Group Title: Journal of Biological Engineering 2009, 3:17
Title: Puncture mechanics of cnidarian cnidocysts: a natural actuator
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Title: Puncture mechanics of cnidarian cnidocysts: a natural actuator
Series Title: Journal of Biological Engineering 2009, 3:17
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Creator: Oppegard SC
Anderson PA
Eddington DT
Publication Date: 40084
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Journal of Biological Engineering BioedCenra


Research


Puncture mechanics of cnidarian cnidocysts: a natural actuator
Shawn C Oppegard', Peter A Anderson2 and David T Eddington*1,3


Address: 'Department of Bioengineering, University of Illinois at Chicago, Chicago, IL 60607, USA, 2Whitney Laboratory for Marine Bioscience
and Department of Physiology and Functional Genomics, University of Florida, St. Augustine, FL 32080, USA and 3Department of
Biopharmaceutical Sciences, University of Illinois at Chicago, Chicago, IL 60612, USA
Email: Shawn C Oppegard soppeg2@uic.edu; Peter A Anderson paa@whitney.ufl.edu; David T Eddington* dte@uic.edu
* Corresponding author



Published: 28 September 2009 Received: 17 March 2009
journal ofBiological Engineering 2009, 3:17 doi:10. 186/1754-16 1 -3-17 Accepted: 28 September 2009
This article is available from: http://www.jbioleng.org/content/3/1/17
2009 Oppegard et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Background: Cnidocysts isolated from cnidarian organisms are attractive as a drug-delivery
platform due to their fast, efficient delivery of toxins. The cnidocyst could be utilized as the means
to deliver therapeutics in a wearable drug-delivery patch. Cnidocysts have been previously shown
to discharge upon stimulation via electrical, mechanical, and chemical pathways. Cnidocysts isolated
from the Portuguese Man O' War jellyfish (Physalia physalis) are attractive for this purpose because
they possess relatively long threads, are capable of puncturing through hard fish scales, and are
stable for years.
Results: As a first step in using cnidocysts as a functional component of a drug delivery system,
the puncture mechanics of the thread were characterized. Tentacle-contained cnidocysts were
used as a best-case scenario due to physical immobilization of the cnidocysts within the tentacle.
Ex vivo tentacle-contained cnidocysts from Physalia possessed an elastic modulus puncture
threshold of approximately 1-2 MPa, based on puncture tests of materials with a gamut of hardness.
Also, a method for inducing discharge of isolated cnidocysts was found, utilizing water as the
stimulant. Preliminary lectin-binding experiments were performed using fluorophore-conjugated
lectins as a possible means to immobilize the isolated cnidocyst capsule, and prevent reorientation
upon triggering. Lectins bound homogeneously to the surface of the capsule, suggesting the lectins
could be used for cnidocyst immobilization but not orientation.
Conclusion: Cnidocysts were found to puncture materials up to I MPa in hardness, can be
discharged in a dry state using water as a stimulant, and bind homogeneously to lectins, a potential
means of immobilization. The information gained from this preliminary work will aid in determining
the materials and design of the patch that could be used for drug delivery.




Background lization, predator defense, and locomotion. Cnidocysts
Cnidarians (including jellyfish, sea anemones, and corals) consist of a rigid capsule wall made of collagen-like pro-
utilize the cnidocyst as a tool to capture prey and inject teins, enclosing a tightly coiled stinging thread that rap-
venom as shown in figure 1. Cnidocysts are produced by idly everts from the capsule upon discharge. The thread
cnidocytes and are typically located in the tentacle of the penetrates the prey's integument and introduces venom.
organism where they serve the functions of prey immobi- This discharge event is one of the fastest processes in the


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Journal of Biological Engineering 2009, 3:17


Figure I
Illustration depicting cnidocyst discharge into prey. The cnidocil is a mechanosensory element on the apical surface of
the cnidocyte. Upon appropriate stimulation, including mechanical stimulation of the cnidocil, the cnidocyst is triggered to rap-
idly evert its stinging thread into the potential predator or prey. Venom is then delivered into the contacting organism.


animal kingdom, characterized by a theoretical thread tip
pressure of nearly 7 GPa and a velocity of over 15 m/s [1].

Cnidocyst discharge is still poorly understood, owing to
the nanosecond duration of the process. Cnidocyte dis-
charge in vivo typically requires the near simultaneous
application of chemical and mechanical stimuli [2-4]. The
mechanosensory aspect of discharge is mediated by stim-
ulation of the cnidocyte cnidocil, which is essentially a
finger-like projection at the apical end of the cell that ini-
tiates an intracellular signaling cascade upon mechanical
perturbation by a predator or prey [5]. The chemosensory
component is less well-understood, but is thought to
include both contact and distance chemoreceptors, and
presumably is required to ensure the organism only dis-
charges its cnidocytes when the probability of capturing
food is high [2].

A few theories have been proposed to explain the rapid
process of discharge once the cnidocyst is stimulated by
the cell. Discharge was initially thought to be mediated by
a high internal osmotic pressure, mechanical energy
stored within the capsule wall, and a sudden increase in
hydrostatic pressure [6]. High internal calcium concentra-
tions have been measured intracellularly, and upon dis-
charge, Ca2+ ions are rapidly exocytosed as the cnidocyst
membrane fuses with the cnidocyte membrane and the
cyst comes into contact with sea water. Within the capsule,
an anionic network of polyglutamic acid is bound and
crosslinked by these calcium ions, maintaining a low


osmotic gradient [7,8]. As a prelude to discharge, the cal-
cium ions are thought to dissociate from the poly-
glutamic peptides and exit the capsule. The poly-Glu pep-
tides remain within the cyst, and the now uncrosslinked
monomers of the anionic network increase the number of
osmotically active species [6,9]. A rapid influx of sea water
occurs, yielding a large, sudden increase in hydrostatic
pressure, inducing the thread to discharge. The force of the
hydrostatic pressure causes the thread to rapidly evert with
a force sufficient to puncture through even the hard scales
of some bony fish.

More recent research suggests that a proton gradient estab-
lished across the cyst wall may explain the extremely quick
initial exocytosis of the thread [10]. The protons unbind
from polymer matrix and leave the capsule, leading to
electrostatic repulsion of the polyglutamic acid chains.
This repulsion ultimately results in an increase in pressure
on the capsular wall and an increase in the capsule vol-
ume. The uptake of inorganic cations and water may
change the pH and osmotic pressure within the capsule
and aid in evagination of the thread due to a conforma-
tional change. However, the rapid speed cannot be
explained by this influx of ions alone and thus is argued
to be due to the fast translocation of protons.

Cnidocysts offer an attractive solution to transdermal
drug delivery as they are essentially a natural microscale
injection module that could be integrated into a patch-
like device. The cnidocysts could be loaded with drug and


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upon discharge would evert their threads, injecting the
patient with the drug. Specifically, the work outlined in
this paper explores the use of cnidocysts isolated from the
siphonophore Physalia physalis (Portuguese Man O' War)
as a first step towards this goal. The use of this animal is
attractive for a number of reasons. The isolated cnidocysts
after removal from the tentacle cnidocytes are capable of
being stored for more than one year in water at 40C (to
minimize bacterial contamination), while still able to dis-
charge upon stimulation (unpublished observations).
Also, the penetration of cnidocyst threads would be pain-
less due to the microscale diameter of the thread. Pain
experienced by a cnidarian sting is due to the injection of
venom as opposed to the mechanical disturbance of the
thread. For example, a 33 gauge needle is the smallest
diameter needle commonly used for clinical application,
possessing about a 90 gm tip diameter. In contrast, the
cnidocyst thread tip diameter is about 2 gm [11,12];
nearly two orders of magnitude smaller. Finally, Physalia
itself is capable of puncturing the exoskeleton of certain
crustaceans and possesses relatively long threads (many
over 1 mm in length). These attributes permit more flexi-
bility in choosing the materials and design of the patch.

As a first step in using cnidocysts isolated from Physalia in
a drug-delivery patch, the discovery of a simple and effec-
tive discharge technique was required. A number of pre-


discharge and discharge solutions were utilized to deter-
mine the best combination, based on literature findings
for cnidocysts isolated from other species. The next step
was to examine the puncture mechanics of the thread, by
assessing its ability to puncture materials with elastic
modulus ranging from 0.02 to 90,000 MPa. The puncture
tests were accomplished using excised tentacles from
Physalia as they afforded the most mechanically stable
platform from which to trigger discharge. To date, the
puncture mechanics of the thread have been only theoret-
ically calculated using mass and velocity information
[13]. Finally, preliminary work was conducted exploring
lectins as a means to immobilize and possibly orient cni-
docysts in the patch. Lectins bind strongly to sugar moie-
ties on the surface of the cnidocyst capsule and have been
used in cell culture as an attachment substrate.

Results
Lectin-binding examination
A variety of fluorophore-conjugated lectins bound to the
surface of the capsule (figure 2, table 1), supporting the
idea that they could be used for cnidocyst immobiliza-
tion. In all positive cases, binding was not localized to a
particular region of the capsule, but rather spread uni-
formly over the entire surface of the cyst at pH 7.0 and
with the ionic conditions utilized. Preliminary work using
Con-A to immobilize cysts was unsuccessful. Con-A was


Figure 2
Fluorophore-conjugated lectin binding to the cnidocyst. Fluorescence micrographs of Physalia tentacles stained with
FITC-conjugated lectins. (A). Lower power image of part of a tentacle stained with FITC-conjugated isolectin B4 lectin. The
two clusters of stained cysts represent two cnidosacs. (B). A higher power image of cysts stained with soybean agglutinin. Two
size classes of cysts are evident in this image.



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Table I: Summary of fluorophore-conjugated lectin-binding to cnidocyst capsules


Abbreviation


Lectin name


Target


Cyst binding


Concanavalin A
Dolichos Biflorus Agglutinin
Datura Stramonium Lectin
Erythrina Cristagalli Lectin
GSL-I isolectin B4
Griffonia Simplicifolia Lectin II
Jacalin
Lens Culinaris Agglutinin
Lycopersicon Esculentum Lectin
Peanut Agglutinin
Phaseolus Vulgaris Erythroagglutinin
Phaseolus Vulgaris Leucoagglutinin
Pisum Sativum Agglutinin
Ricinus Communis Agglutinin I
Soybean Agglutinin
Solanum Tuberosum Lectin
Succinulated Wheat Germ Agglutinin
Ulex Europaeus Agglutinin I
Vicia Villosa Lectin
Wheat Germ Agglutinin


Glu, Man
GalNAc
GlcNAc
Gal
Gal, GalNAx
GlcNAc
Gal
Glu, Man
GlcNAc
Gal
Complex N-glycans
Complex N-glycans
Glu, Man
Gal, GalNAc
Gal, GalNAc
GlcNAc
GluNAc
Fuc
GalNAc
GlcNAc


A variety of fluorophore-conjugated lectins were used to investigate which ones could be potentially used for cnidocyst immobilization.
Gal galactose; Glu glucose; Man mannose; Fuc fucose; GalNAc N-acetyl galactosamine;
GlcNAc N-acetyl glucosamine.
I stained apical end of cnidocyte in intact tentacles, but not cyst.


chosen due to availability and protocols in the literature
for adhering cells [ 14]. The adhesion by way of Con-A was
not sufficient to prevent the cnidocyst rotating to absorb
the mechanical force its thread encountered as it tried to
penetrate something as soft as gelatin. Other lectins were
not examined.

Stimulation of cnidocyst discharge
Cnidocysts isolated from Physalia proved to be capable of
discharge. Discharge was accomplished only by first dry-


Table 2: Summary of the isolated cnidocyst discharge study


ing the cnidocysts in 0.025 M to 1 M EDTA solution. In
their properly dried form, any water-based solution could
be used to stimulate discharge (table 2), as indicated by
plus signs, denoting that over 95% of the isolated cnido-
cyst sample group had discharged. Note that "starting
medium" in table 2 indicates both a wet discharge
medium, or as the solution in which cnidocysts were
dried. Alcohol-based solutions, like methanol, did not
induce discharge, as denoted by a minus sign indicating
less than 1% discharge. EDTA at concentrations below


Discharging Solution (0.1 I M)*


DI Water EGTA


EDTA Alcohols KCI NaSCN Boric Acid HCI


Starting Medium


Sea Water
DI Water
Spring Water
EGTA
EDTA
Alcohols
CaCI


+ +


+ +


Isolated Physalia cnidocyst discharge tests, using concentrations between 0.1 M and I M of the indicated solutions (note that EGTA was also used at
5 mM as described in the literature[34]). Starting medium indicates both the wet medium and drying solutions before stimulation with test
discharging solutions. A positive sign denotes that more than 95% of the cnidocysts discharged. A negative sign indicates less than 5% cnidocyst
discharge. Discharge was only achieved when the cnidocysts were dried in EDTA and rehydrated with an aqueous solution, except in the case of
DTT-induced discharge. Note that the grouping "alcohols" include ethanol, methanol, and methylated spirits (a mixture of ethanol and methanol).
Experiments were conducted on at least three different batches of cnidocysts at a density of approximately I03 cysts per 50 1.
* Note: EGTA was also used at 5 mM[34]


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Con-A
DBA
DSL
ECL
GSL-I
GSL-II
Jacalin
LCA
LEL
IPNA
Pha-E
Pha-L
PSA
RCA120
SBA
STL
S-WGA
UEA I
WA
WGA


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0.025 M coupled with rehydration yielded very few dis-
charged cnidocysts upon resuspension, proportional to
the concentration of EDTA used for drying. Note that all
chemicals were tried with at least two different concentra-
tions to ensure test solutions were not too dilute or
potent, varying between 0.1 M to 1 M, and based off suc-
cessful concentrations used in the literature. Cnidocyst
discharge was essentially "all-or-none" with respect to the
chemicals tested, with no need for a partial discharge cat-
egorization. Unfortunately, the discharged isolated cnido-
cyst threads failed to puncture through the softest material
evaluated (gelatin) due to capsule rotation upon thread
contact with the test sample.

Interestingly, discharge was observed only when the cni-
docytes were exposed fairly quickly to water. In some
experiments, water was added to microwells containing
the dried cnidocysts and allowed to slowly spread across
the surface of the well. In such cases, the cnidocysts would
not discharge, suggesting that a sudden addition of water
and/or a forceful change in surface tension is required for
discharge. This sudden change is not present in the slow
spreading of the water droplet. Primarily water was uti-
lized to examine this fast wetting discharge mechanism,
but no gross differences were observed between water and
liquids of different surface energy (i.e. EGTA). The wetting
experiments were conducted in triplicate with each chem-
ical. Determining the specific mechanism of this wetting
speed phenomenon requires more research.

Addition of 0.5 M DTT solution prepared in methanol
yielded very slow discharge of the threads when the cnido-
cysts were buffered (wet) or dried in EDTA, which was
quite unlike the very fast discharge when exposed to
water. Notably, DTT was the only solution that yielded
discharge of the cnidocyst thread in a "wet" state, suggest-
ing a separate discharging mechanism than with aqueous
solutions. The slow uncoiling of the thread delayed about
1 min-post stimulation and required another minute for
complete uncoiling. In the dried cnidocysts, the threads
only partially everted. Contrastingly, the cnidocysts buff-
ered in EDTA solution (wet) completely discharged the
thread. The unraveling occurred at a slightly faster rate and
resulted in complete separation of the thread from the
capsule.

Electric field stimulation ofcnidocyst discharge proved to
be unsuccessful in all cases; no version of the thread was
observed. Notably, the intact cnidocysts moved when
exposed to an electric field, with a majority migrating
towards the anode, suggesting a net positive charge.

Mechanical puncture tests
The tentacle-contained Physalia cnidocysts were capable of
puncturing PDMS and all materials tested with an elastic


modulus below 1 MPa as shown in table 3. However, no
thread penetration was observed through nitrile or mate-
rials harder than 2 MPa. This suggests that the cnidocyst
thread for this animal has an elastic modulus puncture
threshold of about 1-2 MPa. In all positive penetration
cases, more than 10 threads punctured the material in
each trial, making penetration classification unambigu-
ous. The penetration success rate for all positive cases was
100%.

Discussion
A means of isolated Physalia cnidocyst discharge in a sta-
ble, dried form has been discovered. Upon drying cnido-
cysts in EDTA solution, the cnidocyst could be stimulated
to discharge in any aqueous solution including distilled
water (Figure 3). No other drying solution yielded the
same result, including the more potent calcium chelator
EGTA. The exact mechanism of discharge in this way is
unknown, but could be explained by the difference in
morphology between EDTA-drying and drying in other
solutions. When dried in EDTA, the cnidocysts possess a
pseudo-hydrated morphology where intracapsular
osmotic pressure is still maintained (Figure 4). This is in
stark contrast to the appearance of cnidocyst dried in any
other solution. In the latter case, the cnidocysts shrivel
when dried. Upon addition of an aqueous solution, a
majority of the EDTA-dried cnidocyst discharge their
threads. However, the shriveled cnidocysts dried in other
solutions simply rehydrate upon exposure to water, but
do not discharge. The reason for this incomplete drying of
the cnidocysts in EDTA solution could be explained by
blockage of pores on the surface of the capsule. EDTA is a
chelator for a number of cations besides calcium, includ-
ing magnesium and other metal ions. Perhaps these cati-
ons play an important role in ion translocation across the

Table 3: Summary of tentacle-contained cnidocyst penetration
of various materials


Material

Gelatin
Polyacrylamide
Teflon
Latex
PDMS
Nitrile
Polyvinylchloride
Polycarbonate
Aluminum
Glass


Elastic Modulus (MPa)

0.02
0.06
0.10
0.80
1.00
2.60
250
2,000
70,000
90,000


Penetration

+
+
+
+
+


List of test materials and their respective elastic moduli. The ability of
tentacle-contained cnidocysts from Physalia to puncture these
materials is also listed. Note that the elastic modulus of human skin is
approximately 0.13 MPa [35]. A positive sign denotes successful
penetration of at least some of the threads in all three trials. A
negative sign indicates a lack of successful penetration in all trials. In
positive cases, thread penetration occurred in all three trials.


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Rehydration

I



Figure 3
Images depicting before and after cnidocyst discharge via rehydration with water. Isolated Physalia cnidocyst dried
in EDTA (left). The capsule remains spherical when dried in this solution. The cnidocyst rapidly everts its stinging thread fol-
lowing rehydration with distilled water (right). Note the -I mm thread continues beyond the area depicted in the picture.


surface of the capsule, which is highly permeable to a
number of charged atoms [9]. Due to the maintenance of
the osmotic pressure within the capsule in the pseudo-
hydrated form, the thread is allowed to rapidly evert via
the osmotic pressure mechanism.

Unfortunately, isolated cnidocysts were unable to pene-
trate even gelatin, probably because the unrestrained cap-
sule was rotating to relieve the pressure induced when the
thread encountered the test material. Lectins were consid-
ered as a means to immobilize the capsule and prevent
this reorientation, but did not yield thread penetration


even in gelatin. This penetration failure could be attrib-
uted to the fact that the substrate-lectin bond, while phys-
ically strong, was not sufficient to counter the large
moment generated by the rotational forces created by the
capsule reorienting. Liu et al describe a procedure where
plant lectins (E-PHA) can be covalently bound to a glass
coverslip by compression and photo-crosslinking [15]. A
similar procedure could be used to bind Con-A or STL to
a glass substrate, ensuring a stronger bond. Another possi-
bility for the lack of immobilization is that drying the cni-
docysts unbinds the cnidocysts from the Con-A-coated
substrate. Water molecules have been shown to play an


Figure 4
Comparison of cnidocysts dried in EDTA vs. any other aqueous solution. Brightfield images comparing cnidocysts
dried in EDTA (left) vs. any of the other aqueous drying solutions tested (right). Cnidocysts discharged when in the "pseudo-
hydrated" form depicted on the left.



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I-------I


20 pm
I-


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important role in the binding kinetics of a number of
lectins [16-18]. The cnidocyst discharge event could occur
before the water component of the discharging solution
can restore the lectin bond. Finally, the lectins could be
degraded in a dried form, thus cleaving the capsule attach-
ment to the substrate. More work is required to fully assess
the applicability of lectins for capsule immobilization.
Another possibility that could be explored is whether
there are differences in discharge mechanisms and forces
between the rehydration method of isolated cnidocysts
and physiological discharge in an intact animal.

DTT was used to investigate the importance of disulfide
bonds in cnidocyst thread discharge. DTT prepared in
methanol yielded a slow discharge of the cnidocyst
thread, with the speed dependent on the hydration state
before addition of DTT. The slow uncoiling in both cases
could be explained by the fact that disulfide bonds may be
important in maintaining the tightly coiled structure of
the cnidocyst thread [19], in addition to maintaining the
capsule wall integrity reported elsewhere [12,20]. As the
DTT slowly diffuses into the capsule and cleaves these
bonds, the tension associated with the thread coil over-
whelms the remaining bonds in the capsule wall and
thread itself, as visualized by this slow uncoiling. The lack
of fast and full discharge could be due to the lack of
extreme pressure or electrostatic repulsion forcing the
thread. Thus, DTT-induced discharge may be permitting
observation of the thread tension component of dis-
charge. Surprisingly, DTT did not cause discharge in cni-
docysts buffered or dried in any of the other chemicals
tested, suggesting that the cleavage of disulfide bonds is
not the only mechanism and the EDTA plays an impor-
tant role beyond that of maintaining osmotic pressure.
The mechanism is not purely calcium dependent, as cni-
docysts buffered or dried in EGTA solution did not fire, so
perhaps the affinity of EDTA for other cations plays a role
in permitting thread version.

Fast-occurring biological phenomenon due to structure
tension and calcium translocation is not a new concept in
biological systems. Shin et al quantified the force of a bio-
logical spring in the ability of a Limulus polyphemus sperm
actin bundle to puncture the egg envelope, without the
presence of a build-up of hydrostatic pressure as in the
case of a cnidocyst [21]. The mechanism for bundle exten-
sion involves calcium disassociation from the actin-
crosslinker scruin, which then permits the initially over-
twisted actin bundle to rapidly untwist and therefore
extend [22]. Sperm bundle pressures exceeded 1.6 MPa,
suggesting that the spring-like configuration of the cnido-
cyst thread could play a small role in the 7 GPa thread tip
pressure found in Hydra [13]. The contraction dynamics
of Vorticella convallaria are also very relevant to the present
study. The protozoan attaches to a substrate via a stalk and


rapidly contracts when stimulated. The organism has
shown to change nearly 5000 times its length in less than
a second. As with cnidocysts and horseshoe crab sperm,
high intracellular calcium levels are found within the cell
and mediate the explosive process [23]. When calcium is
bound to the spasmoneme enclosing the stalk, the repul-
sive forces generated by the negatively-charged spasmin
backbone are neutralized, yielding contraction [24]. In
the calcium unbound state, the spasmin protein chains
repel one another, resulting in stalk extension. This proc-
ess is somewhat similar to the proton gradient theory
applied to cnidocysts in that charge repulsion and reloca-
tion of ions causes an explosive biological process [10]. A
subsequent study on Vorticella quantified speed through
viscous media to elucidate the contraction dynamics [25].
Stalk contraction was demonstrated to occur at a faster
rate than the velocity of the cell body, suggesting that the
rate of contraction is ultimately dependent on the power
delivered by the spasmoneme, as well as the rate at which
calcium can be released from the stalk [22].

Cnidocyst discharge by chemical means is not the only
method previously reported and may not even be the
most efficient. Electrical discharge has worked for a
number of groups with a wide variety of cnidarian species
[1,5,26]. Following protocols used for stimulating dis-
charge of the freshwater cnidarian Hydra [13], electrical
stimulation of cnidocyst version was attempted on iso-
lated Physalia cnidocysts, but proved unsuccessful. The
observation that many of the cnidocysts migrated towards
the anode suggests a net positive charge of the cnidocysts,
which corroborates with other observations in the litera-
ture, where researchers discovered that the stinging cap-
sule operculum is positively charged while the basal end
of the cnidocyst is neutral. The positive charge finding
prompted the use of dielectrophoresis patterning of the
cnidocysts as part of a possible patch fabrication method
[27,28]. The results were ultimately not consistent or
effective.

In the present study, the puncture mechanics of the
Physalia cnidocyst thread have been characterized for the
first time. As expected, the threads were capable of punc-
turing relatively hard materials (PDMS = 1 MPa) com-
pared to human skin (0.13 MPa). This information will be
useful in designing the patch. A material used for the
membrane separating the cnidocyst within the well and
the patient's skin should be made of a material with an
elastic modulus of less than 1 MPa, such as PDMS or
something softer.

The binding of lectins to the surface of the cnidocyst cap-
sule is attractive as a possible means of immobilizing the
cnidocysts within the containment wells of the patch. The
cnidocysts could be bound to the separation membrane


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or bottom of the well. Some of the cnidocysts would fire
into through the membrane and into the patient's skin. A
binding molecule that exhibits heterogeneous binding to
the capsule wall is ultimately preferred which would per-
mit pre-orientation of the cnidocysts in addition to
immobilization and ensure that a majority of the cnido-
cysts would discharge in the correct direction. Only
homogeneous binding was observed using the lectins
tested with Physalia. It should be noted that no attempt
was made to confirm that the observed binding patterns
of different lectins mirrored the distribution of the rele-
vant sugar moieties since we were not interested in the
biochemistry of the cells, per se, merely whether or not
one might be able to capitalize on structural heterogeneity
to orient immobilized cysts. Lotan et al suggested the use
of other bioadhesives that could be used, including bio-
logical glues (e.g. BIOBOND'), bacterial fimbrins, and
polylysine meshes, among many other means [29]. The
charge heterogeneity of the cnidocyst capsule could be uti-
lized as well, such as by binding the operculum to a nega-
tively-charged material that would serve as the puncture
membrane of the patch.

The rest of the patch could be made of a harder material,
such as silicon. This would prevent those cnidocysts that
were not oriented from discharging in the wrong direc-
tion. Silicon is a hard material that permits fabrication of
complex designs. A microfluidic network could be incor-
porated into the silicon component of the patch. This net-
work could deliver water to the individual containment
wells, inducing discharge of the cnidocysts. Additionally,
multiple drugs could be independently delivered using
the same patch by localized stimulation of cnidocysts
with the microfluidic network. Ultimately, it is hoped that
the cnidocysts could be genetically re-engineered to not
only deliver but also manufacture the desired drugs for
injection. Creation of an interstitial cell line for cnidocytes
would greatly aid in the re-engineering process but this
would only be applicable to drugs that can be produced
biologically.

Conclusion
The ability to utilize cnidocysts as a functional material is
attractive based on their robust mechanism for thread
version. Tentacles of Physalia were able to penetrate
PDMS, which is a welcomed result due to the ease of fab-
rication. A protocol for discharging isolated cnidocysts
was also identified; however they were not able to pene-
trate any material tested due to the inability to anchor
them to a substrate. To explore means of immobilizing
and possibly orienting the cnidocysts prior to firing, fluor-
ophore-conjugated lectin binding to the capsule wall was
visualized. Several lectins bound homogenously to the
surface of the Physalia capsules, suggesting the possibility
for cnidocyst immobilization but not orientation. The


information determined from this study will be useful in
applying cnidocyst to drug delivery applications.

Methods
Isolation of cnidocysts from Physalia
Physalia physalis specimens (multiple animals) were col-
lected in shallow water off beaches near the University of
Florida Whitney Laboratory for Marine Bioscience,
Marineland, Florida. Specimens were typically about 10 -
15 cm in float length. Excised, full length tentacles from
Physalia were soaked in distilled water at 40C for one
week, with multiple rinses. Because of their density, iso-
lated cysts readily accumulated at the base of the vessel
allowing the supernatant to be removed and the slurry of
cysts to be collected. The slurry was washed three times,
stored in distilled water at 4 C, and used within 6 months
of isolation.

Stimulation of isolated cnidocyst discharge
Numerous combinations of pre-discharge incubation and
discharge-inducing solutions were tested as a way of
inducing discharge of isolated cnidocysts (table 2). The
chemicals were used at various concentrations based on
reports from the literature demonstrating discharge of cni-
docysts in other species [30-32,26,33,34]. The stock cni-
docyst solution used contained a high concentration of
cnidocysts suspended in distilled water. To test the various
"wet" and "dry" discharge solutions, the stock solution
was mixed 1:10 with the test solution of choice for rinsing
purposes. This mixture was then centrifuged at 700 RCF to
pellet the cnidocysts. The supernatant rinsing solution
was removed and the test solution was then added to the
original cnidocyst pellet. Once resuspended, the cnidocyst
solution was pipetted into microwells of a custom-fabri-
cated polydimethylsiloxane (PDMS) well-plate for high-
throughput testing. The well plate was essentially a slab of
PDMS containing 5 mm diameter holes and was oxygen
plasma-bonded to a glass slide. For "wet" discharge stud-
ies, the cnidocysts were allowed to incubate in the test
solution for 1 h and then stimulated to discharge chemi-
cally. In the "dry" studies, the cnidocysts were placed in a
dehumidified room at 45 C to rapidly dry the organelles.
Once dried, the test solutions were pipetted atop the cni-
docysts to induce discharge.

Of note, pure methanol was used as a control for investi-
gating dithiothreitol-induced discharge. Dithiothreitol
(DTT) cleaves disulfide bonds and is a common reducing
agent in molecular biology. As with the rest of the chemi-
cals examined, it was utilized on both wet and dry cnido-
cysts.

Electric current-mediated discharge was also attempted on
isolated Physalia cnidocysts. The recent paper suggesting a
proton gradient and electrostatic repulsion mechanism of


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Microchannel Wall

/


I
PhysaliaTentacle Cnidocyst thread
Physalia Tentacle


Figure 5
Thread penetration into the PDMS microchannel wall. Brightfield macroscope (left) and microscope (right) images
depicting thread penetration into the PDMS in a custom-made microchannel.


discharge encouraged the use of electric fields to stimulate
the cnidocysts [10]. A range of frequencies (0 Hz to 16
MHz) and amplitudes (10 mV to 30 V) were used to
induce discharge of cnidocysts positioned atop electrodes
in both DI water and conductive solutions. For the pilot
experiments, a cnidocyst suspension in sea water was
pipetted between two parallel exposed wires in a setup
similar to Holstein and Tardent [13]. Multiple wire sepa-
rations of between 1 mm and 10 mm were used. A variety
of other solutions (see table 2) were tested to examine the
effects of the medium on discharge. To improve the repro-
ducibility and handling of the experiment, a circuit board
was designed using metal-etching coupled with conven-
tional photolithography. Both 1 mm and 10 mm spacing
were used in designing the photolithography mask. Each
stimulation method (both chemical and electrical) was
applied to large batches of cnidocysts (>103) over 3 trials
(N = 3) to ensure reproducibility.


Preparation of excised tentacles
Small sections of Physalia tentacles (2 cm) were excised
from a single Physalia specimen's tentacles maintained in
an aquarium at the Whitney Laboratory for Marine Bio-
science (Marineland, FL). These were then anaesthetized
by incubation in isotonic (0.37 M) MgCl2 mixed 1:1 with
sea water. Once anaesthetized, the tentacles were further
sectioned into 5 mm pieces. These were then used for the
mechanical puncture tests.

Tentacle-contained cnidocysts puncture tests
Excised tentacles from Physalia were used to characterize
the ability of the cnidocysts to puncture a variety of mate-
rials with a range of elastic moduli (table 3). The materials
ranged from 0.02 MPa (glutaraldehyde-crosslinked gela-
tin) to 90,000 MPa (glass). The silicone elastomer poly-
dimethylsiloxane (PDMS) was used as a starting point.
For reference, human skin ranges from about 0.1-0.15
MPa [35]. PDMS microchannels (600 [tm wide x 300 [tm
tall) were fabricated using conventional photolithography
and replica molding techniques. The ex vivo tentacles were


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Journal of Biological Engineering 2009, 3:17


then pulled within the channels. Discharge stimulation
was induced by addition of the potent calcium chelator
ethylene glycol tetraacetic acid (EGTA) solution, since cal-
cium ion translocation plays a critical role in discharge
[9]. EGTA yielded the most discharged threads for Physalia
and has been used with great effectiveness for discharge
stimulation of other cnidarian species as well, including
isolated capsules [34]. Thread penetration into the sur-
rounding PDMS walls was easily observed with both a dis-
secting and compound microscope (Figure 5).

For testing with all other materials, the excised tentacle
was placed atop a glass slide, and then a 200 |im thick film
of each material (McMaster-Carr, Chicago, IL) was placed
on top of the tentacle. Discharge stimulus occurred via
mechanical stimulation of the cnidocil by tweezer prob-
ing, which was more efficient than EGTA stimulation.
Thread penetration up through the material was visual-
ized using a dissection microscope. Each material was
tested at least 3 times. To be denoted "positive" penetra-
tion, one or more threads must have penetrated the mate-
rial in all three trials.

Lectin-binding
Tentacles were excised from freshly collected Physalia and
anaesthetized in a 1:1 solution of isotonic MgCl2 and nat-
ural, unfiltered sea water for 30 min. They were then
lightly stretched and pinned to a layer of PDMS using cac-
tus spines before being fixed in 4% phosphate-buffered
paraformaldehyde for 2 h at room temperature. Following
four, 30 min rinses in phosphate-buffered saline contain-
ing 0.25% Triton-X 100 (PBS-T) followed by two addi-
tional 30 min rinses in PBS-T containing 1% goat serum,
tentacle fragments were incubated overnight at 40C in
1:1000 dilutions of a range of FITC-conjugated lectins
(Vector Labs) as listed in table 1 at pH 7.0. Following five,
30 min rinses in PBS-T, the tentacles were mounted on a
microscope slide in 90% glycerol containing the anti-fad-
ing agent paraphenylenediamine (Sigma). The protocol
for staining of isolated cysts was the same except that iso-
lated cysts were fixed on gelatin-coated microscope slides
for 1 h. Rinsing protocols were the same as for whole ten-
tacles, but lectin incubation was reduced to 1 h at room
temperature. Stained tissues were examined using either a
Leitz DMRD fluorescence microscope (Wetzlar, Germany)
or a Leica SP2 Confocal (Bannockburn, IL).

Lectins were also evaluated as a means to immobilize the
cnidocysts, preventing capsule rotation during discharge.
Lectins were adhered to a glass slide using a protocol for
neuron culture vessel coating [14]. The cnidocysts sus-
pended in ethylenediaminetetraacetic (EDTA) solution
were allowed to dry and then a wet piece of gelatin was
placed on top to stimulate discharge.


Competing interests
The authors declare that they have no competing interests.

Authors' contributions
SO carried out the experiments and drafted the manu-
script. PA provided materials for the experiments. DE con-
ceived of the study, and participated in its design and
coordination. All authors read and approved the final
manuscript.

Acknowledgements
Technical assistance on tentacle harvesting was provided by Dr. Christelle
Bouchard and Rebecca Price. This project was supported by DARPA
through a U.S. Army Research Office Award (Agreement # W91 I NF-07-
1-0360).

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