Synthesis and gene transfer activities of novel serum compatible cholesterol-based gemini lipids possessing oxyethylene-type spacers

Four novel cholesterol-based gemini cationic lipids differing in the length of oxyethylene-type spacers [-CH2-(CH2-O-CH2)n-CH2-] between each ammonium headgroup have been synthesized. These formed stable suspensions in aqueous media. Cationic liposomes were prepared from each of these lipids individually and as mixtures of cationic lipid and DOPE. These were used as nonviral gene delivery agents. All the cholesterol-based gemini lipids induced better transfection activity than their monomeric counterpart. Inclusion of DOPE in co-liposomal formulation of the cationic gemini lipid potentiates their gene transfer activity significantly. A major characteristic feature of these oxyethylene spacer based cholesterol gemini lipids was that serum does not inhibit the transfection activity of these gemini lipids, whereas the transfection activity of their monomeric counterpart decreased drastically in the presence of serum. One of the cholesterol-based gemini lipids 2a possessing a -CH2-CH2-O-CH2-CH2- spacer showed the highest transfection activity.     

Spacer structure and hydrophobicity influences transfection activity of novel polycationic gemini amphiphiles

Three novel polycationic gemini amphiphiles with different spacers were developed and evaluated in terms of their physiochemical properties and transfection efficiencies. Cationic liposomes formed by these amphiphiles and the helper lipid DOPE were able to successfully condense DNA, as shown by gel mobility shift and ethidium bromide intercalation assays. Transfection activity of the liposomes was superior to Lipofectamine^® 2000 and was dependent on spacer structure, hydrophobicity, and nucleic acid type (pDNA or siRNA). We demonstrated that the cationic liposomes 2X6/DOPE and 2X7/DOPE are potential non-toxic vehicles for gene delivery.     

In vivo cutaneous interferon-gamma gene delivery using novel dicationic (gemini) surfactant-plasmid complexes

BACKGROUND: Localized scleroderma (morphea and linear scleroderma) is a connective tissue disease, accompanied by excessive proliferation and deposition of collagen within the skin, inflammation, vasculopathy and a deranged immune system. Interferon gamma (IFNgamma), an inhibitor of collagen synthesis and an immunomodulator, could be a potential therapeutic agent if it could be delivered into or expressed locally in affected skin in a non-invasive manner. In this study, the feasibility of topical delivery of the IFNgamma gene and expression of IFNgamma were investigated in mice. METHODS: Novel dicationic (gemini) surfactant (spacer length n=2-16; alkyl chain m=12 or 16)-DNA complexes were formulated and characterized by circular dichroism and atomic force microscopy to select gemini analogues with the highest transfection efficiency (TE). Transfection and cellular expression of IFNgamma from the bicistronic pGTmCMV.IFN-GFP plasmid were evaluated in PAM 212 keratinocyte culture by ELISA and fluorescence microscopy. Topical delivery of plasmid using liposomal and nanoemulsion systems, based on gemini surfactant 16-3-16, was evaluated in mice by IFNgamma expression analysis. RESULTS: In vitro TE was found to be dependent on the spacer length of the gemini surfactant, with the C3 spacer showing the highest activity (both 12-3-12 and 16-3-16). Both gemini cationic liposomes and gemini nanoemulsion (3x25 microg DNA/animal) produced significantly higher levels of IFNgamma in the skin (359.4 and 607.24 pg/cm2) compared to naked DNA (135.69 pg/cm2) or a liposomal Dc-chol formulation (82.15 pg/cm2). IFNgamma expression in the lymph nodes was higher in the animals treated with gemini liposomes (422.74 pg/animal) compared to the nanoemulsion formulation (131.27 pg/animal) or the Dc-chol formulation (82pg/animal). CONCLUSIONS: The feasibility of topical delivery of pGTmCMV.IFN-GFP plasmid in mice using gemini cationic surfactant based delivery systems was demonstrated. IFNgamma expression after treatment with gemini-DNA formulations in the skin was 3-5-fold higher compared to the treatment with naked DNA (p<0.05), and 4-6-fold higher than the Dc-chol-DNA complex, indicating a significant advance in topical DNA delivery across intact skin in vivo.     

Structure-activity investigation on the gene transfection properties of cardiolipin mimicking gemini lipid analogues

A structure-activity relationship has been explored on the gene transfection efficiencies of cardiolipin mimicking gemini lipid analogues upon variation of length and hydrophilicity of the spacer between the cationic ammonium headgroups and lipid hydrocarbon chain lengths. All the gemini lipids were found to be highly superior in gene transfer abilities as compared to their monomeric lipid and a related commercially available formulation. Pseudoglyceryl gemini lipids bearing an oxyethylene (-CH2-(CH2-O-CH2)m-CH2-) spacer were found to be superior gene transfecting agents as compared to those bearing polymethylene (-CH2)m-) spacers. The major characteristic feature of the present set of gemini lipids is their serum compatibility, which is most often the major hurdle in liposome-mediated gene delivery.     

Gene transfection efficacies of novel cationic gemini lipids possessing aromatic backbone and oxyethylene spacers

Six novel gemini cationic lipids based on aromatic backbone, bearing n-C14H 29 or n-C16H33 hydrocarbon chains, differing in the length of oxyethylene type spacers -CH2-(CH2-O-CH2)m-CH2- between each ammonium headgroups have been synthesized, where m varies from 1 to 3. Each of these lipids formed stable suspensions in aqueous media. Cationic liposomes were prepared from each of these lipids individually and as mixtures of each cationic lipid and DOPE. These were used as nonviral gene delivery agents. Transfection studies showed that among lipids bearing n-C14H29 chains, the transfection efficacies decreased with the increase in the length of the spacer, whereas in case of lipids bearing n-C 16H33 chains, the transfection efficacies increased with the increase in the length of the spacer. Lipid bearing n-C16H33 hydrocarbon chains with a [-(CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2)-] spacer was found to be a potent gene transfer agent and its transfection was highly serum compatible even in the presence of 50% serum conditions.     

Interaction of oligonucleotides with cationic lipids: the relationship between electrostatics, hydration and state of aggregation

Lipoplexes, which are spontaneously formed complexes between oligonucleotide (ODN) and cationic lipid, can be used to deliver ODNs into cells, both in vitro and in vivo. The present study was aimed at characterizing the interactions associated with the formation of lipoplexes, specifically in terms of electrostatics, hydration and particle size. Large unilamellar vesicles (approximately 100 nm diameter), composed of either DOTAP, DOTAP/cholesterol (mole ratio 1:1) or DOTAP/DOPE (mole ratio 1:1) were employed as a model of cationic liposomes. Neutral vesicles ( approximately 100 nm diameter), composed of DOPC/DOPE (mole ratio 1:1), were employed as control liposomes. After ODN addition to vesicles, at different mole ratios, changes in pH and electrical surface potential at the lipid-water interface were analyzed by using the fluorophore heptadecyl-7-hydroxycoumarin. In separate 'mirror image' experiments, liposomes were added at different mole ratios to fluorescein isothiocyanate-labeled ODNs, thus yielding data about changes in the pH near the ODN molecules induced by the complexation with the cationic lipid. Particle size distribution and turbidity fluctuations were analyzed by the use of photon correlation spectroscopy and static light-scattering, respectively. In additional fluorescent probe studies, TMADPH was used to quantify membrane defects while laurdan was used to measure the level of hydration at the water-lipid interface. The results indicate that mutual neutralization of cationic lipids by ODNs and vice versa is a spontaneous reaction and that this neutralization is the main driving force for lipoplex generation. When lipid neutralization is partial, induced membrane defects cause the lipoplexes to exhibit increased size instability.     

Structural and transfection properties of amine-substituted gemini surfactant-based nanoparticles

BACKGROUND: Increases in DNA transfection efficiencies for non-viral vectors can be achieved through rational design of novel cationic building blocks. Based on previous results examining DNA condensation by polyamines, novel gemini surfactants have been designed that incorporate aza or imino substituents within the spacer group in order to increase interactions with DNA and potentially improve their DNA transfection ability. METHODS: Transfection efficiencies and cell toxicity of gemini nanoparticles constructed from plasmid DNA, gemini surfactant, and a neutral lipid were measured in COS7 cells using a luciferase assay. Structural properties of nanoparticles were examined by using circular dichroism, particle size, zeta potential, and small-angle X-ray scattering (SAXS) measurements. RESULTS: The incorporation of aza and imino substituents within the spacer group was observed to enhance the transfection ability of gemini surfactants. Incorporation of an imino group in the structure of the 1,9-bis(dodecyl)-1,1,9,9-tetramethyl-5-imino-1,9-nonanediammonium dibromide surfactant (12-7NH-12) resulted in a statistically significant (p < 0.01) 9-fold increase in transfection compared to an unsubstituted gemini surfactant and a 3-fold increase compared to the corresponding aza-substituted compound. A pH-dependent transition in size and zeta potential was observed to occur at pH 5.5 for complexes formed from the 12-7NH-12 compound. SAXS results show weakly ordered structures and the presence of multiple phases. CONCLUSIONS: The incorporation of a pH-active imino group within the spacer of the gemini surfactant results in a significant increase in transfection efficiency that can be related to both pH-induced changes in nanoparticle structure and the formation of multiple phases that more readily allow for membrane fusion that may facilitate DNA release.     

Plasmid DNA-encapsulating liposomes: Effect of a spacer between the cationic head group and hydrophobic moieties of the lipids on gene expression efficiency

We have synthesized a series of cationic amino acid-based lipids having a spacer between the cationic head group and hydrophobic moieties and examined the influence of the spacer on a liposome gene delivery system. As a comparable spacer, a hydrophobic spacer with a hydrocarbon chain composed of 0, 3, 5, 7, or 11 carbons, and a hydrophilic spacer with an oxyethylene chain (10 carbon and 3 oxygen molecules) were investigated. Plasmid DNA (pDNA)-encapsulating liposomes were prepared by mixing an ethanol solution of the lipids with an aqueous solution of pDNA. The zeta potentials and cellular uptake efficiency of the cationic liposomes containing each synthetic lipid were almost equivalent. However, the cationic lipids with the hydrophobic spacer were subject to fuse with biomembrane-mimicking liposomes. 1,5-Dihexadecyl-N-lysyl-N-heptyl-l-glutamate, having a seven carbon atom spacer, exhibited the highest fusogenic potential among the synthetic lipids. Increased fusion potential correlated with enhanced gene expression efficiency. By contrast, an oxyethylene chain spacer showed low gene expression efficiency. We conclude that a hydrophobic spacer between the cationic head group and hydrophobic moieties is a key component for improving pDNA delivery.     

Hydration of lipoplexes commonly used in gene delivery: follow-up by laurdan fluorescence changes and quantification by differential scanning calorimetry.

Lipoplexes, which are formed spontaneously between cationic liposomes and negatively charged nucleic acids, are commonly used for gene and oligonucleotide delivery in vitro and in vivo. Being assemblies, lipoplexes can be characterized by various physicochemical parameters, including size distribution, shape, physical state (lamellar, hexagonal type II and/or other phases), sign and magnitude of electrical surface potential, and level of hydration at the lipid-DNA interface. Only after all these variables will be characterized for lipoplexes with a broad spectrum of lipid compositions and DNA/cationic lipid (L(+)) mole (or charge) ratios can their relevance to transfection efficiency be understood. Of all these physicochemical parameters, hydration is the most neglected, and therefore the focus of this study. Cationic liposomes composed of DOTAP without and with helper lipids (DOPC, DOPE, or cholesterol) or of DC-Chol/DOPE were complexed with pDNA (S16 human growth hormone) at various DNA(-)/L(+) charge ratios (0.1-3.2). (DOTAP=N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride; DC-Chol=(3beta-[N-(N',N'-dimethylaminoethane)-carbamoyl]-cholester ol; DOPC=1, 2-dioleoyl-sn-glycero-3-phosphocholine; DOPE=1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine). The hydration levels of the different cationic liposomes and the DNA separately are compared with the hydration levels of the lipoplexes. Two independent approaches were applied to study hydration. First, we used a semi-quantitative approach of determining changes in the 'generalized polarization' (GP) of laurdan (6-dodecanoyl-2-dimethylaminonaphthalene). This method was recently used extensively and successfully to characterize changes of hydration at lipid-water interfaces. Laurdan excitation GP at 340 nm (GP(340)DOTAP. The GP(340) of lipoplexes of all lipid compositions (except those based on DC-Chol/DOPE) was higher than the GP(340) of the cationic liposomes alone and increased with increasing DNA(-)/L(+) charge ratio, reaching a plateau at a charge ratio of 1. 0, suggesting an increase in dehydration at the lipid-water interface with increasing DNA(-)/L(+) charge ratio. Confirmation was obtained from the second method, differential scanning calorimetry (DSC). DOTAP/DOPE lipoplexes with charge ratio 0.44 had 16.5% dehydration and with charge ratio 1.5, 46.4% dehydration. For DOTAP/Chol lipoplexes with these charge ratios, there was 17.9% and 49% dehydration, respectively. These data are in good agreement with the laurdan data described above. They suggest that the dehydration occurs during lipoplex formation and that this is a prerequisite for the intimate contact between cationic lipids and DNA.     

Infrared spectroscopic characterization of the interaction of cationic lipids with plasmid DNA

Fourier transform infrared spectroscopy was used to characterize the interaction of the cationic lipids 1,2-dioleoyl-3-trimethylammonium-propane and dioctadecyldimethylammonium bromide with plasmid DNA. The effect of incorporating the neutral colipids cholesterol and dioleoylphosphatidylethanolamine on this interaction was also examined. Additionally, dynamic and phase analysis light scattering were used to monitor the size and zeta potential of the resulting complexes under conditions similar to the Fourier transform infrared measurements. Results suggest that upon interaction of cationic lipids with DNA, the DNA remains in the B form. Distinct changes in the frequency of several infrared bands arising from the DNA bases, however, suggest perturbation of their hydration upon interaction with cationic lipids. A direct interaction of the lipid ammonium headgroup with and dehydration of the DNA phosphate is observed when DNA is complexed with these lipids. Changes in the apolar regions of the lipid bilayer are minimal, whereas the interfacial regions of the membrane show changes in hydration or molecular packing. Incorporation of helper lipids into the cationic membranes results in increased conformational disorder of the apolar region and further dehydration of the interfacial region. Changes in the hydration of the DNA bases were also observed as the molar ratio of helper lipid in the membranes was increased.     

A multi-step lipid mixing assay to model structural changes in cationic lipoplexes used for in vitro transfection.

Formation of liposome/polynucleotide complexes (lipoplexes) involves electrostatic interactions, which induce changes in liposome structure. The ability of these complexes to transfer DNA into cells is dependent on the physicochemical attributes of the complexes, therefore characterization of binding-induced changes in liposomes is critical for the development of lipid-based DNA delivery systems. To clarify the apparent lack of correlation between membrane fusion and in vitro transfection previously observed, we performed a multi-step lipid mixing assay to model the sequential steps involved in transfection. The roles of anion charge density, charge ratio and presence of salt on lipid mixing and liposome aggregation were investigated. The resonance-energy transfer method was used to monitor lipid mixing as cationic liposomes (DODAC/DOPE and DODAC/DOPC; 1:1 mole ratio) were combined with plasmid, oligonucleotides or Na(2)HPO(4). Cryo-transmission electron microscopy was performed to assess morphology. As plasmid or oligonucleotide concentration increased, lipid mixing and aggregation increased, but with Na(2)HPO(4) only aggregation occurred. NaCl (150 mM) reduced the extent of lipid mixing. Transfection studies suggest that the presence of salt during complexation had minimal effects on in vitro transfection. These data give new information about the effects of polynucleotide binding to cationic liposomes, illustrating the complicated nature of anion induced changes in liposome morphology and membrane behavior.     

Conformational transition of DNA induced by cationic lipid vesicle in acidic solution: spectroscopy investigation

The conformational transition of DNA induced by the interaction between DNA and a cationic lipid vesicle, didodecyldimethylammonium bromide (DDAB), had been investigated by circular dichroism (CD) and UV spectroscopy methods. We used singular value decomposition least squares method (SVDLS) to analyze the experimental CD spectra. Although pH value influenced the conformation of DNA in solution, the results showed that upon binding to double helical DNA, positively charged liposomes induced a conformational transition of DNA molecules from the native B-form to more compact conformations. At the same time, no obvious conformational changes occurred at single-strand DNA (ssDNA). While the cationic lipid vesicles and double-strand DNA (dsDNA) were mixed at a high molar ratio of DDAB vesicles to dsDNA, the conformation of dsDNA transformed from the B-form to the C-form resulting in an increase in duplex stability (DeltaT(m)=8+/-0.4 degrees C). An increasing in T(m) was also observed while the cationic lipid vesicles interacted with ssDNA.     

High transfection efficiency and low toxicity cationic lipids with aminoglycerol–diamine conjugate

The solid phase synthesis of a library of aminoglycerol–diamine conjugate-based transfection agents having urea linkage between diverse length of diamines and various lengths of hydrophobic tails is described. These compounds were characterized and structure–activity relationships were determined for DNA binding and transfection ability when formulated as cationic liposomes. Cationic lipids with short spacer length and short hydrophobic tails bound to DNA and delivered DNA into HEK293 cells more efficient than those with longer ones. Transfection efficiency of some of the cationic liposomes was superior to that of the commercial transfection agents, Effectene^TM_, DOTAP and DC-Chol. The lipids 6Ab and 6Bb did not require the helper lipid DOPE to produce high-efficiency transfection of human cells while displaying minimal cytotoxicity. This suggests that these newly described aminoglycerol-based lipids should be very promising in liposome-mediated gene delivery and illustrate the potential of solid phase synthesis method for non-viral vector discovery.     

Interaction of cationic liposomes and their DNA complexes with monocytic leukemia cells

Cationic liposomes complexed with DNA have been used extensively as non-viral vectors for the intracellular delivery of reporter or therapeutic genes in culture and in vivo. We examined the relationship between the characteristics of the lipoplexes, their mode of interaction with monocytic THP-1 cells and their ability to transfect these cells. We determined the size and zeta potential of cationic liposomes (composed of 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP) and its mixtures with neutral lipids), and lipoplexes at different (+/-) charge ratios. As the (+/-) charge ratio of the lipoplexes decreased to (1/1), a significant reduction in zeta potential and an increase in size was observed. The increase in size resulted from fusion between liposomes promoted by DNA, as demonstrated by a lipid mixing assay, and from aggregation of the complexes. Interaction of liposomes and lipoplexes with THP-1 cells was assessed by monitoring lipid mixing ('fusion') as well as binding and cell association. While no lipid mixing was observed with the 1/2 (+/-) lipid/DNA complexes, lipoplexes with higher (+/-) charge ratios underwent significant fusion in conjunction with extensive cell binding. Liposome binding to cells was dependent on the positive charge of the liposomes, and their fusion could be modulated by the co-lipid. DOTAP/phosphatidylethanolamine (1:1) liposomes fused with THP-1 cells, unlike DOTAP/phosphatidylcholine (1:1) liposomes, although both liposome types bound to the cells to a similar extent. The use of inhibitors of endocytosis indicated that fusion of the cationic liposomes with cells occurred mainly at the plasma membrane level. The presence of serum increased the size of the cationic liposomes, but not that of the lipoplexes. Low concentrations of serum (3%) completely inhibited the fusion of cationic liposomes with cells, while inhibiting binding by only 20%. Our results suggest that binding of cationic liposomes and lipoplexes to cells is governed primarily by electrostatic interactions, whereas their fusion is regulated by the lipid composition and sterically favorable interactions with cell surface molecules. In addition our results indicate no correlation between fusion of the lipoplexes with the plasma membrane and the levels of transfection.     

Membrane fusogenic lysine type lipid assemblies possess enhanced NLRP3 inflammasome activation potency

Lysine (K) type cationic lipid with a propyl spacer and ditetradecyl hydrophobic moieties composing liposomes, K3C14, previously studied for gene delivery, were reported to activate the NLRP3 inflammasomes in human macrophages via the conventional phagolysosomal pathway. In this study, K3C16, a propyl spacer bearing lysine type lipids with dihexadecyl moieties (an extension of two hydrocarbon tail length) were compared with K3C14 as liposomes. Such a small change in tail length did not alter the physical properties such as size distribution, zeta potential and polydispersity index (PDI). The NLRP3 activation potency of K3C16 was shown to be 1.5-fold higher. Yet, the toxicity was minimal, whereas K3C14 has shown to cause significant cell death after 24 h incubation. Even in the presence of endocytosis inhibitors, cytochalasin D or dynasore, K3C16 continued to activate the NLRP3 inflammasomes and to induce IL-1β release. To our surprise, K3C16 liposomes were confirmed to fuse with the plasma membrane of human macrophages and CHO-K1 cells. It is demonstrated that the change in hydrophobic tail length by two hydrocarbons drastically changed a cellular entry route and potency in activating the NLRP3 inflammasomes.     

Synthesis and optimization of cholesterol-based diquaternary ammonium Gemini Surfactant (Chol-GS) as a new gene delivery vector

Amongst a number of potential nonviral vectors, cationic liposomes have been actively researched, with both gemini surfactants and bola amphiphiles reported as being in possession of good structures in terms of cell viability and in vitro transfection. In this study, a cholesterol-based diquaternary ammonium gemini surfactant (Chol-GS) was synthesized and assessed as a novel nonviral gene vector. Chol-GS was synthesized from cholesterol by way of four reaction steps. The optimal efficiency was found to be at a weight ratio of 1:4 of lipid:DOPE (1,2-dioleoyl-L-alpha- glycero-3-phosphatidylethanolamine), and at a ratio of between 10:1~15:1 of liposome:DNA. The transfection efficiency was compared with commercial liposomes and with Lipofectamine, 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE-C), and N-[1-(2,3-dioleoyloxy)propyl]- N,N,N-trimethylammonium chloride (DOTAP). The results indicate that the efficiency of Chol-GS is greater than that of all the tested commercial liposomes in COS7 and Huh7 cells, and higher than DOTAP and Lipofectamine in A549 cells. Confirmation of these findings was observed through the use of green fluorescent protein expression. Chol-GS exhibited a moderate level of cytotoxicity, at optimum concentrations for efficient transfection, indicating cell viability. Hence, the newly synthesized Chol-GS liposome has the potential of being an excellent nonviral vector for gene delivery.     

Surface charge density determines the efficiency of cationic gemini surfactant based lipofection

The efficiencies of the binary liposomes composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine and cationic gemini surfactant, (2S,3R)-2,3-dimethoxy-1,4-bis(N-hexadecyl-N,N-dimethylammonium)butane dibromide as transfection vectors, were measured using the enhanced green fluorescent protein coding plasmid and COS-1 cells. Strong correlation between the transfection efficiency and lipid stoichiometry was observed. Accordingly, liposomes with X(SR-1) > or = 0.50 conveyed the enhanced green fluorescent protein coding plasmid effectively into cells. The condensation of DNA by liposomes with X(SR-1) > 0.50 was indicated by static light scattering and ethidium bromide intercalation assay, whereas differential scanning calorimetry and fluorescence anisotropy of diphenylhexatriene revealed stoichiometry dependent reorganization in the headgroup region of the liposome bilayer, in alignment with our previous Langmuir-balance study. Surface charge density and the organization of positive charges appear to determine the mode of interaction of DNA with (2S,3R)-2,3-dimethoxy-1,4-bis(N-hexadecyl-N,N-dimethylammonium)butane dibromide/1,2-dimyristoyl-sn-glycero-3-phosphocholine liposomes, only resulting in DNA condensation when X(SR-1) > 0.50. Condensation of DNA in turn seems to be required for efficient transfection.     

Microencapsulation of nanoparticulate complexes of DNA with cationic lipids and polymers in pectin beads for targeted gene delivery

Nanoparticulate complexes of plasmid DNA (pDNA) with cationic liposomes/polymer, of approx 200 nm diameter, were encapsulated with a high degree of efficiency within calcium pectinate gel beads. Electron microscopy showed the DNA nanocomplexes to be evenly distributed throughout the gel matrix. Controlled release of pDNA-lipid nanocomplexes was achieved by the action of pectinase enzymes, whereas release of naked and polymer-complexed DNA was found to be more greatly influenced by the swelling behavior of the polysaccharide matrices in buffer alone. Physical degradation of pDNA within pectin beads was found to be accelerated during bead drying, most probably as a result of shear forces generated within the gel matrices by the evaporation of water. Plasmid complexation with cationic liposomes provided a greater degree of protection for the DNA during bead drying than complexation with cationic polymer, and was shown to successfully transfect cultured cells after release from the beads, via the action of pectinase. Observations concerning the physical stability of nanocomplexed pDNA, and its encapsulation within and release from pectin gel beads, are discussed with reference to the electrostatic interactions existing between the various components.