Non-viral vaccination through cationic guanidium polymer-pDNA polyplex mediated gene transfer.

Vaccination through cellular transfection of nucleotide-based vaccines is a powerful approach to combatting disease. Plasmid DNA (pDNA) vaccines are particularly promising vectors for non-viral immunomodulation that afford high degrees of potency and flexibility. Versatile guanidinium-functionalized poly(oxanorbornene)imide (PONI-Guan) homopolymers were used to facilitate non-disruptive pDNA condensation into discrete polyplexes, enabling efficient in vitro transfection of endothelial cells and HD-11 macrophages. Translation of these vectors for vaccination of white leghorn chickens against Newcastle disease virus (NDV) elicited strong humoral immune responses against the virus. This approach presents a highly versatile method for targeted immunomodulation in vivo, with the potential for translatability as a non-viral vaccine platform.

Carbohydrate-interactive pDNA and siRNA gene vectors based on boronic acid functionalized poly(amido amine)s.

In order to evaluate the influence of incorporation of boronic acid groups on the properties of poly(amido amine)s as gene vectors, a novel poly(amido amine) copolymer p(CBA-ABOL/2AMPBA) containing ortho-aminomethylphenylboronic acid (2AMPBA) moieties was prepared by Michael-type polyaddition of a mixture of 1,4-aminobutanol (ABOL) and 2-((4-aminobutylamino)methyl)phenyl boronic acid to N,N'-cystamine bisacrylamide (CBA). It appeared that the presence of the boronic acid moieties as side groups along the polymer chain strongly enhances the stability of the self-assembled nanoparticles and nanosized polyplexes formed from this polymer; no aggregation was observed after storage for 6days at 37°C. This strong stabilization can be attributed to intermolecular Lewis acid-base interactions between the 2AMPBA groups and the alcohol and amine groups present in the polymer, leading to dynamical (reversible) crosslinking in the nanoparticles. Moreover, since the boronic acids can reversibly form boronic esters with vicinal diol groups, the presence of the 2AMPBA groups add carbohydrate-interactive properties to these polymers that strongly influence their behavior as gene delivery vectors. DNA transfection with p(CBA-ABOL/2AMPBA) polyplexes gave transfection efficiencies that were approximately similar to commercial PEI in different cell lines (COS-7, HUH-6 and H1299-Fluc), but lower than those obtained with reference polyplexes from p(CBA-ABOL). It is hypothesized that the uptake of the boronated polyplexes is suppressed by binding to the glycocalyx of the cells. This is supported by the observation that addition of sorbitol or dextran to the transfection medium significantly enhances the transfection efficiency, which can be attributed to increased cellular uptake of the polyplexes due to boronic ester formation with these agents. AFM, SEM and confocal microscopy showed that polyplexes of p(CBA-ABOL/2AMPBA) become decorated with a dextran layer in the presence of 0.9% (w/v) dextran in the polyplex medium. In siRNA-mediated gene silencing experiments the use of p(CBA-ABOL/2AMPBA) as polymeric vector gave a knockdown of luciferase expression in H1229-Fluc cells of 35%. Also in this case addition of 0.9% (w/v) sorbitol or dextran to the transfection medium strongly increased the knockdown efficiency to 59% and 76%, respectively.

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.

Effects of branched or linear architecture of bioreducible poly(amido amine)s on their in vitro gene delivery properties.

In this study, the gene delivery properties of new hyperbranched poly(amido amine)s (PAAs) with disulfide linkages in the main chain were investigated in comparison with their linear analogs. Eight different bioreducible PAAs were prepared by Michael addition of N,N'-bisacryloylpiperazine (BP) with cystamine (CYST) or N,N'-dimethylcystamine (DMC) and of N,N'-cystaminebisacrylamide (CBA) with N,N'-ethylenediamine (EDA) or N,N'-dimethylethylenediamine (DMEDA). In order to study the effect of terminal groups on the transfection efficiency, each polymer was terminated with 4-aminobutanol (ABOL) or with 2-aminoethanol (ETA). The hyperbranched and the linear PAAs generally formed polyplexes with plasmid DNA with sizes around 200nm and positive zeta potentials ranging from +10 to +22mV at polymer/DNA weight ratios equal or higher than 3/1. Remarkably low or no cytotoxicity was observed for both hyperbranched and linear PAAs. Hyperbranched CBA-containing PAAs showed higher gene expression in DNA transfection tests with COS-7 cells than their linear analogs and up to two times higher than linear PEI that was used as the reference polymer. Transfection efficiencies of the branched PAAs were generally enhanced by the presence of serum, which is a promising property for future in vivo studies with these hyperbranched PAAs. In this study the ease of synthetic modification of both linear and hyperbranched poly(amido amide)s and the versatility of hyperbranched PAAs in regulating DNA transfection and cytotoxicity are demonstrated. The results show the large possibilities for this class of polymers to provide polymeric vectors with controllable properties for gene therapy applications.

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.

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.

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.

Novel poly(amido amine)s with bioreducible disulfide linkages in their diamino-units: structure effects and in vitro gene transfer properties.

A series of novel water-soluble, bioreducible poly(amido amine)s containing disulfide linkages in their amino units (SS-PAAs) was synthesized by Michael addition polymerization of N,N'-dimethylcystamine (DMC) with various bisacrylamides. The synthetic route allows large structural variation in the bisacrylamide segments and is complementary to the earlier developed route to SS-PAAs in which the disulfide bond is incorporated in cystamine bisacrylamide units. The physicochemical and biomedical properties of the novel DMC-based polymers were evaluated for their application as non-viral gene delivery vectors and compared with analogs lacking the disulfide moieties. DMC-based SS-PAAs show high buffer capacities in the pH range pH 5.1-7.4, a property that may favorably contribute to the endosomal escape of the polyplexes. The polymers are capable to condense DNA into nanoscaled (<250 nm) and positively charged (>+20 mV) polyplexes which are relatively stable in medium mimicking physiological conditions but rapidly disintegrate in the presence of 2.5 mM DTT, mimicking the intracellular reductive environment. Polyplexes from DMC-based SS-PAAs are capable to transfect COS-7 cells in vitro with transfection efficiencies up to 4 times higher than those of pDMAEMA and PEI, with no or only very low cytotoxicity at the polymer/DNA ratios where the highest transfection is observed. The results show that DMC-based SS-PAAs have very promising properties for the development of potent and non-toxic polymeric gene carriers.