- Email: [email protected]
Multiscale molecular modeling and rational design of polymer based gene delivery vectors
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Abstracts / Journal of Controlled Release 152 (2011) e133–e191
Conclusion We conclude that bisphosphonate-mediated metal surfaces of stents are a suitable platform for a localizable viral vector delivery system that also prevents systemic spread of vector. Gene delivery using bisphosphonate-mediated metal surfaces of stents-based antiDNA antibody tethering of vectors should be suitable for a wide array of single or multiple therapeutic gene strategies, and for further device-based gene delivery therapeutic strategies. References Fig. 1. XPS to detect phosphorus (A) and iron (B) on non-modified and modified steel surfaces demonstrates the appearance of P after PAA-BP treatment with persistent Fe signals.
the emergence of a characteristic P(2p) signal (Fig. 1A) in the XPS of the treated sample, a phosphorus signal is not present in the control 316 L steel (Fig. 1A). Furthermore, the characteristic Fe(2p) peaks of the steel substrate are still present in the XPS from the PAA-BPmodified sample (Fig. 1B), indicating that the thickness of the PAA-BP coordination layer is less than the effective XPS sampling depth (5 nm). SPDP is a hetero-bifunctional cleavable cross-linker. In this study SPDP was used to chemically couple an antibody on the PAA-BP monolayer coating. When putting the PAA-BP monolayer into SPDP solution, the N-hydroxysuccinimide (NHS) residue is specifically reacted with the amine groups in PAA-BP molecules. After rinsing the films to eliminate unbound free SPDP, the intermediate 2-pyridyldisulfide residue ( N S S ) in SPDP is chemically linked on the monolayer coating. When the disulfide-modified film is added into the anti-DNA antibody, which has been designed to contain an appended sulfhydryl group (–SH), the 2-pyridyl-disulfide group further reacts with the –SH in anti-DNA antibody molecules to chemically link the antibody segment onto PAA-BP molecular coatings. PAA-BP monolayer was activated with SPDP to introduce pyridyldisulfide groups to the stent matrix. This was followed by reacting the sulfhydryl-containing antibody fragment with the pyridyl-disulfide residue resulting in covalent attachment of the antibodies. The antiDNA monoclonal antibody fragment was used for specific binding of the gene vector. In cell culture, the stent material showed no detrimental effect on cell growth. The antibody immobilized and GFP tethered stent demonstrated efficient gene transduction in the pig arterial epithelial cells. The stents retrieved from cell culture after 72 h of incubation showed numerous GFP-transduced cells that exclusively infiltrated the PAA-BP coating on the stent, indicating a highly localized and efficient gene delivery. Cell transfection of GFP showed selective expression in pig arterial epithelial cell lines with a transfection rate of 93%. However, the control stents, immobilized with nonspecific antibody and incubated with the same amount of GFP, resulted in very few transduced GFP positive cells on the stent (Fig. 2).
Efficiency of GFP Transduction (%)
100 80 60 40 20 0
specific anti-knob
nonspecific anti-knob
SPDP only
physical adsorption
Fig. 2. Stent with antibody tethered Ad-GFP immobilized on the surface demonstrating restriction of transduction. Specificity of antibody affinity for vector tethering determined by extent of transduction in cell culture.
[1] D.H. Walter, M. Cejna, L. Diaz-Sandoval, Local gene transfer of phVEGF-2 plasmid by gene-eluting stents: an alternative strategy for inhibition of restenosisCirculation 110 (2004) 36–45. [2] S.J. Stachelek, C. Song, I. Alferiev, et al., Localized gene delivery using antibody tethered adenovirus from polyurethane heart valve cusps and intra-aortic implants, Gene Ther. 11 (2004) 15–24.
doi:10.1016/j.jconrel.2011.08.071
Multiscale molecular modeling and rational design of polymer based gene delivery vectors Yongmei Wang, Jesse Ziebarth Department of Chemistry, the University of Memphis, Memphis, TN 38154, USA E-mail address: [email protected] (Y. Wang). Summary We used multiscale modeling techniques and investigated several issues in non-viral based gene delivery using polyethylenimine (PEI). Atomistic molecular dynamic simulations captured the spontaneous complex formation between a short PEI chain or a poly(L-lysine) (PLL) chain with DNA helices. Atomic structures obtained for DNA/PEI and DNA/PLL complexes were compared very well with X-ray diffraction data. A computational Monte Carlo titration model was developed and was used to investigate the protonation state of PEI. The PEI was found to have about 55% of amine groups protonated at physiological condition, thereby supporting the potential hypothesis of the “proton sponge effect” of PEI. Coarse-grained modelings were used to find an optimum design of block copolymer, PEG-b-PEI that would condense DNA and form core–corona structures to provide better biocompatibility. Keywords: Polyethylenimine, Molecular dynamics simulations, Protonation, Copolymers, Multiscale modeling Introduction Positively charged polyethylenimines (PEI) possess great transfection efficiencies as gene delivery vectors [1]. The success of PEI as non-viral based gene delivery vector is attributed to its “proton sponge effect” [2,3]. However, there are still many obstacles that need to be overcome before PEI or its derivatives may be used in clinical trials of gene therapy. What currently impedes the development of polymer based gene delivery systems is a lack of quantitative structure-function relationship, which is primarily due to a lack of structural information of polyplexes formed in gene delivery. Measurement of particle sizes and charges can be done fairly easily. Microscopy can also help to visualize the nanoscopic particles. However, structural information at higher resolutions is difficult to obtain. Also difficult to assess is the stability and dynamic responses of polyplex particles. In order to rationally design polymer based gene delivery vectors, computational modeling can be of great help by providing critical information inaccessible to experiments. We used a multiscale modeling technique and investigated different aspects of problems encountered in gene delivery [4–6].
Abstracts / Journal of Controlled Release 152 (2011) e133–e191
Experimental methods Molecular dynamic simulations. Atomistic molecular dynamic simulations were performed using the AMBER molecular simulation package with AMBER Pamr99 force field. The force fields for the repeating units in PEI, protonated and unprotonated forms were developed based on the AMBER gaff module. The canonical B-form DNA structure was created with the nucgen module of AMBER. The DNA sequence used in all simulations was the self-complementary Drew– Dickerson dodecamer, d(CGCGAATTCGCG)2, with the first 12 bases belonging to strand 1 and the final 12 bases belonging to strand 2. We investigated two types of polycations, poly(L) lysine (PLL) and PEI with the PEI chain modeled in two different protonation states, namely all protonated or 50% protonated. Monovalent counterions were added to the system using LEaP to neutralize the charges on polycations. All systems were solvated in ~ 25,000 TIP3P water molecules in a rectangular box. Coarse-grained molecular dynamic simulations were performed with LAMMPS. The chains were modeled by simple beadspring models in implicit water solvent with explicit counter ions. Computational Monte Carlo titration. A coarse-grained model of PEI chain was first developed based on an atomistic model of the PEI chain. The coarse grained model of PEI was represented by a bead– spring chain with each bead representing a monomer of PEI centered at the nitrogen atom of the amine group. The beads were connected via harmonic bond and harmonic angle bending potentials. The parameters needed in these potentials were obtained by fitting the distributions obtained from the atomistic dynamic simulations. Purely repulsive Lennard–Jones potential was included to prevent overlap between non-bonded beads. The Monte Carlo titration includes the random displacement of the two beads plus random switch of the charge of the two beads. The move is accepted or rejected according to the Metropolis rule by considering the total energy change during the proposed move. If the charge of the beads is switched, the associated energy of changing the charge is governed M
by ΔEtit ¼ ∑Wij qi qj kB T ðln10ÞðpH−pK0 Þ, where Wij is the electrostatic j¼1
interaction energy between sites i and j, M was the total number of beads in the chain, and pK0 was the intrinsic pKa of site i as an isolated amine group. For Wij, we used screened Coulomb interaction with a distance dependent dielectric constant. Results and discussion Fig. 1 presents a typical snapshot of the complexes formed at the end of molecular dynamic simulations. The runs have been repeated for several times. Although the exact atomic contacts vary from run to run, some consistent trends have been observed. We found that most of the interactions in the complexes were between the negatively charged DNA phosphate groups and positively charged amine groups of the polycation; however, there were significant interactions between electronegative sites in the major and minor grooves of DNA and the polycations. The fully protonated PEI(20) chain lines up
Fig. 1. Typical snapshots of complex formed late in the trajectory for (A) PLL, (B) PEI (20), and (C) 50%-PEI(20) simulations. The polycation is shown in a space-filling model, while the DNA is shown as sticks-and-balls.
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with phosphate groups on both strands of the DNA duplex with a section of the polycation forming a bridge across the minor groove. In contrast, a section of the 50% protonated (50%-PEI(20)) chain lies in the minor groove of the DNA. The PLL chain, in which the charged amine groups are on side chains several bonds away from the polymer backbone instead of along the backbone as in PEI, has several amine groups sticking away from the DNA chain, reducing the number of positive charges that are close to the DNA phosphate groups. Fig. 2 presents the results of Monte Carlo titration of a PEI chain with that obtained from experiments by Smits et al. [7]. The X-axis is the averaged fraction of protonated amine sites, α, at the given pH. The computational titration curve at salt concentration Cs = 0.01 M matched well with the experimental curve obtained at Cs = 0.1 M. The salt dependence however was not captured correctly. Monte Carlo titration also provided detailed information as to which amines are protonated. We found that end beads are more likely protonated than the inner beads. At α = 0.5, the chain has nearly an ordered structure, with every other bead being protonated. Coarse-grained molecular dynamic simulations were used to investigate DNA condensation by PEI–PEG block copolymers using generic bead-spring chain models. Coarse-grained molecular dynamic simulations can cover much longer time and length scales. In the coarsegrained simulations, one can readily observe the complex formation and collapse of DNA chain. Details of results can be found in Ref. [5]. In the future, we plan to develop consistent coarse-grained models of three highly investigated polycations, PEI, PAMAM and chitosan. Molecular dynamic simulations of DNA condenstaions with derived coarse-grained models for these three polycations will be performed. The relative structures and stabilities of formed polyplexes will be compared. Conclusion Multiscale molecular modeling can provide critical information inaccessible to experimental studies on the structure and dynamics of polyplex particles. With structural information provided through multiscale modeling, better design of polymer based gene delivery systems can be achieved. Acknowledgments We acknowledge the support provided by Oak Ridge Associated University in partnership with Oak Ridge, National Lab through ORAU/ORNL high performance computing grant. The high performance computing facility at University of Memphis is also acknowledged. Partial financial fund from NIH/NIGMS (R01GM073095-01A2) through a subcontract from Iowa State University is acknowledged. References [1] O. Boussif, F. Lezoualc'h, M.A. Zanta, M.D. Mergny, D. Scherman, B. Demeneix, J.P. Behr, A versatile vector for gene and oligonecleotide transfer into cells in culture an in vivo: polyethylenimineProc. Natl. Acad. Sci. U. S. A. 92 (1995) 7297–7301.
Fig. 2. Overlay of experimental titration curves (shown as symbols) with computational titration curves (shown as lines). The salt concentration for experiments Cs = 0 M (circles) and Cs = 0.1 M (triangles). Salt concentrations for computational curves Cs = 0 M (dot-dashed line), 0.01 M (solid line), 0.1 M (dashed line). pK0 was assumed to be 10.0.
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[2] T. Merdan, K. Kunath, D. Fischer, J. Kopecek, T. Kissel, Intracellular processing of poly (ethylene imine)/ribozyme complexes can be observed in living cells using confocal laser scanning microscopy and inhibitor experiments, Pharm. Res. 19 (2002) 140–146. [3] M. Thomas, A.M. Klibanov, Enhancing polyethylenimine's delivery of plasmid DNA into mammalian cells, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 14640–14645. [4] J. Ziebarth, Y.M. Wang, Molecular dynamics simulations of DNA–polycation complex formation, Biophys. J. 97 (2009) 1971–1983. [5] J. Ziebarth, Y.M. Wang, Coarse-grained molecular dynamics simulations of DNA condensation by block copolymer and formation of core–corona structures, J. Phys. Chem. B 114 (19) (2010) 6225–6232. [6] J.D. Ziebarth, Y.M. Wang, Understanding the protonation behavior of linear polyethylenimine in solutions through monte carlo simulations, Biomacromolecules 11 (2010) 29–38. [7] R.G. Smits, G.J.M. Koper, M. Mandel, The influence of nearest- and next-nearestneighbor interactions on the potentiometric titration of linear poly(ethylenimine), J. Phys. Chem. 97 (1993) 5745–5751.
doi:10.1016/j.jconrel.2011.08.072
Synthesis of oligoethylenimine grafted net-poly(amino ester) and their application in gene delivery Jialiang Xia1,2, Lei Chen1,2, Huayu Tian1, Xuesi Chen1, Atsushi Maruyama3, Tae Gwan Park4 1 Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China 2 Graduate University of Chinese Academy of Sciences, Beijing 100049, China 3 Institute for Materials Chemistry and Engineering, Kyushu University, Fukuoka 812-8581, Japan 4 Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Republic of Korea E-mail address: [email protected] (J. Xia). Summary Novel net-polymers were prepared by grafting oligoethylenimine (OEI) on net-poly(amino ester) (N-P-g-OEI) and applied as gene carriers. N-P-g-OEI can effectively condense DNA to form small-sized complex particles (b150 nm). These cationic polymers showed low toxicity due to PEG-containing backbones and the low molecular weight OEI. The results demonstrated that these cationic polymers have much higher transfection efficiency compared to PEI25k in COS-7 cells. Keywords: Poly(amino ester), Biodegradable, Oligoethylenimine, Gene delivery Introduction Gene therapy has great potential for treating various human diseases [1,2]. Obviously, a major key to successful gene therapy is the development of gene vectors that are effective in vivo. The use of nonviral gene carriers [3] may offer advantages and resolve some of the current problems associated with viral gene carriers, such as safety, immunogenicity, mutagenesis and cost. Polyethylenimine (PEI) has been successfully used for gene delivery in vitro and in vivo. However, further developments of PEI are limited by its high toxicity, low transfection efficiency and non-biodegradability. Many strategies were used to improve polymeric gene carriers based on PEI. Hydrophilic polymers such as PEG and chitosan were introduced into PEI derivatives to make the polymer/DNA complexes stable in the circulation [4,5]. Hydrophobic poly(gamma-benzyl lglutamate) (PBLG) [6] or poly(phenylalanine) segments introduced into PEI could reduce the positive charge density of PEI resulting in weakening of the polymer/DNA interactions and more facile dissociation of DNA in the presence of anionic biological macromolecules. PEI with low molecular weight was introduced to hyperbranched polymer backbones, such as hyperbranched PBLG [7], to gain highly effective gene carriers with low toxicity.
The purpose of this study was to design biodegradable polymeric gene carriers. These polymers based on PEG-contained net-poly (amino ester) backbones and oligoethylenimine (OEI). The preliminary in vitro experiments showed that these polymers had low toxicity and could transfect plasmid DNA (pEGFP-N1) to COS-7 cells efficiently. Experimental methods Synthesis of crosslinked cationic polymer. Linear poly(amino ester) (L-PAE) was firstly synthesized by Michael Addition Reaction between polyethylene glycol diacrylate and aminoethanol under sealed-in condition. Then hexamethylene diisocyanate (HMDI) was used to slightly crosslink the L-PAE to get net-poly(amino ester) (N-P) with active isocyanate groups. Finally, different kinds of OEI were grafted to N-P to produce cationic net-polymers N-P-g-OEI. Relative cell viability assay and in vitro transfection. The cytotoxicity of net-polymers was assessed using the MTT assay. Triplicate MTT assays were performed with COS-7 cells at various polymer concentrations using 96-well plates. Transfection experiments were also performed with COS-7 cells by using plasmid DNA (pEGFP-N1) at different polymer/DNA mass ratios. Both MTT assays and transfection experiments were carried out. PEI-25k was used as control. Results and discussion The synthesis of N-P-g-OEI is shown in Scheme 1. The Michael Addition Reaction was carried out between polyethylene glycol diacrylate (Mn = 258) and aminoethanol. GPC results showed that low molecular polymer L-PAE with molecular weight of 2570 was produced. Such low molecular weight poly(amino ester) can't be used directly as the backbones of gene carriers because of the fast degradability and not very reactive groups. Then, HMDI was used as crosslinker to get higher molecular polymers. The molar feed ratio of HMDI to hydroxyl groups of L-PAE was 2:1. Some HMDI reacted with two hydroxyl groups which led to crosslinked structures, other HMDI just reacted with one hydroxyl group yielding another isocyanate group unreacted. The crosslinking reaction was carried out at different concentrations and produced N-P with different molecular weights. A higher concentration produced higher molecular weight N-P (N-P-1 with molecular weight of 3870 and N-P-2 with molecular weight of 11020, respectively). Finally, two kinds of OEI, linear OEI with molecular weight of 423 (OEI423) and branched OEI with molecular weight of 600 (OEI600) were grafted to N-P-1 and N-P-2. Four kinds of N-P-g-OEI macromolecules: N-P-1-OEI423, N-P-1OEI600, N-P-2-OEI423 and N-P-2-OEI600 were produced, respectively. Different N-P-g-OEI cationic polymers showed different toxicity. As shown in Fig. 1, a lower molecular weight backbone grafted with lower molecular weight OEI led to much lower toxicity. OEI600 grafted P-N showed much higher toxicity than the OEI423 grafted
Scheme 1. The synthesis route to N-P-g-OEI.
Abstracts / Journal of Controlled Release 152 (2011) e133–e191
Conclusion We conclude that bisphosphonate-mediated metal surfaces of stents are a suitable platform for a localizable viral vector delivery system that also prevents systemic spread of vector. Gene delivery using bisphosphonate-mediated metal surfaces of stents-based antiDNA antibody tethering of vectors should be suitable for a wide array of single or multiple therapeutic gene strategies, and for further device-based gene delivery therapeutic strategies. References Fig. 1. XPS to detect phosphorus (A) and iron (B) on non-modified and modified steel surfaces demonstrates the appearance of P after PAA-BP treatment with persistent Fe signals.
the emergence of a characteristic P(2p) signal (Fig. 1A) in the XPS of the treated sample, a phosphorus signal is not present in the control 316 L steel (Fig. 1A). Furthermore, the characteristic Fe(2p) peaks of the steel substrate are still present in the XPS from the PAA-BPmodified sample (Fig. 1B), indicating that the thickness of the PAA-BP coordination layer is less than the effective XPS sampling depth (5 nm). SPDP is a hetero-bifunctional cleavable cross-linker. In this study SPDP was used to chemically couple an antibody on the PAA-BP monolayer coating. When putting the PAA-BP monolayer into SPDP solution, the N-hydroxysuccinimide (NHS) residue is specifically reacted with the amine groups in PAA-BP molecules. After rinsing the films to eliminate unbound free SPDP, the intermediate 2-pyridyldisulfide residue ( N S S ) in SPDP is chemically linked on the monolayer coating. When the disulfide-modified film is added into the anti-DNA antibody, which has been designed to contain an appended sulfhydryl group (–SH), the 2-pyridyl-disulfide group further reacts with the –SH in anti-DNA antibody molecules to chemically link the antibody segment onto PAA-BP molecular coatings. PAA-BP monolayer was activated with SPDP to introduce pyridyldisulfide groups to the stent matrix. This was followed by reacting the sulfhydryl-containing antibody fragment with the pyridyl-disulfide residue resulting in covalent attachment of the antibodies. The antiDNA monoclonal antibody fragment was used for specific binding of the gene vector. In cell culture, the stent material showed no detrimental effect on cell growth. The antibody immobilized and GFP tethered stent demonstrated efficient gene transduction in the pig arterial epithelial cells. The stents retrieved from cell culture after 72 h of incubation showed numerous GFP-transduced cells that exclusively infiltrated the PAA-BP coating on the stent, indicating a highly localized and efficient gene delivery. Cell transfection of GFP showed selective expression in pig arterial epithelial cell lines with a transfection rate of 93%. However, the control stents, immobilized with nonspecific antibody and incubated with the same amount of GFP, resulted in very few transduced GFP positive cells on the stent (Fig. 2).
Efficiency of GFP Transduction (%)
100 80 60 40 20 0
specific anti-knob
nonspecific anti-knob
SPDP only
physical adsorption
Fig. 2. Stent with antibody tethered Ad-GFP immobilized on the surface demonstrating restriction of transduction. Specificity of antibody affinity for vector tethering determined by extent of transduction in cell culture.
[1] D.H. Walter, M. Cejna, L. Diaz-Sandoval, Local gene transfer of phVEGF-2 plasmid by gene-eluting stents: an alternative strategy for inhibition of restenosisCirculation 110 (2004) 36–45. [2] S.J. Stachelek, C. Song, I. Alferiev, et al., Localized gene delivery using antibody tethered adenovirus from polyurethane heart valve cusps and intra-aortic implants, Gene Ther. 11 (2004) 15–24.
doi:10.1016/j.jconrel.2011.08.071
Multiscale molecular modeling and rational design of polymer based gene delivery vectors Yongmei Wang, Jesse Ziebarth Department of Chemistry, the University of Memphis, Memphis, TN 38154, USA E-mail address: [email protected] (Y. Wang). Summary We used multiscale modeling techniques and investigated several issues in non-viral based gene delivery using polyethylenimine (PEI). Atomistic molecular dynamic simulations captured the spontaneous complex formation between a short PEI chain or a poly(L-lysine) (PLL) chain with DNA helices. Atomic structures obtained for DNA/PEI and DNA/PLL complexes were compared very well with X-ray diffraction data. A computational Monte Carlo titration model was developed and was used to investigate the protonation state of PEI. The PEI was found to have about 55% of amine groups protonated at physiological condition, thereby supporting the potential hypothesis of the “proton sponge effect” of PEI. Coarse-grained modelings were used to find an optimum design of block copolymer, PEG-b-PEI that would condense DNA and form core–corona structures to provide better biocompatibility. Keywords: Polyethylenimine, Molecular dynamics simulations, Protonation, Copolymers, Multiscale modeling Introduction Positively charged polyethylenimines (PEI) possess great transfection efficiencies as gene delivery vectors [1]. The success of PEI as non-viral based gene delivery vector is attributed to its “proton sponge effect” [2,3]. However, there are still many obstacles that need to be overcome before PEI or its derivatives may be used in clinical trials of gene therapy. What currently impedes the development of polymer based gene delivery systems is a lack of quantitative structure-function relationship, which is primarily due to a lack of structural information of polyplexes formed in gene delivery. Measurement of particle sizes and charges can be done fairly easily. Microscopy can also help to visualize the nanoscopic particles. However, structural information at higher resolutions is difficult to obtain. Also difficult to assess is the stability and dynamic responses of polyplex particles. In order to rationally design polymer based gene delivery vectors, computational modeling can be of great help by providing critical information inaccessible to experiments. We used a multiscale modeling technique and investigated different aspects of problems encountered in gene delivery [4–6].
Abstracts / Journal of Controlled Release 152 (2011) e133–e191
Experimental methods Molecular dynamic simulations. Atomistic molecular dynamic simulations were performed using the AMBER molecular simulation package with AMBER Pamr99 force field. The force fields for the repeating units in PEI, protonated and unprotonated forms were developed based on the AMBER gaff module. The canonical B-form DNA structure was created with the nucgen module of AMBER. The DNA sequence used in all simulations was the self-complementary Drew– Dickerson dodecamer, d(CGCGAATTCGCG)2, with the first 12 bases belonging to strand 1 and the final 12 bases belonging to strand 2. We investigated two types of polycations, poly(L) lysine (PLL) and PEI with the PEI chain modeled in two different protonation states, namely all protonated or 50% protonated. Monovalent counterions were added to the system using LEaP to neutralize the charges on polycations. All systems were solvated in ~ 25,000 TIP3P water molecules in a rectangular box. Coarse-grained molecular dynamic simulations were performed with LAMMPS. The chains were modeled by simple beadspring models in implicit water solvent with explicit counter ions. Computational Monte Carlo titration. A coarse-grained model of PEI chain was first developed based on an atomistic model of the PEI chain. The coarse grained model of PEI was represented by a bead– spring chain with each bead representing a monomer of PEI centered at the nitrogen atom of the amine group. The beads were connected via harmonic bond and harmonic angle bending potentials. The parameters needed in these potentials were obtained by fitting the distributions obtained from the atomistic dynamic simulations. Purely repulsive Lennard–Jones potential was included to prevent overlap between non-bonded beads. The Monte Carlo titration includes the random displacement of the two beads plus random switch of the charge of the two beads. The move is accepted or rejected according to the Metropolis rule by considering the total energy change during the proposed move. If the charge of the beads is switched, the associated energy of changing the charge is governed M
by ΔEtit ¼ ∑Wij qi qj kB T ðln10ÞðpH−pK0 Þ, where Wij is the electrostatic j¼1
interaction energy between sites i and j, M was the total number of beads in the chain, and pK0 was the intrinsic pKa of site i as an isolated amine group. For Wij, we used screened Coulomb interaction with a distance dependent dielectric constant. Results and discussion Fig. 1 presents a typical snapshot of the complexes formed at the end of molecular dynamic simulations. The runs have been repeated for several times. Although the exact atomic contacts vary from run to run, some consistent trends have been observed. We found that most of the interactions in the complexes were between the negatively charged DNA phosphate groups and positively charged amine groups of the polycation; however, there were significant interactions between electronegative sites in the major and minor grooves of DNA and the polycations. The fully protonated PEI(20) chain lines up
Fig. 1. Typical snapshots of complex formed late in the trajectory for (A) PLL, (B) PEI (20), and (C) 50%-PEI(20) simulations. The polycation is shown in a space-filling model, while the DNA is shown as sticks-and-balls.
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with phosphate groups on both strands of the DNA duplex with a section of the polycation forming a bridge across the minor groove. In contrast, a section of the 50% protonated (50%-PEI(20)) chain lies in the minor groove of the DNA. The PLL chain, in which the charged amine groups are on side chains several bonds away from the polymer backbone instead of along the backbone as in PEI, has several amine groups sticking away from the DNA chain, reducing the number of positive charges that are close to the DNA phosphate groups. Fig. 2 presents the results of Monte Carlo titration of a PEI chain with that obtained from experiments by Smits et al. [7]. The X-axis is the averaged fraction of protonated amine sites, α, at the given pH. The computational titration curve at salt concentration Cs = 0.01 M matched well with the experimental curve obtained at Cs = 0.1 M. The salt dependence however was not captured correctly. Monte Carlo titration also provided detailed information as to which amines are protonated. We found that end beads are more likely protonated than the inner beads. At α = 0.5, the chain has nearly an ordered structure, with every other bead being protonated. Coarse-grained molecular dynamic simulations were used to investigate DNA condensation by PEI–PEG block copolymers using generic bead-spring chain models. Coarse-grained molecular dynamic simulations can cover much longer time and length scales. In the coarsegrained simulations, one can readily observe the complex formation and collapse of DNA chain. Details of results can be found in Ref. [5]. In the future, we plan to develop consistent coarse-grained models of three highly investigated polycations, PEI, PAMAM and chitosan. Molecular dynamic simulations of DNA condenstaions with derived coarse-grained models for these three polycations will be performed. The relative structures and stabilities of formed polyplexes will be compared. Conclusion Multiscale molecular modeling can provide critical information inaccessible to experimental studies on the structure and dynamics of polyplex particles. With structural information provided through multiscale modeling, better design of polymer based gene delivery systems can be achieved. Acknowledgments We acknowledge the support provided by Oak Ridge Associated University in partnership with Oak Ridge, National Lab through ORAU/ORNL high performance computing grant. The high performance computing facility at University of Memphis is also acknowledged. Partial financial fund from NIH/NIGMS (R01GM073095-01A2) through a subcontract from Iowa State University is acknowledged. References [1] O. Boussif, F. Lezoualc'h, M.A. Zanta, M.D. Mergny, D. Scherman, B. Demeneix, J.P. Behr, A versatile vector for gene and oligonecleotide transfer into cells in culture an in vivo: polyethylenimineProc. Natl. Acad. Sci. U. S. A. 92 (1995) 7297–7301.
Fig. 2. Overlay of experimental titration curves (shown as symbols) with computational titration curves (shown as lines). The salt concentration for experiments Cs = 0 M (circles) and Cs = 0.1 M (triangles). Salt concentrations for computational curves Cs = 0 M (dot-dashed line), 0.01 M (solid line), 0.1 M (dashed line). pK0 was assumed to be 10.0.
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Abstracts / Journal of Controlled Release 152 (2011) e133–e191
[2] T. Merdan, K. Kunath, D. Fischer, J. Kopecek, T. Kissel, Intracellular processing of poly (ethylene imine)/ribozyme complexes can be observed in living cells using confocal laser scanning microscopy and inhibitor experiments, Pharm. Res. 19 (2002) 140–146. [3] M. Thomas, A.M. Klibanov, Enhancing polyethylenimine's delivery of plasmid DNA into mammalian cells, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 14640–14645. [4] J. Ziebarth, Y.M. Wang, Molecular dynamics simulations of DNA–polycation complex formation, Biophys. J. 97 (2009) 1971–1983. [5] J. Ziebarth, Y.M. Wang, Coarse-grained molecular dynamics simulations of DNA condensation by block copolymer and formation of core–corona structures, J. Phys. Chem. B 114 (19) (2010) 6225–6232. [6] J.D. Ziebarth, Y.M. Wang, Understanding the protonation behavior of linear polyethylenimine in solutions through monte carlo simulations, Biomacromolecules 11 (2010) 29–38. [7] R.G. Smits, G.J.M. Koper, M. Mandel, The influence of nearest- and next-nearestneighbor interactions on the potentiometric titration of linear poly(ethylenimine), J. Phys. Chem. 97 (1993) 5745–5751.
doi:10.1016/j.jconrel.2011.08.072
Synthesis of oligoethylenimine grafted net-poly(amino ester) and their application in gene delivery Jialiang Xia1,2, Lei Chen1,2, Huayu Tian1, Xuesi Chen1, Atsushi Maruyama3, Tae Gwan Park4 1 Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China 2 Graduate University of Chinese Academy of Sciences, Beijing 100049, China 3 Institute for Materials Chemistry and Engineering, Kyushu University, Fukuoka 812-8581, Japan 4 Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Republic of Korea E-mail address: [email protected] (J. Xia). Summary Novel net-polymers were prepared by grafting oligoethylenimine (OEI) on net-poly(amino ester) (N-P-g-OEI) and applied as gene carriers. N-P-g-OEI can effectively condense DNA to form small-sized complex particles (b150 nm). These cationic polymers showed low toxicity due to PEG-containing backbones and the low molecular weight OEI. The results demonstrated that these cationic polymers have much higher transfection efficiency compared to PEI25k in COS-7 cells. Keywords: Poly(amino ester), Biodegradable, Oligoethylenimine, Gene delivery Introduction Gene therapy has great potential for treating various human diseases [1,2]. Obviously, a major key to successful gene therapy is the development of gene vectors that are effective in vivo. The use of nonviral gene carriers [3] may offer advantages and resolve some of the current problems associated with viral gene carriers, such as safety, immunogenicity, mutagenesis and cost. Polyethylenimine (PEI) has been successfully used for gene delivery in vitro and in vivo. However, further developments of PEI are limited by its high toxicity, low transfection efficiency and non-biodegradability. Many strategies were used to improve polymeric gene carriers based on PEI. Hydrophilic polymers such as PEG and chitosan were introduced into PEI derivatives to make the polymer/DNA complexes stable in the circulation [4,5]. Hydrophobic poly(gamma-benzyl lglutamate) (PBLG) [6] or poly(phenylalanine) segments introduced into PEI could reduce the positive charge density of PEI resulting in weakening of the polymer/DNA interactions and more facile dissociation of DNA in the presence of anionic biological macromolecules. PEI with low molecular weight was introduced to hyperbranched polymer backbones, such as hyperbranched PBLG [7], to gain highly effective gene carriers with low toxicity.
The purpose of this study was to design biodegradable polymeric gene carriers. These polymers based on PEG-contained net-poly (amino ester) backbones and oligoethylenimine (OEI). The preliminary in vitro experiments showed that these polymers had low toxicity and could transfect plasmid DNA (pEGFP-N1) to COS-7 cells efficiently. Experimental methods Synthesis of crosslinked cationic polymer. Linear poly(amino ester) (L-PAE) was firstly synthesized by Michael Addition Reaction between polyethylene glycol diacrylate and aminoethanol under sealed-in condition. Then hexamethylene diisocyanate (HMDI) was used to slightly crosslink the L-PAE to get net-poly(amino ester) (N-P) with active isocyanate groups. Finally, different kinds of OEI were grafted to N-P to produce cationic net-polymers N-P-g-OEI. Relative cell viability assay and in vitro transfection. The cytotoxicity of net-polymers was assessed using the MTT assay. Triplicate MTT assays were performed with COS-7 cells at various polymer concentrations using 96-well plates. Transfection experiments were also performed with COS-7 cells by using plasmid DNA (pEGFP-N1) at different polymer/DNA mass ratios. Both MTT assays and transfection experiments were carried out. PEI-25k was used as control. Results and discussion The synthesis of N-P-g-OEI is shown in Scheme 1. The Michael Addition Reaction was carried out between polyethylene glycol diacrylate (Mn = 258) and aminoethanol. GPC results showed that low molecular polymer L-PAE with molecular weight of 2570 was produced. Such low molecular weight poly(amino ester) can't be used directly as the backbones of gene carriers because of the fast degradability and not very reactive groups. Then, HMDI was used as crosslinker to get higher molecular polymers. The molar feed ratio of HMDI to hydroxyl groups of L-PAE was 2:1. Some HMDI reacted with two hydroxyl groups which led to crosslinked structures, other HMDI just reacted with one hydroxyl group yielding another isocyanate group unreacted. The crosslinking reaction was carried out at different concentrations and produced N-P with different molecular weights. A higher concentration produced higher molecular weight N-P (N-P-1 with molecular weight of 3870 and N-P-2 with molecular weight of 11020, respectively). Finally, two kinds of OEI, linear OEI with molecular weight of 423 (OEI423) and branched OEI with molecular weight of 600 (OEI600) were grafted to N-P-1 and N-P-2. Four kinds of N-P-g-OEI macromolecules: N-P-1-OEI423, N-P-1OEI600, N-P-2-OEI423 and N-P-2-OEI600 were produced, respectively. Different N-P-g-OEI cationic polymers showed different toxicity. As shown in Fig. 1, a lower molecular weight backbone grafted with lower molecular weight OEI led to much lower toxicity. OEI600 grafted P-N showed much higher toxicity than the OEI423 grafted
Scheme 1. The synthesis route to N-P-g-OEI.