Matthias Barz

Overcoming the PEG-addiction: well-defined alternatives to PEG, from structure–property relationships to better defined therapeutics

Synthetic methods in polymer chemistry have evolved tremendously during the last decade. Nowadays more and more attention is devoted to the application of those tools in the development of the next generation of nanomedicines. Nevertheless, poly(ethylene glycol) (PEG) remains the most frequently used polymer for biomedical applications. In this review, we try to summarize recent efforts and developments in controlled polymerisation techniques that may allow alternatives to PEG based systems and can be used to improve the properties of future polymer therapeutics.

Radioactive Labeling of Defined HPMA-Based Polymeric Structures Using [18F]FETos for In Vivo Imaging by Positron Emission Tomography

During the last decades polymer-based nanomedicine has turned out to be a promising tool in modern pharmaceutics. The following article describes the synthesis of well-defined random and block copolymers by RAFT polymerization with potential medical application. The polymers have been labeled with the positron-emitting nuclide fluorine-18. The polymeric structures are based on the biocompatible N-(2-hydroxypropyl)-methacrylamide (HPMA). To achieve these structures, functional reactive ester polymers with a molecular weight within the range of 25,000-110,000 g/mol were aminolyzed by 2-hydroxypropylamine and tyramine (3%) to form (18)F-labelable HPMA-polymer precursors. The labeling procedure of the phenolic tyramine moieties via the secondary labeling synthon 2-[(18)F]fluoroethyl-1-tosylate ([(18)F]FETos) provided radiochemical fluoroalkylation yields of ∼80% for block copolymers and >50% for random polymer architectures within a synthesis time of 10 min and a reaction temperature of 120 °C. Total synthesis time including synthon synthesis, (18)F-labeling, and final purification via size exclusion chromatography took less than 90 min and yielded stable (18)F-labeled HPMA structures in isotonic buffer solution. Any decomposition could be detected within 2 h. To determine the in vivo fate of (18)F-labeled HPMA polymers, preliminary small animal positron emission tomography (PET) experiments were performed in healthy rats, demonstrating the renal clearance of low molecular weight polymers. Furthermore, low metabolism rates could be detected in urine as well as in the blood. Thus, we expect this new strategy for radioactive labeling of polymers as a promising approach for in vivo PET studies.

From Defined Reactive Diblock Copolymers to Functional HPMA-Based Self-Assembled Nanoaggregates

This paper describes the synthesis of functional amphiphilic poly( N-(2-hydroxypropyl) methacrylamide)-block-poly(lauryl methacrylate) copolymers by RAFT polymerization via the intermediate step of activated ester block copolymers (pentafluoro-phenyl methacrylate). Block copolymers with molecular weights from 12000-28000 g/mol and PDIs of about 1.2 have been obtained. The amphiphilic diblock copolymers form stable super structures (nanoaggregates) by self-organization in aqueous solution. The diameters of these particles are between 100 and 200 nm and depend directly on the molecular weight of the block copolymer. Furthermore, we investigated the impact of these nanoaggregates on cell viability and on the motility of adherent cells. Cytotoxicity was investigated by the MTS test and the fluctuation in cell shape was monitored employing ECIS (electrical cell-substrate impedance sensing). In these investigations, the formed particles are not cell toxic up to a concentration of 2 mg/mL. Thus, our polymeric particles offer potential as polymer therapeutics.

The uptake of N-(2-hydroxypropyl)-methacrylamide based homo, random and block copolymers by human multi-drug resistant breast adenocarcinoma cells.

A series of well-defined, fluorescently labelled homopolymers, random and block copolymers based on N-(2-hydroxypropyl)-methacrylamide were prepared by reversible addition-fragmentation chain transfer polymerization (RAFT polymerization). The polydispersity indexes for all polymers were in the range of 1.2-1.3 and the number average of the molar mass (M(n)) for each polymer was set to be in the range of 15-30 kDa. The cellular uptake of these polymers was investigated in the human multi-drug resistant breast adenocarcinoma cell line MCF7/ADR. The uptake greatly depended on the polymer molecular mass and structure. Specifically, smaller polymers (approx. 15 kDa) were taken up by the cells at much lower concentrations than larger polymers (approx. 30 kDa). Furthermore, for polymers of the same molar mass, the random copolymers were more easily internalized in cells than block copolymers or homopolymers. This is attributed to the fact that random copolymers form micelle-like aggregates by intra- and interchain interactions, which are smaller and less stable than the block copolymer structures in which the hydrophobic domain is buried and thus prevented from unspecific interaction with the cell membrane. Our findings underline the need for highly defined polymeric carriers and excipients for future applications in the field of nanomedicine.

Polypeptoid-block-polypeptide Copolymers: Synthesis, Characterization, and Application of Amphiphilic Block Copolypept(o)ides in Drug Formulations and Miniemulsion Techniques

We report the synthesis of polysarcosine-block-polyglutamic acid benzylester (PSar-block-PGlu(OBn)) and polysarcosine-block-polylysine-ε-N-benzyloxycarbonyl (PSar-block-PLys(Z)) copolymers. The novel polypeptoid-block-polypeptide copolymers (Copolypept(o)ides) have been synthesized by ring-opening polymerization (ROP) of N-carboxyanhydrides (NCAs). Polymerization conditions were optimized regarding protecting groups, block sequence and length. While the degree of polymerization of the PSar block length was set to be around 200 or 400, PGlu(OBn) and PLys(Z) block lengths were varied between 20 to 75. The obtained block copolymers had a total degree of polymerization of 220-475 and dispersity indices between 1.1 and 1.2. Having ensured a nontoxic behavior up to a concentration of 3 mg/mL in HEK293 cells, the novel block copolymers have been applied to the synthesis of organic colloids (by miniemulsion polymerization and miniemulsion solvent evaporation process). Colloids of around 100 nm (miniemulsion polymerization) to 200 nm (miniemulsion process) have been prepared. Additionally, PSar-block-PGlu(OBn) copolymers have been used in a drug formulation of an adenylate cyclase inhibitor. Micelles of 28.0 nm (without drug) and 33.0 nm (with drug) diameter have been observed by fluorescence correlation spectroscopy (FCS). The polypeptoid-block-polypeptide formulation increased solubility of the drug and enhances its bioavailability, which leads to a reduction of intracellular cAMP levels in MaMel 91 melanoma cells.

Synthesis and In Vitro Evaluation of Defined HPMA Folate Conjugates: Influence of Aggregation on Folate Receptor (FR) Mediated Cellular Uptake

In this article we report the synthesis and in vitro evaluation of well-defined, folate functionalized and fluorescently labeled polymers based on the clinically approved N-(2-hydroxypropyl)-methacrylamide (HPMA). The polymers were prepared applying the RAFT polymerization method as well as the reactive ester approach. The molecular weights of the polymers synthesized were around 15 and 30 kDa. The total content of conjugated folate varied from 0, 5, and 10 mol %. The cellular uptake of these polymers was investigated in the folate receptor (FR)-positive human nasopharyngeal epidermal carcinoma (KB-3-1) and FR-negative human lung epithelial carcinoma (A549) cancer cell lines. In FR-positive cells, the cellular uptake of polymers depended strongly on the folate content. The conjugates with the highest folate content led to the highest level of cell-associated fluorescence. Regarding influence of molecular weight, nonsignificant differences were observed when total cell uptake was analyzed. The cellular uptake is related to the aggregate formation of the polymer conjugates, which were studied by fluorescence correlation spectroscopy (FCS). For the conjugates, we found aggregates with a diameter ranging from 11-18 nm. Much to our surprise, we found aggregates of the same size for the 30 kDa polymer bearing 5 mol % folate and for the 15 and 30 kDa conjugates with a folate content of 10 mol %. Consequently, a different conformation in solution for the different conjugates was expected. By live cell confocal fluorescence microscopy the receptor-mediated endocytosis process was observed, as colocalization with lysosomal markers was achieved. In addition, cellular uptake was not observed in FR-negative cells (A549) and can be dramatically reduced by blocking the FR with free folic acid. Our findings clearly underline the need for a minimum amount of accessible folate units to target the FR that triggers specific cellular uptake. Furthermore, it has been demonstrated that the targeting vector itself strongly influences the aggregation behavior in solution and thus determines the interaction with cells regarding cellular uptake as well as intracellular localization.

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.

A controlled and versatile NCA polymerization method for the synthesis of polypeptides

A versatile and simple methodology for the preparation of well-defined polyglutamate nanocarriers is described. For the first time ammonium salts with non-nucleophilic tetrafluoroborate anions are used as initiators for the ring opening polymerization of α-N-carboxyanhydrides (NCAs) allowing a multigram scale polyglutamate synthesis with defined molecular weight (up to 800 units), low polydispersity (<1.2), controlled chain end functionality and adequate stereoselectivity and absence of any trace of toxic impurity to allow biomedical applications.

New perspectives of HPMA-based copolymers derived by post-polymerization modification.

Poly[N-(2-hydroxypropyl) methacrylamide] (HPMA) was one of the first polymers applied as polymer drug conjugate in the clinics. Since then many attempts have been made to expand the functionality of HPMA-based copolymers from advanced synthetic pathways to multiple biomedical applications. This Feature Article highlights multifunctional HPMA based copolymers prepared by controlled radical polymerization and subsequent post-polymerization modification of activated ester precursor polymers via aminolysis. This approach combines precise control of the polymers microstructure (molecular weight, dispersity, block copolymer formation, end group functionalization) with an easy introduction of various multifunctional groups. The obtained polymers can be used as versatile targeted drug carriers for sophisticated molecular imaging techniques that provide detailed information about structure property relationships both in vitro as well as in vivo. Moreover, recent studies have shown that such multifunctional HPMA copolymers may have high potential as advanced carriers in the field of tumor immunotherapy.

72/74As-labeling of HPMA based polymers for long-term in vivo PET imaging.

In the context of molecular imaging, various polymers based on the clinically approved N-(2-hydroxypropyl)-methacrylamide (HPMA) have been radio-labeled using longer-living positron emitters 72As t1/2=26 h or 74As t1/2=17.8 d. This approach may lead to non-invasive determination of the long-term in vivo fate of polymers by PET (positron emission tomography). Presumably, the radio label itself will not strongly influence the polymer structure due to the fact that the used nuclide binds to already existing thiol moieties within the polymer structure. Thus, the use of additional charges or bulky groups can be avoided.