Kenneth Howard - Academia.edu (original) (raw)

Papers by Kenneth Howard

Since the late 1990s, the National Severe Storms Laboratory (NSSL) has been actively involved in ... more Since the late 1990s, the National Severe Storms Laboratory (NSSL) has been actively involved in developing, maintaining, and serving spatial data to National Weather Service (NWS) users in support of flash-flood operations. One of the first and largest efforts was the creation of a national seamless dataset of flash-flood-scale basins. These basins provide the spatial framework for calculations of average basin rainfall rate and accumulation in the Flash Flood Monitoring and Prediction [1] (FFMP) program used at the NWS Weather Forecast Offices (WFOs). During the past decade, the dataset has been updated and enhanced with features such as seamless hydrologic-connectivity attributes that allow downstream tracing of flow and upstream drainage area selection. This dataset is currently served to users through NSSL's National FFMP Basin Repository and FFMP Basin Customization Repository websites. As a result of recent NWS Advanced Hydrologic Prediction Service (AHPS) funding, a new ...

Journal of extracellular vesicles, 2014

Cells release a mixture of extracellular vesicles, amongst these exosomes, that differ in size, d... more Cells release a mixture of extracellular vesicles, amongst these exosomes, that differ in size, density and composition. The standard isolation method for exosomes is centrifugation of fluid samples, typically at 100,000×g or above. Knowledge of the effect of discrete ultracentrifugation speeds on the purification from different cell types, however, is limited. We examined the effect of applying differential centrifugation g-forces ranging from 33,000×g to 200,000×g on exosome yield and purity, using 2 unrelated human cell lines, embryonic kidney HEK293 cells and bladder carcinoma FL3 cells. The fractions were evaluated by nanoparticle tracking analysis (NTA), total protein quantification and immunoblotting for CD81, TSG101, syntenin, VDAC1 and calreticulin. NTA revealed the lowest background particle count in Dulbecco's Modified Eagle's Medium media devoid of phenol red and cleared by 200,000×g overnight centrifugation. The centrifugation tube fill level impacted the sedime...

2011 IEEE RadarCon (RADAR), 2011

ABSTRACT The melting of aggregated snow/crystals often results in an enhancement of the reflectiv... more ABSTRACT The melting of aggregated snow/crystals often results in an enhancement of the reflectivity observed by weather radars, and this is commonly referenced as the bright band (BB). The locally high reflectivity often causes overestimation in radar quantitative precipitation estimates (QPE) if no appropriate correction is applied. When the melting layer is high, a complete bright band layer profile including top, peak, and bottom can be observed by the radar and a vertical profile of reflectivity (VPR) correction can be made to reduce the BB impact. When a melting layer is near the ground and the bottom part of the bright band cannot be observed by the radar, a VPR correction can not be made directly from radar observations. This paper presents a new VPR correction method under this situation. From high- resolution precipitation profiler data, an empirical relationship between BB peak and BB bottom is developed. The empirical relationship is combined with the apparent BB peak observed by volume scan radars and the BB bottom is found. Radar QPEs are then corrected based on the estimated BB bottom. The new method is shown to be effective in reducing the radar QPE overestimation under low bright band situations. I. INTRODUCTION When ice crystals or snow flakes fell into warm surroundings below the freezing level, two consequent effects would impact the reflectivity of the particles: 1) The change from ice to water would result in an increase in the reflective properties of the particles so that the radar reflectivity intensity increases; 2) the fall velocity of the flakes is less than that of the resulting water drops so that the number of particles per unit volume decreases continuously. These two effects formed so-called bright band (BB) in radar reflectivity field. Quantitative estimates confirmed that the bright band associated with the freezing level was caused by the coalescence and melting of snowflakes and followed by breakup below. Battan (1973) (2) proposed that the primary cause of the enhancement of weather targets reflectivity was a rapid increase in the dielectric constant of hydrometeors at the top of the melting layer followed by an increase of the fall velocities of particles toward the end of the melting process. Fabry and Zawadzki (1995) (3) performed a detailed analysis of bright band, and suggested that, in addition to the

Cancer research, Jan 15, 2014

Exosomes are small secreted vesicles that can transfer their content to recipient cells. In cance... more Exosomes are small secreted vesicles that can transfer their content to recipient cells. In cancer, exosome secretion has been implicated in tumor growth and metastatic spread. In this study, we explored the possibility that exosomal pathways might discard tumor-suppressor miRNA that restricts metastatic progression. Secreted miRNA characterized from isogenic bladder carcinoma cell lines with differing metastatic potential were uncoupled from binding to target transcripts or the AGO2-miRISC complex. In metastatic cells, we observed a relative increase in secretion of miRNA with tumor-suppressor functions, including miR23b, miR224, and miR921. Ectopic expression of miR23b inhibited invasion, anoikis, angiogenesis, and pulmonary metastasis. Silencing of the exocytotic RAB family members RAB27A or RAB27B halted miR23b and miR921 secretion and reduced cellular invasion. Clinically, elevated levels of RAB27B expression were linked to poor prognosis in two independent cohorts of patients ...

Pharmaceutical Research, 2010

Purpose This work describes the production and application of an aerosolised formulation of chito... more Purpose This work describes the production and application of an aerosolised formulation of chitosan nanoparticles for improved pulmonary siRNA delivery and gene silencing in mice. Methods Aerosolised chitosan/siRNA nanoparticles were pneumatically formed using a nebulising catheter and sized by laser diffraction. In vitro silencing of aerosolised and non-aerosolised formulations was evaluated in an EGFP endogenous-expressing H1299 cell line by flow cytometry. Non-invasive intratracheal insertion of the catheter was used to study nanoparticle deposition by histological detection of Cy3-labeled siRNA and gene silencing in transgenic EGFP mouse lungs using a flow cytometric method. Results Flow cytometric analysis demonstrated minimal alteration in gene silencing efficiency before (68%) and after (62%) aerosolisation in EGFP-expressing H1299 cells. Intratracheal catheter administration in mice resulted in nanoparticle deposition throughout the entire lung in both alveoli and bronchiolar regions using low amounts of siRNA. Transgenic EGFP mice dosed with the aerosolised nanoparticle formulation showed significant EGFP gene silencing (68% reduction compared to mismatch group).

Molecular Therapy, 2006

This work introduces a novel chitosan-based siRNA nanoparticle delivery system for RNA interferen... more This work introduces a novel chitosan-based siRNA nanoparticle delivery system for RNA interference in vitro and in vivo. The formation of interpolyelectrolyte complexes between siRNA duplexes (21-mers) and chitosan polymer into nanoparticles, ranging from 40 to 600 nm, was shown using atomic force microscopy and photon correlation spectroscopy. Rapid uptake (1 h) of Cy5-labeled nanoparticles into NIH 3T3 cells, followed by accumulation over a 24 h period, was visualized using fluorescence microscopy. Nanoparticle-mediated knockdown of endogenous enhanced green fluorescent protein (EGFP) was demonstrated in both H1299 human lung carcinoma cells and murine peritoneal macrophages (77.9% and 89.3% reduction in EGFP fluorescence, respectively). In addition, Western analysis showed approximately 90% reduced expression of BCR/ABL-1 leukemia fusion protein while BCR expression was unaffected in K562 (Ph(+)) cells after transfection using nanoparticles containing siRNA specific to the BCR/ABL-1 junction sequence. Effective in vivo RNA interference was achieved in bronchiole epithelial cells of transgenic EGFP mice after nasal administration of chitosan/siRNA formulations (37% and 43% reduction compared to mismatch and untreated control, respectively). These findings highlight the potential application of this novel chitosan-based system in RNA-mediated therapy of systemic and mucosal disease.

Molecular Therapy, 2002

Gene therapy for systemic diseases requires intravenous administration, but existing vectors are ... more Gene therapy for systemic diseases requires intravenous administration, but existing vectors are not suitable for systemic delivery, often showing rapid elimination from the bloodstream that restricts potential transfection sites to "first-pass" organs. To develop long-circulating vectors, here we have compared polyplexes containing DNA and poly-L-lysine (PLL) or polyethylenimine (PEI), surface-modified with either monovalent polyethylene glycol (PEG) or multivalent copolymers of N-(2-hydroxypropyl)methacrylamide (PHPMA), correlating their biophysical properties with their distribution following intravenous injection. A key difference between the two types of coating is the introduction of lateral stabilization by surface attachment of multivalent PHPMA, in addition to the steric stabilization provided by both types of polymers. The alpha-half-life for bloodstream clearance of polycation/DNA polyplexes (typically <5 minutes in mice) could be extended using multivalent PHPMA coating to >90 minutes. We found that the dose administered, as well as the amount and molecular weight of the coating PHPMA, had important effects on circulation properties. Multivalent PHPMA coating allows, for the first time, considerably extended circulation time using polyplex systems-a prerequisite for systemic gene delivery.

The Journal of Gene Medicine, 2008

Background Small interfering RNAs (siRNAs) can induce specific gene silencing through cytoplasmic... more Background Small interfering RNAs (siRNAs) can induce specific gene silencing through cytoplasmic mRNA cleavage and nuclear transcriptional silencing, necessitating delivery to different cellular compartments. This study presents a reducible copolypeptide (rCPP) carrier containing different molar ratios of a histidine-rich peptide (HRP) and nuclear localization sequence (NLS) peptide to modulate intracellular trafficking of transfected siRNA and primary RNA transcripts (pri-miRNA).

Journal of Climate, 1993

... Precipitación atmosférica. ; Lluvia. ; Dispositivo hiperfrecuencia. ; Detección IR. ; Variaci... more ... Precipitación atmosférica. ; Lluvia. ; Dispositivo hiperfrecuencia. ; Detección IR. ; Variación diurna. ; Variación mensual. ; Variación interanual. ; Circulación general. ; Nube convección. ; México. ; Estados Unidos. ; America central. ; America. ; America del norte. ; Localisation / Location ...

European Journal of Pharmaceutical Sciences, 2014

The application of RNA interference (RNAi) has great therapeutic potential for degenerative disea... more The application of RNA interference (RNAi) has great therapeutic potential for degenerative diseases of cartilaginous tissues by means of fine tuning the phenotype of cells used for regeneration. However, possible non-specific effects of transfection per se might be relevant for future clinical application. In the current study, we selected two synthetic transfection reagents, a cationic lipid-based commercial reagent Lipofectamine RNAiMAX and polyethylenimine (PEI), and two naturally-derived transfection reagents, namely the polysaccharides chitosan (98% deacetylation) and hyaluronic acid (20% amidation), for siRNA delivery into primary mesenchymal cells including nucleus pulposus cells, articular chondrocytes and mesenchymal stem cells (MSCs). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous model gene to evaluate the extent of silencing by 20 nM or 200 nM siRNA at day 3 and day 6 post-transfection. In addition to silencing efficiency, non-specific effects such as cytotoxicity, change in DNA content and differentiation potential of cells were evaluated. Among the four transfection reagents, the commercial liposome-based agent was the most efficient reagent for siRNA delivery at 20 nM siRNA, followed by chitosan. Transfection using cationic liposomes, chitosan and PEI showed some decrease in viability and DNA content to varying degrees that was dependent on the siRNA dose and cell type evaluated, but independent of GAPDH knockdown. Some effects on DNA content were not accompanied by concomitant changes in viability. However, changes in expression of marker genes for cell cycle inhibition or progression, such as p21 and PCNA, could not explain the changes in DNA content. Interestingly, aspecific upregulation of GAPDH activity was found, which was limited to cartilaginous cells. In conclusion, non-specific effects should not be overlooked in the application of RNAi for mesenchymal cell transfection and may need to be overcome for its effective therapeutic application.

Biophysical Journal, 2007

Information on the interaction strength between small interfering RNA (siRNA) and chitosan can co... more Information on the interaction strength between small interfering RNA (siRNA) and chitosan can contribute to the understanding of the formation and stability of chitosan/siRNA nanoparticles used as siRNA delivery systems for gene silencing. In this study, we utilize atomic force microscopy to obtain force spectroscopy results of the interaction strengths between siRNA and chitosan measured in physiological phosphate buffered saline buffer at different pH. The force measurements revealed that the adhesive interactions decreased in force strength and force frequency as the pH was increased from 4.1 to 6.1, 7.4, and 9.5, exhibiting distinct multimodal distributions of the interaction forces between siRNA and chitosan molecules at acidic pH and only negligible adhesive forces were observed at neutral or high pH. The strong pH dependence of siRNAchitosan interactions can provide a convincing rationale for siRNA/chitosan complex formation and nanoparticle stability under low acidic conditions. These findings demonstrate that the use of force spectroscopy for the adhesive force measurements allows an evaluation of the complexing ability between siRNA and chitosan that can be utilized to predict nanoparticle stability.

Biomaterials, 2007

We have previously introduced the use of the biomaterial chitosan to form chitosan/siRNA nanopart... more We have previously introduced the use of the biomaterial chitosan to form chitosan/siRNA nanoparticles for gene silencing protocols. This present study shows that the physicochemical properties (size, zeta potential, morphology and complex stability) and in vitro gene silencing of chitosan/siRNA nanoparticles are strongly dependent on chitosan molecular weight (M w ) and degree of deacetylation (DD). High M w and DD chitosan resulted in the formation of discrete stable nanoparticles 200nminsize.Chitosan/siRNAformulations(N:P50)preparedwithlowMw(200 nm in size. Chitosan/siRNA formulations (N:P 50) prepared with low M w (200nminsize.Chitosan/siRNAformulations(N:P50)preparedwithlowMw(10 kDa) showed almost no knockdown of endogenous enhanced green fluorescent protein (EGFP) in H1299 human lung carcinoma cells, whereas those prepared from higher M w (64.8-170 kDa) and DD ($80%) showed greater gene silencing ranging between 45% and 65%. The highest gene silencing efficiency (80%) was achieved using chitosan/siRNA nanoparticles at N:P 150 using higher M w (114 and 170 kDa) and DD (84%) that correlated with formation of stable nanoparticles of $200 nm. In conclusion, this work confirms the application of chitosan as a non-viral carrier for siRNA and the importance of polymeric properties for the optimisation of gene silencing using chitosan/siRNA nanoparticles. r

Biomaterials, 2008

Standard in vitro gene silencing protocols are performed using aqueous formulations of transfecti... more Standard in vitro gene silencing protocols are performed using aqueous formulations of transfection reagents and small interfering RNAs (siRNA) reconstituted immediately prior to use. In this study, we describe a method for producing gene silencing-active lyophilized cationic polymer (chitosan) or lipid (TransIT-TKO) siRNA formulations. We demonstrate specific and efficient knockdown of enhanced green fluorescent protein (EGFP) in H1299 human lung carcinoma cells transfected in plates pre-coated with both Trans IT-TKO/siRNA ($85%) and a chitosan/siRNA formulation containing sucrose as lyoprotectant ($70%). This method removes the necessity for both siRNA reconstitution immediately prior to use and addition onto cells. Furthermore, silencing activity of the chitosan/ siRNA formulation was shown over the period studied ($2 months) when stored at room temperature. Higher cell viability was observed using the chitosan system compared to the lipid formulation. Silencing of the proinflammatory cytokine tumour necrosis factor (TNF-a) was also demonstrated in the RAW macrophage cell line using the lyophilized chitosan/siRNA system suggesting that the coating can improve the biocompatibility of medical implants. This work describes an efficient gene silencing methodology using freeze-dried formulations with potential applications as a high throughput screening tool for gene function, biocompatible medical implant components and longer shelf-life therapeutics. r

Biomaterials, 2010

This work presents a method for decorating the surface of poly (lactide-co-glycolide) (PLGA) nano... more This work presents a method for decorating the surface of poly (lactide-co-glycolide) (PLGA) nanoparticles with polyethyleneimine (PEI) utilising a cetyl derivative to improve surface functionalisation and siRNA delivery. Sub-micron particles were produced by an emulsion-diffusion method using benzyl alcohol. We demonstrate by x-ray photoelectron spectroscopy (XPS), 2.6 times higher surface presentation of amines using the cetyl derivative compared to non-cetylated-PEI formulations (6.5 and 2.5% surface nitrogen, respectively). The modified particles were shown by spectroscopy, fluorescent microscopy and flow cytometry to bind and mediate siRNA delivery into the human osteosarcoma cell line U2OS and the murine macrophage cell line J774.1. Specific reduction in the anti-apoptotic oncogene BCL-w in U2OS cells was achieved with particles containing cetylated-PEI (53%) with no cellular toxicity. In addition, particles containing cetylated-PEI achieved 64% silencing of TNFa in J774.1 cells. This rapid method for surface modification of PLGA nanoparticles promotes its application for alternative cetylated functional derivatives as a strategy to control specific biological properties of nanoparticles.

ACS Nano, 2009

Magnetic nanoparticles (MNP) can be used as contrast-enhancing agents to visualize tumors by magn... more Magnetic nanoparticles (MNP) can be used as contrast-enhancing agents to visualize tumors by magnetic resonance imaging (MRI). Here we describe an easy synthesis method of magnetic nanoparticles coated with polyethylene glycol (PEG) and demonstrate size-dependent accumulation in murine tumors following intravenous injection. Biocompatible iron oxide MNPs coated with PEG were prepared by replacing oleic acid with a biocompatible and commercially available silane-PEG to provide an easy and effective method for chemical coating. The colloidal stable PEGylated MNPs were magnetically separated into two distinct size subpopulations of 20 and 40 nm mean diameters with increased phagocytic uptake observed for the 40 nm size range in vitro. MRI detection revealed greater iron accumulation in murine tumors for 40 nm nanoparticles after intravenous injection. The enhanced MRI contrast of the larger MNPs in the tumor may be a combined result of the size-dependent extravasation and capture by macrophages in the tumor, providing important considerations for improved bioimaging approaches.

Since the late 1990s, the National Severe Storms Laboratory (NSSL) has been actively involved in ... more Since the late 1990s, the National Severe Storms Laboratory (NSSL) has been actively involved in developing, maintaining, and serving spatial data to National Weather Service (NWS) users in support of flash-flood operations. One of the first and largest efforts was the creation of a national seamless dataset of flash-flood-scale basins. These basins provide the spatial framework for calculations of average basin rainfall rate and accumulation in the Flash Flood Monitoring and Prediction [1] (FFMP) program used at the NWS Weather Forecast Offices (WFOs). During the past decade, the dataset has been updated and enhanced with features such as seamless hydrologic-connectivity attributes that allow downstream tracing of flow and upstream drainage area selection. This dataset is currently served to users through NSSL's National FFMP Basin Repository and FFMP Basin Customization Repository websites. As a result of recent NWS Advanced Hydrologic Prediction Service (AHPS) funding, a new ...

Journal of extracellular vesicles, 2014

Cells release a mixture of extracellular vesicles, amongst these exosomes, that differ in size, d... more Cells release a mixture of extracellular vesicles, amongst these exosomes, that differ in size, density and composition. The standard isolation method for exosomes is centrifugation of fluid samples, typically at 100,000×g or above. Knowledge of the effect of discrete ultracentrifugation speeds on the purification from different cell types, however, is limited. We examined the effect of applying differential centrifugation g-forces ranging from 33,000×g to 200,000×g on exosome yield and purity, using 2 unrelated human cell lines, embryonic kidney HEK293 cells and bladder carcinoma FL3 cells. The fractions were evaluated by nanoparticle tracking analysis (NTA), total protein quantification and immunoblotting for CD81, TSG101, syntenin, VDAC1 and calreticulin. NTA revealed the lowest background particle count in Dulbecco's Modified Eagle's Medium media devoid of phenol red and cleared by 200,000×g overnight centrifugation. The centrifugation tube fill level impacted the sedime...

2011 IEEE RadarCon (RADAR), 2011

ABSTRACT The melting of aggregated snow/crystals often results in an enhancement of the reflectiv... more ABSTRACT The melting of aggregated snow/crystals often results in an enhancement of the reflectivity observed by weather radars, and this is commonly referenced as the bright band (BB). The locally high reflectivity often causes overestimation in radar quantitative precipitation estimates (QPE) if no appropriate correction is applied. When the melting layer is high, a complete bright band layer profile including top, peak, and bottom can be observed by the radar and a vertical profile of reflectivity (VPR) correction can be made to reduce the BB impact. When a melting layer is near the ground and the bottom part of the bright band cannot be observed by the radar, a VPR correction can not be made directly from radar observations. This paper presents a new VPR correction method under this situation. From high- resolution precipitation profiler data, an empirical relationship between BB peak and BB bottom is developed. The empirical relationship is combined with the apparent BB peak observed by volume scan radars and the BB bottom is found. Radar QPEs are then corrected based on the estimated BB bottom. The new method is shown to be effective in reducing the radar QPE overestimation under low bright band situations. I. INTRODUCTION When ice crystals or snow flakes fell into warm surroundings below the freezing level, two consequent effects would impact the reflectivity of the particles: 1) The change from ice to water would result in an increase in the reflective properties of the particles so that the radar reflectivity intensity increases; 2) the fall velocity of the flakes is less than that of the resulting water drops so that the number of particles per unit volume decreases continuously. These two effects formed so-called bright band (BB) in radar reflectivity field. Quantitative estimates confirmed that the bright band associated with the freezing level was caused by the coalescence and melting of snowflakes and followed by breakup below. Battan (1973) (2) proposed that the primary cause of the enhancement of weather targets reflectivity was a rapid increase in the dielectric constant of hydrometeors at the top of the melting layer followed by an increase of the fall velocities of particles toward the end of the melting process. Fabry and Zawadzki (1995) (3) performed a detailed analysis of bright band, and suggested that, in addition to the

Cancer research, Jan 15, 2014

Exosomes are small secreted vesicles that can transfer their content to recipient cells. In cance... more Exosomes are small secreted vesicles that can transfer their content to recipient cells. In cancer, exosome secretion has been implicated in tumor growth and metastatic spread. In this study, we explored the possibility that exosomal pathways might discard tumor-suppressor miRNA that restricts metastatic progression. Secreted miRNA characterized from isogenic bladder carcinoma cell lines with differing metastatic potential were uncoupled from binding to target transcripts or the AGO2-miRISC complex. In metastatic cells, we observed a relative increase in secretion of miRNA with tumor-suppressor functions, including miR23b, miR224, and miR921. Ectopic expression of miR23b inhibited invasion, anoikis, angiogenesis, and pulmonary metastasis. Silencing of the exocytotic RAB family members RAB27A or RAB27B halted miR23b and miR921 secretion and reduced cellular invasion. Clinically, elevated levels of RAB27B expression were linked to poor prognosis in two independent cohorts of patients ...

Pharmaceutical Research, 2010

Purpose This work describes the production and application of an aerosolised formulation of chito... more Purpose This work describes the production and application of an aerosolised formulation of chitosan nanoparticles for improved pulmonary siRNA delivery and gene silencing in mice. Methods Aerosolised chitosan/siRNA nanoparticles were pneumatically formed using a nebulising catheter and sized by laser diffraction. In vitro silencing of aerosolised and non-aerosolised formulations was evaluated in an EGFP endogenous-expressing H1299 cell line by flow cytometry. Non-invasive intratracheal insertion of the catheter was used to study nanoparticle deposition by histological detection of Cy3-labeled siRNA and gene silencing in transgenic EGFP mouse lungs using a flow cytometric method. Results Flow cytometric analysis demonstrated minimal alteration in gene silencing efficiency before (68%) and after (62%) aerosolisation in EGFP-expressing H1299 cells. Intratracheal catheter administration in mice resulted in nanoparticle deposition throughout the entire lung in both alveoli and bronchiolar regions using low amounts of siRNA. Transgenic EGFP mice dosed with the aerosolised nanoparticle formulation showed significant EGFP gene silencing (68% reduction compared to mismatch group).

Molecular Therapy, 2006

This work introduces a novel chitosan-based siRNA nanoparticle delivery system for RNA interferen... more This work introduces a novel chitosan-based siRNA nanoparticle delivery system for RNA interference in vitro and in vivo. The formation of interpolyelectrolyte complexes between siRNA duplexes (21-mers) and chitosan polymer into nanoparticles, ranging from 40 to 600 nm, was shown using atomic force microscopy and photon correlation spectroscopy. Rapid uptake (1 h) of Cy5-labeled nanoparticles into NIH 3T3 cells, followed by accumulation over a 24 h period, was visualized using fluorescence microscopy. Nanoparticle-mediated knockdown of endogenous enhanced green fluorescent protein (EGFP) was demonstrated in both H1299 human lung carcinoma cells and murine peritoneal macrophages (77.9% and 89.3% reduction in EGFP fluorescence, respectively). In addition, Western analysis showed approximately 90% reduced expression of BCR/ABL-1 leukemia fusion protein while BCR expression was unaffected in K562 (Ph(+)) cells after transfection using nanoparticles containing siRNA specific to the BCR/ABL-1 junction sequence. Effective in vivo RNA interference was achieved in bronchiole epithelial cells of transgenic EGFP mice after nasal administration of chitosan/siRNA formulations (37% and 43% reduction compared to mismatch and untreated control, respectively). These findings highlight the potential application of this novel chitosan-based system in RNA-mediated therapy of systemic and mucosal disease.

Molecular Therapy, 2002

Gene therapy for systemic diseases requires intravenous administration, but existing vectors are ... more Gene therapy for systemic diseases requires intravenous administration, but existing vectors are not suitable for systemic delivery, often showing rapid elimination from the bloodstream that restricts potential transfection sites to "first-pass" organs. To develop long-circulating vectors, here we have compared polyplexes containing DNA and poly-L-lysine (PLL) or polyethylenimine (PEI), surface-modified with either monovalent polyethylene glycol (PEG) or multivalent copolymers of N-(2-hydroxypropyl)methacrylamide (PHPMA), correlating their biophysical properties with their distribution following intravenous injection. A key difference between the two types of coating is the introduction of lateral stabilization by surface attachment of multivalent PHPMA, in addition to the steric stabilization provided by both types of polymers. The alpha-half-life for bloodstream clearance of polycation/DNA polyplexes (typically <5 minutes in mice) could be extended using multivalent PHPMA coating to >90 minutes. We found that the dose administered, as well as the amount and molecular weight of the coating PHPMA, had important effects on circulation properties. Multivalent PHPMA coating allows, for the first time, considerably extended circulation time using polyplex systems-a prerequisite for systemic gene delivery.

The Journal of Gene Medicine, 2008

Background Small interfering RNAs (siRNAs) can induce specific gene silencing through cytoplasmic... more Background Small interfering RNAs (siRNAs) can induce specific gene silencing through cytoplasmic mRNA cleavage and nuclear transcriptional silencing, necessitating delivery to different cellular compartments. This study presents a reducible copolypeptide (rCPP) carrier containing different molar ratios of a histidine-rich peptide (HRP) and nuclear localization sequence (NLS) peptide to modulate intracellular trafficking of transfected siRNA and primary RNA transcripts (pri-miRNA).

Journal of Climate, 1993

... Precipitación atmosférica. ; Lluvia. ; Dispositivo hiperfrecuencia. ; Detección IR. ; Variaci... more ... Precipitación atmosférica. ; Lluvia. ; Dispositivo hiperfrecuencia. ; Detección IR. ; Variación diurna. ; Variación mensual. ; Variación interanual. ; Circulación general. ; Nube convección. ; México. ; Estados Unidos. ; America central. ; America. ; America del norte. ; Localisation / Location ...

European Journal of Pharmaceutical Sciences, 2014

The application of RNA interference (RNAi) has great therapeutic potential for degenerative disea... more The application of RNA interference (RNAi) has great therapeutic potential for degenerative diseases of cartilaginous tissues by means of fine tuning the phenotype of cells used for regeneration. However, possible non-specific effects of transfection per se might be relevant for future clinical application. In the current study, we selected two synthetic transfection reagents, a cationic lipid-based commercial reagent Lipofectamine RNAiMAX and polyethylenimine (PEI), and two naturally-derived transfection reagents, namely the polysaccharides chitosan (98% deacetylation) and hyaluronic acid (20% amidation), for siRNA delivery into primary mesenchymal cells including nucleus pulposus cells, articular chondrocytes and mesenchymal stem cells (MSCs). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous model gene to evaluate the extent of silencing by 20 nM or 200 nM siRNA at day 3 and day 6 post-transfection. In addition to silencing efficiency, non-specific effects such as cytotoxicity, change in DNA content and differentiation potential of cells were evaluated. Among the four transfection reagents, the commercial liposome-based agent was the most efficient reagent for siRNA delivery at 20 nM siRNA, followed by chitosan. Transfection using cationic liposomes, chitosan and PEI showed some decrease in viability and DNA content to varying degrees that was dependent on the siRNA dose and cell type evaluated, but independent of GAPDH knockdown. Some effects on DNA content were not accompanied by concomitant changes in viability. However, changes in expression of marker genes for cell cycle inhibition or progression, such as p21 and PCNA, could not explain the changes in DNA content. Interestingly, aspecific upregulation of GAPDH activity was found, which was limited to cartilaginous cells. In conclusion, non-specific effects should not be overlooked in the application of RNAi for mesenchymal cell transfection and may need to be overcome for its effective therapeutic application.

Biophysical Journal, 2007

Information on the interaction strength between small interfering RNA (siRNA) and chitosan can co... more Information on the interaction strength between small interfering RNA (siRNA) and chitosan can contribute to the understanding of the formation and stability of chitosan/siRNA nanoparticles used as siRNA delivery systems for gene silencing. In this study, we utilize atomic force microscopy to obtain force spectroscopy results of the interaction strengths between siRNA and chitosan measured in physiological phosphate buffered saline buffer at different pH. The force measurements revealed that the adhesive interactions decreased in force strength and force frequency as the pH was increased from 4.1 to 6.1, 7.4, and 9.5, exhibiting distinct multimodal distributions of the interaction forces between siRNA and chitosan molecules at acidic pH and only negligible adhesive forces were observed at neutral or high pH. The strong pH dependence of siRNAchitosan interactions can provide a convincing rationale for siRNA/chitosan complex formation and nanoparticle stability under low acidic conditions. These findings demonstrate that the use of force spectroscopy for the adhesive force measurements allows an evaluation of the complexing ability between siRNA and chitosan that can be utilized to predict nanoparticle stability.

Biomaterials, 2007

We have previously introduced the use of the biomaterial chitosan to form chitosan/siRNA nanopart... more We have previously introduced the use of the biomaterial chitosan to form chitosan/siRNA nanoparticles for gene silencing protocols. This present study shows that the physicochemical properties (size, zeta potential, morphology and complex stability) and in vitro gene silencing of chitosan/siRNA nanoparticles are strongly dependent on chitosan molecular weight (M w ) and degree of deacetylation (DD). High M w and DD chitosan resulted in the formation of discrete stable nanoparticles 200nminsize.Chitosan/siRNAformulations(N:P50)preparedwithlowMw(200 nm in size. Chitosan/siRNA formulations (N:P 50) prepared with low M w (200nminsize.Chitosan/siRNAformulations(N:P50)preparedwithlowMw(10 kDa) showed almost no knockdown of endogenous enhanced green fluorescent protein (EGFP) in H1299 human lung carcinoma cells, whereas those prepared from higher M w (64.8-170 kDa) and DD ($80%) showed greater gene silencing ranging between 45% and 65%. The highest gene silencing efficiency (80%) was achieved using chitosan/siRNA nanoparticles at N:P 150 using higher M w (114 and 170 kDa) and DD (84%) that correlated with formation of stable nanoparticles of $200 nm. In conclusion, this work confirms the application of chitosan as a non-viral carrier for siRNA and the importance of polymeric properties for the optimisation of gene silencing using chitosan/siRNA nanoparticles. r

Biomaterials, 2008

Standard in vitro gene silencing protocols are performed using aqueous formulations of transfecti... more Standard in vitro gene silencing protocols are performed using aqueous formulations of transfection reagents and small interfering RNAs (siRNA) reconstituted immediately prior to use. In this study, we describe a method for producing gene silencing-active lyophilized cationic polymer (chitosan) or lipid (TransIT-TKO) siRNA formulations. We demonstrate specific and efficient knockdown of enhanced green fluorescent protein (EGFP) in H1299 human lung carcinoma cells transfected in plates pre-coated with both Trans IT-TKO/siRNA ($85%) and a chitosan/siRNA formulation containing sucrose as lyoprotectant ($70%). This method removes the necessity for both siRNA reconstitution immediately prior to use and addition onto cells. Furthermore, silencing activity of the chitosan/ siRNA formulation was shown over the period studied ($2 months) when stored at room temperature. Higher cell viability was observed using the chitosan system compared to the lipid formulation. Silencing of the proinflammatory cytokine tumour necrosis factor (TNF-a) was also demonstrated in the RAW macrophage cell line using the lyophilized chitosan/siRNA system suggesting that the coating can improve the biocompatibility of medical implants. This work describes an efficient gene silencing methodology using freeze-dried formulations with potential applications as a high throughput screening tool for gene function, biocompatible medical implant components and longer shelf-life therapeutics. r

Biomaterials, 2010

This work presents a method for decorating the surface of poly (lactide-co-glycolide) (PLGA) nano... more This work presents a method for decorating the surface of poly (lactide-co-glycolide) (PLGA) nanoparticles with polyethyleneimine (PEI) utilising a cetyl derivative to improve surface functionalisation and siRNA delivery. Sub-micron particles were produced by an emulsion-diffusion method using benzyl alcohol. We demonstrate by x-ray photoelectron spectroscopy (XPS), 2.6 times higher surface presentation of amines using the cetyl derivative compared to non-cetylated-PEI formulations (6.5 and 2.5% surface nitrogen, respectively). The modified particles were shown by spectroscopy, fluorescent microscopy and flow cytometry to bind and mediate siRNA delivery into the human osteosarcoma cell line U2OS and the murine macrophage cell line J774.1. Specific reduction in the anti-apoptotic oncogene BCL-w in U2OS cells was achieved with particles containing cetylated-PEI (53%) with no cellular toxicity. In addition, particles containing cetylated-PEI achieved 64% silencing of TNFa in J774.1 cells. This rapid method for surface modification of PLGA nanoparticles promotes its application for alternative cetylated functional derivatives as a strategy to control specific biological properties of nanoparticles.

ACS Nano, 2009

Magnetic nanoparticles (MNP) can be used as contrast-enhancing agents to visualize tumors by magn... more Magnetic nanoparticles (MNP) can be used as contrast-enhancing agents to visualize tumors by magnetic resonance imaging (MRI). Here we describe an easy synthesis method of magnetic nanoparticles coated with polyethylene glycol (PEG) and demonstrate size-dependent accumulation in murine tumors following intravenous injection. Biocompatible iron oxide MNPs coated with PEG were prepared by replacing oleic acid with a biocompatible and commercially available silane-PEG to provide an easy and effective method for chemical coating. The colloidal stable PEGylated MNPs were magnetically separated into two distinct size subpopulations of 20 and 40 nm mean diameters with increased phagocytic uptake observed for the 40 nm size range in vitro. MRI detection revealed greater iron accumulation in murine tumors for 40 nm nanoparticles after intravenous injection. The enhanced MRI contrast of the larger MNPs in the tumor may be a combined result of the size-dependent extravasation and capture by macrophages in the tumor, providing important considerations for improved bioimaging approaches.