Meaghan Andreoli, Anna Glynn, Andrew Keller, and Beth...
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Nanoparticle System to Entrap and Deactivate Vesicants Meaghan Andreoli, Anna Glynn, Andrew Keller, and Beth Stuart Advisor: Dr. Cristina Sabliov Department of Biological and Agricultural Engineering, Louisiana State University, Baton Rouge, LA Abstract Materials and Methods Objectives Background Results Conclusions Characterization Acknowledgements Louisiana State University College of Agriculture, Dr. Cristina Sabliov, Carlos Astete, Dr. Daniel Hayes, Dr. Evgueni Nesterov, Ammar Qureshi, Balasubramanian Sundar, and Nipur Patel •Mustard agents are easy to make and a widely used type of chemical warfare agent. •Potential users include researchers and first responders such as firefighters, emergency medical technicians, police, and military personnel. Prior Products Drawbacks Boots, gloves, protective suits Areas of concern: neck, wrists, ankles Topical lotions/creams Application issues Povidone Iodine (PI) Antidote, not prophylactic Anti-inflammatory medication Reduces swelling, but not skin damage Fuller’s earth Dust problems with military equipment Decontaminating Solution 2 (DS2) Corrosive NP System Component Justification Polymer: poly D, L-lactide-co- glycolide (PLGA) FDA approved for human therapy Well-characterized polymer Biocompatible Biodegradable Deactivating Chemical: -Trioctylamine (TOA) -Diisopropyl ethyl amine (DIEA) Hydrophobic amine Reactive for our purposes; Non-nucleophilic base Will not bond with the polymer Surfactant: Polyvinyl alcohol (PVA) Water soluble Excellent emulsifying properties Reduces surface tension Aids in the solubility of vesicant Solvent: Dichloromethane (DCM) Not reactive with deactivating chemical Ability to dissolve all other NP synthesis components Evaporates easily due to its relatively low boiling point (39.6°C) Model vesicant: 2-chloroethyl ethyl sulfide (2-CEES) Comparable analog to mustard gas, but has weaker blistering effects TOA DIEA Mustard gas 2-CEES Show in vitro: 1. The synthesis of nanoparticles loaded with a deactivating chemical. 2. The model vesicant was absorbed by the designed nanoparticle system. 3. The vesicant reacted with deactivating chemical in the nanoparticle system to form non- toxic byproducts. •To provide a safe, yet more efficient method of protection from vesicants that improves the operational effectiveness of first responders and soldiers . •Design a nanoparticle system that will absorb, entrap, and deactivate the target vesicant. Throughout history, terrorism and warfare have motivated the development of weapons in the form of chemical agents. Specifically, mustard gas has maintained a history as a harmful and potentially life-threatening vesicant. Prior products used to combat the threat of mustard gas have been insufficient as a prophylactic approach. This research sought to provide a solution by designing a nanoparticle (NP) system to absorb, entrap, and deactivate mustard gas into safe byproducts. Material selection and methods for the synthesis and testing of the system were developed based on the functional decomposition of previously explored technologies. The nanoparticles were then synthesized using the emulsion- solvent evaporation technique and characterized, which proved that particles with a diameter of approximately 200 nanometers were produced. Using a pH meter, a calibration curve was developed and used as a comparison for the loaded nanoparticle solution to determine an 83% entrapment efficiency. The reaction was tested by measuring the pH change after introduction of the vesicant with free deactivating chemical, blank nanoparticles, and nanoparticles loaded with deactivating chemical. The pH was chosen as an indicator of the deactivation reaction because the hydrolysis of the vesicant produces hydrochloric acid. The nanoparticles proved to be a better deactivator than free chemicals, however the difference between blank and loaded nanoparticles was minimal. 150 mg PVA Purify by dialysis (12 hours with nano-pure H 2 O; change H 2 O every 4 hours) Nanoparticles (in solution) 80mg Trehalose Freeze NP suspension for 3 hours at 5°C Freeze dry for 48 hours under a vacuum at -80°C 100 mL H 2 O 5 mL DCM Nanoparticles (powder form) Evaporate under rotating vacuum (32°C for 6 mins.) Sonicate to form emulsion (10 mins. at 38% amplitude; pulse of 4:2; in an ice bath) 10mL of the continuous phase stock solution 300mg PVA 100mL Nano-pure H 2 O 2.0mL of the discontinuous phase stock solution 100 mg PLGA 5 mg TOA or DIEA 5 mL DCM Nanoparticle System Synthesis: Emulsion-Solvent Evaporation Technique + + 2-CEES TOA Ethylvinyl sulfide Ethyl thioethanol Hydrochloric acid or DIEA Characterization Entrapment Efficiency Reaction Testing •Size: >200 nm to prevent endocytosis •Polydispersity Index (PDI): Monodisperse <0.1 •Zeta Potential: ±30 mV for moderate stability •Successfully entrap >70% of the deactivating chemical in the nanoparticles •NP instantaneous response to presence of 2-CEES •Entrapment and deactivation of 2-CEES •Loaded NP deactivation occurs faster than free chemical reaction with 2-CEES Measurable Objectives Entrapment Efficiency Reaction Testing Loaded Nanoparticles •Average size: 213.1 nm •PDI: 0.142 •Zeta Potential: -8.4 mV Blank Nanoparticles •Average Size: 216.3 nm •PDI: 0.098 •Zeta Potential: -21.7 mV •Visually confirm that nanoparticles were produced as designed using: •Dynamic light scattering •Transmission electron microscopy •Nanoparticle characterization showed successful synthesis results Consistent with measurable objectives for size, polydispersity index, and zeta potential •Entrapment efficiency of deactivating chemical was successfully determined Consistent with measurable objectives •Although no significant change was observed between the loaded and blank nanoparticles, reaction testing showed that nanoparticles reacted faster than free deactivating chemicals Consistent with measureable objectives Blank NP + 0 mg TOA Blank NP + 2.5 mg TOA Blank NP + 1 mg TOA Blank NP + 5 mg TOA 100% 80% 50% 0% Pellet after centrifugation Supernatant pH measured pH=4.10 pH=4.24 pH=4.21 pH=4.17 0 0.0001 0.0002 0.0003 0.0004 0 50 100 150 [H + ] (mol/liter) Time (sec) Comparison of [H + ] Generated by Reactions of Surfactant, Deactivating Chemical, and Vesicant PVA PVA and TOA PVA and DIEA Profile of [H + ] generated after introduction of vesicant to solutions of surfactant and deactivating chemicals. 0 0.0001 0.0002 0.0003 0.0004 0.0005 0.0006 0.0007 0.0008 0 50 100 150 200 [H + ] (mol/liter) Time (sec) Comparison of [H + ] Generated in Reactions of Nanoparticles with Vesicant TOA loaded DIEA loaded Profile of [H + ] generated after introduction of vesicant to solutions of nanoparticles loaded with deactivating chemicals. 0 5 10 15 20 25 Blank NP TOA NP DIEA NP PVA PVA TOA PVA DIEA % Destruction of 2-CEES Comparisons of Reaction Efficacy Nanoparticles Free Chemicals Comparison of percent destruction of vesicant after introduction to various chemical and nanoparticle solutions. Deactivation reaction: •Vesicant undergoes hydrolysis producing HCl, which causes a measurable decrease in pH •Deactivating chemical is not consumed in the reaction Procedure: •5 μL of vesicant reacted with 10 mL free deactivating chemical, blank NPs, and loaded NPs •pH change measured and recorded over time Results: •NPs deactivate vesicant better than free deactivating chemicals •Percent destruction of vesicant by blank and loaded NPs was similar http://www.sciencedirect.com.libezp.lib.lsu. edu/science? 4.00 4.05 4.10 4.15 4.20 4.25 4.30 4.35 0.0000 0.1000 0.2000 0.3000 0.4000 0.5000 0.6000 pH Concentration of TOA in Supernatant (mg/mL) Standard pH Curve based on TOA Concentration in the Supernatant Standard curve used to determine the entrapment efficiency of the deactivating chemical, TOA, loaded inside the nanoparticles. Procedure: •Four batches of blank NPs synthesized •Different amounts of deactivating chemical added to the NP solutions simulating varying entrapment efficiencies •NP system centrifuged for 25 minutes at 35,000 rpm to separate nanoparticles (pellet) from solution (supernatant) •Steady state pH of supernatants was measured Results: •pH of loaded nanoparticle supernatant= 4.16 •Interpolation from standard curve used to determine a concentration of 0.0857 mg/mL •The concentration corresponded to an entrapment efficiency of 82.86% •Consistent with measureable objectives Blank NP
Transcript of Meaghan Andreoli, Anna Glynn, Andrew Keller, and Beth...
Nanoparticle System to Entrap and Deactivate VesicantsMeaghan Andreoli, Anna Glynn, Andrew Keller, and Beth Stuart
Advisor: Dr. Cristina SabliovDepartment of Biological and Agricultural Engineering, Louisiana State University, Baton Rouge, LA
Abstract Materials and Methods
Objectives
Background
Results
Conclusions
Characterization
AcknowledgementsLouisiana State University College of Agriculture, Dr. Cristina Sabliov, Carlos Astete, Dr. Daniel Hayes, Dr. Evgueni Nesterov, Ammar Qureshi, Balasubramanian Sundar, and Nipur Patel
•Mustard agents are easy to make and a widely used type of chemical warfare agent. •Potential users include researchers and first responders such as firefighters, emergency medical technicians, police, and military personnel.
Prior Products Drawbacks
Boots, gloves, protective suits Areas of concern: neck, wrists, ankles
Topical lotions/creams Application issues
Povidone Iodine (PI) Antidote, not prophylactic
Anti-inflammatory medication Reduces swelling, but not skin damage
Fuller’s earth Dust problems with military equipment
Decontaminating Solution 2 (DS2) Corrosive
NP System Component
Justification
Polymer: poly D, L-lactide-co-glycolide (PLGA)
FDA approved for human therapyWell-characterized polymerBiocompatibleBiodegradable
Deactivating Chemical:-Trioctylamine(TOA)-Diisopropyl ethyl amine (DIEA)
Hydrophobic amineReactive for our purposes;Non-nucleophilic base Will not bond with the polymer
Surfactant:Polyvinyl alcohol (PVA)
Water solubleExcellent emulsifying properties Reduces surface tensionAids in the solubility of vesicant
Solvent: Dichloromethane (DCM)
Not reactive with deactivating chemicalAbility to dissolve all other NP synthesis componentsEvaporates easily due to its relatively low boiling point (39.6°C)
Model vesicant: 2-chloroethyl ethylsulfide (2-CEES)
Comparable analog to mustard gas, but has weaker blistering effects
TOA DIEA
Mustard gas
2-CEES
Show in vitro:1. The synthesis of nanoparticles loaded with a deactivating chemical.
2. The model vesicant was absorbed by the designed nanoparticle system.
3. The vesicant reacted with deactivating chemical in the nanoparticle system to form non-toxic byproducts.
•To provide a safe, yet more efficient method of protection from vesicants that improves the operational effectiveness of first responders and soldiers .•Design a nanoparticle system that will absorb, entrap, and deactivate the target vesicant.
Throughout history, terrorism and warfare have motivated the development of weapons in the form of chemical agents. Specifically, mustard gas has maintained a history as a harmful and potentially life-threatening vesicant. Prior products used to combat the threat of mustard gas have been insufficient as a prophylactic approach. This research sought to provide a solution by designing a nanoparticle (NP) system to absorb, entrap, and deactivate mustard gas into safe byproducts. Material selection and methods for the synthesis and testing of the system were developed based on the functional decomposition of previously explored technologies. The nanoparticles were then synthesized using the emulsion-solvent evaporation technique and characterized, which proved that particles with a diameter of approximately 200 nanometers were produced. Using a pH meter, a calibration curve was developed and used as a comparison for the loaded nanoparticle solution to determine an 83% entrapment efficiency. The reaction was tested by measuring the pH change after introduction of the vesicant with free deactivating chemical, blank nanoparticles, and nanoparticles loaded with deactivating chemical. The pH was chosen as an indicator of the deactivation reaction because the hydrolysis of the vesicant produces hydrochloric acid. The nanoparticles proved to be a better deactivator than free chemicals, however the difference between blank and loaded nanoparticles was minimal.
150 mg PVA
Purify by dialysis (12 hours with nano-pure H2O; change H2O every 4
hours)
Nanoparticles
(in solution) 80mg Trehalose
Freeze NP suspension for 3 hours at 5°C
Freeze dry for 48 hours under a vacuum at -80°C
100 mL H2O
5 mL DCM
Nanoparticles
(powder form)
Evaporate under rotating vacuum
(32°C for 6 mins.)
Sonicate to form emulsion (10 mins. at 38% amplitude; pulse of 4:2; in an ice bath)
10mL of the continuous phase stock solution
300mg PVA
100mLNano-pure H2O
2.0mL of the discontinuous phase stock solution
100 mg PLGA
5 mgTOA or DIEA
5 mL DCM
Nanoparticle System Synthesis: Emulsion-Solvent Evaporation Technique + +2-CEES
TOA
Ethylvinylsulfide
Ethylthioethanol
Hydrochloric acid
or
DIEA
Characterization Entrapment Efficiency Reaction Testing
•Size:>200 nm to prevent endocytosis•Polydispersity Index (PDI):Monodisperse <0.1•Zeta Potential:±30 mV for moderate stability
•Successfully entrap >70% of the deactivating chemical in the nanoparticles
•NP instantaneous response to presence of 2-CEES•Entrapment and deactivation of 2-CEES•Loaded NP deactivation occurs faster than free chemical reaction with 2-CEES
Measurable Objectives
Entrapment Efficiency
Reaction Testing
Loaded Nanoparticles •Average size: 213.1 nm•PDI: 0.142•Zeta Potential: -8.4 mV
Blank Nanoparticles•Average Size: 216.3 nm•PDI: 0.098•Zeta Potential: -21.7 mV
•Visually confirm that nanoparticles were produced as designed using:•Dynamic light scattering •Transmission electron microscopy
•Nanoparticle characterization showed successful synthesis resultsConsistent with measurable objectives for size, polydispersityindex, and zeta potential
•Entrapment efficiency of deactivating chemical was successfully determinedConsistent with measurable objectives
•Although no significant change was observed between the loaded and blank nanoparticles, reaction testing showed that nanoparticles reacted faster than free deactivating chemicals
Consistent with measureable objectives
Blank NP
+ 0 mg TOA
Blank NP
+ 2.5 mg TOA
Blank NP
+ 1 mg TOA
Blank NP
+ 5 mg TOA
100% 80% 50% 0%
Pellet after centrifugation
Supernatant pH measured
pH=4.10 pH=4.24pH=4.21pH=4.17
0
0.0001
0.0002
0.0003
0.0004
0 50 100 150
[H+]
(mo
l/li
ter)
Time (sec)
Comparison of [H+] Generated by Reactions of Surfactant, Deactivating Chemical, and Vesicant
PVA
PVA and TOA
PVA and DIEA
Profile of [H+] generated after introduction of vesicant to solutions of surfactant and deactivating chemicals.
00.00010.00020.00030.00040.00050.00060.00070.0008
0 50 100 150 200
[H+ ]
(m
ol/
lite
r)
Time (sec)
Comparison of [H+] Generated in Reactions of Nanoparticles with Vesicant
blank NP
TOA loaded
DIEA loaded
Profile of [H+] generated after introduction of vesicant to solutions of nanoparticles loaded with deactivating chemicals.
0
5
10
15
20
25
Blank NP TOA NP DIEA NP PVA PVA TOA PVA DIEA
% D
est
ruct
ion
of
2-C
EES
Comparisons of Reaction Efficacy
Nanoparticles Free Chemicals
Comparison of percent destruction of vesicant after introduction to various chemical and nanoparticle solutions.
Deactivation reaction: •Vesicant undergoes hydrolysis producing HCl, which causes a measurable decrease in pH•Deactivating chemical is not consumed in the reaction
Procedure:•5 µL of vesicant reacted with 10 mL free deactivating chemical, blank NPs, and loaded NPs•pH change measured and recorded over timeResults:•NPs deactivate vesicant better than free deactivating chemicals•Percent destruction of vesicant by blank and loaded NPs was similar
http://www.sciencedirect.com.libezp.lib.lsu.edu/science?
4.004.054.104.154.204.254.304.35
0.0000 0.1000 0.2000 0.3000 0.4000 0.5000 0.6000
pH
Concentration of TOA in Supernatant (mg/mL)
Standard pH Curve based on TOA Concentration in the Supernatant
Standard curve used to determine the entrapment efficiency of the deactivating chemical, TOA, loaded inside the nanoparticles.
Procedure:•Four batches of blank NPs synthesized•Different amounts of deactivating chemical added to the NP solutions simulating varying entrapment efficiencies •NP system centrifuged for 25 minutes at 35,000 rpm to separate nanoparticles (pellet) from solution (supernatant)•Steady state pH of supernatants was measured
Results:•pH of loaded nanoparticle supernatant= 4.16•Interpolation from standard curve used to determine a concentration of 0.0857 mg/mL•The concentration corresponded to an entrapment efficiency of 82.86%•Consistent with measureable objectives
Blank NP
