Preparation of mechano-nanoswitches for ultrasound-controlled drug activation

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PROTOCOL Special Topic - CONTROLLED NANOMEDICINE
MATERIALS
Preparation of mechano-nanoswitches for ultrasound-controlled
drug activation
Zhihuan Liao1#，Junliang Chen1#，Menghan Xiao1，Shuaidong Huo1 ✉
1FujianProvincialKeyLaboratoryofInnovativeDrugTargetResearch,SchoolofPharmaceuticalSciences,Xiamen
University,Xiamen361102,China
Received:25October2024/Accepted:21November2024
Abstract Chemotherapy is often hindered by issues associated with deficient drug selectivity and ineluctable
toxiceffects.The emerging realm ofmechanochemistryhas demonstrated significant promiseinpre-
cisedrugactivationbyusingultrasound-inducedmechanicalforcestoregulatethechemicalproperties
of compounds at the molecular level. Recently, we proved that the successful introduction of nanos-
tructurestomechanochemistrycouldimprovedrugloadingcapacityandenhancetheirmechanicalre-
sponsiveness.Tofurtherexpandtheapplicationoftheultrasound-responsedrugactivationstrategyin
nanosystems,in this context,weillustratethepreparation of a mechano-nanoswitchforspatiotempo-
ralcontrolofdrugactivation.
Keywords Ultrasound,Mechano-nanoswitch,Nanodimer,Drugactivation,Force-sensitive


INTRODUCTION
Controlleddrugrelease is a promising strategyforim-
provingtherapeuticefficacywhilereducing sideeffects
(Baryakovaet al.2023;Mitchellet al.2021;Stateret al.
2021). In recent years, drug delivery systems respon-
sive to specific stimuli have been developed to regu-
late drug release in response to internal triggers or
external stimuli (Fan et al. 2023; Mi 2020). However,
without precise control over drug activity, these ap-
proachesfacelimitations,suchasprematuredrugleak-
age,lowresponsesensitivity,etc.(Tuet al.2021).
The emergence of mechanochemistry brings new
possibilitiesfor altering drugactivitybyutilizingultra-
sound-induced shear force to trigger specific chemical
bond cleaving or rearranging (O’Neill and Boulatov
2021;Zhaoet al.2021).Recently,wepresentedthefirst
exampleofultrasound-inducedmechanochemical bond
cleavagefor drug activation, demonstrating thepoten-
tial of ultrasound for spatiotemporal control of drug
activity(Huo et al.2021). Later, we showed that com-
bining nanoparticle systems with polymer mecha-
nochemistry improves drug loading capacity and
significantlyenhancesmechanicalresponsiveness(Huo
et al. 2022). Taking the reported gold nanodimer and
anticancer drug doxorubicin (DOX) as an example,
herein,we provide a detaileddescriptionof the proto-
col for constructing a mechano-nanoswitch that selec-
tivelyactivatesdrugsbyultrasound.Thenanoparticles
at both ends serve as the conductive arms of the
sonomechanicalforce,whilethedrugloadingsiteinthe
middleis the mechanophore (force-responsive group).
Intheory,therelevantpartscanalsobeadjusted orre-
placed with other nanostructures and mechanophores
accordingly. Due to the particularity of the nanodimer
couplingand preparation process, the yield ofthenan-
odimerisabout8%.High-puritynanodimerscanbeob-
tained through a straightforward gel electrophoresis,
greatlysimplifyingthepurification process.Thisproto-
col provides an approach for creating mechanosensi-
tive nanosystems, offering more precise control over

# Zhihuan Liao and Junliang Chencontributed equally to this
work.
✉Correspondence:huosd@xmu.edu.cn(S.Huo)
BiophysRep2024,10(X):1−7
https://doi.org/10.52601/bpr.2024.240054 Biophysics Reports
©TheAuthor(s)2024 1|December2024|Volume10|IssueX

drug activity and valuable insights for future applica-
tionsinnanomedicine.

STEP-BY-STEP PROCEDURE

Step 1: Preparation of gold nanoparticles (AuNPs)
[TIMING 2–3 d]
Step1.1:Prepareaquaregiainalargebeakerinafume
cupboard by mixing 3:1 (v:v) concentrated HCl:HNO3.
SoakutensilsthatcomeintocontactwithAuNPsduring
synthesis in aqua regia for at least 15 min. Rinse the
utensils with plenty of deionized water and then with
Milli-Qwater(LiuandLu2006).
[TIP] Obtaining high-quality AuNPs is the first step
towardexperimentalsuccess. Take care toensurethat
no contamination is introduced during the AuNPs
synthesisprocess.
[CAUTION] Be extremely careful when preparing
and using aqua regia. Wear goggles and gloves, and
conduct the experiments in a fume cupboard. Aqua
regia should be prepared freshly and never stored in
closedcontainers.
Step 1.2: Load 30 mL of Milli-Q water in a two-
necked flask. Add 0.51 mL of 25 mmol/L HAuCl4
solution.Place the flask ona hotplate and refluxwhile
stirring vigorously. Wait for the solution to boil, then
add1.8 mL of citricacidsolution(30mmol/L)quickly,
andtheheatingceasesafter20min,thencoolnaturally
toroomtemperature(25°C)toformthe15nmcitrate-
protectedAuNPs(Fig.1).
[TIP] The size of nanoparticles plays an important
roleintheirmechanicalresponse.Ingeneral,thelarger
the particle size, the more pronounced its mechanical
response. The particle size selected in this protocol is
around15nmtoprovideatypicalcasecommonlyused.
Step1.3:AddBis(p-sulfonatophenyl)phenylphosphine
dihydrate dipotassium salt (BSPP) rapidly to the mix-
ture to reach a concentration of 0.2 mg/mL and stir
thoroughly overnight at room temperature (25 °C) to
facilitatethemodificationoftheAuNPssurface.
[TIP]TheprincipalroleofBSPPistofacilitatestabil-
ity and surface charge optimization for AuNPs, thus
providing a stable and negatively charged surface for
subsequentDNAbinding.Thisstepenhancestheeffica-
cyand reproducibility ofexperimental procedures (Jin
et al.2015).

Heat to boiling
Stir overnight
Heat
NaCl
Cool to room
temperature (25°C)
7000 r/min Age
25 mmol/L
HAuCl4
+
Milli-Q water
0.2 mg/mL
Remove supernatant
Resuspend
30 mmol/L
Stir vigorously 20 min
3 min
Incubate
Overnight
O-K+
P
SO
O
P
SO
O
K+O-
S
O
O
P
SO
O
K+O-
O-
K+
S
O
O
S
O
O
O
-
K
+
O
-
K
+
O
OO
O
O
O
O
O
O
O
O
O
Na+O-
O-Na+
OK
KO
P
S
O
O
S
O
O
2H
2
O
O
O
O
HO
Na
+
O
-
O
-
Na
+
Na
+
O
-
Na+O-
Na+O-
Na+O-
O-Na+
O-Na+
O-Na+
Fig. 1TheworkflowofpreparationofAuNPs
PROTOCOL Z.Liaoet al.
2|December2024|Volume10|IssueX ©TheAuthor(s)2024

Step 1.4: Add solid sodium chloride (NaCl) to the
modifiedAuNPsuntilacolorchangefromredtoblueis
observed. The addition of NaCl shields the surface
charge of the AuNPs, resulting in AuNPs aggregation
(solution color changes from red to blue). Then,
incubate the mixture overnight at room temperature
(25 °C) to ensure complete interaction. Centrifuge the
mixtureat7000r/min for 3 min toremovethesuper-
natant.Afterremovingthesaltsolution,resuspendwith
Milli-QwatertocompletetheagingprocessoftheAuNPs.
When NaCl is removed, the surface charge is restored
and the particles are dispersed again (solution color
returnstored).
[TIP] Perform this step directly with a 50 mL cen-
trifugetubetoimprovetheagingefficiency.Atthispoint,
the AuNPs will be collected at the bottom of the cen-
trifuge tube. Carefully remove the supernatant and do
notremovetheAuNPs.
Step 2: Preparation of Au-DNA dimer mechano-
nanoswitches [TIMING 2–3 d]
Step2.1:Dissolve the terminally double-thiolatedDNA
aptamerinannealingbuffer(200mmol/LKCl,4mmol/L
MgCl2, and 28 mmol/L Tris-HCl), and then anneal at
100 °C for 5 min. Cool the mixture slowly to 25 °C to
form a double-stranded DNA structure (closed con-
figuration)(Fig.2).
[TIP] Before dissolving with buffer solutions,
centrifugethe test tube containing aptamerpowder at
4000r/min andcollecttothebottom ofthetube.Once
thestockingsolutionisprepared,it shouldbestoredat
4°C.
Step 2.2: Prepare Au-DNA conjugates by mixing
AuNPs with the aptamer (TCEP treated) in a molar
ratioof3:1,andincubatethemixturein0.5×TBEbuffer
(containing 50 mmol/L NaCl) for 12 h. Adding TBE
buffer may cause AuNPs aggregation, and the solution
may turn red to blue. Following incubation, centrifuge
the solution at 7000 r/min for 2 min to facilitate the
separation of the supernatant, which is then carefully
removed. After eliminating the salt solution, concen-
tratetheremainingsolutiontoapproximately20μL.
[TIP] Before coupling with AuNPs, introduce tris(2-
carboxyethyl)phosphine hydrochloride (TCEP) to re-
duce the disulfide bonds present in the primer. This
step is crucial to prevent potential cross-linking be-
tweentheprimers’ terminal sulfhydryl groupsandthe
polymerduringsubsequentannealingoperations.
Step 3: Purification of Au-DNA dimer mechano-
nanoswitches [TIMING 2-3 d]
Step3.1: Weigh 1.2 gof agarose powderand mix with
40mLof0.5×TBEbuffertoachievea3%(w/v)agarose
concentration. Shake the mixture thoroughly and then
heatinamicrowaveovenfor2minuntiltheagaroseis
completelydissolved.Afterheating,coolthesolutionto
approximately 65 °C. Subsequently, pour the agarose
solution meticulously into a casting tray and cool
naturally.Insert acombverticallyintothegelto create
wells,andsolidifythegelat25°Cfor2h(Fig.3A).

Annealing buffer TE buffer
100 °C
+
TCEP
+
Anneal 0.5× TBE buffer
12 h
7000 r/min
2 min
DNA
DNA
(open)
5 min
25 °C
DNA
(closed)
Fig. 2TheworkflowofpreparationofAu-DNAdimermechano-nanoswitches
Preparationofmechano-nanoswitchesforultrasound-controlleddrugactivation PROTOCOL
©TheAuthor(s)2024 3|December2024|Volume10|IssueX

[TIP]Cleanthegelatinemoldmeticulouslyeachtime
toprevent contamination. It isessentialto ensure that
themeltedagaricisadequatelyshakenandmeticulous-
ly poured onto the gelatin plate, as this will minimize
theriskofobtainingsuboptimalresultslater.Theselec-
tionof anappropriateagaroseconcentration isofcriti-
cal importance for the successful isolation of Au-DNA
dimers.Based ontheresultsof ourexperimentaltrials,
3%(w/v)agarosewasidentifiedastheoptimalgelcon-
centration.
Step3.2:Mix15μLAu-DNAconjugatessolutionwith
5μLglycerinandloadgentlyintoanagarosegel.Then,
carefullyload thesolutionintothewells oftheagarose
gel.As a control, AuNPs that haveundergonetheaging
processbutwithoutmodificationwithaptamerarealso
included in the experiment. The electrophoresis is
conductedatavoltageof130Vfor30minina0.5×TBE
runningbufferwithintheelectrophoresistank(Fig.3B).
[TIP] The electrophoresis tank provides sufficient
coverageof the agarosegel, with approximately 20 μL
per well. Furthermore, the inclusion of glycerol in the
mixtureisrecommendedtopreventthecontentsofthe
wells from migrating out during the electrophoresis
process. This precaution is to maintain the samples’
integrityandachieveclearanddistincttargetbandsfor
analysis.
Step3.3:Followingelectrophoresis,meticulouslyre-
move the agarose gel and carefully cut the third tar-
get band. Subsequently, recover the cut band with a
D-Tube™ electroelution accessory kit through another

Agarose powder
0.5× TBE buffer
Heat
2 min
Cool to 65 °C
Solidify for 2 h
25 °C
A
B
Glycerin
5 μL
Load
130 V
5 min
130 V
30 min
Charaterization
Electrophoresis
Dialysis
Electrophoresis
Excise the
third band
Control
Fig. 3Theworkflowofagarosegelpreparation(A)andpurificationofAu-DNAdimermechano-nanoswitches(B)
PROTOCOL Z.Liaoet al.
4|December2024|Volume10|IssueX ©TheAuthor(s)2024

gel electrophoresis at 130 V for 5 min. Place the kit
horizontallyintheelectrophoresisbath,andaddMilli-Q
wateruntilthegelbandsarefullysubmerged.
[TIP]Theelectrophoresistimefortheelectroelution
accessory kit is not excessive to prevent the gel from
dissolving, which could have a detrimental impact on
subsequentcharacterization.

Step 4: Doxorubicin intercalation into mechano-
nanoswitches [TIMING 1 d]
Step 4.1: For the doxorubicin (DOX) intercalation,
incubateDOX with the Au-DNA dimer at a molar ratio
of 20:1 and shake gently in the dark for 1 h in an ice
bath. The DOX is capable of intercalating into double-
stranded 5’-GC-3’or 5’-CG-3’ (Zhu et al. 2013). After
that,centrifugethecomplextoremove excessfreeDOX
(Fig.4).
TheDOXloadingefficiencyiscalculatedbasedonthe
followingformula:
DOX loading efficiency (%) = [1 − (Fluofree − Fluobuffer)/
(Fluototal−Fluobuffer)]×100%
[TIP]Theincubationratio and time can beadjusted
accordinglywiththenumberofdrugintercalationsites
inthedesignedDNAsequence.

Step 5: Ultrasound-controlled drug activation
experiments [TIMING 1-2 d]
Step5.1:Performan ultrasonication experiment of the
DOX-loaded mechano-nanoswitches in a 1 mL heavy-
walledultrasonicationvesselwithasonicatorequipped
witha3mmdiametermicrotipprobe(A12628PRB20).
Perform sonication using pulsed ultrasound (1.0 s on,
1.0 s off at 50% amplitude) at f = 20 kHz. Place the
vessel in an ice bath to maintain a temperature inside
thevesselof6–9°Cthroughoutsonication(Fig.5).
[TIP] To prevent the thermal effects of ultra-
sound from influencing the structure of mechano-
nanoswitches,thesampleshouldbe keptinanicebath
throughoutthesonicationexperiment.
Step 5.2: For studying the mechanochemical re-
sponseof the drug (DOX)release,measurethefluores-
cenceemission intensity of thesupernatant at λ= 591
nmimmediately after ultrasonication, underanexcita-
tionwavelengthλ=488nmat25°C.
Step 5.3: For studying the US-induced structure
change of the mechano-switches, deposit 4 μL of the
post-dialysis solution onto a copper mesh, evaporate,
and dry naturally. Then observe the sample under a
transmission electron microscope (TEM). Meanwhile,
determine the particle size change through dynamic
lightscattering(DLS)analysis.
[TIP]Conductcelltoxicity or animal experiments to
verify the activity of the released drugs. Select other
characterizationmethods to analyze the regulatory be-
havior of sonomechanical force on drug activity based
onthetypeandcharacteristicsofthedrugsloaded.

MATERIALS AND EQUIPMENT

Materials
•Chloroauricacid(Sigma)CASNo.27988-77-8
•Sodiumcitrate(Sigma)CASNo.6132-04-3
•Bis(p-sulfonatophenyl)phenylphosphine dihydrate
dipotassiumsalt(Sigma)CASNo.308103-66-4

Remove
supernatant
Shake in the
dark for 1 h
Ice bath Resuspend
7000 r/min 2 min
Remove free DOX
+
DOX
A
C
G
T
3'5'
O
O
HO
O O
O
OH
O
OH
HO
H
2
N
HO
HCl
=
Fig. 4Theworkflowofdoxorubicinintercalationintomechano-nanoswitches
Preparationofmechano-nanoswitchesforultrasound-controlleddrugactivation PROTOCOL
©TheAuthor(s)2024 5|December2024|Volume10|IssueX

•Sodiumchloride(Sigma)CASNo.7647-14-5
•Agarose(SangonBiotech)CASNo.9012-36-6
•DNA (SH-5’-AAAAAAAAAAAAAAAAAAAAGGAGGAGG
AGGAGGAAAAATCCTCCTCCTCCTCCAAAAAAAAAAA
AAAAAAAAA-3’-SH)
•Doxorubicin hydrochloride (Sigma) CAS No. 25316-
40-9
•TBEbuffer(SangonBiotech),5×
•Tris(2-carboxyethyl)phosphine hydrochloride (Sangon
Biotech)CASNo.51805-45-9
•Ultrasonication vessel (Test tube heavy-walled,
2775/2,Assistant)
•D-Tube™ electroelution accessory kit (D-Tube™
DialyzerMidi,Merk)

Equipment
•Magneticheatingagitator(IKA)
•Centrifugalmachine(HC-3016R)
•Horizontalelectrophoresisapparatus(BIO-RAD)
•Transmission electron microscopy (JEOL JEM-
2100plus)
•NanoZSZetasizer(25°C,Malvern,England)
•Gelimagersystem(Tanon)
•QsonicaQ125sonicator(USA)

AcknowledgementsThis work was supported by the Natural
Science Foundation of Fujian Province (2023J06006), the
National Natural Science Foundation of China (32371436), and
the Nanqiang Outstanding Young Talents Program from Xiamen
University.

Compliance with Ethical Standards

Conflict of interestZhihuan Liao, Junliang Chen, MenghanXiao
and Shuaidong Huo declare that they have no conflict of
interests.

Human and animal rights and informed consentThis article
does not contain any studies with human or animal subjects
performedbyanyoftheauthors.
Open AccessThis article is licensed under a CreativeCommons
Attribution4.0International (CC BY 4.0) License, which permits
use, sharing, adaptation, distribution and reproduction in any
mediumorformat, as long as you give appropriate credittothe
original author(s) and the source, provide a link to the Creative
Commons licence, and indicate if changes were made. The
images or other third party material in this article are included
in the article’s Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not
included in the article’s Creative Commons licence and your
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the permitted use, you will need to obtain permission directly
from the copyright holder. To view a copy of this licence, visit
http://creativecommons.org/licenses/by/4.0/.
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Preparationofmechano-nanoswitchesforultrasound-controlleddrugactivation PROTOCOL
©TheAuthor(s)2024 7|December2024|Volume10|IssueX