has to be cleaved from PLL in the cell to exhibit an antiviral effect.     

Heparin removal from blood using poly(L-lysine) immobilized hollow fiber

Based on the negative charge density characteristics of heparin, an affinity adsorption technique has been developed for the removal of heparin from blood. Poly(L-lysine) . HBr (PLL . HBr), a polycation, was immobilized with the help of cyanogen bromide (BrCN) onto poly(ethylene-vinyl alcohol) (PEVAL) copolymer coated polyethylene (PE) hollow fibers. Heparin bound rapidly onto PLL . HBr imobilized surface in buffer, plasma, and blood. The heparin binding capacity of PLL immobilized surface increased sevenfold as compared to a non-PLL-treated control. When heparinized blood was recirculated through a PLL immobilized PEVAL hollow fiber cartridge, the anticoagulant activity of heparin decreased by 85% from initial activity in 25 min. Moreover, circulation of blood through PLL immobilized hollow fiber did not show any adverse effects; no hemolysis was observed and no significant loss of plasma proteins was noted during the heparin removal process. These results suggest that PLL immobilized surface may be utilized for rapid and effective removal of heparin from blood. (c) 1992 John Wiley & Sons, Inc.     

Effects of poly(L-lysine) on the structural and thermotropic properties of dipalmitoylphosphatidylglycerol bilayers.

The effects of poly(L-lysine) on the structural and thermotropic properties of dipalmitoylphosphatidylglycerol (DPPG) bilayers were studied with differential scanning calorimetry (DSC), X-ray diffraction and freeze-fracture electron microscopy. For thermal behavior, in the DPPG/poly(L-lysine) system the main transition temperature rises to 45.7 degrees C and the pretransition disappears in opposition to pure DPPG vesicles. An additional transition appears approximately at 36 degrees C for the DPPG/poly(L-lysine) system after incubation at 4 degrees C for two months. The incubated sample gives a X-ray diffraction pattern having several additional reflections in the range of 0.2-0.9 nm at 15 degrees C. These results suggest that even in the presence of poly(L-lysine) the DPPG bilayers form the subgel (Lc) phase after the long incubation at a low temperature. The X-ray diffraction measurements indicate that the structure of the Lc phase for DPPG/poly(L-lysine) system is different from that of pure DPPG bilayers. On the other hand, in the gel (L beta') phase, the wide-angle X-ray diffraction pattern suggests that the presence of poly(L-lysine) hardly affects the packing of hydrocarbon chains in the DPPG bilayers. The small-angle X-ray diffraction and freeze-fracture electron microscopy exhibit that the DPPG/poly(L-lysine) system forms a tightly packed multilamellar structure in which the poly(L-lysine) is intercalated between the subsequent DPPG bilayers.     

New amphiphilic poly(2-ethyl-2-oxazoline)/ poly(L-lactide) triblock copolymers

A new series of cationic, thermo-sensitive, and biodegradable poly(L-lactide)-poly(2-ethyl-2-oxazoline)-poly(L-lactide) (PLLA-PEOz-PLLA) triblock copolymers were synthesized by ring-opening polymerization. With increasing molecular weight and crystallinity of hydrophobic PLLA blocks, the critical micellization concentrations (CMC) occurred at lower concentration. The PLLA-PEOz-PLLA aqueous solution was transparent at room temperature. Heating the solution resulted in precipitations, which were caused by the combination of dehydration of water around PEOz and the aggregations of PLLA segments. Acid/base titration profiles indicated that PLLA-PEOz-PLLA were protonated at neutral and acidic conditions. Considerable buffering capacity was found over the entire pH range. The specific PLLA-PEOz-PLLA triblock copolymers with thermal- and pH-sensitive properties can be tailored by varying the compositions and can be applied as controlled release carries for biomedical applications.     

Molecular dynamics simulation of coarse-grained poly(L-lysine) dendrimers

Poly(L-lysine) (PLL) dendrimer are amino acid based macromolecules and can be used as drug delivery agents. Their branched structure allows them to be functionalized by various groups to encapsulate drug agents into their structure. In this work, at first, an attempt was made on all-atom simulation of PLL dendrimer of different generations. Based on all-atom results, a course-grained model of this dendrimer was designed and its parameters were determined, to be used for simulation of three generations of PLL dendrimer, at two pHs. Similar to the all-atom, the coarse-grained results indicated that by increasing the generation, the dendrimer becomes more spherical. At pH 7, the dendrimer had larger size, whereas at pH 12, due to back folding of branching chains, they had the tendency to penetrate into the inner layers. The calculated radial probability and radial distribution functions confirm that at pH 7, the PLL dendrimer has more cavities and as a result it can encapsulate more water molecules into its inner structure. By calculating the moment of inertia and the aspect ratio, the formation of spherical structure for PLL dendrimer was confirmed.     

Use of N-terminal modified poly(L-lysine)-antibody conjugate as a carrier for targeted gene delivery in mouse lung endothelial cells.

A DNA targeted delivery and expression system has been designed based on an N-terminal modified poly(L-lysine) (NPLL)-antibody conjugate, which readily forms a complex with plasmid DNA. Monoclonal antibodies against the cell-surface thrombomodulin conjugated with NPLL were used for targeted delivery of foreign plasmid DNA to an antigen-expressing mouse lung endothelial cell line in vitro and to mouse lungs in vivo. In both cases significant amounts of DNA can be specifically bound to the target cells or tissues. Specific gene expression was observed in the treated mouse lung endothelial cells.