Peptide-labeled quantum dots for imaging GPCRs in whole cells and as single molecules

We report a robust and practical method for the preparation of water-soluble luminescent quantum dots (QDs) selectively coupled through an amine or thiol linkage to peptide ligands targeted to G-protein coupling receptors (GPCRs) and demonstrate their utility in whole-cell and single-molecule imaging. We utilized a low molecular weight ( approximately 1200 Da) diblock copolymer with acrylic acids as hydrophilic segments and amido-octyl side chains as hydrophobic segments for facile encapsulation of QDs (QD 595 and QD 514) in aqueous solutions. As proof of principle, these QDs were targeted to the human melanocortin receptor (hMCR) by chemoselectively coupling the polymer-coated QDs to either a hexapeptide analog of alpha-melanocyte stimulating hormone or to the highly potent MT-II ligand containing a unique amine. To label QDs with ligands lacking orthogonal amines, the diblock copolymers were readily modified with water-soluble trioxa-tridecanediamine to incorporate freely available amine functionalities. The amine-functionalized QDs underwent facile reaction with the bifunctional linker NHS-maleimide, allowing for covalent coupling to GPCR-targeted ligands modified with unique cysteines. We demonstrate the utility of these maleimide-functionalized QDs by covalent conjugation to a highly potent Deltorphin-II analog that allowed for selective cell-surface and single-molecule imaging of the human delta-opioid receptor (hDOR).     

Synthesis of novel folic acid-functionalized biocompatible block copolymers by atom transfer radical polymerization for gene delivery and encapsulation of hydrophobic drugs

Two synthetic routes to folic acid (FA)-functionalized diblock copolymers based on 2-(methacryloyloxy)ethyl phosphorylcholine [MPC] and either 2-(dimethylamino)ethyl methacrylate [DMA] or 2-(diisopropylamino)ethyl methacrylate [DPA] were explored. The most successful route involved atom transfer radical polymerization (ATRP) of MPC followed by the tertiary amine methacrylate using a 9-fluorenylmethyl chloroformate (Fmoc)-protected ATRP initiator. Deprotection of the Fmoc groups produced terminal primary amine groups, which were conjugated with FA to produce two series of novel FA-functionalized biocompatible block copolymers. Nonfunctionalized MPC-DMA diblock copolymers have been previously shown to be effective synthetic vectors for DNA condensation; thus, these FA-functionalized MPC-DMA diblock copolymers appear to be well suited to gene therapy applications based on cell targeting strategies. In contrast, the FA-MPC-DPA copolymers are currently being evaluated as pH-responsive micellar vehicles for the delivery of highly hydrophobic anticancer drugs.     

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.     

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.     

人工纤维小体骨架蛋白的分子设计与自组装

纤维小体是厌氧微生物分泌的能够高效降解木质纤维素的一种多酶复合体。其骨架蛋白质的大量重组合成是人工纤维小体构建的关键。为了构建全长的结构复杂的能招募大量酶亚基的骨架蛋白质,本研究对解纤维梭菌Ruminiclostridum cellulolyticum骨架蛋白CipC进行分子模块化设计,并分别与肽-蛋白质共价偶联系统SpyTag/SpyCatcher和SnoopTag/SnoopCatcher融合表达。然后分别通过非固定化和固定化组装策略对人工纤维小体骨架蛋白质进行了初步组装。结果显示,SpyTag和SpyCatcher、SnoopTag和SnoopCatcher之间自发形成稳定的共价异肽键,而且SpyTag和SnoopCatcher, SnoopTag和SpyCacther之间无交叉反应。最终成功组装出含有不同数量黏附域的骨架蛋白质,实现骨架蛋白链的延伸。本研究为构建结构更复杂、活性更高的人工纤维小体提供了新的设计思路并奠定了其技术基础。     

pH-responsive micelles and vesicles nanocapsules based on polypeptide diblock copolymers

The self-assembly of well-defined polypeptide-based diblock copolymers into micelles and vesicles is presented. The stimuli-responsive behavior of polypeptides to pH and ionic strength is used to produce stimuli-responsive nanoparticles with controlled size and shape. Results focusing on micelles and vesicles obtained from polypeptide-based diblock copolymers that are particularly promising for biomedical applications are detailed by means of static and dynamic light scattering analysis, UV circular dichroism, NMR and small angle neutron scattering experiments. Also systems able to form vesicles with a narrow size distribution at basic and acid pH going through a single molecule intermediate state are presented. These nanoparticles are particularly interesting for encapsulation and delivery purpose at a controlled pH.     

Amine-reactive biodegradable diblock copolymers

A new class of diblock copolymers was synthesized from biodegradable poly(lactic acid) and poly(ethylene glycol)minus signmonoamine. These polymers were activated by covalently attaching linkers such as disuccinimidyl tartrate or disuccinimidyl succinate to the hydrophilic polymer chain. The polymers were characterized by (1)H NMR spectroscopy, (13)C NMR spectroscopy and gel permeation chromatography (GPC). These investigations indicated that the polymers were obtained with the correct composition, in high purities, and the expected molecular weight. By using dyes containing primary amine groups such as 5-aminoeosin as model substrates, it was possible to show that the polymers are able to bind such compounds covalently. The diblock copolymers were developed to suppress unspecific protein adsorption and allow the binding of bioactive molecules by instant surface modification. The polymers are intended to be used for tissue engineering applications where surface immobilized cell adhesion peptides or growth factors are needed to control cell behavior.     

Kinetically controlled nanostructure formation in self-assembled globular protein-polymer diblock copolymers

Aqueous processing of globular protein-polymer diblock copolymers into solid-state materials and subsequent solvent annealing enables kinetic and thermodynamic control of nanostructure formation to produce block copolymer morphologies that maintain a high degree of protein fold and function. When model diblock copolymers composed of mCherry-b-poly(N-isopropylacrylamide) are used, orthogonal control over solubility of the protein block through changes in pH and the polymer block through changes in temperature is demonstrated during casting and solvent annealing. Hexagonal cylinders, perforated lamellae, lamellae, or hexagonal and disordered micellar phases are observed, depending on the coil fraction of the block copolymer and the kinetic pathway used for self-assembly. Good solvents for the polymer block produce ordered structures reminiscent of coil-coil diblock copolymers, while an unfavorable solvent results in kinetically trapped micellar structures. Decreasing solvent quality for the protein improves long-range ordering, suggesting that the strength of protein interactions influences nanostructure formation. Subsequent solvent annealing results in evolution of the nanostructures, with the best ordering and the highest protein function observed when annealing in a good solvent for both blocks. While protein secondary structure was found to be almost entirely preserved for all processing pathways, UV-vis spectroscopy of solid-state films indicates that using a good solvent for the protein block enables up to 70% of the protein to be retained in its functional form.     

Spontaneous self-assembly of diblock copolymers in nanoconfined geometries by dissipative particle dynamics

We performed dissipative particle dynamics (DPD) simulations to reproduce phase separation morphologies of diblock copolymers for directed self-assembly (DSA) lithography. DSA is a promising technique to overcome the current photolithography resolution limit. The Flory–Huggins χ parameter estimated from a small-angle X-ray scattering experiment was used for the DPD simulations owing to the multiple degrees of coarse-graining. The degree of coarse-graining and the bond parameter for the spring force were optimised to represent the experimental result of the number of lamellar layers formed in a trench guide. The DPD simulations using these parameters can also represent the diameter of the central cylinder domain that is formed by phase separation in the cylindrical hole. It was found that the bond parameter considering the spreading of polymer segments in a coarse-grained particle gives good quantitative agreement between the results of the simulations and the experimental data.     

Intramolecular cross-linking of single-stranded copolymers of 4-thiouridine and cytidine

Highly polymerized copolymers of 4-thiouridine and cytidine were prepared by direct thiolation of polycytidylic acid. Irradiation with 300–400 nm light of these copolyribonucleotides results in the covalent linkage of about 10% of the thiouridines with cytidines. No such photoreaction occurs with the corresponding mixture of nucleosides, nor with either the thiolated CpC dinucleotide or the double-stranded complexes formed by the copolymers when associated with poly(I) or poly(G): the thiouridine-cytidine covalent links are formed between $\text{non adjacent residues in the folded single-stranded chains}$.     

Synthesis of biodegradable poly-epsilon-caprolactone microspheres by dispersion ring-opening polymerization in supercritical carbon dioxide

A series of fluorinated diblock and triblock copolymers of poly(epsilon-caprolactone) and poly(heptadecafluorodecylacrylate) were prepared by combining ring-opening polymerization of epsilon-CL and atom transfer radical polymerization of the acrylate. These copolymers with well-controlled molecular weight and composition were characterized by (1)H NMR spectroscopy and used as stabilizers for the dispersion ring-opening polymerization of epsilon-CL in supercritical carbon dioxide. The effect of composition and architecture of the polymeric stabilizers on the stabilization of PCL microspheres was investigated. Finally, purification of PCL was successfully implemented by reactive supercritical fluid extraction of the tin catalyst.     

Study of the stability of long-range-ordered lamellar structures for directed self-assembly lithography,performed using dissipative particle dynamics

We performed dissipative particle dynamics (DPD) simulations to obtain long-range-ordered lamellar structures for directed self-assembly lithography. The self-assembled structure of diblock copolymers (DBCs) depends on the length of the different blocks and the difference in their solubility parameters. In the DPD simulations, the DBCs were formed from coarse-grained particles, and the difference between the solubility parameters was represented by a repulsion parameter. We examined the phase separation morphology of the DBCs, which were confined using a trench model system. The repulsion parameter for the assembly of the lamellar structures from the DBC particles was chosen from six types of parameters. The orientation of the lamellar structure was controlled by the repulsion parameter that described the repulsion between the particles and the wall of the system. We changed the width of the trench, and examined the probability for the formation of the lamellar structure. The lamellar structure could not be obtained by increasing the width. To increase the probability, we placed a ridge at the centre of the bottom wall. It was found that the presence of the ridge increased the probability for the formation of the long-range-ordered lamellar structures.     

Phosphonium-containing diblock copolymers for enhanced colloidal stability and efficient nucleic Acid delivery

RAFT polymerization successfully controlled the synthesis of phosphonium-based AB diblock copolymers for nonviral gene delivery. A stabilizing block of either oligo(ethylene glycol(9)) methyl ether methacrylate or 2-(methacryloxy)ethyl phosphorylcholine provided colloidal stability, and the phosphonium-containing cationic block of 4-vinylbenzyltributylphosphonium chloride induced electrostatic nucleic acid complexation. RAFT polymerization generated well-defined stabilizing blocks (M(n) = 25000 g/mol) and subsequent chain extension synthesized diblock copolymers with DPs of 25, 50, and 75 for the phosphonium-containing block. All diblock copolymers bound DNA efficiently at ± ratios of 1.0 in H(2)O, and polyplexes generated at ± ratios of 2.0 displayed hydrodynamic diameters between 100 and 200 nm. The resulting polyplexes exhibited excellent colloidal stability under physiological salt or serum conditions, and they maintained constant hydrodynamic diameters over 24 h. Cellular uptake studies using Cy5-labeled DNA confirmed reduced cellular uptake in COS-7 and HeLa cells and, consequently, resulted in low transfection in these cell lines. Serum transfection in HepaRG cells, which are a predictive cell line for in vivo transfection studies, showed successful transfection using all diblock copolymers with luciferase expression on the same order of magnitude as Jet-PEI. All diblock copolymers exhibited low cytotoxicity (>80% cell viability). Promising in vitro transfection and cytotoxicity results suggest future studies involving the in vivo applicability of these phosphonium-based diblock copolymer delivery vehicles.     

One-step synthesis of amphiphilic diblock copolymers from bacterial poly([R]-3-hydroxybutyric acid)

Catalyzed transesterification in the melt is used to produce diblock copolymers of poly([R]-3-hydroxybutyric acid), PHB, and monomethoxy poly(ethylene glycol), mPEG, in a one-step process. Bacterial PHB of high molecular weight is depolymerized by consecutive and partly simultaneous reactions: pyrolysis and transesterification. The formation of diblocks is accomplished by the nucleophilic attack from the hydroxyl end-group of the mPEG catalyzed by bis(2-ethylhexanoate) tin. The resulting diblock copolymers are amphiphilic and self-assemble into sterically stabilized colloidal suspensions of PHB crystalline lamellae.     

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.     

Computer simulation of polymer and biopolymer self-assembly for drug delivery

Molecular simulation is an emerging tool to bridge relevant time- and length-scales in self-assembly and interfacial processes in soft matter and biological systems. In this review, we highlight mesoscale and coarse-grained molecular dynamics simulation techniques as applied to poly(ethylene oxide)-based diblock copolymer self-assembly. Moreover, we review recent progress pertaining to diblock copolymer and biopolymer self-assembly, stability, and finally, interactions of hydrophobic drugs with polymer membranes. We expect that these computational investigations should provide a useful complement to experimental studies that address open questions in the field of polymeric drug delivery.     

Polymersome encapsulated hemoglobin: a novel type of oxygen carrier

Bovine hemoglobin (Hb) was encapsulated inside polymer vesicles (polymersomes) to form polymersome encapsulated Hb (PEH) dispersions. PEH particles are 100% surface PEGylated with longer PEG chains and possess thicker hydrophobic membranes as compared to conventional liposomes. Polymersomes were self-assembled from poly(butadiene)-poly(ethylene glycol) (PBD-PEO) amphiphilic diblock copolymers with PBD-PEO molecular weights of 22-12.6, 5-2.3, 2.5-1.3, and 1.8-0.9 kDa. The first two diblock copolymers possessed linear hydrophobic PBD blocks, while the later possessed branched PBD blocks. PEH dispersions were extruded through 100 and 200 nm pore radii membranes. The size distribution, Hb encapsulation efficiency, P(50), cooperativity coefficient, and methemoglobin (metHb) level of PEH dispersions were consistent with values required for efficient oxygen delivery in the systemic circulation. The influence of different molecular weight diblock copolymers on the physical properties of PEH dispersions was analyzed. PBD-PEO copolymers with molecular weights of 22-12.6 and 2.5-1.3 kDa completely dissolved in aqueous solution to form polymersomes, while the other two copolymers formed a mixture of solid copolymer precipitates and polymersomes. PEHs self-assembled from 22-12.6 and 2.5-1.3 kDa PBD-PEO copolymers possessed Hb loading capacities greater than PEG-LEHs, PEGylated actin-containing LEHs, and nonmodified LEHs, although their sizes were smaller and their hydrophobic membranes were thicker. The Hb loading capacities of these polymersomes were also higher than lipogel encapsulated hemoglobin particles and nanoscale hydrogel encapsulated hemoglobin particles. PEH dispersions exhibited average radii larger than 50 nm and exhibited oxygen affinities comparable to human erythrocytes. Polymersomes did not induce Hb oxidation. The interaction between Hb and the membrane of 2.5-1.3 kDa PBD-PEO polymersomes improved the monodispersity of these particular PEH dispersions. These results suggest that PEHs could serve as efficient oxygen therapeutics.     

Additional stabilization of penicillin G acylase-agarose derivatives by controlled chemical modification with formaldehyde.

We have tested the effect of chemical modifications with formaldehyde on the activity/stability of immobilized derivatives of the enzyme penicillin G acylase (PGA). These derivatives were previously stabilized through enzyme-support multipoint covalent attachment. We carried out very different chemical treatments of our derivatives by testing the effect of different variables which control the intensity and the nature of these amine-formaldehyde reactions. The variables tested were: formaldehyde concentration, pH, time, and temperature. We also developed a colorimetric titration of the free amine groups on immobilized PGA in order to evaluate the extension of the reaction between formaldehyde and the amine groups of the enzyme. As a consequence of these studies, we have been able to get additional stabilizations of our previously stabilized-immobilized derivatives: e.g. a factor of 24-fold was achieved in terms of stabilization against irreversible thermal inactivation. The integrated effect of additional chemical modification plus previous multipoint covalent attachment has allowed us to prepare PGA derivatives which are 50,000 more thermostable than native PGA as well as most of the commercial PGA derivatives.     

Effect of methylamine on the reaction of alpha 2-macroglobulin with enzymes.

The kinetics of reaction of alpha 2-macroglobulin (alpha 2M) with thrombin and with trypsin were studied in the presence and absence of methylamine. The rate of enzyme-induced thiol release was found to be the same whether or not amine was present. The result suggests that covalent bond formation and enzyme-catalyzed amine incorporation proceed via a common (enzyme-dependent) rate-determining step. The reaction of lysyl-modified enzymes (which show poor covalent binding with alpha 2M) was similarly unaffected by amine, indicating that enzyme-catalyzed steps were also rate determining for hydrolysis of the thiol ester. The products of the reactions were analyzed by native and denaturing gel electrophoresis. Methylamine did not affect the total binding of enzyme to alpha 2M but did cause a substantial decrease in covalent binding. Surprisingly, not all covalent complexes were affected by the presence of amine: complexes in which enzyme was covalently bound to one half-molecule increased compared to the reaction with no amine; complexes in which two half-molecules are cross-linked by two bonds to a single enzyme were substantially reduced, however. The results are consistent with a mechanism of reaction in which an enzyme-dependent step is rate determining. This step is accompanied by activation of two thiol esters. One of these reacts immediately with the bound enzyme (or may be hydrolyzed if the enzyme amine groups are blocked). The other activated center is capable of reaction with external nucleophiles such as methylamine.     

Versatile method for production and controlled polymer-immobilization of biologically active recombinant proteins

The immobilization of a protein by covalent attachment to a support matrix should involve only functional groups of the protein that are not essential for its biological activity. A general strategy for obtaining recombinant proteins designed for oriented covalent grafting onto copolymers was investigated. The rationale involves the definition of seven p24-derived recombinant proteins as fused to either distant or adjacent tags comprising primary amine rich tag consisting of six contiguous lysines suitable for oriented covalent immobilization and a hexa-histidine tag suitable for metal chelate affinity purification. High-level expression, efficient affinity purification, and coupling yields onto maleic anhydride-alt-methyl vinyl ether copolymers higher than 95% were obtained for all proteins. Afterwards, an investigation of the biological features of the immobilized vs. nonimmobilized protein onto the copolymer allowed us to select one bioconjugate which was used in a diagnostic context, i.e., as a capture antigen in an ELISA format test. Sera from 107 HIV-seropositive individuals at various stages of HIV infection, including two seroconversion panels and 104 healthy HIV-seronegative controls, were tested using either RH24 or RK24H-copolymer coated onto the microtiter plate. These assays showed that the use of such a protein-copolymer bioconjugate allowed detection of lower antibody titers than the RH24 protein, illustrating the potential of applications of such doubly tagged proteins. Thus, a set of expression vectors was designed containing four different combinations of hexa-lysine and hexa-histidine tags and a multiple cloning site, allowing the production of different recombinant fusion proteins suitable for biological reactivity conservation after immobilization.