l-Proline is the only, out of 20 essential, amino acid that contains a cyclized substituted α-amino group (is formally an imino acid), which restricts its conformational shape. The synthesis of well-defined homo- and copolymers of l-proline has been plagued either by the low purity of the monomer or the inability of most initiating species to polymerize the corresponding N-carboxy anhydride (NCA) because they require a hydrogen on the 3-N position of the five-member ring of the NCA, which is missing. Herein, highly pure l-proline NCA was synthesized by using the Boc-protected, rather than the free amino acid. The protection of the amine group as well as the efficient purification method utilized resulted in the synthesis of highly pure l-proline NCA. The high purity of the monomer and the use of an amino initiator, which does not require the presence of the 3-N hydrogen, led for the first time to well-defined poly(l-proline) (PLP) homopolymers, poly(ethylene oxide)-b-poly(l-proline), and poly(l-proline)-b-poly(ethylene oxide)-b-poly(l-proline) hybrids, along with poly(γ-benzyl-l-glutamate)-b-poly(l-proline) and poly(Boc-l-lysine)-b-poly(l-proline) copolypeptides. The combined characterization (NMR, FTIR, and MS) that results for the l-proline NCA revealed its high purity. In addition, all synthesized polymers exhibit high molecular and compositional homogeneity.

The hierarchical self-assembly and dynamics of poly(γ-benzyl-L-glutamate)-b-poly(L-proline) (PBLG-b-PLP) polypeptides are investigated with X-rays and solid state NMR. Both blocks possess helices stabilized solely either by hydrogen bonds (PBLG) or by steric hindrance (PLP) and are further packed in two different hexagonal cells. We report a trans/cis conformational change of PLP upon confinement that mimics the isomerization of isolated proline residues in proteins. These cis PLP conformations reside primarily at the PLP/PBLG interface, alleviate the packing frustration, and permit PBLG and PLP helices to pack with their bulk properties.

Internal bleeding is an injury that can be difficult to localize and effectively treat without invasive surgeries. Injectable polymeric nanoparticles have been developed that can reduce clotting times and blood loss, but they have yet to incorporate sufficient diagnostic capabilities to assist in identifying bleeding sources. Herein, polymeric nanoparticles were developed to simultaneously treat internal bleeding while incorporating tracers for visualization of the nanoparticles by standard clinical imaging modalities. Addition of 1,1′-dioctadecyl-3,3,3′,3′tetramethylindodicarbocyanine perchlorate (DiD; a fluorescent dye), biotin functionality, and gold nanoparticles to hemostatic polymeric nanoparticles resulted in nanoparticles amenable to imaging with near-infrared (NIR) imaging, immunohistochemistry, and X-ray computed tomography (CT), respectively. Following a lethal liver resection injury, visualization of accumulated nanoparticles by multiple imaging methods was achieved in rodents, with the highest accumulation observed at the liver injury site, resulting in improved survival rates. Tracer addition to therapeutic nanoparticles allows for an expansion of their applicability, during stabilization by first responders to diagnosis and identification of unknown internal bleeding sites by clinicians using standard clinical imaging modalities.

Well-defined amphiphilic polymers of the ABA and ABC type are synthesized, where A is poly(L-lysine hydrochloride) (PLL), B is poly(γ-benzyl-(d7) L-glutamate) (PBLG(-d7)), and C is poly(ethylene oxide) (PEO). The two polymers exhibit similar PBLG(-d7) composition, while in the ABC, the volume fraction of PEO block is higher than that of PLL. Both polymers form polymersomes in water. The polymersomes are loaded with doxorubicin or paclitaxel. It is found that in the ABC, due to asymmetry of the two hydrophilic blocks, PEO is always on the outer periphery and the dimensions of the vesicles are smaller. The release of the vesicles is temperature- and pH-dependent. In vivo toxicity tests of the empty vesicles show that they are not toxic. In vitro activity of the loaded vesicles against human pancreatic cancer cell lines reveals comparable activity to Myocet for the ABA loaded with doxorubicin, while lower activity is observed for the ABC.

Gelation of the left helical N-substituted homopolypeptide poly(l-proline) (PLP) in water was explored, employing rheological and small-angle scattering studies at different temperatures and concentrations in order to investigate the network structure and its mechanical properties. Stiff gels were obtained at 10 wt % or higher at 5 °C, the first time gelation has been observed for homopolypeptides. The secondary structure and helical rigidity of PLP has large structural similarities to gelatin but as gels the two materials show contrasting trends with temperature. With increasing temperature in D2O, the network stiffens, with broad scattering features of similar correlation length for all concentrations and molar masses of PLP. A thermoresponsive transition was also achieved between 5 and 35 °C, with moduli at 35 °C higher than gelatin at 5 °C. The brittle gels could tolerate strains of 1% before yielding with a frequency-independent modulus over the observed range, similar to natural proline-rich proteins, suggesting the potential for thermoresponsive or biomaterial-based applications.

Intestinal barrier derangement allows intestinal bacteria and their products to translocate to the systemic circulation. Pseudomonas aeruginosa (PA) superimposed infection in critically ill patients increases gut permeability and leads to gut-driven sepsis. PA infections are challenging due to multi-drug resistance (MDR), biofilms, and/or antibiotic tolerance. Inhibition of the quorum-sensing transcriptional regulator MvfR(PqsR) is a desirable anti-PA anti-virulence strategy as MvfR controls multiple acute and chronic virulence functions. Here we show that MvfR promotes intestinal permeability and report potent anti-MvfR compounds, the N-Aryl Malonamides (NAMs), resulting from extensive structure-activity-relationship studies and thorough assessment of the inhibition of MvfR-controlled virulence functions. This class of anti-virulence non-native ligand-based agents has a half-maximal inhibitory concentration in the nanomolar range and strong target engagement. Using a NAM lead in monotherapy protects murine intestinal barrier function, abolishes MvfR-regulated small molecules, ameliorates bacterial dissemination, and lowers inflammatory cytokines. This study demonstrates the importance of MvfR in PA-driven intestinal permeability. It underscores the utility of anti-MvfR agents in maintaining gut mucosal integrity, which should be part of any successful strategy to prevent/treat PA infections and associated gut-derived sepsis in critical illness settings. NAMs provide for the development of crucial preventive/therapeutic monotherapy options against untreatable MDR PA infections.

A simple approach to enhancing the activity and stability of organophosphorous hydrolase (OPH) is developed based on interactions between the hydrophobic poly(propylene oxide) (PPO) block of amphiphillic Pluronics and the enzyme. This strategy provides an efficient route to new formulations for decontaminating organophosphate neurotoxins.

We describe the synthesis and self-assembly of particularly high periodicity of diblock copolymers composed of poly(benzyl-l-hydroxyproline) (PBLHyP) and poly(γ-benzyl-l-glutamate) (PBLG), that is, two polypeptide blocks with dissimilar helical structures. The robust helicity of the PBLHyP block is driven by steric constraints of the repeat units, while PBLG forms α-helices driven by hydrogen bonding, allowing defects and deformations. Herein, high-molecular-weight diblock copolypeptides of PBLG-b-PBLHyP with three different volume fractions of the PBLHyP-blocks are discussed. For shorter PBLHyP blocks, hexagonal packing of PBLHyP helices is observed, while by increasing the length of the PBLHyP block, keeping at a similar PBLG block length, the packing is distorted. Zig-zag lamellar structures were obtained due to the mismatch in the packing periodicities of the PBLG and PBLHyP helices. The frustration that takes place at the interface leads the PBLHyP to tilt to match the PBLG periodicity. The zig-zag morphology is reported for the first time for high-molecular-weight helix-helix (rod-rod) copolypeptides, and the self-assembled periodicity is uncommonly large.