Skip to main content

Full text of "DTIC AD1045209: A Nanolayer Copper Coating for Prevention Nosocomial Multi-Drug Resistant Infections"

See other formats


AWARD NUMBER: W81XWH-15-2-0066 


TITLE: A Nanolayer Copper Coating for Prevention Nosocomial Multi-drug Resistant 

Infections 


PRINCIPAL INVESTIGATOR: Aaron D. Strickland 

CONTRACTING ORGANIZATION: iFyber, LLC 

Ithaca, NY 14850 


REPORT DATE: October 2016 


TYPE OF REPORT: Annual 


PREPARED FOR: U.S. Army Medical Research and Materiel Command 
Fort Detrick, Maryland 21702-5012 


DISTRIBUTION STATEMENT: Approved for Public Release; 

Distribution Unlimited 


The views, opinions and/or findings contained in this report are those of the author(s) and should 
not be construed as an official Department of the Army position, policy or decision unless so 
designated by other documentation. 



REPORT DOCUMENTATION PAGE 


Form Approved 
OMB No. 0704-0188 


Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the 
data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing 
this burden to Department of Defense, Washington Headquarters Services, Directorate for Information Operations and Reports (0704-0188), 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA 22202- 
4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to any penalty for failing to comply with a collection of information if it does not display a currently 
valid OMB control number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. 


1. REPORT DATE 

October 2016 


2. REPORT TYPE 

Annual 


3. DATES COVERED 

15 Sept 2015 to 14 Sept 2016 


4. TITLE AND SUBTITLE 

A Nanolayer Copper Coating for Prevention Nosocomial 
Multi-drug Resistant Infections 


5a. CONTRACT NUMBER 


5b. GRANT NUMBER 

W81XWH-15-2-0066 


5c. PROGRAM ELEMENT NUMBER 


6. AUTHOR(S) 

Aaron D. Strickland 


5d. PROJECT NUMBER 


5e. TASK NUMBER 


E-Mail: 


astrick@ifyber.com 


5f. WORK UNIT NUMBER 


7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 

iFyber, LLC 

2415 N. Triphammer Rd. 

Ithaca, NY 14850 


8. PERFORMING ORGANIZATION REPORT 
NUMBER 

4 


9. SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES) 

U.S. Army Medical Research and Materiel Command 
Fort Detrick, Maryland 21702-5012 


10. SPONSOR/MONITOR’S ACRONYM(S) 


11. SPONSOR/MONITOR’S REPORT 
NUMBER(S) 


12. DISTRIBUTION / AVAILABILITY STATEMENT 

Approved for Public Release; Distribution Unlimited 


13. SUPPLEMENTARY NOTES 


14. ABSTRACT 

This project aims to establish an an antimicrobial and biocompatible copper coating for a 
standard issue antimicrobial combat wound dressing. This initial one-year project was done 
to provide important foundational data that will guide subsequent research efforts and 
position the technology for commercial development. As part of these efforts, iFyber 
established a number of coating parameters a number of candidate dressings that have a range 
of in vitro antimicrobial efficacy and biocompatibility performance metrics. Best performing 
candidates were shown to be biocompatible through an in vivo sensitization study and 
effective against a number of clinical bacterial isolates. 


15. SUBJECT TERMS 


16. SECURITY CLASSIFICATION OF: 

17. LIMITATION 

OF ABSTRACT 

18. NUMBER 
OF PAGES 

19a. NAME OF RESPONSIBLE PERSON 
USAMRMC 

a. REPORT 

Unclassified 

b. ABSTRACT 

Unclassified 

c. THIS PAGE 

Unclassified 

Unclassified 

20 

19b. TELEPHONE NUMBER (include area 
code) 


Standard Form 298 (Rev. 8-98) 

Prescribed by ANSI Std. Z39.18 




Table of Contents 


Page 


1. Introduction.4 

2. Keywords.4 

3. Accomplishments.4 

4. Impact.17 

5. Changes/Problems.17 

6. Products.17 

7. Participants & Other Collaborating Organizations.18 

8. Special Reporting Requirements.19 


9. Appendices 


20 












1.0 Introduction 

The long-term goal of this project is to establish an enabling antimicrobial technology aimed at 
providing a range of anti-infective materials for use by the military. To accomplish this goal, 
iFyber’s immediate research and development centered on identifying and optimizing an 
antimicrobial and biocompatible copper coating for a standard issue antimicrobial combat wound 
dressing. This initial one-year project was done to provide important foundational data that will 
guide subsequent research efforts and position the technology for commercial development. As 
part of these efforts, iFyber established a number of coating parameters to produce a range of 
candidate dressings for evaluation in in vitro antimicrobial efficacy assays and biocompatibility 
assays using mammalian cells. Best performing candidates were then taken on to in vivo 
biocompatibility studies to foundational data regarding the safety of the copper coating. This 
annual report details the major accomplishments of this study, and outlines next steps in the 
development of an antimicrobial combat wound dressing. 

2.0 Keywords 

antimicrobial, copper, cotton, wound dressing, biocompatibility 

3.0 Accomplishments 

3.1 Goals of the Project 

The long-term goal of this effort is to establish an enabling antimicrobial technology aimed at 
providing a range of anti-infective materials for use by the military. iFyber’s immediate research 
and development goals set forth in the current effort are centered on identifying and optimizing 
an antimicrobial and biocompatible copper coating for a standard issue wound dressing. Below 
is a list of technical objectives that were proposed for this one-year project aimed at developing a 
prototype antimicrobial combat dressing. Efforts in these research tasks are expected to provide 
important foundational data that is expected to guide subsequent research efforts that will 
position this technology for commercial development of a new standard issue antimicrobial 
combat wound dressing. 

Specific Objectives and Tasks of the Project: The following specific objectives and tasks 

identified for this project have been designed to allow iterative feedback enabling optimization 
of the copper coating process and development of a prototype antimicrobial wound dressing that 
may be utilized throughout the military health care system as a standard issue product. 

Objective 1. Produce a candidate copper-coated dressing that balances antimicrobial efficacy 
and mammalian cell function, in vitro. 

Task 1 . Adjust copper coating parameters to produce a range of candidate 

dressings. A range of candidate copper-coated dressings will be 


4 



manufactured by altering specific coating parameters including reagent 
concentrations and soaking/dwell times. 

Task 2. Assess the in vitro antimicrobial efficacy and cytotoxicity potential of 

candidate copper-coated dressings. Each candidate dressing will be 
assessed for in vitro antimicrobial efficacy and mammalian cell 
cytotoxicity potential using standardized assays that are approved by the 
Food and Drug Administration (FDA) 

Objective 2. Assess the in vivo biocompatibility status of candidate copper-coated dressing. 
To remain in line with regulatory requirements, prototype dressings will be put forward for 
rigorous in vivo biocompatibility assessment as outlined in the below tasks, and will be 
conducted under Good Faboratory Practice guidelines. 

Task 3. Determine the dermal irritation potential of iFyber copper-coated dressing. 

Task 4. Determine the allergic response potential of iFyber copper-coated 

dressing. 

3.2 Major Accomplishments of the Project 

3.2.1 Development of a Cu-based antimicrobial wound dressing 

In this initial study, iFyber focused research efforts on refining the copper coating process to 
provide an antimicrobial coating with a range of efficacy with respect to standard in vitro 
antimicrobial assays. As part of these efforts, iFyber has established a collaboration with Cotton 
Inc., the main research and marketing company representing the US cotton industry 
( www.cottoninc.com) , to develop a chemical treatment for cotton that supports the iFyber copper 
coating, and is amenable to the cotton gauze wound dressing that has been selected for 
prototyping. Independent of coating optimization, the base prototype dressing material was 
selected in collaboration with our partners at H&H Associates (www.buyhandh.com), a leading 
vendor of standard issue military wound dressings, which is a 100% cotton gauze bearing a 
crinkle-weave pattern that provides a thick dressing substrate and a self-adherent property. 

The chemical treatment and copper coating process is illustrated in Figure 1. Using a model 
cotton substrate for coating optimization, iFyber has refined the Cu-coating technology to 
provide a range of copper loadings onto the cotton substrate. An iterative process was used to 
establish a thorough understanding of the parameters that are important to optimizing the copper 
coating process (outlined below). 

• Pre-treatment to provide Anionic Cotton: Concentration and time components for the 

incorporation of anionic aqueous Cu chelation chemistries to the cotton dressing are key to 
the development of Cu-coated cotton (e.g., anionic acetate or sulfonate groups for modified 
cotton surfaces). Together with our partners at Cotton Inc. who have state of the art 
equipment for evaluating chemical treatments of cotton, we have established a robust and 


5 



scalable process for covalently attaching acetate groups to the cotton surface. While we 
have also evaluated chemistries for attaching sulfonate groups to the cotton, sulfonated 
cotton prepared for this effort did not exhibit sufficient Cu binding properties compared to 
the acetate functionality. Using the acetate chemistry, we have established parameters for 
applying an antimicrobial Cu coating to a model substrate (outlined below), and have also 
established that this chemistry is compatible with the selected crinkle-weave prototype 
cotton-based dressing. Briefly, the process involves the steps of pad application of aqueous 
sodium hydroxide (NaOH), followed by pad application of chloroacetic acid and subsequent 
oven baking to complete the transformation to carboxymethylcellulose (i.e., cotton 
functionalized with acetate groups). The resulting substrate is washed to remove excess 
reagents and then further modified with copper as outlined below. 


Production of an Antimicrobial Cu-based nanocoating 
on a Cotton Dressing Substrate 


Anionic cellulose 



or 



Cu 2+ 




- Cu 2+ 
-Cu 2+ 
-Cu 2+ 
-Cu 2+ 
Cu 2+ 


Positive metal 
ions 


Metal ion - cotton 
fiber complex 


NaBH 4 


Air 


> 


Cu/Cu 2 0 


Oxidation » 

Metal-Metal oxide - 
coated cotton fibers 


Figure 1. Schematic representation of the copper coating process used in the formation of a Cu-coated 


2+ o | 

• Copper ion (Cu ) treatment: The copper coating process starts with formation of a Cu - 

2 | 

carboxymethylcellulose complex, which is predominately controlled by the Cu 
concentration and dwell time. The coating process is fairly basic and involves soaking the 
carboxymethylcellulose cotton in a buffered aqueous solution of Cu (e.g., in the form of 
copper sulfate; CUSO 4 ) for a specified amount of time at room temperature. While we have 
established that Cu loading can be controlled by varying the dwell time, in the current effort, 
we have determined that changing the Cu concentration provides better control over this 
parameter. This is largely due to the fact that Cu binding to the cotton substrate occurs 
rapidly, with the majority of binding occurring in the first 5 minutes, and complete binding 
occurring at 30 minutes (refer to Figure 2; left plot). 

• Copper coating: The final step in the process is reducing the Cu -carboxymethylcellulose 
complex to a composite thin film of copper/copper oxide on the surface of the cotton fibers. 
The reducing agent selected for the current effort is sodium borohydride (NaBH 4 ), which we 
have used in previous efforts and is a common reagent used in industrial textile coating 
processes. We have established that complete reduction of the Cu -carboxymethylcellulose 
intermediate occurs within 10 minutes to yield a Cu° metal coating, which rapidly oxidizes 
upon drying in to yield a green composite coating of Cu °/Cu 20 . 


6 










Copper loading as a function of [Cu] and 
dwell time 



5 Min 30 min 60 min 

Dwell Time 


Loadings on current Cu-coated 



1 mM 5 mM 50 mM 

Concentration of CuS04 


Figure 2. (left) Cu loading data for early efforts to establish the effect of Cu 2+ concentration and dwell 
time on the Cu loading of Cu-coated dressings, (right) Cu loadings using established Cu coating process. 


iFyber has shared the Cu coating process with 
researchers at Cotton Inc. and we are confident 
that the parameters are amenable to production 
at an industrial scale without the need for 
specialty equipment or overly burdensome 
processing steps. With these baseline 
parameters set, iFyber has evaluated the 
antimicrobial coating process on the prototype 
crinkle-weave cotton gauze, and have confirmed 
that this substrate will support Cu loading. 

Figure 3 contains images of the cotton taken 
throughout the coating process starting from the 
unmodified native gauze. Note the faint yellow 
color in the anionic gauze, which is due to the 
carboxymethlycellulose treatment, and the 
characteristic faint green color of the Cu-coated 
gauze. 

Using the model cotton substrate (i.e., not 
gauze), iFyber also established the baseline 
antimicrobial performance for the Cu coating 
against three common wound pathogens, before 
and after sterilization (refer to Sections 3.1.4 
and 3.1.5). 

3.2.2 In vitro antimicrobial performance testing of model Cu-coated dressings 

Concurrent with the selection and chemical pre-treatment of the prototype cotton gauze wound 
dressing, iFyber established the baseline performance metrics of the antimicrobial copper coating 



Figure 3. Photographs of the prototype cotton 
gauze dressing taken throughout the coating 
process. 


7 














































using a model Cu-coated cotton substrate and a number of standard antimicrobial assays. 
Antimicrobial efficacy was tested against lab strains of three common wound pathogens: 
Enterococcus faecalis (ATCC 700802 ), Staphylococcus aureus (ATCC 29213), 
and Pseudomonas aeruginosa PAOl (PAO-JP1). Antimicrobial efficacy was established 
relative to initial bacterial inoculum (e.g., varying colony forming units per milliliter; CFU/mL), 
exposure time (2-24 h), and Cu-loading (samples treated with 1 mM, 5 mM, and 50 mM Cu ; 
refer to Fig 2; right plot). Efficacy testing was done in the presence of both bacterial growth 
media (i.e., tryptic soy broth and agar; TSB and TSA) and in physiological buffer (i.e., phosphate 
buffered saline (PBS), pH 7.4) at 37°C. To date, little or no antimicrobial efficacy has been 
observed when growth medium is present in the assay (TSA or TSB), and although this result is 
noteworthy, antimicrobial agents typically fail under these conditions as these media are 
optimized for bacterial growth and do not represent typical conditions. Therefore, most studies 
reporting on the antimicrobial activity of a given substance are done in physiological buffer (with 
the exception of many antibiotics as these require active metabolism for efficacy). This can be 
done using a standard antimicrobial assay defined in ASTM method E2149-01 for determining 
antibacterial activity of immobilized agents under dynamic contact conditions, which is briefly 
described below. 

The baseline antimicrobial properties for the various Cu-coated dressings described in Section 
3.2.1 were compared using a method adapted from ASTM method E2149-01. Briefly, in a 24 
well culture plate, 7 mm cotton disks were placed in 1 mL of PBS and subsequently inoculated 
with either 10 or 10 CFU of the aforementioned bacteria. Samples were then incubated for set 
time periods, and the surviving bacteria in each sample well were enumerated using standard 
methods (serial dilution and plate counts). These data are summarized in Figure 4, which shows 
results of plate counts (represented as CFU/mL) obtained from experiments having the following 
variables: 

• Cotton disk: Cu-coated (experimental sample), Ag + -coated (positive control), non-coated 
(negative control). 

• Cu-loading: reported as disks loaded with 1 mM, 5 mM, and 50 mM Cu , which 
provides a differential Cu loading as reported in Fig 2. 

• Time: contact times of 2, 6, and 24 h. 

• Bacteria: E. faecalis, S. aureus, and P. aeruginosa 

• Inoculum: 10 6 or 10 8 CFU 


8 



Cell Viability (2 h) 


Cell Viability (6 h) 


1.00E+09 
1.00E+08 
1.00E+07 
1.00E+06 
E 1.00E+05 
2 1.00E+04 
° 1.00E+03 
1.00E+02 
1.00E+01 
1.00E+00 


1 




L 




li 



i 

T 1 




i 

1 



■ — 


■ 


i 

— 

— 


ImM 5mM 50 mM Ag+ CNTL PBS 
Cu Cu Cu CNTL Cotton CNTL 


1.00E+08 
1.00E+07 
1.00E+06 
■J 1.00E+05 
D 1.00E+04 
U 1.00E+03 
1.00E+02 
1.00E+01 
1.00E+00 



ImM 5 mM 50 mM Ag+ CNTL PBS 
Cu Cu Cu CNTL Cotton CNTL 


■ S. aureus ■ P aeruginosa ■ E. faecalis 


S. aureus ■ E. faecalis 


Cell Viability (24 h) 


1.00E+09 
1.00E+08 
1.00E+07 
1.00E+06 
E 1.00E+05 
£ 1.00E+04 
° 1.00E+03 
1.00E+02 
1.00E+01 
1.00E+00 








= 



T 


. 
































. 








:: 




I 


1 




ImM 5 mM 50 mM Ag+ CNTL PBS 
Cu Cu Cu CNTL Cotton CNTL 


■ S. aureus ■ P. aeruginosa ■ E. faecalis 


Figure 4. Comparative antimicrobial activity 
data showing the effects of various Cu and 
control dressings over time on the viability of 
planktonic bacteria in PBS media. In these plots, 
results are presented as viable colony forming 
units per mL (CFU/mL) determined after 
exposure times of 2, 6, and 24 h from standard 
plating and colony counts. 


From the results presented in Fig 4, the iFyber copper coating shows excellent antimicrobial 
activity against the three wound pathogens tested. In general, the trends in efficacy as a function 
of Cu loading and time make sense (i.e., more kill with higher Cu loading and longer exposure 
times) with the exception of P. aeruginosa. Specifically, results obtained for P. aeruginosa 
show that this species is very sensitive to copper at early time points (e.g., 2 h), but is able to 
recover sometime after 6 hours to reach levels close to controls after 24 hours of exposure. This 
results is remarkable given that the cell are in an environment that should cause metabolic 
quiescence; however, there is literature precedent for the ability of Gram negative microbes (e.g., 
E. coir, refer to J. Bacteriol. 1955; 69(4): 393-398) to grow in buffer and even distilled water, 
presumably by feeding off dead cells and cell debris. We will continue to monitor this effect 
over the course of this project. Another interesting result from this assay is effect of copper on 
bacterial cell viability compared to silver. Although we have not yet quantified the silver 
loadings in the control samples, Cu-coated dressings at all loadings exhibited a greater level of 
efficacy than the silver dressings for all bacteria tested. Our studies also indicate that there is a 
dose response for the Cu-coated dressings in all species, which suggests that our coating process 
can be tuned to provide a range antimicrobial efficacy. This last point may be important when 
we begin to establish the biocompatibility profile for the coating against mammalian cells - 


9 
















































































where we anticipate being able to tune the Cu coating to maximize antimicrobial activity while 
minimizing negative impacts on mammalian cells, in vitro. 

3.2.3 In vitro antimicrobial activity testing against multi-drug resistant clinical isolates 

The prototype Cu dressing was also tested against multidrug-resistant pathogenic microbial 
clinical isolates from patients, which were made available to this effort by our clinical 
collaborators at the State of New York Upstate Medical University. These isolates include 
vancomycin resistant E. faecium (VRE) from a penile wound, E. coli from biliary fluid, P. 
aeruginosa from an anterior tibia wound, Group B Streptococcus from penile discharge, and 
Methicillin-resistant S. aureus from an arm wound. For these studies, antimicrobial efficacy was 
established using a starting inoculum of ~10 6 CFU, the ASTM E2149-01 test method, and PBS 
as the medium. Figure 5 summarizes the results from these studies and indicate that the 
prototype Cu dressings is effective against all clinical isolates, with a 4-7 log reduction in 
viability bacteria for a 24 hr exposure time using two Cu dressing doses (reported as total Cu 
content in micrograms). These results establish that the Cu dressing is a broad spectrum 
antimicrobial, which is expected to help in limiting nosocomial infections on the battlefield and 
help to prevent microbial infections in wounded soldiers. These results have been shared with 
our partners at H&H associates who have expressed interest in this technology as a standard 
issue antimicrobial combat wound dressing. 


Antimicrobial efficacy against clinical isolates 


1.00E+07 


1.00E+06 


1.00E+05 


1.00E+04 

E 

3 


o 


1.00E+03 


1.00E+02 


1.00E+01- 


1.00E+00 


~5-7 log reduction 
in viable 

pathogens after 24 
hours 




1 ll 

I'; . 



Cu-CW Cu-CW Control Cotton 
3.37^yg/mL 10.1 /ig/mL 


Vancomycin resistent E. 
faecium (VRE) 


■E. coli 


P. aeruginosa 


-Group B Streptococcus 


Methicillin-resistent S. aureus 
(MRSA) 


10 CFU/mL = 
Detection limit 


Figure 5. Results from antimicrobial activity testing against multidrug-resistant 
clinical isolates after 24 h exposure to two doses of Cu dressing and control 
dressings in PBS media. 


10 


























3.2.4 Effects of sterilization on in vitro antimicrobial efficacy 

While many marketed antimicrobial dressings do not require sterilization, we aimed to study the 
effect of gamma irradiated samples with respect to antimicrobial efficacy. Antimicrobial testing 
was done against lab strains of three common wound pathogens: Enterococcus faecalis (ATCC 
700802), Staphylococcus aureus (ATCC 29213), and Pseudomonas aeruginosa PAO1 (PAO- 
JP1). Using the ASTM method E2149-01 dynamic contact assay previously described, the new 
batch of prototype Cu dressing showed significant antimicrobial activity against all three lab 
strains when the assay is conducted in PBS. In general, no adverse effects were observed after 
exposure to a gamma dose of 28-30 kGy, suggesting that the end product could be sterilized 
if desired. 

3.2.5 In vitro biocompatibility testing of the prototype Cu dressing 

Determining the cytotoxic status of a potential wound dressing is a key component to regulatory 
approval, and is governed by specific testing protocols outlined in the International Standards 
Organization (ISO) document 10993-5 Biological Evaluation of Medical Devices - Tests for 
Cytotoxicity. These standardized, FDA accepted assays include assessment of multiple aspects of 
mammalian cell function upon exposure to the test article. To remain in line with our proposed in 
vivo biocompatibility testing, MEM elution assays were done to determine the cytotoxic 
potential of the prototype Cu dressing. This approach is widely accepted for determining the 
cytotoxic potential of various materials that may come into contact with human tissue. 

Monolayer cultures of L929 fibroblasts were propagated under standard culture parameters 
(37°C, 5% C02) in MEM media containing 10% horse serum. At 80 - 90% confluency, 
fibroblasts were detached from the tissue culture dish using accutase treatment and plated into 96 
well tissue culture plates at a density of 10 cells/well. At the same time, dressings were extracted 
into cell culture media at 37°C for 24 h, after which serial dilutions of the dressing extracts were 
then exposed to the now semi-confluent monolayer cultures of L929 murine fibroblast cells. 
After a further 24 h incubation, cellular responses were quantified using the well- known MTT 
assay. 

The MTT assay is a recognized method for quantifying cell metabolic status as a function of 
mitochondrial reductase activity. Briefly, in the MTT assay, a tetrazolium compound is 
introduced to cells in culture, and reduced to insoluble formazan crystals by metabolically active 
cells. The crystals are then solubilized by addition of a solvent (e.g., isopropanol), producing a 
purple solution. Results were quantified spectrophotometrically at a wavelength of 570 nm. 
Following guidelines laid out in ISO-10993-12 Biological evaluation of medical devices, 100 mg 
quantities of the Cu dressing were extracted for these baseline studies. Specimens were extracted 
with both MEM media alone and MEM media containing serum (done in triplicate), and the 
resulting 24 h extracts were applied to L929 cells and assessed for viability via MTT. Results 
from these studies are reported in Figure 6, and indicate that the extracts at 100% strength are 
toxic to cells while subsequent dilution of the extracts show significantly lower levels of toxicity 


11 



relative to controls. It is important to note that a benchmark for success, as detailed in ISO 
10993-5, is maintenance of cell viability at or above 70% of control cells (i.e., vehicle only). 
Also important is the fact that at the 25% dilution of the extracts, which is similar in copper 
content (e.g., 9.9 ug total Cu) that was used in the antimicrobial efficacy studies reported in 
Sections 3.2.2 - 3.2.3, exhibits a very low level of toxicity. Taken together with the high 
antimicrobial activity of the dressing at this dose, these results are very promising and provide 
excellent baseline data for the prototype Cu dressing that were compared to the in vivo 
compatibility studies reported in Section 3.2.6. 

_ 100.0 

~o 

CD 

rS 80.0 

o> 

1 60.0 

M- 

o 

40.0 

1 20.0 

E 

= o.o 

= 100 % 

O 

“full 

Figure 6. Results of the MTT assays showing the effect of the cotton extract on the survival of L929 cells 
as determined through their metabolic activity (average of three independent experiments). 

3.2.6 Determination of the dermal sensitization potential of iFyber copper-coated 
dressing. 

To determine if iFyber copper coated dressings stimulate the immune system to produce an 
allergic reaction, the following in vivo sensitization will be conducted. The Guinea Pig 
Maximization Sensitization Test (GPMST) is an adjuvant sensitization test requiring intradermal 
injections of the test substance (extracted into appropriate polar and non-polar solvents) along 
with Freund’s Complete Adjuvant (FCA), which enhances the potential of weak sensitizers by 
non-specifically stimulating the immune system of the animals. A group of 17 Hartley guinea 
pigs (11 test subjects 

and 6 vehicle controls) Table 1- Injection protocol for Induction 1. 
will be used in the test 
arm. WuXi AppTec 
carries out positive 

control testing every 

three months, as per 
their standard operating 
procedures. If this cycle 
coincides with our 
testing schedule, an 


Preparation 

Volume 
Injected 
Per Site 

Syringe Contents 

Ratio 

(V/ 

V) 

Test Group 

Syringe 1 

0.1 mL 

FCA + 0.9% Sterile Saline 

1:1 

Syringe 2 

0.1 mL 

Test Extract 

NA 

Syringe 3 

0.1 mL 

FCA + 0.9% Sterile Saline (1 :1) 

+ Test extract 

1:1 


Negative Control Group 

Syringe 1 

0.1 mL 

FCA + 0.9% Sterile Saline 

1:1 

Syringe 2 

0.1 mL 

Control Vehicle 

NA 

Syringe 3 

0.1 mL 

FCA + 0.9% Sterile Saline (1:1) 

+ Control Vehicle 

1:1 



75% 50% 25% 

Extract strenght 


media extract ■serum-free media extract 


12 


































additional 17 guinea pigs will be used (11 positive control and 6 vehicle control). For positive 
control testing, the known sensitizer dinitrochlorobenzene (0.3%) is used, with ethanol as a 
vehicle. The positive control assessment will be carried out using the same procedure as used for 
the experimental test arm. 

Induction 1 (intradermal injection). On day 0 the shoulder region of the animals will be clipped 
of hair, a nd three pairs of intradermal injections will be made simultaneously on either side of 
the midline. This is commonly referred to as Induction 1. Two solvents will be used for 
extraction of Cu prototype dressings, with two rows of injections being placed on either side of 
the midline of the animal (one polar extraction, one non-polar extraction on each side). The 
injection details are summarized in Table 1. 

Induction 2 (topical application). On day 6, the injection site areas will be treated with the 
detergent, and known sensitizer, sodium lauryl sulfate (10% in mineral oil). On day 7, fresh Cu 
dressing extract will be applied to filter paper and taped over the injection site. Control animals 
will receive vehicle only. 


Induction 


Intradermal injection 
Day 0 

• FCA + saline 

Exposed . Extract in vehicle 
animals • Extract in vehicle + FCA 


Topical application 
Day 7 

• Extract applied to 
filter paper and secured 
over injection site 


Challenge 

Naive skin patch 
testing Day 21 

• Extract and vehicle 
applied to filter paper 
and secured over naive skin 



Control 

animals 


• Vehicle applied to 
filter paper and secured 
over injection site 


• Extract and vehicle 
applied to filter paper 
and secured over naive skin 


Challenge phase. On day 21 the challenge procedure will be initiated on test and control animals. 
Fur will be clipped from both the left and right flank/dorsum region to uncover naive skin. 
Freshly prepared Cu dressing 
extract will applied to filter paper 
and placed in contact with naive 
skin on the right flank, and vehicle 
controls will be applied in a similar 
fashion to the left fla nk . Both test 
and control animals will receive the 
same treatments. The patches will 
be held in place by an occlusive 
dressing for 24 hours, after which 
the challenge skin areas will be 
observed for signs of sensitization 
as indicated by erythema and 
edema. The grading scales are 
detailed in the table shown in 
Figure 7. 

Main Test Results Reported by 
WuXi Apptech 41923: None of 
the negative control animals 
challenged with the control vehicles 
were observed with a sensitization 

Figure 7. Experimental design of the Guinea Pig maximization 

response greater than 0. None of ... .. . . , . , , . . , . . ,, 

* ° sensitization test (above) and associated scoring table that will be 

the test animals challenged with the used to qualify results from this test. 




Patch Test Reaction 

Grading 

scale 

No visible change - No erythema or edema 

0 

Discrete or patchy erythema 

1 

Moderate and confluent erythema 

2 

Intense ery thema and/or edema 

3 


13 






test article extracts were observed with a sensitization response greater than 'O'. A negative 
sensitization incidence was interpreted for all test animals. See Table 2 for individual animal 
scores. 


Table 2. Daily Challenge Observations 


Normal Saline (NS) 


Animal # 

24 Hour Scores 

48 Hour Scores 

Results 

(*)or(-) 

Test Group 

Control 

Vehicle 

Test 

Extract 

Control 

Vehicle 

Test 

Extract 

1642 

0 

0 

0 

0 

- 

1644 

0 

0 

0 

0 


1645 

0 

0 

0 

0 

- 

1647 

0 

0 

0 

0 

- 

1648 

0 

0 

0 

0 

• 

1649 

0 

0 

0 

0 

- 

1650 

0 

0 

0 

0 

- 

1651 

0 

0 

0 

0 

- 

1652 

0 

0 

0 

0 

- 

1654 

0 

0 

0 

0 

- 

1655 

0 

0 

0 

0 

- 

Animal# 

Negative Control Group 

Results 

worn 

Control 

Vehicle 

Test 

Extract 

Control 

Vehicle 

Test 

Extract 

1635 

0 

0 

0 

0 

- 

1636 

0 

0 

0 

0 

- 

1637 

0 

0 

0 

0 

- 

55947 

0 

0 

0 

0 

- 

1640 

0 

0 

0 

0 

- 

1641 

0 

0 

0 

0 

- 


Sesame Oil (SO) 

Animal# 

24 Hour Scores 

48 Hour Scores 

Results 
(♦) or (-) 

Test Group 

Control 

Vehicle 

Test 

Extract 

Control 

Vehicle 

Test 

Extract 

1662 

0 

0 

0 

0 

- 

1663 

0 

0 

0 

0 

- 

1664 

0 

0 

0 

0 


1494 

0 

0 

0 

0 


1672 

0 

0 

0 

0 


1690 

0 

0 

0 

0 


1708 

0 

0 

0 

0 


1726 

0 

0 

0 

0 


1746 

0 

0 

0 

0 


1764 

0 

0 

0 

0 

- 

1782 

0 

0 

0 

0 1 

- 

H 

Negative Control Group 

Results 
(+) or (-) 

Control 

Vehicle 

Test 

Extract 

Control 

Vehicle 

Test 

Extract 

ll 1656 

0 

0 

0 

0 

- 


0 

0 

0 

0 

- 

1658 

0 

H o 

0 

0 

- 

1659 

0 

0 

0 

0 

- 

1660 

0 

o 

0 

0 

- 

1661 

0 

0 

0 

0 

- 


Positive Control: WuXi AppTec completes positive controls every 3 months. A positive control 
was completed 08/25/16 (see Table 3 for individual animal scores). The methods for the positive 
control assay are similar to the methods described above in the "Experimental Summary." 
Guinea pigs utilized for positive control studies are of the Hartley strain and are supplied by the 
same vendor as animals used for general testing (Charles River Laboratories). For the Induction I 
and Induction II phases, a known sensitizer, 0.3% dinitrochlorobenzene (DNCB) in ethanol, was 
used. For the challenge phase, 0.15% DNCB in acetone was used. The negative control animals 
were exposed to the appropriate vehicle (ethanol was used for the Inductions I and II and acetone 
was used for the challenge). 

At 24 hours and 48 hours after challenge patch removal, all animals in the test group were 
observed with discrete or patchy erythema (scores of T), moderate and confluent erythema 
(scores of'2') as well as intense erythema (scores of'3') at the challenge sites treated with 0.15% 
w/v DNCB in acetone. By contrast, none of the animals in the control group exhibited erythema 
at the challenge sites treated with 0.15% w/v DNCB in acetone (scores of '0') at either the 24- 


14 










hour or 48-hour scoring periods, with the exception of control animal #53808 which was 
observed with discrete or patchy erythema 
(score of T) at the 24 hour scoring period. 

However, this was not sustained at the 48 
hour scoring period, indicating that this was 
temporary irritation. Therefore, a 0% 
sensitization incidence was interpreted for 
the control group animals at the 48 hour 
scoring period. Per the evaluation criteria of 
the assay, the strength of the response in the 
test group compared to the negative control 
gro up indicates a sensitization response due 
to the repeated applications of the DNCB. 

Since grades of'3', '2' or T were observed in 
the test group animals at the 24 hour and 48 
hour scoring periods, these represented 
sensitization reactions (100% sensitization 
incidence). Therefore, based on the results 
obtained, this test methodology demonstrated 
dermal sensitization in guinea pigs using 
DNCB, a known sensitizer. 

Analysis and Conclusions: None of the negative control animals challenged with the control 
vehicles were observed with a sensitization response greater than 'O'. None of the animals 
challenged with the test article extracts were observed with a sensitization response greater than 
'O'. The normal saline extract of the test material had a sensitization response of 'O' under valid 
test conditions. The sesame oil extract of the test material had a sensitization response of 'O' 
under valid test conditions. Under the conditions of this protocol, the test article did not 
elicit a sensitization response. 

3.3 Project Status after Year 1 Performance Period 

The following table is used to help track the status of the project relative to the goals stated in 
Section 3.1 and the major technical objectives and tasks that are needed to achieve these goals. 
For each technical objective the overall status of the work is qualified, the major findings are 
listed for year of the project, and the estimated timelines for completing the remaining objectives 
are presented. At this point in the project, one technical objective remains; that is, an in vivo 
irritation study to further assess the in vivo biocompatibility of the copper dressing. A no-cost 
extension was granted in order to complete this study. 


Table 3. Test (ONCB) and Control Daily Challenge 
Observations Complete on 08/25/16 


24 Hour Scores 

48 Hour Scores 

Results 
(+) or (-) 

Test Group 

Animal # 

Control 

Vehicle 

DNCB 

Solution 

Control 

Vehicle 

DNCB 

Solution 

53809 

0 

3 

0 

2 

+ 

53810 

0 

2 

0 

2 

+ 

53811 

0 

2 

0 

1 

+ 

53812 

0 

2 

0 

1 

+ 

53813 

0 

3 

0 

2 

+ 

53814 

0 

3 

0 

2 

+ 

53815 

0 

2 

0 

1 

+ 

53816 

0 

2 

0 

2 

+ 

53817 

0 

3 

0 

2 

+ 

53818 

0 

1 

0 

1 

+ 

53819 

0 

2 

0 

2 

+ 

Control Group 

Results 
(♦) or (-) 

Animal # 

Control 

Vehicle 

DNCB 

Solution 

Control 

Vehicle 

DNCB 

Solution 

53803 

0 

0 

0 

0 

- 

53804 

0 

0 

0 

0 

- 

53805 

0 

0 

0 

0 

- 

53806 

0 

0 

0 

0 

- 

53807 

0 

0 

0 

0 

- 

53808 

0 

1 a 

0 

0 

- 


15 













































Technical Objective 1 - 

Produce prototype copper- 
coated dressings that 
balance antimicrobial 
efficacy and mammalian 
cell function, in vitro. 

Overall Status 

Major Findings (Ql) 

Timeline for 
Completion 

Tasks 1 and 2 




Subtask 1: Establish coating 
parameters that produce 
candidate dressings that 
offer maximal antimicrobial 
action without comp¬ 
romising mammalian cell 
viability. 

Complete 

Baseline coating parameters 
are set and will allow 
coating adjustments to be 
made to maximize 
antimicrobial performance 
and minimize effects to 
mammalian cells, in vitro 

• Established cotton 

pre-treatment 

process 

• Established 
important parameters 
for changing Cu 
loadings 

• Established Cu 
coating on prototype 
gauze dressing 

Complete 

Ql 

Subtask 2: Establish 

baseline antimicrobial 
performance metrics for 
copper-coated candidate 
dressings prepared in 

Subtask 1. 

Complete 

Baseline performance has 
been established for 3 
wound pathogens as well as 
for 5 mulidrug-resistant 
clinical isolates. 

• Cu coatings effective 
against 3 common 
wound pathogens 
and 5 multidrug- 
resistant clinical 

isolates 

• 4-7 log reduction in 
viable bacteria can 

be achieved within 2 
hr of exposure. 

Complete 

Q2 

Subtask 3: Establish 
baseline biocompatibility 
performance metrics for 
copper-coated candidate 
dressings prepared in 

Subtask 1. 

Complete 

Baseline cytotoxicity profile 
has been established for the 
prototype Cu dressing 

• The Cu coating 
shows some 
cytotoxicity potential 
at high loading, but 
is within FDA 
accepted criteria at 
the loadings used to 
establish 

antimicrobial 

activity. 

Complete 

Q3 

Sub task 4: Conduct 
antimicrobial assays on 
sterilized samples to 
determine if sterilization 

Pending 

(Q4) 


Complete 

Q4 


16 




alters dressing 
performance. 




Technical Objective 2 - 

Establish the 

biocompatibility profile of 
an optimized prototype 
copper-coated dressing, in 
vivo. 




Tasks 3 and 4 




Subtask 1: Obtain IACUC 

and USAMRMC ACURO 
approvals for animal studies 
to be performed in Subtasks 
2-3 below. 

Partially complete 

(sensitization) 

IACUC and ACURO 

document have been 
submitted for irritation study 


Expected 

Completion 

(irritation) 

07/16 

Subtask 2: Assess the 
dermal irritation potential 
of the prototype copper- 
coated dressing. 

Pending 

Year 2 


Expected 

Completion 

11/16-12/16 

Subtask 3 Assess the 
allergic potential of the 
sterilized copper-coated 
prototype dressing. 

Complete 

Cu dressings did not elicit a 
sensitization effect 


Complete 

Q4 


4.0 Impact 

Nothing to Report 


5.0 Changes/Problems 

Nothing to Report 


6.0 Products 

Nothing to Report 


17 




7.0 Participants and Other Collaborating Organizations 


Name: 

Project Role: 

Nearest person month worked: 
Contribution to Project: 


Aaron D. Strickland 
Principal Investigator / Project Manager 
1 

Dr. Strickland has been responsible for all experimental 
designs and has participated in data analysis and 
interpretation of results. Dr. Strickland has organized 
communication with partners and H&H Associates and 
Cotton Inc. 


Name: 

Project Role: 

Nearest person month worked: 
Contribution to Project: 


Name: 

Project Role: 

Nearest person month worked: 
Contribution to Project: 


Name: 

Project Role: 

Nearest person month worked: 
Contribution to Project: 


Name: 

Project Role: 

Nearest person month worked: 
Contribution to Project: 


Name: 

Project Role: 

Nearest person month worked: 
Contribution to Project: 


Deborah Diehl 

Research Scientist (Biology) 

1 

Mrs. Diehl has been responsible for conducting the bulk of 
the antimicrobial assays and has also participated in the 
production and chemical analysis of the model Cu wound 
dressings. 

Alison Schug 
Technician 
1 

Ms. Schug has performed work on the production and 
assessment of metal loading content of the model Cu 
wound dressings. 

Nina Bionda 

Sr. Research Scientist (Biology) 

1 

Dr. Bionda collaborated with Dr. Strickland to manage the 
day to day laboratory activities and design of experiments. 
Dr. Bionda has also led the in vitro biocompatibility efforts. 

Matt Farrell, PhD 

Manager, Textile Chemistry Research at Cotton, Inc. 

1 

Dr. Farrell works for Cotton Inc, and collaborated with Dr. 
Strickland to produce anionic cotton dressing material for 
the attachment of iFyber Cu coating on cotton. 

Joseph DeCorta 

Vice President at H&H Medical, Inc. 

1 

Mr. DaCorta is a medical device expert and has help to 
guide iFyber efforts towards a relevant cotton-based 
standard issue combat dressing current offered by H&H. 


18 



8.0 SECTION 5 - Special Reporting Requirements 

Quad chart attached 


19 




"0 


> 


QD 

O 


3 


D 

C/) 

#—K 

o' 

7T 

0) 

3 

Q_ 


"0 

=T 

D 


o 

co 


*< 

C7 

0 


O 


? 


0) 

Q. 


> 

3 


o 

c 

3 


-G9 


4^ 

00 

00 

o 



20 


A Nanolayer Copper Coating for Prevention of Nosocomial Multi-drug Resistant Infections 
ERMS/Log Number 12117004 
Award Number: W81XWH-15-2-0066