Polymerization Of N-carboxyanhydrides Research Articles

"Grafting-from" synthesis and characterization of poly (2-ethyl-2-oxazoline)-b-poly (benzyl L-glutamate) micellar nanoparticles for potential biomedical applications.

Introduction: Recent advances in the field of poly (2-oxazolines) as bio-inspired synthetic pseudopeptides have proven their potential biomedical applications such as drug delivery and tissue engineering. Methods: In order to fabricate a biodegradable micellar nanoparticle of poly (2-ethyl 2-oxazoline)-b-poly (benzyl L-glutamate) or pEOx-b-pBLG, "grafting-from" synthesis approach was used involving consecutive steps of cationic ring-opening polymerization of 2-ethyl-2-oxazoline, amine functionalization of pEOx using 1-Boc-piperazine and N-carboxyanhydride polymerization of γ-benzyl- L-glutamate. Following hydrolysis of the copolymer, the protecting γ-benzyl groups were removed yielding a double-hydrophilic block ionomer of pEOx-b-poly (L-glutamic acid). The polymers were characterized by FTIR, 1H-NMR, size exclusion chromatography and differential scanning calorimetry (DSC). Aqueous assembly of the polymers was investigated by pyrene assay, dynamic light scattering, and transmission electron microscopy. MTT cytotoxicity assay was also performed to determine the cytocompatibility in various tumor cell lines. Results: The polymeric micelles presented a uni-modal size distribution with mean hydrodynamic diameter of 149.8 ± 10.6 nm and critical aggregation concentration of 60 µg/mL. The average molecular weight of pEOx increased from ~ 14 to 20 kDa for pEOx-b-poly (L-glutamic acid) as determined by light scattering (Debye plot), indicating a successful copolymerization. MTT assay showed little to no practical cytotoxicity at concentrations below 1 mg/mL. Conclusion: Multi-step synthesis of pEOx-b-pBLG and subsequent alkaline hydrolysis were performed to obtain the block ionomer pEOx-b-poly (L-glutamic acid). Both pEOx-based copolymers can be considered for various potential applications such as loading and delivery of drugs, genes, and contrast agents either by chemical conjugation or physical loading.

Glycopolypeptide-Grafted Bioactive Polyionic Complex Vesicles (PICsomes) and Their Specific Polyvalent Interactions.

Glycopolypeptide-based self-assembled nano-/microstructures with surface-tethered carbohydrates are excellent mimics of glycoproteins on the cell surface. To expand the broad repertoire of glycopolypeptide-based supramolecular soft structures such as polymersomes formed via self-assembly of amphiphilic polymers, we have developed a new class of polyionic complex vesicles (PICsomes) with glycopolypeptides grafted on the external surface. Oppositely charged hydrophilic block copolymers of glycopolypeptide20-b-poly-l-lysine100 and PEG2k-b-poly-l-glutamate100 [PEG = poly(ethylene glycol)] were synthesized using a combination of ring-opening polymerization of N-carboxyanhydrides and “click” chemistry. Under physiological conditions, the catiomer and aniomer self-assemble to form glycopolypeptide-conjugated PICsomes (GP-PICsomes) of micrometer dimensions. Electron and atomic force microscopy suggests a hollow morphology of the PICsomes, with inner aqueous pool (core) and peripheral PIC (shell) regions. Owing to their relatively large (∼micrometers) size, the hollowness of the supramolecular structure could be established via fluorescence microscopy of single GP-PICsomes, both in solution and under dry conditions, using spatially distributed fluorescent probes. Furthermore, the dynamics of single PICsomes in solution could be imaged in real time, which also allowed us to test for multivalent interactions between PICsomes mediated by a carbohydrate (mannose)-binding protein (lectin, Con-A). The immediate association of several GP-PICsomes in the presence of Con-A and their eventual aggregation to form large insoluble aggregate clusters reveal that upon self-assembly carbohydrate moieties protrude on the outer surface which retains their biochemical activity. Challenge experiments with excess mannose reveal fast deaggregation of GP-PICsomes as opposed to that in the presence of excess galactose, which further establishes the specificity of lectin-mediated polyvalent interactions of the GP-PICsomes.

Simultaneous Polymerization and Polypeptide Particle Production via Reactive Spray-Drying.

A method for producing polypeptide particles via in situ polymerization of N-carboxyanhydrides during spray-drying has been developed. This method was enabled by the development of a fast and robust synthetic pathway to polypeptides using 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as an initiator for the ring-opening polymerization of N-carboxyanhydrides. The polymerizations finished within 5 s and proved to be very tolerant toward impurities such as amino acid salts and water. The formed particles were prepared by mixing the monomer, N-carboxyanhydride of l-glutamic acid benzyl ester (NCAGlu) and the initiator (DBU) during the atomization process in the spray-dryer and were spherical with a size of ∼1 μm. This method combines two steps; making it a straightforward process that facilitates the production of polypeptide particles. Hence, it furthers the use of spray-drying and polypeptide particles in the pharmaceutical industry.

Synthesis and Mechanism Insight of a Peptide-Grafted Hyperbranched Polymer Nanosheet with Weak Positive Charges but Excellent Intrinsically Antibacterial Efficacy.

Antimicrobial resistance is an increasingly problematic issue in the world and there is a present and urgent need to develop new antimicrobial therapies without drug resistance. Antibacterial polymers are less susceptible to drug resistance but they are prone to inducing serious side effects due to high positive charge. Herein we report a peptide-grafted hyperbranched polymer which can self-assemble into unusual nanosheets with highly effective intrinsically antibacterial activity but weak positive charges (+ 6.1 mV). The hyperbranched polymer was synthesized by sequential Michael addition-based thiol-ene and free radical mediated thiol-ene reactions, and followed by ring-opening polymerization of N-carboxyanhydrides (NCAs). The nanosheet structure was confirmed by transmission electron microscopy (TEM) and atomic force microscopy (AFM) studies. Furthermore, a novel "wrapping and penetrating" antibacterial mechanism of the nanosheets was revealed by TEM and it is the key to significantly decrease the positive charges but have a very low minimum inhibitory concentration (MIC) of 16 μg mL(-1) against typical Gram-positive and Gram-negative bacteria. Overall, our synthetic strategy demonstrates a new insight for synthesizing antibacterial nanomaterials with weak positive charges. Moreover, the unique antibacterial mechanism of our nanosheets may be extended for designing next-generation antibacterial agents without drug resistance.

Tuning the conformation of synthetic co-polypeptides of serine and glutamic acid through control over polymer composition

Ring opening polymerization (ROP) of N-carboxy anhydride (NCA) amino acids presents a rapid way to synthesize high molecular weight polypeptides with different amino acid compositions. The compositional and functional versatility of polypeptides make these materials an attractive choice for biomaterials. The functional performance of polypeptide materials is equally linked to their conformation which is determined by the amino acid sequence in the polymer chains. Here, the interplay between composition and conformation of synthetic polypeptides obtained by NCA polymerization was explored. Various copolypeptides from Glu(Bzl) and Ser(Bzl) were prepared to investigate how polypeptide composition affected the conformation of the resulting copolymer. Polymerization kinetics indicated that the copolymerization of Glu(Bzl) and Ser(Bzl) preferentially yielded alternating copolymers. Both the polydispersity and the conformation of the polypeptides were dependent on the Ser(Bzl) content in the polymer, demonstrating that polypeptide functionalities could be tuned directly by altering the relative amounts of amino acids in the chain. This work presents the first step toward an improved understanding and control over polypeptide conformation through modulating the amino acid composition of the material. Understanding this sequence–functionality relationship is essential to advancing the use of ROP as a technique to design smart polypeptide based materials with specific functions. © 2016 The Authors. Journal of Polymer Science Part A: Polymer Chemistry Published by Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2016, 54, 2331–2336

Heterofunctional Glycopolypeptides by Combination of Thiol-Ene Chemistry and NCA Polymerization.

Glycopolypeptides are prepared either by the polymerization of glycosylated amino acid N-carboxyanhydrides (NCAs) or by the post-polymerization functionalization of polypeptides with suitable functional groups. Here we present a method for the in-situ functionalization and (co-) polymerization of allylglycine N-carboxyanhydride in a facile one-pot procedure, combining radical thiol-ene photochemistry and nucleophilic ring-opening polymerization techniques, to yield well-defined heterofunctional glycopolypeptides.

PH-Responsive biodegradable polymeric micelles with anchors to interface magnetic nanoparticles for MR imaging in detection of cerebral ischemic area.

A novel type of pH-responsive biodegradable copolymer was developed based on methyloxy-poly(ethylene glycol)-block-poly[dopamine-2-(dibutylamino) ethylamine-l-glutamate] (mPEG-b-P(DPA-DE)LG) and applied to act as an intelligent nanocarrier system for magnetic resonance imaging (MRI). The mPEG-b-P(DPA-DE)LG copolymer was synthesized by a typical ring opening polymerization of N-carboxyanhydrides (NCAs-ROP) using mPEG-NH2 as a macroinitiator, and two types of amine-terminated dopamine groups and pH-sensitive ligands were grafted onto a side chain by a sequential aminolysis reaction. This design greatly benefits from the addition of the dopamine groups to facilitate self-assembly, as these groups can act as high-affinity anchors for iron oxide nanoparticles, thereby increasing long-term stability at physiological pH. The mPEG moiety in the copolymers helped the nanoparticles to remain well-dispersed in an aqueous solution, and pH-responsive groups could control the release of hydrophobic Fe3O4 nanoparticles in an acidic environment. The particle size of the Fe3O4-loaded mPEG-b-P(DPA-DE)LG micelles was measured by dynamic light scattering (DLS) and cryo-TEM. The superparamagnetic properties of the Fe3O4-loaded mPEG-b-P(DPA-DE)LG micelles were confirmed by a superconducting quantum interference device (SQUID). T2-weighted magnetic resonance imaging (MRI) of Fe3O4-loaded mPEG-b-P(DPA-DE)LG phantoms exhibited enhanced negative contrast with an r2 relaxivity of approximately 106.7 mM(-1) s(-1). To assess the ability of the Fe3O4-loaded mPEG-P(DE-DPA)LG micelles to act as MRI probes, we utilized a cerebral ischemia disease rat model with acidic tissue. We found that a gradual change in contrast in the cerebral ischemic area could be visualized by MRI after 1 h, and maximal signal loss was detected after 24 h post-injection. These results demonstrated that the Fe3O4-loaded mPEG-b-P(DPA-DE)LG micelles can act as pH-triggered MRI probes for diagnostic imaging of acidic pathological tissues.

Chemically tunable mucin chimeras assembled on living cells

Mucins are a family of secreted and transmembrane glycoproteins characterized by a massive domain of dense O-glycosylation on serine and threonine residues. Mucins are intimately involved in immunity and cancer, yet elucidation of the biological roles of their glycodomains has been complicated by their massive size, domain polymorphisms, and variable glycosylation patterns. Here we developed a synthetic route to a library of compositionally defined, high-molecular weight, dual end-functionalized mucin glycodomain constructs via N-carboxyanhydride polymerization. These glycopolypeptides are the first synthetic analogs to our knowledge to feature the native α-GalNAc linkage to serine with molecular weights similar to native mucins, solving a nearly 50-year synthetic challenge. Physical characterization of the mimics revealed insights into the structure and properties of mucins. The synthetic glycodomains were end-functionalized with an optical probe and a tetrazine moiety, which allowed site-specific bioorthogonal conjugation to an engineered membrane protein on live mammalian cells. This strategy in protein engineering will open avenues to explore the biological roles of cell surface mucins.

Star-Shaped Polypeptides: Synthesis and Opportunities for Delivery of Therapeutics.

Significant advances in the synthesis of polypeptides by N-carboxyanhydride (NCA) polymerisation over the last decade have enabled the design of advanced polypeptide architectures such as star-shaped polypeptides. These materials combine the functionality offered by amino acids with the flexibility of creating stable nanoparticles with adjustable cargo space for therapeutic delivery. This review highlights recent advances in the synthesis of star polypeptides by NCA polymerisation followed by a critical review of the applications of this class of polymer in the delivery of therapeutic agents. This includes examples of traditional small-molecule drugs as well as the emerging class of biologics such as genetic therapeutics (gene delivery).

Synthesis of Amphiphilic Block Copolypept(o)ides by Bifunctional Initiators: Making PeptoMicelles Redox Sensitive.

In this work, the synthesis of polypeptoid-block-polypeptide copolymers (block copolypept(o)ides) based on bifunctional initiators is described, which introduces a distinct chemical entity at the connection between both blocks. With a view towards redox-sensitive block copolypept(o)ides, a cystamine-based initiator was used to synthesize polysarcosine macroinitiators with degrees of polymerization (Xn) between 100 and 200 displaying monomodal molecular weight distributions and dispersities (Đ) around 1.1 as determined by size exclusion chromatography. Block copolypept(o)ides with a poly(γ-t-butyloxycarbonyl-L-glutamate) (PGlu(O(t) Bu)) block (Xn = 25 or 50) were synthesized by controlled N-carboxyanhydride polymerization. Resulting block copolymers possess monomodal molecular weight distributions, dispersities around 1.2 and were applied to degradation studies. While block copolypept(o)ides are stable at 10 × 10(-6) M, they degrade over time at GSH concentrations of 10 × 10(-3) and 100 × 10(-3) M. Furthermore, these disulfide-containing block copolymers form PeptoMicelles, which degrade at intracellular GSH concentrations while remaining stable at extracellular GSH levels.

Controlled synthesis of phosphorylcholine derivatives of poly(serine) and poly(homoserine).

We report methods for the synthesis of polypeptides that are fully functionalized with desirable phosphorylcholine, PC, groups. Because of the inherent challenges in the direct incorporation of the PC group into α-amino acid N-carboxyanhydride (NCA) monomers, we developed a synthetic approach that combined functional NCA polymerization with efficient postpolymerization modification. While poly(L-phosphorylcholine serine) was found to be unstable upon synthesis, we successfully prepared poly(L-phosphorylcholine homoserine) with controlled chain lengths and found these to be water-soluble with disordered chain conformations.

Block copolypeptide nanoparticles for the delivery of ocular therapeutics.

Self-assembling block copolypeptides were prepared by sequential ring-opening polymerization of N-carboxyanhydride (NCA) derivatives of γ-benzyl-L-glutamic acid and ε-carbobenzyloxy-L-lysine, followed by selective deprotection of the benzyl glutamate block. The synthesized polymers had number average molecular weights close to theoretical values, and had low dispersities (ĐM = 1.15-1.28). Self-assembly of the amphiphilic block copolymers into nanoparticles was achieved using the "solvent-switch" method, whereby the polymer was dissolved in THF and water and the organic solvent removed by rotary evaporation. The type of nanostructures formed varied from spherical micelles to a mixture of spherical and worm-like micelles, depending on copolymer composition. The spherical micelles had an average diameter of 43 nm by dynamic light scattering, while the apparent diameter of the mixed phase system was around 200 nm. Reproducibility of nanoparticle preparation was demonstrated to be excellent; almost identical DLS traces were obtained over three repeats. Following qualitative dye-solubilization experiments, the nanoparticles were loaded with the ocular anti-inflammatory drug dexamethasone. Loading efficiency of the nanoparticles was 90% and the cumulative drug release was 94% over 16 d, with a 20% burst release in the first 24 h.

Self-assembled micelles of amphiphilic poly(L-phenylalanine)-b-poly(L-serine) polypeptides for tumor-targeted delivery.

The aim of this work was to design, synthesize, and characterize self-assembled micelles based on polypeptides as a potential antitumor drug carrier. Amphiphilic poly(l-phenylalanine)-b-poly(l-serine) (PFS) polypeptides were obtained through the polymerization of N-carboxyanhydride. As a novel hydrophilic segment, poly(l-serine) was utilized to enhance tumor targeting due to a large demand of tumors for serine. PFS could self-assemble into micelles with an average diameter of 110–240 nm and a slightly negative charge. PFS polypeptides adopted random coil in pH 7.4 phosphate-buffered saline and could partly transform to α-helix induced by trifluoroethanol. PFS micelles with a low critical micelle concentration of 4.0 μg mL−1 were stable in pH 5–9 buffers and serum albumin solution. PFS micelles had a loading capacity of 3.8% for coumarin-6 and exhibited a sustained drug release. Coumarin-6 loaded rhodamine B isothiocyanate-labeled PFS micelles were incubated with Huh-7 tumor cells to study the correlation between drugs and carriers during endocytosis. The uptake of drugs was consistent with the micelles, illustrating that the intracellular transport of drugs highly depended on the micelles. PFS micelles diffused in whole cytoplasm while coumarin-6 assumed localized distribution, suggesting that the micelles could release the loaded drugs in particular areas. The internalization mechanism of PFS micelles was involved with clathrin-mediated endocytosis and macropinocytosis. Excess serine inhibited the uptake of PFS micelles, which demonstrated that serine receptors played a positive role in the internalization of PFS. The more interesting thing was that the uptake inhibition impacted on normal cells but not on tumor cells at the physiological concentration of serine. The difference in the uptake of PFS micelles was fourfold as high between the tumor cells and the normal cells, which indicated that PFS micelles had good tumor targeting in vitro. In conclusion, PFS micelles reported in this work were a promising drug delivery system for tumor targeting therapy.

Glycosylated star polypeptides from NCA polymerization: selective binding as a function of degree of branching and glycosylation.

Star-shaped polypeptides were synthesized via N-carboxyanhydride (NCA) polymerization initiated from various generations of PPI dendrimers. Molecular weight, arm length, and arm density were readily controlled to afford a series of star-shaped poly(glutamic acid) derivatives. Glycosylation of star-shaped poly(glutamic acid) resulted in the formation of a diverse range of glycopolypeptide architectures with tuneable degree of sugar conjugation. The secondary structure of the branched glycopolypeptides was studied as a function of the degree glycosylation. The bioactivity of the described glycopoly-peptides toward the lectin ConA was investigated and was shown to be architecture dependent.

Cover Picture: Macromol. Biosci. 10/2014

Front Cover: A series of well-defined polypeptide-polypeptoid block copolymers based on the body's own amino acids sarcosine and lysine are prepared by K. Fischer, M. Bros, M. Barz, and co-workers on page 1380 by ring opening polymerization of N-carboxyanhydrides. The obtained block ionomers are used to complex pDNA resulting in the formation of polyplexes (PeptoPlexes). The PeptoPlexes can be successfully applied in the transfection of HEK293T cells and are able to transfect up to 50% of cells in vitro (FACS assay), while causing no detectable toxicity in an Annexin V assay. These findings are a first indication that PeptoPlexes may be a suitable alternative to PEG based non-viral transfection systems.

A new strategy to synthesize bottlebrushes with a helical polyglutamate backbone via N-carboxyanhydride polymerization and RAFT.

Polyglutamate bottlebrushes with poly(oligo(ethylene glycol)acrylate) as side chains were successfully prepared for the first time by combination of N-carboxyanhydride polymerization and reversible addition-fragmentation chain transfer (RAFT) polymerization. This work has provided a metal-free polymerization strategy and can be applicable to synthesize well-defined polypeptide bottlebrushes for bio-application purposes.

Guanidinated multi-arm star polyornithines with a polyethylenimine core for gene delivery

Multi-arm star polyornithines PEI-P(Orn)n are prepared by grafting polyornithine arms onto branched polyethylenimine (PEI) with an Mw of 600 via the ring-opening polymerization of N-carboxyanhydride of benzyloxycarbonyl ornithine. To enhance gene delivery efficiency and reduce cytotoxicity, the amino side groups on the polyornithine arms are partially guanidinated that transforms the ornithine units to arginine units. Thus, the guanidinated products G-PEI-P(Orn)n contain multiple poly(ornithine-co-arginine) arms. PEI-P(Orn)n and G-PEI-P(Orn)71 mediate the transfection of pGL3 plasmid to 293T cells almost as efficient as 25 kDa PEI in serum-free medium. Notably, in contrast to the dramatically lowered efficiency of 25 kDa PEI in the presence of serum, the efficiency of G-PEI-P(Orn)71 can be retained or even enhanced in the medium containing 10% serum. The improved serum-compatibility and high efficiency of the guanidinium-modified star polyornithines make them promising for gene delivery.

Introducing PeptoPlexes: polylysine-block-polysarcosine based polyplexes for transfection of HEK 293T cells.

A series of well-defined polypeptide-polypeptoid block copolymers based on the body's own amino acids sarcosine and lysine are prepared by ring opening polymerization of N-carboxyanhydrides. Block lengths were varied between 200-300 for the shielding polysarcosine block and 20-70 for the complexing polylysine block. Dispersity indexes ranged from 1.05 to 1.18. Polylysine is polymerized with benzyloxycarbonyl as well as trifluoroacetyl protecting groups at the ϵ-amine group and optimized deprotection protocols for both groups are reported. The obtained block ionomers are used to complex pDNA resulting in the formation of polyplexes (PeptoPlexes). The PeptoPlexes can be successfully applied in the transfection of HEK 293T cells and are able to transfect up to 50% of cells in vitro (FACS assay), while causing no detectable toxicity in an Annexin V assay. These findings are a first indication that PeptoPlexes may be a suitable alternative to PEG based non-viral transfection systems.

Amine-Tethered Adsorbents Based on Three-Dimensional Macroporous Silica for CO2 Capture from Simulated Flue Gas and Air

New covalently tethered CO2 adsorbents are synthesized through the in situ polymerization of N-carboxyanhydride (NCA) of l-alanine from amine-functionalized three-dimensional (3D) interconnected macroporous silica (MPS). The interconnected macropores provide low-resistant pathways for the diffusion of CO2 molecules, while the abundant mesopores ensure the high pore volume. The adsorbents exhibit high molecular weight (of up to 13058 Da), high amine loading (more than 10.98 mmol N g(-1)), fast CO2 capture kinetics (t1/2 < 1 min), high adsorption capacity (of up to 3.86 mmol CO2 g(-1) in simulated flue gas and 2.65 mmol CO2 g(-1) in simulated ambient air under 1 atm of dry CO2), as well as good stability over 120 adsorption-desorption cycles, which allows the overall CO2 capture process to be promising and sustainable.

Terpolymerization kinetics of amino acid N‐carboxy anhydrides

ABSTRACTBased on their versatility with respect to amino acid type and sequence, polypeptides have become attractive for a number of biological applications such as drug delivery, biomineralization, and drugs. N‐carboxy anhydride (NCA) polymerization is a convenient way to rapidly prepare high‐molecular weight polypeptides with good control over molecular weight and polydispersity. However, the kinetics of the incorporation of NCA monomers into copolypeptides during random copolymerization are poorly understood. Here, kinetic data is presented that allows insight into the NCA polymerization of a terpolymer composed of three commercially relevant amino acids, namely, glutamic acid, lysine, and tyrosine. Furthermore, kinetic data and copolymerization parameters from the copolymerization of binary mixtures of these three amino acid NCAs is used to make predictions of the terpolymer composition. This study provides access to the information necessary to prepare functional copolypeptides with better‐defined sequence architecture that will be essential for the future development of polypeptide‐based materials. © 2014 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2014, 52, 1228–1236