Cationic amphiphilic star and linear block copolymers: synthesis, self-assembly, and in vitro gene transfection

A series of amphiphilic star and linear block copolymers were synthesized using ATRP. The core consisted of either polystyrene (PS) or poly(n-butyl acrylate) (PBuA), having different glass-transition (T(g)) values. These polymers were used as macroinitiators in the polymerization of the cationic 2-(dimethylamino)ethyl methacrylate (DMAEMA). The polymers were used to study the effects of polymer architecture and flexibility on the self-assembling properties, DNA complexation, and transfection. All polymers formed core-shell micelles in aqueous solutions and condensed plasmid DNA. Linear PDMAEMA-PBuA-PDMAEMA has transfection efficiency comparable to PEI25K in ARPE19 cell line. Glassy state of the micellar core and star-shaped architecture decreased the DNA transfection compared with the rubbery and linear polymer structures. The polymers showed low cellular toxicity at low nitrogen/phosphate (n/p) ratios.     

Folate-conjugated thermoresponsive block copolymers: highly efficient conjugation and solution self-assembly

A combination of controlled radical polymerization and azide-alkyne click chemistry was employed to prepare temperature-responsive block copolymer micelles conjugated with biological ligands with potential for active targeting of cancer tissues. Block copolymers of N-isopropylacrylamide (NIPAM) and N,N-dimethylacrylamide (DMA) were synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization with an azido chain transfer agent (CTA). Pseudo-first-order kinetics and linear molecular weight dependence on conversion were observed for the RAFT polymerizations. CuI-catalyzed coupling with propargyl folate resulted in folic acid residues being efficiently conjugated to the alpha-azido chain ends of the homo and block copolymers. Temperature-induced self-assembly resulted in aggregates capable of controlled release of a model hydrophobic drug. CuI-catalyzed azide-alkyne cycloaddition has proven superior to conventional methods for conjugation of biological ligands to macromolecules, and the general strategy presented herein can potentially be extended to the preparation of folate-functionalized assemblies with other stimuli susceptibility (e.g., pH) for therapeutic and imaging applications.     

Solution self-assembly of hybrid block copolymers containing poly(ethylene glycol) and amphiphilic beta-strand peptide sequences

The self-assembly in aqueous solution of hybrid block copolymers consisting of amphiphilic beta-strand peptide sequences flanked by one or two PEG chains was investigated by means of circular dichroism spectroscopy, small-angle X-ray scattering, and transmission electron microscopy. In comparison with the native peptide sequence, it was found that the peptide secondary structure was stabilized against pH variation in the di- and tri-block copolymers with PEG. Small-angle X-ray scattering indicated the presence of fibrillar structures, the dimensions of which are comparable to the estimated width of a beta-strand (with terminal PEG chains in the case of the copolymers). Transmission electron microscopy on selectively stained and dried specimens shows directly the presence of fibrils. It is proposed that these fibrils result from the hierarchical self-assembly of peptide beta-strands into helical tapes, which then stack into fibrils.     

Bacterial synthesis of PHA block copolymers

Polyhydroxyalkanoates (PHA) containing block copolymers were synthesized in Cupriavidus necator using periodic substrate addition. Poly(3-hydroxybutyrate) (PHB) segments were formed during fructose utilization. Pulse feeds of pentanoic acid resulted in the synthesis of 3-hydroxyvalerate monomers, forming poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) random copolymer. PHA synthesis was controlled using analysis of oxygen uptake and carbon evolution rates from the bioreactor off-gas. A combination of characterization techniques applied to the polymer batches strongly suggests the presence of block copolymers: (i) Thermodynamically stable polymer samples obtained by fractionation and analyzed by differential scanning calorimetry (DSC) and nuclear magnetic resonance spectroscopy (NMR) indicate that some fractions, representing approximately 30% of the total polymer sample, exhibit melting characteristics and nearest-neighbor statistics indicative of block copolymers, (ii) preliminary rheology experiments indicate additional mesophase transitions only found in block copolymer materials, (iii) dynamic mechanical analysis shows extension of the rubbery plateaus in block copolymer samples, and (iv) uniaxial extension tests result in differences in mechanical properties (modulus and elongation at failure) expected of similarly prepared block copolymer and single polymer type materials.     

Dynamic light scattering study of self-assembly of HPMA hybrid graft copolymers

The time course of self-assembly of a hybrid hydrogel system was investigated using dynamic light scattering (DLS) techniques. The self-assembling system consisted of a hydrophilic synthetic N-(2-hydroxypropyl)methacrylamide (HPMA) polymer backbone and a pair of oppositely charged peptide grafts (CCE and CCK). These two distinct pentaheptad peptides were anticipated to act as physical cross-linkers by the formation of antiparallel coiled-coil heterodimers. Equimolar mixture of HPMA graft copolymers CCE-P and CCK-P solutions (where P is the HPMA copolymer backbone) with total concentration from 1.25 to 10 mg/mL were measured at a scattering angle 90 degrees and room temperature. A critical extension of average relaxation time was observed with increasing concentration and incubation time. To reveal the role of coiled-coil grafts in the self-assembly process, a pair of modified random coil peptides, CCEw and CCKy, was designed. The DLS evaluation of HPMA copolymer conjugates (CCEw-P and CCKy-P) at total concentration of 10 mg/mL demonstrated that no association occurred after 28 h of incubation. Moreover, addition of a competing peptide (CCK) or a denaturant (guanidium chloride, GndHCl) to the self-assembled CCE-P/CCK-P hydrogels resulted in partial disassembly or collapse of the hydrogel clusters. These results correlated to changes in the secondary structure of peptides (grafts) as measured by circular dichroism spectroscopy (CD). These investigations supported the hypothesis that the self-assembly of CCE-P/CCK-P into hybrid hydrogels is mediated by the formation of coiled-coil heterodimers.     

Hydrogel biopolymer created from the self-assembly of a designed protein containing a four-helix bundle forming motif

A protein hydrogel system based on the assembly of a four-helix bundle motif was proposed and synthesized by genetic engineering methods. This new polypeptide, named GBH1, consists of identical amphipathic helices of 22 residues in length oriented in opposite fashion to one another at each end of a polypeptide with a total length of 227 amino acids. The middle portion of the polypeptide (residues 79-147) is an unstructured random coil. The region between the amphipathic and unstructured segment is an α-helical stretch (23-78 and 148-204) not possessing a sequence compatible with a coiled-coil conformation, but rather possesses regions that have overwinding of the helix. The thermal unfolding of GBH1 shows more than one inflection point (T(m1) = 30.5 °C, T(m2) = 64.6 °C), indicative of a partially unfolded intermediate and, thus, multiple interactions in the folded state. A qualitative assessment of hydrogel formation with varying pH showed that acidic conditions did not support the gel state, indirectly indicating that the proposed four-helix bundle is the major cross-linking structure and not a leucine zipper motif. Scanning electron microscopy reveals a network of interacting protein molecules forming a spongelike matrix with numerous pores that would be occupied with water molecules.     

Synthesis and characterization of self-assembling block copolymers containing bioadhesive end groups

3,4-Dihydroxyphenyl-L-alanine (DOPA) is an unusual amino acid found in mussel adhesive proteins (MAPs) that is believed to lend adhesive characteristics to these proteins. In this paper, we describe a route for the conjugation of DOPA moieties to poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) block copolymers. Hydroxyl end groups of PEO-PPO-PEO block copolymers were activated by N,N'-disuccinimidyl carbonate and then reacted with DOPA or its methyl ester with high coupling efficiencies from both aqueous and organic solvents. DOPA-modified PEO-PPO-PEO block copolymers were freely soluble in cold water, and dye partitioning and differential scanning calorimetry analysis of these solutions revealed that the copolymers aggregated into micelles at a characteristic temperature that was dependent on block copolymer composition and concentration in solution. Oscillatory rheometry demonstrated that above a block copolymer concentration of approximately 20 wt %, solutions of DOPA-modified PEO-PPO-PEO block copolymers exhibited sol-gel transitions upon heating. The gelation temperature could be tailored between approximately 23 and 46 degrees C by changing the composition, concentration, and molecular weight of the block copolymer. Rheological measurement of the bioadhesive interaction between DOPA-modified Pluronic and bovine submaxillary mucin indicated that DOPA-modified Pluronic was significantly more bioadhesive than unmodified Pluronic.     

Poly(ethylene glycol)-poly(ester-carbonate) block copolymers carrying PEG-peptidyl-doxorubicin pendant side chains: synthesis and evaluation as anticancer conjugates

Water soluble polymer anticancer conjugates can improve the pharmacokinetics of covalently bound drugs by limiting cellular uptake to the endocytic route, thus prolonging plasma circulation time and consequently facilitating tumor targeting by the enhanced permeability and retention (EPR) effect. Many of the first generation antitumor polymer conjugates used nonbiodegradable polymeric carriers which limits the molecular weight that can be safely used to <40,000 g/mol. The aim of this ambitious study was to synthesize and evaluate a novel, prototype biodegradable polymeric system based on high molecular weight, water-soluble functionalized polyesters. The main polymeric platform was prepared from bis(4-hydroxy)butyl maleate (DBM) and poly(ethylene glycol) (PEG4000) blocks to give the polymer DBM2-PEG4000 containing biodegradable carbonate bonds and having a M(w) of 100,000-190,000 g/mol; M(n) of 37,000-53,000 g/mol, and M(w)/M(n) of 3.0-3.7. Using thioether linkages, this polymer was then grafted with HS-PEG3000-Gly-Phe-Lue-Gly doxorubicin (HS-PEG3000-GFLG-Dox) pendant side chains ( approximately 30 per DBM2-PEG chain). The final construct, DBM2-PEG4000-S-PEG3000-GFLG-Dox had a total Dox content of 3-4 wt % and a free Dox content of < or = 0.7% total Dox. During incubation with isolated lysosomal enzymes, the rate of Dox release from the polymer backbone was relatively slow (<5% release over 5 h) compared to that seen for PEG5000-GFLG-Dox alone (>20% over 5 h). The in vitro cytotoxicity was assessed using B16F10 murine melanoma (MTT assay). DBM2-PEG4000-S-PEG3000-GFLG-Dox was 10-20-fold less toxic than free Dox. In vivo antitumor activity of the DBM2-PEG4000-S-PEG3000-GFLG-Dox conjugates was assessed using a subcutaneous (s.c.) B16F10 murine melanoma model, and an intraperitoneal (i.p.) L1210 leukaemia model. The increased toxicity (attributed to poor solubility) and low antitumor activity of DBM2-PEG4000-S-PEG3000-GFLG-Dox conjugates compared to PEG5000-GFLG-Dox and HPMA copolymer-Dox conjugates was attributed to the slow rate of Dox release. The DBM2-PEG4000-S-PEG3000-GFLG-Dox conjugates were considered unfavorable as candidates for further development. However, the successful scale-up synthesis of DBM2-PEG4000-S-PEG3000 constructs suggest that they are worthy of further investigation as carriers for controlled release and targeting of less hydrophobic agents.     

Novel synthesis and characterization of bioconjugate block copolymers having oligonucleotides

Novel and efficient synthesis of polymers terminated with nucleotides via the phosphoramidite method has been developed. A hydroxyl-terminated polymer was converted into a polymer capped with a nucleotide in three steps, where the conversion of the reactions was very high, almost 100%. By repetition of this synthetic method, a block copolymer composed of a synthetic polymer, polystyrene, and biological oligonucleotides with thymidine units has been successfully synthesized. A microphase-separated structure of this block copolymer was observed by both transmission electron microscopy and small-angle X-ray scattering, and a cylindrical structure was confirmed.     

Calorimetric dissection of thermal unfolding of OspA,a predominantly beta-sheet protein containing a single-layer beta-sheet

Outer surface protein A (OspA) from Borrelia burgdorferi is a predominantly beta-sheet protein comprised of beta-strands beta1-beta21 and a short C-terminal alpha-helix. It contains two globular domains (N and C-terminal domains) and a unique single-layer beta-sheet (central beta-sheet) that connects the two domains. OspA contains an unusually large number of charged amino acid residues. To understand the mechanism of stabilization of this unique beta-sheet protein, thorough thermodynamic investigations of OspA and its truncated mutant lacking a part of the C-terminal domain were conducted using calorimetry and circular dichroism. The stability of OspA was found to be sensitive to pH and salt concentration. The heat capacity curve clearly consisted of two components, and all the thermodynamic parameters were obtained for each step. The thermodynamic parameters associated with the two transitions are consistent with a previously proposed model, in which the first transition corresponds to the unfolding of the C-terminal domain and the last two beta-strands of the central beta-sheet, and the second transition corresponds to that of the N-terminal domain and the first beta-strand of the central beta-sheet in the second peak. The ratio of calorimetric and van't Hoff enthalpies indicates that the first peak includes another thermodynamic intermediate state. Large heat capacity changes were observed for both transitions, indicative of large changes in the exposure of hydrophobic surfaces associated with the transitions. This observation demonstrates that hydrophobic parts are buried efficiently in the native structure in spite of the low content of hydrophobic residues in OspA. By decomposing the enthalpy, entropy, and Gibbs free energy into contributions from different interactions, we found that the enthalpy changes for hydrogen bonding and polar interactions are exceptionally large, indicating that OspA maintains its stability by making full use of its unique beta-sheet and high content of polar residues. These thermodynamic analyses demonstrated that it is possible to maintain protein tertiary structure by making effective use of an unusual amino acid composition.     

Unexpected multivalent display of proteins by temperature triggered self-assembly of elastin-like polypeptide block copolymers

We report herein the unexpected temperature triggered self-assembly of proteins fused to thermally responsive elastin-like polypeptides (ELPs) into spherical micelles. A set of six ELP block copolymers (ELP(BC)) differing in hydrophilic and hydrophobic block lengths were genetically fused to two single domain proteins, thioredoxin (Trx) and a fibronectin type III domain (Fn3) that binds the α(v)β(3) integrin. The self-assembly of these protein-ELP(BC) fusions as a function of temperature was investigated by UV spectroscopy, light scattering, and cryo-TEM. Self-assembly of the ELP(BC) was unexpectedly retained upon fusion to the two proteins, resulting in the formation of spherical micelles with a hydrodynamic radius that ranged from 24 to 37 nm, depending on the protein and ELP(BC). Cryo-TEM images confirmed the formation of spherical particles with a size that was consistent with that measured by light scattering. The bioactivity of Fn3 was retained when presented by the ELP(BC) micelles, as indicated by the enhanced uptake of the Fn3-decorated ELP(BC) micelles in comparison to the unimer by cells that overexpress the α(v)β(3) integrin. The fusion of single domain proteins to ELP(BC)s may provide a ubiquitous platform for the multivalent presentation of proteins.     

Biodegradable polycarbonate-b-polypeptide and polyester-b-polypeptide block copolymers: synthesis and nanoparticle formation towards biomaterials

The amino poly(trimethylene carbonate)-NHt-Boc (PTMC-NHt-Boc) and poly(epsilon-caprolactone)-NH -Boc (PCL-NHt-Boc) were synthesized by ring-opening polymerization (ROP) of TMC or CL and subsequently deprotected into the corresponding PTMC-NH2 and PCL-NH2. These functional homopolymers were used as macroinitiators for the ROP of gamma-benzyl-L-glutamate N-carboxyanhydride (BLG), consequently, giving the respective diblock copolymers PTMC-b-PBLG and PCL-b-PBLG in almost quantitative yields. The (co)polymers have been characterized by NMR and SEC analyses. DSC and IR studies confirmed the block structure of the copolymers and highlighted a phase separation between the rigid peptide (alpha-helix conformation) and the more flexible polyester segments. The self-assembly and the degradation behaviors of the copolymers depended on the nature of the polyester block and on the copolymer composition. Nanoparticles obtained from PBLG block copolymers were twice smaller ( RH < 100 nm) than those formed from PTMC and PCL homopolymers. Finally, their enzymatic degradation revealed that PTMC nanoparticles degraded faster than those made of PCL.     

Chemo-enzymatic synthesis and characterization of graft copolymers from lignin and acrylic compounds

Chemo-enzymatic initiation of graft copolymerization of acrylic compounds onto different technical lignosulfonates (LS) was compared to a Fenton-like system (ferrous ion, t-BHP). The enzyme tested was a phenoloxidase laccase (EC 1.10.3.2) from the white rot basidomycete Trametes versicolor. Most applied lignins were successfully grafted, resulting in a polymer yield of more than 90%. The effect of initiator concentration and the lignin/monomer ratio on the yield and M(w) of enzymatically grafted polymers were studied. The homopolymer proportion in the enzymatically produced grafts of Ca-LS and acrylic acid was 5 to 6x lower than those initiated by the Fenton-like reagent; no such differences were observed for Na-LS.     

Synthesis and ordered structure of amphipatic block copolymers with a saccharide and a peptide block

Amphipatic block copolymers (OβEb) with a hydrophilic saccharide block and a hydrophobic polypeptide block were synthesized. In these copolymers the saccharide block is the glyco-amino acid Oβ from ovomucoid and the peptide block (Eb) is a poly(γ-benzyl-L -glutamate) block. Copolymers OβEb exhibit, in the solid state and in Me₂SO concentrated solutions, mesomorphic lamellar structures where the polypeptide chains are in an α-helical conformation. Depending on the molecular weight of the polypeptide block, three types of lamellar structures are obtained, and they differ by the mode of organization of the polypeptide chains in their lamellae and by the T or Y conformation of the saccharide block.     

Controlling the aggregation of conjugates of streptavidin with smart block copolymers prepared via the RAFT copolymerization technique

Block copolymers containing stimuli-responsive segments provide important new opportunities for controlling the activity and aggregation properties of protein-polymer conjugates. We have prepared a RAFT block copolymer of a biotin-terminated poly(N-isopropylacrylamide) (PNIPAAm)-b-poly(acrylic acid) (PAA). The number-average molecular weight (M(n)) of the (PNIPAAm)-b-(PAA) copolymer was determined to be 17.4 kDa (M(w)/M(n) = 1.09). The PNIPAAm block had an M(n) of 9.5 kDa and the poly(acrylic acid) (PAA) block had an M(n) of 7.9 kDa. We conjugated this block copolymer to streptavidin (SA) via the terminal biotin on the PNIPAAm block. We found that the usual aggregation and phase separation of PNIPAAm-SA conjugates that follow the thermally induced collapse and dehydration of PNIPAAm (the lower critical solution temperature (LCST) of PNIPAAm is 32 degrees C in water) is prevented through the shielding action of the PAA block. In addition, we show that the cloud point and aggregation properties (as measured by loss in light transmission) of the [(PNIPAAm)-b-(PAA)]-SA conjugate also depended on pH. At pH 7.0 and at temperatures above the LCST, the block copolymer alone was found to form particles of ca. 60 nm in diameter, while the bioconjugate exhibited very little aggregation. At pH 5.5 and 20 degrees C, the copolymer alone was found to form large aggregates (ca. 218 nm), presumably driven by hydrogen bonding between the -COOH groups of PAA with other -COOH groups and also with the -CONH- groups of PNIPAAm. In comparison, the conjugate formed much smaller particles (ca. 27 nm) at these conditions. At pH 4.0, however, large particles were formed from the conjugate both above and below the LCST (ca. 700 and 540 nm, respectively). These results demonstrate that the aggregation properties of the block copolymer-SA conjugate are very different from those of the free block copolymer, and that the outer-oriented hydrophilic block of PAA shields the intermolecular aggregation of the block copolymer-SA bioconjugate at pH values where the -COOH groups of PAA are significantly ionized.     

New ferrocene containing peptide conjugates: synthesis and effect on human leukemia (HL-60) cells

Data reported in this article describe the synthesis of Arg-rich oligopeptide conjugates of ferrocenecarboxylic acid on solid support with two different strategies and for the first time, the successful preparation of peptide conjugates of ferrocenylacrylic acid in solution. The antitumor effect of conjugates was analyzed by MTT assay in vitro. We demonstrated that ferrocenylacrylic acid possessing an enone (--CH==CH--CO--) moiety exhibited remarkable antiproliferative effect against human leukemia cells (HL-60) in vitro, but its effect was not improved by conjugation with hexa- or octaarginines. However, we observed highly increased water-solubility. In contrast, the results provide evidence that conjugation of ferrocenecarboxylic acid to Arg(n) (n = 6, 8) improved not only its water-solubility, but also antitumor effect on human leukemia cells in vitro.     

A conformational alpha-helix to beta-sheet transition accompanies racemic self-assembly of polylysine: an FT-IR spectroscopic study

The self-assembly of polylysine chains with opposite chiral senses is an intriguing phenomenon, suggesting that subtle hydrational effects may be a driving force of protein aggregation. We have used FT-IR spectroscopy to characterize the alpha-helix-to-beta-sheet conformational transition that accompanies the aggregation of single and mixed enantiomers of polylysine. The preferential racemic self-assembly not only takes place at a lower temperature, but is also less prone to repulsive electrostatic interactions between lysine charged side chains, caused by decreasing pH (pD). While the process is generally irreversible, it yet appears to proceed in a stepwise manner through a sequence of thermodynamically, rather than kinetically controlled events involving gradual destabilization of alpha-helices. Interestingly, although the alpha/beta-transition is in either case (single or mixed enantiomers) an endothermic process, it may also be induced by freezing of water, which leads to markedly more complete (and irreversible) aggregation of the mixed enantiomers. Relevance of these findings has been discussed in the context of protein aggregation studies.     

Self-assembling diblock copolymers of poly[N-(2-hydroxypropyl)methacrylamide] and a beta-sheet peptide

The self-assembly of hybrid diblock copolymers composed of poly(HPMA) and beta-sheet peptide P11 (CH(3)CO-QQRFQWQFEQQ-NH(2)) blocks was investigated. Copolymers were synthesized via thiol-maleimide coupling reaction, by conjugation of semitelechelic poly(HPMA)-SH with maleimide-modified beta-sheet peptide. As expected, CD and CR binding studies showed that the peptide block imposed its beta-sheet structural arrangement on the structure of diblock copolymers. TEM and AFM proved that peptide and these copolymers had the ability to self-assemble into fibrils.     

Sweet block copolymer nanoparticles: preparation and self-assembly of fully oligosaccharide-based amphiphile

The preparation of biocompatible nanocarriers that have potential applications in the cosmetic and health industries is highly desired. The self-assembly of amphiphilic block copolymers displaying biosourced polysaccharides at the surface is one of the most promising approaches. In the continuity of our works related to the preparation of "hybrid" amphiphilic oligosaccharide-based block copolymers, we present here the design of a new generation of self-assembled nanoparticles composed entirely of oligosaccharide-based amphiphilic block co-oligomers (BCO). These systems are defined by a covalent linkage of the two saccharidic blocks through their reducing end units, resulting in a sweet "head-to-head" connection. As an example, we have prepared and studied a BCO in which the hydrophilic part is composed of a free maltoheptaosyl derivative clicked to a hydrophobic part composed of a peracetylated maltoheptaosyl derivative. This amphiphilic BCO self-assembles to form spherical micelles in water with an average diameter of 30 nm. The efficient enzymatic hydrolysis of the maltoheptaose that constitutes the shell of the micelles was followed by light scattering and colorimetric methods.