Supporting Information
for
Adv. Mater.
, DOI 10.1002/adma.202307106
Truly Tiny Acoustic Biomolecules for Ultrasound Imaging and Therapy
Bill Ling, Bilge Gungoren, Yuxing Yao, Przemysław Dutka, Reid
Vassallo, Rohit Nayak,
Cameron A. B. Smith, Justin Lee, Margaret B. Swift and Mikhail G
. Shapiro*
1
Supplementary Information
Truly tiny acoustic biomolecules for ultrasound diagnostics
and therapy
Bill Ling
1
, Bilge Gungoren
1
, Yuxing Yao
1
,
Przemysław Dutka
1,2
,
Reid Vassallo
3
, Rohit
Nayak
1
,
Cameron A. B. Smith
1
,
Justin Lee
2
,
Margaret B. Swift
1
,
Mikhail G. Shapiro
1,
4
,
5
,*
1. Division of Chemistry and Chemical Engineering, California Institute of Technology,
Pasadena, CA, USA
2. Division of Biology and Bioengineering, California Institute of Technology, Pasadena,
CA, USA
3. School of Biomedical Engineering, University of British Columbia, Vancouver, BC,
Canada
4
. Division of Engineering and Applied Science, California Institute of Technology,
Pasadena, CA, USA
5
. Howard Hughes Medical Institute, California Institute of Technology,
Pasadena, CA,
USA
2
Figure S1
. Impact of gene deletions on GV morphology.
a
)
Representative TEM images
of GVs expressed using the indicated gene cluster. A2C represents the full GV cluster
[1]
.
Deletions are indicated by ∆. Scale bars, 100 nm.
b
)
Mean length and diameter of
individual particles measured in images from panel A.
N
= 50
-
100 particles. Error bars, ±
SD.
3
Figure S2.
Bicone suspensions are stable for at least 5 weeks at 4°C.
a)
Representative TEM images of purified bicones. Scale bars, 200 nm.
b)
Intensity
-
weighted DLS measurements. Samples from the same bicone preparation are
connected by a black line.
N
= 5.
Paired t
-
test, (*, p < 0.05; n.s, p ≥ 0.05).
c)
Representative BURST images of samples after the indicated time at 4°C. Scale bars, 1
mm.
d)
BURST signal within wells of samples from panel c. Samples from the same
preparation are connected by a black line.
N
= 6. Welch’s t
-
test, (n.s, p ≥ 0.05).
4
Figure S
3
.
To estimate the physical properties of bicones
, we modeled each particle as
two identical right circular cones joined at their bases.
Total volume (0.0298 aL) was
calculated u
sing the mean length and diameter from our cryo
-
EM measurements (Fig.
1d)
. We then assumed a uniform, 2
-
nm thick protein shell, yielding
an inner gas volume
of 0.0228 aL.
By further assuming the shell density to be 1.4 g mL
-
1
, we calculated a
mass of approximately
9.86e
-
15 mg, equivalent to a molecular weight of 5.94 MDa
[2]
.
Neglecting contributions from other potential structural components, t
his translates to
~800 GvpA monomers per particl
e.
Figure S
4
.
Comparison of hydrostatic (
a
) and acoustic (
b
) collapse midpoints for
bicones relative to GVs isolated from
H. salinarum
and
A. flos
-
aquae
[3]
, or
recombinantly
expressed in
E. coli
using a cluster native to
B. megaterium
[4]
.
5
Figure S
5
.
a)
Representative B
-
mode images of bicones loaded into an agarose
phantom at various
particle
concentrations. Scale bars, 1 mm.
b)
Quantification of
signal intensities. Data were fit by linear regression, slope = 0.064, r
2
= 0.528.
N
= 24.
Error bars, ± SEM.
6
Figure S
6
.
BURST images of U
-
87 MG tumors acquired 1 h after IV injection of
16.8
pmol b
icones, overlaid on a B
-
mode image
to show anatomy
. A second acquisition
(Post
-
Collapse) at the same location was used to verify that signal was specific to intact
bicones.
Two sets of images were acquired at different planes within each tumor
.
N
= 3.
Scale bars, 1 mm.
7
Figure S
7
.
a
)
Representative
power spectral density
of emissions from
PBS, pre
-
collapsed, and intact bicones (5 nM)
insonated with a single
30 cycle
FUS
pulse at 0.
5
MPa PNP.
b
)
Mean PCD signal
of samples from panel A, calculated
by integrating
spectra from 8 MHz to 27 MHz.
PBS,
N
= 16; collapsed,
N
= 6; intact,
N
= 24.
Error
bars, ±
SEM. Welch’s t
-
test (*
*
*, p < 0.0
0
1
; ****, p < 0.0001
).
8
Figure S
8
.
Bicones produce comparable PCD signal to
Anabaena
GVs at the same gas
volume.
a
-
b)
Representative power spectral densities of acoustic emissions following
insonation of bicones (4.2 nM) and
Anabaena
GVs (OD 0.25) with a single FUS pulse at
0.3 MPa PNP (
a
) or 0.5 MPa PNP (
b
).
c
-
d
)
Mean PCD signal following insonation at 0.3
MPa PNP (
c
) or 0.5 MPa PNP (
d
). Spectra were integrated from 8
-
27 MHz.
N
= 3
-
24.
Error bars, ± SEM. Welch’s t
-
test, (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p <
0.0001; n.s, p ≥ 0.05).
9
Figure S
9
.
Power spectral density of emissions from intact bicones (5 nM) insonated
with a single 60 cycle 670 kHz FUS pulse at 0.3 MPa PNP.
N
= 3. Individual trials are
shown in gray, and the mean is shown in black. Harmonic signals indicative of stable
cavitation are annotated with blue arrows.
Figure S
10
.
ANS fluoresces in the presence of collapsed bicones.
a
)
Representative
fluorescence emission spectra (ex. 380 nm) of bicone samples with 100 μM ANS
following collapse by bath sonication or hydrostatic pressure.
b
)
Fluorescence intensity
at 480 nm in spectra from panel A.
N
= 4
-
6.
Error bars, ±
SEM. Welch’s t
-
test (*, p
<
0.05; ***, p
<
0.001).
10
Figure S
11
.
Fluorescence of suspensions containing 100 μM ANS mixed with the
indicated concentration of collapsed bicones. Data were fit by linear regression, slope =
0.3447
, r
2
= 0.99
.
N
= 4. Error bars, ±
SEM.
11
Figure S
12
.
Bicones remain intact following FUS insonation.
a)
Representative BURST
images of
samples loaded into an agarose phantom following a 30 s exposure to FUS
at various PNPs. Scale bars, 1 mm.
b)
Quantification of BURST signal in images from
panel a.
N
= 4. Error bars, ± SEM.
c)
Representative TEM images.
Images in the
bottom row
are
at a higher magnification.
Scale bars, 200 nm.
d)
Diameters of particles,
measured manually from TEM images.
N
= 400.
12
Figure S
13
.
Anabaena
GVs collapse under FUS insonation.
a
)
Representative
emission spectra of OD 0.25
Anabaena
GVs with 100 μM ANS following insonation at
0.65 MPa PNP, 500 Hz pulse repetition frequency.
b
)
Relative
ANS fluorescence at 480
nm of GVs after
exposure to
1, 10, or 15,000
FUS pulses at
0.65 MPa PNP, normalized
to fluorescence of intact and pre
-
collapsed samples.
N
= 4. Error bars, ±
SEM.
Figure S
14
.
PEGylated bicones are colloidally stable.
a)
Representative number
-
weighted size distributions of particles before
(
blue
)
and after
(red)
5 weeks of storage
at 4 °C, as measured by DLS.
b)
Representative TEM image of PEGylated bicones
after 5 weeks at 4 °C
. Small clusters are present but most particles remain dispersed.
Scale bars, 200 nm.
13
Figure S
1
5
.
Flow cytometry gating strategy for iRGD targeting experiments. Debris was
excluded based on SSC
-
A vs FSC
-
A. Single cells were selected from FSC
-
H vs FSC
-
A.
Bicone positive cells
(dN+)
were defined relative to a cell
-
only control sample.
Figure S
1
6
.
Targeting improves with iRGD loading.
a
)
Representative confocal
microscopy images of cells after incubation with bicones
carrying the indicated
maximum stoichiometry
of iRGD peptides
. Scale bars, 50 μm.
b
)
Mean fluorescence
intensity in the bicone channel of cell regions in images from panel
A
. U
-
87 MG (top)
and HT
-
1080 (bottom) cells were defined by thresholding on autofluorescence in the
DAPI channel.
N
= 4. Error bars, ±
SEM. Welch’s t
-
test, (*, p < 0.0
5
).
14
Figure S1
7
.
SDS
-
PAGE
of
bicones (200 μg
mL
-
1
) and
Anabaena
GVs (OD 10). GvpC
(indicated with an arrow) is not found on bicones.
Lanes from the same gel have been
spliced together to remove empty regions.
Figure S1
8
.
Anti
-
FLAG dot blot of purified bicones containing the indicated FLAG
-
fusion
protein before and after treatment with 6 M urea to remove surface
-
bound proteins.
The
top row (A2C
-
C) shows full
-
size GVs which are known to contain GvpC.
15
Figure S1
9
.
GvpJ and GvpS can also be used to genetically functionalize larger GVs.
a
)
A2C gene cluster. The SpyTag peptide was appended to the end of
G
vpC,
G
vpJ, or
G
vpS. Purified particles were then reacted with SC
-
mNG.
b
)
Fluorescence of purified
samples after conjugation.
N
= 4. Error bars, ±
SEM. Welch’s t
-
test (***, p
<
0.001; ****,
p
<
0.0001).
Table S1.
Physical properties of bicones compared to
Anabaena
GVs.