PEI-based vesicle-polymer hybrid gene delivery system with improved biocompatibility

Wider use of the transfection agent polymer polyethylenimine (PEI) in vivo has been hampered by its toxicity. In order to examine whether material combining properties of polymers and lipid type of carriers would have improved characteristics, four PEI derivatives were synthesised: The methylation of the branched PEI (25 kDa) created a permanently charged quaternary ammonium derivative. Acylation of these backbones using pendant palmitic acid chains created amphiphilic PEI variants which formed nanoparticles or vesicles. Finally hydrophilic groups were added to the polymer backbone by PEGylation. The materials were characterised and their in vitro and in vivo properties were tested. The modifications improved the materials biocompatibility markedly when compared to the starting material but also reduced transfection efficiency. The material bearing ammonium and palmitoyl groups was 10x less toxic while retaining about 30% of the transfection efficiency in vitro. After intravenous administration in a mouse model the materials also gave rise to GFP transgene expression in the liver. The synthetic strategy altered complex physicochemistry and improved biocompatibility while maintaining in vitro gene expression for most formulations. The strategy of combination of complementary properties of cationic lipids and polymers into a hybrid material may also be applicable to other materials.     

Gene transfer with three amphiphilic glycol chitosans--the degree of polymerisation is the main controller of transfection efficiency

A number of studies have examined the possibility of delivering genes for the treatment of genetic diseases using various polymers and lipids. We have previously demonstrated the gene transfer ability of amphiphilic polymers (a soluble amine polymer covalently bound to lipid pendant groups). In the current communication we explore the gene transfer activity of amphiphilic glycol chitosans. Glycol chitosan was acid depolymerised to give polymers of various molecular weights. Palmitoyl or hexadecyl and in some cases additional N-methyl quaternary ammonium groups were attached to the polymers. DNA binding was studied by measuring the reduced fluorescence of ethidium bromide and the polyplex particle size and zeta potential. Biological characterisation of the polyplexes involved haemolysis, cytotoxicity and gene transfer assays. For the 22 polymers tested, DNA binding was optimum at a nitrogen to phosphate ratio of 2:1 and above. Polyplexes were 200-500 nm in diameter with a neutral or positive zeta potential. The haemolytic activity of the N-methyl polymers was studied and no haemolysis was detected up to a concentration of 10 mg ml-1. Cytotoxicity studies showed that the biocompatibility of glycol chitosan was adversely affected by a combination of a palmitoyl group and depolymerisation and that biocompatibility was subsequently restored with the introduction of N-methyl groups. In vitro transfection efficiency superior to the cationic lipid formulation N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulphate (DOTAP) was seen with depolymerised glycol chitosan in the A431 cell line only and with the depolymerised N-methyl quaternary ammonium amphiphilic derivatives in both the A431 and A549 cell lines. Degree of polymerisation (DP) was the most important controller of transfection efficiency and transfection resided within polymers with a DP of 73-171. High DP polymers diminished DNA-cell association, the first step in the cellular gene transfer process, thus apparently diminishing cell uptake. In vivo transfection with the N-methyl quaternary ammonium amphiphile was best at a DP of 86 and this glycol chitosan amphiphile gave superior liver and heart gene expression levels when compared to both Exgen 500 (linear polyethylenimine) and Superfect (a polyamidoamine dendrimer).     

Gene Transfer with Three Amphiphilic Glycol Chitosans—the Degree of Polymerisation is the Main Controller of Transfection Efficiency

A number of studies have examined the possibility of delivering genes for the treatment of genetic diseases using various polymers and lipids. We have previously demonstrated the gene transfer ability of amphiphilic polymers (a soluble amine polymer covalently bound to lipid pendant groups). In the current communication we explore the gene transfer activity of amphiphilic glycol chitosans. Glycol chitosan was acid depolymerised to give polymers of various molecular weights. Palmitoyl or hexadecyl and in some cases additional N-methyl quaternary ammonium groups were attached to the polymers. DNA binding was studied by measuring the reduced fluorescence of ethidium bromide and the polyplex particle size and zeta potential. Biological characterisation of the polyplexes involved haemolysis, cytotoxicity and gene transfer assays. For the 22 polymers tested, DNA binding was optimum at a nitrogen to phosphate ratio of 2:1 and above. Polyplexes were 200–500 nm in diameter with a neutral or positive zeta potential. The haemolytic activity of the N-methyl polymers was studied and no haemolysis was detected up to a concentration of 10 mg ml^?1. Cytotoxicity studies showed that the biocompatibility of glycol chitosan was adversely affected by a combination of a palmitoyl group and depolymerisation and that biocompatibility was subsequently restored with the introduction of N-methyl groups. In vitro transfection efficiency superior to the cationic lipid formulation N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulphate (DOTAP) was seen with depolymerised glycol chitosan in the A431 cell line only and with the depolymerised N-methyl quaternary ammonium amphiphilic derivatives in both the A431 and A549 cell lines. Degree of polymerisation (DP) was the most important controller of transfection efficiency and transfection resided within polymers with a DP of 73–171. High DP polymers diminished DNA–cell association, the first step in the cellular gene transfer process, thus apparently diminishing cell uptake. In vivo transfection with the N-methyl quaternary ammonium amphiphile was best at a DP of 86 and this glycol chitosan amphiphile gave superior liver and heart gene expression levels when compared to both Exgen 500 (linear polyethylenimine) and Superfect (a polyamidoamine dendrimer).     

Physicochemical and biological evaluation of cationic polymethacrylates as vectors for gene delivery.

We report here the physicochemical and biological evaluation of a series of polymethacrylates with side groups of different pK(a) values, such as tertiary amines, pyridine groups, acid functions and imidazole groups as synthetic vectors for gene delivery. The ability of the different polymers to condense DNA was studied by ethidium bromide exclusion tests and agarose gel electrophoresis. The results show that all polymers are able to condense DNA. Both the molecular weight and the chemical composition of the polymers have an influence on the DNA condensation process. Furthermore, the biological properties of the polymer-DNA complexes were investigated, including their haemolytic activity, cytotoxicity and in vitro transfection efficiency. Complexes based on polymers containing only tertiary amines, have a transfection efficiency similar to that of poly(ethyleneimine) (PEI). Polymers containing pyridine groups have a reduced transfection efficiency compared to polymers containing tertiary amines. Introduction of imidazole groups or acid functions results in a loss of the transfection efficiency of the corresponding complexes with DNA. In general, the viability of cells incubated with complexes based on the polymethacrylates is higher than with PEI. Polymers with high transfection efficiency induce erythrocyte lysis.     

Muscarinic ganglionic stimulants: conformationally restrained analogues related to [4-[[N-(3-chlorophenyl)carbamoyl]oxy]-2-butynyl]trimethylammonium chloride

The synthesis of a series of tertiary and quaternary cyclic analogues (isoarecolinol, dihydroisoarecolinol, arecolinol, and 3-pyrroline-3-carbinol derivatives) of [4-[[N-(3-chlorophenyl)carbamoyl]oxy]-2-butynyl]trimethylammonium chloride (McN-A-343) (1), a selective stimulant of muscarinic receptors in sympathetic ganglia (so-called M1 receptors), is reported. The compounds 3-10 were tested for muscarinic ganglion-stimulating activity by recording blood pressure responses in pithed rats. All tertiary compounds tested had no ganglion-stimulating activity. Among the series of quaternary derivatives, only the isoarecolinol analogues 4a and 4b showed considerable ganglion-stimulating effects, whereas the dihydroisoarecolinol (8), the arecolinol (6a, 6b), and the 3-pyrroline-3-carbinol derivatives (10) were much less potent. Our experiments therefore demonstrate that in this series a quaternary nitrogen atom, unsaturation at C2 of the ammonium side chain, and a certain spatial arrangement of the ammonium and the phenylcarbamate groups are essential for potent M1-receptor stimulating activity.     

Neuromuscular blocking agents. Replacement of quaternary ammonium groups in bis-onium compounds by amidinium, guanidinium, thiouronium, sulphonium and sulphoxonium groups

The quaternary ammonium groups of suxamethonium have been replaced by amidinium, guanidinium and thiouronium groups. The guanidine compound exhibited competitive neuromuscular activity of a low order of potency; the other compounds were ineffective. Replacement of the quaternary ammonium groups in decamethonium by ethylmethylsulphonium or dimethylsulphoxonium groups gave less potent, depolarizing agents.     

Permeation enhancer effect of chitosan and chitosan derivatives: Comparison of formulations as soluble polymers and nanoparticulate systems on insulin absorption in Caco-2 cells.

In this study four quaternized derivatives of chitosan: trimethyl chitosan (TMC), dimethylethyl chitosan (DMEC), diethylmethyl chitosan (DEMC) and triethyl chitosan (TEC) with degree of substitution of approximately 50+/-5% were synthesized and their effect on the permeability of insulin across intestinal Caco-2 monolayers was studied and compared with chitosan both in free-soluble form and in nanoparticulate systems. Transepithelial electrical resistance (TEER) studies revealed that all four chitosan derivatives in free-soluble forms were able to decrease the TEER value in the following order TMC>DMEC>DEMC=TEC>chitosan, indicating their abilities to open the tight junctions. Recovery studies on the TEER showed that the effect of the polymers on Caco-2 cell monolayer is reversible and proves the viability of cells after incubation with all polymers. A similar rank order was also observed when measuring the zeta-potentials of the various polymers in solution form. Transport studies of insulin together with the soluble polymers across Caco-2 cell layers showed the following ranking: TMC>DMEC>DEMC>TEC>chitosan which is in agreement with the strength of the cationic charge of the polymer. In comparison to the free-soluble polymers, the nanoparticles prepared by ionic gelation of the chitosan and its quaternized derivatives had much lower effect on decreasing the TEER by opening of the tight junctions. This can be explained by the reduced available amount of positive charge at the surface of the nanoparticles. In accordance with these results, the insulin loaded nanoparticles showed much less permeation across the Caco-2 cell monolayer in comparison to the free-soluble polymers. Mass balance transport studies revealed that a substantial amount of the nanoparticles has been entrapped into the Caco-2 monolayer or attached to the cell surface. It can thus be stated that while free-soluble polymers can reversibly open the tight junctions and increase the permeation of insulin, the nanoparticles had basically only a low effect on the opening of the tight junction and the paracellular transport of insulin across the Caco-2 cell monolayer. These data convincingly show that nanoparticles consisting of chitosan and its quaternary ammonium derivatives loaded with insulin are less effective in facilitating paracellular transport across Caco-2 cell monolayers than the corresponding free polymers.     

Anisamido-Polyethylenimines as Efficient Nonviral Vectors for the Transport of Plasmid DNA to Sigma Receptor–Bearing Cells In Vitro

Site-specific delivery of therapeutics promises better outcomes in the treatment of diseases. A small ligand, anisamide, has been shown to specifically bind sigma receptors highly overexpressed on prostate cancer cells, one of the leading cancers causing deaths worldwide. Here, anisamide-tethered polyethylenimine polymers (AP) have been synthesized and evaluated for their capability to transport nucleic acid across the cell membrane. A series of modified polymers (AP-1 to AP-4) was synthesized, physicochemically characterized, and evaluated for their transfection efficiency and cytotoxicity. Postconjugation, there was a marginal decrease in the buffering capacity; however, it did not diminish the ultimate objective of the study rather improved the transfection efficiency and decreased the cytotoxicity making these polymers as efficient and safe vectors for nucleic acid delivery. All the modified polymers displayed enhanced capability to deliver DNA inside the cells. Among the series, the modified polymer, AP-4 (10% attempted substitution), exhibited the highest transfection in HEK293 cells having abundant sigma receptors with minimal cytotoxicity. The projected polymer also showed complete protection of bound DNA against enzymatic degradation. Altogether, the results demonstrated targeting ability of the proposed polymers to deliver nucleic acid to sigma receptor–bearing cells in vitro.     

Synthesis and radioligand binding studies of methoxylated 1,2,3,4-tetrahydroisoquinolinium derivatives as ligands of the apamin-sensitive Ca2+-activated K+ channels

Several methoxylated 1,2,3,4-tetrahydroisoquinoliniums derived from N-methyl-laudanosine and N-methyl-noscapine were synthesized and evaluated for their affinity for apamin-sensitive binding sites. The quaternary ammonium derivatives have a higher affinity with regard to the tertiary amines. 6,7-Dimethoxy analogues possess a higher affinity than the 6,8- and 7,8-dimethoxy isomers. A 3,4-dimethoxybenzyl or a 2-naphthylmethyl moiety in C-1 position are more favorable than a 3,4-dimethoxyphenethyl group. Smaller groups such as propyl or isobutyl are unfavorable. In 6,7-dimethoxy analogues, increasing the size and lipophilicity with a naphthyl group in the C-1 position leads to a slight increase of affinity, while the same group in the 6,7,8-trimethoxy series is less favorable. The 6,7,8-trimethoxy derivative 3f is the first tertiary amine in the series to possess an affinity close to that of N-methyl-laudanosine and N-methyl-noscapine. Moreover, electrophysiological studies show that the most effective compound 4f blocks the apamin-sensitive afterhyperpolarization in rat dopaminergic neurons.     

Tumour gene expression from C12 spermine amphiphile gene delivery systems

Gene therapy requires safe and efficient gene delivery systems. Towards this aim both the gene formulation and tumour transfection ability of C12 spermine amphiphiles were tested. Five amphiphiles were synthesised and characterised: 1-[N,N-bis(3-aminopropyl)-1,4-butane diamine] dodecane (12G0--a C12 spermine amphiphile), a poly(ethylene glycol) (PEG, MW = 2 kDa) derivative of 12G0, 1,12-[N,N-bis(3-aminopropyl)-1,4-butane diamine] dodecane (12G1--a C12 spermine bolaamphiphile) and N-methyl quaternary ammonium derivatives of both 12G0 (12QG0) and 12G1 (12QG1). All amphiphiles except 12G0, which precipitates, yield nanoparticles in aqueous media with and without DNA. Thus when 12G0 is substituted with either quaternary ammonium or PEG groups it forms nanoparticles both with and without DNA. The minimum nitrogen, phosphate ratio required to completely condense DNA (NP) was inversely proportional to the particles' zeta potential (zeta), NP = 1626/zeta(0.98). Biological testing showed that both PEG and quaternary ammonium groups diminished the membrane lytic ability of these C12 amphiphiles. On intratumoural injection, while PEG groups hamper gene transfer, the quaternary ammonium amphiphile (12QG0) produces tumour confined gene expression that is 80% of that produced by linear poly(ethylenimine) (LPEI, MW = 22 kDa); while the intratumoural injection of LPEI produced significant gene expression in the liver and lung, making 12QG0 suitable for the administration of cytotoxic tumouricidal genes.     

Effects of physicochemical characteristics of poly(2-(dimethylamino)ethyl methacrylate)-based polyplexes on cellular association and internalization

The cationic polymer poly(2-(dimethylamino)ethyl methacrylate) (p(DMAEMA)) is able to efficiently bind and condense DNA and to mediate transfection of a variety of cell types. In this study, fluorescence activated cell sorting (FACS), confocal laser fluorescence microscopy (CSLM) and electron microscopy (EM) techniques were used to investigate in vitro the cellular interaction of p(DMAEMA)-based polyplexes with human ovarian carcinoma cells (OVCAR-3). Cellular association and subsequent internalization only occurred when the polyplexes exhibited a positive zeta potential. Small-sized polyplexes have an advantage over large-sized complexes regarding cellular entry. The effect of the presence of tertiary amine groups versus the presence of quatenary amine groups was evaluated by comparing p(DMAEMA) with its quaternary ammonium analogue poly(2-(trimethylamino)ethyl methacrylate) (p(TMAEMA)). The combined cellular interaction and transfection results suggest that the latter polymer does not have an intrinsic endosomal escape property, in contrast to the 'proton sponge' effect proposed for p(DMAEMA). PEGylation of p(DMAEMA) effectively shielded the surface charge and yielded a notably lower degree of cellular interaction. Data on the effects of the presence of endocytosis inhibitors and an endosome-disruptive peptide in the culture medium on the cellular interaction and transfection activity of p(DMAEMA)-based polyplexes support endocytosis as being the principal pathway for intracellular delivery of plasmid. Both the CLSM and EM studies did not reveal the presence of polyplexes or plasmid outside the endocytic vesicles or within the nucleus, suggesting that intracellular trafficking from the endosomes to the nucleus is a very inefficient process.     

Transport characteristics of propantheline across rat intestinal brush border membrane

The transport mechanism of propantheline, an anti-acetylcholine quaternary ammonium compound, has been studied using brush border membrane vesicles isolated from rat small intestine. The uptake of propantheline was facilitated by the transmembrane electrical potential difference (cell interior negative) induced by NaSCN, NaI or valinomycin. But this effect was a secondary action; in the initial phase of propantheline uptake (less than 5 min), there was no facilitating effect. When the transmembrane potential difference was induced after propantheline uptake had reached a steady state, there was an overshoot of the drug. Therefore, it is suggested that the transport of propantheline across the brush border membrane consists of at least two processes. In the first, propantheline rapidly binds to the brush border membrane, in the second it enters into epithelium driven by the negative transmembrane electrical potential difference. Cationic tertiary amines such as chlorpromazine, imipramine and promethazine markedly inhibited propantheline uptake. These results suggest that there is a common absorption process for tertiary amines and quaternary ammonium compounds.     

血吸虫病化学治疗的研究Ⅺ.芳氧烷基胺化合物的制备

苯环上带有氯或硝基取代的ω-溴代芳氧烷烃分别与氮己环、甲基氮己环、吗啉、甲基吗啉、三甲胺、邻苯二甲酰亚胺、邻磺酰苯甲酰亚胺等作用,获得一系列芳氧烷基胺、季铵、或酰胺化合物,动物试验对日本血吸虫没有作用。     

pH dependence of the blocking activity of carbisocaine and its derivatives

The blocking activity and pH dependence of carbanilate local anaesthetics, carbisocaine and its homologues, were tested on isolated rat sciatic nerves at pH 6.0, 7.2 and 8.4. Carbisocaine blocked the compound action potential more strongly than the derivatives with shorter alkoxysubstituents. The blocking potency of shorter derivatives increased with the rise of external medium pH, whereas the activity of carbisocaine increased with decreasing external pH. Quaternary compounds applied in the external medium were able to block the action potential, but in higher concentrations and with a longer half-time than their tertiary analogues. The blocking potency of quaternary derivatives correlated well with the length of the alkoxysubstituent and thus also with their lipophilicity. Extracellular pH did not consistently change the inhibitory effect of quaternized derivatives. These observations support the view that the lipophilicity of local anaesthetics is one of the possible factors determining their anaesthetic activity and the pH dependence of their effect.     

Assessment of the combined approach of N-alkylation and salt formation to enhance aqueous solubility of tertiary amines using bupivacaine as a model drug.

Quaternary prodrug types of poorly water-soluble tertiary amines have been shown to exhibit significantly enhanced solubilities as compared to the parent amine. In the present study the combined effect of N-alkylation and salt formation to enhance aqueous solubility of tertiary amines have been investigated using bupivacaine as a model compound. X-ray structure analyses of selected salts were included to investigate the potential existence of correlations between salt solubility and crystal packing modes. Alkyl groups were methyl, ethyl, propyl, and butyl and the derivatives were isolated as their iodide salts. Chloride, mesylate, formate, acetate, glycolate, and tosylate salts were obtained by anion exchange of the N-methyl-bupivacaine derivative. N-Alkylation and salt formation afforded quaternary ammonium salts possessing pH-independent aqueous solubilities far exceeding that of the parent tertiary amine (up to a factor of 3200 at pH 8). A moderate reduction in solubility with increasing length of the alkyl chain was observed for the iodide salts of the N-alkylated bupivacaine derivatives. In case of the N-methyl-bupivacaine derivative variation of the counterion had a significant impact on the solubility with the iodide salt being 200 times less soluble than the chloride salt. X-ray analysis revealed that both the alkyl substituent and the anionic counterion influenced salt packing modes, however, in an unpredictable manner making establishment of quantitative correlations between crystal packing and solubility difficult even for a series of closely related derivatives.     

Steroidal Ammonium Compounds as New Neuromuscular Blocking Agents

Neuromuscular blocking agents are widely used as an anesthesia auxiliary in surgery, which induce relaxation of skeletal muscles by blocking signal transmission at the neuromuscular junction. Many neuromuscular blocking agents s were developed over the past decades, but none of them fully meets the needs of the clinic by various reasons. In this study, a series of quaternary ammonium steroidal neuromuscular blocking agents were synthesized and evaluated on isolated mouse phrenic nerve–hemidiaphragms for their bioactivities. The initial separation of mono‐ and bis‐quaternary ammonium compounds turned out to be very challenging on regular silica gel chromatography. Therefore, a facile purification method, in which the silica gel was pretreated with methanolic sodium bromide solution, was finally achieved. Compounds 3g (0.36 μm ) and 4g (0.37 μm ) exhibited excellent neuromuscular blocking activities, which were about sixfold to sevenfold higher in potency than that of rocuronium (2.50 μm ). In addition, other bis‐quaternized compounds also showed good potencies close to that of rocuronium. Furthermore, the preliminary structure–activity relationship of this series was also elucidated. Benzyl group was found to be a promising quaternary group in this series.     

Novel transmucosal absorption enhancers obtained by aminoalkylation of chitosan

Literature data suggest that quaternized chitosans have a transmucosal drug absorption enhancing property depending on their MW, quaternization degree and other structural features. With the purpose of preparing novel effective promoters, a chitosan (Ch) from crab shell (ChC; viscometric MW, 800 kDa; deacetylation: 90%, IR; 84%, NMR) and one from shrimp shell (ChS; viscometric MW, 590 kDa; deacetylation: 90%, IR; 82%, NMR) were reacted with 2-diethylaminoethyl chloride (DEAE-Cl) and novel derivatives containing different percentages of pendant quaternary ammonium groups were obtained. NMR analysis, based on HSQC, COSY, TOCSY and ROESY maps, indicated that three partially substituted N,O-[N,N-diethylaminomethyl(diethyldimethylene ammonium)(n)]methyl chitosans, coded N(+)-ChS-2 (degree of substitution, DS=40%; n=1.6), N(+)-ChS-4 (DS=132%; n=2.5), and N(+)-ChC-4 (DS=85%; n=1.7) resulted from the reaction, depending on whether the DEAE-Cl/Ch repeating unit molar ratio, was 2:1 or 4:1. The effects of the derivatives on the permeability of rhodamine 123 (Rh-123), hydrophobic, marker of the transcellular absorption route, and of fluorescein sodium (NaFlu), polar, marker of the paracellular route, across excised porcine cheek epithelium were assessed, using Franz type diffusion cells. Rh-123 permeability was enhanced by N(+)-ChS-4 (enhancement ratio, ER=8.4) and by N(+)-ChC-4 (ER=3.9), whereas N(+)-ChS-2 was ineffective. NaFlu permeability was enhanced by N(+)-ChS-2 (ER=7.2), N(+)-ChS-4 (ER=7.4) and N(+)-ChC-4 (ER=6.6). In conclusion, the three derivatives, whichever their DS, promote paracellular transport, while transcellular transport is substantially accelerated only by the most substituted one.     

Cationic lipids containing cyclen and ammonium moieties as gene delivery vectors

In this study, two novel cationic lipids containing protonated cyclen and quaternary ammonium moieties were designed and synthesized as non-viral gene delivery vectors. The structures of the two lipids differ in their hydrophobic region (cholesterol or diosgenin). Cationic liposomes were easily prepared from the lipids individually or from the mixtures of each cationic lipid and dioleoylphosphatidylethanolamine. Several studies including DLS, gel retardation assay, and ethidium bromide intercalation assay suggest that these amphiphilic molecules are able to bind and compact DNA into nanometer particles which can be used as non-viral gene delivery agents. Our results from in vitro transfection show that in association with dioleoylphosphatidylethanolamine, two cationic lipids can induce effective gene transfection in human embryonic kidney 293 cells, although the gene transfection efficiencies of two cationic lipids were found to be lower than that of lipofectamine 2000(TM) . Besides, different cytotoxicity was found for two lipoplexes. This study demonstrates that the title cationic lipids have large potential to be efficient non-viral gene vectors.     

Modified poly(propylene imine) dendrimers as effective transfection agents for catalytic DNA enzymes (DNAzymes)

The major bottleneck in gene therapy remains the issue of delivery. In this work, various modified poly(propylene imine) (PPI) dendrimers are introduced as gene transfection agents. Commercially available PPI-dendrimers have been modified (i) at the exterior primary amines with acetyl groups or glycol gallate (PEG-like) groups, and (ii) at the interior tertiary amines with methyl iodide (MeI) or MeCl to produce multiple quaternized cationic sites in the core of the dendrimer. The prepared materials have been tested with respect to their binding capabilities to DNA, their toxicity in cell cultures, their in vitro transfection efficiency and their in vivo delivery possibilities. In all cases, a 33-mer oligonucleotide (DNAzyme) was used. Polyacrylamide gel electrophoresis (PAGE) studies have demonstrated strong but reversible binding, where the quarternized and higher generation dendrimer species have shown more potent binding. Typically, for the modified fourth PPI-dendrimers, binding is observed at a concentration of about 4 microM DNA and a dendrimer-DNA charge ratio of around 2:1-1:1. All the tested PPI-dendrimers display a low cellular toxicity, especially when higher serum contents are used in the culture medium. For example, most of the prepared fourth generation PPI-dendrimers are not or hardly toxic up to at least 20 microM in 20% serum. An in vitro characterization has revealed a high dendrimer-mediated intracellular uptake of the DNAzyme: all the tested fourth generation PPI-dendrimers display transfection efficiencies close to or exceeding 80%, even when the concentration of serum in the medium is increased from 10 to 40%. Finally, the potential of using modified PPI-dendrimers for in vivo gene therapy experiments is demonstrated. Injecting a G4-PEG(MeI)-ssDNA complex intravenously into Nude mice has resulted in a high nuclear uptake as confirmed by co-localization studies.     

Modified poly(propylene imine) dendrimers as effective transfection agents for catalytic DNA enzymes (DNAzymes)

The major bottleneck in gene therapy remains the issue of delivery. In this work, various modified poly(propylene imine) (PPI) dendrimers are introduced as gene transfection agents. Commercially available PPI-dendrimers have been modified (i) at the exterior primary amines with acetyl groups or glycol gallate (PEG-like) groups, and (ii) at the interior tertiary amines with methyl iodide (MeI) or MeCl to produce multiple quaternized cationic sites in the core of the dendrimer. The prepared materials have been tested with respect to their binding capabilities to DNA, their toxicity in cell cultures, their in vitro transfection efficiency and their in vivo delivery possibilities. In all cases, a 33-mer oligonucleotide (DNAzyme) was used. Polyacrylamide gel electrophoresis (PAGE) studies have demonstrated strong but reversible binding, where the quarternized and higher generation dendrimer species have shown more potent binding. Typically, for the modified fourth PPI-dendrimers, binding is observed at a concentration of about 4 μM DNA and a dendrimer-DNA charge ratio of around 2:1–1:1. All the tested PPI-dendrimers display a low cellular toxicity, especially when higher serum contents are used in the culture medium. For example, most of the prepared fourth generation PPI-dendrimers are not or hardly toxic up to at least 20 μM in 20% serum. An in vitro characterization has revealed a high dendrimer-mediated intracellular uptake of the DNAzyme: all the tested fourth generation PPI-dendrimers display transfection efficiencies close to or exceeding 80%, even when the concentration of serum in the medium is increased from 10 to 40%. Finally, the potential of using modified PPI-dendrimers for in vivo gene therapy experiments is demonstrated. Injecting a G4-PEG(MeI)–ssDNA complex intravenously into Nude mice has resulted in a high nuclear uptake as confirmed by co-localization studies.