Jeffrey Hughes - Academia.edu (original) (raw)

Papers by Jeffrey Hughes

Research paper thumbnail of Effect of Lyophilization and Freeze-thawing on the Stability of siRNA-liposome Complexes

Aaps Pharmscitech, Dec 29, 2007

The purpose of this research was to describe the application of lyophilization in the delivery of... more The purpose of this research was to describe the application of lyophilization in the delivery of siRNA using cationic lipids by addressing the long-term formulation/stability issues associated with cationic lipids and to understand the mechanism of lyoprotection. siRNA liposomes complexes were formed in different potential cyro/lyoprotectants and subjected to either lyophilization or freeze thaw cycles. siRNA, liposomes and/or lipoplexes were tested for activity, SYBR Green I binding, cellular uptake and particle size. The lipoplexes when lyophilized in the presence of sugars as lyoprotectants could be lyophilized and reconstituted without loss of transfection efficacy but in ionic solutions they lost 65-75% of their functionality. The mechanism of this loss of activity was further investigated. The lyophilization process did not alter siRNA's intrinsic biological activity as was evident by the ability of lyophilized siRNA to retain functionality and SYBR green I binding ability. While the lipoplex size dramatically increased (∼50-70 times) after lyophilization in the absence of non-ionic lyoprotectants. This increase in size correlated to the decrease in cellular accumulation of siRNA and a decrease in activity. In conclusion, siRNAs can be applied in cationic lipid lyophilized formulations and these complexes represent a potential method of increasing the stability of pre-formed complex.

Research paper thumbnail of A Novel Piggyback Strategy for mRNA Delivery Exploiting Adenovirus Entry Biology

Viruses, Sep 30, 2022

This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY

Research paper thumbnail of Surface properties, more than size, limiting convective distribution of virus-sized particles and viruses in the central nervous system

Journal of Neurosurgery, 2005

Object. Achieving distribution of gene-carrying vectors is a major barrier to the clinical applic... more Object. Achieving distribution of gene-carrying vectors is a major barrier to the clinical application of gene therapy. Because of the blood—brain barrier, the distribution of genetic vectors to the central nervous system (CNS) is even more challenging than delivery to other tissues. Direct intraparenchymal microinfusion, a minimally invasive technique, uses bulk flow (convection) to distribute suspensions of macromolecules widely through the extracellular space (convection-enhanced delivery [CED]). Although acute injection into solid tissue is often used for delivery of oligonucleotides, viruses, and liposomes, and there is preliminary evidence that certain of these large particles can spread through the interstitial space of the brain by the use of convection, the use of CED for distribution of viruses in the brain has not been systematically examined. That is the goal of this study. Methods. Investigators used a rodent model to examine the influence of size, osmolarity of bufferi...

Research paper thumbnail of A Novel Piggyback Strategy for mRNA Delivery Exploiting Adenovirus Entry Biology

Molecular therapies exploiting mRNA vectors embody enormous potential, as evidenced by the utilit... more Molecular therapies exploiting mRNA vectors embody enormous potential, as evidenced by the utility of this technology for the context of the COVID-19 pandemic. None-the-less, broad implementation of these promising strategies has been restricted by the limited repertoires of delivery vehicles capable of mRNA transport. On this basis, we explored a strategy based on exploiting the well characterized entry biology of adenovirus. To this end, we studied an adenovirus-polylysine (AdpL) that embodied “piggyback” transport of the mRNA on the capsid exterior of adenovirus. We hypothesized that the efficient steps of Ad binding, receptor-mediated entry, and capsid-mediated endosome escape could provide an effective pathway for transport of mRNA to the cellular cytosol for transgene expression. Our studies confirmed that AdpL could mediate effective gene transfer of mRNA vectors in vitro and in vivo. Facets of this method may offer key utilities to feasibilize the promise of mRNA-based thera...

Research paper thumbnail of Kinetic modeling of plasmid DNA degradation in rat plasma

AAPS PharmSci, 1999

A major obstacle in gene delivery is the transport of intact plasmid DNA (pDNA) to target sites. ... more A major obstacle in gene delivery is the transport of intact plasmid DNA (pDNA) to target sites. We sought to investigate the kinetic processes underlying the degradation of pDNA in a rat plasma model, as this is one of the main components responsible for the clearance of pDNA after intravenous administration. We further sought to construct a complete kinetic model to describe the degradation of all three topoforms (supercoiled, open circular, and linear) of pDNA in a rat plasma model. Supercoiled pDNA was incubated in isolated rat plasma at 37 o C in vitro. At various time points, the plasma was assayed by electrophoresis for the amounts of supercoiled, open circular, and full-length linear pDNA remaining. The calculated amounts remaining were fit to linear differential equations describing this process. In this model, pDNA degradation is considered to be a unidirectional process, with supercoiled degrading to open circular and then to the linear topoform. The calculated kinetic parameters suggested that supercoiled pDNA degrades in rat plasma with a half-life of 1.2 minutes, open circular pDNA degrades with a half-life of 21 minutes, and linear pDNA degrades with a half-life of 11 minutes. Complexation of pDNA with liposomes resulted in a portion of the supercoiled plasmid remaining detectable through 5.5 hours.

Research paper thumbnail of The protective effects of plasma gelsolin on stroke outcome in rats

Experimental & translational stroke medicine, Jan 2, 2011

To date, recombinant tissue plasminogen activator (rtPA) is the only approved drug for ischemic s... more To date, recombinant tissue plasminogen activator (rtPA) is the only approved drug for ischemic stroke. It is intravenously administered functioning as a thrombolytic agent and is used to obtain reperfusion of the affected area of the brain. Excitotoxicity, inflammation and apoptosis are all involved in delayed neuronal death following stroke and offer multiple opportunities to intervene with neuroprotective agents. Gelsolin (GSN) is an actin- and calcium-binding protein mediating the disassembly of actin filaments and activity of calcium channels. It also functions as a regulator of apoptosis and inflammatory responses. This study tests the hypothesis that increasing the concentration of the form of GSN known as plasma GSN (pGSN) near an infarct will provide neuroprotection following ischemic stroke. We induced middle cerebral artery occlusion (MCAO) in male rats via intracranial injection of endothelin-1 (ET-1), a potent vasoconstrictor, and then treated with local delivery of pGS...

Research paper thumbnail of Rationally Designed Synthetic Vectors for Gene Delivery

The Open Drug Delivery Journal, 2007

Vector development is one of the most important challenges facing the successful use of genes for... more Vector development is one of the most important challenges facing the successful use of genes for treatment of diseases. Although chemically produced vectors offer distinct advantages over biological systems such as viruses, there are still some hurdles that have to be overcome before synthetic gene delivery vectors can be successfully implemented. This brief review discusses the biological barriers that limit current delivery strategies and reviews currently employed strategies for plasmid delivery. Nanoparticle-based gene delivery is reviewed along with methods for their characterization, physiochemical properties and toxicity. Finally a prospectus is provided for future development of an ideal synthetic gene delivery vector.

Research paper thumbnail of α7 Nicotinic receptor gene delivery into mouse hippocampal neurons leads to functional receptor expression, improved spatial memory-related performance, and tau hyperphosphorylation

Neuroscience, 2007

Brain ␣7 nicotinic receptors have become therapeutic targets for Alzheimer's disease (AD) based o... more Brain ␣7 nicotinic receptors have become therapeutic targets for Alzheimer's disease (AD) based on their memory-enhancing and neuroprotective actions. This study investigated the feasibility of increasing neuronal ␣7 receptor functions using a gene delivery approach based on neuron-selective recombinant adeno-associated virus (rAAV)derived vectors. In order to determine whether ␣7 receptormediated cytotoxicity was dependent on receptor density, rat ␣7 nicotinic receptors were expressed at high concentrations in GH4C1 cells as measured with nicotine-displaceable [ 3 H]methyllycaconitine (MLA) binding. The potency of GTS-21 (an ␣7 receptor agonist) to induce cell loss was similar in these cells to that seen in pheochromocytoma (PC12) cells expressing nine-times-lower receptor levels, suggesting that cytotoxicity was more dependent on agonist concentration than receptor density. Hippocampal transduction with rat ␣7 nicotinic receptors increased [ 3 H]MLA binding in this region in wild type and ␣7 receptor-knockout (KO) mice without apparent cytotoxicity. No difference was observed in Kd values for MLA binding between endogenous and transgenic receptors. Single cell recordings demonstrated that dentate granule cells that normally have no ␣7 receptor response did so following ␣7 receptor gene delivery in wild type mice. Recovery of ␣7 function was also observed in stratum oriens and stratum radiatum neurons of KO mice following gene delivery. Wild type mice exhibited improved acquisition performance in the Morris water task 1 month after bilateral hippocampal transductions with the rat ␣7 receptor gene compared with green fluorescent protein-transduced controls. However, both groups reached similar training levels and there was no difference in subsequent probe performance. Finally, this gene delivery approach was used to test whether ␣7 receptors affect tau-phosphorylation. Chronic (i.e. 2 month but not 2 week) expression of high levels of ␣7 receptors in hippocampus increased AT8 staining characteristic of hyperphosphorylated tau in that region, indicating that endogenous agonist-mediated receptor activation may be able to modulate this process.

Research paper thumbnail of Adenovirus-Facilitated Nuclear Translocation of Adeno-Associated Virus Type 2

Journal of Virology, 2002

We examined cytoplasmic trafficking and nuclear translocation of adeno-associated virus type 2 (A... more We examined cytoplasmic trafficking and nuclear translocation of adeno-associated virus type 2 (AAV) by using Alexa Fluor 488-conjugated wild-type AAV, A20 monoclonal antibody immunocytochemistry, and subcellular fractionation techniques followed by DNA hybridization. Our results indicated that in the absence of adenovirus (Ad), AAV enters the cell rapidly and escapes from early endosomes with a t 1/2 of about 10 min postinfection. Cytoplasmically distributed AAV accumulated around the nucleus and persisted perinuclearly for 16 to 24 h. Viral uncoating occurred before or during nuclear entry beginning about 12 h postinfection, when viral protein and DNA were readily detected in the nucleus. Few, if any, intact AAV capsids were found in the nucleus. In the presence of Ad, however, cytoplasmic AAV quickly translocated into the nucleus as intact particles as early as 40 min after coinfection, and this facilitated nuclear translocation of AAV was not blocked by the nuclear pore complex ...

Research paper thumbnail of Alpha 1-Antitrypsin Therapy Mitigated Ischemic Stroke Damage in Rats

Journal of Stroke and Cerebrovascular Diseases, 2014

Currently, the only effective therapy for acute ischemic stroke is the thrombolytic agent recombi... more Currently, the only effective therapy for acute ischemic stroke is the thrombolytic agent recombinant tissue plasminogen activator. α 1-Antitrypsin, an endogenous inhibitor of serine proteinases and a primary acute phase protein with potent anti-inflammatory, anti-apoptotic, antimicrobial and cytoprotective activities, could be beneficial in stroke.. The goal of this study was to test whether α 1-antitrypsin could improve ischemic stroke outcome in an established rat model. Middle cerebral artery occlusion was induced in male rats via intracranial microinjection of endothelin-1. Five to ten minutes following stroke induction rats received either intracranial or intravenous delivery of human α 1-antitrypsin. Cylinder and vibrissae tests were used to evaluate sensorimotor function before and 72 hours after middle cerebral artery occlusion. Infarct volumes were examined via either 2,3,5-triphenyltetrazolium chloride assay or magnetic resonance imaging 72 hours after middle cerebral artery occlusion. Despite equivalent initial strokes, at 72 hours the infarct volumes of the human α 1-antitrypsin treatment groups (local and systemic injection) were statistically significantly reduced by 83% and 63% (p<0.0001 and p < 0.05 respectively) compared with control rats. Human α 1-antitrypsin significantly limited sensory motor systems deficits. Human α 1-antitrypsin could be a potential novel therapeutic drug for the protection against

Research paper thumbnail of Effect of Lyophilization and Freeze-thawing on the Stability of siRNA-liposome Complexes

AAPS PharmSciTech, 2007

The purpose of this research was to describe the application of lyophilization in the delivery of... more The purpose of this research was to describe the application of lyophilization in the delivery of siRNA using cationic lipids by addressing the long-term formulation/stability issues associated with cationic lipids and to understand the mechanism of lyoprotection. siRNA liposomes complexes were formed in different potential cyro/lyoprotectants and subjected to either lyophilization or freeze thaw cycles. siRNA, liposomes and/or lipoplexes were tested for activity, SYBR Green I binding, cellular uptake and particle size. The lipoplexes when lyophilized in the presence of sugars as lyoprotectants could be lyophilized and reconstituted without loss of transfection efficacy but in ionic solutions they lost 65-75% of their functionality. The mechanism of this loss of activity was further investigated. The lyophilization process did not alter siRNA's intrinsic biological activity as was evident by the ability of lyophilized siRNA to retain functionality and SYBR green I binding ability. While the lipoplex size dramatically increased (∼50-70 times) after lyophilization in the absence of non-ionic lyoprotectants. This increase in size correlated to the decrease in cellular accumulation of siRNA and a decrease in activity. In conclusion, siRNAs can be applied in cationic lipid lyophilized formulations and these complexes represent a potential method of increasing the stability of pre-formed complex.

Research paper thumbnail of Characterization of a pH-sensitive surfactant, dodecyl-2-(1′-imidazolyl) propionate (DIP), and preliminary studies in liposome mediated gene transfer

Biochimica et Biophysica Acta (BBA) - Biomembranes, 1998

BIOCHIMICA ET BIOPHYSICA ACTA 88N Characterization of a pH-sensitive surfactant, dodecyl-2-(l'-im... more BIOCHIMICA ET BIOPHYSICA ACTA 88N Characterization of a pH-sensitive surfactant, dodecyl-2-(l'-imidazolyl) propionate (DIP), and preliminary studies in liposome mediated gene transfer

Research paper thumbnail of Liposomal siRNA Delivery

Methods in Molecular Biology, 2009

With the recent discovery of small interfering RNA (siRNA), to silence the expression of genes in... more With the recent discovery of small interfering RNA (siRNA), to silence the expression of genes in vitro and in vivo, there has been a need to deliver these molecules to the cell nucleus. Forming a lipid/nucleic acid complex has become a solution and is explored here. Certain methods and ideas are used, such as: the positive/negative electrostatic interaction with a cationic lipid and an anionic RNA molecule, the size of the lipid vesicle aiding the uptake target tissues, targeted lipoplexes which can increase efficiency, and the protection of the siRNA molecule from the natural defenses of the immune system. Many lipid formulations exist and can be experimented with to achieve varying results depending on the application.

Research paper thumbnail of Alpha 1-Antitrypsin Therapy Mitigated Ischemic Stroke Damage in Rats

Journal of Stroke and Cerebrovascular Diseases, 2014

Our objective is to develop a new therapy for the treatment of stroke. Currently, the only effect... more Our objective is to develop a new therapy for the treatment of stroke. Currently, the only effective therapy for acute ischemic stroke is the thrombolytic agent recombinant tissue plasminogen activator. α1-Antitrypsin (AAT), a serine proteinase inhibitor with potent anti-inflammatory, anti-apoptotic, antimicrobial, and cytoprotective activities, could be beneficial in stroke. The goal of this study is to test whether AAT can improve ischemic stroke outcome in an established rat model. Middle cerebral artery occlusion was induced in male rats via intracranial (i.c.) microinjection of endothelin-1. Five to 10 minutes after stroke induction, rats received either i.c. or intravenous delivery of human AAT. Cylinder and vibrissae tests were used to evaluate sensorimotor function before and 72 hours after middle cerebral artery occlusion. Infarct volumes were examined via either 2,3,5-triphenyltetrazolium chloride assay or magnetic resonance imaging 72 hours after middle cerebral artery occlusion. Despite equivalent initial strokes, at 72 hours, the infarct volumes of the human AAT treatment groups (local and systemic injection) were statistically significantly reduced by 83% and 63% (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; .0001 and P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; .05, respectively) compared with control rats. Human AAT significantly limited sensory motor system deficits. Human AAT could be a potential novel therapeutic drug for the protection against neurodegeneration after ischemic stroke, but more studies are needed to investigate the protective mechanisms and efficacy in other animal models.

Research paper thumbnail of Effect of Lyophilization and Freeze-thawing on the Stability of siRNA-liposome Complexes

Aaps Pharmscitech, Dec 29, 2007

The purpose of this research was to describe the application of lyophilization in the delivery of... more The purpose of this research was to describe the application of lyophilization in the delivery of siRNA using cationic lipids by addressing the long-term formulation/stability issues associated with cationic lipids and to understand the mechanism of lyoprotection. siRNA liposomes complexes were formed in different potential cyro/lyoprotectants and subjected to either lyophilization or freeze thaw cycles. siRNA, liposomes and/or lipoplexes were tested for activity, SYBR Green I binding, cellular uptake and particle size. The lipoplexes when lyophilized in the presence of sugars as lyoprotectants could be lyophilized and reconstituted without loss of transfection efficacy but in ionic solutions they lost 65-75% of their functionality. The mechanism of this loss of activity was further investigated. The lyophilization process did not alter siRNA's intrinsic biological activity as was evident by the ability of lyophilized siRNA to retain functionality and SYBR green I binding ability. While the lipoplex size dramatically increased (∼50-70 times) after lyophilization in the absence of non-ionic lyoprotectants. This increase in size correlated to the decrease in cellular accumulation of siRNA and a decrease in activity. In conclusion, siRNAs can be applied in cationic lipid lyophilized formulations and these complexes represent a potential method of increasing the stability of pre-formed complex.

Research paper thumbnail of A Novel Piggyback Strategy for mRNA Delivery Exploiting Adenovirus Entry Biology

Viruses, Sep 30, 2022

This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY

Research paper thumbnail of Surface properties, more than size, limiting convective distribution of virus-sized particles and viruses in the central nervous system

Journal of Neurosurgery, 2005

Object. Achieving distribution of gene-carrying vectors is a major barrier to the clinical applic... more Object. Achieving distribution of gene-carrying vectors is a major barrier to the clinical application of gene therapy. Because of the blood—brain barrier, the distribution of genetic vectors to the central nervous system (CNS) is even more challenging than delivery to other tissues. Direct intraparenchymal microinfusion, a minimally invasive technique, uses bulk flow (convection) to distribute suspensions of macromolecules widely through the extracellular space (convection-enhanced delivery [CED]). Although acute injection into solid tissue is often used for delivery of oligonucleotides, viruses, and liposomes, and there is preliminary evidence that certain of these large particles can spread through the interstitial space of the brain by the use of convection, the use of CED for distribution of viruses in the brain has not been systematically examined. That is the goal of this study. Methods. Investigators used a rodent model to examine the influence of size, osmolarity of bufferi...

Research paper thumbnail of A Novel Piggyback Strategy for mRNA Delivery Exploiting Adenovirus Entry Biology

Molecular therapies exploiting mRNA vectors embody enormous potential, as evidenced by the utilit... more Molecular therapies exploiting mRNA vectors embody enormous potential, as evidenced by the utility of this technology for the context of the COVID-19 pandemic. None-the-less, broad implementation of these promising strategies has been restricted by the limited repertoires of delivery vehicles capable of mRNA transport. On this basis, we explored a strategy based on exploiting the well characterized entry biology of adenovirus. To this end, we studied an adenovirus-polylysine (AdpL) that embodied “piggyback” transport of the mRNA on the capsid exterior of adenovirus. We hypothesized that the efficient steps of Ad binding, receptor-mediated entry, and capsid-mediated endosome escape could provide an effective pathway for transport of mRNA to the cellular cytosol for transgene expression. Our studies confirmed that AdpL could mediate effective gene transfer of mRNA vectors in vitro and in vivo. Facets of this method may offer key utilities to feasibilize the promise of mRNA-based thera...

Research paper thumbnail of Kinetic modeling of plasmid DNA degradation in rat plasma

AAPS PharmSci, 1999

A major obstacle in gene delivery is the transport of intact plasmid DNA (pDNA) to target sites. ... more A major obstacle in gene delivery is the transport of intact plasmid DNA (pDNA) to target sites. We sought to investigate the kinetic processes underlying the degradation of pDNA in a rat plasma model, as this is one of the main components responsible for the clearance of pDNA after intravenous administration. We further sought to construct a complete kinetic model to describe the degradation of all three topoforms (supercoiled, open circular, and linear) of pDNA in a rat plasma model. Supercoiled pDNA was incubated in isolated rat plasma at 37 o C in vitro. At various time points, the plasma was assayed by electrophoresis for the amounts of supercoiled, open circular, and full-length linear pDNA remaining. The calculated amounts remaining were fit to linear differential equations describing this process. In this model, pDNA degradation is considered to be a unidirectional process, with supercoiled degrading to open circular and then to the linear topoform. The calculated kinetic parameters suggested that supercoiled pDNA degrades in rat plasma with a half-life of 1.2 minutes, open circular pDNA degrades with a half-life of 21 minutes, and linear pDNA degrades with a half-life of 11 minutes. Complexation of pDNA with liposomes resulted in a portion of the supercoiled plasmid remaining detectable through 5.5 hours.

Research paper thumbnail of The protective effects of plasma gelsolin on stroke outcome in rats

Experimental & translational stroke medicine, Jan 2, 2011

To date, recombinant tissue plasminogen activator (rtPA) is the only approved drug for ischemic s... more To date, recombinant tissue plasminogen activator (rtPA) is the only approved drug for ischemic stroke. It is intravenously administered functioning as a thrombolytic agent and is used to obtain reperfusion of the affected area of the brain. Excitotoxicity, inflammation and apoptosis are all involved in delayed neuronal death following stroke and offer multiple opportunities to intervene with neuroprotective agents. Gelsolin (GSN) is an actin- and calcium-binding protein mediating the disassembly of actin filaments and activity of calcium channels. It also functions as a regulator of apoptosis and inflammatory responses. This study tests the hypothesis that increasing the concentration of the form of GSN known as plasma GSN (pGSN) near an infarct will provide neuroprotection following ischemic stroke. We induced middle cerebral artery occlusion (MCAO) in male rats via intracranial injection of endothelin-1 (ET-1), a potent vasoconstrictor, and then treated with local delivery of pGS...

Research paper thumbnail of Rationally Designed Synthetic Vectors for Gene Delivery

The Open Drug Delivery Journal, 2007

Vector development is one of the most important challenges facing the successful use of genes for... more Vector development is one of the most important challenges facing the successful use of genes for treatment of diseases. Although chemically produced vectors offer distinct advantages over biological systems such as viruses, there are still some hurdles that have to be overcome before synthetic gene delivery vectors can be successfully implemented. This brief review discusses the biological barriers that limit current delivery strategies and reviews currently employed strategies for plasmid delivery. Nanoparticle-based gene delivery is reviewed along with methods for their characterization, physiochemical properties and toxicity. Finally a prospectus is provided for future development of an ideal synthetic gene delivery vector.

Research paper thumbnail of α7 Nicotinic receptor gene delivery into mouse hippocampal neurons leads to functional receptor expression, improved spatial memory-related performance, and tau hyperphosphorylation

Neuroscience, 2007

Brain ␣7 nicotinic receptors have become therapeutic targets for Alzheimer's disease (AD) based o... more Brain ␣7 nicotinic receptors have become therapeutic targets for Alzheimer's disease (AD) based on their memory-enhancing and neuroprotective actions. This study investigated the feasibility of increasing neuronal ␣7 receptor functions using a gene delivery approach based on neuron-selective recombinant adeno-associated virus (rAAV)derived vectors. In order to determine whether ␣7 receptormediated cytotoxicity was dependent on receptor density, rat ␣7 nicotinic receptors were expressed at high concentrations in GH4C1 cells as measured with nicotine-displaceable [ 3 H]methyllycaconitine (MLA) binding. The potency of GTS-21 (an ␣7 receptor agonist) to induce cell loss was similar in these cells to that seen in pheochromocytoma (PC12) cells expressing nine-times-lower receptor levels, suggesting that cytotoxicity was more dependent on agonist concentration than receptor density. Hippocampal transduction with rat ␣7 nicotinic receptors increased [ 3 H]MLA binding in this region in wild type and ␣7 receptor-knockout (KO) mice without apparent cytotoxicity. No difference was observed in Kd values for MLA binding between endogenous and transgenic receptors. Single cell recordings demonstrated that dentate granule cells that normally have no ␣7 receptor response did so following ␣7 receptor gene delivery in wild type mice. Recovery of ␣7 function was also observed in stratum oriens and stratum radiatum neurons of KO mice following gene delivery. Wild type mice exhibited improved acquisition performance in the Morris water task 1 month after bilateral hippocampal transductions with the rat ␣7 receptor gene compared with green fluorescent protein-transduced controls. However, both groups reached similar training levels and there was no difference in subsequent probe performance. Finally, this gene delivery approach was used to test whether ␣7 receptors affect tau-phosphorylation. Chronic (i.e. 2 month but not 2 week) expression of high levels of ␣7 receptors in hippocampus increased AT8 staining characteristic of hyperphosphorylated tau in that region, indicating that endogenous agonist-mediated receptor activation may be able to modulate this process.

Research paper thumbnail of Adenovirus-Facilitated Nuclear Translocation of Adeno-Associated Virus Type 2

Journal of Virology, 2002

We examined cytoplasmic trafficking and nuclear translocation of adeno-associated virus type 2 (A... more We examined cytoplasmic trafficking and nuclear translocation of adeno-associated virus type 2 (AAV) by using Alexa Fluor 488-conjugated wild-type AAV, A20 monoclonal antibody immunocytochemistry, and subcellular fractionation techniques followed by DNA hybridization. Our results indicated that in the absence of adenovirus (Ad), AAV enters the cell rapidly and escapes from early endosomes with a t 1/2 of about 10 min postinfection. Cytoplasmically distributed AAV accumulated around the nucleus and persisted perinuclearly for 16 to 24 h. Viral uncoating occurred before or during nuclear entry beginning about 12 h postinfection, when viral protein and DNA were readily detected in the nucleus. Few, if any, intact AAV capsids were found in the nucleus. In the presence of Ad, however, cytoplasmic AAV quickly translocated into the nucleus as intact particles as early as 40 min after coinfection, and this facilitated nuclear translocation of AAV was not blocked by the nuclear pore complex ...

Research paper thumbnail of Alpha 1-Antitrypsin Therapy Mitigated Ischemic Stroke Damage in Rats

Journal of Stroke and Cerebrovascular Diseases, 2014

Currently, the only effective therapy for acute ischemic stroke is the thrombolytic agent recombi... more Currently, the only effective therapy for acute ischemic stroke is the thrombolytic agent recombinant tissue plasminogen activator. α 1-Antitrypsin, an endogenous inhibitor of serine proteinases and a primary acute phase protein with potent anti-inflammatory, anti-apoptotic, antimicrobial and cytoprotective activities, could be beneficial in stroke.. The goal of this study was to test whether α 1-antitrypsin could improve ischemic stroke outcome in an established rat model. Middle cerebral artery occlusion was induced in male rats via intracranial microinjection of endothelin-1. Five to ten minutes following stroke induction rats received either intracranial or intravenous delivery of human α 1-antitrypsin. Cylinder and vibrissae tests were used to evaluate sensorimotor function before and 72 hours after middle cerebral artery occlusion. Infarct volumes were examined via either 2,3,5-triphenyltetrazolium chloride assay or magnetic resonance imaging 72 hours after middle cerebral artery occlusion. Despite equivalent initial strokes, at 72 hours the infarct volumes of the human α 1-antitrypsin treatment groups (local and systemic injection) were statistically significantly reduced by 83% and 63% (p<0.0001 and p < 0.05 respectively) compared with control rats. Human α 1-antitrypsin significantly limited sensory motor systems deficits. Human α 1-antitrypsin could be a potential novel therapeutic drug for the protection against

Research paper thumbnail of Effect of Lyophilization and Freeze-thawing on the Stability of siRNA-liposome Complexes

AAPS PharmSciTech, 2007

The purpose of this research was to describe the application of lyophilization in the delivery of... more The purpose of this research was to describe the application of lyophilization in the delivery of siRNA using cationic lipids by addressing the long-term formulation/stability issues associated with cationic lipids and to understand the mechanism of lyoprotection. siRNA liposomes complexes were formed in different potential cyro/lyoprotectants and subjected to either lyophilization or freeze thaw cycles. siRNA, liposomes and/or lipoplexes were tested for activity, SYBR Green I binding, cellular uptake and particle size. The lipoplexes when lyophilized in the presence of sugars as lyoprotectants could be lyophilized and reconstituted without loss of transfection efficacy but in ionic solutions they lost 65-75% of their functionality. The mechanism of this loss of activity was further investigated. The lyophilization process did not alter siRNA's intrinsic biological activity as was evident by the ability of lyophilized siRNA to retain functionality and SYBR green I binding ability. While the lipoplex size dramatically increased (∼50-70 times) after lyophilization in the absence of non-ionic lyoprotectants. This increase in size correlated to the decrease in cellular accumulation of siRNA and a decrease in activity. In conclusion, siRNAs can be applied in cationic lipid lyophilized formulations and these complexes represent a potential method of increasing the stability of pre-formed complex.

Research paper thumbnail of Characterization of a pH-sensitive surfactant, dodecyl-2-(1′-imidazolyl) propionate (DIP), and preliminary studies in liposome mediated gene transfer

Biochimica et Biophysica Acta (BBA) - Biomembranes, 1998

BIOCHIMICA ET BIOPHYSICA ACTA 88N Characterization of a pH-sensitive surfactant, dodecyl-2-(l'-im... more BIOCHIMICA ET BIOPHYSICA ACTA 88N Characterization of a pH-sensitive surfactant, dodecyl-2-(l'-imidazolyl) propionate (DIP), and preliminary studies in liposome mediated gene transfer

Research paper thumbnail of Liposomal siRNA Delivery

Methods in Molecular Biology, 2009

With the recent discovery of small interfering RNA (siRNA), to silence the expression of genes in... more With the recent discovery of small interfering RNA (siRNA), to silence the expression of genes in vitro and in vivo, there has been a need to deliver these molecules to the cell nucleus. Forming a lipid/nucleic acid complex has become a solution and is explored here. Certain methods and ideas are used, such as: the positive/negative electrostatic interaction with a cationic lipid and an anionic RNA molecule, the size of the lipid vesicle aiding the uptake target tissues, targeted lipoplexes which can increase efficiency, and the protection of the siRNA molecule from the natural defenses of the immune system. Many lipid formulations exist and can be experimented with to achieve varying results depending on the application.

Research paper thumbnail of Alpha 1-Antitrypsin Therapy Mitigated Ischemic Stroke Damage in Rats

Journal of Stroke and Cerebrovascular Diseases, 2014

Our objective is to develop a new therapy for the treatment of stroke. Currently, the only effect... more Our objective is to develop a new therapy for the treatment of stroke. Currently, the only effective therapy for acute ischemic stroke is the thrombolytic agent recombinant tissue plasminogen activator. α1-Antitrypsin (AAT), a serine proteinase inhibitor with potent anti-inflammatory, anti-apoptotic, antimicrobial, and cytoprotective activities, could be beneficial in stroke. The goal of this study is to test whether AAT can improve ischemic stroke outcome in an established rat model. Middle cerebral artery occlusion was induced in male rats via intracranial (i.c.) microinjection of endothelin-1. Five to 10 minutes after stroke induction, rats received either i.c. or intravenous delivery of human AAT. Cylinder and vibrissae tests were used to evaluate sensorimotor function before and 72 hours after middle cerebral artery occlusion. Infarct volumes were examined via either 2,3,5-triphenyltetrazolium chloride assay or magnetic resonance imaging 72 hours after middle cerebral artery occlusion. Despite equivalent initial strokes, at 72 hours, the infarct volumes of the human AAT treatment groups (local and systemic injection) were statistically significantly reduced by 83% and 63% (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; .0001 and P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; .05, respectively) compared with control rats. Human AAT significantly limited sensory motor system deficits. Human AAT could be a potential novel therapeutic drug for the protection against neurodegeneration after ischemic stroke, but more studies are needed to investigate the protective mechanisms and efficacy in other animal models.