Sustained Release of Functional Antibiotics from a Keratin Hydrogel
Roy
1,2
D ,
Burmeister
2
D,
Saul
3
J,
Meng
3
H,
Ellenburg
1
M,
Burnett
1
L,
Tomblyn
1
S,
Christy
2
R
1KeraNetics,
LLC, Winston-Salem, NC 27101
2United States Army Institute of Surgical Research, Fort Sam Houston, TX 78234
3Miami University, Oxford, OH 45056
Methods
Results
(cont’d)
Abstract
Wound infection resulting from battlefield injury can
lead to impaired healing, increased hospitalization
time, limb amputation, and psychological disorders.
Current approaches to prevent burn infection include
wound debridement and multiple applications of silvercontaining antibiotic dressings, which have been
associated with delayed healing [1]. Therefore, the
need exists for an aggressive, point-of-care antibiotic
delivery system that prevents infection and infection
recurrence while aiding in the regeneration process.
Keratins are promising biomaterials for tissue
engineering applications due to their ability to support
cell attachment, proliferation, and migration [2,3]. Here
we show that hydrogels composed of 15% (w/v)
keratin supported the sustained release of several
antibiotics including Ciprofloxacin, Cefazolin,
Streptomycin and Neomycin. Specifically,
Ciprofloxacin-loaded keratin hydrogels completely
prevented both Gram-positive and Gram-negative
bacteria growth for at least 2 weeks in vitro. The ability
of Ciprofloxacin-loaded keratin hydrogels to restore
healing of infected, full-thickness skin wounds is being
investigated in a porcine model. These studies suggest
antibiotic-loaded keratin hydrogels have the potential
to prevent infection resulting from battlefield injury.
.
Group
A
B
C
D
E
Treatment Groups for Excision Study
Tissue Wounds
Test Material
Harvest per Pig
Day 3
3
Infected
Day 7
3
Keratin Gel + 20 mg/ml Ciprofloxacin
Day 11
3
Day 3
3
Infected
Day 7
3
Keratin Gel + 10 mg/ml Ciprofloxacin
Day 11
3
Day 3
3
Infected
Day 7
3
Keratin Gel + 5 mg/ml Ciprofloxacin
Day 11
3
Day 3
3
Infected
Day 7
3
Saline
Day 11
3
Day 3
3
Uninfected
Day 7
3
Saline
Day 11
3
Conclusions
(A)
(B)
Figure 1. Keratin hydrogels support sustained
release of several antibiotics. 100 l aliquots of the
keratin hydrogels containing either Ciprofloxacin (2 mg/ml),
Cefazolin (20 mg/ml), Streptomycin (20 mg/ml) or Neomycin
(20 mg/ml) were placed in microcentrifuge tubes and overlaid
with 100 l DPBS. DPBS was exchanged and analyzed at
regular intervals. Antibiotics were quantified by fluorescence
(Ciprofloxacin), light absorption (Cefazolin), size exclusion
chromatography followed by UV absorption (Streptomycin),
and ELISA (Neomycin, Europromixa, Netherlands). Data are
presented as mean values +/- SEM of 3 separate gels.
(A)
Figure 5. Ciprofloxacin-loaded keratin hydrogels
do not disrupt granulation tissue deposition. Full-
Figure 3. Keratin hydrogels loaded with increasing
Ciprofloxacin concentrations display increased
antibiotic release and decreased gel degradation.
100 l aliquots of the keratin hydrogels containing either 20,
10, 5, 2, or 0 mg/ml Ciprofloxacin were placed in
microcentrifuge tubes and overlaid with 100 l DPBS. DPBS
was exchanged and analyzed at regular intervals. (A)
Ciprofloxacin was quantified by fluorescence. (B) Total protein
was quantified using a BCA assay. Data are presented as
mean values +/- SEM of 3 separate gels. *, significant from all
other treatments, p<0.05 (ANOVA).
Methods
Keratin Gel Preparation – Keratin was obtained by
oxidative extraction of human hair and purified by
KeraNetics using a patented process in a 21CFR820
validated facility. The lyophilized extract was weighed
using a 95:5 a:g keratin ratio and hydrated with an
aqueous solution containing antibiotics to achieve a
15% weight-to-volume ratio. Hydrogels formed
overnight at 37 C.
Porcine Excision Model – Full-thickness excision
wounds were made on the dorsum of a pig using a 10
mm biopsy punch. Wounds were either infected with
2.5 x 104 colony-forming units (cfu) of Pseudomonas
aeruginosa or uninfected (saline-treated) and allowed
to heal. On days 1 and 3 post-surgery, infected
wounds were treated with 200 l of antibiotic-loaded
keratin hydrogels or saline. Biopsies were collected on
days 3, 7, and 11 post-surgery for bacteria
quantification and histological analysis.
Results
Results
(B)
(A)
thickness wounds were infected with P. aeruginosa and
treated with Ciprofloxacin (Cipro)-loaded keratin hydrogels as
described in ‘Methods’. Wound tissue was biopsied on day 11
post-surgery, formalin-fixed, embedded in paraffin and
sectioned. Sections through the center of the wound were
stained using Masson’s Trichrome. Images represent 1 of 3
separate wounds. Arrows indicate wound edge. Bar = 1 mm.
Conclusions
• Keratin hydrogels support the sustained release of
several antibiotics.
• Ciprofloxacin-loaded keratin hydrogels inhibit Grampositive and Gram-negative bacteria growth for 2
weeks in vitro.
• Ciprofloxacin-loaded keratin hydrogels decrease
bacteria growth in a porcine excision wound model
without interfering with healing.
• Antibiotic-loaded keratin hydrogels make
promising candidates to prevent infection
resulting from battlefield injury.
Acknowledgements
(B)
The authors would like to thank Sandra Becerra, Chris Bell, Sean
Christy, and Nicole Wrice for their excellent technical assistance and Dr.
Shanmugasundaram Natesan for helpful scientific discussion. This work
was possible due to generous funding from the US Army, contracts #
W81XWH-12-C-0004 and # W81XWH-10-C-0165.
References
Figure 2. Antibiotic-loaded keratin hydrogels
inhibit bacteria growth. (A) Mueller-Hinton broth was
inoculated with a single colony of P. aeruginosa and diluted to
a concentration of 105 cfu/ml. The 105 cfu/ml broth was added
to 1 ml of keratin hydrogels with or without the antibiotics,
sampled daily, and serially diluted to determine the number of
cfu/ml present in the broth. Data are presented as mean +/SEM of 3 separate gels. *, significant from ‘Keratin Only’,
p<0.05 (ANOVA). (B) Similar studies to that described in (A)
were carried out with Staphylococcus aureus and Fascioloides
magna. After day 7, the bacterial culture was continued, and
samples were diluted to 104 cfu/ml. Data represents the day
in which colonies were observed at this dilution, indicating the
failure of the antibiotic to control the bacteria growth. Data are
presented as mean values +/- SEM of 3 separate gels.
Figure 4. Ciprofloxacin-loaded keratin hydrogels
decrease bacteria growth in vivo. Full-thickness
wounds were infected with P. aeruginosa and treated with
Ciprofloxacin (Cipro)-loaded keratin hydrogels as described in
‘Methods’. Wound tissue was biopsied on days 7 and 11,
homogenized, serially diluted and plated onto either (A)
Mueller-Hinton agar or (B) Pseudomonas-selective agar to
determine viable cfu. Data are presented as mean values +/SEM of 3 separate wounds. *, significant from ‘Saline’, p<0.05
(ANOVA).
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2.Verma V et al. Preparation of scaffolds from human hair proteins for
tissue-engineering applications. Biomed Mater. 2008; 3(2).
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Animal Statement
This study has been conducted in compliance with the Animal Welfare
Act, the implementing Animal Welfare Regulations, and the principles of
the Guide for the Care and Use of Laboratory Animals.
Department of Defense Disclaimer
The opinions or assertions contained herein are the private views of the
author and are not to be construed as official or as reflecting the views of
the Department of the Army or the Department of Defense.