Tomáš Strašák

Phosphonium carbosilane dendrimers for biomedical applications – synthesis, characterization and cytotoxicity evaluation

We report the synthesis and cytotoxicity evaluation of a completely new class of cationic carbosilane dendrimers functionalized with several different phosphonium peripheral groups and an ammonium functionalised one as a reference. The carbosilane dendrimers with NMe3, PMe3, P(Et2)2(CH2)3OH, PBu3, P(C6H4-OMe)3 and P(Ph)3 peripheral substituents were synthesized, thoroughly characterized and modelled by computer simulations. The cytotoxicities of the dendrimers were investigated in vitro on three model cell lines (B14, BRL and NRK cells) by MTT and CV assay methods. Generally, the cytotoxicities of PMe3 carbosilane dendrimers were similar or slightly lower when compared with NMe3 dendrimers. The substitution of methyl groups in PMe3 carbosilane dendrimers with more hydrophobic and bulky alkyl substituents (PBu3 and P(Et2)2(CH2)3OH dendrimers) resulted in an increase of cytotoxicity. The P(C6H4-OMe)3 dendrimer showed exceptionally low cytotoxicity across all cell lines or assay methods used. Generally, phosphonium carbosilane dendrimers could represent a valuable alternative to ammonium ones in gene therapy applications due to comparable or lower cytotoxicities, the presence of positive charge for nucleic acid electrostatic binding and in the cases of P(C6H4-OMe)3 and P(Ph)3 dendrimers high potential of mitochondrial targeting.

Synthesis and characterization of carbosilane dendrimer–sodium montmorillonite clay nanocomposites. Experimental and theoretical studies

Novel organic/inorganic hybrid materials with carbosilane fillers were developed. New carbosilane dendrimers of the first (Dm1) and second (Dm2) generations bearing four and eight cationic ammonium groups on their periphery were synthesized first. Their structure was elucidated by NMR spectroscopy and ESI-HRMS whereas their thermal stability was confirmed by TGA. Both dendrimers were used as organic components in the preparation of organoclays. A series of nanocomposites Dm1Mt and Dm2Mt with varied contents of dendrimers were prepared and characterized by a wide variety of analytical techniques. The analytical data show that the structure of organoclays and the ability of dendrimers to effectively interact with montmorillonite are strongly affected by their generation. Both species Dm1 and Dm2 intercalated into the interlayer space of montmorillonite increase correspondingly the basal spacing of Mt. In contrast to Dm2, increased loadings of Dm1 in suspension during the intercalation process have a negligible effect on d-spacing in the studied concentration range, as confirmed by XRD analysis. Besides unmodified Mt we also studied the interactions of dendrimers with plasma treated Mt and the results are presented. Computer modelling based mainly on Molecular Dynamics has shown the non-linear dependence of the d-spacing on the amount of dendrimers in the interlayer space within the studied dendrimer/montmorillonite mass ratio interval. The filling capacity of plasma unmodified montmorillonite with respect to both dendrimers was estimated using a combination of experimental and theoretical results.

Organoclays with carbosilane dendrimers containing ammonium or phosphonium groups

New composite materials could reveal attractive capabilities and favourable properties. Herein, we present novel layered nanocomposites consisting of inorganic clay (montmorillonite) and cationic carbosilane dendrimers. A comparative study was performed to evaluate cationic moieties (ammonium or phosphonium) and various generations of dendrimers (first, second and third) and their influence on the characteristics of resulting materials. The target nanocomposites were characterized by XRD, TEM, TGA, IR and MAS NMR. Interestingly, with multivalent dendritic cations, as opposed to monovalent cations, we detected a new connection of cationic species to the inorganic matrix by both ammonium and phosphonium dendrimers. To evaluate the mechanism of thermal degradation of the prepared materials, non-oxidative pyrolysis coupled with analysis of the resulting products by IR and MS was performed. To illustrate the catalytic activity of the as-prepared materials, selected samples were tested as catalysts in the cycloaddition of CO2 with epoxide (allyl glycidyl ether).

Evaluation of toxicological and teratogenic effects of carbosilane glucose glycodendrimers in zebrafish embryos and model rodent cell lines

Abstract Glycodendrimers (Glyco-DDMs) represent a rapidly growing class of nanoparticles with promising properties for biomedical applications but concerns regarding the impact on human health and environment are still justified. Here we report, for the first time, the comparative study of in vivo developmental toxicity of carbosilane Glyco-DDMs and their cytotoxicity in vitro. Carbosilane Glyco-DDMs (generation 1–3) containing 4, 8, and 16 β-d-glucopyranosyl units at the periphery (DDM1Glu, DDM2Glu, and DDM3Glu) were synthesized and characterized by 1H, 13C and 29Si NMR, mass spectrometry, dynamic light scattering, atomic force microscopy (AFM), and computer modeling. In vitro cytotoxicity assay (MTT) of DDM1–3Glu was performed on three different rodent cell lines (Cricetulus griseus) – B14 (ATCC, CCL-14.1), BRL 3A (ATCC, CRL-1442), and NRK 52E (ATCC, CRL-1571). Overall, very low cytotoxicity was observed with calculated IC50 in mM range with slight difference between each cell line and DDM generation investigated. Modified fish embryo test (FET) was further used for DDM3Glu developmental toxicity testing on zebrafish (Danio rerio) embryos. While seemingly harmless to intact embryos, adverse effects of DDMs on the embryonic development become evident after chorion removal (LD50=2.78 µM at 96 hpe). We summarized that the modified FET test showed a two to three orders of magnitude difference between the in vitro cytotoxicity and in vivo developmental toxicity of DDM3Glu. While, in general, the Glyco-DDMs show great promises as efficient vectors in targeted drug delivery or as therapeutic molecules itself, we suggest that their developmental toxicity should be thoroughly investigated to exclude safety risks associated with their potential biomedical use.

ESI-TOF mass spectrometry of cationic carbosilane dendrimers: A potent tool for characterization of structural defects

Macromolecular polyelectrolytes are gaining considerable attention for the application in medicine that implies their detailed characterization. We have successfully applied electrospray ionization mass spectrometry (ESI MS) to the analysis of defects in the structure of three generations of polycationic carbosilane dendrimers bearing series of quarternary phosphonium groups at their periphery. Besides expected defects caused by incomplete conversion of particular reaction steps during the synthesis of dendritic scaffold and subsequent peripheral functionalization, also, several products of side reactions were observed together with defects created in the course of measurement (particularly ion exchange products). Defective molecules can be to some extent separated by means of gel permeation chromatography that proves that they are not products of in source fragmentation processes. Within the reaction sequence used for the synthesis of dendrimers under study, hydrosilylation was the source of most defects; the effectivity of quarternization depends on the type of phosphine. Results confirm high sensitivity of ESI MS towards defects, stability of the carbosilane skeleton towards fragmentation under the conditions of ESI ionization, and capability to detect both lower- and higher-molecular weight impurities arising from the synthetic sequence in the same m/z range as the target dendrimer, thus providing valuable view of the polydispersity.