Martin Piest

Effects of charge density and hydrophobicity of poly(amido amine)s for non-viral gene delivery.

High cationic charge densities in polymeric vectors result in tight DNA condensation, leading to small highly positively charged polyplexes which show generally high cellular uptake in vitro. However, high cationic charge densities also introduce membrane-disruptive properties to the polymers, thereby frequently causing high cytotoxities. We previously developed poly(amido amine)s with repetitive disulfide linkages in the main chain (SS-PAAs) that are significantly less toxic than PEI, due to fast intracellular degradation of these polymers by bioreductive cleavage of the disulfide bonds. In this study we have investigated the effects of variation in charge density and hydrophobicity on the gene delivery properties of these polymers by varying the degree of acetylation and benzoylation in SS-PAAs with aminobutyl side chains. It was found that introduction of hydrophobic benzoyl groups results in higher transfection efficiencies, both in the absence and presence of serum.

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.

pH-responsive, dynamically restructuring hydrogels formed by reversible crosslinking of PVA with phenylboronic acid functionalised PPO–PEO–PPO spacers (Jeffamines®)

Dynamically restructuring (“self-healing”) hydrogels were prepared by reversible formation of boronic-ester crosslinks between α,ω-phenylboronic acid terminated PPO–PEO–PPO spacers and poly(vinyl alcohol) (PVA). For this purpose two different bis-(phenylboronic acid) functionalised crosslinkers with Jeff–2AMPBA and Jeff–4AMPBA were synthesised via reductive coupling of 2- and 4-formylphenylboronic acid to Jeffamine® ED1900. Due to the different positions of the secondary amino group with respect to the boronic acid moiety (ortho or para) the pKa of the amino and boronic acid groups at the spacer endgroups differ significantly, thereby influencing the pH-dependency of boronic ester formation and gel properties. Upon mixing the boronic acid spacers with PVA (72–195 kDa) in the pH range 3–9 hydrogels were immediately formed. The gel strength and relaxation time of Jeff–2AMPBA gels increase with increasing acidity of the solution whereas those of Jeff–4AMPBA increase with increasing basicity. The pH-tunable properties could be explained in terms of pH effects on intra-molecular and inter-molecular B–N interactions in the Jeff–2AMPBA gels and Jeff–4AMPBA gels, respectively.

Role of boronic acid moieties in poly(amido amine)s for gene delivery

The effects of the presence of two different types of phenylboronic acids as side groups in disulfide-containing poly(amido amine)s (SS-PAA) were investigated in the application of these polymers as gene delivery vectors. To this purpose, a para-carboxyphenylboronic acid was grafted on a SS-PAA with pending aminobutyl side chains, resulting in p(DAB-4CPBA) and an ortho-aminomethylphenylboronic acid was incorporated through copolymerization, resulting in p(DAB-2AMPBA). Both polymers have 30% of phenylboronic acid side chains and 70% of residual aminobutyl side chains and were compared with the non-boronated benzoylated analogue p(DAB-Bz) of similar M(w). It was found that the presence of phenylboronic acid moieties improved polyplex formation with plasmid DNA since smaller and more monodisperse polyplexes were formed as compared to their non-boronated counterparts. The transfection efficiency of polyplexes of p(DAB-4CPBA) was approximately similar to that of p(DAB-Bz) and commercial PEI (Exgen), both in the absence and the presence of serum, indicating that p(DAB-4CPBA) and p(DAB-Bz) are potent gene delivery vectors. However, the polymers with phenylboronic acid functionalities showed increased cytotoxicity, which is stronger for the ortho-aminophenylboronic acid containing polyplexes of p(DAB-2AMPBA) than for the p(DAB-4CPBA) analog. The cytotoxic effect may be caused by increased membrane disruptive interaction as was indicated by the increased hemolytic activity observed for these polymers.

Modulating rheological and degradation properties of temperature-responsive gelling systems composed of blends of PCLA–PEG–PCLA triblock copolymers and their fully hexanoyl-capped derivatives☆

In this study, the ability to modulate rheological and degradation properties of temperature-responsive gelling systems composed of aqueous blends of poly(ε-caprolactone-co-lactide)-b-poly(ethylene glycol)-b-poly(ε-caprolactone-co-lactide) (PCLA-PEG-PCLA) triblock copolymers (i.e. uncapped) and their fully capped derivatives was investigated. Uncapped and capped PCLA-PEG-PCLA triblock copolymers, abbreviated as degree of modification 0 and 2 (DM0 and DM2, respectively), were composed of identical PCLA and PEG blocks but different end groups: namely hydroxyl and hexanoyl end groups. DM0 was synthesized by ring opening polymerization of l-lactide and ε-caprolactone in toluene using PEG as initiator and tin(II) 2-ethylhexanoate as the catalyst. A portion of DM0 was subsequently reacted with an excess of hexanoyl chloride in solution to yield DM2. The cloud point and phase behaviour of DM0 and DM2 in buffer as well as that of their blends were determined by light scattering in a diluted state and by vial tilting and rheological measurements in a concentrated state. Degradation/dissolution properties of temperature-responsive gelling systems were studied in vitro at pH 7.4 and 37°C. The cloud points of DM0/DM2 blends were ratio-dependent and could be tailored from 15 to 40°C for blends containing 15 to 100wt.% DM0. Vial tilting and rheological experiments showed that, with solid contents between 20 and 30wt.%, DM0/DM2 blends (15/85 to 25/75w/w) had a sol-to-gel transition temperature at 10-20°C, whereas blends with less than 15wt.% DM0 formed gels below 4°C and the ones with more than 25wt.% DM0 did not show a sol-to-gel transition up to 50°C. Complete degradation of temperature-responsive gelling systems took ∼100days, independent of the DM0 fraction and the initial solid content. Analysis of residual gels in time by GPC and (1)H-NMR showed no chemical polymer degradation, but indicated gel degradation by dissolution. Preferential dissolution of lactoyl-rich polymers induced enrichment of the residual gels in caproyl-rich polymers. To the best of our knowledge, degradation of temperature-responsive gelling systems by dissolution has not been reported or hypothesized as being the consequence of acylation of polymers. In conclusion, blending of PCLA-PEG-PCLA triblock polymers composed of identical backbones but different end groups provides for a straightforward preparation of temperature-responsive gelling systems with well-characterized rheological properties and potential in drug delivery. Furthermore, acylation of triblock copolymers may allow for the design of bioerodible systems with control over degradation by polymer dissolution.

Unraveling the cellular uptake of bioreducible poly(amido amine)--gene complexes in cells of the retinal pigment epithelium.

In vitro endocytosis of gene complexes composed of a bioreducible polyamidoamine CBA ABOL and plasmid DNA, in cells of the retinal pigment epithelium (RPE) was studied, the latter being an interesting target for ocular gene therapy. We found that cationic CBA ABOL DNA polyplexes attach to cell surface proteoglycans of these RPE cells and get subsequently internalized via a phagocytosis-like mechanism, as well as Flotillin dependent endocytosis. Introduction Proper delivery of therapeutic genes to designated cells and their availability at the intracellular site of action are crucial requirements for successful gene therapy. To this end, typically sub-micron sized particles are made by combining the therapeutic genes with a carrier material, such as cationic polymers, that aid in delivering the genes to the target site. In this work, for the first time, we have evaluated the ability of the highly promising bioreducible polymer carrier cystamin bisacrylamid aminobutanol (CBA ABOL) [1] (Fig. 1) to deliver plasmid DNA in cultured cells of the retinal pigment epithelium (ARPE-19) and characterized in vitro the cellular interactions with these target cells. These studies are of crucial importance since the further design and functionalization of polymeric gene carriers depend strongly on our understanding of the mechanisms involved in cellular adhesion, intracellular uptake and intracellular processing of the polyplexes. Experimental methods ARPE-19 cells (retinal pigment epithelial cell line; ATCC number CRL-2302) were cultured in DMEM:F12 supplemented with 10% fetal bovine serum, 2 mM l-glutamine, and 2% penicillin-streptomycin. All cells were grown at 37 °C in a humidified atmosphere containing 5% CO2. CBA ABOL gene complexes with an average hydrodynamic diameter of 130 nm and an average zeta potential of + 45 mV in 20 mM HEPES buffer were obtained by adding the polymer in a mass ratio of 48/1 in 20 mM HEPES to the plasmid and vortexing the mixture for 10 min. For every transfection, fresh polyplexes were prepared and applied to the cells within 30 min after complexation. For all uptake studies, YOYO-1™ (λex = 491 nm, λem = 509 nm, Molecular Probes, Merelbeke, Belgium) labeled pGL4.13 plasmid (Promega, Leiden, The Netherlands) was used. For all transfection studies, gWiz™GFP plasmid (Aldevron, Freiburg, Germany) was used. siRNAs were all purchased from Dharmacon and transfected in cells with the help of LipofectaminRNAiMAX (Invitrogen, Merelbeke, Belgium). Protein knockdown was assessed on Western Blot. Uptake of polyplexes or endocytic markers or GFP expression was measured on a FACS Calibur Flow Cytometer (Beckton Dickinson, Erembodegem, Belgium). For genetic labeling of endosomes, cells were transfected with GFP-fusion proteins. For fluorescence colocalization studies with GFP labeled cellular structures, cells were transfected with GFP-fusion proteins using Lipofectamin2000 (Invitrogen, Merelbeke, Belgium) and 24 h later exposed to red fluorescent labeled polyplexes. For this, CBA ABOL was complexed with pGL4.13 plasmid, covalently labeled with Cy5 (Label IT Nucleic Acid Labeling Kit, Mirus Bio Corporation, WI, USA). Live cell fluorescence colocalization was then performed on a custom built laser epi-fluorescence microscope set-up. A Nikon Plan Apo VC 100× 1.4 NA oil immersion objective lens (Nikon Belux, Brussels, Belgium) was used for imaging. GFP and Cy5 were excited with 491 nm and 636 nm laser light and emission was detected on an EMCCD camera (Roper Scientific, Nieuwegein, The Netherlands). For live cell imaging the cells were placed in a stage top incubation chamber (Tokai Hit, Shizuoka, Japan), set at 37 °C, 5% CO2 and 100% humidity. Result and discussion First, we found evidence that these net positively charged CBA ABOL polyplexes adhere to the negatively charged heparan sulfate proteoglycans (HSPGs) at the cell surface and that polyplex internalization is blocked by antibodies against Toll-like receptor 9