Characterization of neurocalcin delta membrane binding by biophysical methods.

Neurocalcin delta (NCALD) is a member of the neuronal calcium sensors protein family. In the retina, NCALD is expressed by ganglion and amacrine cells. NCALD is composed of 4 EF-hand motifs but only 3 of them may bind calcium. The binding of calcium induces a conformational change of the protein which leads to the extrusion of its N-terminal myristoyl group as well as some hydrophilic residues. This mechanism, named calcium-myristoyl switch, is presumably involved in its membrane binding. The parameters responsible for the interaction of NCALD with membranes are only partially known. The purpose of this study was thus to gather more information on the membrane binding behavior of NCALD using lipid monolayers, including the influence of the lipid composition, the calcium and the myristoyl group. NCALD was injected underneath different lipid monolayers and this model membrane allowed the determination of the binding parameters as maximum insertion pressure (MIP) and synergy. The values of MIP are larger when monolayers were composed of a saturated phospholipid with phosphoethanolamine polar head. This trend is confirmed by polarization modulation infrared reflection absorption spectroscopy measurements. Moreover, the observations by fluorescence microscopy show that NCALD preferentially interacts with phospholipids which are in the liquid-condensed physical state, as found in membrane microdomains. This observation could explain the changes of NCALD expression level in the brains of patients suffering from Alzheimer's disease because of the alteration of lipid composition in microdomains structures.

Efficient Shielding of Polyplexes Using Heterotelechelic Polysarcosines.

Shielding agents are commonly used to shield polyelectrolyte complexes, e.g., polyplexes, from agglomeration and precipitation in complex media like blood, and thus enhance their in vivo circulation times. Since up to now primarily poly(ethylene glycol) (PEG) has been investigated to shield non-viral carriers for systemic delivery, we report on the use of polysarcosine (pSar) as a potential alternative for steric stabilization. A redox-sensitive, cationizable lipo-oligomer structure (containing two cholanic acids attached via a bioreducible disulfide linker to an oligoaminoamide backbone in T-shape configuration) was equipped with azide-functionality by solid phase supported synthesis. After mixing with small interfering RNA (siRNA), lipopolyplexes formed spontaneously and were further surface-functionalized with polysarcosines. Polysarcosine was synthesized by living controlled ring-opening polymerization using an azide-reactive dibenzo-aza-cyclooctyne-amine as an initiator. The shielding ability of the resulting formulations was investigated with biophysical assays and by near-infrared fluorescence bioimaging in mice. The modification of ~100 nm lipopolyplexes was only slightly increased upon functionalization. Cellular uptake into cells was strongly reduced by the pSar shielding. Moreover, polysarcosine-shielded polyplexes showed enhanced blood circulation times in bioimaging studies compared to unshielded polyplexes and similar to PEG-shielded polyplexes. Therefore, polysarcosine is a promising alternative for the shielding of non-viral, lipo-cationic polyplexes.

Secondary-Structure-Driven Self-Assembly of Reactive Polypept(o)ides: Controlling Size, Shape, and Function of Core Cross-Linked Nanostructures.

Achieving precise control over the morphology and function of polymeric nanostructures during self-assembly remains a challenge in materials as well as biomedical science, especially when independent control over particle properties is desired. Herein, we report on nanostructures derived from amphiphilic block copolypept(o)ides by secondary-structure-directed self-assembly, presenting a strategy to adjust core polarity and function separately from particle preparation in a bioreversible manner. The peptide-inherent process of secondary-structure formation allows for the synthesis of spherical and worm-like core-cross-linked architectures from the same block copolymer, introducing a simple yet powerful approach to versatile peptide-based core-shell nanostructures.

Synthesis and Characterization of Stimuli-Responsive Star-Like Polypept(o)ides: Introducing Biodegradable PeptoStars.

Star-like polymers are one of the smallest systems in the class of core crosslinked polymeric nanoparticles. This article reports on a versatile, straightforward synthesis of three-arm star-like polypept(o)ide (polysarcosine-block-polylysine) polymers, which are designed to be either stable or degradable at elevated levels of glutathione. Polypept(o)ides are a recently introduced class of polymers combining the stealth-like properties of the polypeptoid polysarcosine with the functionality of polypeptides, thus enabling the synthesis of materials completely based on endogenous amino acids. The star-like homo and block copolymers are synthesized by living nucleophilic ring opening polymerization of the corresponding N-carboxyanhydrides (NCAs) yielding polymeric stars with precise control over the degree of polymerization (Xn = 25, 50, 100), Poisson-like molecular weight distributions, and low dispersities (D = 1.06-1.15). Star-like polypept(o)ides display a hydrodynamic radius of 5 nm (µ2 < 0.05) as determined by dynamic light scattering (DLS). While star-like polysarcosines and polypept(o)ides based on disulfide containing initiators are stable in solution, degradation occurs at 100 × 10-3 m glutathione concentration. The disulfide cleavage yields the respective polymeric arms, which possess Poisson-like molecular weight distributions and low dispersities (D = 1.05-1.12). Initial cellular uptake and toxicity studies reveal that PeptoStars are well tolerated by HeLa, HEK 293, and DC 2.4 cells.

Combining Orthogonal Reactive Groups in Block Copolymers for Functional Nanoparticle Synthesis in a Single Step.

We report on the synthesis of polysarcosine-block-poly(S-alkylsulfonyl)-l-cysteine block copolymers, which combine three orthogonal addressable groups enabling site-specific conversion of all reactive entities in a single step. The polymers are readily obtained by ring-opening polymerization (ROP) of corresponding α-amino acid N-carboxyanhydrides (NCAs) combining azide and amine chain ends, with a thiol-reactive S-alkylsulfonyl cysteine. Functional group interconversion of chain ends using strain-promoted azide-alkyne cycloaddition (SPAAC) and activated ester chemistry with NHS- and DBCO-containing fluorescent dyes could be readily performed without affecting the cross-linking reaction between thiols and S-alkylsulfonyl protective groups. Eventually, all three functionalities can be combined in the formation of multifunctional disulfide core cross-linked nanoparticles bearing spatially separated functionalities. The simultaneous attachment of dyes in core and corona during the formation of core-cross-linked nanostructures with controlled morphology is confirmed by fluorescence cross-correlation spectroscopy (FCCS).

Phosphatidylserine Allows Observation of the Calcium-Myristoyl Switch of Recoverin and Its Preferential Binding.

Recoverin undergoes a calcium-myristoyl switch during visual phototransduction. Indeed, calcium binding by recoverin results in the extrusion of its myristoyl group, which allows its membrane binding. However, the contribution of particular lipids and of specific amino acids of recoverin in its membrane binding has not yet been demonstrated. In the present work, the affinity of recoverin for the negatively charged phosphatidylserine has been clearly shown to be governed by a cluster of positively charged residues located in its N-terminal segment. Moreover, the calcium-myristoyl switch of recoverin was only observed upon binding onto monolayers of phosphatidylserine and not in the case of other anionic phospholipids. Fluorescence microscopy experiments with mixed lipid monolayers allowed confirmation of the specific binding of myristoylated recoverin to phosphatidylserine, whereas the extent of penetration of recoverin in phosphatidylserine monolayers was estimated by ellipsometry. A model has thus been proposed for the membrane binding of myristoylated recoverin in the presence of calcium.

Polypept(o)ides: Hybrid Systems Based on Polypeptides and Polypeptoids.

Polypept(o)ides combine the multifunctionality and intrinsic stimuli-responsiveness of synthetic polypeptides with the "stealth"-like properties of the polypeptoid polysarcosine (poly(N-methyl glycine)). This class of block copolymers can be synthesized by sequential ring opening polymerization of α-amino acid N-carboxy-anhydrides (NCAs) and correspondingly of the N-substituted glycine N-carboxyanhydride (NNCA). The resulting block copolymers are characterized by Poisson-like molecular weight distributions, full end group integrity, and dispersities below 1.2. While polysarcosine may be able to tackle the currently arising issues regarding the gold standard PEG, including storage diseases in vivo and immune responses, the polypeptidic block provides the functionalities for a specific task. Additionally, polypeptides are able to form secondary structure motives, e.g., α-helix or ß-sheets, which can be used to direct self-assembly in solution. In this feature article, we review the relatively new field of polypept(o)ides with respect to synthesis, characterization, and first data on the application of block copolypept(o)ides in nanomedicine. The summarized data already indicates the great potential of polypept(o)ides.

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 (D) 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.