International Journal of Pharmaceutics 494 (2015) 146151

Contents lists available at ScienceDirect

International Journal of Pharmaceutics

journal homepage: www.elsevier.com/locate/ijpharm

Preservation of imaging capability in sensitive ultrasound contrast

agents after indirect plasma sterilization
Lorenzo Albalaa , Utku K. Ercanb , Suresh G. Joshic , John R. Eisenbreyd ,
Nutte Teraphongphoma , Margaret A. Wheatleya,*
a

School of Biomedical Engineering, Science and Health Systems, Drexel University, Philadelphia, PA 19104, USA
Department of Biomedical Engineering, Izmir Katip Celebi University, Izmir, Turkey
Center for Surgical Infection and Biolm, Department of Microbiology & Immunology, College of Medicine, Drexel University, Philadelphia, PA 19102, USA
d
Department of Radiology, Thomas Jefferson University, Philadelphia, PA 19107, USA
b
c

A R T I C L E I N F O

A B S T R A C T

Article history:
Received 8 February 2015
Received in revised form 24 May 2015
Accepted 26 July 2015
Available online 1 August 2015

Many injectables are not amenable to standard sterilization methods, which destroy sensitive materials.
This is particularly true for ultrasound contrast agents (UCA) consisting of gas bubbles stabilized by a
surfactant or polymer shell. We investigated a new method to achieve safe and effective sterilization in
production by introducing dielectric-barrier discharge non-thermal plasma. A dielectric-barrier
discharge was generated to rst produce plasma-treated phosphate-buffered saline (PTPBS), which
was used as a sterilant solution for our UCA SE61, avoiding direct heat, pressure, chemicals, or radiation.
Treated samples were tested for acoustic properties in vitro and in a ow phantom, and for sterility by
standard methods. Three minutes plasma treatment of phosphate-buffered saline (PBS) proved effective.
The samples showed signicant inactivation of inoculated bacteria upon PTPBS treatment as compared to
un-treated-PBS (p = 0.0022). The treated and untreated samples showed no statistical signicance
(p > 0.05) in acoustic response or bubble diameter (mean
SEM: 2.52
0.31 mm). Nile Red was used to
model intercalation of drug in the hydrophobic shell, intercalated successfully into SE61, and was
unaffected by plasma treatment. The PTPBS completely sterilized suspensions of UCA, and it did not
compromise the acoustic properties of the agent or its ability to retain a hydrophobic compound.
2015 Elsevier B.V. All rights reserved.

Keywords:
Ultrasound contrast agents
Nonthermal plasma
Sterilization
Microbubble
Surfactant

1. Introduction
The majority of currently FDA-approved sterilization techniques have drawbacks when considering fragile entities such as
ultrasound contrast microbubbles. These include negative alteration of material properties, such as molecular weight, volume,
and morphology, and destruction of imaging capabilities. Some
classical methods require high temperatures or structure-altering
irradiation, while others require vacuum chambers or toxic and
highly reactive gases such as formaldehyde or ethylene oxide
(Agalloco et al., 2004).
The FDA identies only two available technologies for
temperature and moisture-sensitive medical devices: ethylene
oxide (ETO) gas sterilization and hydrogen peroxide gas plasma
sterilization (Rutala and Weber, 2008). However, ETO is absorbed
by many materials, is quite toxic, and requires several hours to take
effect, making its application unsuitable for the UCA. The latter is

* Corresponding author. Fax: +1 215 895 4983.

http://dx.doi.org/10.1016/j.ijpharm.2015.07.064
0378-5173/ 2015 Elsevier B.V. All rights reserved.

highly oxidizing and requires a deep vacuum to function, which

would introduce destructive pressure changes into the
manufacturing process (Rutala and Weber, 2008). One possible
non-destructive technique is the direct use of cold plasma
(Fridman et al., 2007). Plasma is dened as the fourth state of
the matter and composed of ionized gas, reactive oxygen and
nitrogen species, electrically excited species and free electrons
(Fridman et al., 2008). Utilization of cold plasma in biomedicine
has taken attention due to its novel and effective applications such
as sterilization, wound healing, dental applications, plasmaeukaryotic cell interactions and built a new eld called as plasma
medicine (Fridman et al., 2008; Dobrynin et al., 2009). Plasma
sterilization has become one of the most widely studied biological
applications of non-thermal plasma since last decade since its
capabilities for disinfecting delicate materials and excellent
antimicrobial efcacy even on multi-drug resistant bacterial
strains. Direct application of cold plasma has been used against
gram positive and gram-negative bacteria, biolm-producing
bacteria, virus, fungi and spores, and even prions for sterilizing
surfaces and for would healing (Heinlin et al., 2011; Elmoualij et al.,

L. Albala et al. / International Journal of Pharmaceutics 494 (2015) 146151

2012). In addition to direct plasma disinfection effect where the

plasma or plasma after glow comes in direct contact with
microorganisms, also antimicrobial effects through plasma treated
materials is widely reported. Plasma treatment of water and waterbased solutions, including PBS and saline solution, demonstrated
that liquid acquires antimicrobial properties and can be used for
sterilization of delicate materials as discussed present study
(Oehmigen et al., 2011; Ercan et al., 2013).
The other alternative is the use of pre-sterilized components
and aseptic technique in cleanroom manufacturing, currently the
standard for sterile injectables (US Food and Drug Administration,
2004; Woodcock and Wosinska, 2013). The advantage of cleanroom use is that there is no additional sterilization step after the
bubbles are made, minimizing possible losses in acoustic properties. The disadvantage of cleanroom use is that it is cumbersome,
and requires compliance with current good manufacturing
practice (cGMP) and standards such as ISO 14644 and 14698 for
airborne particulate cleanliness and maintenance. Also, clean
facilities not only require a large initial investment for installation,
but they can be expensive in upkeep, personnel, training,
regulation, and compliance costs. These costs can be proscriptive
to smaller institutions and those in academic settings. Here we
show that indirect, non-thermal plasma is an attractive option.
This study deals with sterilization of ultrasound contrast agents
(UCA) with a delicate surfactant-stabilized shell structure, SE61.
The UCA are shell-stabilized microbubbles less than 6 mm in
diameter that enhance an ultrasound image by strongly reecting
the impinging ultrasound beam back to the collecting transducer.
Composed of a sonicated mixture Span 60 and water-soluble
vitamin E entrapping perurocarbon gas, SE61 microbubbles can
successfully enhance contrast in diagnostic and therapeutic
ultrasound (US) imaging by upwards of 20 dB (Methachan,
2012). Unfortunately, its fragile shell ensures that it cannot
tolerate the conditions of conventional sterilization methods
(autoclave, ionizing radiation, gas sterilization). A sterilization step
with the least physical or chemical stress on the microbubble UCA
is required.
Non-thermal plasma used in this study can be created costeffectively and in various settings: it is generated safely at room
temperature, using air at atmospheric pressure, via dielectric
barrier discharge (Cooper et al., 2010). More importantly, the
antimicrobial properties of the plasma have been shown to transfer
over to a liquid, such as phosphate buffered saline (PBS), which can
retain these properties over a period of months to years, and
function as a sterilant solution to inactivate a wide range of multidrug resistant bacteria and fungal pathogens in planktonic and
biolm forms within 15 min of contact (Ercan et al., 2013).
The processes within the plasma that are responsible for such
decontamination efciency, and that can be applied to a liquid, are
in part acidication and introduction of increased concentrations
of NO2, NO3, and hydrogen peroxide (Oehmigen et al., 2010).
There has been a recent interest in UCA for ultrasoundtriggered drug delivery from both surfactant and polymerstabilized agents (Eisenbrey et al., 2010). Nile Red (9-diethylamino-5H-benzo[alpha]phenoxazine-5-one) is used in this study as
a model hydrophobic drug to test the UCAs ability to retain drug
after sterilization because its uorescence is strongly dependent
on the polarity of its environment: in aqueous media, it is relatively
insoluble and the uorescence is strongly quenched.
In this paper we describe a study to evaluate the effects of using
plasma-treated PBS (PTPBS) to effectively sterilize the surfactantstabilized microbubble contrast agent SE61, without compromising its acoustic properties, size or drug delivery capacity. We
suggest this may be an excellent method for sterilization of many
delicate biologics and contrast agents.

147

2. Materials and methods

2.1. Microbubble contrast agent fabrication
UCA were manufactured via procedures published by our lab,
outlined in United States patents (Goldberg et al., 1994) and
literature (Wheatley and Singhal, 1995; Solis et al., 2010) with
slight modications. In summary, calculated quantities of nonionic
surfactants TPGS and Span 60 purchased from Eastman (Kingsport,
TN) and SigmaAldrich (St. Louis, MO), respectively, were added to
50 mL of PBS and heated to boiling. To test the intercalation of a
hydrophobic material, 0.5 mg of Nile Red (SigmaAldrich, St. Louis,
MO) were added to the solution which was heated once more to a
boil, then allowed to cool with stirring.
The surfactant mixtures were autoclaved for 30 min in order to
further decrease the particle size of Span 60, followed by a cooling
phase with continuous magnetic bar stirring. The cooled mixture
was placed in an ice bath and continuously sonicated for 3 min at
110 W using a 0.5-inch probe horn (Misonix Inc. CL4 tapped horn
probe with 0.500 tip, Farmingdale, NY). The solution was purged
with a steady stream of octauoropropane (American Gas Group,
Toledo, OH) before and during the sonication. Microbubbles were
extracted from the solution via gravity separation in a 250 mL glass
separation funnel and washed 3 times with cold (4  C) PBS every
120 min using the same separation funnel. While in the funnel, the
solution forms layers, of which the bottom is discarded. The
intermediate layer formed after the 3rd wash was collected as the
microbubble fraction. One-mL aliquots of native bubble suspension were pipetted with a pipet specically designed for viscous
uids (Gilson Pipetman, Middleton, WI) into 15 mL lyophilization
vials obtained from West Pharmaceutical Services (Lionville, PA).
Upon preparing the lyophilization vials with native UCA suspension, 0.5 mL of 400 mM glucose was added to each vial to act as
cryoprotectant (Solis et al., 2010).
2.2. Non-thermal oating electrode dielectric-barrier discharge (FEDBD) plasma
The DBD-generating setup (fabricated at Drexel University)
used in this study is reported earlier (Fridman et al., 2006). The
plasma generated in this manner is cold to the touch, and is
generated under normal atmospheric conditions and room air (no
separate gases used or added). The method for plasma treatment of
the PBS used in this study was as described by Joshi et al. (2010)
with some modications (Ercan et al., 2013). In essence, PBS (1 mL)
was pipetted into the custom-built glass liquid container of the
DBD, and the upper electrode (dielectric barrier) was lowered into
position (2 mm above uid surface) and plasma was continuously
generated for 1 min, 2 min, or 3 min. The pulse waveform had 5 ms
pulse duration at 31.4 kV and a frequency of 1.5 kHz where
0.29 RED/cm2 power density was generated.
After plasma treatment, 0.5 mL of the resulting PTPBS was
added to each lyophilization vial to be sterilized using aseptic
technique, and the vial stoppered in preparation for freezedrying.
A 0.5 mL aliquot of sterile, un-treated PBS was added to the
remaining vials to form the control group.
2.3. Bacterial inoculation
Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC
25923) strains purchased from American Type Culture Collection
(ATCC, Manassas, VA) were revived, maintained, and used as
overnight cultures in trypticase soy broth (TSB) for primary
inoculations according to the suppliers guidelines. The known
biocide agent was 70% ethyl alcohol. In the experiments that
featured addition of bacterial load to the samples, a given pathogen

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L. Albala et al. / International Journal of Pharmaceutics 494 (2015) 146151

(10 mL inoculate) was incubated overnight in Trypticase Soy Broth

(Soybean-Casean Digest) medium (10 mL), and incubated at 37  C
for 4 h on an orbital shaker incubator. The optical density at 600 nm
(OD600) was adjusted to 0.2 before use, and the colony forming
units determined each time by colony count assay (Ercan et al.,
2013; Joshi et al., 2010). A 100 mL suspension of 104 CFU/mL was
added to the lyophilization vials (already containing 1 mL of UCA
and 0.5 mL of glucose solution), and nally, 0.5 mL of PTPBS was
added to the vials. Bacterial exposure to active PTPBS lasted until
freezedrying occurred (several hours), and plating was done 24 h
later (once the sample was lyophilized).
2.4. Freezedrying and reconstituting UCA
The 2 mL samples in lyophilization vials were freezedried as
per procedures published by our lab (Solis et al., 2010). In brief,
Fluortec lyophilization stoppers were inserted into vials to the
rst groove (leaving a gap for air to escape). The samples were
ash-frozen in liquid nitrogen for 5 min and subsequently freeze
dried for 24 h on a Virtis Benchtop freezedrier (Gardiner, NY),
tted with a two-shelf sealing/stoppering assembly kept at 80  C.
The conditions during this process were 76.5  C (in the vacuum
drier chamber) and 1720 mBar. Before removing the samples, a
piston was lowered that depressed the upper shelf, thereby closing
the stoppers and sealing a vacuum inside the vials.
The vial of the freezedried (FD) mixture was further sealed by
wrapping the stopper with paralm tape. The vials are lled with
sterile octauoropropane gas under an aseptic laminar ow hood
by passage through a sterile 0.22 mm lter (Nalgene, Rochester, NY)
using a sterile syringe needle to puncture the septum of the vial.
2.5. Characterization of UCA
Samples were reconstituted with sterile de-ionized (DI) water
(2 mL), so that 1:1 dilution was maintained relative to native UCA,
and they were tested for microbial load and acoustic properties.
The average diameter of the microbubbles collected was analyzed
using a Zetasizer nano ZS (Malvern Instruments, Worcestershire,
UK) in Z-average mode, using a dynamic light scattering technique.
Dynamic light scattering is the scattering of laser light caused by
the Brownian motion of particles in suspension; analysis of
intensity uctuations yields Brownian motion velocity and thus
particle size using the StokesEinstein relationship. Testing was
repeated 3 times.
2.6. Acoustic testing of UCA in vitro
A custom-built acrylic plastic vessel with a clear acoustic
window (1.5  1.5 in) was placed in a tank lled with 75 L of
distilled (DI) water (temperature-controlled to 37  C) for in vitro
acoustic testing of the UCA. Within the vessel, a stir-bar
continuously stirred 50 mL of PBS at 37  C, directly in the line of
sight of a single Panametric (Waltham, MA) 5 MHz transducer
(12.7 mm diameter, 6 dB bandwidth of 91% and focal length of
50.8 mm) (Eisenbrey et al., 2008b). The transducer was focused
through the acoustic window using an xy positioning system
(Edmund Scientic, Barrington, NJ) and a 5072 pulser-receiver
(Waltham, MA) was used to generate acoustic pressures with a
pulse repetition frequency of 100 Hz. Reected signal from the UCA
was detected by the same transducer and amplied 40 dB before
being read by a digital oscilloscope (LeCroy 9350A, LeCroy
Corporation, Chestnut Ridge, NY). Data acquisition and processing
was done on a computer with LabView 7.1 (National Instruments,
Austin, TX).
To obtain a dose response curve, samples were pipetted into the
sample holder at 60 s intervals with increments of 2 mL for the rst

two, and 5 mL for each subsequent dose. Cumulative dose response

was constructed to obtain maximum enhancement that could be
obtained from the UCA sample. A cumulative approach was
considered acceptable because readings required 1 min (i.e., 1 min
intervals between dose application), wherein only a small
proportion of the existing microbubbles was destroyed (half-life
was seen to be much greater than 1 min).
To construct a time response curve (the variation of enhancement with time in the presence of an ultrasound beam), UCA doses
were chosen on the rise of the respective dose-enhancement
curves generated for each sample, usually taking 2434 mL. The
values obtained were normalized with respect to initial value to
compare sample half-life value. Testing was repeated 3 times.
2.7. In vitro imaging
Response of the agent to a clinical US scanner was measured
using a Logiq 9 ultrasound scanner with 9 L probe operating in
nonlinear contrast imaging mode (GE Healthcare, Milwaukee, WI).
A ow phantom (model 524; ATS Laboratories, Bridgeport, CT)
with a 6-mm diameter vessel embedded at a depth of 2 cm in
urethane rubber was charged with diluted agent using a roller
pump set at 250 mL/min. and drawing from a reservoir of 800 mL
PBS into which was added 800 mL of agent at t = 0. Ultrasound
images were then manually stored approximately every 10 s for
60 s.
2.8. Agar plating and microbial evaluation
Following the freezedrying process, the vials were reconstituted with 2 mL sterile DI water, and 100 mL out of that volume
(initial dilution factor of 20) was extracted to create 1:1, 1:10, and
1:100 dilutions with sterile DI water addition. Subsequently
100 mL were spread on Trypticase Soy Agar plates and set to
incubate at 37  C for 24 h. Plating was done in triplicate. The plates
that did not show any visible growth or colony formation were
returned to the incubator for up to 92 h to rule out dormancy. After
this incubation period, colony-forming units were counted to
quantify bacterial load of each sample. Bacterial evaluation was
repeated 4 times.
2.9. Light microscopy
Light microscopy (Olympus IX71 uorescence microscope) was
used to assess Nile Red uorescence to visualize intercalation into
the shell using excitation/emission, 552/636 nm. The images were
obtained using SPOT Advanced software.
2.10. Statistical analysis
All data were expressed as mean
standard error about the
mean, and were analyzed using the KolmogorovSmirnov for
acoustic response and bubble sizing, and using KruskalWallis for
the analysis of microbial contamination.
3. Results
Direct application of non-thermal plasma to the UCA, which
does not have many of the drawbacks of classical sterilization
techniques, has already been successfully tested in our lab for
sterilization of poly-lactic acid UCA (Eisenbrey et al., 2009).
However this direct approach was not successful with the more
fragile SE61 shell composition (data not shown) leading us to
investigate an indirect method.

L. Albala et al. / International Journal of Pharmaceutics 494 (2015) 146151

149

3.1. Preliminary testing to establish treatment time

Fig. 1 compares the cumulative dose response of freshly
prepared SE61 (prior to freezedrying) that had been mixed with
PBS treated for 1, 2, or 3 min durations with an untreated PBS
control in order to establish a treatment time that did not produce
PTPBS that damaged the surfactant components of SE61.
The maximum echogenicity of over 20 dB was achieved for all
treatments at identical doses to those for the control. Since the
echogenicity of the 3-min sample was excellent, and the longer
treatment time probably produced PTPBS with a higher ability to
sterilize, the 3-min treatment time was taken as the standard and
used throughout.
3.2. Acoustic properties using 3-min PTPBS
The dose response curves for UCA SE61 that had undergone the
full manufacturing process including freezedrying and resuspension are shown in Fig. 2. Samples include SE61 without
indirect plasma effects and those with PTPBS (Plasma treated).
There is no signicant difference between the two curves
(p = 0.5696), with the trend of the control to be at a slightly lower
enhancement per dose.
The time-response curve, (ultrasound enhancement of a single
dose of microbubbles under constant insonation) allowed for an
estimation of the in vitro acoustic half-life of the UCA. As evident in
Fig. 3, the time response for the control and treated UCA are not
signicantly different (p = 0.9251). This indicated that PTPBS
treatment did not destabilize the UCA bubbles. Furthermore, the
half-life is longer than 15 min, more than acceptable for in vivo use.
3.3. Flow phantom images
In vitro ultrasound enhancement of PTPBS sterilized SE61 was
conrmed using a clinical ultrasound scanner in a ow phantom
(example images shown in Fig. 4). Example images are provided
showing B-mode ultrasound (left half of images in gray) and
nonlinear contrast mode (right half of images in gold) at baseline
(A), 20 s post contrast injection (B), and 60 s post injection (C).
Ultrasound contrast enhancement is clearly visible within the
lumen post injection (demonstrated by strong nonlinear signals in
gold in the contrast mode in panel B). Full lling of the vessel
lumen is apparent, indicating a sufcient microbubble population
to provide adequate enhancement under circulatory ow conditions. No decrease in signal enhancement was observed 60 s post
injection (panel C), reinforcing the stability of PTPBS sterilized
SE61 observed in time response experiments.

Fig. 2. Effect of plasma treatment on enhancement of ultrasound signal for SE61.

(*) Control, no treatment (&) PTPBS treated. N = 3, error bars = SEM, p > 0.05.

Fig. 3. Time response for SE61 with and without indirect plasma sterilization.
Insonation by 5 MHz transducer. (*) Control, no treatment (&) PTPBS treated. N = 3,
error bars = SEM, p > 0.05.

3.4. Size
UCA microbubble sizing was determined using a Malvern Nano
Zetasizer. Untreated mean
SEM: 2.11
0.59 mm, PTPBS-treated
mean
SEM: 2.52
0.31 mm (N = 4, p = 0.7714 exact). Poly-dispersity index (PDI), a dimensionless value calculated from the data
that indicates the breadth of the size distribution, was 0.85
0.11
and 0.57
0.15 (n = 4 for both), for the untreated and treated
samples, respectively, indicating a narrowing of the size distribution after treatment. A PDI of 0.7 or greater indicates a broad size
distribution. The PTPBS does not cause a signicant increase in the
UCA average diameter compared to the control. The average
diameter of the UCA is smaller than 3 microns. Even with the broad
size distribution, the overall diameter of the population is well
under 6 microns, the criterion for intravenous injection. However,
it must be born in mind that the Zeta sizer does not adequately take
into account the nano-sized bubble population.
3.5. Microbial evaluation
The results comparing the bacterial load in the various treated
UCA samples using a bacterial survival assays are shown in Fig. 5.
The addition of PTPBS to the manufacturing process wholly
decontaminates the sample to which it is added, even with
combined contamination density of initial manufacturing microbial load and inoculation of gram-negative or gram-positive
bacteria (p = 0.002).
3.6. Retention of incorporated Nile Red

Fig. 1. Effect of duration of plasma treatment of PBS on the dose response curves of
SE61.
(*) PBS control, (*) 1 min, (4) 2 min, (&) 3 min, N = 3, errors bars = SEM.

When Nile Red, acting as a model drug, was added to the SE61
mixture after the autoclave step and before sonication, uorescence data indicated that it successfully became intercalated in the

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L. Albala et al. / International Journal of Pharmaceutics 494 (2015) 146151

Fig. 4. Flow phantom images 20 and 60 s post-injection using the 9 L probe at 6 MHz, showing contrast enhancement within the lumen in B-mode (left) and coded harmonic
imaging (right).

Fig. 5. Log 10 of bacterial load of UCA under different conditions. N = 4, error

bar = SEM, p = 0.0022.

microbubble shell, as shown in Fig. 6 which tracks the fate of the

Nile Red by uorescent microscopy. Shown are (A) the control
(treated with regular PBS) microbubble, and (B) the UCA treated
with PTPBS. The uorescence is retained after PTPBS treatment,
indicating the presence of Nile Red in the hydrophobic tail portion
of the SE61 shell before and after treatment.
4. Discussion
The initial concerns regarding the use of the PTPBS were
centered on the potential destructive effects that the products of
plasma treatment (acidication and reactive oxygen species)
might have on the antioxidant TPGS that is an integral part of the
UCA shell. The 3-min treatment, unlike the shorter treatment
times, did not show shadowing (where high UCA concentrations

completely reect the incident ultrasound beam, paradoxically

reducing enhancement). This indicated that the total concentration of bubbles was probably reduced by the 3-min treatment,
possibly due to the loss of larger bubbles, as reected in the
lowering of the PDI.
The enhancement and half-life data support the proposition
that PTPBS treatment does not negatively affect the contrast
properties of the UCA and does not destabilize the surfactant shell.
We have found that a value of 20 dB measured in vitro produces
excellent images in vivo (Methachan, 2012). This is also supported
by the ow phantom data, which uses a clinical scanner. It is also
noteworthy that during the ow phantom testing time frames, the
acoustic properties of sterilized UCA were not destroyed by mixing
into the 800 mL reservoir, or by the action of the roller pump.
The uorescent dye, Nile Red, was successfully incorporated
and retained after sterilization, suggesting the absence of
structural changes to the conguration of the microbubble shell
that maintained the capacity retain a hydrophobic species. If Nile
Red were released from the shell during sterilization, its emission
would be immediately quenched in the aqueous medium. The two
images in Fig. 6 provide qualitative assurance that a hydrophobic
compound can be loaded into SE61, and that it is not removed or
noticeably affected by PTPBS. The large uorescent bubbles in the
images are a depth of eld effect caused by agent that is out of focus
and very close to the lens.
Compared to for example polymer microbubble (Eisenbrey
et al., 2008a), the SE61 microbubble is highly sensitive to its
environment, making it an excellent subject for testing a novel
sterilization modality. Here, DBD plasma activation of PBS has been
shown to be a successful method for the sterilization of a delicate
material, conrmed via direct measurement of the materials
performance.
Further research focusing on imaging and therapeutic delivery
potential can be explored in vivo with this UCA, now that a
sterilization method has been developed. Indirect plasma

Fig. 6. Light uorescence microscopy (40x, with 1.6x eyepiece/camera magnication) of SE61 w/ nile red intercalated. Size bar = 5 microns.

L. Albala et al. / International Journal of Pharmaceutics 494 (2015) 146151

sterilization has a plethora of potential uses and applications;

indeed, sterile injectables reduce the need for intensive antibiotic
usage. This is crucial in a healthcare system where the emergence
and explosion of multidrug-resistant (MDR) bacteria is an intrinsic
component of a legacy of indiscriminate antibiotic use and
transmission of resistance within and between individuals
(Andersson, 2003; Guillemot, 1999).
5. Conclusions
The synthesis process described in this work matches surfactant-stabilized microbubble preparation with an indirect plasma
sterilization technique that did not alter morphology, stability,
size, or enhancement in vitro, while providing an inexpensive and
effective approach for production of a sterile, shelf stable UCA. The
successful use of this novel sterilization approach demonstrates its
potential use for an alternative to traditional cleanroom
manufacturing.
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