Functionalized amphiphilic hyperbranched polymers for targeted drug delivery

Amphiphilic hyperbranched core-shell polymers with folate moieties as the targeting groups were synthesized and characterized. The core of the amphiphilic polymers was hyperbranched aliphatic polyester Boltorn H40. The inner part and the outer shell of the amphiphilic polymers were composed of hydrophobic poly(epsilon-caprolactone) segments and hydrophilic poly(ethylene glycol) (PEG) segments, respectively. To achieve tumor cell targeting property, folic acid was further incorporated to the surface of the amphiphilic polymers via a coupling reaction between the hydroxyl group of the PEG segment and the carboxyl group of folic acid. The polymers were characterized by (1)H NMR, (13)C NMR, and combined size-exclusion chromatography and multiangle laser light scattering analysis. The nanoparticles of the amphiphilic polymers prepared by dialysis method were characterized by transmission electron microscopy and particle size analysis. Two antineoplastic drugs, 5-fluorouracil and paclitaxel, were encapsulated into the nanoparticles. The drug release property and the targeting of the drug-loaded nanoparticles to different cells were evaluated in vitro. The results showed the drug-loaded nanoparticles exhibited enhanced cell inhibition because folate targeting increased the cytotoxicity of drug-loaded nanoparticles against folate receptor expressing tumor cells.     

Nanoscale amphiphilic macromolecules as lipoprotein inhibitors: the role of charge and architecture

A series of novel amphiphilic macromolecules composed of alkyl chains as the hydrophobic block and poly(ethylene glycol) as the hydrophilic block were designed to inhibit highly oxidized low density lipoprotein (hoxLDL) uptake by synthesizing macromolecules with negatively charged moieties (ie, carboxylic acids) located in the two different blocks. The macromolecules have molecular weights around 5,500 g/mol, form micelles in aqueous solution with an average size of 20-35 nm, and display critical micelle concentration values as low as 10(-7) M. Their charge densities and hydrodynamic size in physiological buffer solutions correlated with the hydrophobic/ hydrophilic block location and quantity of the carboxylate groups. Generally, carboxylate groups located in the hydrophobic block destabilize micelle formation more than carboxylate groups in the hydrophilic block. Although all amphiphilic macromolecules inhibited unregulated uptake of hoxLDL by macrophages, inhibition efficiency was influenced by the quantity and location of the negatively charged-carboxylate on the macromolecules. Notably, negative charge is not the sole factor in reducing hoxLDL uptake. The combination of smaller size, micellar stability and charge density is critical for inhibiting hoxLDL uptake by macrophages.     

Report on the use of poly(organophosphazenes) for the design of stimuli-responsive vesicles

A novel family of amphiphilic temperature- and pH-sensitive poly(organophosphazenes) with varying ratios of ethylene oxide, alkyl chains and free acid units was synthesized by living cationic polymerization. Depending on their composition, these poly(organophosphazenes) exhibited lower critical solution temperatures ranging from 32 to 44 degrees C, which were pH-dependent for copolymers bearing carboxylic acid groups. The alkylated copolymers were then anchored into phospholipid bilayers to obtain stimuli-responsive liposomes that released their content upon a change in temperature or pH. Such polymer/vesicle complexes could find practical applications for site-specific and intracellular drug delivery.     

From multifunctionalized poly(ethylene imine)s toward antimicrobial coatings

Primary amine groups of branched poly(ethylene imine) (PEI) were functionalized with quaternary ammonium groups, alkyl chains of different length, allylic and benzylic groups in a one-step reaction, using a carbonate coupler. The structure of the obtained amphiphilic polymers was determined by means of 1H and 13C NMR spectroscopy. Depending on their hydrophilic/hydrophobic balance, the obtained polymers can be used as water-soluble disinfectants and for antimicrobial coating materials. The bactericidal properties of some of the amphiphilic polymers against Gram-negative and Gram-positive bacteria were investigated. Minimal inhibitory concentrations (log 4 reduction of bacterial growth) against Escherichia coli and Bacillus subtilis were determined in the range of 0.3-0.4 mg/mL and 0.03-0.04 mg/mL for water-soluble polymers. Glass slides coated with functionalized PEIs showed a reduction of colony forming units of at least 95%, at best 99.9%, against E. coli and B. subtilis.     

Physicochemical characterization of thermoresponsive poly(N-isopropylacrylamide)-poly(ethylene imine) graft copolymers

Synthetic polycations have shown promise as gene delivery vehicles but suffer from an unacceptable toxicity and low transfection efficiency. Novel architectures are being explored to increase transfection efficiency, including copolymers with a thermoresponsive character. The physicochemical characterization of a family of copolymers comprising a core of the cationic polymer poly(ethylene imine) (PEI) with differing thermoresponsive poly( N-isopropylacrylamide) (PNIPAM) grafts has been carried out using pulsed-gradient spin-echo NMR (PGSE-NMR) and small-angle neutron scattering (SANS). For the copolymers that have longer chain PNIPAM grafts, there is clear evidence of the collapse of the grafts with increasing temperature and the associated emergence of an attractive interpolymer interaction. These facets depend on the number of PNIPAM grafts attached to the PEI core. While a collapse in the smaller PNIPAM grafts is observed for the third polymer, there is no appearance of the interpolymer attractive interaction. These observations provide further insight into the association behavior of these copolymers, which is fundamental to developing a full understanding of how they interact with nucleic acids. Furthermore, the differing behaviors of the three copolymers over temperatures in which the PNIPAM blocks undergo coil-to-globule transitions is indicative of changes in the presentation of charged-core and hydrophobic chain components, which are key factors affecting nucleic acid binding and, ultimately, cell transfection ability.     

Mechanism of polymer-induced hemolysis: nanosized pore formation and osmotic lysis

Hemolysis induced by antimicrobial polymers was examined to gain an understanding of the mechanism of polymer toxicity to human cells. A series of cationic amphiphilic methacrylate random copolymers containing primary ammonium groups as the cationic functionality and either butyl or methyl groups as hydrophobic side chains have been prepared by radical copolymerization. Polymers with 0-47 mol % methyl groups in the side chains, relative to the total number of monomeric units, showed antimicrobial activity but no hemolysis. The polymers with 65 mol % methyl groups or 27 mol % butyl groups displayed both antimicrobial and hemolytic activity. These polymers induced leakage of the fluorescent dye calcein trapped in human red blood cells (RBCs), exhibiting the same dose-response curves as for hemoglobin leakage. The percentage of disappeared RBCs after hemolysis increased in direct proportion to the hemolysis percentage, indicating complete release of hemoglobin from fractions of RBCs (all-or-none leakage) rather than partial release from all cells (graded leakage). An osmoprotection assay using poly(ethylene glycol)s (PEGs) as osmolytes indicated that the PEGs with MW > 600 provided protection against hemolysis while low molecular weight PEGs and sucrose had no significant effect on the hemolytic activity of polymers. Accordingly, we propose the mechanism of polymer-induced hemolysis is that the polymers produce nanosized pores in the cell membranes of RBCs, causing an influx of small solutes into the cells and leading to colloid-osmotic lysis.     

Synthesis of novel cationic poly(ethylene glycol) containing lipids.

In this paper, the synthesis of novel divalent cationic lipids with poly(ethylene glycol) segments is described. The lipids consist of an unsaturated double-chain hydrophobic moiety based on 3, 4-dihydroxy benzoic acid, attached to a hydrophilic poly(ethylene glycol) spacer which contains a divalent cationic end group. As poly(ethylene glycol) spacers monodisperse triethylene glycol and telechelic poly(ethylene glycol)s with an average degree of polymerization of 9, 23, and 45 were used. The divalent cationic end group was attached by coupling a protected dibasic amino acid to the PEG spacer and following cleavage of the protecting groups. These novel class of cationic lipids is of particular interest for nonviral gene delivery applications.     

pH-triggered reversible "stealth" polycationic micelles

Amphiphilic polycations with a "stealth" cationic nature have been designed and synthesized by the PEGylation of polycationic amphiphile via a novel pH responsible benzoic imine linker. The linkage is stable in aqueous solution at physiological pH but cleaves in slight acidic conditions such as the extracellular environment of solid tumor and endosomes. The polymeric micelle formed from the amphiphilic "stealth" polycation contains a pH-switchable cationic surface driven by the reversible detachment/reattachment of the shielding PEG chains due to the cleavage/formation process of the imine linkage. At physiological pH, the micellar surface was shielded by the PEG corona, leading to lower cytotoxicity and less hemolysis, whereas in a mild acidic condition like in endosomes or solid tumors, the deshielding of the PEG chains exposed the positive charge on the micellar surface and retained the membrane disrupting ability. The amphiphilic "stealth" polycation is potentially useful as a drug targeting system toward tumors via endocytosis and trafficked through the endosomal pathway.     

Structural effects on the biodistribution and positron emission tomography (PET) imaging of well-defined (64)Cu-labeled nanoparticles comprised of amphiphilic block graft copolymers

The synthesis of poly(methyl methacrylate-co-methacryloxysuccinimide-graft-poly(ethylene glycol)) (PMMA-co-PMASI-g-PEG) via living free radical polymerization provides a convenient route to well-defined amphiphilic graft copolymers having a controllable number of reactive functional groups, variable length PEG grafts, and low polydispersity. These copolymers were shown to form PMMA-core/PEG-shell nanoparticles upon hydrophobic collapse in water, with the hydrodynamic size being defined by the molecular weight of the backbone and the PEG grafts. Functionalization of these polymeric nanoparticles with a 1,4,7,10-tetraazacyclododecanetetraacetic acid (DOTA) ligand capable of chelating radioactive 64Cu nuclei enabled the biodistribution and in vivo positron emission tomography of these materials to be studied and directly correlated to the initial structure. Results indicate that nanoparticles with increasing PEG chain lengths show increased blood circulation and low accumulation in excretory organs, suggesting the possible use of these materials as stealth carriers for medical imaging and systemic administration.     

The antibacterial activity of 4,4'-bipyridinium amphiphiles with conventional, bicephalic and gemini architectures

Dialkyl 4,4'-bipyridinium compounds are widely employed for their useful redox properties, and are commonly known as viologens due to their intense coloration upon reduction. Despite their prevalence and amphiphilic nature, the antibacterial activity of these compounds remains largely unreported. We have thus prepared a series of mono- and bis-alkylated analogs of 4,4'-bipyridine to investigate structure-activity relationships in their inhibition of a battery of Gram positive and Gram negative bacteria. The prepared cationic compounds were conventional (one cationic head, one non-polar tail), bicephalic (two heads, one tail), or gemini (two heads, two tails) in their amphiphilic structure. Additionally, an isomeric series of six bis-alkylated compounds ranging from symmetric (PQ-11,11) to highly asymmetric (PQ-20,2) were prepared. Four themes of bioactivity emerged: (1) the most bioactive compounds were gemini in structure; (2) 22 carbons in the alkyl chains, with little to modest asymmetry, led to optimal activity; (3) bicephalic compounds were generally comparable to conventional amphiphiles, though only about 12 carbons in the alkyl chains were solubilized in water by each cationic nitrogen; (4) the effects of counterion identity were not evident between chlorides and bromides; however, the presence of the iodide counterion inhibited dissolution in all compounds tested. Three isomeric compounds with little to no asymmetry in tail length, PQ-11,11, PQ-12,10, and PQ-14,8, prepared as the bromide salts, showed comparable and highly potent activity, with MIC levels around 2 μM against 3 of 4 bacteria tested. The simple (one- to two-step) syntheses of potent antimicrobials portend well for future optimization.     

Mass spectrometric analysis of permethylated glycosphingolipids I. Sequence analysis of two blood-group B active glycosphingolipids from human B erythrocyte membranes.

Two blood group B active glycosphingolipids (B-I and B-II) formerly isolated and purified from human B erythrocytes (16) were investigated by mass spectrometry after permethylation. B-I yielded fragments up to m/e 1266 and B-II up to m/e 1495, showing the sequence of six and seven carbohydrate residues respectively. In combination with additional experimental evidence (18) the glycosphingolipids are demonstrated to be a gal-[ fuc ]-gal-glcNAc-gal-glc-ceramide (B-I) and a gal-[ fuc ]-gal-glcNAc-gal-glcNAc-gal-glc-ceramide (B-II). Mass spectrometric evidence for the ceramide residues are also obtained indicating besides spingosine C24-,C24:1-, and C22-fatty acids as main constituents.     

Identification of novel small-molecule histone deacetylase inhibitors by medium-throughput screening using a fluorigenic assay

Cationic peptides, known to disrupt bacterial membranes, are being developed as promising agents for therapeutic intervention against infectious disease. In the present study, we investigate structure-activity relationships in the bacterial membrane disruptor betapep-25, a peptide 33-mer. For insight into which amino acid residues are functionally important, we synthesized alanine-scanning variants of betapep-25 and assessed their ability to kill bacteria (Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus) and to neutralize LPS (lipopolysaccharide). Activity profiles were found to vary with the bacterial strain examined. Specific cationic and smaller hydrophobic alkyl residues were crucial to optimal bactericidal activity against the Gram-negative bacteria, whereas larger hydrophobic and cationic residues mediated optimal activity against Gram-positive Staph. aureus. Lysine-substituted norleucine (n-butyl group) variants demonstrated that both charge and alkyl chain length mediate optimal activity. In terms of LPS neutralization, activity profiles were essentially the same against four species of LPS (E. coli 055 and 0111, Salmonella enterica serotype Typhimurium and Klebsiella pneumoniae), and different for two others (Ps. aeruginosa and Serratia marcescens), with specific hydrophobic, cationic and, surprisingly, anionic residues being functionally important. Furthermore, disulfide-bridged analogues demonstrated that an anti parallel beta-sheet structure is the bioactive conformation of betapep-25 in terms of its bactericidal, but not LPS endotoxin neutralizing, activity. Moreover, betapep-25 variants, like the parent peptide, do not lyse eukaryotic cells. This research contributes to the development and design of novel antibiotics.     

Proteinase inhibitors from a mimosoideae legume, Albizzia julibrissin. Homologues of soybean trypsin inhibitor (Kunitz)

Four proteinase inhibitors, A-II, A-III, B-I, and B-II, were isolated from seeds of Albizzia julibrissin (silk tree) of the subfamily Mimosoideae, which is often regarded as the most primitive group of the Leguminosae plants. They were all of the high-molecular weight type (21,600 for A-II and A-III, and 19,000 for B-I and B-II), and composed of two polypeptide chains, linked together by a disulfide bond. A-II (A-III) inhibited bovine trypsin and alpha-chymotrypsin probably at an identical site. B-I (BII) inactivated bovine alpha-chymotrypsin and porcine elastase. Sequence analyses of A-II and B-II revealed a considerable homology with soybean trypsin inhibitor (Kunitz) but suggested the presence of an about 20-amino acid insertion in the A-II molecule.     

Synthesis and evaluation of amphiphilic cationic quinine-derived for antibacterial activity against methicillin-resistant Staphylococcus aureus

Several representative amphiphilic cationic quinine-derived have been synthesized and evaluated against methicillin- resistant Staphylococcus aureus. This is the first reported antibacterial activity of this class of compounds. In vitro the minimal inhibitory concentration values of the best compound Q7 ranged from 0.4 to 1.6 microg/mL (MBC<3.2 microg/mL).     

Self-assembled micelles of biodegradable triblock copolymers based on poly(ethyl ethylene phosphate) and poly(-caprolactone) as drug carriers

A series of novel amphiphilic triblock copolymers of poly(ethyl ethylene phosphate) and poly(-caprolactone) (PEEP-PCL-PEEP) with various PEEP and PCL block lengths were synthesized and characterized. These triblock copolymers formed micelles composed of a hydrophobic core of poly(-caprolactone) (PCL) and a hydrophilic shell of poly(ethyl ethylene phosphate) (PEEP) in aqueous solution. The micelle morphology was spherical, determined by transmission electron microscopy. It was found that the size and critical micelle concentration values of the micelles depended on both hydrophobic PCL block length and PEEP hydrophilic block length. The in vitro degradation characteristics of the triblock copolymers were investigated in micellar form, showing that these copolymers were completely biodegradable under enzymatic catalysis of Pseudomonas lipase and phosphodiesterase I. These triblock copolymers were used for paclitaxel (PTX) encapsulation to demonstrate the potential in drug delivery. PTX was successfully loaded into the micelles, and the in vitro release profile was found to be correlative to the polymer composition. These biodegradable triblock copolymer micelles are potential as novel carriers for hydrophobic drug delivery.     

The influence of the polar head and the hydrophobic chain on the skin penetration enhancement effect of poly(ethylene glycol) derivatives

The effect of a homologue series of nonionic surfactants, namely poly(ethylene glycol) (PEG) fatty acid esters, differing in oxyethylene (PEG 8, PEG 12, and PEG 40) and fatty acid (stearate, mono and di-laurate, and mono and di-oleate) chain lengths, on in vitro skin permeability of ketoprofen (KTP) vehicled in plasters was investigated. The drug diffusion through hairless mouse skin as well as the effect of the surfactant type and strength was studied by Franz diffusion cells and ATR-FTIR spectroscopy. The use of PEG stearate series revealed that the surfactant with the largest polar head, namely PEG 40, was ineffective in enhancing the skin permeation of KTP, independently of the plaster concentrations. The effect of the hydrophobic chain was investigated only by using the shortest oxyethylene chains. The experimental results revealed that the oxyethylene chain length of surfactants appeared to be more influent than the alkyl chain. The prediction of the absorption enhancing capability of these PEG derivatives appeared related to the vehicle other than the proper combination of the number of ethylene oxide groups and alkyl groups.     

Design, synthesis and antibacterial activity studies of model peptidess of proline/arginine-rich region in bactenecin7

Bactenecin 7 (Bac7), a cationic antibacterial peptide, contains a repeating region of Xaa-Pro-Arg-Pro (Xaa = hydrophobic residue). To investigate the structure and property of a Pro/Arg-rich region, e synthesized a series of peptides, Xaa-Pro-Arg-Pro (Xaa = Gly, Arg, Leu, Ile, and Phe) as models and characterized . The conformational preferences of these peptides in water and trifluoroethanol were examined by circular dichroism. The results suggest the presence of largely poly(Pro)-II helical conformation in aqueous and trifluoroethanol solutions. Their antibacterial activity against gram-negative bacteria such as Escherichia coli, Klebsiella Pneumoniae, Pseudomonas aeruginosa, and Escherichia coliHB101, and gram-positive bacteria such as Staphylococcus aureus were measured at various peptide concentrations. Two of our synthetic tetrapeptide fragments containing Gly and Arg were efficiently killed with gram-positive bacteria, Staphylococcus aureus, at the concentration level of 200 microg/mL.     

Synthesis and characterization of six-arm star poly(delta-valerolactone)-block-methoxy poly(ethylene glycol) copolymers

Novel amphiphilic six-arm star diblock copolymers based on biocompatible and biodegradable poly(delta-valerolactone) (PVL) and methoxy poly(ethylene glycol) (MePEG) were synthesized by a two-step process. First, the hydrophobic star-shaped PVL with hydroxyl terminated functional groups was synthesized using a multifunctional alcohol, dipentaerythritol (DPE), as the initiator and fumaric acid as the catalyst. The amphiphilic six-arm star copolymer of poly(delta-valerolactone)-b-methoxy poly(ethylene glycol), (PVL-b-MePEG)(6), was then synthesized by coupling the hydroxyl terminated six-arm PVL homopolymer with alpha-methoxy-omega-chloroformate-poly(ethylene glycol) (MePEG-COCl). (1)H NMR and GPC analyses confirmed the successful synthesis of star-shaped copolymers with predicted compositions and narrow molecular weight distributions. DSC analysis revealed that the glass transition temperatures of the star PVL homopolymers with M(n) between 5000 and 49 000 are not dependent on their molecular weights, whereas the melting temperatures of both the PVL homopolymers and the amphiphilic (PVL-b-MePEG)(6) copolymers increase with an increase in the PVL molecular weight. Micelles were prepared from the (PVL-b-MePEG)(6) copolymers via the dialysis method and found to have effective mean diameters ranging from 10 to 45 nm, depending on the copolymer composition. In addition, the (PVL-b-MePEG)(6) copolymers having lower PVL content were found to form micelles with a narrow monomodal size distribution, whereas the copolymers having higher PVL content tended to form aggregates with a bimodal size distribution. The noncytotoxicity of the copolymers was also confirmed in CHO-K1 fibroblast cells using a cell viability assay, indicating that the (PVL-b-MePEG)(6) copolymers are suitable for biomedical applications such as drug delivery.     

Synthesis,biodistribution and antitumor activity of hematoporphyrin-platinum(II) conjugates

A new series of platinum(II) complexes of pegylated hematoporphyrin derivatives with controlled hydrophobic/hydrophilic balance were synthesized by introducing different kinds of poly(ethylene glycol) and amine ligands to the porphyrin ring. The antitumor activity of the porphyrin-platinum(II) conjugates was assayed in vitro and in vivo against leukemia L1210 cell line and various human tumor cell lines. The present complexes exhibited high antitumor activity and improved water solubility as well as considerable lipophilicity. In particular, complex 16 showed not only higher in vivo activity (T/C%=258) than cisplatin (T/C%=184) and carboplatin (T/C%=168), but also excellent solubility in water and organic solvent. The antitumor activity of complex 20 was superior to that of carboplatin against all human tumor cell lines tested. Moreover, some amphiphilic complexes (7 and 12) exhibited elevated tumor-localizing effect (tumor/muscle ratio>2).     

Amphiphilic block copolymer/poly(dimethylsiloxane) (PDMS) blends and nanocomposites for improved fouling-release

Amphiphilic diblock copolymers, Sz6 and Sz12, consisting of a poly(dimethylsiloxane) block (average degree of polymerisation?=?132) and a PEGylated-fluoroalkyl modified polystyrene block (Sz, average degree of polymerisation?=?6, 12) were prepared by atom transfer radical polymerization (ATRP). Coatings were obtained from blends of either block copolymer (1-10 wt%) with a poly(dimethylsiloxane) (PDMS) matrix. The coating surface presented a simultaneous hydrophobic and lipophobic character, owing to the strong surface segregation of the lowest surface energy fluoroalkyl chains of the block copolymer. Surface chemical composition and wettability of the films were affected by exposure to water. Block copolymer Sz6 was also blended with PDMS and a 0.1 wt% amount of multiwall carbon nanotubes (CNT). The excellent fouling-release (FR) properties of these new coatings against the macroalga Ulva linza essentially resulted from the inclusion of the amphiphilic block copolymer, while the addition of CNT did not appear to improve the FR properties.