Surface-modified and internally cationic polyamidoamine dendrimers for efficient siRNA delivery

A novel internally quaternized and surface-acetylated poly(amidoamine) generation four dendrimer (QPAMAM-NHAc) was synthesized and evaluated for intracellular delivery of siRNA. The proposed dendrimer as a nanocarrier possesses the following advantages: (1) modified neutral surface of the dendrimer for low cytotoxicity and enhanced cellular internalization; (2) existence of cationic charges inside the dendrimer (not on the outer surface) resulting in highly organized compact nanoparticles, which can potentially protect nucleic acids from degradation. The properties of this dendrimer were compared with PAMAM-NH 2 dendrimer, possessing surface charges, and with an internally quaternized charged and hydroxyl-terminated QPAMAM-OH dendrimer. Atomic force microscopy studies revealed that internally charged and surface neutral dendrimers, QPAMAM-OH and QPAMAM-NHAc, formed well-condensed, spherical particles (polyplexes) with siRNA, while PAMAM-NH 2 resulted in the formation of nanofibers. The modification of surface amine groups to amide significantly reduced cytotoxicity of dendrimers with QPAMAM-NHAc dendrimer showing the lowest toxicity. Confocal microscopy demonstrated enhanced cellular uptake and homogeneous intracellular distribution of siRNA delivered by the proposed QPAMAM-NHAc nanocarrier. The results clearly demonstrated distinct advantages of developed QPAMAM-NHAc/siRNA polyplexes over the existing nucleic acid dendrimeric carriers.     

Endosomal escape of polymeric gene delivery complexes is not always enhanced by polymers buffering at low pH

One of the crucial steps in gene delivery with cationic polymers is the escape of the polymer/DNA complexes ("polyplexes") from the endosome. A possible way to enhance endosomal escape is the use of cationic polymers with a pKa around or slightly below physiological pH ("proton sponge"). We synthesized a new polymer with two tertiary amine groups in each monomeric unit [poly(2-methyl-acrylic acid 2-[(2-(dimethylamino)-ethyl)-methyl-amino]-ethyl ester), abbreviated as pDAMA]. One pKa of the monomer is approximately 9, providing cationic charge at physiological pH, and thus DNA binding properties, the other is approximately 5 and provides endosomal buffering capacity. Using dynamic light scattering and zeta potential measurements, it was shown that pDAMA is able to condense DNA in small particles with a surface charge depending on the polymer/DNA ratio. pDAMA has a substantial lower toxicity than other polymeric transfectants, but in vitro, the transfection activity of the pDAMA-based polyplexes was very low. The addition of a membrane disruptive peptide to pDAMA-based polyplexes considerably increased the transfection efficiency without adversely affecting the cytotoxicity of the system. This indicates that the pDAMA-based polyplexes alone are not able to mediate escape from the endosomes via the proton sponge mechanism. Our observations imply that the proton sponge hypothesis is not generally applicable for polymers with buffering capacity at low pH and gives rise to a reconsideration of this hypothesis.     

DNA compaction into new DNA vectors based on cyclodextrin polymer: surface enhanced Raman spectroscopy characterization

The ability of DNA to bind polycation yielding polyplexes is widely used in nonviral gene delivery. The aim of the present study was to evaluate the DNA compaction with a new DNA vector using Raman spectroscopy. The polyplexes result from an association of a beta-cyclodextrin polymer (polybeta-CD), an amphiphilic cationic connector (DC-Chol or adamantane derivative Ada2), and DNA. The charge of the polymeric vector is effectively controlled by simple addition of cationic connector in the medium. We used surface enhanced Raman spectroscopy (SERS) to characterize this ternary complex, monitoring the accessibility of adenyl residues to silver colloids. The first experiments were performed using model systems based on polyA (polyadenosine monophosphate) well characterized by SERS. This model was then extended to plasmid DNA to study polybeta-CD/Ada2/DNA and polybeta-CD/DC-Chol/DNA polyplexes. The SERS spectra show a decrease of signal intensity when the vector/DNA charge ratio (Z+/-) increases. At the highest ratio (Z+/- = 10) the signal is 6-fold and 3-fold less intense than the DNA reference signal for Ada2 and DC-Chol polyplexes, respectively. Thus adenyl residues have a reduced accessibility as DNA is bound to the vector. Moreover, the SERS intensity variations are in agreement with gel electrophoresis and zeta potential experiments on the same systems. The overall study clearly demonstrates that the cationic charges neutralizing the negative charges of DNA result in the formation of stable polyplexes. In vitro transfection efficiency of those DNA vectors are also presented and compared to the classical DC-Chol lipoplexes (DC-Chol/DNA). The results show an increase of the transfection efficiency 2-fold higher with our vector based on polybeta-CD.     

Structure-activity examination of poly(glycoamidoguanidine)s: glycopolycations containing guanidine units for nucleic acid delivery

In this study we synthesized a new series of polymers known as poly(glycoamidoguanidine)s (PGAGs). These new polymer structures were synthesized by copolymerizing a carbohydrate monomer (diester; galatarate or tartarate) with a diamine incorporating guanidine or methylguanidine as a charge center to create a polyamide backbone. These materials were strategically designed and compared to our previously studied DNA delivery vehicles, poly(glycoamidoamine)s (PGAAs), which contain secondary amines as the charge groups along the polymer backbone to examine the effect of charge center type on the cellular delivery efficiency of plasmid DNA (pDNA). The guanidine moieties within the PGAGs facilitate electrostatic binding with the negatively charged phosphate backbone of plasmid DNA (pDNA). Stable polymer-pDNA complexes (polyplexes) with sizes in the range of 60-200 nm are formed at polymer/pDNA charge ratios (N/P) of 5 and above. When the PGAGs are complexed with Cy5-labeled pDNA (Cy5-pDNA) at N/P ratios of 10 and 25, between 80 and 95% of HeLa cells were positive for Cy5 fluorescence, indicating effective cellular internalization of the polyplexes. The toxicity of both PGAA and PGAG polyplexes was studied via MTT assays, and over 95% cell survival was observed at N/P ratios of 5, 10, 15, 20, 25, and 30 in HeLa cells. Transgene expression was examined via luciferase assays at various N/P ratios in the absence and presence of serum. In the absence of serum, the PGAG polyplexes revealed similar transgene expression when compared to polyplexes formed with their analogous PGAA structures. In the presence of serum, one analog (Gg) consisting of galactarate copolymerized with the guanidine monomer yielded gene expression similar to the positive control, Glycofect Transfection Reagent. This new series of guanidine-containing oligomers are promising as a new design strategy to incorporate an alternative charge center type within the backbone of glycopolymer-based nucleic acid delivery vehicles.     

The transfection of Jurkat T-leukemic cells by use of pH-sensitive immunoliposomes

An effective pH-sensitive gene transfer vector has been developed utilising anionic liposomes with various formulations as a carrier of plasmid DNA (pEGlacZ, 7.6 kb) to transfect CD3 T+ lymphocytes (Jurkat cells). The plasmid DNA was condensed using poly-l-lysines with a range of molecular masses to form polyplexes that were interacted with several anionic liposome formulations to form lipopolyplexes. For targeting to the Jurkat cells, distearoylphosphatidylethanolamine (DSPE) linked to poly (ethylene glycol) molecular mass 2000 and coupled to anti-CD3 antibody was incorporated in the liposomes. The polyplexes and lipopolyplexes were characterised in terms of size, zeta potential, gel electrophoresis and electron microscopy. The gene transfer activity of the lipopolyplexes, assessed from beta-galactosidase expression, depended on the charge ratio (NH(3)+/PO(4)-) of the component polyplex and the lipid/DNA weight ratio of the lipopolyplex.     

Deposition gene transfection using bioconjugates of DNA and thermoresponsive cationic homopolymer

A poly(N,N-dimethylaminoethylmethacrylate) (PDMAEMA) homopolymer with both thermoresponsive and cationic characteristics was applied to a vector for use in deposition transfection. PDMAEMA with a molecular weight of 2.5 × 10(5) g mol(-1) was synthesized by photoinduced radical polymerization. Polyplexes approximately 750 nm in size were formed by mixing PDMAEMA with luciferase-encoding plasmid DNA. The polyplexes had a lower critical solution temperature (LCST) of approximately 30 °C. In addition, they exhibited excellent adsorption and durability on a polystyrene surface, as confirmed by a surface chemical compositional analysis. When HeLa cells and primary cells were cultured on a substrate coated with the polyplexes, high transgene expression and cell viability of more than 90% were obtained at low charge ratios (PDMAEMA/plasmid DNA ratio) ranging from 2 to 8. In addition, transgene expression was sustained for over 2 weeks post-transfection whereas decreased expression was observed 5 days post-transfection when the conventional solution-mediated transfection method was used. Thus, high and sustained transgene expression as well as high cell viability can be realized by using small amounts of PDMAEMA as a deposition transfection material.     

Influence of chitosan structure on the formation and stability of DNA-chitosan polyelectrolyte complexes

The interactions between DNA and chitosans varying in fractional content of acetylated units (FA), degree of polymerization (DP), and degree of ionization were investigated by several techniques, including an ethidium bromide (EtBr) fluorescence assay, gel retardation, atomic force microscopy, and dynamic and electrophoretic light scattering. The charge density of the chitosan and the number of charges per chain were found to be the dominating factors for the structure and stability of DNA-chitosan complexes. All high molecular weight chitosans condensed DNA into physically stable polyplexes; however, the properties of the complexes were strongly dependent on FA, and thereby the charge density of chitosan. By employing fully charged oligomers of constant charge density, it was shown that the complexation of DNA and stability of the polyplexes is governed by the number of cationic residues per chain. A minimum of 6-9 positive charges appeared necessary to provide interaction strength comparable to that of polycations. In contrast, further increase in the number of charges above 9 did not increase the apparent binding affinity as judged from the EtBr displacement assay. The chitosan oligomers exhibited a pH-dependent interaction with DNA, reflecting the number of ionized amino groups. The complexation of DNA and the stability of oligomer-based polyplexes became reduced above pH 7.4. Such pH-dependent dissociation of polyplexes around the physiological pH is highly relevant in gene delivery applications and might be one of the reasons for the high transfection activity of oligomer-based polyplexes observed.     

Gene transfection of Toxoplasma gondii using PEI/DNA polyplexes

The purpose of this study was to explore the potential of using cationic polyethylenimine (PEI) to deliver green fluorescent protein (GFP) to protozoan parasite Toxoplasma gondii. PEI/DNA polyplexes were formed using branched PEI and pEGFP-N1 plasmid with various N/P ratios that ranged from 5 to 50. With the increment of N/P ratio, the average size of formed PEI/DNA polyplexes determined by dynamic light scattering analysis decreased from 306 to 203nm, while the surface charge of polyplexes obtained by zeta potential measurements increased from 20.2 to 36.7mV. Gene transfection efficiency modulated by N/P ratio was determined, indicating PEI/DNA polyplexes were capable of transfecting parasites. The maximal GFP expression was observed 8h post-transfection using N/P ratio of 30. To demonstrate the infectivity and potential use of GFP-expressing T. gondii, transfected parasites were inoculated to the monolayer of human foreskin fibroblast (HFF) cells. GFP-expressing tachyzoites were observed in intracellular milieu of the infected HFF cells one day after the infection. After 12-day culture, the bradyzoites expressing GFP within cysts were clearly visualized extracellularly. Our results revealed that PEI can be harnessed as an effective and inexpensive reagent to construct GFP-expressing T. gondii which has potential uses such as the study of interconversion stages and antimicrobial drug screening.     

Reversibly shielded DNA polyplexes based on bioreducible PDMAEMA-SS-PEG-SS-PDMAEMA triblock copolymers mediate markedly enhanced nonviral gene transfection

Reversibly shielded DNA polyplexes based on bioreducible poly(dimethylaminoethyl methacrylate)-SS-poly(ethylene glycol)-SS-poly(dimethylaminoethyl methacrylate) (PDMAEMA-SS-PEG-SS-PDMAEMA) triblock copolymers were designed, prepared and investigated for in vitro gene transfection. Two PDMAEMA-SS-PEG-SS-PDMAEMA copolymers with controlled compositions, 6.6-6-6.6 and 13-6-13 kDa, were obtained by reversible addition-fragmentation chain transfer (RAFT) polymerization of dimethylaminoethyl methacrylate (DMAEMA) using CPADN-SS-PEG-SS-CPADN (CPADN: 4-cyanopentanoic acid dithionaphthalenoate; PEG: 6 kDa) as a macro-RAFT agent. Like their nonreducible PDMAEMA-PEG-PDMAEMA analogues, PDMAEMA-SS-PEG-SS-PDMAEMA triblock copolymers could effectively condense DNA into small particles with average diameters less than 120 nm and close to neutral zeta potentials (0 ～ +6 mV) at and above an N/P ratio of 3/1. The resulting polyplexes showed excellent colloidal stability against 150 mM NaCl, which contrasts with polyplexes of 20 kDa PDMAEMA homopolymer. In the presence of 10 mM dithiothreitol (DTT), however, polyplexes of PDMAEMA-SS-PEG-SS-PDMAEMA were rapidly deshielded and unpacked, as revealed by significant increase of positive surface charges as well as increase of particle sizes to over 1000 nm. Release of DNA in response to 10 mM DTT was further confirmed by gel retardation assays. These polyplexes, either stably or reversibly shielded, revealed a low cytotoxicity (over 80% cell viability) at and below an N/P ratio of 12/1. Notably, in vitro transfection studies showed that reversibly shielded polyplexes afforded up to 28 times higher transfection efficacy as compared to stably shielded control under otherwise the same conditions. Confocal laser scanning microscope (CLSM) studies revealed that reversibly shielded polyplexes efficiently delivered and released pDNA into the perinuclei region as well as nuclei of COS-7 cells. Hence, reduction-sensitive reversibly shielded DNA polyplexes based on PDMAEMA-SS-PEG-SS-PDMAEMA are highly promising for nonviral gene transfection.     

Layer-by-layer deposition of oppositely charged polyelectrolytes on the surface of condensed DNA particles.

DNA can be condensed with an excess of poly-cations in aqueous solutions forming stable particles of submicron size with positive surface charge. This charge surplus can be used to deposit alternating layers of polyanions and polycations on the surface surrounding the core of condensed DNA. Using poly-L-lysine (PLL) and succinylated PLL (SPLL) as polycation and polyanion, respectively, we demonstrated layer-by-layer architecture of the particles. Polyanions with a shorter carboxyl/backbone distance tend to disassemble binary DNA/PLL complexes by displacing DNA while polyanions with a longer carboxyl/backbone distance effectively formed a tertiary complex. The zeta potential of such complexes became negative, indicating effective surface recharging. The charge stoichiometry of the DNA/PLL/SPLL complex was found to be close to 1:1:1, resembling poly-electrolyte complexes layered on macrosurfaces. Recharged particles containing condensed plasmid DNA may find applications as non-viral gene delivery vectors.     

Nanostructure of polyplexes formed between cationic diblock copolymer and antisense oligodeoxynucleotide and its influence on cell transfection efficiency

Although various cationic polymers have been used to condense anionically charged DNA to improve their transfection efficiency, there is still a lack of fundamental understanding about how to control the nanostructure and charge of the polyplexes formed and how to relate such information to cell transfection efficiency. In this work, we have synthesized a weak cationic and phosphorylcholine-containing diblock copolymer and used it as a model vector to deliver an antisense oligodeoxynucleotide (ODN) into HeLa cells. Small angle neutron scattering (SANS) was used to determine the copolymer/ODN polyplex structure. The SANS data revealed the formation of polyplex nanocylinders at high copolymer (N)/ODN (P) charge ratios, where N symbolizes the amine groups on the copolymer and P symbolizes the phosphate groups. However, the cylindrical lengths remained constant, indicating that the ODN binding over this region did not alter the cylindrical shape of the copolymer in solution. As the N/P ratio decreased and became close to unity the polyplex diameters remained constant, but their lengths increased substantially, suggesting the end-to-end bridging by ODN binding between copolymer cylinders. As the N/P ratios went below unity (with ODN in excess), the polyplex diameters increased substantially, indicating different ODN bridging to bundle the small polyplexes together. Transfection studies from HeLa cells indicated a steady increase in transfection efficiency with increasing cationic charge and decreasing polyplex size. Cell growth inhibition assay showed significant growth inhibition by the polyplexes coupled with weak cytotoxicity, indicating effective ODN delivery. While this study has confirmed the overall charge effect, it has also revealed progressive structural changes of the polyplexes against varying charge ratio, thereby providing useful insight into the mechanistic process behind the ODN delivery.     

PAMAM-PEG-PAMAM: novel triblock copolymer as a biocompatible and efficient gene delivery carrier

A novel triblock copolymer, PAMAM-block-PEG-block-PAMAM was synthesized and applied as a gene carrier. PAMAM dendrimer is proven to be an efficient gene carrier itself, but it is associated with certain problems such as low water solubility and considerable cytotoxicity. Therefore, we introduced PEG to engineer a nontoxic and highly transfection efficient polymeric gene carrier because PEG is known to convey water-solubility and biocompatibility to the conjugated copolymer. This copolymer could achieve self-assembly with plasmid DNA, forming compact nanosized particles with a narrow size distribution. Fulfilling our expectations, the copolymer was found to form highly water-soluble polyplexes with plasmid DNA, showed little cytotoxicity despite its poor degradability, and finally achieved high transfection efficiency comparable to PEI in 293 cells. Consequently, these data show that an approach involving the introduction of PEG to create a tree-like cationic copolymer possesses a great potential for use in gene delivery systems.     

Comparative biodistribution of PAMAM dendrimers and HPMA copolymers in ovarian-tumor-bearing mice

The biodistribution profile of a series of linear N-(2-hydroxylpropyl)methacrylamide (HPMA) copolymers was compared with that of branched poly(amido amine) dendrimers containing surface hydroxyl groups (PAMAM-OH) in orthotopic ovarian-tumor-bearing mice. Below an average molecular weight (MW) of 29 kDa, the HPMA copolymers were smaller than the PAMAM-OH dendrimers of comparable molecular weight. In addition to molecular weight, hydrodynamic size and polymer architecture affected the biodistribution of these constructs. Biodistribution studies were performed by dosing mice with (125)iodine-labeled polymers and collecting all major organ systems, carcass, and excreta at defined time points. Radiolabeled polymers were detected in organ systems by measuring gamma emission of the (125)iodine radiolabel. The hyperbranched PAMAM dendrimer, hydroxyl-terminated, generation 5 (G5.0-OH), was retained in the kidney over 1 week, whereas the linear HPMA copolymer of comparable molecular weight was excreted into the urine and did not show persistent renal accumulation. PAMAM dendrimer, hydroxyl-terminated, generation 6.0 (G6.0-OH), was taken up by the liver to a higher extent, whereas the HPMA copolymer of comparable molecular weight was observed to have a plasma exposure three times that of this dendrimer. Tumor accumulation and plasma exposure were correlated with the hydrodynamic sizes of the polymers. PAMAM dendrimer, hydroxyl-terminated, generation 7.0 (G7.0-OH), showed extended plasma circulation, enhanced tumor accumulation, and prolonged retention with the highest tumor/blood ratio for the polymers under study. Head-to-head comparative study of HPMA copolymers and PAMAM dendrimers can guide the rational design and development of carriers based on these systems for the delivery of bioactive and imaging agents.     

An acid-labile block copolymer of PDMAEMA and PEG as potential carrier for intelligent gene delivery systems

Intelligent gene delivery systems based on physiologically triggered reversible shielding technology have evinced enormous interest due to their potential in vivo applications. In the present work, an acid-labile block copolymer consisting of poly(ethylene glycol) and poly(2-(dimethylamino)ethyl methacrylate) segments connected through a cyclic ortho ester linkage (PEG- a-PDMAEMA) was synthesized by atom transfer radical polymerization of DMAEMA using a PEG macroinitiator with an acid-cleavable end group. PEG- a-PDMAEMA condensed with plasmid DNA formed polyplex nanoparticles with an acid-triggered reversible PEG shield. The pH-dependent shielding/deshielding effect of PEG chains on the polyplex particles were evaluated by zeta potential and size measurements. At pH 7.4, polyplexes generated from PEG- a-PDMAEMA exhibited smaller particle size, lower surface charge, reduced interaction with erythrocytes, and less cytotoxicity compared to PDMAEMA-derived polyplexes. At pH 5.0, zeta potential of polyplexes formed from PEG- a-PDMAEMA increased, leveled up after 2 h of incubation and gradual aggregation occurred in the presence of bovine serum albumin (BSA). In contrast, the stably shielded polyplexes formed by DNA and an acid-stable block copolymer, PEG- b-PDMAEMA, did not change in size and zeta potential in 6 h. In vitro transfection efficiency of the acid-labile copolymer greatly increased after 6 h incubation at pH 5.0, approaching the same level of PDMAEMA, whereas there was only slight increase in efficiency for the stable copolymer, PEG- b-PDMAEMA.     

DNA complexing with polyamidoamine dendrimers: implications for transfection.

DNA and polyamidamine (PAMAM) dendrimers form complexes on the basis of the electrostatic interactions between negatively charged phosphate groups of the nucleic acid and protonated (positively charged) amino groups of the polymers. Charge neutralization of both components and subsequent increases of the net positive charge of the complex result in changes in the physicochemistry and biological properties of the complexes. The formation of soluble, low-density and insoluble, high-density complexes was analyzed using UV light absorption and measurements of radioactive labeled DNA. Formation of high molecular weight and high-density complexes depended mainly on the DNA concentration and was enhanced by increasing the dendrimer-DNA charge ratio. Electrostatic charge related effects (attraction or repulsion of charged particles) appeared to be modulated by the generation of dendrimer (size of the polymer). With the progressive increases in the dendrimer-DNA charge ratio (above 20), an increase in the amount of low-density, soluble complexes was observed. Functional analysis revealed that the great majority (>90%) of transfection is carried by low-density, soluble, complexes which only represent approximately 10-20% of total complexed DNA. The ability of the dendrimer to complex and form aggregates with DNA is crucial for efficient transfection and the function of the complexed DNA.     

Stabilized nanocarriers for plasmids based upon cross-linked poly(ethylene imine)

Stabilized PEI/DNA polyplexes were generated by cross-linking PEI with biodegradable disulfide bonds. The reaction conversion of different PEIs with the amine reactive cross-linker dithiobis(succinimidyl propionate) (DSP) was investigated, and the molecular weight of the reaction products was identified. Light scattering and microelectrophoresis were employed to assess size and zeta potential of the resulting polyplexes. Polyplex morphology and mechanic stability were investigated using atomic force microscopy. Finally, albumin and erythrocyte interactions and stability against polyanions and high ionic strength were checked. Polyplexes of PEI and DNA were prepared by two different formulation methods, either using pre-cross-linked polymers or by cross-linking polyplexes after complexation. Only the latter method yielded small (100-300 nm) polyplexes with a positive zeta potential when HMW PEI was used, whereas cross-linked LMW PEI resulted in polyplexes with increased size (>1000 nm) and zeta potentials down to -20 mV. In addition, only cross-linking after polyplex formation was able to enhance resistance against polyanion exchange and high ionic strength. AFM images revealed no changes in the morphology of cross-linked HWM PEI polyplexes, and indentation force measurements using AFM significantly increased mechanical stability of cross-linked HMW PEI polyplexes. These polyplexes also displayed significantly reduced interactions with major blood components like albumin and erythrocytes. The resulting biocompatible particles offer a means of combining enhanced polyplex stability with redox-triggered activation for in vivo application.     

Reduction-degradable linear cationic polymers as gene carriers prepared by Cu(I)-catalyzed azide-alkyne cycloaddition

Linear reduction-degradable cationic polymers with different secondary amine densities (S2 and S3) and their nonreducible counterparts (C2 and C3) were synthesized by Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) step-growth polymerization of the dialkyne-oligoamine monomers and the diazide monomers. These polymers were studied with a goal of developing a set of new gene carriers. The buffering capacity and DNA binding ability of these polymers were evaluated by acid-base titration, gel retardation, and ethidium bromide (EB) exclusion assay. The polymers with lower amine density exhibit a weaker DNA-binding ability but a stronger buffering capacity in the range of pH 5.1 and 7.4. Particle size and zeta-potential measurements demonstrate that the polymers with higher amine density condense pDNA to form polyplexes with smaller sizes, while the disulfide bond in the backbone shows a negative effect on the condensing capability of the polymers, resulting in the formation of polyplexes with large size and nearly neutral surface. The reduction-sensitive polyplexes formed by polymer S2 or S3 can be disrupted by dithiothreitol (DTT) to release free DNA, which has been proven by the combination of gel retardation, EB exclusion assay, particles sizing, and zeta potential measurements. Cell viability measurements by MTT assay demonstrate that the reduction-degradable polymers (S2 and S3) have little cytotoxicity while the nonreducible polymers (C2 and C3) show obvious cytotoxicity, in particular, at high N/P ratios. In vitro transfection efficiencies of these polymers were evaluated using EGFP and luciferase plasmids as the reporter genes. Polymers S3 and S2 show much higher efficiencies than the nonreducible polymers C3 and C2 in the absence of 10% serum; unexpectedly, the lowest transfection efficiency has been observed for polymer S3 in the presence of serum.     

Electrostatic theory of the assembly of PAMAM dendrimers and DNA

The electrostatic interactions mediated by counterions between a cationic PAMAM dendrimer, modelized as a sphere of radius and cationic surface charge highly increasing with generation, and a DNA, modelized as an anionic elastic line, are analytically calculated in the framework of condensation theory. Under these interactions the DNA is wrapped around the sphere. For excess phosphates relative to dendrimer primary amines, the free energy of the DNA‐dendrimer complex displays an absolute minimum when the complex is weakly negatively overcharged. This overcharging opposes gene delivery. For a highly positive dendrimer and a DNA fixed by experimental conditions to a number of phosphates less than the number of dendrimer primary amines, excess amine charges, the dendrimer may at the same time bind stably DNA and interact with negative cell membranes to activate cell transfection in fair agreement with molecular simulations and experiments. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 276–286, 2016.     

Epidermal growth factor-PEG functionalized PAMAM-pentaethylenehexamine dendron for targeted gene delivery produced by click chemistry

Aim of this study was the site-specific conjugation of an epidermal growth factor (EGF)-polyethylene glycol (PEG) chain by click chemistry onto a poly(amido amine) (PAMAM) dendron, as a key step toward defined multifunctional carriers for targeted gene delivery. For this purpose, at first propargyl amine cored PAMAM dendrons with ester ends were synthesized. The chain terminal ester groups were then modified by oligoamines with different secondary amino densities. The oligoamine-modified PAMAM dendrons were well biocompatible, as demonstrated in cytotoxicity assays. Among the different oligoamine-modified dendrons, PAMAM-pentaethylenehexamine (PEHA) dendron polyplexes displayed the best gene transfer ability. Conjugation of PAMAM-PEHA dendron with PEG spacer was conducted via click reaction, which was performed before amidation with PEHA. The resultant PEG-PAMAM-PEHA copolymer was then coupled with EGF ligand. pDNA transfections in HuH-7 hepatocellular carcinoma cells showed a 10-fold higher efficiency with the polyplexes containing conjugated EGF as compared to the ligand-free ones, demonstrating the concept of ligand targeting. Overall gene transfer efficiencies, however, were moderate, suggesting that additional measures for overcoming subsequent intracellular bottlenecks in delivery have to be taken.     

Role of biophysical parameters on ex vivo and in vivo gene transfer to the airway epithelium by polyethylenimine/albumin complexes

Efficient gene transfer to the airways by nonviral vectors is a function of different parameters, among which the size and the charge of the transfecting particles. The aim of this study was to determine the transfection efficiency of polyethylenimine (PEI)/albumin polyplexes in ex vivo and in vivo models of respiratory epithelium and to correlate it with biophysical characteristics of the particles. Complexes were obtained by adding different amounts of human serum albumin (HSA) to PEI polyplexes preformed in saline. The presence of HSA caused the formation of bigger and more negative polyplexes and increased PEI transfection efficiency in primary respiratory epithelial cells by 4-6-fold. For in vivo administration to the lung, PEI polyplexes were formed in water and optimized with respect to the N/ P ratio. PEI/pC-Luc complexes gave the highest luciferase expression at N/ P 15 when administered through the trachea. At this N/ P ratio, the size and the surface charge of albumin-containing polyplexes were not different as compared with plain PEI polyplexes. Formulation of PEI polyplexes in the presence of HSA or murine serum albumin (MSA) resulted in a 2-fold increase in luciferase expression. In mice treated with PEI or PEI/MSA polyplexes containing the nuclear beta-gal gene, X-gal staining revealed that transfected cells localized at the bronchiolar epithelium and that PEI/MSA transfected four times as many cells as PEI ( p < 0.05). Finally, double administration of PEI/MSA polyplexes resulted in a further enhancement of transfection of the lung. Our data show that serum albumin enhances PEI-mediated gene transfer to airway epithelial cells in vivo, likely facilitating the uptake of polyplexes, and indicate that this formulation would fulfill the requirement of repeated administration, as necessary in chronic lung diseases like cystic fibrosis.