Cell penetrating peptide-mediated transport enables the regulated secretion of accumulated cargoes from mast cells

Article abstract of DOI:10.1016/j.jconrel.2015.02.005

The in vivo utility of technologies employing cell penetrating peptides and bioportides may be compromised by the general capacity of polycationic peptides to activate mast cell secretion. Moreover, the same technologies could be exploited in a clinical setting either to directly modulate intrinsic exocytotic mechanisms or to load mast cells with bioactive cargoes. Comparative investigations identified two cell penetrating vectors, Tat and C105Y, which readily translocate into mast cells without inducing receptor-independent exocytosis. Efficient Tat transduction also enabled the intracellular delivery and accumulation of cargoes within discrete intracellular compartments. A tetramethylrhodamine-Tat conjugate is effectively translocated into the secretory lysosomes of RBL-2H3 cells. In contract, the intracellular delivery of avidin, as a non-covalent complex with a biotinylated Tat vector, is also efficient but the protein is predominantly accumulated outside of secretory lysosomes. Significantly, both cargoes can be subsequently released following mast cell stimulation either by mastoparan, a wasp venom secretagogue, or by the physiological mechanism of antigen-induced aggregation of high affinity IgE receptors. These studies indicate that mast cells could be exploited to direct the delivery of bioactive agents to disease sites as an innovative cell-mediated therapy.

Full text of DOI:10.1016/j.jconrel.2015.02.005

Journal of Controlled Release 202 (2015) 108–117
Contents lists available at ScienceDirect
Journal of Controlled Release
journal homepage: www.elsevier.com/locate/jconrel
Cell penetrating peptide-mediated transport enables the regulated
secretion of accumulated cargoes from mast cells
⁎
John Howl , Sarah Jones
Research Institute in Healthcare Science, Faculty of Science and Engineering, University of Wolverhampton, Wulfruna Street, Wolverhampton WV1 1LY, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
The in vivo utility of technologies employing cell penetrating peptides and bioportides may be compromised by
the general capacity of polycationic peptides to activate mast cell secretion. Moreover, the same technologies
could be exploited in a clinical setting either to directly modulate intrinsic exocytotic mechanisms or to load
mast cells with bioactive cargoes. Comparative investigations identified two cell penetrating vectors, Tat and
C105Y, which readily translocate into mast cells without inducing receptor-independent exocytosis. Efficient
Tat transduction also enabled the intracellular delivery and accumulation of cargoes within discrete intracellular
compartments. A tetramethylrhodamine–Tat conjugate is effectively translocated into the secretory lysosomes of
RBL-2H3 cells. In contract, the intracellular delivery of avidin, as a non-covalent complex with a biotinylated Tat
vector, is also efficient but the protein is predominantly accumulated outside of secretory lysosomes. Significant-
ly, both cargoes can be subsequently released following mast cell stimulation either by mastoparan, a wasp
venom secretagogue, or by the physiological mechanism of antigen-induced aggregation of high affinity IgE
receptors. These studies indicate that mast cells could be exploited to direct the delivery of bioactive agents to
disease sites as an innovative cell-mediated therapy.
Received 20 December 2014
Received in revised form 30 January 2015
Accepted 3 February 2015
Available online 07 February 2015
Keywords:
Bioportide
Cell penetrating peptide
Mast cell
Mastoparan
Secretion
Tat transduction
© 2015 Elsevier B.V. All rights reserved.
1. Introduction
stimulation of mast cell (MC) secretion. This potentially disastrous lim-
itation, associated with the administration of polycationic peptides into
Cell penetrating peptides, or protein transduction domains, are
proven vehicles for the efficient intracellular delivery of bioactive
agents that include small drugs, peptides, proteins and oligonucleo-
tides [1–3]. Some of the first examples of CPPs that were clearly ef-
fective pharmacokinetic modifiers included sequences related to
penetratin (RQIKIWFQNRRMKWKK [4]) and Tat (GRKKRRQRRRPPQ
[5]), polycationic peptides identified within helical domains of
insect- and virally-encoded transcription factors respectively. More
recently, CPPs with intrinsic bioactivities, now designated as
bioportides [6], have also attracted significant attention as biomedi-
cal research tools and putative diagnostics and/or therapeutics
(reviewed in [7]).
Some of the potential caveats associated with the development of
peptide and peptide-like drugs, including both CPP vectors and
biologically-active bioportide technologies, have been extensively doc-
umented [8,9]. Fortunately, many practical limitations often associated
with the utilisation of peptides in a clinical setting, particularly pharma-
cokinetic challenges due to rapid proteolysis and the need to employ
undesirable routes of delivery, are now more commonly surmountable.
Yet many in vivo applications of CPPs and bioportides may be compro-
mised by a consistent problem that is the acute and undesirable
the cardiovascular system, is a consequence of the exposure of circulat-
ing MCs to peptides that are potentially potent secretagogues. As key
mediators of inflammatory and allergic responses, MCs are exquisitely
sensitive to some polycationic and/or amphiphilic sequences including
a plethora of venom peptides and the neuropeptide substance P
(RPKPQQFFGLM), an undecapeptide containing just two cationic side
chains [10,11]. Moreover, this mode of action of polycationic peptides
is commonly receptor-independent and may arise in part from the di-
rect activation of intracellular heterotrimeric G proteins [10,11]. Thus,
it is reasonable to assume that cell penetrant CPPs and bioportides
may have an enhanced propensity to promote MC secretion by a variety
of mechanisms that could include the perturbation of plasma mem-
brane integrity, direct activation of G proteins and the modulation of
protein–protein interactions in signalling pathways.
The RBL-2H3 cell line is widely employed in studies of regulated se-
cretory phenomena [12–16] and is, therefore, an accepted MC model
system. However, this neoplastically-transformed line is clearly of baso-
philic origin [17,18] and consequently exhibits some phenotypic and
functional characteristics that differ from primary MC cultures. Of
particular relevance to this study, the secretory granules of RBL-2H3
are of lysosomal nature, acidic and accessible to endosomal markers
[15,16]. Moreover, in the absence of a reliable and readily-cultured
human cell line, RBL-2H3 is commonly employed to study functional
FcεR1 type IgE receptors intrinsically coupled to regulated secretory
⁎
Corresponding author.
E-mail address: J.Howl@wlv.ac.uk (J. Howl).
http://dx.doi.org/10.1016/j.jconrel.2015.02.005
0168-3659/© 2015 Elsevier B.V. All rights reserved.

J. Howl, S. Jones / Journal of Controlled Release 202 (2015) 108–117
109
mechanisms [12–18]. Indeed, the exocytosis of β‐hexosaminidase, a se-
cretory lysosomal marker, is a convenient assay of regulated secretion in
RBL-2H3 cells that has been previously employed to determine the effi-
cacies of polybasic peptide secretagogues [12–14]. Thus, we utilised
RBL-2H3 cells in these studies to further address fundamental questions
that relate to potential applications of CPPs and bioportides in vivo. Our
investigations provide evidence that some CPPs, including Tat and
C105Y, can be gainfully employed to specifically deliver bioactive moie-
ties into secretory lysosomes or other compartments of MCs without
promoting receptor-independent exocytosis. Furthermore, our results
support the hypothesis that MC degranulation could be readily
exploited to achieve the targeted release of bioactive agents within a
specific site of tissue inflammation.
AA coupling reactions were accomplished with a 4-fold molar excess
of Fmoc-protected AA with HCTU and diisopropylethylamine
(DIPEA), molar ratio of 1:1:2 (AA/HCTU/DIPEA), in 4 ml for 10 min
at 25 W/75 °C. The coupling of arginine was performed in two stages,
30 min 0 W/~25 °C followed by 5 min at 17 W/75 °C, and routinely
repeated to ensure 100% incorporation that was confirmed by a
qualitative ninhydrin test. Amino terminal acylation with biotin or
6-carboxytetramethylrhodamine (TAMRA) generated biotinylated
(Btn) and fluorescent (TAMRA) analogues respectively.
As previously reported (see references in Table 1), all peptides were
purified to apparent homogeneity by semi-preparative scale high per-
formance liquid chromatography, and their predicted masses (average
M + H+) were confirmed to an accuracy of 1.0 by matrix-assisted
laser desorption ionization (MALDI) time of flight mass spectrometry
operated in positive ion mode using α-cyano-4-hydroxycinnamic acid
(Sigma) as a matrix [6,23,24].
2. Materials and methods
2.1. Peptide selection and synthesis
2.2. Culture of RBL-2H3 cells
2.1.1. Chemical diversity of CPPs and bioportides
When selecting polycationic peptides for these investigations, our
intention was to evaluate a chemically diverse set of sequences that
comprised both commonly utilised CPPs [4,5,19,20] and bioportides
[4,5]. As indicated in Table 1, we compared common CPP vectors (Tat
[5], penetratin [4], C105Y [19] and transportan 10 (TP10; [20])); mast
cell secretagogues mastoparan (MP; [21]) and mitoparan (MitP; [22]);
human cytochrome c-derived sequences (Cyt c5–13 and Cyt c77–101
[23]); the anti-angiogenic bioportide nosangiotide [6]; camptide [6], a
known activator of G-proteins; and polycationic sequences within the
primary structure of leucine rich repeat kinase 2 (LRRK21322–1340 and
LRRK22413–2427). The sequences and sources of these peptides are
provided in Table 1 and the references therein.
RBL-2H3 cells were maintained in DMEM containing L-glutamine
(0.1 mg ml−1) supplemented with fetal bovine serum (10% v/v),
penicillin (100 U ml−1) and streptomycin (100 μg ml−1) in a humidi-
fied atmosphere of 5% CO2 at 37 °C [13].
2.3. Exocytosis assays
2.3.1. Secretory efficacies of polycationic CPPs and bioportides
RBL-2H3 cells were cultured in 24-well plates. To compare the
secretory efficacies of cationic peptides, the lysosomal enzyme β-hexos-
aminidase was assayed in samples of cell medium following exogenous
application of peptides to RBL-2H3 cells in HAMS F12 medium [13,14,
27,28]. 5 μl aliquots of medium were transferred into 96 well plates
and incubated with ρ-nitrophenyl-N-acetyl-β-D-glucosamide (20 μl of
1 mM in 0.1 M sodium citrate buffer, pH 4.5) for 1 h at 37 °C. Na2CO3/
NaHCO3 buffer (200 μl of 0.1 M, pH 10.5) was then added and β-hexos-
aminidase activity determined by colourimetric analysis at 405 nm.
2.1.2. Peptide synthesis
The syntheses, purification by high performance liquid
chromatography and mass determination of a majority of the peptides
employed in this study have been described previously where indicated
in Table 1. Polycationic LRRK2-derived sequences were synthesized on
a 0.1 mmol scale using a using a Discover SPS Microwave Peptide Syn-
thesizer (CEM Microwave Technology Ltd, Buckingham, UK; [24]). The
solid phase was Rink amide 4-methylbenzhydrylamine resin pre-
loaded with the first amino acid (AnaSpec, Inc. Cambridge Bioscience
Ltd., Cambridge, UK) and employed an N-α-Fmoc protection strategy
with (2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HCTU) activation. Deprotection with 7 ml of
20% piperidine was performed for 3 min at 50 W/75 °C. A majority of
2.3.2. Regulated exocytosis of translocated peptide conjugates
To analyse the release of accumulated peptide-conjugates following
Tat transduction, exocytosis was stimulated by FcɛR1 activation [14] or
by the receptor-independent secretagogue MP (Table 1; [13,14,21]).
RBL-2H3 cells were cultured in 24-well plates as above and pre-
treated for 1 h with either TAMRA–Tat (3 μM) or a conjugate of Btn–
Tat plus Texas Red-conjugated Avidin (Avidin–TXR; Life Technologies
Ltd., Paisley, UK) in HAMS F12 medium without phenol red (Stratech
Scientific Ltd., Newmarket, UK). To ensure Btn–Tat–Avidin–TXR com-
plex formation, 1 μM Btn–Tat was mixed with Avidin–TXR at a 3:1
molar ratio for 1 h prior to incubation with cells [24]. Subsequently, exo-
cytosis was either stimulated with increasing concentrations of MP
(0.1–30 μM, for 15 min) or by antigen–antibody-mediated crosslinking
of FcɛR1. Both exocytotic stimuli were performed in a final volume of
250 μl in HAMS F12 medium without phenol red. For antigen stimula-
tion, cells were initially sensitized with 1 μg/ml anti-dinitrophenol
(DNP) IgE (clone SPE-7, Sigma) for 2 h then stimulated with
100 ng/ml DNP-BSA (Albumin from Bovine Serum (BSA), 2,4-
Dinitrophenylated, Life Technologies, Paisley, UK) for the time periods
indicated.
Following stimulation, both extracellular (exocytosed) and intracel-
lular fractions were assayed so as to establish the percentage exocytosis
of accumulated peptide conjugates. Thus, to measure exocytosed fluo-
rescent peptide conjugates, 200 μl medium samples were transferred
to black 96 well plates and analysed using a ThermoFischer Scientific
Fluoroskan Ascent FL fluorescence spectrophotometer (λAbs 544 nm/
λEm 590 nm). 5 μl medium samples were also collected and assayed
for concomitant release of β-hexosaminidase as above. To measure
the intracellular fraction of fluorescent peptide conjugates, cells were
Table 1
Primary sequences of CPPs and bioportides employed in this study. All peptides were pre-
pared with an amidated C-terminus. Sources and synthetic details, where previously pub-
lished, are provided in the references indicated.
Peptide
Sequence
Source
Synthesis
Secretagogues
Mastoparan (MP)
Mitoparan (MitP)
INLKALAALAKKIL
INLKKLAKL(Aib)KKIL
[21]
[22]
[14]
[22]
CPPs
Tat
GRKKRRQRRRPPQ
[5]
[4]
[19]
[25]
[20]
[24]
[24]
[24]
[23]
[13]
Penetratin
C105Y
Cyt c5–13
TP10
RQIKIWFQNRRMKWKK
CSIPPEVKFNKPFVYLI
KGKKIFIMK
AGYLLGKINLKALAALAKKIL
Bioportides
Cyt c77–101
GTKMIFVGIKKKEERADLIKKA
RKKTFKEVANAVKISA
RKLTTIFPLNWKYRKALSLG
LQQRLKKAVPYNRMKLMIV
RVKTLCLQKNTALWI
[25]
[6]
[6]
[26]
[26]
[23]
[6]
[6]
[24]
[24]
Nosangiotide
Camptide
LRRK21322–1340
LRRK22413–2427

110
J. Howl, S. Jones / Journal of Controlled Release 202 (2015) 108–117
treated with 0.1% (v/v) Triton X-100 (250 μl/well, for 5 min on ice) and
200 μl lysate samples collected for fluorimetric analyses. 5 μl cell lysate
samples were also collected and assayed to determine the intracellular
content of β-hexosaminidase.
3. Results
3.1. Comparative secretory efficacies of CPPs and bioportides
To further address the question of whether all polycationic CPPs and
bioportides stimulate the regulated secretion of β-hexosaminidase, our
initial experiments compared the secretory efficacies of peptides with
diverse sequences and biological activities (Table 1). As indicated
(Fig. 1), three CPPs that contain sequences related to MP, a wasp
venom peptide containing multiple lysine residues (MP, MitP and
TP10), all promoted β-hexosaminidase secretion when exogenously
applied at a concentration of 10 μM. Penetratin, Cyt c77–101, camptide
and LRRK22413–2427 were also secretagogues whilst other polycationic
sequences, including the vectors Tat and C105Y, were inert in these
assays.
We have previously reported that relatively low concentrations of
TP10 (≤3 μM) can be used to deliver bioactive peptides to RBL-2H3
cells without promoting secretory responses [13]. Thus, we also deter-
mined the concentration-dependent secretory efficacies of selected
CPPs to positively identify vector sequences that could be used to
deliver bioactive payloads to MCs. As indicted (Fig. 2), these compara-
tive investigations identified C105Y as a most suitable inert CPP vector.
Similar studies also confirmed that the Tat sequence failed to promote
β-hexosaminidase secretion even at a maximum concentration of
30 μM. These data also confirmed that secretory responses to TP10
become significant at concentrations N 3 μM.
2.4. Live cell scanning confocal microscopy
RBL-2H3 cells were transferred to 35 mm sterile glass base dishes
(IWAKI, Japan) and grown to ~75% confluence. Immediately prior to
the addition of fluorescent peptides and/or other materials, cells were
washed with and transferred into phenol red free HAMS F12 medium.
During the period of exposure to peptides, cell layers were maintained
at 37 °C in a humidified atmosphere of 5% CO2. Immediately prior to ob-
servation, cells were then washed gently with phenol red free HAMS
F12 (8×) and analysed with a Carl Zeiss LSM510Meta confocal micro-
scope equipped with a live cell imaging chamber.
For the visualization of acidic secretory lysosomes, cells were incu-
bated with LysoSensor™ Green DND-189 (1 μM), a pH-dependent lyso-
somal stain (pKa 5.2) which is brightly fluorescent at acidic pH
(Molecular Probes, Invitrogen, Paisley, UK). To visualize the intracellular
localization of Tat-delivered Avidin, 1 μM Btn–Tat was mixed with Avi-
din–TXR at a 3:1 molar ratio as described above and subsequently incu-
bated with RBL-2H3 cells for 1 h. To visualize the TAMRA chromophore
conjugated to Tat and other CPPs in this study, cells were treated with
3 μM of TAMRA-labelled peptide for the time periods indicated. All con-
focal analyses were carried out in phenol red free HAMS F12, except for
those cells treated with TP10. Establishment of the intracellular distri-
bution of TP10 was carried out in phenol red free DMEM so as to
avoid complications associated with secretory events.
All quantitative co-localization analyses were performed using the
Carl Zeiss analysis software incorporating an interactive scatter plot
and data table linked to the images [22]. Data were collected from 4 en-
tire images and from 3 independent experiments. Co-localization
(coloc) coefficients were calculated for the two different fluorophores
according to the following equations: C1 = pixels channel 1 coloc/pixels
channel 1 total, C2 = pixels channel 2 coloc/pixels channel 2 total and
defined as relative number of co-localizing pixels in channel 1
(designated rhodamine) or 2 (designated fluorescein), respectively, as
compared to the total number of pixels above threshold. Co-
localization coefficients generated values from 0 to 1.0 whereby, a
value of 1.0 for channel 1 (designated rhodamine) and a value of 0.4
for channel 2 (designated fluorescein) would indicate that 100% rhoda-
mine pixels co-localized with fluorescein pixels and 40% fluorescein
pixels co-localized with rhodamine.
7
*
6
5
4
*
3
2.5. Quantitative analyses of CPP translocation
*
*
*
Translocation efficacies of both TAMRA–Tat and the Btn–Tat–Avidin–
TXR non-covalent complex were determined by quantitative uptake
analyses of fluorescently labelled moieties in CPP-treated and control
cells [23]. RBL-2H3 cells were transferred to six-well plates and grown
to 80% confluence. After washing in HAMS F12 medium without phenol
red, cells were incubated with 1 μM TAMRA–Tat or a Btn–Tat–Avidin–
TXR complex (see Section 2.3.2. above) in phenol red-free HAMS F12
medium for 1 h at 37 °C in a humidified atmosphere of 5% CO2. To
fully discount any fluorescence arising from membrane-associated Avi-
din–TXR, control cells were also treated with Avidin–TXR (0.33 μM)
alone, fluorescence values for which were subtracted from those values
obtained for cells treated with Btn–Tat–Avidin–TXR. After 1 hour incu-
bation with CPP conjugates cells were washed four times, detached
with 300 μl of 1% (w/v) trypsin at 37 °C, collected by centrifugation
and lysed in 300 μl 0.1 M NaOH for 2 h on ice. 250 μl aliquots of cell ly-
sates were transferred to black 96-well plates and analysed using a
ThermoFischer Scientific Fluoroskan Ascent FL fluorescence spectro-
photometer (λAbs 544 nm/λEm 590 nm).
2
1
0
*
*
0
2
t
4
1 3
e
e
0
P
n
1 0 1
-
t
i
5 -
d
i
1 3
2 4 2 7
1
ta
d
i
t
M P
0 5 Y
ti
7 7
a
c
M
1 3 -
4
r
1
p t
o
T P
t
t
3 2 2 -
C
1
m
t
c
n e
C y
n g i
2
c a
a
C y
K 2
K
p e
o s
R
n
R R
L R
L
Fig. 1. Comparative secretory activities of CPPs and bioportides. RBL-2H3 cells were treat-
ed with peptides (10 μM) for 15 min. Data are means S.E.M., n = 6, from 2 independent
experiments. Significant enhancements in the secretion of β-hexosaminidase,
here expressed a fold/basal (cells treated with vehicle only), are indicated by an asterisk
(*, P b 0.005, unpaired, non-parametric, Mann Whitney test).

J. Howl, S. Jones / Journal of Controlled Release 202 (2015) 108–117
111
1 0
9
C 105Y
cam p tid e
T P 10
p e n e tra tin
8
C y t c 7 7 -1 0 1
T a t
7
6
5
4
3
2
1
0
-7 .0
-6 .5
-6 .0
-5 .5
-5 .0
-4 .5
-4 .0
lo g { [p e p tid e ] (M ) }
Fig. 2. Concentration-dependent secretory activities of selected CPPs. RBL-2H3 cells were
treated with increasing concentrations of CPPs for 15 min at the concentrations indicated.
Data points are means S.E.M., n = 6, from 2 independent experiments.
Fig. 3. Calcium sensitivity of peptide-induced secretion. RBL-2H3 cells were treated with
10 μM peptide in the presence (filled bars) and absence (open bars) of 5 mM EGTA.
Data points are mean S.E.M., n = 6, from 2 independent experiments.
3.2. Calcium sensitivity reveals two mechanisms of CPP-induced β-hexosa-
minidase secretion
The exogenous application of ethylene glycol-bis(2-
aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA; 5 mM), to
greatly reduce [Ca2+]o, markedly influenced CPP-induced secretion of
β-hexosaminidase (Fig. 3). These data indicate that the secretory effica-
cies of Cyt c77–101, penetratin and camptide are entirely dependent
upon Ca2+-influx. In contract, a component of the secretory response
to MP-related sequences is independent of Ca2+o and almost certainly
a consequence of G protein activation [10–12].
(mean
S.E.M.) Avidin–TXR was coincident with these structures.
These images also indicate that a component of the fluorescent signal
derived from Avidin–TXR is associated with the plasma membrane.
Thus, in order to quantify the Btn–Tat (1 μM)-mediated accumulation
of Avidin–TXR (0.33 μM) into RBL-2H3 cells, translocation efficacies
were quantitatively determined [23]. Analyses of the intracellular accu-
mulation of TAMRA–Tat (1 μM) were also performed to provide compar-
ative data. Translocation efficacies (mean
S.E.M. n = 6) were
3.3. Intracellular accumulation of Btn–Tat–Avidin–TXR conjugates and
calculated to be 4.1 1.2 for TAMRA–Tat and 4.9 0.8 for Btn–Tat–Av-
idin–TXR. These data indicate that Tat transduction of both TAMRA and
avidin are highly efficient processes in RBL-2H3 cells and confirm that
both cargoes are efficiently delivered to intracellular compartments as
observed by confocal analyses.
TAMRA-labelled CPPs
3.3.1. Tat-conjugates are effectively delivered and differentially distributed
in intracellular compartments of RBL-2H3 cells
The secretory granules of RBL-2H3 cells are of lysosomal nature,
acidic and readily visualized by endosomal markers such as
LysoSensor™ Green DND-189 [15,16]. Fig. 4a,b clearly demonstrates
that TAMRA–Tat (3 μM), within 15 min, whilst adopting a punctate
and vesicular distribution, preferentially locates within acidic secre-
tory granules following membrane translocation. Moreover, co-
localization coefficient analyses further demonstrated that 95.3
3.3.2. Temporal accumulation of TAMRA-conjugated CPPs into secretory
granules of RBL-2H3 cells
As an extension of the above findings, RBL-2H3 cells were also treat-
ed with a range of TAMRA-conjugated CPPs (3 μM) and acidic secretory
granules were similarly identified using LysoSensor™ Green DND-189
(1 μM). As with TAMRA–Tat (Fig. 4), the CPP conjugate TAMRA–C105Y
rapidly, within 15 min, accumulated within secretory granules
(96.8 0.9%, mean S.E.M.; Fig. 5a, b). In contrast, TAMRA–penetratin
demonstrated only a modest degree of lysosomal colocalization
0.8% (mean
S.E.M.) of intracellular TAMRA–Tat was coincident
with these structures.
As a non-covalent complex with Btn–Tat (1 μM), Avidin–TXR
(0.33 μM) is also efficiently delivered into the interior of RBL-2H3 cells
(Fig. 4c,d). However, and in contrast to TAMRA–Tat, this large protein as-
sumes a punctate and vesicular distribution which predominantly accu-
(47.3
colocalization (94.2
Moreover, following 30 min of treatment, TAMRA–TP10 showed
3.3%, mean + S.E.M.) after 30 min (Fig. 5c) and a maximal
1.9%, mean S.E.M.) after 55 min (Fig. 5d).
mulates outside of secretory lysosomes, whereby only 24.0
6.0%

112
J. Howl, S. Jones / Journal of Controlled Release 202 (2015) 108–117
LysoSensorTM Green
Merge
TAMRA-Tat
Merge + DIC
a
b
LysoSensorTM Green
Merge
Btn-Tat:Avidin TXR
c
d
Fig 4. Differential intracellular distributions of TAMRA–Tat and Btn–Tat–Avidin–TXR conjugates in RBL-2H3 cells. Cells were treated with LysoSensor™ Green DND-189 (1 μM) so as to vi-
sualize secretory lysosomal structures. Further incubation with TAMRA–Tat (3 μM) and subsequent visualization by live confocal cell imaging demonstrated that TAMRA–Tat readily (with-
in 15 min) accumulated within secretory granules as designated by yellow colocalization (a, merge). A more detailed visualization of this colocalization event is presented using digital
zoom (b). In contrast (c, d), the intracellular distribution of Btn–Tat–Avidin–TXR (1 μM) demonstrated a predominantly cytoplasmic location outside secretory lysosomal structures. Con-
focal images are also presented here merged with images taken under transmission differential interference contrast (DIC) so as to assist with visualization of cellular morphology, cellular
integrity and subcellular distribution of TAMRA–Tat (a, b merge + DIC) and Btn–Tat–Avidin–TXR (d). Scale bar = 20 μm in all figures. (For interpretation of the references to color in this
figure legend, the reader is referred to the web version of this article.)
minimal colocalization (15.84 5.4%, mean S.E.M.) with secretory
granules (Fig. 5e) and at 60 min demonstrated a maximal colocalization
of 38.0 4.1% (mean S.E.M.; Fig. 5f). From these observations, it is
noteworthy that those CPPs such as Tat and C105Y, which do not induce
mast cell degranulation, readily accumulate within acidic secretory
vesicles of RBL-2H3. In contrast TP10, a CPP with significant secretory
efficacy at concentrations N 3 μM, predominantly accumulated outside
of these intracellular structures.
3.4.1. MP-induced secretion
MP is a potent peptide secretagogue that promotes the exocytosis of
both β-hexosaminidase, and histamine from RBL-2H3 [12,14]. Signifi-
cantly, the identification of differential peptide secretagogues [14] and
other biochemical assays (see Section 4) indicate that histamine, like
Tat-translocated Avidin–TXR, maintains a predominantly cytoplasmic
localisation in RBL-2H3 cells. Fig. 6a indicates that exogenous application
of MP to cells loaded with TAMRA–Tat produced a concentration-
dependent exocytosis of the TAMRA cargo (EC50 ~ 8 μM). TAMRA
release reached ~80% at 30 μM MP. The concomitant exocytosis of β-hex-
osaminidase (Fig. 6b) was also concentration-dependent but with an
increased EC50 of ~30 μM and a maximum release of ~50% at 30 μM MP.
MP also induced a concentration-dependent secretion of accumulat-
ed Avidin–TXR (EC50 ~ 25 μM; Fig. 6c) with maximum secretion of ~55%
3.4. Exocytosis of accumulated cargoes
The main objective of these studies was to evaluate whether cargoes
predominantly located within (TAMRA) and outside (avidin) secretory
lysosomes could be released by exocytosis.

J. Howl, S. Jones / Journal of Controlled Release 202 (2015) 108–117
113
LysoSensorTM Green
Merge
Merge + DIC
TAMRA-CPP
C105Y (15 min)
a
b
c
d
e
f
C105Y (15 min)
Penetratin
(30 min)
Penetratin
(55 min)
TP10 (30 min)
TP10 (60 min)
Fig. 5. Temporal accumulation of TAMRA-conjugated CPPs into secretory granules of RBL-2H3 cells. Cells were treated with a range of TAMRA-conjugated CPPs (3 μM) and LysoSensor™
Green DND-189 (1 μM), so as to visualize acidic secretory lysosomes. Cells were subsequently viewed by live confocal cell imaging analysis. Colocalization of TAMRA-conjugated CPPs with
secretory lysosomal structures is shown as yellow (merge). As with TAMRA–Tat (Fig. 4), the CPP C105Y rapidly, within 15 min, accumulated within secretory granules (a). A more detailed
visualization of this colocalization event is presented using digital zoom (b). In contrast, TAMRA–penetratin demonstrated only a minor degree of lysosomal colocalization after 30 min
(c) and a maximal colocalization after 55 min (d). Moreover, following 30 min of treatment, TAMRA–TP10 does not colocalize with secretory granules (e) and only after 60 min minimal
colocalization is detected (f). Confocal images are presented here merged with images taken under transmission differential interference contrast (DIC) so as to assist with visualization of
cellular morphology, cellular integrity and subcellular distribution of TAMRA-conjugated CPP. Scale bar in figures a, c, d and e = 20 μm, b and f = 10 μm. (For interpretation of the refer-
ences to color in this figure legend, the reader is referred to the web version of this article.)
of total at 30 μM MP. The concomitant release of β-hexosaminidase in
these experiments is indicated in Fig. 6d (EC50 ~ 17 μM, maximum
release ~95% at 30 μM MP). Comparison of Fig. 6b and d reveals that
both the magnitude and efficacy of MP-induced β-hexosaminidase
exocytosis is reduced in cells treated with TAMRA–Tat compared with
cells treated with Btn–Tat–Avidin–TXR.
3.4.2. IgE/DNP-induced secretion
The degranulation of MCs is usually a consequence of antigen-
induced aggregation of the multi-subunit high-affinity IgE receptor
(FcεRI). Thus, we also investigated whether this endogenous pathway
of MC activation would induce the exocytosis of accumulated cargoes
from RBL-2H3 cells. As indicated (Fig. 7a), the cross-linking of FcεRI

114
J. Howl, S. Jones / Journal of Controlled Release 202 (2015) 108–117
90
80
70
60
50
40
30
20
10
0
50
40
30
20
10
0
-6.0
-5.5
-5.0
-4.5
-4.0
-6.0
-5.5
lo g
-5.0
-4.5
-4.0
{
[M P ] (M ) }
lo g { [M P ] (M ) }
60
50
40
30
20
10
0
100
80
60
40
20
0
-6.0
-5.5
-5.0
-4.5
-4.0
-6.0
-5.5
-5.0
-4.5
-4.0
lo g { [M P ] (M ) }
lo g { [M P ] (M ) }
Fig. 6. MP-induced exocytosis of accumulated cargoes. Fig. 6a (Top left panel), RBL-2H3 cells were treated with TAMRA–Tat (3 μM) for 1 h then stimulated with increasing concentrations of
MP. Fig. 6b (Top right panel), concomitant release of β-hexosaminidase in the same experiments as Fig. 6a. Fig. 6c (Bottom left panel), RBL-2H3 cells were treated with Btn–Tat–Avidin–TXR
(1 μM) for 1 h then stimulated with increasing concentrations of MP. Fig. 6d. (Bottom right panel), concomitant release of β-hexosaminidase in the same experiments as Fig. 6c. Data points
are means S.E.M., n = 6 (Fig. 6a, c and d) or n = 12 (Fig. 6b), from 2 independent experiments.
released accumulated TAMRA–Tat in a time-dependent mechanism over
a period of 0–30 min. Concomitant release of β-hexosaminidase follow-
ed a near identical time course under the same conditions (Fig. 7b). Sim-
ilarly, a time-dependent efflux of Avidin–TXR was also observed in
response to FcεRI cross-linking (Fig. 7c) that was again also accompa-
nied by an efflux of β-hexosaminidase over the same time period
(Fig. 7d).
response initiated by the degranulation of MCs (reviewed in [29]). In
concordance with these findings, the regulated-secretion of β-hexosa-
minidase from RBL-2H3 is induced by MP and its structural analogues
but is relatively insensitive to a diverse range of membrane imperme-
able polycationic peptides including hormones and neuropeptides
[14]. As CPPs and bioportides are intrinsically cell permeable there is
an obviously increased likelihood that these sequences could act like
MP to promote exocytosis through the interaction with intracellular G
proteins or other targets intrinsic to regulated secretory mechanisms
[14,29]. It is known that TP-10, a chimeric CPP that includes the
sequence of MP as the carboxyl-terminal [20], can be used to affect
the intracellular delivery of bioactive peptide cargoes into RBL-2H3
in the absence of secretion when exogenously employed at
concentrations ≤ 3 μM [13]. However, until we completed the studies
described herein, it was unknown whether other CPPs could be used
4. Discussion
The observation that exocytosis from MCs can be stimulated in a
receptor-independent fashion by cationic peptides was first reported
more than three decades ago [10,11]. Moreover, MP [21] and related
polycationic peptide components of wasp venoms have seemingly
evolved to exploit this mechanism and trigger a localized inflammatory

J. Howl, S. Jones / Journal of Controlled Release 202 (2015) 108–117
115
16
50
40
30
20
10
0
12
8
4
0
0
5
10
15
20
25
30
35
0
10
20
30
T im e (m in )
T im e (m in )
50
40
30
20
10
0
30
20
10
0
0
10
20
T im e (m in )
30
0
10
20
T im e (m in )
30
Fig. 7. Time course of IgE/DNP stimulated-exocytosis of accumulated cargoes. Fig. 7a (Top left panel), cells were treated with TAMRA–Tat (3 μM) for 1 h prior to FcɛR1-stimulated exocy-
tosis. Fig. 7b (Top right panel), concomitant release of β-hexosaminidase in the same experiments as Fig. 7a. Fig. 7c (Bottom left panel), cells were treated with Btn–Tat–Avidin–TXR (1 μM)
for 1 h prior to FcɛR1-stimulated exocytosis. Fig. 7d (Bottom right panel), concomitant release of β-hexosaminidase in the same experiments as Fig. 7c. Data points are means S.E.M., n =
6, from 2 independent experiments.
to deliver payloads to RBL-2H3 in a relatively inert fashion. This was an
important question given that some CPPs, specifically those which ele-
vate [Ca2+]i, can trigger lysosomal exocytosis from cells such as the
HeLa lines that are considered non-secretory [30]. In concordance with
our results, the Tat CPP has no influence upon β-hexosaminidase release
from HeLa cells even at a concentration of 20 μM [30]. Our data also
identify C105Y as a very promising CPP that could be employed to deliv-
er cargoes into MCs without promoting exocytosis [19]. Indeed, com-
parative investigations in spermatozoa [30] have also identified C105Y
as a highly efficient CPP despite a relatively modest cationic charge den-
sity. Clearly, rationally-designed bioportides could also be employed
both to decipher and modulate the exocytotic mechanisms of MCs
that are fundamental to their multiple roles in acquired and innate
immunity.
Data presented herein also distinguish two modes of action by
which CPPs and bioportides induce β-hexosaminidase secretion. MP,
MitP and TP10 interact with intracellular protein targets, most likely
heterotrimeric G proteins of the Gi subtype [10–12,31], and secretory
responses to these peptides are only partially attenuated by the addition
of EGTA to reduce [Ca2+]o. In contract, the secretory actions of
penetratin, Cyt c77–101 and camptide are clearly dependent upon
[Ca2+]o and probably the consequence of a generalised Ca2+-influx in
response to plasma membrane perturbation [30,32]. The secretory
activity of camptide, a bioportide known to activate G proteins and
elevate the second messenger cyclic adenosine monophosphate [6],
can be explained by a selective activation of Gs rather than Gi.
The differential accumulation of TAMRA–Tat and Btn–Tat–Avidin–
TXR, following Tat transduction, provides anatomical evidence to sup-
port the contention that RBL-2H3 contains distinct pools of bioactive
mediators regulated by different signalling pathways. The secretion of
the enzyme β-hexosaminidase, a discrete marker of the relatively
small irregularly-sized secretory lysosomes in RBL-2H3 [36], is a phos-
pholipase D (PLD)-dependent mechanism [14]. In contrast, RBL-2H3
cells maintain a more freely accessible “cytoplasmic” pool of 5-HT
[28], the secretion of which does not correlate with PLD activity [14].
The differential release of 5-HT and histamine from rat peritoneal
mast cells [37] provides additional evidence that other MCs also main-
tain discrete pools of secretory mediators, the release of which does
not necessarily correlate with the exocytosis of secretory granules. Our
confocal studies confirmed that secretory lysosomes are freely accessi-
ble to Tat when conjugated to a relatively small cargo like TAMRA, but
less so to other CPPs including penetratin and TP10. However, the intra-
cellular distribution of Btn–Tat–Avidin–TXR did not co-localize with the
same organelles despite the fact that the delivery of avidin as non-

116
J. Howl, S. Jones / Journal of Controlled Release 202 (2015) 108–117
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covalent complex with Btn–Tat is a relatively efficient process in RBL-
2H3 cells. This latter observation is curious considering that RBL-2H3 se-
cretory lysosomes are accessible to endocytotic markers [15,16] and
that endocytosis, in its various guises, is expected to be the predominant
mechanism of entry of Tat-conjugated proteins [33–35]. Fragments of
IgE are known to slowly accumulate in secretory lysosomes after
endocytosis and these can be secreted upon further stimulation [16].
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important to note that, in comparison with RBL-2H3 cells [15,16,38],
primary human MCs possess multiple heterogeneous types of secretory
vesicle [38,39] some of which can accumulate and degranulate large
molecular weight components of Major Histocompatibility Complex
Class II proteins [39]. Thus, one can reasonably predict that the distribu-
tion of Tat-translocated cargoes in other MC types could differ from the
discrete pattern observed in RBL-2H3. Nevertheless, we anticipate that
Tat transduction, or indeed the delivery of bioactive cargoes by other
inert CPPs including C105Y, could be readily adapted to load human
MCs with an exocytosis-competent cargo without inducing exocytosis.
As we demonstrate herein, both small molecular weight cargoes
(TAMRA) and much larger proteins (avidin) are effectively released by
both FcɛR1-dependent and receptor-independent secretory stimuli. In
these studies, the measurement of β-hexosaminidase secretion was
performed essentially as a control to confirm that RBL-2H3 cells
remained exocytosis competent after Tat transduction. Moreover, our
experimental protocols enabled us to routinely assay the enzymatic ac-
tivity of β-hexosaminidase in the very same samples used to determine
the efflux of TAMRA–Tat or Avidin–TXR. A variety of factors may be re-
sponsible for the variations recorded in the kinetics of β-hexosamini-
dase release from cells stimulated with either MP (Fig. 6) or IGE/DNP
(Fig. 7). Firstly, the efflux of Avidin–TXR is from a predominant cytoplas-
mic store whilst β-hexosaminidase is a well-documented secretory
lysosome marker [11–16]. Thus, secretagogues may have a differential
efficacy to promote secretion from these different pools [14].
Polycationic CPPs or even the avidin protein might also interfere with
regulated secretory mechanisms at a variety of proximal and/or distal
sites leading either to a modulation in β-hexosaminidase secretion or
changes in enzymatic activity. Nevertheless, it is certain that Tat trans-
duction can be employed to deliver bioactive cargoes into MCs which
are stored and subsequently released in response to exocytotic stimuli.
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cargoes are not detrimentally modified by other macromolecules stored
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Acknowledgments
Support for the synthesis of LRRK2-derived peptides was provided
by the Michael J Fox Foundation in New York.
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