Sathya Srinivasachari - Academia.edu (original) (raw)

Papers by Sathya Srinivasachari

Biomaterials, Feb 1, 2009

Herein, we report the efficient synthesis of high molecular weight polymers (up to 331 kDa) that ... more Herein, we report the efficient synthesis of high molecular weight polymers (up to 331 kDa) that contain b-cyclodextrin within the polymer backbone and the examination of these structures for pDNA delivery within cultured mammalian cells. Two series of polymers were synthesized, one with variation in oligoethyleneamine stoichiometry, Cd1 46 , Cd2 44 , Cd3 49 , and Cd4 47 (1-4 oligoethyleneamines in the repeat unit, respectively and similar degree of polymerization, n w ¼ 44-49) and another with variation in polymer length (four ethyleneamines in the repeat unit), Cd4 27 , Cd4 47 , Cd4 93 , and Cd4 200 [n w ¼ 27, 47, 93, 200] via the ''click reaction''. The two series of polymers revealed efficient pDNA binding and compaction through gel electrophoresis, dynamic light scattering, and transmission electron microscopy experiments. The DNase protection assay showed a decrease in pDNA degradation with an increase in the polymer amine stoichiometry, where polymer Cd3 49 and all of the Cd4 analogs completely protected pDNA for up to 8 h in serum. The cellular uptake and gene expression profiles were examined in HeLa cells, which similarly demonstrated that both the series of polymers had high pDNA delivery where, Cd3 49 and Cd4 93 had the most effective luciferase gene expression. In addition, the cell viability profiles were quite high with all of the structures.

Topics in current chemistry, 2010

Carbohydrates have been investigated and developed as delivery vehicles for shuttling nucleic aci... more Carbohydrates have been investigated and developed as delivery vehicles for shuttling nucleic acids into cells. In this review, we present the state of the art in carbohydrate-based polymeric vehicles for nucleic acid delivery, with the focus on the recent successes in preclinical models, both in vitro and in vivo. Polymeric scaffolds based on the natural polysaccharides chitosan, hyaluronan, pullulan, dextran, and schizophyllan each have unique properties and potential for modification, and these results are discussed with the focus on facile synthetic routes and favorable performance in biological systems. Many of these carbohydrates have been used to develop alternative types of biomaterials for nucleic acid delivery to typical polyplexes, and these novel materials are discussed. Also presented are polymeric vehicles that incorporate copolymerized carbohydrates into polymer backbones based on polyethylenimine and polylysine and their effect on transfection and biocompatibility. Unique scaffolds, such as clusters and polymers based on cyclodextrin (CD), are also discussed, with the focus on recent successes in vivo and in the clinic. These results are presented with the emphasis on the role of carbohydrate and charge on transfection. Use of carbohydrates as molecular recognition ligands for cell-type specific delivery is also briefly reviewed. We contend that carbohydrates have contributed

ABSTRACTIn bacteria that live in hosts whose terminal sugar is a sialic acid, Glucosamine 6-phosp... more ABSTRACTIn bacteria that live in hosts whose terminal sugar is a sialic acid, Glucosamine 6-phosphate deaminase (NagB) catalyzes the last step in the conversion of sialic acid into Fructose-6-phosphate, which enters the glycolytic pathway. The enzyme exists as a hexamer in Gram-negative bacteria and is shown to be allosterically regulated. In Gram-positive bacteria, it exists as a monomer and lacks allosteric regulation. Our identification of a dimeric Gram-negative bacterial NagB motivated us to characterize the structural basis of the various oligomeric forms. We characterized the crystal structures of NagB from two Gram-negative pathogens, Haemophilus influenzae (Hi) and Pasturella multocida (Pm). The Hi-NagB is active as a hexamer, while Pm-NagB is active as a dimer. We confirm that this is not a crystallographic artifact by cryo-electron microscopy. Both Hi-NagB and Pm-NagB contain the C-terminal helix, and the residues in the interface involved in oligomerization are conserved...

Biomaterials, 2007

Cationic polymers are currently being studied as non-viral vectors to deliver therapeutic DNA int... more Cationic polymers are currently being studied as non-viral vectors to deliver therapeutic DNA into cells. In this study, a series of trehalose-based glycopolymers containing four secondary amines in the repeat unit were synthesized via the 'click reaction' [degrees of polymerization (n w) ¼ 35, 53, 75, or 100] to elucidate how the polymer length affects the bioactivity. The four structures bound and charge-neutralized pDNA with similar affinity that was independent of the length, as determined through gel electrophoresis, heparin competitive displacement, and isothermal titration calorimetric assays. Dynamic light scattering measurements revealed that the polyplexes formed with the longer polymers (n w ¼ 53, 75, or 100) inhibited flocculation in media containing serum, whereas the polyplexes formed with the shorter polymer (n w ¼ 35) aggregated rapidly. Similar results were observed via transmission electron microscopy studies, where the nanoparticles formed with the polymers having longer degrees of polymerization showed discrete particles in media containing 10% serum. Transfection experiments revealed that the polymers exhibited low cytotoxicity at low N/P ratios and could facilitate high cellular uptake and gene expression in HeLa and H9c2(2-1) cells, and the results were dependent on the degrees of polymerization (longer polymers yielded higher transfection and toxicity).

Microbial biotechnology, 2018

The process of obtaining a well-expressing, soluble and correctly folded constructs can be made e... more The process of obtaining a well-expressing, soluble and correctly folded constructs can be made easier and quicker by automating the optimization of cloning, expression and purification. While there are many semiautomated pipelines available for cloning, expression and purification, there is hardly any pipeline that involves complete automation. Here, we achieve complete automation of all the steps involved in cloning and in vivo expression screening. This is demonstrated using 18 genes involved in sialic acid catabolism and the surface sialylation pathway. Our main objective was to clone these genes into a His-tagged Gateway vector, followed by their small-scale expression optimization in vivo. The constructs that showed best soluble expression were then selected for purification studies and scaled up for crystallization studies. Our technique allowed us to quickly find conditions for producing significant quantities of soluble proteins in Escherichia coli, their large-scale purifi...

Topics in Current Chemistry, 2010

Carbohydrates have been investigated and developed as delivery vehicles for shuttling nucleic aci... more Carbohydrates have been investigated and developed as delivery vehicles for shuttling nucleic acids into cells. In this review, we present the state of the art in carbohydrate-based polymeric vehicles for nucleic acid delivery, with the focus on the recent successes in preclinical models, both in vitro and in vivo. Polymeric scaffolds based on the natural polysaccharides chitosan, hyaluronan, pullulan, dextran, and schizophyllan each have unique properties and potential for modification, and these results are discussed with the focus on facile synthetic routes and favorable performance in biological systems. Many of these carbohydrates have been used to develop alternative types of biomaterials for nucleic acid delivery to typical polyplexes, and these novel materials are discussed. Also presented are polymeric vehicles that incorporate copolymerized carbohydrates into polymer backbones based on polyethylenimine and polylysine and their effect on transfection and biocompatibility. Unique scaffolds, such as clusters and polymers based on cyclodextrin (CD), are also discussed, with the focus on recent successes in vivo and in the clinic. These results are presented with the emphasis on the role of carbohydrate and charge on transfection. Use of carbohydrates as molecular recognition ligands for cell-type specific delivery is also briefly reviewed. We contend that carbohydrates have contributed

Journal of the American Chemical Society, 2008

Herein, a novel series of multivalent polycationic beta-cyclodextrin &amp... more Herein, a novel series of multivalent polycationic beta-cyclodextrin "click clusters" with discrete molecular weight have been synthesized, characterized, and examined as therapeutic pDNA carriers. The materials were creatively designed based on a beta-cyclodextrin core to impart a biocompatible multivalent architecture and oligoethyleneamine arms to facilitate pDNA binding, encapsulation, and cellular uptake. An acetylated-per-azido-beta-cyclodextrin (4) was reacted with series of alkyne dendrons (7a-e) (containing one to five ethyleneamine units) using copper-catalyzed 1,3-dipolar cycloaddition, to form a series of click clusters (9a-e) bearing 1,2,3-triazole linkers. Gel electrophoresis experiments, dynamic light scattering, and transmission electron microscopy revealed that the macromolecules bind and compact pDNA into spherical nanoparticles in the size range of 80-130 nm. The polycations protect pDNA against nuclease degradation, where structures 9c, 9d, and 9e did not allow pDNA degradation in the presence of serum for up to 48 h. The cellular uptake profiles were evaluated in Opti-MEM and demonstrate that all the click clusters efficiently deliver Cy5-labeled pDNA into HeLa and H9c2 (2-1) cells, and compounds 9d and 9e yielded efficacy similar to that of the positive controls, Jet-PEI and Superfect. Furthermore, the luciferase gene delivery experiments revealed that the level of reporter gene expression increased with an increase in oligoethyleneamine number within the cluster arms. The cytotoxicity profiles of these materials were evaluated by protein, MTT, and LDH assays, which demonstrate that all the click clusters remain nontoxic within the expected dosage range while the positive controls, Jet PEI and Superfect, were highly cytotoxic. In particular, 9d and 9e were the most effective and promising polycationic vehicles to be further optimized for future systemic delivery experiments.

Biomaterials, 2007

Cationic polymers are currently being studied as non-viral vectors to deliver therapeutic DNA int... more Cationic polymers are currently being studied as non-viral vectors to deliver therapeutic DNA into cells. In this study, a series of trehalose-based glycopolymers containing four secondary amines in the repeat unit were synthesized via the 'click reaction' [degrees of polymerization (n w) ¼ 35, 53, 75, or 100] to elucidate how the polymer length affects the bioactivity. The four structures bound and charge-neutralized pDNA with similar affinity that was independent of the length, as determined through gel electrophoresis, heparin competitive displacement, and isothermal titration calorimetric assays. Dynamic light scattering measurements revealed that the polyplexes formed with the longer polymers (n w ¼ 53, 75, or 100) inhibited flocculation in media containing serum, whereas the polyplexes formed with the shorter polymer (n w ¼ 35) aggregated rapidly. Similar results were observed via transmission electron microscopy studies, where the nanoparticles formed with the polymers having longer degrees of polymerization showed discrete particles in media containing 10% serum. Transfection experiments revealed that the polymers exhibited low cytotoxicity at low N/P ratios and could facilitate high cellular uptake and gene expression in HeLa and H9c2(2-1) cells, and the results were dependent on the degrees of polymerization (longer polymers yielded higher transfection and toxicity).

Biomaterials, 2009

Herein, we report the efficient synthesis of high molecular weight polymers (up to 331 kDa) that ... more Herein, we report the efficient synthesis of high molecular weight polymers (up to 331 kDa) that contain b-cyclodextrin within the polymer backbone and the examination of these structures for pDNA delivery within cultured mammalian cells. Two series of polymers were synthesized, one with variation in oligoethyleneamine stoichiometry, Cd1 46 , Cd2 44 , Cd3 49 , and Cd4 47 (1-4 oligoethyleneamines in the repeat unit, respectively and similar degree of polymerization, n w ¼ 44-49) and another with variation in polymer length (four ethyleneamines in the repeat unit), Cd4 27 , Cd4 47 , Cd4 93 , and Cd4 200 [n w ¼ 27, 47, 93, 200] via the ''click reaction''. The two series of polymers revealed efficient pDNA binding and compaction through gel electrophoresis, dynamic light scattering, and transmission electron microscopy experiments. The DNase protection assay showed a decrease in pDNA degradation with an increase in the polymer amine stoichiometry, where polymer Cd3 49 and all of the Cd4 analogs completely protected pDNA for up to 8 h in serum. The cellular uptake and gene expression profiles were examined in HeLa cells, which similarly demonstrated that both the series of polymers had high pDNA delivery where, Cd3 49 and Cd4 93 had the most effective luciferase gene expression. In addition, the cell viability profiles were quite high with all of the structures.

Journal of the American Chemical Society, 2006

All reagents used in the synthesis, if not specified, were obtained from Aldrich Chemical Co. (Mi... more All reagents used in the synthesis, if not specified, were obtained from Aldrich Chemical Co. (Milwaukee, WI) and used without further purification. Methanol, triethylamine (TEA) and dichloromethane used in all experiments were purified according to conventional methods. 1 Plasmid DNA (pDNA, pCMVβ) was purchased from Plasmidfactory (Bielefeld, Germany) and gWiz-luc DNA was purchased from Aldevron (Fargo, ND). Fluorescein-labeled pDNA was purchased from Mirus (Madison, WI). Mass spectra were obtained with an IonSpec ESI mass spectrometer in positive ion mode. IR spectra were measured with a Perkin Elmer Spectrum One Fourier transform infrared spectrometer as KBr pellets. 1 H and 13 C NMR were S2 recorded on a Bruker AV 400 MHz spectrometer. The 1 H NMR data are reported as follows: chemical shift (δ ppm), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, bs = broad singlet, bm = broad multiplet), J coupling constant (Hz), and the peak integration. Elemental analyses were conducted by the Microanalysis Laboratory at the University of Illinois (Urbana, IL). Melting points were determined with a MEL−TEMP (Laboratory Devices Inc. USA) apparatus. Fluorescence measurements were performed on a Varian Cary spectrofluorometer. Cell culture media and supplements were purchased from Gibco/Invitrogen (Carlsbad, CA). HeLa cells were purchased from ATCC (Rockville, MD). The luciferase assays were completed with a Promega Luciferase Assay kit (Madison, WI). 6, 6'-Diiodo-6, 6'-dideoxy-D-trehalose 2. 2 To a stirred solution of anhydrous α,α'-D-trehalose (1) (8.00 g, 23.4 mmol) in dry DMF (200 mL), triphenylphosphine (30.65 g, 117 mmol) and I 2 (23.75 g, 93.6 mmol) were added under N 2. The mixture was heated to 80 °C for 1.5 h and then evaporated to about 2/3 the volume of DMF with a rotary evaporator. Methanol (300 mL) was added and the solution was adjusted to a pH of 9 with solid NaOMe. This mixture was stirred at room temperature for 30 min and neutralized with Amberlyst 15 (H +) resin. The resin was filtered off and washed with MeOH. The combined filtrates were concentrated, triturated with water (250 mL), and the white precipitate was filtered off. The aqueous solution was evaporated to yield an amorphous solid (9.30 g, 71%).

Herein, we report the efficient synthesis of high molecular weight polymers (up to 331 kDa) that ... more Herein, we report the efficient synthesis of high molecular weight polymers (up to 331 kDa) that contain b-cyclodextrin within the polymer backbone and the examination of these structures for pDNA delivery within cultured mammalian cells. Two series of polymers were synthesized, one with variation in oligoethyleneamine stoichiometry, Cd1 46 , Cd2 44 , Cd3 49 , and Cd4 47 (1-4 oligoethyleneamines in the repeat unit, respectively and similar degree of polymerization, n w ¼ 44-49) and another with variation in polymer length (four ethyleneamines in the repeat unit), Cd4 27 , Cd4 47 , Cd4 93 , and Cd4 200 [n w ¼ 27, 47, 93, 200] via the ''click reaction''. The two series of polymers revealed efficient pDNA binding and compaction through gel electrophoresis, dynamic light scattering, and transmission electron microscopy experiments. The DNase protection assay showed a decrease in pDNA degradation with an increase in the polymer amine stoichiometry, where polymer Cd3 49 and all of the Cd4 analogs completely protected pDNA for up to 8 h in serum. The cellular uptake and gene expression profiles were examined in HeLa cells, which similarly demonstrated that both the series of polymers had high pDNA delivery where, Cd3 49 and Cd4 93 had the most effective luciferase gene expression. In addition, the cell viability profiles were quite high with all of the structures.

Biomaterials, Feb 1, 2009

Herein, we report the efficient synthesis of high molecular weight polymers (up to 331 kDa) that ... more Herein, we report the efficient synthesis of high molecular weight polymers (up to 331 kDa) that contain b-cyclodextrin within the polymer backbone and the examination of these structures for pDNA delivery within cultured mammalian cells. Two series of polymers were synthesized, one with variation in oligoethyleneamine stoichiometry, Cd1 46 , Cd2 44 , Cd3 49 , and Cd4 47 (1-4 oligoethyleneamines in the repeat unit, respectively and similar degree of polymerization, n w ¼ 44-49) and another with variation in polymer length (four ethyleneamines in the repeat unit), Cd4 27 , Cd4 47 , Cd4 93 , and Cd4 200 [n w ¼ 27, 47, 93, 200] via the ''click reaction''. The two series of polymers revealed efficient pDNA binding and compaction through gel electrophoresis, dynamic light scattering, and transmission electron microscopy experiments. The DNase protection assay showed a decrease in pDNA degradation with an increase in the polymer amine stoichiometry, where polymer Cd3 49 and all of the Cd4 analogs completely protected pDNA for up to 8 h in serum. The cellular uptake and gene expression profiles were examined in HeLa cells, which similarly demonstrated that both the series of polymers had high pDNA delivery where, Cd3 49 and Cd4 93 had the most effective luciferase gene expression. In addition, the cell viability profiles were quite high with all of the structures.

Topics in current chemistry, 2010

Carbohydrates have been investigated and developed as delivery vehicles for shuttling nucleic aci... more Carbohydrates have been investigated and developed as delivery vehicles for shuttling nucleic acids into cells. In this review, we present the state of the art in carbohydrate-based polymeric vehicles for nucleic acid delivery, with the focus on the recent successes in preclinical models, both in vitro and in vivo. Polymeric scaffolds based on the natural polysaccharides chitosan, hyaluronan, pullulan, dextran, and schizophyllan each have unique properties and potential for modification, and these results are discussed with the focus on facile synthetic routes and favorable performance in biological systems. Many of these carbohydrates have been used to develop alternative types of biomaterials for nucleic acid delivery to typical polyplexes, and these novel materials are discussed. Also presented are polymeric vehicles that incorporate copolymerized carbohydrates into polymer backbones based on polyethylenimine and polylysine and their effect on transfection and biocompatibility. Unique scaffolds, such as clusters and polymers based on cyclodextrin (CD), are also discussed, with the focus on recent successes in vivo and in the clinic. These results are presented with the emphasis on the role of carbohydrate and charge on transfection. Use of carbohydrates as molecular recognition ligands for cell-type specific delivery is also briefly reviewed. We contend that carbohydrates have contributed

ABSTRACTIn bacteria that live in hosts whose terminal sugar is a sialic acid, Glucosamine 6-phosp... more ABSTRACTIn bacteria that live in hosts whose terminal sugar is a sialic acid, Glucosamine 6-phosphate deaminase (NagB) catalyzes the last step in the conversion of sialic acid into Fructose-6-phosphate, which enters the glycolytic pathway. The enzyme exists as a hexamer in Gram-negative bacteria and is shown to be allosterically regulated. In Gram-positive bacteria, it exists as a monomer and lacks allosteric regulation. Our identification of a dimeric Gram-negative bacterial NagB motivated us to characterize the structural basis of the various oligomeric forms. We characterized the crystal structures of NagB from two Gram-negative pathogens, Haemophilus influenzae (Hi) and Pasturella multocida (Pm). The Hi-NagB is active as a hexamer, while Pm-NagB is active as a dimer. We confirm that this is not a crystallographic artifact by cryo-electron microscopy. Both Hi-NagB and Pm-NagB contain the C-terminal helix, and the residues in the interface involved in oligomerization are conserved...

Biomaterials, 2007

Cationic polymers are currently being studied as non-viral vectors to deliver therapeutic DNA int... more Cationic polymers are currently being studied as non-viral vectors to deliver therapeutic DNA into cells. In this study, a series of trehalose-based glycopolymers containing four secondary amines in the repeat unit were synthesized via the 'click reaction' [degrees of polymerization (n w) ¼ 35, 53, 75, or 100] to elucidate how the polymer length affects the bioactivity. The four structures bound and charge-neutralized pDNA with similar affinity that was independent of the length, as determined through gel electrophoresis, heparin competitive displacement, and isothermal titration calorimetric assays. Dynamic light scattering measurements revealed that the polyplexes formed with the longer polymers (n w ¼ 53, 75, or 100) inhibited flocculation in media containing serum, whereas the polyplexes formed with the shorter polymer (n w ¼ 35) aggregated rapidly. Similar results were observed via transmission electron microscopy studies, where the nanoparticles formed with the polymers having longer degrees of polymerization showed discrete particles in media containing 10% serum. Transfection experiments revealed that the polymers exhibited low cytotoxicity at low N/P ratios and could facilitate high cellular uptake and gene expression in HeLa and H9c2(2-1) cells, and the results were dependent on the degrees of polymerization (longer polymers yielded higher transfection and toxicity).

Microbial biotechnology, 2018

The process of obtaining a well-expressing, soluble and correctly folded constructs can be made e... more The process of obtaining a well-expressing, soluble and correctly folded constructs can be made easier and quicker by automating the optimization of cloning, expression and purification. While there are many semiautomated pipelines available for cloning, expression and purification, there is hardly any pipeline that involves complete automation. Here, we achieve complete automation of all the steps involved in cloning and in vivo expression screening. This is demonstrated using 18 genes involved in sialic acid catabolism and the surface sialylation pathway. Our main objective was to clone these genes into a His-tagged Gateway vector, followed by their small-scale expression optimization in vivo. The constructs that showed best soluble expression were then selected for purification studies and scaled up for crystallization studies. Our technique allowed us to quickly find conditions for producing significant quantities of soluble proteins in Escherichia coli, their large-scale purifi...

Topics in Current Chemistry, 2010

Carbohydrates have been investigated and developed as delivery vehicles for shuttling nucleic aci... more Carbohydrates have been investigated and developed as delivery vehicles for shuttling nucleic acids into cells. In this review, we present the state of the art in carbohydrate-based polymeric vehicles for nucleic acid delivery, with the focus on the recent successes in preclinical models, both in vitro and in vivo. Polymeric scaffolds based on the natural polysaccharides chitosan, hyaluronan, pullulan, dextran, and schizophyllan each have unique properties and potential for modification, and these results are discussed with the focus on facile synthetic routes and favorable performance in biological systems. Many of these carbohydrates have been used to develop alternative types of biomaterials for nucleic acid delivery to typical polyplexes, and these novel materials are discussed. Also presented are polymeric vehicles that incorporate copolymerized carbohydrates into polymer backbones based on polyethylenimine and polylysine and their effect on transfection and biocompatibility. Unique scaffolds, such as clusters and polymers based on cyclodextrin (CD), are also discussed, with the focus on recent successes in vivo and in the clinic. These results are presented with the emphasis on the role of carbohydrate and charge on transfection. Use of carbohydrates as molecular recognition ligands for cell-type specific delivery is also briefly reviewed. We contend that carbohydrates have contributed

Journal of the American Chemical Society, 2008

Herein, a novel series of multivalent polycationic beta-cyclodextrin &amp... more Herein, a novel series of multivalent polycationic beta-cyclodextrin "click clusters" with discrete molecular weight have been synthesized, characterized, and examined as therapeutic pDNA carriers. The materials were creatively designed based on a beta-cyclodextrin core to impart a biocompatible multivalent architecture and oligoethyleneamine arms to facilitate pDNA binding, encapsulation, and cellular uptake. An acetylated-per-azido-beta-cyclodextrin (4) was reacted with series of alkyne dendrons (7a-e) (containing one to five ethyleneamine units) using copper-catalyzed 1,3-dipolar cycloaddition, to form a series of click clusters (9a-e) bearing 1,2,3-triazole linkers. Gel electrophoresis experiments, dynamic light scattering, and transmission electron microscopy revealed that the macromolecules bind and compact pDNA into spherical nanoparticles in the size range of 80-130 nm. The polycations protect pDNA against nuclease degradation, where structures 9c, 9d, and 9e did not allow pDNA degradation in the presence of serum for up to 48 h. The cellular uptake profiles were evaluated in Opti-MEM and demonstrate that all the click clusters efficiently deliver Cy5-labeled pDNA into HeLa and H9c2 (2-1) cells, and compounds 9d and 9e yielded efficacy similar to that of the positive controls, Jet-PEI and Superfect. Furthermore, the luciferase gene delivery experiments revealed that the level of reporter gene expression increased with an increase in oligoethyleneamine number within the cluster arms. The cytotoxicity profiles of these materials were evaluated by protein, MTT, and LDH assays, which demonstrate that all the click clusters remain nontoxic within the expected dosage range while the positive controls, Jet PEI and Superfect, were highly cytotoxic. In particular, 9d and 9e were the most effective and promising polycationic vehicles to be further optimized for future systemic delivery experiments.

Biomaterials, 2007

Cationic polymers are currently being studied as non-viral vectors to deliver therapeutic DNA int... more Cationic polymers are currently being studied as non-viral vectors to deliver therapeutic DNA into cells. In this study, a series of trehalose-based glycopolymers containing four secondary amines in the repeat unit were synthesized via the 'click reaction' [degrees of polymerization (n w) ¼ 35, 53, 75, or 100] to elucidate how the polymer length affects the bioactivity. The four structures bound and charge-neutralized pDNA with similar affinity that was independent of the length, as determined through gel electrophoresis, heparin competitive displacement, and isothermal titration calorimetric assays. Dynamic light scattering measurements revealed that the polyplexes formed with the longer polymers (n w ¼ 53, 75, or 100) inhibited flocculation in media containing serum, whereas the polyplexes formed with the shorter polymer (n w ¼ 35) aggregated rapidly. Similar results were observed via transmission electron microscopy studies, where the nanoparticles formed with the polymers having longer degrees of polymerization showed discrete particles in media containing 10% serum. Transfection experiments revealed that the polymers exhibited low cytotoxicity at low N/P ratios and could facilitate high cellular uptake and gene expression in HeLa and H9c2(2-1) cells, and the results were dependent on the degrees of polymerization (longer polymers yielded higher transfection and toxicity).

Biomaterials, 2009

Herein, we report the efficient synthesis of high molecular weight polymers (up to 331 kDa) that ... more Herein, we report the efficient synthesis of high molecular weight polymers (up to 331 kDa) that contain b-cyclodextrin within the polymer backbone and the examination of these structures for pDNA delivery within cultured mammalian cells. Two series of polymers were synthesized, one with variation in oligoethyleneamine stoichiometry, Cd1 46 , Cd2 44 , Cd3 49 , and Cd4 47 (1-4 oligoethyleneamines in the repeat unit, respectively and similar degree of polymerization, n w ¼ 44-49) and another with variation in polymer length (four ethyleneamines in the repeat unit), Cd4 27 , Cd4 47 , Cd4 93 , and Cd4 200 [n w ¼ 27, 47, 93, 200] via the ''click reaction''. The two series of polymers revealed efficient pDNA binding and compaction through gel electrophoresis, dynamic light scattering, and transmission electron microscopy experiments. The DNase protection assay showed a decrease in pDNA degradation with an increase in the polymer amine stoichiometry, where polymer Cd3 49 and all of the Cd4 analogs completely protected pDNA for up to 8 h in serum. The cellular uptake and gene expression profiles were examined in HeLa cells, which similarly demonstrated that both the series of polymers had high pDNA delivery where, Cd3 49 and Cd4 93 had the most effective luciferase gene expression. In addition, the cell viability profiles were quite high with all of the structures.

Journal of the American Chemical Society, 2006

All reagents used in the synthesis, if not specified, were obtained from Aldrich Chemical Co. (Mi... more All reagents used in the synthesis, if not specified, were obtained from Aldrich Chemical Co. (Milwaukee, WI) and used without further purification. Methanol, triethylamine (TEA) and dichloromethane used in all experiments were purified according to conventional methods. 1 Plasmid DNA (pDNA, pCMVβ) was purchased from Plasmidfactory (Bielefeld, Germany) and gWiz-luc DNA was purchased from Aldevron (Fargo, ND). Fluorescein-labeled pDNA was purchased from Mirus (Madison, WI). Mass spectra were obtained with an IonSpec ESI mass spectrometer in positive ion mode. IR spectra were measured with a Perkin Elmer Spectrum One Fourier transform infrared spectrometer as KBr pellets. 1 H and 13 C NMR were S2 recorded on a Bruker AV 400 MHz spectrometer. The 1 H NMR data are reported as follows: chemical shift (δ ppm), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, bs = broad singlet, bm = broad multiplet), J coupling constant (Hz), and the peak integration. Elemental analyses were conducted by the Microanalysis Laboratory at the University of Illinois (Urbana, IL). Melting points were determined with a MEL−TEMP (Laboratory Devices Inc. USA) apparatus. Fluorescence measurements were performed on a Varian Cary spectrofluorometer. Cell culture media and supplements were purchased from Gibco/Invitrogen (Carlsbad, CA). HeLa cells were purchased from ATCC (Rockville, MD). The luciferase assays were completed with a Promega Luciferase Assay kit (Madison, WI). 6, 6'-Diiodo-6, 6'-dideoxy-D-trehalose 2. 2 To a stirred solution of anhydrous α,α'-D-trehalose (1) (8.00 g, 23.4 mmol) in dry DMF (200 mL), triphenylphosphine (30.65 g, 117 mmol) and I 2 (23.75 g, 93.6 mmol) were added under N 2. The mixture was heated to 80 °C for 1.5 h and then evaporated to about 2/3 the volume of DMF with a rotary evaporator. Methanol (300 mL) was added and the solution was adjusted to a pH of 9 with solid NaOMe. This mixture was stirred at room temperature for 30 min and neutralized with Amberlyst 15 (H +) resin. The resin was filtered off and washed with MeOH. The combined filtrates were concentrated, triturated with water (250 mL), and the white precipitate was filtered off. The aqueous solution was evaporated to yield an amorphous solid (9.30 g, 71%).

Herein, we report the efficient synthesis of high molecular weight polymers (up to 331 kDa) that ... more Herein, we report the efficient synthesis of high molecular weight polymers (up to 331 kDa) that contain b-cyclodextrin within the polymer backbone and the examination of these structures for pDNA delivery within cultured mammalian cells. Two series of polymers were synthesized, one with variation in oligoethyleneamine stoichiometry, Cd1 46 , Cd2 44 , Cd3 49 , and Cd4 47 (1-4 oligoethyleneamines in the repeat unit, respectively and similar degree of polymerization, n w ¼ 44-49) and another with variation in polymer length (four ethyleneamines in the repeat unit), Cd4 27 , Cd4 47 , Cd4 93 , and Cd4 200 [n w ¼ 27, 47, 93, 200] via the ''click reaction''. The two series of polymers revealed efficient pDNA binding and compaction through gel electrophoresis, dynamic light scattering, and transmission electron microscopy experiments. The DNase protection assay showed a decrease in pDNA degradation with an increase in the polymer amine stoichiometry, where polymer Cd3 49 and all of the Cd4 analogs completely protected pDNA for up to 8 h in serum. The cellular uptake and gene expression profiles were examined in HeLa cells, which similarly demonstrated that both the series of polymers had high pDNA delivery where, Cd3 49 and Cd4 93 had the most effective luciferase gene expression. In addition, the cell viability profiles were quite high with all of the structures.