72 results on '"Pack DW"'
Search Results
2. Polymer/Nanoceria Hybrid Polyplexes for Gene and Antioxidant Delivery.
- Author
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Mott L, Hancock M, Grulke EA, and Pack DW
- Subjects
- Humans, HeLa Cells, DNA genetics, DNA metabolism, Polymers, Antioxidants
- Abstract
Various diseases, including cancers and inflammatory diseases, are characterized by a disruption of redox homeostasis, suggesting the need for synergistic treatments involving co-delivery of gene therapies and free radical scavengers. In this report, polyethylenimine (PEI), nanoceria (NC), and DNA were complexed to form nanoparticles providing simultaneous delivery of a gene and an antioxidant. NC was coated in citric acid to provide stable, 4 nm particles that electrostatically bound PEI/DNA polyplexes. The resulting ternary particles transfected HeLa cells with similar efficiency to that of ternary polyplexes comprising 15 kDa poly-l-α-glutamic acid/PEI/DNA while providing smaller particle sizes by more than 100 nm. NC/PEI/DNA polyplexes exhibited enhanced radical-scavenging activity compared to free NC, and oxidative stress from the superoxide-generating agent, menadione, could be completely reversed by the delivery of NC/PEI/DNA polyplexes. Transfection by NC/PEI/DNA polyplexes was demonstrated to occur efficiently through caveolin-mediated endocytosis and macropinocytosis. Co-delivery of genes encoding reactive oxygen species-scavenging proteins, transcription factors, growth factors, tumor suppressors, or anti-inflammatory genes with NC, therefore, may be a promising strategy in synergistic therapeutics.
- Published
- 2023
- Full Text
- View/download PDF
3. Enhanced Gene Delivery and CRISPR/Cas9 Homology-Directed Repair in Serum by Minimally Succinylated Polyethylenimine.
- Author
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Uddin N, Warriner LW, Pack DW, and DeRouchey JE
- Subjects
- Gene Knock-In Techniques, HEK293 Cells, HeLa Cells, Humans, Polyethyleneimine analogs & derivatives, Recombinational DNA Repair, CRISPR-Cas Systems genetics, Gene Transfer Techniques, Genetic Therapy methods, Polyethyleneimine chemistry
- Abstract
Gene therapy aims to treat patients by altering or controlling gene expression. The field of gene therapy has had increasing success in recent years primarily using viral-based approaches; however, there is still significant interest toward the use of polymeric materials due to their potential as flexible, low-cost scaffolds for gene delivery that do not suffer the mutagenesis and immunogenicity concerns of viral vectors. To address the challenges of efficiency and biocompatibility, a series of zwitterion-like polyethylenimine derivatives (zPEIs) were produced via the succinylation of 2-11.5% of polyethylenimine (PEI) amines. With increasing modification, zPEI polyplexes exhibited decreased serum-protein aggregation and dissociated more easily in the presence of a competitor polyanion when compared to unmodified PEI. Surprisingly, the gene delivery mediated in the presence of serum showed that succinylation of as few as 2% of PEI amines resulted in transgene expression 260- to 480-fold higher than that of unmodified PEI and 50- to 65-fold higher than that of commercial PEI-PEG
2k in HEK293 and HeLa cells, respectively. Remarkably, the same zPEIs also produced 16-fold greater efficiency of CRISPR/Cas9 gene knock-in compared to unmodified PEI in the presence of serum. In addition, we show that 2% succinylation does not significantly decrease polymer/DNA binding ability or serum protein interaction to a significant extent, yet this small modification is still sufficient to provide a remarkable increase in transgene expression and gene knock-in in the presence of serum.- Published
- 2021
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4. Cyanuric chloride as the basis for compositionally diverse lipids.
- Author
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Nardo D, Akers CM, Cheung NE, Isom CM, Spaude JT, Pack DW, and Venditto VJ
- Abstract
Cyanuric chloride has been utilized in the development of new synthetic lipid compounds using two differing schemes. The resulting lipids, presented in this manuscript, were characterized and evaluated for their ability to form nanoparticles and subsequently tested for their utility in various biological applications, including gene delivery and immunization. Of the 12 lipids synthesized, 8 formed nanoparticles that remained stable, based on dynamic light scattering, for at least one month. The compounds were then assessed for their toxicity, and subsequently tested for their ability to encapsulate drugs, genes and peptides. While the compounds did not seem to encapsulate carboxyfluorescein, we demonstrate that these lipids are capable of plasmid delivery in vitro , and inducing antibody profiles similar to other hydrophobic anchors in liposomal peptide vaccines. This strategy for accessing diverse lipid compounds offers a way to easily optimize lipid-based therapeutics for research in an expedited manner., Competing Interests: There are no conflicts of interest to declare., (This journal is © The Royal Society of Chemistry.)
- Published
- 2021
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5. Succinylated Polyethylenimine Derivatives Greatly Enhance Polyplex Serum Stability and Gene Delivery In Vitro.
- Author
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Warriner LW, Duke JR 3rd, Pack DW, and DeRouchey JE
- Subjects
- Breast Neoplasms pathology, Cell Survival, DNA chemistry, Female, Gene Transfer Techniques, Genetic Therapy, HeLa Cells, Humans, Plasmids administration & dosage, Plasmids chemistry, Tumor Cells, Cultured, Breast Neoplasms genetics, DNA administration & dosage, Polyethyleneimine chemistry, Polymers chemistry, Serum chemistry, Succinic Acid chemistry, Transfection methods
- Abstract
Polymeric materials provide particularly attractive scaffolds for the creation of supramolecular bioconjugates for the delivery of nucleic acids but typically lack the efficiency and biocompatibility to be clinically relevant. To address both issues, we produced zwitterion-like derivatives of polyethylenimine via succinylation of primary and secondary amines (zPEI). Polymers were generated with 9-55% of the amines modified (zPEI X, where X indicates the percentage of amines succinylated). Characterization of polymer/DNA interactions revealed that the presence of succinyl groups decreased the protonation constant of zPEI, resulting in both a decreased buffering capacity and polyplexes that dissociated in the presence of lower amounts of a competing counteranion compared to unmodified PEI. zPEI polyplexes also exhibited decreased aggregation in the presence of serum proteins. In the absence of serum, transfections with zPEI/DNA polyplexes exhibited similar or slightly improved transgene expression compared to unmodified PEI/DNA polyplexes. More importantly, zPEI 9-25 increased transgene expression up to 51-fold upon transfection in the presence of serum compared to PEI/DNA, while higher succinylation decreased gene delivery activity. Gene delivery mediated by zPEI 9/DNA polyplexes in the presence of serum was equal to or greater than unmodified PEI/DNA polyplexes in the absence of serum. The data suggest that succinylation increased gene transfection by decreasing polymer/DNA interaction strength, which may allow for more facile polyplex unpackaging, and/or increased stability of polyplex size and inhibition of aggregation in the presence of serum. However, it appears there exists a balance between the positive effects of succinylation and the need for sufficient polymer/DNA binding to condense and protect the cargo.
- Published
- 2018
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6. Evaluation of FOXC1 as a therapeutic target for basal-like breast cancer.
- Author
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Mott L, Su K, and Pack DW
- Subjects
- Animals, Cell Line, Tumor, Female, Mice, Neoplasm Metastasis, Forkhead Transcription Factors biosynthesis, Forkhead Transcription Factors genetics, Gene Expression Regulation, Neoplastic, Mammary Neoplasms, Animal genetics, Mammary Neoplasms, Animal metabolism, Mammary Neoplasms, Animal pathology, Mammary Neoplasms, Animal therapy, Neoplasm Proteins biosynthesis, Neoplasm Proteins genetics
- Abstract
Basal-like breast cancer (BLBC) is a malignant carcinoma with aggressive motility and rapid growth. Accounting for 15% of breast cancers, BLBC often exhibits a poor prognosis and tends to metastasize to the brain and lungs. Because most BLBC display a triple-negative phenotype (ER-, PR-, and HER2-), conventional cytotoxic chemotherapy remains the only treatment option despite poor success and high rate of relapse. The overexpression of the forkhead-box transcription factor C1 (FOXC1) was recently identified as a biomarker of BLBC. Increased expression of FOXC1 was linked to excessive mobility and growth of BLBC cell lines, suggesting FOXC1 as a therapeutic target. In this study, siRNA-mediated knockdown of FOXC1 was confirmed to decrease the proliferation rate, migration, and invasion in a model BLBC-like cell line (4T1). 4T1 and 4T1-∆FOXC1 cells lacking FOXC1 expression (generated by CRISPR/Cas9) were used to evaluate the effects of FOXC1 expression in an orthotopic murine model of BLBC. No statistically significant difference in tumor volume was observed between 4T1 and 4T1-∆FOXC1 tumors. Furthermore, tumors metastasized to the liver and lungs to a similar degree regardless of FOXC1 expression. These data suggest that, despite positive results in vitro, FOXC1 may not be a promising therapeutic target for BLBC.
- Published
- 2018
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7. Prospects of siRNA applications in regenerative medicine.
- Author
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Mottaghitalab F, Rastegari A, Farokhi M, Dinarvand R, Hosseinkhani H, Ou KL, Pack DW, Mao C, Dinarvand M, Fatahi Y, and Atyabi F
- Subjects
- RNA, Small Interfering pharmacology, Regenerative Medicine, Tissue Engineering
- Abstract
Small interfering RNA (siRNA) has established its reputation in the field of tissue engineering owing to its ability to silence the proteins that inhibit tissue regeneration. siRNA is capable of regulating cellular behavior during tissue regeneration processes. The concept of using siRNA technology in regenerative medicine derived from its ability to inhibit the expression of target genes involved in defective tissues and the possibility to induce the expression of tissue-inductive factors that improve the tissue regeneration process. To date, siRNA has been used as a suppressive biomolecule in different tissues, such as nervous tissue, bone, cartilage, heart, kidney, and liver. Moreover, various delivery systems have been applied in order to deliver siRNA to the target tissues. This review will provide an in-depth discussion on the development of siRNA and their delivery systems and mechanisms of action in different tissues., (Copyright © 2017 Elsevier B.V. All rights reserved.)
- Published
- 2017
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8. Rapid and facile quantitation of polyplex endocytic trafficking.
- Author
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Lazebnik M and Pack DW
- Subjects
- Endosomes metabolism, HeLa Cells, Humans, Polyamines analysis, Polyelectrolytes, Polyethyleneimine analysis, RNA Interference, RNA, Small Interfering analysis, RNA, Small Interfering genetics, RNA, Small Interfering pharmacokinetics, Endocytosis, Polyamines metabolism, Polyethyleneimine metabolism, RNA, Small Interfering administration & dosage, Transfection
- Abstract
Design of safe and effective synthetic nucleic acid delivery vectors such as polycation/DNA or polycation/siRNA complexes (polyplexes) will be facilitated by quantitative understanding of the mechanisms by which such materials escort cargo from the cell surface to the nucleus. In particular, the mechanisms of cellular internalization by various endocytosis pathways and subsequent endocytic vesicle trafficking have been shown to strongly affect nucleic acid delivery efficiency. Fluorescence microscopy and subcellular fractionation methods are commonly employed to follow intracellular trafficking of biomolecules and nanoparticulate delivery systems such as polyplexes. However, it is difficult to obtain quantitative data from microscopy and subcellular fractionation is experimentally difficult and low throughput. We have developed a method for quantifying the transport of polyplexes through important endocytic vesicles. The method is based on polymerization of 3,3'-diaminobenzidine by endocytosed horseradish peroxidase, causing an increase in the vesicle density, resistance to being solubilized by detergent and quenching of fluorophores within the vesicles, which makes them easy to separate and quantify. Using this method in HeLa cells, we have observed polyethylenimine/siRNA polyplexes initially appearing in early endosomes and rapidly moving to other compartments within 30min post-transfection. At the same time, we observed the kinetics of accumulation of the polyplexes in lysosomes at a similar rate. The results from the new method are consistent with similar measurements by confocal fluorescence microscopy and subcellular fractionation of endocytic vesicles on a Percoll gradient. The relative ease of this new method will aid investigation of gene delivery mechanisms by providing the means to rapidly quantify endocytic trafficking of polyplexes and other vectors., (Copyright © 2017 Elsevier B.V. All rights reserved.)
- Published
- 2017
- Full Text
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9. Endocytic Transport of Polyplex and Lipoplex siRNA Vectors in HeLa Cells.
- Author
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Lazebnik M, Keswani RK, and Pack DW
- Subjects
- Cell Culture Techniques, Cell Survival, Cholesterol chemistry, Drug Delivery Systems, Drug Liberation, Endocytosis, Fatty Acids, Monounsaturated metabolism, Gene Silencing, HeLa Cells, Humans, Liposomes, Phosphatidylethanolamines metabolism, Quaternary Ammonium Compounds metabolism, Fatty Acids, Monounsaturated chemistry, Phosphatidylethanolamines chemistry, Polyethyleneimine chemistry, Quaternary Ammonium Compounds chemistry, RNA, Small Interfering metabolism
- Abstract
Purpose: siRNA may be delivered as electrostatic complexes with cationic lipids (lipoplexes) or polycations (polyplexes). The purpose of this project was to determine the effect of cellular internalization mechanism(s) on siRNA-mediated gene silencing efficiency., Methods: Lipoplexes were formed comprising siRNA and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP), cholesterol and dioleoyl phosphatidylethanolamine (DOPE), and polyplexes comprised siRNA with polyethylenimine (PEI). During transfections, specific uptake mechanisms were inhibited by pharmacological agents and RNAi-mediated knockdown of proteins involved in various endocytosis pathways. Confocal fluorescence microscopy further elucidated the predominant endocytic pathways of siRNA delivery via colocalization of vectors with endocytic vesicle markers., Results: Inhibition of macropinocytosis (MP), caveolin-mediated endocytosis (CvME), flotillin-mediated endocytosis (FME) and knockdown of ARF6 significantly decreased PEI/siRNA-mediated gene silencing. Inhibition of endocytosis pathways, however, had negligible effect on lipoplex uptake and gene silencing mediated by lipoplexes. Rather, internalization of lipoplexes and subsequent siRNA-mediated gene silencing occurred via an energy-independent process., Conclusions: MP, CvME and FME, but not the acidified clathrin-mediated pathway, lead to effective gene silencing by PEI/siRNA polyplexes. Lipoplexes, in contrast, deliver siRNA primarily by direct fusion of the liposomal and cellular membranes. These results provide a new understanding of the mechanisms of siRNA delivery materials in HeLa cells and may aid in design of more effective RNAi strategies.
- Published
- 2016
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10. Derivation of an Analytical Solution to a Reaction-Diffusion Model for Autocatalytic Degradation and Erosion in Polymer Microspheres.
- Author
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Ford Versypt AN, Arendt PD, Pack DW, and Braatz RD
- Subjects
- Catalysis, Polylactic Acid-Polyglycolic Acid Copolymer, Drug Carriers chemistry, Drug Delivery Systems, Lactic Acid chemistry, Microspheres, Models, Theoretical, Polyglycolic Acid chemistry
- Abstract
A mathematical reaction-diffusion model is defined to describe the gradual decomposition of polymer microspheres composed of poly(D,L-lactic-co-glycolic acid) (PLGA) that are used for pharmaceutical drug delivery over extended periods of time. The partial differential equation (PDE) model treats simultaneous first-order generation due to chemical reaction and diffusion of reaction products in spherical geometry to capture the microsphere-size-dependent effects of autocatalysis on PLGA erosion that occurs when the microspheres are exposed to aqueous media such as biological fluids. The model is solved analytically for the concentration of the autocatalytic carboxylic acid end groups of the polymer chains that comprise the microspheres as a function of radial position and time. The analytical solution for the reaction and transport of the autocatalytic chemical species is useful for predicting the conditions under which drug release from PLGA microspheres transitions from diffusion-controlled to erosion-controlled release, for understanding the dynamic coupling between the PLGA degradation and erosion mechanisms, and for designing drug release particles. The model is the first to provide an analytical prediction for the dynamics and spatial heterogeneities of PLGA degradation and erosion within a spherical particle. The analytical solution is applicable to other spherical systems with simultaneous diffusive transport and first-order generation by reaction.
- Published
- 2015
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11. Intracellular trafficking of hybrid gene delivery vectors.
- Author
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Keswani RK, Lazebnik M, and Pack DW
- Subjects
- Blood Proteins metabolism, Caveolae metabolism, Chitosan chemistry, Cholesterol chemistry, Clathrin-Coated Vesicles metabolism, Fatty Acids, Monounsaturated chemistry, HEK293 Cells, HeLa Cells, Humans, Kinetics, Liposomes, Microscopy, Confocal, Moloney murine leukemia virus genetics, Phosphatidylethanolamines chemistry, Pinocytosis, Quaternary Ammonium Compounds chemistry, Temperature, Virion genetics, Virion metabolism, Chitosan metabolism, Cholesterol metabolism, Endocytosis drug effects, Fatty Acids, Monounsaturated metabolism, Genetic Vectors, Moloney murine leukemia virus metabolism, Phosphatidylethanolamines metabolism, Quaternary Ammonium Compounds metabolism, Transfection methods
- Abstract
Viral and non-viral gene delivery vectors are in development for human gene therapy, but both exhibit disadvantages such as inadequate efficiency, lack of cell-specific targeting or safety concerns. We have recently reported the design of hybrid delivery vectors combining retrovirus-like particles with synthetic polymers or lipids that are efficient, provide sustained gene expression and are more stable compared to native retroviruses. To guide further development of this promising class of gene delivery vectors, we have investigated their mechanisms of intracellular trafficking. Moloney murine leukemia virus-like particles (M-VLPs) were complexed with chitosan (Chi) or liposomes (Lip) comprising DOTAP, DOPE and cholesterol to form the hybrid vectors (Chi/M-VLPs and Lip/M-VLPs, respectively). Transfection efficiency and cellular internalization of the vectors were quantified in the presence of a panel of inhibitors of various endocytic pathways. Intracellular transport and trafficking kinetics of the hybrid vectors were dependent on the synthetic component and used a combination of clathrin- and caveolar-dependent endocytosis and macropinocytosis. Chi/M-VLPs were slower to transfect compared to Lip/M-VLPs due to the delayed detachment of the synthetic component. The synthetic component of hybrid gene delivery vectors plays a significant role in their cellular interactions and processing and is a key parameter for the design of more efficient gene delivery vehicles., (Copyright © 2015 Elsevier B.V. All rights reserved.)
- Published
- 2015
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12. Dependence of PEI and PAMAM Gene Delivery on Clathrin- and Caveolin-Dependent Trafficking Pathways.
- Author
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Hwang ME, Keswani RK, and Pack DW
- Subjects
- Dose-Response Relationship, Drug, Gene Expression Regulation, Genes, Reporter, HeLa Cells, Humans, Luciferases, Firefly biosynthesis, Luciferases, Firefly genetics, Plasmids chemistry, Caveolins metabolism, Clathrin metabolism, Dendrimers chemistry, Endocytosis drug effects, Plasmids metabolism, Polyethyleneimine chemistry, Transfection methods
- Abstract
Purpose: Non-viral gene delivery vehicles such as polyethylenimine and polyamidoamine dendrimer effectively condense plasmid DNA, facilitate endocytosis, and deliver nucleic acid cargo to the nucleus in vitro. Better understanding of intracellular trafficking mechanisms involved in polymeric gene delivery is a prerequisite to clinical application. This study investigates the role of clathrin and caveolin endocytic pathways in cellular uptake and subsequent vector processing., Methods: We formed 25-kD polyethylenimine (PEI) and generation 4 (G4) polyamidoamine (PAMAM) polyplexes at N/P 10 and evaluated internalization pathways and gene delivery in HeLa cells. Clathrin- and caveolin-dependent endocytosis inhibitors were used at varying concentrations to elucidate the roles of these important pathways., Results: PEI and PAMAM polyplexes were internalized by both pathways. However, the amount of polyplex internalized poorly correlated with transgene expression. While the caveolin-dependent pathway generally led to effective gene delivery with both polymers, complete inhibition of the clathrin-dependent pathway was also deleterious to transfection with PEI polyplexes. Inhibition of one endocytic pathway may lead to an overall increase in uptake via unaffected pathways, suggesting the existence of compensatory endocytic mechanisms., Conclusions: The well-studied clathrin- and caveolin-dependent endocytosis pathways are not necessarily independent, and perturbing one mechanism of trafficking influences the larger trafficking network.
- Published
- 2015
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13. The effect of glycosaminoglycan content on polyethylenimine-based gene delivery within three-dimensional collagen-GAG scaffolds.
- Author
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Hortensius RA, Becraft JR, Pack DW, and Harley BA
- Subjects
- Collagen metabolism, Gene Transfer Techniques, Luciferases genetics, Luciferases metabolism, Tendons chemistry, Tissue Engineering, Tissue Scaffolds, Transfection, Biocompatible Materials chemistry, Collagen genetics, Glycosaminoglycans chemistry, Polyethyleneimine chemistry, Tendons cytology
- Abstract
The design of biomaterials for increasingly complex tissue engineering applications often requires exogenous presentation of biomolecular signals. Integration of gene delivery vectors with a biomaterial scaffold offers the potential to bypass the use of expensive and relatively inefficient growth factor supplementation strategies to augment cell behavior. However, integration of cationic polymer based gene delivery vectors within three-dimensional biomaterials, particularly matrices which can carry significant surface charge, remains poorly explored. We examined the potential of polyethylenimine (PEI) as a gene delivery vector for three-dimensional collagen-glycosaminoglycan (CG) scaffolds under development for tendon repair. While acetylated versions of PEI have demonstrated improved transfection efficiency in 2D culture assays, we investigated translation of this effect to a 3D biomaterial that contains significant electrostatic charge. A reporter gene was used to examine the impact of polymer modification, polymer:DNA ratio, and the degree of sulfation of the biomaterial microenvironment on gene delivery in vitro. We observed highest transgene expression in acetylated and unmodified PEI at distinct polymer:DNA ratios; notably, the enhancement often seen in two-dimensional culture for acetylated PEI did not fully translate to three-dimensional scaffolds. We also found highly sulfated heparin-based CG scaffolds showed enhanced initial luciferase expression but not prolonged activity. While PEI constructs significantly reduced tenocyte metabolic health during the period of transfection, heparin-based CG scaffolds showed the greatest recovery in tenocyte metabolic health over the full 2 week culture. These results suggest that the electrostatic environment of three-dimensional biomaterials may be an important design criterion for cationic polymer-based gene delivery.
- Published
- 2015
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14. Pulsatile protein release from monodisperse liquid-core microcapsules of controllable shell thickness.
- Author
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Xia Y and Pack DW
- Subjects
- Biodegradable Plastics chemistry, Drug Compounding methods, Molecular Weight, Particle Size, Polymers chemistry, Serum Albumin, Bovine chemistry, Capsules chemistry, Polyglactin 910 chemistry, Proteins chemistry
- Abstract
Purpose: Pulsatile delivery of proteins, in which release occurs over a short time after a period of little or no release, is desirable for many applications. This paper investigates the effect of biodegradable polymer shell thickness on pulsatile protein release from biodegradable polymer microcapsules., Methods: Using precision particle fabrication (PPF) technology, monodisperse microcapsules were fabricated encapsulating bovine serum albumin (BSA) in a liquid core surrounded by a drug-free poly(lactide-co-glycolide) (PLG) shell of uniform, controlled thickness from 14 to 19 μm., Results: When using high molecular weight PLG (Mw 88 kDa), microparticles exhibited the desired core-shell structure with high BSA loading and encapsulation efficiency (55-65%). These particles exhibited very slow release of BSA for several weeks followed by rapid release of 80-90% of the encapsulated BSA within 7 days. Importantly, with increasing shell thickness the starting time of the pulsatile release could be controlled from 25 to 35 days., Conclusions: Biodegradable polymer microcapsules with precisely controlled shell thickness provide pulsatile release with enhanced control of release profiles.
- Published
- 2014
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15. Efficient in vitro gene delivery by hybrid biopolymer/virus nanobiovectors.
- Author
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Keswani R, Su K, and Pack DW
- Subjects
- Genetic Vectors genetics, HEK293 Cells, Humans, Leukemia Virus, Murine genetics, Virion genetics, Virion ultrastructure, Chitosan chemistry, Genetic Vectors chemistry, Leukemia Virus, Murine chemistry, Transfection methods, Virion chemistry
- Abstract
Recombinant retroviruses provide highly efficient gene delivery and the potential for sustained gene expression, but suffer from significant disadvantages including low titer, expensive production, poor stability and limited flexibility for modification of tropism. In contrast, polymer-based vectors are more robust and allow cell- and tissue-specific deliveries via conjugation of ligands, but are comparatively inefficient. The design of hybrid gene delivery agents comprising both virally derived and synthetic materials (nanobiovectors) represents a promising approach to development of safe and efficient gene therapy vectors. Non-infectious murine leukemia virus-like particles (M-VLPs) were electrostatically complexed with chitosan (χ) to replace the function of the viral envelope protein. At optimal fabrication conditions and compositions, ranging from 6 to 9μg chitosan/10(9) M-VLPs at 10×10(9)M-VLPs/ml to 40μg chitosan/10(9) M-VLPs at 2.5×10(9)M-VLPs/ml, χ/M-VLPs were ~300-350nm in diameter and exhibited efficient transfection similar to amphotropic MLV vectors. In addition, these nanobiovectors were non-cytotoxic and provided sustained transgene expression for at least three weeks in vitro. This combination of biocompatible synthetic agents with inactive viral particles to form a highly efficient hybrid vector is a significant extension in the development of novel gene delivery platforms., (Copyright © 2014 Elsevier B.V. All rights reserved.)
- Published
- 2014
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16. Modelling intravascular delivery from drug-eluting stents with biodurable coating: investigation of anisotropic vascular drug diffusivity and arterial drug distribution.
- Author
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Zhu X, Pack DW, and Braatz RD
- Subjects
- Anisotropy, Diffusion, Pharmacokinetics, Coronary Vessels metabolism, Drug-Eluting Stents, Models, Biological
- Abstract
In-stent restenosis occurs in coronary arteries after implantation of drug-eluting stents with non-uniform restenosis thickness distribution in the artery cross section. Knowledge of the spatio-temporal drug uptake in the arterial wall is useful for investigating restenosis growth but may often be very expensive/difficult to acquire experimentally. In this study, local delivery of a hydrophobic drug from a drug-eluting stent implanted in a coronary artery is mathematically modelled to investigate the drug release and spatio-temporal drug distribution in the arterial wall. The model integrates drug diffusion in the coating and drug diffusion with reversible binding in the arterial wall. The model is solved by the finite volume method for both high and low drug loadings relative to its solubility in the stent coating with varied isotropic-anisotropic vascular drug diffusivities. Drug release profiles in the coating are observed to depend not only on the coating drug diffusivity but also on the properties of the surrounding arterial wall. Time dependencies of the spatially averaged free- and bound-drug levels in the arterial wall on the coating and vascular drug diffusivities are discussed. Anisotropic vascular drug diffusivities result in slightly different average drug levels in the arterial wall but with very different spatial distributions. Higher circumferential vascular diffusivity results in more uniform drug loading in the upper layers and is potentially beneficial in reducing in-stent restenosis. An analytical expression is derived which can be used to determine regions in the arterial with higher free-drug concentration than bound-drug concentration.
- Published
- 2014
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17. Controlled protein release from monodisperse biodegradable double-wall microspheres of controllable shell thickness.
- Author
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Xia Y, Ribeiro PF, and Pack DW
- Subjects
- Animals, Cattle, Microspheres, Particle Size, Polyesters, Polylactic Acid-Polyglycolic Acid Copolymer, Polymers chemistry, Porosity, Biocompatible Materials chemistry, Delayed-Action Preparations chemistry, Lactic Acid chemistry, Polyglycolic Acid chemistry, Serum Albumin, Bovine administration & dosage
- Abstract
Biodegradable polymer microparticles are promising delivery depots for protein therapeutics due to their relatively simple fabrication and facile administration. Double-wall microspheres (DWMS) comprising a core and shell made of two distinct polymers may provide enhanced control of the drug release profiles. Using precision particle fabrication (PPF) technology, monodisperse DWMS were fabricated with model protein bovine serum albumin (BSA)-loaded poly(lactide-co-glycolide) (PLG) core and drug-free poly(d,l-lactic acid) (PDLL) shell of uniform thickness. Monolithic single-wall microspheres were also fabricated to mimic the BSA-loaded PLG core. Using ethyl acetate and dichloromethane as shell- and core-phase solvents, respectively, BSA was encapsulated selectively in the core region within DWMS with higher loading and encapsulation efficiency compared to using dichloromethane as core and shell solvents. BSA in vitro release rates were retarded by the presence of the drug-free PDLL shell. Moreover, increasing PDLL shell thickness resulted in decreasing BSA release rate. With a 14-μm thick PDLL shell, an extended period of constant-rate release was achieved., (© 2013.)
- Published
- 2013
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18. Coaxial electrohydrodynamic atomization process for production of polymeric composite microspheres.
- Author
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Xu Q, Qin H, Yin Z, Hua J, Pack DW, and Wang CH
- Abstract
Polymeric composite microspheres consisting of a poly(D,L-lactic-co-glycolic acid) (PLGA) core surrounded by a poly(D,L-lactic acid) (PDLLA) shell layer were successfully fabricated by coaxial electrohydrodynamic atomization (CEHDA) process. Process conditions, including nozzle voltage and polymer solution flow rates, as well as solution parameters, such as polymer concentrations, were investigated to ensure the formation of composite microspheres with a doxorubicin-loaded PLGA core surrounded by a relatively drug-free PDLLA shell layer. Various microsphere formulations were fabricated and characterized in terms of their drug distribution, encapsulation efficiency and in vitro release. Numerical simulation of CEHDA process was performed based on a computational fluid dynamics (CFD) model in Fluent by employing the process conditions and fluid properties used in the experiments. The simulation results were compared with the experimental work to illustrate the capability of the CFD model to predict the production of consistent compound droplets, and hence, the expected core-shell structured microspheres.
- Published
- 2013
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19. Optimal Control of One-dimensional Cellular Uptake in Tissue Engineering.
- Author
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Kishida M, Ford Versypt AN, Pack DW, and Braatz RD
- Abstract
A control problem motivated by tissue engineering is formulated and solved in which control of the uptake of growth factors (signaling molecules) is necessary to spatially and temporally regulate cellular processes for the desired growth or regeneration of a tissue. Four approaches are compared for determining 1D optimal boundary control trajectories for a distributed parameter model with reaction, diffusion, and convection: (i) basis function expansion, (ii) method of moments, (iii) internal model control (IMC), and (iv) model predictive control (MPC). The proposed method-of-moments approach is computationally efficient while enforcing a non-negativity constraint on the control input. While more computationally expensive than methods (i)-(iii), the MPC formulation significantly reduced the computational cost compared to simultaneous optimization of the entire control trajectory. A comparison of the pros and cons of each of the four approaches suggests that an algorithm that combines multiple approaches is most promising for solving the optimal control problem for multiple spatial dimensions.
- Published
- 2013
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20. Combined modality doxorubicin-based chemotherapy and chitosan-mediated p53 gene therapy using double-walled microspheres for treatment of human hepatocellular carcinoma.
- Author
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Xu Q, Leong J, Chua QY, Chi YT, Chow PK, Pack DW, and Wang CH
- Subjects
- Carcinoma, Hepatocellular pathology, Caspase 3 metabolism, Cell Death drug effects, Cell Survival drug effects, Combined Modality Therapy, DNA metabolism, Doxorubicin pharmacology, Fluorescent Antibody Technique, Hep G2 Cells, Humans, Liver Neoplasms pathology, Luciferases metabolism, Nanoparticles chemistry, Tumor Suppressor Protein p53 therapeutic use, Carcinoma, Hepatocellular drug therapy, Chitosan chemistry, Doxorubicin therapeutic use, Genetic Therapy, Liver Neoplasms drug therapy, Microspheres, Tumor Suppressor Protein p53 genetics
- Abstract
The therapeutic efficiency of combined chemotherapy and gene therapy on human hepatocellular carcinoma HepG2 cells was investigated using double-walled microspheres that consisted of a poly(D,L-lactic-co-glycolic acid) (PLGA) core surrounded by a poly(L-lactic acid) (PLLA) shell layer and fabricated via the precision particle fabrication (PPF) technique. Here, double-walled microspheres were used to deliver doxorubicin (Dox) and/or chitosan-DNA nanoparticles containing the gene encoding the p53 tumor suppressor protein (chi-p53), loaded in the core and shell phases, respectively. Preliminary studies on chi-DNA nanoparticles were performed to optimize gene transfer to HepG2 cells. The transfection efficiency of chi-DNA nanoparticles was optimal at an N/P ratio of 7. In comparison to the 25-kDa branched polyethylenimine (PEI), chitosan showed no inherent toxicity towards the cells. Next, the therapeutic efficiencies of Dox and/or chi-p53 in microsphere formulations were compared to free drug(s) and evaluated in terms of growth inhibition, and cellular expression of tumor suppressor p53 and apoptotic caspase 3 proteins. Overall, the combined Dox and chi-p53 treatment exhibited enhanced cytotoxicity as compared to either Dox or chi-p53 treatments alone. Moreover, the antiproliferative effect was more substantial when cells were treated with microspheres than those treated with free drugs. High p53 expression was maintained during a five-day period, and was largely due to the controlled and sustained release of the microspheres. Moreover, increased activation of caspase 3 was observed, and was likely to have been facilitated by high levels of p53 expression. Overall, double-walled microspheres present a promising dual anticancer delivery system for combined chemotherapy and gene therapy., (Copyright © 2013 Elsevier Ltd. All rights reserved.)
- Published
- 2013
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21. Design of hybrid lipid/retroviral-like particle gene delivery vectors.
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Keswani RK, Pozdol IM, and Pack DW
- Subjects
- Animals, Cell Line, Cholesterol chemistry, Fatty Acids, Monounsaturated chemistry, Gene Expression, HEK293 Cells, Humans, Leukemia Virus, Murine ultrastructure, Liposomes chemistry, Liposomes ultrastructure, Mice, Phosphatidylethanolamines chemistry, Quaternary Ammonium Compounds chemistry, Transfection, Gene Transfer Techniques, Genetic Vectors chemistry, Leukemia Virus, Murine genetics, Liposomes administration & dosage
- Abstract
Recombinant retroviruses provide highly efficient gene delivery and the potential for stable gene expression. The retroviral envelope protein, however, is the source of significant disadvantages such as immunogenicity, poor stability (half-life of transduction activity of 5-7 h at 37 °C for amphotropic murine leukemia virus), and difficult production and purification. To address these problems, we report the construction of efficient hybrid vectors through the association of murine leukemia virus (MLV)-like particles (M-VLP) with synthetic liposomes comprising DOTAP, DOPE, and cholesterol (φ/M-VLP). We conclude that the lipid composition is a significant determinant of the transfection efficiency and uptake of φ/M-VLP in HEK293 cells with favorable compositions for transfections being those with low DOTAP, low DOPE, and high cholesterol content. Cellular uptake, however, was dependent on DOTAP content alone. By extrusion of liposomes prior to vector assembly, the size of these hybrid vectors could also be decreased to ≈300 nm, as confirmed via DLS and TEM. φ/M-VLP were also robust on storage in terms of vector size and transfection efficiency and provided stable transgene expression over a period of three weeks. We conclude that the noncovalent combination of biocompatible synthetic lipids with inactive retroviral particles to form a highly efficient hybrid vector is a significant extension to the development of novel gene delivery platforms.
- Published
- 2013
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22. Mechanism of drug release from double-walled PDLLA(PLGA) microspheres.
- Author
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Xu Q, Chin SE, Wang CH, and Pack DW
- Subjects
- Drug Compounding, Fluorescence, Microscopy, Electron, Scanning, Molecular Weight, Optical Imaging, Polylactic Acid-Polyglycolic Acid Copolymer, Porosity, Rheology drug effects, Time Factors, Doxorubicin pharmacology, Lactic Acid chemistry, Microspheres, Polyglycolic Acid chemistry
- Abstract
The drug release and degradation behavior of two double-walled microsphere formulations consisting of a doxorubicin-loaded poly(d,l-lactic-co-glycolic acid) (PLGA) core (∼46 kDa) surrounded by a poly(d,l-lactic acid) (PDLLA) shell layer (∼55 and 116 kDa) were examined. It was postulated that different molecular weights of the shell layer could modulate the erosion of the outer coating and limit the occurrence of water penetration into the inner drug-loaded core on various time scales, and therefore control the drug release from the microspheres. For both microsphere formulations, the drug release profiles were observed to be similar. The degradation of the microspheres was monitored for a period of about nine weeks and analyzed using scanning electron microscopy, laser scanning confocal microscopy, and gel permeation chromatography. Interestingly, both microsphere formulations exhibited occurrence of bulk erosion of PDLLA on a similar time scale despite different PDLLA molecular weights forming the shell layer. The shell layer of the double-walled microspheres served as an effective diffusion barrier during the initial lag phase period and controlled the release rate of the hydrophilic drug independent of the molecular weight of the shell layer., (Copyright © 2013 Elsevier Ltd. All rights reserved.)
- Published
- 2013
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23. Protein encapsulation in and release from monodisperse double-wall polymer microspheres.
- Author
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Xia Y, Xu Q, Wang CH, and Pack DW
- Subjects
- Animals, Cattle, Microspheres, Particle Size, Polyesters, Delayed-Action Preparations chemistry, Lactic Acid chemistry, Polymers chemistry, Serum Albumin, Bovine administration & dosage
- Abstract
Biodegradable polymer double-wall microspheres (DWMS) are promising vehicles for macromolecular therapeutics such as proteins and peptides. Using precision particle fabrication (PPF) technology, uniform DWMS with outer diameter approximately 55 μm were fabricated comprising poly(lactide-co-glycolide) cores encapsulating bovine serum albumin (BSA) and approximately 10 μm thick, drug-free, poly(lactic acid) (PLA) shells of varying PLA molecular weight. Also, monolithic single-wall microspheres (SWMS) were fabricated to mimic the BSA-loaded core. The use of relatively fast-extracting ethyl acetate and slowly extracting dichloromethane as shell- and core-phase solvents, respectively, was found to produce DWMS with well-defined core-shell structure, high BSA encapsulation efficiency, and the desired localization of protein in the particle core. Initial protein distribution, particle erosion, and in vitro protein release from DWMS and SWMS were examined. The presence of a BSA-free shell in DWMS decreased the protein release rate and extended the duration of release from approximately 50 days to 70-80 days, demonstrating the capacity of such DWMS to provide enhanced control of protein delivery rates., (Copyright © 2013 Wiley Periodicals, Inc.)
- Published
- 2013
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24. Mathematical modeling of drug delivery from autocatalytically degradable PLGA microspheres--a review.
- Author
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Ford Versypt AN, Pack DW, and Braatz RD
- Subjects
- Microspheres, Polylactic Acid-Polyglycolic Acid Copolymer, Drug Delivery Systems, Lactic Acid chemistry, Models, Theoretical, Polyglycolic Acid chemistry
- Abstract
PLGA microspheres are widely studied for controlled release drug delivery applications, and many models have been proposed to describe PLGA degradation and erosion and drug release from the bulk polymer. Autocatalysis is known to have a complex role in the dynamics of PLGA erosion and drug transport and can lead to size-dependent heterogeneities in otherwise uniformly bulk-eroding polymer microspheres. The aim of this review is to highlight mechanistic, mathematical models for drug release from PLGA microspheres that specifically address interactions between phenomena generally attributed to autocatalytic hydrolysis and mass transfer limitation effects. Predictions of drug release profiles by mechanistic models are useful for understanding mechanisms and designing drug release particles., (Copyright © 2012 Elsevier B.V. All rights reserved.)
- Published
- 2013
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25. Monodisperse double-walled microspheres loaded with chitosan-p53 nanoparticles and doxorubicin for combined gene therapy and chemotherapy.
- Author
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Xu Q, Xia Y, Wang CH, and Pack DW
- Subjects
- Drug Carriers chemistry, Drug Delivery Systems, Genetic Therapy, Lactic Acid chemistry, Microspheres, Plasmids, Polyglycolic Acid chemistry, Polylactic Acid-Polyglycolic Acid Copolymer, Antibiotics, Antineoplastic chemistry, Chitosan chemistry, DNA chemistry, Doxorubicin chemistry, Nanoparticles chemistry, Tumor Suppressor Protein p53 genetics
- Abstract
We have designed and evaluated a dual anticancer delivery system to provide combined gene therapy and chemotherapy. Double-walled microspheres consisting of a poly(d,l-lactic-co-glycolic acid) (PLGA) core surrounded by a poly(lactic acid) (PLA) shell were fabricated via the precision particle fabrication (PPF) technique. We make use of the advantages of double-walled microspheres to deliver chitosan-DNA nanoparticles containing the gene encoding the p53 tumor suppressor protein (chi-p53) and/or doxorubicin (Dox), loaded in the shell and core phases, respectively. Different molecular weights of PLA were used to form the shell layer for each formulation. The microspheres were monodisperse with a mean diameter of 65 to 75 μm and uniform shell thickness of 8 to 17 μm. Blank and Dox-loaded microspheres typically exhibited a smooth surface with relatively few small pores, while chi-microspheres containing p53 nanoparticles, with and without Dox, presented rough and porous surfaces. The encapsulation efficiency of Dox was significantly higher when it was encapsulated alone compared to co-encapsulation with chi-p53 nanoparticles. The encapsulation efficiency of chi-p53 nanoparticles, on the other hand, was not affected by the presence of Dox. As desired, chi-p53 nanoparticles were released first, followed by simultaneous release of chi-p53 nanoparticles and Dox at a near zero-order rate. Thus, we have demonstrated that the PPF method is capable of producing double-walled microspheres and encapsulating dual agents for combined modality treatment, such as gene therapy and chemotherapy., (Copyright © 2012 Elsevier B.V. All rights reserved.)
- Published
- 2012
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26. The effects of PVP(Fe(III)) catalyst on polymer molecular weight and gene delivery via biodegradable cross-linked polyethylenimine.
- Author
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Shum VW, Gabrielson NP, Forrest ML, and Pack DW
- Subjects
- Catalysis, Cell Line, Tumor, Cross-Linking Reagents chemistry, DNA genetics, Female, HeLa Cells, Humans, Polyethyleneimine chemical synthesis, Polyethyleneimine metabolism, Polyvinyls chemistry, DNA administration & dosage, Ferric Compounds chemistry, Polyethyleneimine chemistry, Transfection
- Abstract
Purpose: Crosslinked, degradable derivatives of low-molecular-weight polyethylenimine (PEI) are relatively efficient and non-cytotoxic gene delivery agents. To further investigate these promising materials, a new synthetic approach was developed using a poly(4-vinylpyridine)-supported Fe(III) catalyst (PVP(Fe(III))) that provides more facile synthesis and enhanced control of polymer molecular weight., Methods: Biodegradable polymers (D.PEI) comprising 800-Da PEI crosslinked with 1,6-hexanediol diacrylate and exhibiting molecular weights of 1.2, 6.2, and 48 kDa were synthesized utilizing the PVP(Fe(III)) catalyst. D.PEI/DNA polyplexes were characterized using gel retardation, ethidium bromide exclusion, heparan sulfate displacement, and dynamic light scattering. In vitro transfection, cellular uptake, and cytotoxicity of the polyplexes were tested in human cervical cancer cells (HeLa) and human breast cancer cells (MDA-MB-231)., Results: D.PEIs tightly complexed plasmid DNA and formed 320- to 440-nm diameter polyplexes, similar to those comprising non-degradable, 25-kDa, branched PEI. D.PEI polyplexes mediated 2- to 5-fold increased gene delivery efficacy compared to 25-kDa PEI and exhibited 20% lower cytotoxicity in HeLa and no toxicity in MDA-MB-231. In addition, 2- to 7-fold improved cellular uptake of DNA was achieved with D.PEI polyplexes., Conclusions: PVP(Fe(III)) catalyst provided a more controlled synthesis of D.PEIs, and these materials demonstrated improved in vitro transfection efficacy and reduced cytotoxicity .
- Published
- 2012
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27. Degradable self-assembling dendrons for gene delivery: experimental and theoretical insights into the barriers to cellular uptake.
- Author
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Barnard A, Posocco P, Pricl S, Calderon M, Haag R, Hwang ME, Shum VW, Pack DW, and Smith DK
- Subjects
- DNA metabolism, Mass Spectrometry, Models, Molecular, Molecular Dynamics Simulation, Polyethyleneimine metabolism, Transgenes, Anthracenes, Gene Transfer Techniques
- Abstract
This paper uses a combined experimental and theoretical approach to gain unique insight into gene delivery. We report the synthesis and investigation of a new family of second-generation dendrons with four triamine surface ligands capable of binding to DNA, degradable aliphatic-ester dendritic scaffolds, and hydrophobic units at their focal points. Dendron self-assembly significantly enhances DNA binding as monitored by a range of experimental methods and confirmed by multiscale modeling. Cellular uptake studies indicate that some of these dendrons are highly effective at transporting DNA into cells (ca. 10 times better than poly(ethyleneimine), PEI). However, levels of transgene expression are relatively low (ca. 10% of PEI). This indicates that these dendrons cannot navigate all of the intracellular barriers to gene delivery. The addition of chloroquine indicates that endosomal escape is not the limiting factor in this case, and it is shown, both experimentally and theoretically, that gene delivery can be correlated with the ability of the dendron assemblies to release DNA. Mass spectrometric assays demonstrate that the dendrons, as intended, do degrade under biologically relevant conditions over a period of hours. Multiscale modeling of degraded dendron structures suggests that complete dendron degradation would be required for DNA release. Importantly, in the presence of the lower pH associated with endosomes, or when bound to DNA, complete degradation of these dendrons becomes ineffective on the transfection time scale-we propose this explains the poor transfection performance of these dendrons. As such, this paper demonstrates that taking this kind of multidisciplinary approach can yield a fundamental insight into the way in which dendrons can navigate barriers to cellular uptake. Lessons learned from this work will inform future dendron design for enhanced gene delivery., (© 2011 American Chemical Society)
- Published
- 2011
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28. Acid-Sensitive Magnetic Nanoparticles as Potential Drug Depots.
- Author
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Wuang SC, Neoh KG, Kang ET, Leckband DE, and Pack DW
- Abstract
Superparamagnetic magnetic nanoparticles were successfully functionalized with poly(methacrylic acid) via atom transfer radical polymerization, followed by conjugation to doxorubicin (Dox). Because of pH-sensitive hydrazone linkages, the rate and extent of Dox release from the particles was higher at a lower pH and/or a higher temperature than at physiological conditions. Appropriate changes to the pH and temperature can increase the drug release from the particles. Because of the released drug, the particles were found to be cytotoxic to human breast cancer cells in vitro. Such magnetic nanoparticles, with the potential to retain drug under physiological conditions and release the drug in conditions where the pH is lower or temperature is higher, may be useful in magnetic drug targeting by reducing the side effects of the drug caused to healthy tissues. In addition, they may serve as hyperthermia agents where the high temperatures used in hyperthermia can trigger further drug release.
- Published
- 2011
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29. Hydrophobically modified dendrons: developing structure-activity relationships for DNA binding and gene transfection.
- Author
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Jones SP, Gabrielson NP, Wong CH, Chow HF, Pack DW, Posocco P, Fermeglia M, Pricl S, and Smith DK
- Subjects
- Cells, Cultured, DNA chemistry, Dendrimers chemical synthesis, Genetic Vectors administration & dosage, Humans, Luciferases metabolism, Micelles, Models, Molecular, Molecular Structure, Plasmids genetics, Polyethyleneimine, Spermine metabolism, Spermine toxicity, Structure-Activity Relationship, Transfection, DNA administration & dosage, DNA metabolism, Dendrimers administration & dosage, Dendrimers chemistry, Gene Transfer Techniques, Spermine administration & dosage, Spermine chemistry
- Abstract
This paper develops a structure-activity relationship understanding of the way in which surfactant-like dendrons with hydrophilic spermine surface groups and a variety of lipophilic units at their focal points can self-assemble and subsequently bind to DNA with high affinity. The choice of functional group at the focal point of the dendron and the high tunability of the molecular structure have a very significant impact on DNA binding. Mesoscale modeling of the mode of dendron self-assembly provides a direct insight into how the mode of self-assembly exerts its effect on the DNA binding process. In particular, the hydrophobic unit controls the number of dendrons in the self-assembled micellar structures, and hence their diameters and surface charge density. The DNA binding affinity correlates with the surface charge density of the dendron aggregates. Furthermore, these structure-activity effects can also be extended to cellular gene delivery, as surface charge density plays a role in controlling the extent of endosomal escape. It is reported that higher generation dendrons, although binding DNA less strongly than the self-assembling lower generation dendrons, are more effective for transfection. The impact of the lipophilic group at the focal point is less significant for the DNA binding ability of these larger dendrons, which is predominantly controlled by the spermine surface groups, but it does modify the levels of gene transfection. Significant synergistic effects on gene delivery were observed when employing combinations of the dendrons and polyethyleneimine (PEI, 25 kDa), with transfection becoming possible at low loading levels where the two components would not transfect individually, giving practically useful levels of gene delivery.
- Published
- 2011
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30. Effect of serum on transfection by polyethylenimine/virus-like particle hybrid gene delivery vectors.
- Author
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Drake DM, Keswani RK, and Pack DW
- Subjects
- Blood, Drug Carriers, Polyethyleneimine, Transfection
- Abstract
Purpose: Murine leukemia virus-like particles (M-VLP) complexed with polymers to promote cellular uptake and endosomal escape represent a new class of effective gene delivery vectors. Building upon recent studies of viral-synthetic hybrid vectors, we report the effects of serum on the formation, activity and stability of PEI/M-VLP complexes., Methods: M-VLP were produced by cells grown in serum-supplemented media (M-VLP-S), serum-free media (M-VLP-SF) or serum-free Opti-MEM® I (M-VLP-OM). PEI/M-VLP stoichiometry was varied to investigate complex formation and optimal transfection conditions. The effects of prolonged storage, freeze-thaw cycles, and ultracentrifugation of M-VLP on the stability of vector transduction efficiency were also observed., Results: M-VLP-S required more PEI to form infective complexes than M-VLP-SF and M-VLP-OM. The stoichiometry of PEI/M-VLP-S was dependent on total PEI concentration (7-8 μg/100 μL M-VLP supernatant), while optimal infectivity of PEI/M-VLP-SF and PEI/M-VLP-OM depended on PEI/M-VLP ratios (12-17 μg and 10-14 μg PEI/10(9) M-VLP, respectively). PEI/M-VLP-SF and PEI/M-VLP-OM complexes were significantly more efficient than PEI/M-VLP-S. Stability of the hybrid vectors was not significantly affected by serum., Conclusions: PEI/M-VLP complexes exhibiting increased efficiency were constructed by producing M-VLP in serum-free media. M-VLP could be stored by freezing or refrigeration and concentrated by ultracentrifugation without unacceptable loss of infectivity.
- Published
- 2010
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31. Polymeric carriers for gene delivery: chitosan and poly(amidoamine) dendrimers.
- Author
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Xu Q, Wang CH, and Pack DW
- Subjects
- Animals, Cell Line, Chitosan administration & dosage, Chitosan therapeutic use, Dendrimers administration & dosage, Dendrimers therapeutic use, Drug Carriers administration & dosage, Drug Carriers chemistry, Drug Carriers therapeutic use, Humans, Polymers administration & dosage, Polymers therapeutic use, Chitosan chemistry, Dendrimers chemistry, Drug Design, Gene Transfer Techniques, Polymers chemistry
- Abstract
Gene therapy is a potential medical solution that promises new treatments and may hold the cure for many different types of diseases and disorders of the human race. However, gene therapy is still a growing medical field and the technology is still in its infancy. The main challenge for gene therapy is to find safe and effective vectors that are able to deliver genes to the specific cells and get them to express inside the cells. Due to safety concerns, synthetic delivery systems, rather than viral vectors, are preferred for gene delivery and significant efforts have been focused on the development of this field. However, we are faced with problems like low gene transfer efficiency, cytotoxicity and lack of cell-targeting capability for these synthetic delivery systems. Over the years, we have seen a variety of new and effective polymers which have been designed and synthesized specifically for gene delivery. Moreover, various strategies that aimed at enhancing their physicochemical properties, improving transfection efficiency, reducing cytotoxicity as well as incorporating functional groups that offer better targetability and higher cellular uptake are established. Here, we look at two potential polymeric carriers, chitosan and poly(amidoamine) dendrimers, which have been widely reported for gene delivery. For chitosan, the interest arises from their availability, excellent non-cytotoxicity profile, biodegradability and ease of modification. For poly(amidoamine) dendrimers, the interest arises from their ease of synthesis with controlled structure and size, minimal cytotoxicity, biodegradability and high transfection efficiencies. The latest developments on these polymers for gene delivery will be the main focus of this article.
- Published
- 2010
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32. A top-down approach for construction of hybrid polymer-virus gene delivery vectors.
- Author
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Ramsey JD, Vu HN, and Pack DW
- Subjects
- Chimera, Genes, Viral, Humans, Lysine genetics, Moloney murine leukemia virus genetics, Polyethyleneimine, Polymers, Transgenes, Viral Envelope Proteins genetics, Virion genetics, Virus Diseases genetics, Viruses genetics, Gene Transfer Techniques, Genetic Therapy methods, Genetic Vectors
- Abstract
Safe and efficient delivery of therapeutic nucleic acids remains the primary hurdle for human gene therapy. While many researchers have attempted to re-engineer viruses to be suited for gene delivery, others have sought to develop non-viral alternatives. We have developed a complementary approach in which viral and synthetic components are combined to form hybrid nanoparticulate vectors. In particular, we complexed non-infectious retrovirus-like particles lacking a viral envelope protein, from Moloney murine leukemia virus (M-VLP) or human immunodeficiency virus (H-VLP), with poly-L-lysine (PLL) or polyethylenimine (PEI) over a range of polymer/VLP ratios. At appropriate stoichiometry (75-250 microg polymer/10(6) VLP), the polymers replace the function of the viral envelope protein and interact with the target cell membrane, initiate cellular uptake and facilitate escape from endocytic vesicles. The viral particle, once in the cytosol, efficiently completes its normal infection process including integration of viral genes with the host genome as demonstrated by long-term (at least 5 weeks) transgene expression. In addition, hybrid vectors comprising H-VLP were shown to be capable of infecting non-dividing cells., (Copyright 2010 Elsevier B.V. All rights reserved.)
- Published
- 2010
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33. Efficient polyethylenimine-mediated gene delivery proceeds via a caveolar pathway in HeLa cells.
- Author
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Gabrielson NP and Pack DW
- Subjects
- Caveolin 1 genetics, Chlorpromazine pharmacology, Clathrin genetics, Dopamine Antagonists pharmacology, Down-Regulation, Endocytosis drug effects, Enzyme Inhibitors pharmacology, Folic Acid chemistry, Folic Acid metabolism, HeLa Cells, Humans, Macrolides pharmacology, RNA, Small Interfering genetics, Transfection, Transferrin chemistry, Transferrin metabolism, Caveolin 1 metabolism, Clathrin metabolism, Gene Transfer Techniques, Polyethyleneimine chemistry, Polyethyleneimine metabolism
- Abstract
Most in vivo gene therapies will require cell-specific targeting. Although vector targeting through ligand attachment has met with success in generating gene delivery particles that are capable of specific cellular interactions, little attention has been given to the possible effects of such ligands on subsequent intracellular processing. In this study, we examine the impact of targeting two distinct endocytic routes-the caveolar and clathrin pathways-on polyethylenimine-mediated gene delivery in HeLa cells. Targeting complexes to the caveolar pathway with folic acid and the clathrin pathway with transferrin yields enhanced gene delivery relative to unmodified polyethylenimine. Colocalization studies with caveolin-1 and clathrin heavy chain indicate that the ligands successfully deliver their cargo to the intended pathways. However, inhibition of only the caveolar pathway-whether through the use of small molecule drugs or RNA interference-reduces gene delivery efficiency, suggesting that successful polyethylenimine-mediated gene delivery proceeds via a caveolar pathway in HeLa cells. Transfections in the presence of chloroquine and pH tracking studies reveal that a contributing factor to the success of the caveolar pathway is avoidance of lysosomes. Collectively, these data demonstrate that uptake mechanism and subsequent endocytic processing are important design parameters for gene delivery materials.
- Published
- 2009
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34. Synergistic effects on gene delivery--co-formulation of small disulfide-linked dendritic polycations with Lipofectamine 2000.
- Author
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Hardy JG, Love CS, Gabrielson NP, Pack DW, and Smith DK
- Subjects
- Drug Synergism, Genetic Vectors, Dendrimers, Lipids, Polylysine, Transfection methods
- Abstract
This paper describes the application of gene delivery vectors based on connecting together two well-defined low-generation poly(L-lysine) (PLL) dendrons using a disulfide-containing linker unit. We report that the transfection ability of these vectors in their own right is relatively low, because the low-generation number limits the endosomal buffering capacity. Importantly, however, we demonstrate that when applied in combination with Lipofectamine 2000, a vector from the cationic lipid family, these small cationic additives significantly enhance the levels of gene delivery (up to four-fold). Notably, the cationic additives have no effect on the levels of transfection observed with a cationic polymer, such as DEAE dextran. We therefore argue that the synergistic effects observed with Lipofectamine 2000 arise as a result of combining the delivery advantages of two different classes of vector within a single formulation, with our dendritic additives providing a degree of pH buffering within the endosome. As such, the data we present indicate that small dendritic structures, although previously largely overlooked for gene delivery owing to their inability to transfect in their own right, may actually be useful well-defined additives to well-established vector systems in order to enhance the gene delivery payload.
- Published
- 2009
- Full Text
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35. The effect of chitosan on the migration of neutrophil-like HL60 cells, mediated by IL-8.
- Author
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Park CJ, Gabrielson NP, Pack DW, Jamison RD, and Wagoner Johnson AJ
- Subjects
- Acetylation drug effects, Chitosan chemistry, HL-60 Cells, Humans, Hydrophobic and Hydrophilic Interactions, Neutrophils drug effects, Superoxides metabolism, Cell Movement drug effects, Chitosan pharmacology, Interleukin-8 metabolism, Neutrophils cytology, Neutrophils metabolism
- Abstract
Research interest in chitosan stems in part from the demonstrated wound healing properties. The benefits of chitosan as a therapeutic agent appear to be paradoxical because chitosan also elicits neutrophil infiltration indicative of an inflammatory response. While the affinity between chitosan and neutrophils has been well documented, the underlying mechanism is unclear. To our knowledge, no studies have investigated the consequences of chitosan-neutrophil interaction to explain neutrophil migration. To that end, transwell migration assays to chitosan of varying extent of acetylation were conducted using a differentiated model cell line (HL60-PMN) in order to assess the effect of chitosan chemistry and the resultant physical properties such as charge and hydrophobicity on neutrophil migration. As chitosan N-acetylation increased, neutrophil migration increased and chitosan became less positively charged and more hydrophobic. Moreover, HL60-PMN cells secreted the potent neutrophil chemokine IL-8, also known as CXCL8, when exposed to chitosan and IL-8 levels increased with N-acetylation, and migration was inhibited by anti-IL-8 antibodies. Collectively these results suggest that chitosan-neutrophil interaction is encouraged by material properties, results in IL-8 secretion, and causes migration of neutrophils to chitosan. The implication is that the wound healing properties of chitosan may be enhanced through the attenuation of overabundant neutrophils, and thus the inflammatory response, simply by changing chitosan N-acetylation.
- Published
- 2009
- Full Text
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36. Synergistic effects in gene delivery-a structure-activity approach to the optimisation of hybrid dendritic-lipidic transfection agents.
- Author
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Jones SP, Gabrielson NP, Pack DW, and Smith DK
- Subjects
- Cell Line, Cell Survival drug effects, DNA drug effects, Dendrimers chemical synthesis, Dendrimers pharmacology, Dose-Response Relationship, Drug, Drug Design, Drug Synergism, Genetic Vectors chemical synthesis, Genetic Vectors pharmacology, Humans, Molecular Structure, Structure-Activity Relationship, Transfection, Cholesterol chemistry, Dendrimers chemistry, Drug Delivery Systems, Gene Transfer Techniques, Genetic Vectors chemistry, Spermine chemistry
- Abstract
Novel gene delivery agents based on combining cholesterol units with spermine-functionalised dendrons exhibit enhanced transfection ability-we report significant synergistic effects in mixed (hybrid) systems which combine aspects of both main classes of synthetic vectors, i.e., cationic polymers and lipids.
- Published
- 2008
- Full Text
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37. HER-2-mediated endocytosis of magnetic nanospheres and the implications in cell targeting and particle magnetization.
- Author
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Wuang SC, Neoh KG, Kang ET, Pack DW, and Leckband DE
- Subjects
- Antibodies, Monoclonal pharmacology, Antibodies, Monoclonal, Humanized, Antineoplastic Agents pharmacology, Breast Neoplasms pathology, Breast Neoplasms ultrastructure, Cell Line, Tumor, Cell Survival drug effects, Dose-Response Relationship, Drug, Female, Formazans metabolism, Genetic Therapy, Humans, Light, Nanospheres ultrastructure, Particle Size, Scattering, Radiation, Tetrazolium Salts metabolism, Trastuzumab, Endocytosis physiology, Magnetics, Nanospheres chemistry, Receptor, ErbB-2 metabolism
- Abstract
Polypyrrole-Fe3O4 nanospheres were synthesized via an emulsion polymerization method with hyaluronic acid as the surfactant. Hyaluronic acid offers the advantages of biocompatibility, cell adhesive property and the availability of functional groups for attachment of other molecules. The nanospheres were further functionalized with herceptin, and the efficacy of uptake of the functionalized nanospheres by human breast cancer cells was evaluated. It is envisioned that the combination of hyaluronic acid with its cell adhesive property and herceptin would result in high efficacy of internalization of the nanospheres by the cancer cells via a HER-2-mediated endocytosis. Our results showed that this is indeed the case and that the high concentration of herceptin-functionalized magnetic nanospheres in the cancer cells offers great potential in cancer cell targeting and treatment. In addition, the magnetic property of these nanospheres was also critically investigated and the magnetization was found to be affected by the particles' environment. The combination of these cell-targeting magnetic carriers with chemotherapeutic agents will be highly advantageous for the preferential killing of cancer cells in hyperthermia treatment.
- Published
- 2008
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38. Biochemical investigation of active intracellular transport of polymeric gene-delivery vectors.
- Author
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Drake DM and Pack DW
- Subjects
- Actins metabolism, Biological Transport, Active, Cell Line, Cytochalasin D pharmacology, Dyneins antagonists & inhibitors, Humans, Kinesins antagonists & inhibitors, Microtubules physiology, Genetic Therapy, Genetic Vectors, Polyethyleneimine pharmacokinetics
- Abstract
To design safe, efficient synthetic gene therapy vectors, it is desirable to understand the intracellular mechanisms that facilitate their delivery from the cell surface to the nucleus. Elements of the cytoskeleton and molecular motor proteins are known to play a pivotal role in most intracellular active transport processes. The actin depolymerizer cytochalasin D and microtubule effectors colchicine and paclitaxel were used to evaluate the function of these components of the cytoskeleton in the trafficking of polyethylenimine (PEI)-DNA complexes. In addition, ATPase inhibitors erythro-9[3-(2-hydroxynonyl)] adenine (EHNA), vanadate, adenylylimidodiphosphate (AMP-PNP), and rose bengal lactone (RBL), which have inhibitory activity against dynein and kinesin, were used to examine to the effects of these molecular motors on PEI-DNA delivery. Disruption of microfilaments decreased the delivery efficiency of PEI polyplexes 60-80%, though cytochalasin D did not significantly inhibit uptake. Depolymerization of microtubules by colchicine decreased transfection efficiency by 75%. Microtubule stabilization with paclitaxel, however, facilitated a 20-fold increase in gene expression. Treatment with EHNA and vanadate caused 50% and 80% decreases in transfection efficiency, respectively. Transfection efficiency was also decreased by RBL (80%) and AMP-PNP (98%). Our findings confirm the importance of microfilament- and microtubule-based active transport of PEI-DNA complexes. Further, the strong decrease in transfection efficiency caused by ATPase inhibitors that possess inhibitory activity against kinesin implies an unexpected role for these motors in gene delivery., (2007 Wiley-Liss, Inc)
- Published
- 2008
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39. DNA nanoparticles encapsulated in 3D tissue-engineered scaffolds enhance osteogenic differentiation of mesenchymal stem cells.
- Author
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Hosseinkhani H, Hosseinkhani M, Gabrielson NP, Pack DW, Khademhosseini A, and Kobayashi H
- Subjects
- Acetylation, Animals, Biocompatible Materials chemistry, Male, Polyethyleneimine chemistry, Rats, Rats, Inbred F344, Swine, Cell Differentiation physiology, DNA, Mesenchymal Stem Cells cytology, Nanoparticles, Osteogenesis physiology, Tissue Scaffolds
- Abstract
In this study, we enhanced the expression of a plasmid DNA in mesenchymal stem cells (MSC) by the combination of three-dimensional (3D) tissue-engineered scaffold and nonviral gene carrier. To function as an enhanced delivery of plasmid DNA, acetic anhydride was reacted with polyethylenimine (PEI) to acetylate 80% of the primary and 20% of the secondary amines (PEI-Ac(80)). This acetylated PEI has been demonstrated to show enhanced gene-delivery efficiency over unmodified PEI. Collagen sponges reinforced by incorporating of poly(glycolic acid) (PGA) fibers were used as the scaffold material. DNA nanoparticles formed through simple mixing of plasmid DNA encoding bone morphogenetic protein-2 (BMP-2) and PEI-Ac(80) solutions were encapsulated within these scaffolds. MSC were seeded into each scaffold and cultured for several weeks. Within these scaffolds, the level of BMP-2 expression by transfected MSC was significantly enhanced compared to MSC transfected by DNA nanoparticles in solution (in 2D tissue culture plates). Homogeneous bone formation was histologically observed throughout the sponges seeded with transfected MSC by using DNA nanoparticles after subcutaneous implantation into the back of rats. The level of alkaline phosphatase activity and osteocalcin content at the implanted sites of sponges seeded with transfected MSC by using DNA nanoparticles were significantly higher when compared with those seeded with other agents., (Copyright 2007 Wiley Periodicals, Inc.)
- Published
- 2008
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40. Engineering of a stable retroviral gene delivery vector by directed evolution.
- Author
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Vu HN, Ramsey JD, and Pack DW
- Subjects
- Animals, Cell Line, Humans, Mice, Models, Biological, Moloney murine leukemia virus genetics, Moloney murine leukemia virus growth & development, Mutation, NIH 3T3 Cells, Retroviridae growth & development, Directed Molecular Evolution methods, Genetic Engineering methods, Genetic Vectors genetics, Retroviridae genetics
- Abstract
The lack of safe and effective delivery vectors continues to be a critical limitation facing human gene therapy. Viruses offer excellent efficiency but can be difficult and expensive to produce and purify. For example, the production and efficiency of murine leukemia virus (MLV) are limited by its inherent instability; the half-life of infectivity is 5-8 hours at 37 degrees C. In order to generate a stable MLV, we randomly mutated the virus genome and selected for infectivity after prolonged incubation at 37 degrees C. After seven rounds of incubation and infection, we isolated a pool of MLV variants with double the half-life of wild-type MLV. Remarkably, a single mutation in the viral protease (PR), G119E, was responsible for the enhanced stability. Saturation mutagenesis at residue 119 revealed variants with half-lives of approximately 24 hours at 37 degrees C. Double mutants combining the changes at position 119 of the PR and substitutions in the PR substrate-binding pocket exhibited half-lives of up to approximately 40 hours. MLV variants provided two- to fourfold higher viral titers and exhibited increased stability with various wild-type envelope proteins. The improved stability of the variant MLVs will provide more facile virus production and increased transduction efficiency.
- Published
- 2008
- Full Text
- View/download PDF
41. Influence of particle size and antacid on release and stability of plasmid DNA from uniform PLGA microspheres.
- Author
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Varde NK and Pack DW
- Subjects
- Biocompatible Materials administration & dosage, DNA administration & dosage, DNA metabolism, Delayed-Action Preparations, Drug Compounding, Lactic Acid administration & dosage, Magnesium Hydroxide chemistry, Microscopy, Electron, Scanning, Microspheres, Nucleic Acid Conformation, Particle Size, Plasmids administration & dosage, Polyglycolic Acid administration & dosage, Polylactic Acid-Polyglycolic Acid Copolymer, Polymers administration & dosage, Porosity, Surface Properties, Antacids chemistry, Biocompatible Materials chemical synthesis, DNA chemical synthesis, Lactic Acid chemical synthesis, Plasmids chemical synthesis, Polyglycolic Acid chemical synthesis, Polymers chemical synthesis
- Abstract
PLGA microspheres are attractive DNA delivery vehicles due to their controlled release capabilities. One major problem with PLGA microspheres is that they develop an acidic microclimate as the polymer degrades, lowering the intraparticle pH, and potentially damaging the DNA. Antacids have recently shown promise in buffering this acidic microclimate and enhancing protein stability. We manufactured uniform plasmid DNA-encapsulating PLGA microspheres of two sizes (47, 80 microm diameter) and antacid concentrations (0, 3% Mg(OH)2). Microspheres with antacid had a homogeneous surface coverage of small pores, which resulted in a significant reduction of the burst effect. The 47 microm microspheres exhibited complete release of plasmid DNA over the course of two months. Incomplete release was observed from 80 microm spheres, though microspheres with 3% Mg(OH)2 showed a higher cumulative release, suggesting that the antacid at least partially aids in increasing the stability of DNA. SEM was used to visualize the surface pore evolution and cross-sectional microsphere structure over time. Subsequent image analysis was used to quantify the increase of surface pore sizes. Cross-sectional images showed increasing internal degradation and erosion, which resulted in a hollowing-out of microspheres. Our studies show that the incorporation of antacid into the microsphere structure has potential in addressing some of the major problems associated with DNA encapsulation and release in PLGA microspheres.
- Published
- 2007
- Full Text
- View/download PDF
42. Macromolecule release from monodisperse PLG microspheres: control of release rates and investigation of release mechanism.
- Author
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Berkland C, Pollauf E, Raman C, Silverman R, Kim K', and Pack DW
- Subjects
- Chemistry, Pharmaceutical, Delayed-Action Preparations, Dextrans chemistry, Diffusion, Drug Compounding, Fluoresceins chemistry, Fluorescent Dyes chemistry, Kinetics, Microscopy, Confocal, Microscopy, Electron, Scanning, Particle Size, Polylactic Acid-Polyglycolic Acid Copolymer, Porosity, Rhodamines chemistry, Serum Albumin, Bovine chemistry, Solubility, Surface Properties, Technology, Pharmaceutical methods, Drug Carriers, Lactic Acid chemistry, Microspheres, Polyglycolic Acid chemistry, Polymers chemistry
- Abstract
Novel macromolecular therapeutics such as peptides, proteins, and DNA are advancing rapidly toward the clinic. Because of typically low oral bioavailability, macromolecule delivery requires invasive methods such as frequently repeated injections. Parenteral depots including biodegradable polymer microspheres offer the possibility of reduced dosing frequency but are limited by the inability to adequately control delivery rates. To control release and investigate release mechanisms, we have encapsulated model macromolecules in monodisperse poly(D,L-lactide-co-glycolide) (PLG) microspheres using a double-emulsion method in combination with the precision particle fabrication technique. We encapsulated fluorescein-dextran (F-Dex) and sulforhodamine B-labeled bovine serum albumin (R-BSA) into PLG microspheres of three different sizes: 31, 44, and 80 microm and 34, 47, and 85 microm diameter for F-Dex and R-BSA, respectively. The in vitro release profiles of both compounds showed negligible initial burst. During degradation and release, the microspheres hollowed and swelled at critical time points dependant upon microsphere size. The rate of these events increased with microsphere size resulting in the largest microspheres exhibiting the fastest overall release rate. Monodisperse microspheres may represent a new delivery system for therapeutic proteins and DNA and provide enhanced control of delivery rates using simple injectable depot formulations., ((c) 2007 Wiley-Liss, Inc. and the American Pharmacists Association.)
- Published
- 2007
- Full Text
- View/download PDF
43. Monodisperse liquid-filled biodegradable microcapsules.
- Author
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Berkland C, Pollauf E, Varde N, Pack DW, and Kim KK
- Subjects
- Algorithms, Dextrans chemistry, Fatty Acids, Monounsaturated chemistry, Fluorescent Dyes chemistry, Hexanes chemistry, Lactic Acid chemistry, Methylene Chloride chemistry, Microscopy, Electron, Scanning, Oxazines chemistry, Particle Size, Polyanhydrides chemistry, Polyglycolic Acid chemistry, Polylactic Acid-Polyglycolic Acid Copolymer, Polymers chemistry, Pulsatile Flow, Rapeseed Oil, Rhodamines chemistry, Serum Albumin, Bovine chemistry, Silicone Oils chemistry, Solubility, Solutions chemistry, Water chemistry, Biocompatible Materials chemistry, Capsules chemistry, Drug Compounding methods, Emulsifying Agents chemistry
- Abstract
Purpose: Encapsulation of liquids into biodegradable polymer microcapsules has been a challenging task due to production limitations stemming from solution viscosity, phase stabilization, molecular localization, and scalable production. We report an extension of Precision Particle Fabrication (PPF) technology for the production of monodisperse liquid-filled microcapsules containing an oil or aqueous core and contrast these results to double-walled microspheres., Materials and Methods: PPF technology utilizes a coaxial nozzle to produce a liquid core jet surrounded by a polymer annular jet, which is further encompassed by a non-solvent carrier stream, typically 0.5% wt/vol polyvinyl alcohol in water. Jet diameters are controlled by the volumetric flow rate of each phase. The compound jet is then disrupted into uniform core/shell droplets via a controllable acoustic wave and shell material is hardened by solvent extraction., Results: Monodisperse polymeric microcapsules demonstrated a narrow size distribution and the formation of a continuous shell leading to efficient encapsulation of various liquid cores. The intermingling of core and shell phases and the localization of different molecular probes (fluorescent dyes and fluorescently labeled proteins) to the core or shell phase provided additional evidence of phase separation and molecular partitioning, respectively. We also demonstrate the pulsatile release of bovine serum albumin encapsulated in an aqueous core., Conclusions: PPF technology provided exceptional control of the overall size and shell thickness of microcapsules filled with various types of oil or water. This technique may enable advanced delivery profiles of pharmaceuticals or nutraceuticals.
- Published
- 2007
- Full Text
- View/download PDF
44. Acetylation of polyethylenimine enhances gene delivery via weakened polymer/DNA interactions.
- Author
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Gabrielson NP and Pack DW
- Subjects
- Acetylation, Animals, Cell Line, Humans, Mice, Surface Plasmon Resonance, Gene Transfer Techniques, Plasmids chemistry, Polyethyleneimine chemistry
- Abstract
We previously reported that gene delivery efficiency of 25-kDa, branched polyethylenimine (PEI) increased upon acetylation of up to 43% of the primary amines with acetic anhydride. In the present work, we investigated the effects of further increasing the degree of acetylation and elucidated the source of the higher gene delivery efficiency. Despite reduced buffering capacity, gene delivery activity continued to increase (up to 58-fold in HEK293) with acetylation of up to 57% of primary amines but decreased at higher degrees of acetylation. Characterization of polymer-DNA interactions showed that acetylated polymers bind less strongly to DNA. Further, a fluorescence resonance energy transfer assay showed that increasing acetylation causes polyplexes to unpackage inside cells to a higher degree than polyplexes formed with unmodified PEI. Overall, the data suggest that the increased gene delivery activity may be attributable to an appropriate balance between polymer buffering capacity and strength of polymer/DNA interactions.
- Published
- 2006
- Full Text
- View/download PDF
45. Use of thermodynamic parameters for design of double-walled microsphere fabrication methods.
- Author
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Pollauf EJ and Pack DW
- Subjects
- Chromatography, Gel, Lactic Acid chemistry, Molecular Weight, Polyglycolic Acid chemistry, Polylactic Acid-Polyglycolic Acid Copolymer, Polymers chemistry, Thermodynamics, Microspheres
- Abstract
Double-walled microspheres (DWMS), with drug localized to the particle core, present a promising route for control of drug release rates, for example, by varying the degradation rate or erosion mechanism of the polymer used to form the shell or the thickness of the shell. DWMS are often difficult to fabricate, however. Thermodynamic descriptions for polymer-polymer immiscibility, drug distribution between phases and polymer-solution spreading coefficient provide predictions of appropriate solvents and polymer concentrations for efficiently producing well-formed DWMS. As an example, thermodynamic parameters for a polyphosphoester/poly(D,L-lactide-co-glycolide) (PLG) DWMS system, encapsulating piroxicam, have been calculated and the predictions tested experimentally. Appropriate choices of solvents and initial polymer concentrations resulted in DWMS with the desired polyphosphoester shells and piroxicam located selectively in PLG cores.
- Published
- 2006
- Full Text
- View/download PDF
46. Dendrons with spermine surface groups as potential building blocks for nonviral vectors in gene therapy.
- Author
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Hardy JG, Kostiainen MA, Smith DK, Gabrielson NP, and Pack DW
- Subjects
- Animals, Cell Line, Cell Line, Tumor, Cell Survival, DNA chemistry, DNA metabolism, Dendrimers chemical synthesis, Dendrimers toxicity, Genetic Therapy, Humans, Mice, Plasmids genetics, Spermine chemistry, Spermine toxicity, Transfection, DNA administration & dosage, Dendrimers administration & dosage, Genetic Vectors, Spermine administration & dosage
- Abstract
This paper investigates a series of dendrons based on the Newkome dendritic scaffold that displays a naturally occurring polyamine (spermine) on their surface. These dendrons have previously been shown to interact with DNA in a generation dependent manner with the more highly branched dendrons exhibiting a strong multivalency effect for the spermine surface groups. In this paper, we investigate the ability of these dendrons to transfect DNA into cells (human breast carcinoma cells, MDA-MB-231, and murine myoblast cells, C2C12) as determined by the luciferase assay. Although the dendrons are unable to transfect DNA in their own right, they are capable of delivering DNA in vitro when administered with chloroquine, which assists with escape from endocytic vesicles. The cytotoxicity of the dendrons was determined using the XTT assay, and it was shown that the dendrons were nontoxic either alone or in the presence of DNA. However, when administered with DNA and chloroquine, the most highly branched dendron did exhibit some cytotoxicity. This paper elucidates the relationship between in vitro transfection efficiency and toxicity. While transfection efficiencies are modest, the low toxicity of the dendrons, both in their own right, and in the presence of DNA, provides encouragement that this type of building block, which has a relatively high affinity for DNA, will provide a useful starting point for the further synthetic development of more effective gene transfection agents.
- Published
- 2006
- Full Text
- View/download PDF
47. In vitro degradation of polyanhydride/polyester core-shell double-wall microspheres.
- Author
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Pollauf EJ, Berkland C, Kim KK, and Pack DW
- Subjects
- Chromatography, Gel, Drug Compounding, Drug Stability, Excipients, Microscopy, Electron, Scanning, Microscopy, Fluorescence, Molecular Weight, Particle Size, Polylactic Acid-Polyglycolic Acid Copolymer, Spectroscopy, Fourier Transform Infrared, Hexanes chemistry, Lactic Acid chemistry, Microspheres, Polyglycolic Acid chemistry, Polymers chemistry
- Abstract
Double-wall microspheres (DWMS), comprising distinct polymer core and shell phases, are useful and interesting for controlled-release drug delivery. In particular, the presence of a surface-eroding polymer core may be expected to limit water penetration and, therefore, delay degradation of the core phase and drug release. In this study, solid microspheres and DWMS were fabricated using a surface-eroding polymer (poly[1,6-bis(p-carboxyphenoxy)hexane]; PCPH) and a bulk-eroding polymer (poly(D,L-lactide-co-glycolide); PLG). Erosion of the particles was observed by optical and electron microscopy, while polymer degradation was followed by gel permeation chromatography, during incubation in buffer at 37 degrees C. Degradation and erosion were very different depending on which polymer formed the particle shell. Nevertheless, the relatively thin (approximately 5 microm) PCPH shells could not prevent water penetration, and the PLG cores completely eroded by 6 weeks of incubation.
- Published
- 2005
- Full Text
- View/download PDF
48. Small-molecule release from poly(D,L-lactide)/poly(D,L-lactide-co-glycolide) composite microparticles.
- Author
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Pollauf EJ, Kim KK, and Pack DW
- Subjects
- Chemistry, Pharmaceutical, Delayed-Action Preparations, Microscopy, Confocal, Microscopy, Electron, Scanning, Particle Size, Piroxicam chemistry, Polylactic Acid-Polyglycolic Acid Copolymer, Solubility, Time Factors, Drug Carriers chemistry, Lactic Acid chemistry, Microspheres, Polyesters chemistry, Polyglycolic Acid chemistry, Polymers chemistry
- Abstract
Addition of biodegradable polymer shells surrounding polymeric, drug-loaded microparticles offers the opportunity to control drug release rates. A novel fabrication method was used to produce microparticles with precise control of particle diameter and the thickness of the polymer shell. The effect of shell thickness on release of a model drug, piroxicam, has been clearly shown for 2- to 15-microm thick shells of poly(D,L-lactide) (PDLL) surrounding a poly(D,L-lactide-co-glycolide) (PLG) core and compared to pure PLG microspheres loaded with piroxicam. Furthermore, the core-shell microparticles are compared to microspheres containing blended polymers in the same mass ratios to demonstrate the importance of the core-shell morphology. Combining PDLL(PLG) microcapsules of different shell thicknesses allows nearly constant release rates to be attained for a period of 6 weeks.
- Published
- 2005
- Full Text
- View/download PDF
49. Meteoritic dust from the atmospheric disintegration of a large meteoroid.
- Author
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Klekociuk AR, Brown PG, Pack DW, ReVelle DO, Edwards WN, Spalding RE, Tagliaferri E, Yoo BB, and Zagari J
- Abstract
Much of the mass of most meteoroids entering the Earth's atmosphere is consumed in the process of ablation. Larger meteoroids (> 10 cm), which in some cases reach the ground as meteorites, typically have survival fractions near 1-25 per cent of their initial mass. The fate of the remaining ablated material is unclear, but theory suggests that much of it should recondense through coagulation as nanometre-sized particles. No direct measurements of such meteoric 'smoke' have hitherto been made. Here we report the disintegration of one of the largest meteoroids to have entered the Earth's atmosphere during the past decade, and show that the dominant contribution to the mass of the residual atmospheric aerosol was in the form of micrometre-sized particles. This result is contrary to the usual view that most of the material in large meteoroids is efficiently converted to particles of much smaller size through ablation. Assuming that our observations are of a typical event, we suggest that large meteoroids provide the dominant source of micrometre-sized meteoritic dust at the Earth's surface over long timescales.
- Published
- 2005
- Full Text
- View/download PDF
50. Design and development of polymers for gene delivery.
- Author
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Pack DW, Hoffman AS, Pun S, and Stayton PS
- Subjects
- Animals, Cyclodextrins, Drug Design, Genetic Therapy, Genetic Vectors, Humans, Imidazoles chemistry, Gene Transfer Techniques, Polymers
- Abstract
The lack of safe and efficient gene-delivery methods is a limiting obstacle to human gene therapy. Synthetic gene-delivery agents, although safer than recombinant viruses, generally do not possess the required efficacy. In recent years, a variety of effective polymers have been designed specifically for gene delivery, and much has been learned about their structure-function relationships. With the growing understanding of polymer gene-delivery mechanisms and continued efforts of creative polymer chemists, it is likely that polymer-based gene-delivery systems will become an important tool for human gene therapy.
- Published
- 2005
- Full Text
- View/download PDF
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