Johan F. J. Engbersen

Dinuclear metallo-phosphodiesterase models: application of calix[4]arenes as molecular scaffolds

An important goal in supramolecular chemistry is the synthesis of molecules that exhibit catalytic activity analogous to the activity of enzymes. In this respect, studies toward the biomimetic catalysis of phosphate diester cleavage have received particular attention. In nature this process is catalyzed by enzymes that possess often two or three divalent metal ions in their active sites. In order to mimic the active sites of these metallo-phosphodiesterases chemists generally attempt to connect ligated metal ions by a molecular spacer in such a way that the metal–metal distance matches with the anionic pentacoordinate phosphorus transition state. However, in contrast to enzymes, which bind the transition state by multiple contacts via an induced fit mechanism, many of the low molecular weight model systems exhibit only minor catalytic activity due to lack of catalytic groups and too much rigidity or flexibility. Our approach is to use calix[4]arenes as a molecular scaffold for the dynamic preorganization of multiple catalytic groups. In this review models for dinuclear metallo-phosphodiesterases and the use of calix[4]arenes in such models are described.

Responsive layer-by-layer materials for drug delivery

Due to its versatility and ease of use the layer-by-layer (LbL) assembly technique has been under intensive investigation for drug and gene delivery applications. Especially the development of responsive LbL materials has advanced significantly in recent years. Responsiveness plays an important role in many delivery applications, either for loading of therapeutics or controlled and triggered release. In general four basic mechanisms within responsive LbL films have been identified: disruption of layer interactions, degradation of the LbL film, multilayer destruction via physical stimuli, and phase transitions or polymer rearrangements within the LbL film. This review will outline these different mechanisms and highlight recent advances in these fields.

Effect of chemical functionalities in poly(amido amine)s for non-viral gene transfection.

The development of safe and efficient gene delivery vectors is an essential prerequisite for successful gene therapy. As viral vectors suffer from inherent disadvantages, cationic polymers as non-viral vectors have great potential in gene delivery, but their practical application so far is seriously hampered due to their relatively low transfection efficiency caused by multiple extra- and intracellular gene delivery barriers. Therefore, it is important to provide cationic polymers with functionalities that can seriously influence polymeric properties which are important to overcome gene delivery barriers. In this paper, we aim to contribute to the understanding of the effect of functionalities in cationic polymers on their gene delivery properties and transfection activity. As poly(amido amine)s can be easily provided with a large variety of chemical functionalities, we have focused on this class of cationic polymers. It is shown that various structural characteristics in these peptidomimetic polymers such as charge density, rigidity, basicity, hydrophilicity/hydrophobicity, degradability and type of amino groups influence one or more gene delivery properties such as DNA binding capability, colloidal stability, endosomal escape (buffer capacity), vector unpacking, cytotoxicity, and eventual transfection efficiency. Optimal combination of the functionalities in the poly(amido amine)s may lead to significant increase of the level of gene expression. This indicates that multifunctionalized polymers like the poly(amido amine)s can evolve to the next generation of non-viral gene delivery system for gene therapy.

Specific RNA Dinucleotide Cleavage by a Synthetic Calix[4]arene‐Based Trinuclear Metallo(II)‐phosphodiesterase

Large rate enhancements and considerable nucleobase specificity in the catalytic transesterification of RNA dinucleotides are observed with the calix[4]arene enzyme mimic 1-Zn(3) (the computer-generated model of the complex with GpG is shown). The higher activity toward GpG over ApA (factor of 160) originates from favorable substrate binding and cooperative catalytic action of all three Zn(II) ions. The heterotrinuclear metallo-phosphoesterase mimic 1-Zn(2)Cu is even more active.

Cyclodextrin dimers as receptor molecules for steroid sensors

The dansyl-modified dimer 9 complexes strongly with the steroidal bile salts. Relative to native beta-cyclodextrin, the binding of cholate (1a) and deoxycholate (1b) salts is especially enhanced. These steroids bind exclusively in a 1:1 fashion. For other bile salts (1c-1e) both 1:1 and 1:2 complexes were observed with stabilities similar to those of native beta-cyclodextrin. This indicates that only one cavity is used, with a small contribution from the second. The difference is attributed to the absence of a 12-hydroxy group in the second group of steroids. Comparison with a dimer that lacks the dansyl moiety (6) shows that this group especially hinders the cooperative binding of la and 1b. The smaller interference in the binding of the other steroids indicates that self-inclusion of the dansyl moiety hardly occurs. This weak self-inclusion is supported by fluorescence studies. The dansyl fluorescence of dimer 9 is less blue-shifted than that of other known dansyl-appended cyclodextrin derivatives; this is indicative of a more polar micro-environment. Addition of guests causes a change in fluorescence intensity.

Addition of cyanide ion to nicotinamide cations in acetonitrile. Formation of non-productive charge-transfer complexes

The mixing of equal volumes of 0.2 mmol dm–3 1 -benzylnicotinamide ion and 2 mmol dm–3 cyanide ion results in the immediate formation of a transient absorption band at 375 nm which can be ascribed to a charge–transfer complex. This complex disappears within ca. 0.2 s with the formation of the 1,6-addition product which, in turn, is rapidly converted into the thermodynamically more stable 1,4-adduct. Methyl substitution at the 6-position of the nicotinamide ring inhibits the formation of the 1,6-adduct, resulting in an increase in the lifetime of the charge–transfer complex. Subsequently a mixture of the 1,4-cyanide adduct and, most likely, the 1,2-adduct is formed. Rate effects with variation of substituents in the 1-benzyl group reveal that charge-transfer complex formation is counter-productive to the formation of addition products.

Dynamic Colocalization Microscopy To Characterize Intracellular Trafficking of Nanomedicines

To gain a better understanding of intracellular processing of nanomedicines, we employed quantitative live-cell fluorescence colocalization microscopy to study endosomal trafficking of polyplexes in retinal pigment epithelium cells. A new, dynamic colocalization algorithm was developed, based on particle tracking and trajectory correlation, allowing for spatiotemporal characterization of internalized polyplexes in comparison with endosomal compartments labeled with EGFP constructs. This revealed early trafficking of the polyplexes specifically to Rab5- and flotillin-2-positive vesicles and subsequent delivery to Rab7 and LAMP1-labeled late endolysosomes where the major fraction of the polyplexes remains entrapped for days, suggesting the functional loss of these nanomedicines. Colocalization of polyplexes with the autophagy marker LC3 suggests for the first time that the process of xenophagy could play an important role in the persistent endosomal entrapment of nanomedicines.

Shielding the cationic charge of nanoparticle-formulated dermal DNA vaccines is essential for antigen expression and immunogenicity

Nanoparticle-formulated DNA vaccines hold promise for the design of in vivo vaccination platforms that target defined cell types in human skin. A variety of DNA formulations, mainly based on cationic liposomes or polymers, has been investigated to improve transfection efficiency in in vitro assays. Here we demonstrate that formulation of DNA into both liposomal and polymeric cationic nanoparticles completely blocks vaccination-induced antigen expression in mice and ex vivo human skin. Furthermore, this detrimental effect of cationic nanoparticle formulation is associated with an essentially complete block in vaccine immunogenicity. The blocking of DNA vaccine activity may be explained by immobilization of the nanoparticles in the extracellular matrix, caused by electrostatic interactions of the cationic nanoparticles with negatively charged extracellular matrix components. Shielding the surface charge of the nanoparticles by PEGylation improves in vivo antigen expression more than 55 fold. Furthermore, this shielding of cationic surface charge results in antigen-specific T cell responses that are similar as those induced by naked DNA for the two lipo- and polyplex DNA carrier systems. These observations suggest that charge shielding forms a generally applicable strategy for the development of dermally applied vaccine formulations. Furthermore, the nanoparticle formulations developed here form an attractive platform for the design of targeted nanoparticle formulations that can be utilized for in vivo transfection of defined cell types.

Flotillin-dependent endocytosis and a phagocytosis-like mechanism for cellular internalization of disulfide-based poly(amido amine)/DNA polyplexes

Extensive research is currently performed on designing safe and efficient non-viral carriers for gene delivery. To increase their efficiency, it is essential to have a thorough understanding of the mechanisms involved in cellular attachment, internalization and intracellular processing in target cells. In this work, we studied in vitro the cellular dynamics of polyplexes, composed of a newly developed bioreducible poly(amido amine) carrier, formed by polyaddition of N,N-cystamine bisacrylamide and 1-amino-4-butanol (p(CBA-ABOL)) on retinal pigment epithelium (RPE) cells, which are attractive targets for ocular gene therapy. We show that these net cationic p(CBA-ABOL)/DNA polyplexes require a charge-mediated attachment to the sulfate groups of cell surface heparan sulfate proteoglycans in order to be efficiently internalized. Secondly, we assessed the involvement of defined endocytic pathways in the internalization of the polyplexes in ARPE-19 cells by using a combination of endocytic inhibitors, RNAi depletion of endocytic proteins and live cell fluorescence colocalization microscopy. We found that the p(CBA-ABOL) polyplexes enter RPE cells both via flotillin-dependent endocytosis and a PAK1 dependent phagocytosis-like mechanism. The capacity of polyplexes to transfect cells was, however, primarily dependent on a flotillin-1-dependent endocytosis pathway.

Physicochemical and Biological Evaluation of siRNA Polyplexes Based on PEGylated Poly(amido amine)s

ABSTRACTPurposeUse of RNA interference as novel therapeutic strategy is hampered by inefficient delivery of its mediator, siRNA, to target cells. Cationic polymers have been thoroughly investigated for this purpose but often display unfavorable characteristics for systemic administration, such as interactions with serum and/or toxicity.MethodsWe report the synthesis of a new PEGylated polymer based on biodegradable poly(amido amine)s with disulfide linkages in the backbone. Various amounts of PEGylated polymers were mixed with their unPEGylated counterparts prior to polyplex formation to alter PEG content in the final complex.ResultsPEGylation effectively decreased polyplex surface charge, salt- or serum-induced aggregation and interaction with erythrocytes. Increasing amount of PEG in formulation also reduced its stability against heparin displacement, cellular uptake and subsequent silencing efficiency. Yet, for polyplexes with high PEG content, significant gene silencing efficacy was found, which was combined with almost no toxicity.ConclusionsPEGylated poly(amido amine)s are promising carriers for systemic siRNA delivery in vivo.