Engineering and Technology Degree Level: PhD Abstract ID# 855
Graduate Category: Engineering and Technology Degree Level: PhD Abstract ID# 855
Silver Nanoparticle-Embedded Polymersome Nanocarriers for the Treatment of Antibiotic-Resistant Infections Benjamin M. Geilich1, Anne L. van de Ven2, Gloria L. Singleton3, Srinivas Sridhar2, and Thomas J. Webster3 1Departments of Bioengineering, 2Physics, and 3Chemical Engineering Abstract
Particle Design & Synthesis
Nanoscale
This study explored the development and op8miza8on of a polymersome nanocarrier formed from a biodegradable diblock copolymer to overcome bacterial an8bio8c resistance. Here, polymersomes were synthesized containing silver nanopar8cles embedded in the hydrophobic compartment, and ampicillin in the hydrophilic compartment. Results showed, for the first 8me, that these silver nanopar8cle-‐ embedded polymersomes (AgPs) inhibited the growth of Escherichia coli transformed with a gene for ampicillin resistance (bla) in a dose-‐dependent fashion. Free ampicillin, AgPs without ampicillin, and ampicillin polymersomes without silver nanopar8cles had no effect on bacterial growth. The rela8onship between the silver nanopar8cles and ampicillin was determined to be synergis8c and produced complete growth inhibi8on at a silver-‐to-‐ampicillin ra8o of 1 : 0.64. In this manner, this study introduces a novel nanomaterial that can effec8vely treat problema8c, an8bio8c-‐resistant infec8ons in an improved capacity which should be further examined for a wide range of medical applica8ons.
Nanoscale
Paper
.
A
Bacterial Growth Inhibition
Paper
Transmission Electron Micrograph of AgPs (x50,000)
Background
An8bio8cs have been extensively used since their commercializa8on in the 1930s to treat pa8ents suffering from a wide variety of infec8ous diseases. When u8lized Fig. 1 Particle synthesis. A solution of mPEG-PDLLA copolymer and hydrophobically functionalized silver nanoparticles in organic solvent (red) is B C correctly, these drugs are extremely effec8ve at reducing mortality rates and healing injected through a syringe atomizer into a stirring aqueous solution of ampicillin sodium salt in PBS (blue). The organic solvent and any unencapsulated drug are subsequently removed by dialysis. Schema8c Representa8on of the AgP Structure 8me, which makes them essen8al in the clinic today. Unfortunately, however, an8bio8cs have been used so prevalently over the last 80 years that the bacteria they years, making it one of the most widely studied prokaryotic 48 hours. In the absence of silver nanoparticles, no bacteriowere designed to kill have begun to evolve and adapt, rendering these drugs organisms and thus ideal for a proof-of-concept application. static effect was observed for all ampicillin concentrations First, E. coli was transformed with a plasmid containing the tested. This suggests that the presence of silver potentiates the ineffec8ve. Infec8ons caused by Gram-‐nega8ve bacteria are par8cularly difficult to bla gene encoding for the enzyme TEM-1 β-lactamase using therapeutic efficacy of ampicillin. AgPs without ampicillin likecalcium chloride and heat-shock. TEM-1 is the most wise produced no therapeutic benefit. Additionally, no signifitreat because their robust and hydrophobic outer lipopolysaccharide membrane helps common β-lactamase found in enterobacteriaceae, and confers cant differences were observed between bacteria treated with resistance to multiple antibiotics including the narrow-spec- free ampicillin (200 µg mL ), PBS, AgPs without ampicillin, to impede the influx of drugs into the cell.
D
20
−1
trum cephalosporins, cefamandole, cefoperazone, and all of the penicillins except for temocillin.21 The growth and proliferation of a 106 colony forming units mL−1 (CFU mL−1) suspension of ampicillin-resistant E. coli was examined by measuring the optical density at 600 nm (OD600) for 24 hours following treatment with volumes of AgPs containing a silver : ampicillin (Ag : Amp) ratio of 1 : 0.28 (Fig. 3A), 1 : 0.44 (Fig. 3B), or 1 : 0.64 (Fig. 3C). Ampicillinloaded AgPs displayed significant bacteriostatic action against the E. coli, manifesting as a delay in the time taken to reach exponential growth phase. This response was dose-dependent, with higher concentrations of ampicillin producing a longer delay in bacterial growth. Bacteria treated with ampicillin concentrations above 55 µg mL−1 failed to proliferate within
and ampicillin loaded polymersomes (200 µg mL−1) without silver nanoparticles. When bacteria were treated with suboptimal concentrations of AgPs, bacterial growth was always observed within 17 hours. The time to exponential phase was found to vary with both silver concentration and ampicillin loading (Fig. 3D). A Bliss Model was utilized to determine the degree of synergy for different silver and ampicillin combinations.22,23 Drug interactions were found be synergistic (S > 0) in all cases where ampicillin was supplied at concentrations of 24 µg mL−1 and above (Fig. 3E). At lower concentrations, no synergism was observed (S = 0). The degree of synergy was dose-dependent and increased with both silver and ampicillin concentrations. The therapeutic benefit of ampicillin reached a plateau at 50
Physiochemical characteriza0on. (A) Transmission electron micrographs reveal the presence of 5 nm silver nanopar8cles (dark dots) embedded within the larger polymersome par8cles. Scale bars = 100 nm. (B) The size distribu8on of the AgPs was measured using dynamic light scaFering at 25 °C. Results indicate an average par8cle size of 104.3 nm ± 15.6 nm. (C) Examina8on of TEM images showed that an average of 9.29 ± 6.07 silver nanopar8cles This journal is © The Royal Society of Chemistry Nanoscale present in AgPs was 1 Ag:0.28 Amp, 1 Ag:0.44 were loaded per p2015 olymersome. (D) The final concentra8on of ampicillin View Article Online Amp, and 1 Ag:0.64 Amp, corresponding to a loading efficiency of 23%, 22%, and 20% respec8vely. 6 −1 Paper Nanoscale Fig. 3 Bacterial growth inhibition. The proliferation of a 10 CFU mL suspension of antibiotic-resistant E. coli was measured over 24 hours in the Bacterial growth inhibi0on. The prolifera8on of a 106 CFU mL−1 suspension of an8bio8c-‐resistant presence of different concentrations witho(A) (B)p1resence Ag:0.44oAmp, and (C)concentra8ons 1 Ag:0.64 Amp.oValues E. cof oli AgPs was loaded measured ver 1 2Ag:0.28 4 hours Amp, in the f different f AgPs represent loaded wthe ith mean ± Bacteria-Particle Interactions standard deviation, N = 3. (D) The time taken for the bacteria to reach exponential phase was compared between treatment groups. (E, F) The in vitro (A) 1 Ag:0.28 Amp, (B) 1 Ag:0.44 Amp, and (C) 1 Ag:0.64 Amp. (D) The 8me taken for the bacteria to synergy between the silver and ampicillin during the log-linear growth phase was determined at 13.3 hours by the Bliss Independence model. reach exponen8al phase was compared between treatment groups. (E, F) The in vitro synergy Synergism is observed for increasing concentrations of (E) ampicillin and (F) silver nanoparticles. Transmission Electron Micrograph of AgPs and E. coli (x12,000) between the silver and ampicillin during the log-‐linear growth phase was determined at 13.3 hours by the Bliss Independence model.
Mechanisms of An8bio8c Resistance7
Timeline of An8bio8c Resistance8
ortheastern University on 25/03/2015 16:30:08.
Cytotoxicity
Par0cle cytotoxicity. The viability of CCL-‐110 h u m a n d e r m a l fibroblast cells following 24 and 48 hours as a s s e s s e d v i a M T S assays. The treatment concentra8on displayed corresponds to the highest bacterial of t r e a t m e n t concentra8on tested (1 A g : 0 . 6 4 A m p ) . N o significant cytotoxicity was observed. Fig. 4
Bacteria-‐par0cle interac0ons. 22,24,25 to disulfide bond disruption.T r a n s Regions m i s s i oof n the e l ecell c t envelr o n ope with little to no AgPs contact appeared morphologically micrographs of whole bacteria (A– normal (Fig. 4F–H; black arrows). D) and thin bacteria sec8ons (E–H) aler AgPs treatment. The cytotoxicity of AgPs to mammalian cells was (A–D) investiof the bacterial cell gated using CCL-110 human Indenta8on dermal fibroblasts (Fig. 5). Cells membrane was observed were treated with different concentrations of AgPs for 24in or regions of AgPs contact (white 48 hours, and cell viability was measured using MTS assays. arrows). Silver nanopar8cles inside No significant cytotoxicity was observed over a range of 0–80 A g P s −1 e x h i b i t e d s i g n s o f −1 µg mL ampicillin and 0–125pµg o l amL r i z a 8silver. o n i n d i c a 8 v e o f
Significance mely effective permeability barrier.26–28 In addition, E. coli This work efflux has many important long-‐las8ng, possess pumps whichimplica8ons. can help toA remove antibiotics dual-‐mechanism treatment in one par8cle able to overcome from the cell.29 When bacteria possess genetic resistance, the an8bio8c resistance would be extremely antibiotics in the environment are also beneficial subjectedin to the hydrolysis clinic, both as a systemic treatment and as a localized by β-lactamase enzymes, which can be secreted by the cell.30 treatment for implanted medical devices. It is also important This study showed for the first time that it is possible to overto note that this delivery system could be easily customized come drug resistance through the combined and delivery to encapsulate other therapeu8c nanopar8cles novel of silver nanoparticles cand ampicillin. Co-encapsulation resulted in a pharmaceu8cal ompounds. synergistic activity sufficient to delay or inhibit the growth of bacteria,Acknowledgement even when the gene for ampicillin resistance hydrophobic interac8ons (yellow wasThis present. The authorsby postulate the effectiveness of arrows). (E–H) Bacteria in contact work was supported the IGERT that Nanomedicine with AgPs displayed significant this formulation is most likely dueNSF/ to aDGE-‐096843. mechanism which Science & T echnology P rogram a t N EU, Discussion and conclusion p r o t e i n a g g r e g a 8 o n a n d exploits the reactive properties of nanoscale silver in order to disrup8on. of Webster anomedicine disrupt Nthe integrityLaboratory of the outer cell membrane. This is Prevention of cellular access membrane of a drug is critical toRegions antibiotic the outer membrane with liFle Egan Research Center supported by the observation that silver nanoparticles can resistance, especially for Gram-negative bacteria because their 225 A g P s c o n t a c t a p p e a r e d Northeastern University robust outer lipopolysaccharide membrane provides extre- cause morphological changes and indentations in the bacteria morphologically normal an(black [email protected] arrows). Scale bars = 100 nm (A–D, www.websternano.org F–H) and 500 nm (E). This journal is © The Royal Society of Chemistry 2015
Bacteria-particle interactions. Transmission electron micrographs of whole bacteria (A–D) and thin bacteria sections (E–H) after AgPs treat-
Nanoscale
Silver Nanoparticle-Embedded Polymersome Nanocarriers for the Treatment of Antibiotic-Resistant Infections Benjamin M. Geilich1, Anne L. van de Ven2, Gloria L. Singleton3, Srinivas Sridhar2, and Thomas J. Webster3 1Departments of Bioengineering, 2Physics, and 3Chemical Engineering Abstract
Particle Design & Synthesis
Nanoscale
This study explored the development and op8miza8on of a polymersome nanocarrier formed from a biodegradable diblock copolymer to overcome bacterial an8bio8c resistance. Here, polymersomes were synthesized containing silver nanopar8cles embedded in the hydrophobic compartment, and ampicillin in the hydrophilic compartment. Results showed, for the first 8me, that these silver nanopar8cle-‐ embedded polymersomes (AgPs) inhibited the growth of Escherichia coli transformed with a gene for ampicillin resistance (bla) in a dose-‐dependent fashion. Free ampicillin, AgPs without ampicillin, and ampicillin polymersomes without silver nanopar8cles had no effect on bacterial growth. The rela8onship between the silver nanopar8cles and ampicillin was determined to be synergis8c and produced complete growth inhibi8on at a silver-‐to-‐ampicillin ra8o of 1 : 0.64. In this manner, this study introduces a novel nanomaterial that can effec8vely treat problema8c, an8bio8c-‐resistant infec8ons in an improved capacity which should be further examined for a wide range of medical applica8ons.
Nanoscale
Paper
.
A
Bacterial Growth Inhibition
Paper
Transmission Electron Micrograph of AgPs (x50,000)
Background
An8bio8cs have been extensively used since their commercializa8on in the 1930s to treat pa8ents suffering from a wide variety of infec8ous diseases. When u8lized Fig. 1 Particle synthesis. A solution of mPEG-PDLLA copolymer and hydrophobically functionalized silver nanoparticles in organic solvent (red) is B C correctly, these drugs are extremely effec8ve at reducing mortality rates and healing injected through a syringe atomizer into a stirring aqueous solution of ampicillin sodium salt in PBS (blue). The organic solvent and any unencapsulated drug are subsequently removed by dialysis. Schema8c Representa8on of the AgP Structure 8me, which makes them essen8al in the clinic today. Unfortunately, however, an8bio8cs have been used so prevalently over the last 80 years that the bacteria they years, making it one of the most widely studied prokaryotic 48 hours. In the absence of silver nanoparticles, no bacteriowere designed to kill have begun to evolve and adapt, rendering these drugs organisms and thus ideal for a proof-of-concept application. static effect was observed for all ampicillin concentrations First, E. coli was transformed with a plasmid containing the tested. This suggests that the presence of silver potentiates the ineffec8ve. Infec8ons caused by Gram-‐nega8ve bacteria are par8cularly difficult to bla gene encoding for the enzyme TEM-1 β-lactamase using therapeutic efficacy of ampicillin. AgPs without ampicillin likecalcium chloride and heat-shock. TEM-1 is the most wise produced no therapeutic benefit. Additionally, no signifitreat because their robust and hydrophobic outer lipopolysaccharide membrane helps common β-lactamase found in enterobacteriaceae, and confers cant differences were observed between bacteria treated with resistance to multiple antibiotics including the narrow-spec- free ampicillin (200 µg mL ), PBS, AgPs without ampicillin, to impede the influx of drugs into the cell.
D
20
−1
trum cephalosporins, cefamandole, cefoperazone, and all of the penicillins except for temocillin.21 The growth and proliferation of a 106 colony forming units mL−1 (CFU mL−1) suspension of ampicillin-resistant E. coli was examined by measuring the optical density at 600 nm (OD600) for 24 hours following treatment with volumes of AgPs containing a silver : ampicillin (Ag : Amp) ratio of 1 : 0.28 (Fig. 3A), 1 : 0.44 (Fig. 3B), or 1 : 0.64 (Fig. 3C). Ampicillinloaded AgPs displayed significant bacteriostatic action against the E. coli, manifesting as a delay in the time taken to reach exponential growth phase. This response was dose-dependent, with higher concentrations of ampicillin producing a longer delay in bacterial growth. Bacteria treated with ampicillin concentrations above 55 µg mL−1 failed to proliferate within
and ampicillin loaded polymersomes (200 µg mL−1) without silver nanoparticles. When bacteria were treated with suboptimal concentrations of AgPs, bacterial growth was always observed within 17 hours. The time to exponential phase was found to vary with both silver concentration and ampicillin loading (Fig. 3D). A Bliss Model was utilized to determine the degree of synergy for different silver and ampicillin combinations.22,23 Drug interactions were found be synergistic (S > 0) in all cases where ampicillin was supplied at concentrations of 24 µg mL−1 and above (Fig. 3E). At lower concentrations, no synergism was observed (S = 0). The degree of synergy was dose-dependent and increased with both silver and ampicillin concentrations. The therapeutic benefit of ampicillin reached a plateau at 50
Physiochemical characteriza0on. (A) Transmission electron micrographs reveal the presence of 5 nm silver nanopar8cles (dark dots) embedded within the larger polymersome par8cles. Scale bars = 100 nm. (B) The size distribu8on of the AgPs was measured using dynamic light scaFering at 25 °C. Results indicate an average par8cle size of 104.3 nm ± 15.6 nm. (C) Examina8on of TEM images showed that an average of 9.29 ± 6.07 silver nanopar8cles This journal is © The Royal Society of Chemistry Nanoscale present in AgPs was 1 Ag:0.28 Amp, 1 Ag:0.44 were loaded per p2015 olymersome. (D) The final concentra8on of ampicillin View Article Online Amp, and 1 Ag:0.64 Amp, corresponding to a loading efficiency of 23%, 22%, and 20% respec8vely. 6 −1 Paper Nanoscale Fig. 3 Bacterial growth inhibition. The proliferation of a 10 CFU mL suspension of antibiotic-resistant E. coli was measured over 24 hours in the Bacterial growth inhibi0on. The prolifera8on of a 106 CFU mL−1 suspension of an8bio8c-‐resistant presence of different concentrations witho(A) (B)p1resence Ag:0.44oAmp, and (C)concentra8ons 1 Ag:0.64 Amp.oValues E. cof oli AgPs was loaded measured ver 1 2Ag:0.28 4 hours Amp, in the f different f AgPs represent loaded wthe ith mean ± Bacteria-Particle Interactions standard deviation, N = 3. (D) The time taken for the bacteria to reach exponential phase was compared between treatment groups. (E, F) The in vitro (A) 1 Ag:0.28 Amp, (B) 1 Ag:0.44 Amp, and (C) 1 Ag:0.64 Amp. (D) The 8me taken for the bacteria to synergy between the silver and ampicillin during the log-linear growth phase was determined at 13.3 hours by the Bliss Independence model. reach exponen8al phase was compared between treatment groups. (E, F) The in vitro synergy Synergism is observed for increasing concentrations of (E) ampicillin and (F) silver nanoparticles. Transmission Electron Micrograph of AgPs and E. coli (x12,000) between the silver and ampicillin during the log-‐linear growth phase was determined at 13.3 hours by the Bliss Independence model.
Mechanisms of An8bio8c Resistance7
Timeline of An8bio8c Resistance8
ortheastern University on 25/03/2015 16:30:08.
Cytotoxicity
Par0cle cytotoxicity. The viability of CCL-‐110 h u m a n d e r m a l fibroblast cells following 24 and 48 hours as a s s e s s e d v i a M T S assays. The treatment concentra8on displayed corresponds to the highest bacterial of t r e a t m e n t concentra8on tested (1 A g : 0 . 6 4 A m p ) . N o significant cytotoxicity was observed. Fig. 4
Bacteria-‐par0cle interac0ons. 22,24,25 to disulfide bond disruption.T r a n s Regions m i s s i oof n the e l ecell c t envelr o n ope with little to no AgPs contact appeared morphologically micrographs of whole bacteria (A– normal (Fig. 4F–H; black arrows). D) and thin bacteria sec8ons (E–H) aler AgPs treatment. The cytotoxicity of AgPs to mammalian cells was (A–D) investiof the bacterial cell gated using CCL-110 human Indenta8on dermal fibroblasts (Fig. 5). Cells membrane was observed were treated with different concentrations of AgPs for 24in or regions of AgPs contact (white 48 hours, and cell viability was measured using MTS assays. arrows). Silver nanopar8cles inside No significant cytotoxicity was observed over a range of 0–80 A g P s −1 e x h i b i t e d s i g n s o f −1 µg mL ampicillin and 0–125pµg o l amL r i z a 8silver. o n i n d i c a 8 v e o f
Significance mely effective permeability barrier.26–28 In addition, E. coli This work efflux has many important long-‐las8ng, possess pumps whichimplica8ons. can help toA remove antibiotics dual-‐mechanism treatment in one par8cle able to overcome from the cell.29 When bacteria possess genetic resistance, the an8bio8c resistance would be extremely antibiotics in the environment are also beneficial subjectedin to the hydrolysis clinic, both as a systemic treatment and as a localized by β-lactamase enzymes, which can be secreted by the cell.30 treatment for implanted medical devices. It is also important This study showed for the first time that it is possible to overto note that this delivery system could be easily customized come drug resistance through the combined and delivery to encapsulate other therapeu8c nanopar8cles novel of silver nanoparticles cand ampicillin. Co-encapsulation resulted in a pharmaceu8cal ompounds. synergistic activity sufficient to delay or inhibit the growth of bacteria,Acknowledgement even when the gene for ampicillin resistance hydrophobic interac8ons (yellow wasThis present. The authorsby postulate the effectiveness of arrows). (E–H) Bacteria in contact work was supported the IGERT that Nanomedicine with AgPs displayed significant this formulation is most likely dueNSF/ to aDGE-‐096843. mechanism which Science & T echnology P rogram a t N EU, Discussion and conclusion p r o t e i n a g g r e g a 8 o n a n d exploits the reactive properties of nanoscale silver in order to disrup8on. of Webster anomedicine disrupt Nthe integrityLaboratory of the outer cell membrane. This is Prevention of cellular access membrane of a drug is critical toRegions antibiotic the outer membrane with liFle Egan Research Center supported by the observation that silver nanoparticles can resistance, especially for Gram-negative bacteria because their 225 A g P s c o n t a c t a p p e a r e d Northeastern University robust outer lipopolysaccharide membrane provides extre- cause morphological changes and indentations in the bacteria morphologically normal an(black [email protected] arrows). Scale bars = 100 nm (A–D, www.websternano.org F–H) and 500 nm (E). This journal is © The Royal Society of Chemistry 2015
Bacteria-particle interactions. Transmission electron micrographs of whole bacteria (A–D) and thin bacteria sections (E–H) after AgPs treat-
Nanoscale
