Cationic amphiphilic non-hemolytic synthetic polymers as potential agents to combat bacteria with antibiotics resistance
A.Punia, E. He, K. Lee and N.-L. Yang
Center for Engineered Polymeric Materials and Chemistry Ph.D. Program. City University of New York ,N.Y. USA
*E-mail: [email protected] Professor Nan-Loh Yang
CEPM Diretor & Chair Ph.D Program in Polymer ChemistyCity University of New York, CUNY
[email protected], 1 718 982 3926the New York State Office of Science, Technology, and Academic Research
Dec 15, 2016
Nulastin
CEPM Industrial Sponsors
23,000 people killed every year in United Stateswww.nih.gov
Nearly half of E. coli infections in India are practically untreatablewww.abc.net.au
2 million people infected each year with drug resistant bacteria nationwidewww.nih.gov
Common surgeries will soon become impossible to perform
~ 11,000 deaths/year from methicillin resistant S. aureuswww.nih.gov
~450,000 new cases of MDR-TB each year – 1/3rd
diewww.who.int
Beforeitsnews.com
www.sciencephoto.com
floridamedicalmalpracticelaws.com
www.examiner.com
D. Jabes, Curr. Opin. Microbiol. 2011, 14, 1; www.cdc.gov; www.who.int; www.reuters.com.
The superbug, carbapenem-resistant enterobacteriaceae (CRE), was found after researchers swabbed the floors and walls of the farm’s pens and collected fecal samples during a five-month period in 2015. Journal Antimicrobial Agents and Chemotherapy. Dec. 5, 2016, by Ohio State
antibiotic drugs targeting The Ribosome
the
The ribosome, one of the ubiquitous molecular machines in living cells, is responsible for the critical task of translating the genetic code into functional proteins ( U of Ill)
The story of Magainins..Michael ZasloffProc. Natl. Acad. Sci. USAVol. 84, 1987, 5449-5453
African clawed frog Xenopus laeviso After performing surgery under non-
sterile conditions, the frogs were beingplaced in microbially contaminatedwater tanks
o Surgical wounds never developedinfections
o Normal healing of scar always occurred
o Potent antimicrobial peptides werepurified from frog’s skin and named“Magainins” http://www.upenn.edu/gazette
Surgery
No infection!!!
Positive Charge and facialamphiphilicity being the commonfeature of AMPs
AMPs generally manifest onehydrophobic & one hydrophilic facealong their backbones
Direct action against bacterialmembrane
Development of bacterial resistance isimprobable
α-Helical Cationic AMP
Cationic
Hydrophobic
Membrane rupturePore formation
Leakage of cellular componentMembrane potentialbreakdown
Natural Antimicrobial Peptides (AMPs)
e.g. Magainin (African clawed frog) LL-37 (humans)Cecropins (Silk moth)
Bacterial CellDeath
M. Zasloff, Nature, 2002, 415, 389.K. Kuroda, G. A. Caputo, WIREs: Nanomed. Nanobiotechnol., 2012, 5(1), 49
Chem. Eur. J. 2009, 15, 11784 – 11800
E. coli (ampicillin resistant) E. coli after polymer treatment
Figures of Merits from Biological AvtivitityMeasurement
1) MIC (µg/mL) Minimum Inhibitory Concentration MIC is expressed as the minimum polymers concentration that resulted in
100% inhibition of bacterial growth after an incubation period of 18 hours.
2) HC50 (µg/mL) , RBCsHemolytic activities of polymers were determined in terms of hemolytic concentration-50% (HC50), the minimum polymer concentration resulting in 50% lyses of mouse RBCs within an incubation period of 1 hour.
Selectivity : HC50/MIC; MIC /MIC’
Focus on the effects of topological placement of hydrophilic and lipophilic structure segments. Various structural parameters examined included: amphiphilic balance, spatial arrangement of cationic and hydrophobic groups, random copolymer and homopolymer architecture, and cationic charge density
Structural Isomers
Structural Isomers
Why Synthetic Polymers Large scale implementations of AMPs is a challenge
1) Large scale isolation or manufacturing of complex AMPs would be extremely costly and time consuming
2) Decreased activity of AMPs, once introduced into complex serum milieu
3) Efficient digestion of protein/peptide material by gastrointestinal tract leads to lower oral availability of AMPs
4) Limited broad availability will result from intravenous delivery
Synthetic mimics of antimicrobial peptides??
Spacer arm design strategyPalermo, E. F.; Vemparala, S.; Kuroda, K. Biomacromolecules2012, 13(5)
OO
NH3
OO
NH3
OO
NH3
2-ca
rbon
6-ca
rbon
4-ca
rbon
MIC (E. coli) 1000 2000 10 MIC (S. aureus) 63 250 7.8
HC50 >2000 >2000 2.6
Highly antibacterial Highly hemolytic
All values in µ/mL)
S
O
O
OO
NH3
q
Effect of spacer arm length (Kuroda & Co-workers)
S
O
O
OO
NH3
OO
MIC MIC HC50 E. coli S. aureus RBCs(µg/mL) (µg/mL) (µg/mL)
10 7.8 2.6
7.8 7.8 2.8
6.5 10 2.3
Antibacterial but extremely hemolytic
27% Ethyl
40% Ethyl
Palermo, E. F.; Vemparala, S.; Kuroda, K. Biomacromolecules 2012, 13(5)
HONH2
(Boc)2O, room temp.
1M NaOH : THF (1:1)24 hrs
HO
HN
Boc
OCl
Triethyl amine, CH2Cl20 oC
room temp, 24 h
O
O
NH
Boc M6
HO
HN
HON
Boc
(Boc)2O, 34 oC
H2O, 3 hrs
O
O
N
Boc
OCl
N,N-diisopropylethyl amine0 oC
room temp, CH2Cl224 h
M2
a) Monomer Synthesis
Experimental
1
10
100
1000
10000
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
MIC
(µg/
mL
)
Mole % of M6 Monomer
Polymer Avg-MIC(µg/mL)
PM6-0% 1428
PM6-10% 809
PM6-20% 250
PM6-30% 125
PM6-40% 62
PM6-50% 52
PM6-60% 41
PM6-70% 26
PM6-80% 15
PM6-90% 7.8
PM6-100% 5.8
S
O
O
OO
NH3
q
Antibacterial activity towards E. coli *
Increasing antibacterial activity
*Values reported here are the averages of three separate experiments performed on different days. Error bars represent standard deviation.
S
OO
NH2
p
O
O
S
O
O
OO
NH3
OO
NH2
q1-q
1
10
100
1000
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
MIC
(µg/
mL
)
Mole % of M6 Monomer
Polymer Avg-MIC(µg/mL)
PM6-0% 104
PM6-10% 62
PM6-20% 62
PM6-30% 52
PM6-40% 52
PM6-50% 52
PM6-60% 31
PM6-70% 31
PM6-80% 26
PM6-90% 15
PM6-100% 15
S
O
O
OO
NH3
q
Antibacterial activity towards S. aureus
S
OO
NH2
p
O
O S
O
O
OO
NH3
OO
NH2
q1-q
1
10
100
1000
10000
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
HC
50
(µg/
mL
)
Mole % of M6 Monomer
Polymer HC-50(µg/mL)
PM6-0% >2000
PM6-10% >2000
PM6-20% >2000
PM6-30% >2000
PM6-40% 1619
PM6-50% >2000
PM6-60% >2000
PM6-70% >2000
PM6-80% >2000
PM6-90% 1619
PM6-100% <1.9
S
O
O
OO
NH3
q
Hemolytic activity towards mouse Red Blood Cells*
S
OO
NH2
p
O
O
S
O
O
OO
NH3
OO
NH2
q1-q
* Values reported here are the averages of 3 experiments. Error bar represents standard deviation. RBCs were obtained from freshly drawn mouse blood.
1
10
100
1000
10000
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
MIC
or H
C50
(µg/
mL)
Mole % of M6 Monomer
E. coli
S. aureus
HC50
PolymerSelectivityHC50/MIC
E. coli S. aureus
PM6-0%
PM6-10%
PM6-20%
PM6-30%
PM6-40%
PM6-50%
PM6-60%
PM6-70%
PM6-80%
PM6-90%
PM6-100%
>1.4
>2.5
>8
>16
26
>38
>49
>80
>133
208
<0.33
>19
>32
>32
>38
31
>38
>64
>64
>80
108
<0.13
Biological activity of polymers: Highly selective polymers
S. aureus > E. coli
E. coli ~ S. aureus
Doubly selective polymers: Bacteria over RBCsBacteria A over Bacteria B (switchable) Punia, A.; Yang, N. U.S. Provisional Patent 61889401, 2013
Punia, A.; He, H.; Lee, K.; Banerjee, P.; Yang, N-L., Chem. Commun. 2014, 50, 7071-7074
0
20
40
60
80
100
120
0 2 4 6 8 10
Frac
tiona
l Cel
l Sur
viva
l (%
)
Time (h)
1×MIC
2×MIC
a b c d
e f g h
Time (h)
E. coli
1 × MIC 2 × MIC Control
0
1
2
4
6
8
6.0 × 104
3.0 × 104
4.7 × 103
6 × 102
nil
nil
2.3 × 104
3.5 × 103
2 × 102
nil
nil
nil
0.9 × 105
1.5 × 105
4.8 × 105
4.8 × 106
1.1 × 108
3.6 × 108
Time dependent killing efficiency study
positive control and is shown in a) at 1 h, b) at 6 h time interval. E. coli treated withPM6-90% at c) 0 h, d) 1h, e) 2 h, f) 4 h, g) 6 h, and h) 8 h time interval
Viable cells (CFU/mL) are shown after incubating with PM6-90% or without PM6-90% (control), for various time intervals
S
O
O
OO
NH3
OO
NH2
0.90.1
PM6-90%
N
Cetyltrimethylammonium bromide
N
Cetylpyridinium chloride
NR
Alkyl dimethylbenzyl ammonium chloride(C: 12 to 18)
HC50 = 0.192 µg/mLMIC = 25 µg/mL (gram negative E. coli)R =12
MIC = 4 µg/mL (E. coli)
HC50 ~ 0.23 µg/mL
Quaternary Ammonium Compounds (QACs)Vs
Amphiphilic Polymers
MIC = 7.8 µg/mL (E. coli)HC50 > 1619 µg/mL
S
O
O
OO
NH3
OO
NH2
0.90.1
>7000 times less hemolyticthan Cetyltrimethylammoniumbromide
Terpolymer
•
Time-Kill for Terpolymer
0
20
40
60
80
100
120
0 1 2 3 4 5 6Time (h)
E. coli Pe
rcen
tcel
l sur
vica
l 1XMIC (PolyE-12%)
2XMIC (PolyE-12%)
c)
0
20
40
60
80
100
120
0 2 4 6 8 10
Frac
tiona
l Cel
l Sur
viva
l (%
)
Time (h)
1×MIC (PM6-70-300peg)
2×MIC (PM6-70-300peg)
1×MIC (PM6-90-950peg)
2×MIC (PM6-90-950peg)
0
20
40
60
80
100
120
0 2 4 6 8 10
Frac
tiona
l Cel
l Sur
viva
l (%
)
Time (h)
1×MIC (PM6-70-300peg)
2×MIC (PM6-70-300peg)
1×MIC (PM6-90-950peg)
2×MIC (PM6-90-950peg)
Time dependent killing efficiency study
E. coli
S. aureus
Confirmation of bactericidal activity of PM6-90-PEG950 at various time intervals using Agarplates. The final polymers concentration was equal to MIC value. E. coli without polymertreatment was used as positive control and is shown in a) at 1 h, b) at 6 h time interval. E. colitreated with PM6-90-PEG950 at c) 0 h, d) 1h, e) 2 h, f) 4 h, g) 6 h, and h) 8 h time interval
PM6-90-PEG950 at 1×MIC against E. coli
a b c d
e f g h
PM6-90-PEG950 at 2×MIC against S. aureus
Confirmation of bactericidal activity of PM6-90-PEG950 against S. aureus at various timeintervals using Agar plates. The final polymers concentration was equal to 2×MIC value. S.aureus without polymer treatment was used as positive control and is shown in a) at 1 h, b) at 8 htime interval. E. coli treated with PM6-90-PEG950 at c) 0 h, d) 1h, e) 2 h, f) 4 h, g) 6 h, and h) 8 htime interval
2014-2015 - Our Publications and Non-Provisional Patents on
Cationic amphiphilic non-hemolytic polyacrylates with superior antibacterial activity
Royal Society of Chemistry, Chem. Commun., 2014,50, 7071-7074American Chemical Society , Macro Letters 2015, 4, 426 – 430. Royal Society of Chemistry Advances. 2015, 5, 80318-80324. Royal Society of ChemistryRSC Advances 2015, DOI: 10.1039/C5RA17875DRoyal Society of Chemistry Advances, 2015,5, 95300 - 95306International Journal of Molecular Sciences 2015, 16(10), 23867-23880.
US non-provisional Patent App. No.14/510,686 YANG and PUNIA
Polymer Killing Action
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