Molecular dynamics simulations of PEI mediated DNA aggregation

Understanding the molecular mechanism of polycation induced DNA aggregation and condensation is important for optimal design of gene delivery carriers. In this work, we performed a series of all-atom molecular dynamics (MD) simulations to investigate polyethylenimine (PEI) mediated DNA aggregation. We found that PEIs condense DNA through two mechanisms: polyion bridging and electrostatic screening of the DNA charges. At PEI/DNA charge ratio >1, PEIs can completely neutralize DNAs at a short distance (～12 ? from the C1' atoms), and this distance is found to be insensitive to the exact value of the charge ratio. When excess PEIs are added to a formed DNA-PEI aggregate, they are found to bind to the aggregate and increase its cationic charge. The added PEIs can also replace the PEIs previously bound to the aggregate. The excess PEIs, however, do not change the spacing of the DNAs in the aggregates. Our simulation results shed light on the mechanisms of PEI, and more generally polycation, mediated DNA aggregation and condensation.     

The role of PEI structure and size in the PEI/liposome-mediated synergism of gene transfection

Polyethylenimines (PEIs) and cationic liposomes are widely used for nonviral gene delivery. When PEIs have been used alone, the transfection efficiency has been higher for larger or linear than smaller or branched PEIs. We have reported previously that a combination of small PEIs and liposomes results in a potentiation of transfection efficiency in vitro. Here, the role of PEI size and structure in this synergism has been clarified further. Therefore, two structurally different high MW PEIs, i.e. the linear PEI22K and branched PEI25K, were studied in the SMC cells. We found that both linear PEI22K and branched PEI25K resulted in a similar synergism and comparable transfection efficiencies. However, the potentiation for larger PEIs found in the present study was weaker than that for smaller PEIs obtained in our previous studies. In conclusion, our present and previous results demonstrate that the increment of PEI/liposome-mediated gene transfection by different types of PEIs in vitro is a common attribute that is rather associated with their size than the structure. Interestingly, the effect of PEI size seems to be opposite when combined with liposome or given alone, i.e. the small PEIs are more effective when combined and less effective when alone than the larger ones.     

Disulfide cross-linked polyethylenimines (PEI) prepared via thiolation of low molecular weight PEI as highly efficient gene vectors

Ring-opening reaction of low molecular weight polyethylenimine with an Mw of 800 Da (800 Da PEI) with methylthiirane produced thiolated polyethylenimine (PEI-SHX ). The thiolation degree X, which is the average number of thiol groups on a PEI molecule, was readily adjusted by the methylthiirane/PEI ratio. Oxidation of the thiolated PEIs with DMSO afforded disulfide cross-linked PEIs (PEI-SSX ). The molecular weights of PEI-SS X were estimated by viscosity measurement to be 7100, 8000, and 8400 for X=2.6, 6.5, and 9.4, respectively. The PEI-SSX series can bind and condense plasmid DNAs effectively forming nanosized polyplexes. The size of dry polyplexes is less than 100 nm on the TEM pictures. In solution, the size of the polyplexes was measured by DLS to be about 400 nm. In vitro experiments showed that the PEI-SS X series have a lower cytotoxicity and higher gene transfection efficiency compared with the high molecular weight PEI with Mw of 25 KDa. The presence of fetal bovine serum did not decrease the transfection efficiency. The results proved the hypothesis that reductively degradable disulfide-containing PEIs could possesses simultaneously higher gene transfection efficiency and lower cytotoxicity than the nondegradable ones.     

Gene transfer with modified polyethylenimines

Branched and linear polyethylenimines (PEIs) have proven to be efficient and versatile agents for gene delivery in vitro. In addition, systemic administration of positively charged DNA/PEI complexes results in significant reporter gene expression in lungs. However, re-targeting of complexes to organs other than the lung is hampered by non-specific interactions of polyplexes with blood components and non-target cells. Thus, despite considerable transfectional activity, the properties of PEIs need to be further improved. Therefore, various modifications of PEIs have been explored in recent years. For example, to increase the circulation half-life of the DNA complexes, the surface charge of the particles was shielded by grafting hydrophilic polymers such as polyethylene glycols (PEGs) onto their surface. Alternatively, incorporation of certain ligands into the DNA complexes also resulted in charge shielding even without PEGylation. Herein, I review the most recent PEI derivatives, with a special focus on PEGylated and targeted polymers.     

Conformation and reactivity of DNA in the complex with protein. IV. Circular dichroism of poly-L-histidine model complexes with DNA polymers and specificity of the interaction.

The CD study of the DNA-poly-L-histidine complex at high degree of protonation revealed that complex formation is already observable at 2 M NaCl. The influence of salt together with 5 M urea suggests that in addition to electrostatic interactions probably hydrogen bonding may favour specific complexes. Affinity of protonated histidines to AT-rich regions is strongly supported by the complexes formed with (dA.dT)-containing polymers. The psi-type structure occurs with poly(dA-dT)-poly(dA-dT) while poly(dA)-poly(dT) is restricted to form a similar psi-state on interaction with highly protonated poly-L-histidine. Differences in the helix winding properties due to variation in the sequence is suggested as a possible factor in the formation of the psi-type complexes. The mechanism of interaction including hydrogen bonding of histidine side-chains with an AT pair at high degree of protonation and with GC-regions at lower degree of protonation in the polypeptide structure is discussed.     

Star-shaped poly(ethylene glycol)-block-polyethylenimine copolymers enhance DNA condensation of low molecular weight polyethylenimines

Star-shaped poly(ethylene glycol)-block-polyethylenimine [star-(PEG-b-PEI)] significantly enhance plasmid DNA condensation of low molecular weight (MW) PEIs. The star-block copolymers were prepared via a facile synthesis route using hexamethylene diisocyanate as linker between PEG and PEI blocks. NMR and FT-IR spectroscopy confirmed the structures of intermediately activated PEG and final products. Furthermore, the copolymers were characterized by size exclusion chromatography, static light scattering, and viscosimetry. Their molecular weights (M(w) 19-26 kDa) were similar to high MW PEI (25 kDa). Thermoanalytical investigations (thermogravimetric analysis, differential scanning calorimetry) were also performed and verified successful copolymer synthesis. DNA condensation with the low MW PEIs (800 and 2000 Da) and their 4- and 8-star-block copolymers was studied using atomic force microscopy, dynamic light scattering, zeta-potential measurements, and ethidium bromide (EtBr) exclusion assay. It was found that low MW PEIs formed huge aggregates (500 nm to 2 microm) in which DNA is only loosely condensed. By contrast, the star-block copolymers yielded small (80-110 nm), spherical and compact complexes that were stable against aggregation even at high ionic strength and charge neutrality. Furthermore, as revealed in the EtBr exclusion assay these star-block copolymers exhibited a DNA condensation potential as high as high MW PEI. Since these star-(PEG-block-PEI) copolymers are composed of relatively nontoxic low MW PEI and biocompatible PEG, their potential as gene delivery agents merits further investigations.     

PEI在动物皮肤组织基因转化中的应用研究

用低分子量的聚乙亚胺(PEI)开发一种新的非病毒基因转染系统,为基因在皮肤组织中的有效转染提供一种可靠、廉价的方法。将带有绿色荧光蛋白报告基因(gfp)的真核表达质粒与阳离子聚合物聚乙亚胺结合,用肝癌细胞株CM7721试验,研究其转染效率及可能引起的细胞毒性;进一步转染小鼠皮肤组织,研究转染基因的表达位置及持续表达时间。结果发现,低分子量PEI介导的细胞转染效率最高可达55%,转染效率与PEI结构无关(P>0·05),但是随着PEI分子量的增加,其转染活性略有下降。同时,随着分子量的增加,PEI对细胞的毒性也相应加大;小鼠皮肤转染实验显示,转染24h后,gfp即可在皮肤组织的毛囊、汗腺、皮脂腺等处高效表达,表达可持续7~9d;进一步对皮肤用氮酮、维甲酸处理后,gfp可在皮肤组织的颗粒层细胞中高效表达。PEI是一种高效、有用的非病毒基因转染载体,能够在体外培养的动物细胞及动物皮肤组织中进行基因转移,这对皮肤疾病的基因治疗具有潜在的应用价值。     

Thermodynamics of Polyethylenimine-DNA Binding and DNA Condensation

In this study, polyethylenimine (PEI) binding to DNA was examined by isothermal titration calorimetry. Two types of binding modes were found to describe the interactions between these polyelectrolytes in buffers and in water. One type of binding involves PEI binding to the DNA groove because the enthalpy change of this binding mode is positive, and PEI is deprotonated to bind to DNA. Another likely binding mode involves external binding of PEI to the DNA phosphate backbone, accompanied with DNA condensation. The enthalpy change is negative and PEI is protonated when it binds to DNA in this mode. The intrinsic enthalpy change of first binding mode is 1.1 kJ/mol and −0.88 kJ/mol for the second binding mode. This result implies that the PEI is rearranged from the groove to the phosphate backbone of DNA when DNA is condensed. The mechanism of DNA condensation caused by PEI is discussed in this study.     

Density functional and molecular dynamics simulations of local anesthetics in 0.9% NaCl solution

We have made density functional calculations and molecular dynamics (MD) simulations to investigate the structure and pharmacological action of local anesthetics: tetracaine, procaine and lidocaine. The MD simulations were made in a NPT ensemble, in a 0.9% NaCl solution, on both unprotonated and protonated forms of the molecules. The radial distribution function was used to study solvent effects in different regions of the molecules. Although all three anesthetics have different degrees of hydrophobicity, the amino-terminals were the mostly affected by the protonation yielding hydrophilic regions. The charged amino-esters present hydrophilicity on the ester as well as amine terminals. Cl^?  from the solvent solution forms hydrogen bonds via protonated hydrogen attached to nitrogen, yielding neutral molecules, which could, in principle, penetrate the membranes and loose Cl^?  to act in the protonated form. Density functional theory calculations indicated a change in the electrostatic potential and showed that Cl^?  weakly binds to the amine hydrogen, what suggests it is a favorable interaction and supports the existence of the hydrochloric forms of these local anesthetics.     

Size reduction of galactosylated PEI/DNA complexes improves lectin-mediated gene transfer into hepatocytes.

Hepatocytes are interesting targets for gene therapy applications. Several hepatocyte-directed gene delivery vectors have been described. For example, simple galactosyl residues coupled to polyethylenimine (PEI) gave an efficient vector which selectively transfected hepatocytes via the asialoglycoprotein receptor-mediated endocytosis [Zanta, M. A., et al. (1997) Bioconjugate Chem. 8, 839-844]. However, the large size of these galactosylated PEI/DNA complexes prevented their use in vivo. We have investigated the role of the saccharide length on the size of glycosylated-PEI/DNA particles. When 5% of the PEI nitrogens were grafted with a linear tetragalactose structure (lGal4), small and stable particles were formed upon complexation with plasmid DNA. These particles were essentially toroids having a size of 50-80 nm and a zeta-potential close to neutrality. Moreover, these slightly charged PEI-lGal4/DNA complexes were as selective as the previously described galactosylated-PEI vector to transfect hepatocytes, but in addition, they were more efficient. It is expected that the properties of the PEI-lGal4/DNA complexes may increase their diffusion into the liver and their efficiency to transfect hepatocytes.     

Evaluation of hyperbranched poly(amino ester)s of amine constitutions similar to polyethylenimine for DNA delivery

New hyperbranched poly(amino ester)s were synthesized via A3 + 2BB'B' ' approach, represented by the Michael addition polymerization of trimethylol-propane triacrylate (TMPTA) (A3-type monomers) with a double molar 1-(2-aminoethyl)piperazine (AEPZ) (BB'B'-type monomer) performed in chloroform at ambient temperature. The results obtained by in situ monitoring the polymerization using NMR and MS indicated that hyperbranched poly(TMPTA1-AEPZ2) was formed via a A(B'B')2 intermediate, and the B' ' (the formed 2 degrees amine) was kept intact in the reaction. Therefore, poly(TMPTA1-AEPZ2) contained secondary and tertiary amines in the core and primary amines in the periphery similar to polyethylenimine (PEI). The chemistry of protonated poly(TMPTA1-AEPZ2) was further confirmed by 13C NMR, and the molecular weight, the radius of gyration (Rg), and the hydrodynamic radius (Rh) were determined using GPC, small-angle X-ray scattering (SAXS), and laser dynamic light scattering (LDLS), respectively. The ratio of Rg/Rh of ca. 1.1 verified the hyperbranched structure. Protonated hyperbranched poly(TMPTA1-AEPZ2) is degradable and less cytotoxic as compared with PEI (25 K). Gel electrophoresis reflected that stable complexes could be formed from protonated hyperbranched poly(TMPTA1-AEPZ2) and DNA, and the size and xi-potential of the complexes were characterized. Remarkably, protonated hyperbranched poly(TMPTA1-AEPZ2) showed transfection efficiency comparable to PEI (25 k) for in vitro DNA delivery.     

Dilution of reporter gene with stuffer DNA does not alter the transfection efficiency of polyethylenimines

BACKGROUND: Polyethylenimines (PEIs) are among the most efficient non-viral gene transfer agents developed so far. However, transfections with these polymers were shown to require a very high copy number of plasmid DNA per cell to achieve gene expression. Here, we investigate whether it is possible to reduce the amount of plasmid DNA while keeping a high transfection efficiency. METHODS: Transfection experiments were performed under various conditions in order to study the interdependence between the amount of reporter DNA, the amine-to-phosphate ratio and the transfection efficiency. RESULTS: When suboptimal amounts of linear PEI 22 kDa/DNA complexes were used for transfection, a severe reduction in reporter gene expression was observed. On the other hand, for optimal amounts of PEI/DNA complexes more than half of the reporter gene can be replaced by carrier DNA or polyglutamic acid without substantially decreasing the transfection efficiency of the polymer both in cultured cells and after systemic administration in mice. When used under the same in vitro experimental conditions, the lipospermine DOGS, but not the monocationic lipid DOTAP, gave similar results. CONCLUSIONS: Taken together, our data suggest that the activity of compounds with endosome-buffering capacities, such as PEIs and lipospermines, requires a threshold amount of transfection agent. In addition, our results indicate that, in many gene transfer situations, it will be possible to lower the dose of active plasmid thus reducing costs and the risk of immune stimulation triggered by bacterial DNA.     

pH-responsive sulfonamide/PEI system for tumor specific gene delivery: an in vitro study

The feasibility of pH-sensitive polymeric nanoparticles that effectively target the acidic extracellular matrix of tumors is demonstrated. Plasmid DNA was complexed with polyethyleneimine (PEI) and further with a pH-sensitive diblock copolymer, poly(methacryloyl sulfadimethoxine) (PSD)-block-PEG (PSD-b-PEG), to obtain naonparticles. The shielding/deshielding of nanoparticles was tested along with cell viability and transfection efficiency at physiological and tumor pH. The nanoparticles composed of DNA/PEI/PSD-b-PEG were 300 nm in size and showed low cytotoxicity and transfection at pH 7.4 due to shielding of PEI by PSD-b-PEG. The PSD-b-PEG bound to PEI/DNA complex decreased the interaction of PEI positive charges with cells and reduced the cytotoxicity by 60%. At pH 6.6, the nanoparticles demonstrated high cytotoxicity and transfection, indicating PSD-b-PEG detachment from the nanoparticles and permit PEI to interact with cells. PSD-b-PEG is able to discern the small difference in pH between normal and tumor tissues and hence has remarkable potential in drug targeting to tumor areas.     

Exploring polyethylenimine-mediated DNA transfection and the proton sponge hypothesis

BACKGROUND: The relatively high transfection efficiency of polyethylenimine (PEI) vectors has been hypothesized to be due to their ability to avoid trafficking to degradative lysosomes. According to the proton sponge hypothesis, the buffering capacity of PEI leads to osmotic swelling and rupture of endosomes, resulting in the release of the vector into the cytoplasm. METHODS: The mechanism of PEI-mediated DNA transfer was investigated using quantitative methods to study individual steps in the overall transfection process. In addition to transfection efficiency, the cellular uptake, local pH environment, and stability of vectors were analyzed. N-Quaternized (and therefore non-proton sponge) versions of PEI and specific cell function inhibitors were used to further probe the proton sponge hypothesis. RESULTS: Both N-quaternization and the use of bafilomycin A1 (a vacuolar proton pump inhibitor) reduced the transfection efficiency of PEI by approximately two orders of magnitude. Chloroquine, which buffers lysosomes, enhanced the transfection efficiency of N-quaternized PEIs and polylysine by 2-3-fold. In contrast, chloroquine did not improve the transfection efficiency of PEI. The measured average pH environment of PEI vectors was 6.1, indicating that they successfully avoid trafficking to acidic lysosomes. Significantly lower average pH environments were observed for permethyl-PEI (pH 5.4), perethyl-PEI (pH 5.1), and polylysine (pH 4.6) vectors. Cellular uptake levels of permethyl-PEI and perethyl-PEI vectors were found to be 20 and 90% higher, respectively, than that of parent PEI vectors, indicating that the reduction in transfection activity of the N-quaternized PEIs is due to a barrier downstream of cellular uptake. A polycation/DNA-binding affinity assessment showed that the more charge dense N-quaternized PEIs bind DNA less tightly than PEI, demonstrating that poor vector unpackaging was not responsible for the reduced transfection activity of the N-quaternized PEIs. CONCLUSIONS: The results obtained are consistent with the proton sponge hypothesis and strongly suggest that the transfection activity of PEI vectors is due to their unique ability to avoid acidic lysosomes.     

Low molecular weight polyethylenimines linked by beta-cyclodextrin for gene transfer into the nervous system

BACKGROUND: Polyethylenimines (PEIs) with high molecular weights are effective nonviral gene delivery vectors. However, the in vivo use of these PEIs can be hampered by their cellular toxicity. In the present study we developed and tested a new PEI polymer synthesized by linking less toxic, low molecular weight (MW) PEIs with a commonly used, biocompatible drug carrier, beta-cyclodextrin (CyD). METHODS AND RESULTS: The terminal CyD hydroxyl groups were activated by 1,1'-carbonyldiimidazole. Each activated CyD then linked two branched PEI molecules with MW of 600 Da to form a CyD-containing polymer with MW of 61 kDa, in which CyD served as a part of the backbone. The PEI-CyD polymer developed was soluble in water and biodegradable. In cell viability assays with sensitive neurons, the polymer performed similarly to low-MW PEIs and displayed much lower cellular cytotoxicity compared to PEI 25 kDa. The gene delivery efficiency of the polymer was comparable to, and at higher polymer/DNA ratios even higher than, that offered by PEI 25 kDa in neural cells. Attractively, intrathecal injection of plasmid DNA complexed by the polymer into the rat spinal cord provided levels of gene expression close to that offered by PEI 25 kDa. CONCLUSIONS: The polymer reported in the current study displayed improved biocompatibility over non-degradable PEI 25 kDa and mediated gene transfection in cultured neurons and in the central nervous system effectively. The new polymer would be worth exploring further as an in vivo delivery system of therapeutic genetic materials for gene therapy of neurological disorders.     

Conjugation of a new two-photon fluorophore to poly(ethylenimine) for gene delivery imaging

We report herein the molecular engineering of an efficient two-photon absorbing (TPA) chromophore based on a donor-donor bis-stilbenyl entity to allow conjugation with biologically relevant molecules. The dye has been functionalized using an isothiocyanate moiety to conjugate it with the amine functions of poly(ethylenimine) (PEI), which is a cationic polymer commonly used for nonviral gene delivery. Upon conjugation, the basic architecture and photophysical properties of the active TPA chromophore remain unchanged. At the usual N/P ratio (ratio of the PEI positive charges to the DNA negative charges) of 10 used for transfection, the transfection efficiency and cytotoxicity of the labeled PEI/DNA complexes were found to be comparable to those of the unlabeled PEI/DNA complexes. Moreover, when used in combination with unlabeled PEI (at a ratio of 1 labeled PEI to 3 unlabeled PEI), the labeled PEI does not affect the size of the complexes with DNA. The labeled PEI was successfully used in two-photon fluorescence correlation spectroscopy measurements, showing that at N/P = 10 most PEI molecules are free and the diffusion coefficient of the complexes is consistent with the 360 nm size measured by quasielastic light scattering. Finally, two-photon images of the labeled PEI/DNA complexes confirmed that the complexes enter into the cytoplasm of HeLa cells by endocytosis and hardly escape from the endosomes. As a consequence, the functionalized TPA chromophore appears to be an adequate tool to label the numerous polyamines used in nonviral gene delivery and characterize their complexes with DNA in two-photon 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.     

Different strategies for formation of pegylated EGF-conjugated PEI/DNA complexes for targeted gene delivery.

With the aim of generating gene delivery systems for tumor targeting, we have synthesized a conjugate consisting of polyethylenimine (PEI) covalently modified with epidermal growth factor (EGF) peptides. Transfection efficiency of the conjugate was evaluated and compared to native PEI in three tumor cell lines: KB epidermoid carcinoma cells, CMT-93 rectum carcinoma cells, and Renca-EGFR renal carcinoma cells. Depending on the tumor cell line, incorporation of EGF resulted in an up to 300-fold increased transfection efficiency. This ligand-mediated enhancement and competition with free EGF strongly suggested uptake of the complexes through the EGF receptor-mediated endocytosis pathway. Shielded particles being crucial for systemic gene delivery, we studied the effect of covalent surface modification of EGF-PEI/DNA complexes with a poly(ethylene glycol) (PEG) derivative. An alternative way for the formation of PEGylated EGF-containing complexes was also evaluated where EGF was projected away from PEI/DNA core complexes through a PEG linker. Both strategies led to shielded particles still able to efficiently transfect tumor cells in a receptor-dependent fashion. These PEGylated EGF-containing complexes were 10- to 100-fold more efficient than PEGylated complexes without EGF.