Philipp Heller

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 bodys 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.

Directed Interactions of Block Copolypept(o)ides with Mannose-binding Receptors: PeptoMicelles Targeted to Cells of the Innate Immune System

Core-shell structures based on polypept(o)ides combine stealth-like properties of the corona material polysarcosine with adjustable functionalities of the polypeptidic core. Mannose-bearing block copolypept(o)ides (PSar-block-PGlu(OBn)) have been synthesized using 11-amino-3,6,9-trioxa-undecyl-2,3,4,6-tetra-O-acetyl-O-α-D-mannopyranoside as initiator in the sequential ring-opening polymerization of α-amino acid N-carboxyanhydrides. These amphiphilic block copolypept(o)ides self-assemble into multivalent PeptoMicelles and bind to mannose-binding receptors as expressed by dendritic cells. Mannosylated micelles showed enhanced cell uptake in DC 2.4 cells and in bone marrow-derived dendritic cells (BMDCs) and therefore appear to be a suitable platform for immune modulation.

Synthesis and sequential deprotection of triblock copolypept(o)ides using orthogonal protective group chemistry.

The synthesis of triblock copolymers based on polysarcosine, poly-N-ε-t-butyloxycarbonyl-l-lysine, and poly-N-ε-t-trifluoroacetyl-l-lysine by ring-opening polymerization of the corresponding α-amino acid N-carboxyanhydrides (NCAs) is described. For the synthesis of N-ε-t-butyloxycarbonyl-l-lysine (lysine(Boc)) NCAs, an acid-free method using trimethylsilylchloride/triethylamine as hydrochloric acid (HCl) scavengers is presented. This approach enables the synthesis of lysine(Boc) NCA of high purity (melting point 138.3 °C) in high yields. For triblock copolypept(o)ides, the degree of polymerization (Xn ) of the polysarcosine block is varied between 200 and 600; poly-N-ε-t-butyloxycarbonyl-l-lysine and poly-N-ε-t-trifluoroacetyl-l-lysine blocks are designed to have a Xn in the range of 10-50. The polypeptide-polypeptoid hybrids (polypept(o)ides) can be synthesized with precise control of molecular weight, high end group integrity, and dispersities indices between 1.1 and 1.2. But more important, the use of tert-butyloxycarbonyl- and trifluoroacetyl-protecting groups allows the selective, orthogonal deprotection of both blocks, which enables further postpolymerization modification reactions in a block-selective manner. Therefore, the presented synthetic approach provides a versatile pathway to triblock copolypept(o)ides, in which functionalities can be separated in specific blocks.

Evaluating chemical ligation techniques for the synthesis of block copolypeptides, polypeptoids and block copolypept(o)ides: a comparative study

In this work, we describe the synthesis of block copolypeptides, polypeptoids and block copolypept(o)ides by chemical ligation techniques. Polysarcosine (PSar), poly(N-e-trifluoroacetyl-L-lysine) (PLys(TFA)) and poly(γ-benzyl-L-glutamate) (PGlu(OBzl)) homopolymers of different polarities and end group functionalities but with similar average degrees of polymerization (Xn = 50 and 100) could be obtained by ring opening polymerization (ROP) of α-amino acid N-carboxyanhydrides (NCA) and postpolymerization modification reactions. In the next step, these polymers were applied to copper(I)-catalyzed azide–alkyne coupling (CuAAC), strain-promoted azide–alkyne coupling (SPAAC) and native chemical ligation (NCL). Our results suggest that all the employed ligation techniques can be used for the synthesis of block copolypeptides, polypeptoids and block copolypept(o)ides. SPAAC displayed, for most conditions, the highest ligation efficiencies (up to 86%) and, from a practical point of view, is the most feasible method. NCL, however, performed very well for short hydrophilic polymers (up to 88%) and is favourable for the ligation of peptides from solid phase peptide synthesis (SPPS) to polysarcosine. As a proof of principle, we report a protocol for an efficient NCL coupling of polysarcosine to the T-cell receptor core peptide (TCR CP), which is known to inhibit IL-2 production in antigen-stimulated T cells and, therefore, to suppress inflammation. In a comparative study, the ligation method, which directly relates to the chemical nature of the ligation site, neither influenced cytotoxicity nor complement activation. To conclude, chemical ligation techniques represent a complementary synthetic approach to the well-established sequential ring opening polymerization of NCAs.

Combining reactive triblock copolymers with functional cross-linkers: A versatile pathway to disulfide stabilized-polyplex libraries and their application as pDNA vaccines

ABSTRACT Therapeutic nucleic acids such as pDNA hold great promise for the treatment of multiple diseases. These therapeutic interventions are, however, compromised by the lack of efficient and safe non‐viral delivery systems, which guarantee stability during blood circulation together with high transfection efficiency. To provide these desired properties within one system, we propose the use of reactive triblock copolypept(o)ides, which include a stealth‐like block for efficient shielding, a hydrophobic block based on reactive disulfides for cross‐linking and a cationic block for complexation of pDNA. After the complexation step, bifunctional cross‐linkers can be employed to bio‐reversibly stabilize derived polyplexes by disulfide bond formation and to introduce endosomolytic moieties at the same time. Cross‐linked polyplexes show no aggregation in human blood serum. Upon cellular uptake and cleavage of disulfide bonds, the cross‐linkers can interact with the endosomal membrane, leading to lysis and efficient endosomal translocation. In principal, the approach allows for the combination of one polymer with various different cross‐linkers and thus enables the fast forward creation of a polyplex library. Here, we provide a first insight into the potential of this concept and use a screening strategy to identify a lead candidate, which is able to transfect dendritic cells with a model DNA vaccine.

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 (Đ = 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 (Đ = 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.

The Influence of Block Ionomer Microstructure on Polyplex Properties: Can Simulations Help to Understand Differences in Transfection Efficiency?

Gene therapies enable therapeutic interventions at gene transcription and translation level, providing enormous potential to improve standards of care for multiple diseases. Nonviral transfection agents and in particular polyplexes based on block ionomers are-besides viral vectors and cationic lipid formulations-among the most promising systems for this purpose. Block ionomers combine a hydrophilic noncharged block, e.g., polyethylene glycol (PEG), with a hydrophilic cationic block. For efficient transfection, however, endosomolytic moieties, e.g., imidazoles, are additionally required to facilitate endosomal escape, which raises the general question how to distribute these functionalities within the block copolymer. Combining molecular dynamics simulation with physicochemical and biological characterization, this work aims to provide a first rational for the influence of block ionomer microstructure on polyplex properties, e.g., size, shape, and transfection efficiency. Our findings underline that a triblock microstructure is most efficient in compacting pDNA, which reduces polyplex size, enhances stability against degradation by DNase I, and thus provides better transfection performance.