Stable Polyplexes Research Articles

Evaluation of Pharmacokinetics of Bioreducible Gene Delivery Vectors by Real-time PCR

To investigate pharmacokinetics of reversibly stabilized DNA nanoparticles (rSDN) using a single-step lysis RT-PCR. rSDN were prepared by coating bioreducible polycation/DNA polyplexes with multivalent N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers. Targeted polyplexes were formulated by linking cyclic RGD ligand (c(RGDyK)) to the HPMA surface layer of rSDN. The pharmacokinetic parameters in tumor-bearing mice were analyzed by PKAnalyst. The pharmacokinetics of naked plasmid DNA, simple DNA polyplexes, rSDN, and RGD-targeted rSDN exhibited two-compartment model characteristics with area under the blood concentration-time curve (AUC) increasing from 1,102 ng x ml(-1) x min(-1) for DNA to 3,501 ng x ml(-1) x min(-1) for rSDN. Non-compartment model analysis revealed increase in mean retention time (MRT) from 4.5 min for naked DNA to 22.9 min for rSDN. RT-PCR is a sensitive and convenient method suitable for analyzing pharmacokinetics and biodistribution of DNA polyplexes. Surface stabilization of DNA polyplexes can significantly extend their MRT and AUC compared to naked DNA. DNA degradation in rSDN in blood circulation, due to a combined effect of disulfide reduction and competitive reactions with charged molecules in the blood, contributes to DNA elimination.

Comparison between arginine conjugated PAMAM dendrimers with structural diversity for gene delivery systems

Mono- and di-arginine conjugated PAMAM dendrimers (G = 3 or 4) were synthesized to examine the structure-activity relationships of arginine conjugation for gene delivery systems. Number of conjugated arginines was examined by 1H NMR (PAMAM3-R: 31, PAMAM4-R: 60, PAMAM3-R2: 59, PAMAM4-R2: 116). They could retard pDNA at a charge ratio of 2 and form polyplexes with sizes less than 250 nm from a charge ratio of 4. Di-arginine conjugated dendrimers showed higher Zeta-potential values and polyplex stabilities than mono-arginine conjugates. PAMAM3-R and PAMAM4-R showed low cytotoxicities even at high concentration but PAMAM3-R2 and PAMAM4-R2 exhibited significant cytotoxicities at high concentration. PAMAM3-R2 displayed greater transfection efficiency than PAMAM3-R, although the transfection efficiency of PAMAM4-R2 was not higher than that of PAMAM4-R in all condition. PAMAM3-R2 and PAMAM4-R2 polyplexes were observed to show the good intra-nuclear localization in comparison with PAMAM3-R and PAMAM4-R. It is concluded that di-arginine conjugation to PAMAM dendrimers can improve polyplex stability, intra-nuclear localization, and transfection efficiency but also induce charge density- and generation-dependent cytotoxicity. Therefore, a novel strategy for highly densed arginine conjugation maintaining low cytotoxicity will be needed for the development of efficient gene delivery carriers.

Synthesis and Characterization of a Sterically Stabilized Polyelectrolyte Using Isophorone Diisocyanate as the Coupling Reagent

The aim of the present study was to synthesize and characterize a stabilized polymeric gene delivery carrier, a poly(ethylene glycol)-co-poly(ethylenimine) (PEG-PEI) co-polymer, using isophorone diisocyanate (IPDI) as the coupling reagent. IPDI containing two different active isocyanate functional groups could satisfy the need for a selective conjugation in the two-step coupling reaction procedure. In the first step, methoxy-poly(ethylene glycol) (mPEG) (5 kDa) was combined with one isocyanate group of IPDI to form reactivated pre-polymers, NCO-terminated mPEGs. Then branched polyethylenimine (b-PEI) (25 kDa) reacted with the isocyanate group of a varying number of NCO-terminated mPEGs, leading to PEG-PEI co-polymers with urea bonds. PEG segments in the co-polymer were designed as the stabilizers for steric stabilization of polyplexes. PEG-PEI co-polymers were identified by Fourier transform infrared spectroscopy, nuclear magnetic resonance spectroscopy and gel-permeation chromatography. The thermal properties of PEG-PEI co-polymers were characterized by differential scanning calorimetry and thermogravimetric analysis. The results indicated the existence of hydrogen bonding interaction and partial compatibility between the two phases in the PEG-PEI co-polymers. Finally, this synthetic method allowed us to prepare PEG-PEI co-polymers with the modification degree of PEG varying between 17.8% and 82.6 wt%. This new process was easy to optimize and PEG-PEI co-polymers with high yield could be obtained.

Effects of cell‐penetrating peptides and pegylation on transfection efficiency of polyethylenimine in mouse lungs

Cell-penetrating peptides (CPPs) could potentially be used as vectors for intracellular delivery of proteins, peptides and nucleic acids. The present study examined different CPPs, such as TAT-derived and arginine rich sequences, as well as model amphiphilic peptide, with respect to transfection efficiency of pegylated polyethylenimine (PEI) in A549, Calu-3 cells and in mice after intra-tracheal administration. The conjugates were prepared by the coupling of CPPs to PEI via a heterobifunctional polyethylene glycol (PEG) linker, resulting in the bioconjugates CPP-PEG-PEI. Structures were successfully confirmed by (1)H-nuclear magnetic resonance and diffusion-ordered spectroscopy. Unmodified PEI 25 kDa was compared with pegylated PEI, and aggregation tendency in cell culture medium, interaction with mucin and stability against heparin was assayed. After evaluating transfection efficiency of the polymers in two different lung cell lines, luciferase reporter gene expression was determined in mouse lungs. All conjugates showed superior transfection efficiency compared to unmodified PEI 25 kDa. The conjugates sizes were generally < 300 nm, thus enabling them to penetrate through the mucus lining of the lung and reach the target cells. Coupling of CPPs to PEG-PEI, however, did not significantly improve transfection efficiency in A549 cells, calu-3 cells and in mouse lungs. We show that small and stable polyplex size achieved by pegylation is favourable for successful pulmonary gene delivery. Compared to PEI 25 kDa, pegylated PEI and CPP-PEG-PEI displayed enhanced transfection efficiency both in vitro and in vivo.

Simple Modifications of Branched PEI Lead to Highly Efficient siRNA Carriers with Low Toxicity

Polymer carriers like PEI which proved their efficiency in DNA delivery were found to be far less effective for the applications with siRNA. In the current study, we generated a number of nontoxic derivates of branched PEI through modification of amines by ethyl acrylate, acetylation of primary amines, or introduction of negatively charged propionic acid or succinic acid groups to the polymer structure. The resulting products showed high efficiency in siRNA-mediated knockdown of target gene. In particular, succinylation of branched PEI resulted in up to 10-fold lower polymer toxicity in comparison to unmodified PEI. Formulations of siRNA with succinylated PEI were able to induce remarkable knockdown (80% relative to untreated cells) of target luciferase gene at the lowest tested siRNA concentration of 50 nM in Neuro2ALuc cells. The polyplex stability assay revealed that the efficiency of formulations which are stable in physiological saline is independent of the affinity of siRNA to the polymer chain. The improved properties of modified PEI as siRNA carrier are largely a consequence of the lower polymer toxicity. In order to achieve significant knockdown of target gene, the PEI-based polymer has to be applied at higher concentrations, required most probably for sufficient accumulation and proton sponge effects in endosomes. Unmodified PEI is highly toxic at such polymer concentrations. In contrast, the far less toxic modified analogues can be applied in concentrations required for the knockdown of target genes without side effects.

Construction of caged polyplexes with a reversible intracellular unpacking property to improve stability and transfection

Cross-linking of protein macromonomers accompanies the assembly of viral particles, which provides the virus with high stability in the host. Following inspiration, caged polyplexes were fabricated via a biomimetic cross-linker. Thiolated polyethylenimine was synthesized and showed sufficient DNA condensation ability. Spherical particles with a diameter of about 150 nm were formed at an N/P ratio of 10. Shell-cross-linked polyplexes were then constructed by the oxidation of thiol groups in air. All the results indicate that the cross-linking shell via disulfide bonds could improve the stability of polyplexes in the physiological condition and showed a reversible unpacking property at the intracellular GSH concentration. By selecting the proper preparation conditions, polyplexes caged via a biomimetic cross-linker could efficiently release DNA for transfection.

Quantitative Comparison of Intracellular Unpacking Kinetics of Polyplexes by a Model Constructed From Quantum Dot-FRET

A major challenge for non-viral gene delivery is gaining a mechanistic understanding of the rate-limiting steps. A critical barrier in polyplex-mediated gene delivery is the timely unpacking of polyplexes within the target cell to liberate DNA for efficient gene transfer. In this study, the component plasmid DNA and polymeric gene carrier were individually labeled with quantum dots (QDs) and Cy5 dyes, respectively, as a donor and acceptor pair for fluorescence resonance energy transfer (FRET). The high signal-to-noise ratio in QD-mediated FRET enabled sensitive detection of discrete changes in polyplex stability. The intracellular uptake and dissociation of polyplexes through QD-FRET was captured over time by confocal microscopy. From quantitative image-based analysis, distributions of released plasmid within the endo/lysosomal, cytosolic, and nuclear compartments formed the basis for constructing a three-compartment first-order kinetics model. Polyplex unpacking kinetics for chitosan, polyethylenimine, and polyphosphoramidate were compared and found to correlate well with transfection efficiencies. Thus, QD-FRET-enabled detection of polyplex stability combined with image-based quantification is a valuable method for studying mechanisms involved in polyplex unpacking and trafficking within live cells. We anticipate that this method will also aid the design of more efficient gene carriers.

NOVEL HYBRID GENE VECTOR STABILIZED BY CROSS-LINKING WITH GOLD NANOPARTICLES

Enhanced stability of polyplexes in physiological condition was an important prerequisite for successful systemic gene delivery. Herein novel method was reported to develop stable gene vector by nanotechnology. Thiolated polyplexes were constructed and then cross-linked with gold nanoparticles (AuNPs) by gold-thiol interactions. TEM pictures showed that AuNPs were attached to the shell of spherical polyplexes. The hybrid gene vector was stable enough in physiological condition and maintained efficient transfection, which showed great potential in gene delivery research and application.

Gene delivery using chitosan, trimethyl chitosan or polyethylenglycol-graft-trimethyl chitosan block copolymers: Establishment of structure–activity relationships in vitro

Chitosan, trimethyl chitosan or polyethylenglycol-graft-trimethyl chitosan/DNA complexes were characterized concerning physicochemical properties such as hydrodynamic diameter, condensation efficiency and DNA release. Furthermore, cytotoxicity of polymers and uptake- and transfection efficiency of polyplexes were evaluated in vitro. Under conditions found in cell culture, formation of aggregates of ∼ 1000 nm and strongly decreased DNA condensation efficiency was observed in the case of chitosan polyplexes. These characteristics resulted in only 7% cellular uptake in NIH/3T3 cells and low transfection efficiencies in 4 different cell lines. By contrast, quaternization of chitosan strongly reduced aggregation tendency and pH dependency of DNA complexation. Accordingly, cellular uptake was increased 8.5-fold compared to chitosan polyplexes resulting in up to 678-fold increased transfection efficiency in NIH/3T3 cells. Apart from reduction of the cytotoxicity, PEGylation led to improved colloidal stability of polyplexes and significantly increased cellular uptake compared to unmodified trimethyl chitosan. These improvements resulted in a significant, up to 10-fold increase of transfection efficiency in NIH/3T3, L929 and MeWo cells compared to trimethyl chitosan. This study not only highlights the importance of investigating polyplex stability under different pH- and ionic strength conditions but also elucidates correlations between physicochemical characteristics and biological efficacy of the studied polyplexes.

(LYS) 16-based reducible polycations provide stable polyplexes with anionic fusogenic peptides and efficient gene delivery to post mitotic cells

Extracellular stability, endocytic escape, intracellular DNA release and nuclear translocation of DNA are all critical properties of non-viral vector/DNA particles. We have evaluated a (Lys) 16-based linear, reducible polycation (RPC) in combination with an acid-dependent, anionic fusogenic peptide for gene delivery to dividing and post-mitotic cells. The RPC was formed from Cys(Lys) 16Cys monomers. Molecular weight was 24,000 Da, corresponding to an average of 10.5 peptide monomers per RPC. Non-reducible polylysine (PLL) (27,000 Da) and monomeric (Lys) 16 peptide were evaluated for comparison. (Lys) 16/DNA particles were disrupted at fusogenic peptide concentrations well below those used for gene delivery. By contrast, RPC/DNA an PLL/DNA particles were stable in the presence of high concentrations of the anionic peptide. Addition of 10% serum virtually abolished the transfection ability of (Lys) 16/DNA/fusogenic peptide particles, but had little effect on RPC/DNA/fusogenic peptide particles. RPC/DNA/fusogenic peptide particles were highly effective for gene delivery to both cell lines and post-mitotic corneal endothelium. PLL/DNA/fusogenic peptide particles were moderately effective on cell lines, but gave no gene delivery with corneal endothelial cells. We conclude that (Lys) 16-based RPC/DNA/fusogenic peptide particles provide a gene delivery system which is potentially stable in the extracellular environment and, on reductive depolymerisation, can release DNA plasmids for nuclear translocation.

Biodegradable Poly(2-Dimethylamino Ethylamino)Phosphazene for In Vivo Gene Delivery to Tumor Cells. Effect of Polymer Molecular Weight

PurposePreviously, we have shown that complexes of plasmid DNA with the biodegradable polymer poly(2-dimethylamino ethylamino)phosphazene (p(DMAEA)-ppz) mediated tumor selective gene expression after intravenous administration in mice. In this study, we investigated the effect of p(DMAEA)-ppz molecular weight on both in vitro and in vivo tumor transfection, as well as on complex induced toxicity.Materials and Methodsp(DMAEA)-ppz with a broad molar mass distribution was fractionated by preparative size exclusion chromatography. Polyplexes consisting of plasmid DNA and the collected polymer fractions were tested for biophysical properties, (cyto)toxicity and transfection activity.ResultsFour p(DMAEA)-ppz fractions were collected with weight average molecular weights ranging from 130 to 950 kDa, and with narrow molecular mass distributions (Mw/Mn from 1.1 to 1.3). At polymer-to-DNA (N/P) ratios above 6, polyplexes based on these polymers were all positively charged (zeta potential 25–29 mV), and had a size of 80–90 nm. The in vitro cytotoxicity of the polyplexes positively correlated with polymer molecular weight. The in vitro transfection activity of the different polyplexes depended on their N/P ratio, and was affected by the degree of cytotoxicity, as well as the colloidal stability of the different polyplexes. Intravenous administration of polyplexes based on the high molecular weight polymers led to apparent toxicity, as a result of polyplex-induced erythrocyte aggregation. On the other hand, administration of polyplexes based on low molecular weight p(DMAEA)-ppz’s (Mw 130 kDa) did not show signs of toxicity and resulted in tumor selective gene expression.ConclusionPolymer molecular weight fractionation enabled us to optimize the transfection efficiency/toxicity ratio of p(DMAEA)-ppz polyplexes for in vitro and in vivo tumor transfection.

A facile entrapment approach to construct PEGylated polyplexes for improving stability in physiological condition

PEGylated polyplexes had been proved to improve the stability of DNA complexes. However, the conjugation reaction might reduce the capacity of efficient DNA complexation. Herein we described an easy and favorable approach to construct PEGylated polyplexes via entrapping poly(ethylene glycol) cholesterol ether (CPEG) into polyplexes. It was of interest to find the addition sequence of CPEG had great effect on the stability of polyplexes in physiological salt concentration. The addition of CPEG into the formed PEI 25k/DNA polyplexes had no effect to improve the stability. Whereas by the “CPEG first” method of adding CPEG and PEI 25k mixture into the DNA solution, the PEI 25k/CPEG/DNA polyplexes showed excellent anti-aggregation effect and enhanced transfection efficiency in physiological condition. The difference performance might be explained by the possibility of CPEG entrapment. By the “CPEG first” method, PEGylated polyplexes was constructed due to the hydrophobic interaction between the cholesterol group of CPEG and hydrophobic charged-compensated core. The PEG coating significantly improved the stability of polyplexes in physiological condition. This facile entrapment approach to prepare PEGylated polyplexes might have great potential in non-viral gene delivery research and application.

Synthesis and Characterization of a Novel Arginine-Grafted Dendritic Block Copolymer for Gene Delivery and Study of Its Cellular Uptake Pathway Leading to Transfection

We synthesized a novel arginine-grafted dendritic block copolymer, R-PAMAM-PEG-PAMAM-R G5 (PPP5-R) for gene delivery systems. Its Mw was measured as 2.74 x 104 Da by MALDI-TOF, and approximately 36 arginine residues are found to be grafted to the polymer by 1H NMR. PPP5-R was able to form polyplexes with plasmid DNA, the average size of which was about 200 nm. Positive zeta-potential values (+22 to +28 mV) of PPP5-R polyplex indicate the formation of positively charged stable polyplex particles and suggest that large dendritic blocks with high positive charge may not be fully shielded by PEG chains even after PEG-coated complex formation. PPP5-R polyplex shows enhanced water solubility due to the polymer's PEG core and also shows low cytotoxicity, representing the potential for in vivo application. We identified the greatly enhanced transfection efficiency of PPP5-R in comparison with that of native PPP5 on various cell lines. Moreover, in view of the result of various cellular uptake inhibitor treatments during a transfection step, the cellular uptake of PPP5-R polyplex leading to effective transfection is thought to be not dependent on one exclusive pathway and to have the possibility of multiple pathways (caveolae-, clathrin-, and macropinocytosis-mediated pathways), contrary to the caveolae-dependent uptake of the PPP5 polyplex lacking arginine residues.

Crosslinked nanocarriers based upon poly(ethylene imine) for systemic plasmid delivery: In vitro characterization and in vivo studies in mice

Crosslinked poly(ethylene imine) (PEI) polyplexes for intracellular DNA release were generated using a low molecular weight crosslinking reagent, Dithiobis(succinimidyl propionate) (DSP). Disulfide bonds of the crosslinked polyplexes were susceptible to intracellular redox conditions and DNA release was observed using an ethidium bromide exclusion assay and dynamic light scattering. Transfection experiments were performed to elucidate the effect of extra- and intracellular redox conditions. Pharmacokinetics and organ accumulation of uncrosslinked and crosslinked polyplexes were compared and gene expression patterns were measured in mice 24 h after intravenous injection. Crosslinked PEI and plasmid DNA formed stable polyplexes in a size range of 100–300 nm, with zeta potentials between + 16.4 and + 26.1 mV. DNA release occurred after cleavage of the disulfide bonds. Cell culture experiments under reducing conditions as well as with glutathione loaded cells confirmed the proposed intracellular activation. A significant influence of the intracellular glutathione status on the transfection efficiency was observed. Pharmacokinetic profiles of crosslinked PEI/DNA polyplexes in mice after intravenous administration showed higher blood levels for crosslinked polyplexes. These polyplexes accumulated mainly in the liver and the lungs. In vivo transfection data revealed significantly reduced (unwanted) lung transfection while liver transfection predominated. These studies suggest that crosslinked polyplexes are more stable in circulation and retain their transfection efficiency after intravenous administration.

Poly(glycoamidoamine)s for Gene Delivery. Structural Effects on Cellular Internalization, Buffering Capacity, and Gene Expression

The study of polymeric nucleic acid delivery vehicles has recently grown because of their promise for many biomedical applications. In an effort to understand how the chemical traits of polymers affect the biological mechanisms of nucleic acid delivery, we have calculated the buffering capacity in the physiological pH range of a series of 10 poly(glycoamidoamine)s with systematic structural variations in the amine stoichiometry (from 1 to 4), carbohydrate moiety (d-glucarate or l-tartarate), and amine spacer (ethylene or butylene) within their repeat units. In addition, we have compared the buffering capacity of these polymeric vectors to their polyplex (polymer-DNA complex) stability, cellular internalization, and gene expression profiles to understand the parameters that are important for increasing gene delivery efficiency. The results indicate that the buffering capacity is not always the primary characteristic that determines the gene delivery efficiency for all the poly(glycoamidoamine)s. We have found that the buffering capacity may affect the gene delivery efficiency only when analogous structures containing the same number of amines but different carbohydrates are compared. We reveal that the cellular internalization is the key step in the gene delivery process with systems containing different amine stoichiometry. Also, increasing the number of methylene groups between the secondary amines increases toxicity to a large degree. This systematic and heuristic approach of studying the correlations between structural variables and gene delivery efficiency will facilitate the development of effective synthetic vectors for specific nucleic acid delivery applications.

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.

Biodegradable cationic polyester as an efficient carrier for gene delivery to neonatal cardiomyocytes

Viral-mediated gene delivery has been explored for the treatment and protection of cardiomyocytes, but so far there is only one report using cationic polymer for gene delivery to cardiomyocytes in spite of many advantages of polymer-mediated gene delivery. In this study, a cationic poly(beta-amino ester) (PDMA) with a degradable backbone and cleavable side chains was synthesized by Michael addition reaction. The toxicity of PDMA to neonatal mouse cardiomyocytes (NMCMs) was significantly lower than that of polyethyleneimine (PEI). PDMA formed stable polyplexes with pEGFP. The dissociation of the polyplexes could be triggered by PDMA degradation, and the dissociation time was tunable via the polymer/pEGFP ratio. In vitro transfection showed that PDMA was an effective and low toxic gene delivery carrier for NMCMs. The PDMA/pEGFP polyplexes transfected EGFP gene to NMCMs with about 28% efficiency and caused little death. In contrast, a significant portion of cardiomyocytes cultured with PEI/pEGFP died.

Influence of Polyethylene Glycol Chain Length on the Physicochemical and Biological Properties of Poly(ethylene imine)-graft-Poly(ethylene glycol) Block Copolymer/SiRNA Polyplexes

Polyplexes between siRNA and poly(ethylene imine) (PEI) derivatives are promising nonviral carriers for siRNA. The polyplex stability is of critical importance for efficient siRNA delivery to the cytoplasm. Here, we investigate the effect of PEGylation at a constant ratio ( approximately 50%) on the biophysical properties of the polyplexes. Particle size, zeta potential, and stability against heparin as well as RNase digestion and reporter gene knockdown under in vitro conditions of different siRNA polyplexes were characterized. Stability and size of siRNA polyplexes were clearly influenced by PEI-PEG structure, and high degrees of substitution such as PEI(25k)-g-PEG(550)(30) resulted in large (300-400 nm), diffuse complexes (AFM) which showed condensation behavior only at high N/P ratios. All other polyplexes and the PEI control showed similar sizes (150 nm) and compact structures in AFM, with complete condensation reached at N/P ratio of 3. Stability of siRNA polyplexes against heparin displacement and RNase digestion could be modified by PEGylation. Protection against RNase digestion was highest for PEI(25k)-g-PEG(5k)(4) and PEI(25k)-g-PEG(20k)(1), while siRNA/PEI provided insufficient protection. In knockdown experiments using NIH/3T3 fibroblasts stably expressing beta-galactosidase, it was shown that PEG chain length had a significant influence on biological activity of siRNA. Polyplexes with siRNA containing PEI(25k)-g-PEG(5k)(4) and PEI(25k)-g-PEG(20k)(1) yielded similar efficiencies of ca. 70% knockdown as lipofectamine controls. Confocal microscopy demonstrated enhanced cellular uptake of siRNA into cytosol by polyplexes formation with PEI copolymers. In conclusion, both the chain length and graft density of PEG were found to strongly influence siRNA condensation and stability and hence affect the knockdown efficiency of PEI-PEG/siRNA polyplexes.

Fluorescence resonance energy transfer: Evaluation of the intracellular stability of polyplexes

The investigation of intracellular mechanisms of non-viral nucleic acid delivery systems has provided great impetus for the improvement of their efficacy. Especially the intracellular release of the nucleic acid from the non-viral carrier system may be a relevant criterion for the high transfection efficiency of certain polymers. Therefore, we evaluated fluorescence resonance energy transfer (FRET) in combination with confocal laser scanning microscopy or flow cytometry as tool to determine the intracellular disintegration of polyplexes built with plasmid DNA and linear polyethylenimine. In microscopy, which allowed for an observation of polyplexes within single cells, sensitized emission measurement and acceptor photobleaching have been tested towards quantitative FRET analysis. In contrast, the whole cell population was analyzed by the flow cytometry-based method. We suggest that FRET is a useful tool to evaluate the intracellular disintegration of polyplexes built with various polymers.

Biocleavable Polyrotaxane−Plasmid DNA Polyplex for Enhanced Gene Delivery

A biocleavable polyrotaxane, having a necklace-like structure consisting of many cationic alpha-cyclodextrins (alpha-CDs) and a disulfide-introduced poly(ethylene glycol) (PEG), was synthesized and examined as a nonviral gene carrier. The polyrotaxane formed a stable polyplex having positively charged surface even at low charge ratio. This is likely to be due to structural factors of the polyrotaxane, such as the mobile motion of alpha-CDs in the necklace-like structure. Rapid endosomal escape was observed 90 min after transfection. The positively charged surface and the good buffering capacity are advantageous to show the proton sponge effect. The pDNA decondensation occurred through disulfide cleavage of the polyrotaxane and subsequent supramolecular dissociation of the noncovalent linkages between alpha-CDs and PEG. Transfection of the DMAE-SS-PRX polyplex is independent of the amount of free polycation. Those properties played a key role for delivery of pDNA clusters to the nucleus. Therefore, the polyplex nature and the supramolecular dissociation of the polyrotaxane contributed to the enhanced gene delivery.