pH-sensitive size-shrinking
nanoparticles for cancer therapy

Literature seminar #1
B4 Shinpei Takamaru
2023/01/19 (Thu)ContentsØIntroduc)on§ What are Drug Delivery Systems ?
§ What are Nanopar7cles ?
§ Current problems

ØpH-sensi)ve size-changeable nanopar)cles§ pH-sensi7ve linker cleavage type§ pH-sensi7ve protona7on typeØSummary and perspec)ve2

Drug Delivery Systems

introduc)onØWhat are Drug Delivery Systems?
the method or process of deliver drugs• at the targeted site
• in the desired dose
• at the appropriate time and speed
to achieve therapeutic effects
by improving pharmacokinetics and pharmacodynamics of drugs
Tiwari, G., et al. INTERNATIONAL JOURNAL OF PHARMACEUTICAL INVESTIGATION 2012, 2(1), 2-11.
Retrieved from h9ps://www.sigmaaldrich.com/JP/en/applicaCons/materials-science-and-engineering/drug-delivery

3General objec,ves of DDS

introduc)onblood vessel

effective

concentra)on of drugconcentration of drugside eﬀect

tumor

side eﬀecteﬀec)ve
normal tissue

)me

)me

absorp&on
enhancement

controlled
releasedrug

targe&ng

Okamoto, K. Yakugaku DDS -DDS seizai to sono yakubutsu chiryou- [薬学DDS -DDS製剤と、その薬物治療-] 2019, Kyoto, Japan: Kyoto hirokawa shoten [京都廣川書店].;
Hashida, M. Membrane 1995, 20(1), 55-64.; Retrieved from hJps://www.phar.kyushu-u.ac.jp/lab/gaiyou_10.php 4

Drug Delivery Systems: Examples

introduc)on< absorp)on enhancement >< controlled release >new administra&on routesustained release

Prodrug

drug

lipid soluble

O
R

O
OH

R

O

Okamoto, K. Yakugaku DDS -DDS seizai to sono yakubutsu chiryou- [薬学DDS -DDS製剤と、その薬物治療-] 2019, Kyoto, Japan: Kyoto hirokawa shoten [京都廣川書店].

5

Drug Delivery
Systems: Examples
REVIEWS REVIEWS
Polymeric< ac)ve targe)ng >REVIEWS

Antibody-Drug Conjugate

introduc)on

PolymericInorganic< passive targe)ng >Inorganic

nanopar&cles
Polymersome

PolymericPolymersome
Polymer Dendrimer
micelleDendrimer
Polymersome

NP
Nanosphere
Polymer Silica
micelleSilica NP
Dendrimer

Inorganic

QuantumIronoxidedotNP
Nanosphere

Quantum Silica
dot NP

Lipid-based

LiposomeGold
NP oxide NP
Iron

Liposo
Quant

Lipid
N
Gold

• Precise control of particle
Unique electrical, magnetic
and electrical, magne• Form• Precise control of•particle• Uniquecharacteristics
optical properties
characteristics
optical properties a ran• Payload ﬂexibility for hydrophilic
Variability
in size, structure
prop• Payload ﬂexibility•forhydrophilic• Variability in
Oilsize, structu
and hydrophobic cargo and hydrophobic cargo
and geometry•Highand geometry• Easy surface modiﬁcation• Well suited for theranostic• Paylo• Easy surface modiﬁcation• Well suited for theranosti• Possibility for aggregation
applications• Low• Possibility for aggregation
applications
Polymer
micelleand
Nanosphere
Iron
oxideJapan:
NP • Kyoto
Gold
NP solubility
EmulsionOkamoto, K. Yakugaku DDS -DDS seizai to sono yakubutsu chiryou[薬学DDS-DDS製剤と、その薬物治療-]
2019,
Kyoto,
hirokawa
shoten [京都廣川書店].;
toxicity
Toxicity
andlimitations
andtoxicity• Toxicity andsolubilityeﬃci
lim
Mitchell, M. J., Billingsley, M. M., et al. Nature Reviews Drug Discovery 2021, 20(2), 101–124. 6• Precise control ofparticle• Unique electrical, magnetic and• Formulation simplicity with
Fig. 2 | Classesof NPs. Eachof nanoparticle(NP)classfeaturesmultiplesubclasses,
withmultip
some|opticalFig. 2classClassesof NPs. Eachof nanoparticle(NP)offeaturescharacteristics
properties
a rangephysicochemical
highlighted
here. Each classhas
numerousbroadadvantages
and disadvantages
regardingcar• Payload ﬂexibility
for hydrophilic• Variability
in size,structureproperties
highlighted
here.
Each
class
has numerous
broadadvantages
and disa
and hydrophobic
cargo
and
geometry•Highbioavailability
response.
response.
• Easy surface modiﬁcation• Well suited for theranostic• Payload ﬂexibility• Possibility for aggregation
applications• Low encapsulationand toxicity• Toxicity and solubility limitations
eﬃciency

Nanoparticles for Drug Delivery System (DDS)
Advantages of nanopar7clesØimprove stability and solubilityØvariable drug capacityØgood biocompa7bilityØEPR eﬀectØcustomizable(surface modiﬁca7on)

introduction

Enhanced permea2on and reten2on eﬀect(EPR eﬀect)
normal )ssue

blood vessel

tumor tissue

Tagami, T., & Ozeki, T. Organ Biology 2017, 24(1), 54–60.; Mitchell, M. J., et al. Nature Reviews Drug Discovery 2021, 20(2), 101–124.;
Mousa, S. A., & Bharali, D. J. Cancers 2011, 3(3), 2888-2903.; Ichikawa, H. Journal of The Surface Science Society of Japan 2010, 31(10), 543-550. 7

can cause NPs to preferentially distribute to certain exogenous
tissues. For example, a poly(β- amino- ester) (PBAE) and therefo
ter- polymer/PEG lipid conjugate was optimized for the trigger
lung localization, achieving efficacy two orders of this respon
magnitude above the pre-optimized form both in vitro and other n
One exand in vivo179. Other PBAE polymers have been developed that preferentially target glioblastoma cells over stimuli- re
introduc)on
optimized down into
healthy cells in vitro200. Even AuNPs can beto passively target triple- negative breast cancer cells, biologicalinitial size
the bloods
Surface andTargeting
NP extrav
material properties
At the tum
system int
Carbohydrates
have impro(glucose, galactose,mannose, dextran)
Selfof the plat
peptidescells204. Th
Antibodies
ditional inadministra
Responsiveness
PEGylation
Cell surface
by 10%, wh
Endogenous triggers:
receptors• Acidic/hypoxic
up to 95% ienvironment
commonly
Cell• Enzyme levels
Vitamins
in the kidn
coating• Metabolic shifts(vitamin D,
Exogenous triggers:
this system
folate)• Light
triggers an• SoundDensity andhave great• Force (physical
Aptamers
toughnessor magnetic)
their desig(peptides)
to the tum
AnotheArchitecturepoly(acrylSize
can be moapplication
Shapetransducti+
Charge –or degrada
platform w
modificat
Fig. 5 | Commonly engineered NP surface properties that allow for enhanced
cule, pepti
Mitchell, M. J., Billingsley,M., etandal. material
Nature Reviews
Drug
Discovery
2021, 20(2),101–124.
8
delivery.M.
Surfaceproperties,
architecture,targetingmoietiesandor recogni
responsiveness are all attributes of nanoparticles (NPs) that can be altered in intelligent
a similar, mdesigns to tailor the platform to a specific application. Different combinations of theseto deliver mfour properties allows for seemingly endless permutations of NP features and platforms.
respond to
PEG, poly(ethylene glycol).

nanoparticles can change various moieties.
Advantages of nanopar7clesØimprove stability and solubilityØvariable drug capacityØgood biocompatibilityØEPR effectØcustomizable(surface modification)

VIEWS

What are nanopar,cles?
PolymericPolymersome

Dendrimer

introduc)on
Inorganic

Silica NP

Quantum dot

Lipid-based

Liposome

Lipid NP

Oil

Polymer micelle

Nanosphereü biocompa)bility• Precise control of particle
characteristics
ü precisecontrolfor
ofhydrophilicpar)cles• Payload ﬂexibility
and hydrophobiccargo
ü easysurface modiﬁca)on• Easy surface modiﬁcation
× possibility
aggrega)on• Possibility forforaggregationand toxicity
and toxicity

Iron oxide NP

Gold NPü •uniqueelectrical,
magne)c
Unique electrical,
magnetic
andoptical propertiesop)mal•and
Variability
in size,proper)es
structureand geometry
ü biocompa)bility,
stability• Well suited for theranostic
× low
solubility and toxicity of
applications•heavyToxicity metaland solubility limitations

Emulsionü• Formulation
formula)on
simplicity
simplicity
with
a
range
of
physicochemicalü properties
biocompa)bility
bioavailabilityü• Highself-assembly• Payload ﬂexibility
×• Lowlowencapsulation
encapsula)on eﬃciency
× eﬃciency
biodistribu)on

Fig. 2 | Classes of NPs. Each class of nanoparticle (NP) features multiple subclasses, with some of the most common
highlighted here. Each class has numerous broad advantages and disadvantages regarding cargo, delivery and patient
response.

Mitchell, M. J., Billingsley, M. M., et al. Nature Reviews Drug Discovery 2021, 20(2), 101–124.; Maitani, Y., & Nagai, T. DDS 2002, 17(4), 314-320. 9(PEG) and poly(dimethylsiloxane) (PDMS). Polymeric
micelles, which are also typically responsive block

makes them useful in cancer medicine, gene therapy
and diagnostics. However, disadvantages of polymericIntroduc5on: nanopar5cle-size-dilemmalarge size (20~100nm)
need for EPR effectintroduc)on

small size (~20nm)
need for cell penetration

normal 1ssue

blood vessel

tumor 1ssue

Ichikawa, H. Journal of The Surface Science Society of Japan 2010, 31(10), 543-550.; Cheng, G., Wu, D., et al. Nano Today 2021, 38, 101208. 10

How to shrink the size

introduc)onØ large size un)l distribu)on to tumors
Ø small size a;er tumor distribu)on in order topenetrate tumor cells→ size-change strategy
EPR
eﬀect

cell
penetrate

Ø Trigger of shrinking at only tumor )ssues→use tumor microenvironment or exogenous s)muli
Cheng, G., Wu, D., et al. Nano Today 2021, 38, 101208. 11

ng pH-instable clustered nanoparticle (iCluster/Pt)
m coassembly of PCL-CDM-PAMAM/Pt, PEG-

size-shrinking strategy

introduc)on

Figure 7. (A) Illustration of construction and pH-responsive size shrinkage of DMA-based nano
Sciences. (B) Schematic illustration of DMA-based PNV, with charge reversal for dissociation.
polymer-Dox inside the tumor. Copyright 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinh

enzymepH
PCL-CDM-PAMAM/Pttelf-assembly

G

000

redox
0h

Cumulative release (%)

F

Fig. 1. Preparation and physicochemical propertiesof the clustered nanoparticles. (A) Chemical structure
4h
24 h
PB
of PCL-CDM-PAMAM/Pt.
(B) Self-assembly
andshrinking
struc-size to 10 nm QDs by cleaving away the
80
Figure 8. (A) Schematicof 100 nm QDGelNPs
pH 6.8National
Academyof
Sciences.
(B)
Schematic
illustrationof the design and synergistic eﬀects forturalchangeofiCluster/Pt
in
response
to
tumor
60
IDDHN.
Copyright
2018
ElsevierLtd.
acidity and intracellular reductive environment.
40 PB
PAMAM
(C and E) inhibition).
DLS distributionsof withiCluster/Pt
and HAase
pH 7.4Pt drugamylase-digested hyd
Unlike MMPsa range of(C)substrates,
20
Cluster/Pt (E).
(D
and
F)
TEM
imagesofiCluster/Pt
exclusively hydrolyzes HA to short chains or modiﬁed glucose
depegylation.141 Th
0
Thetreatednatural negative
chargeselectivity owing tounits.131 (F)(D) and Cluster/Pt
in PB at
pH on
6.8HA
formakes
0, 4,itself easy
0
4
8
12
16
to conjugate (Scalepositively
chargedmaterials,
andthethe speciﬁc
which is essential fo
Time (h)and 24 h, respectively.
bar,
100 nmand forcombination between HA and CD44 also provides HA with
reducing the adver
Inset images, 50 nm.) (G and 132−135
H) PAMAM (green line)
Ascorbic acidFor application of superior HA,
active-targeting ability.
uneven levels of enz
5 mM, pH 7.4
and platinumline) releasefromiCluster(G) DGL to
4h
24 h
100
ourdruggroup(redcross-linked
HA withpositively
chargedthe application scopand Clusterform(H)size-shrinkable
under threedifferent conditions,4.3. Size Shrink
HAase-responsive
nanoparticles (Figure
80
PB
Since
the GSH leve
8B)
that
exhibited
extraordinary
penetration
abilitywiththewhich include PB at pH 7.4 to mimic a neutral envipH 6.8
60
136,137
its
responsivenessisfurther
assistance
of
an
NO
donor
on
an
HA
shell.
ronment, PB at pH 6.8 to mimic a tumor extracellular
40 PB
PAMAM
As instanced, the enzyme-triggered size shrinkage is
Guo et al. constructenvironment,
ascorbic
acid
solution(5 mM,
pH6(2),
7.4) 100–116. 12
Li, H., Du, J., et al. PNAS 2016,
113(15), 4164-4169.; Yu, W., Liu,
R., Zhou, commonlyY.,and& Gao,H.
ACS
CentralScience2020,
achieved by shell dissociation of core−shell
polylactide (PEG−P
Pt drug
20 pH 7.4
to mimic annanoparticles
intracellularand
redox
environment.
PAMAM
detachmentofsmall-sizeddecorations(PEI−DMA),142 wh
0138
release wasfrom
quantified
by HPLC, whereas
The rule isplatinum
always applicable,
PEG−PLA−S−S-PE
large nanoparticles.
0
4
8
12
16
Time (h)andarecent-reportnanosystemisalsorepresentative,
which
Figure 9A). The hig
release was determined by ICP-MS.
100

H

Ascorbic acid
5 mM, pH 7.4

light

Cumulative release (%)

0h

enzymeIntracellular
Reduction

Tumor acidity

D

00

)

released upon cleavage of the amide bond at pH 6.8. In contrast,
Cluster/Pt maintained its initial morphology and size underidentical conditions (Fig. 1F).
Next, high-performance liquid chromatography (HPLC) wasused to monitor and compare the release kinetics of PAMAM

MEDICAL

S1). The resultant amide bond is acid labile
be cleaved at pHe to release PAMAM at

can release small-sized cargo nanoparticles when lactateoxidase digests its shell.139 Other enzymes-dependent nanoparticles
also obeyed
principle,
such as α| vol.this| no. 15
| 4165PNAS | April
12, 2016
113designing

the disulﬁde bond an
size shrinkage allow
nucleus (Figure 9B)

ContentsØIntroduc)on§ What are Drug Delivery Systems ?
§ What are Nanopar7cles ?
§ Current problems

ØpH-sensi)ve size-changeable nanopar)cles§ pH-sensi7ve linker cleavage type§ pH-sensi7ve protona7on typeØSummary and perspec)ve
13

pH-sensi,ve size-shrinking strategy

researchlinker cleavagem., Vol. 14, No. 1, 2003

Rozema et al.

R3

O

N

+
R1

R2

R1

R2

H 2N R 3

Rozema, D. B., et al. Bioconjugate Chemistry 2003, 14(1), 51–57.

maleamate derivatives

schiff baseamino polymers

Li, H. J., Du, J. Z., et al. ACS Nano 2016, 10, 6753−6761.; Yu, W., et al. ACS Central Science 2020, 6(2), 100–116.;
14
Zhou, K., Wang, Y., et al. Angew. Chem., Int. Ed. 2011, 50, 6109−6114.

ally conﬂicting attributes of using nanoparticles forreatment.

improved cancer treatme
were employed to trigg

eic anhydride and maleamate derivatives: maleic R1 and R2dH, dimethyl maleic R1 and R2 ) CH3, citraconic
1 ) CH3, cis-aconitic R1or2 ) H and R2or1 ) CH2CO2H, and 2-propionic-3-methylmaleic (CDM) R1or2 ) CH3 and(B) Synthesis of 2-propionic-3-methylmaleic anhydride (CDM): a Horner-Emmons reaction between dimethyriethyl-2-phosphonopropionate, followed by saponification of the ester groups.

ed from https://www.pnas.org by UNIVERSITY OF TOKYO on January 10, 2023 from IP address 133.11.46.191.

acterized (SI Appendix, Figs. S1 and S2). For PCL-CDM-PAMAM/Pt
synthesis, PCL was first reacted with 2-propionic-3-methylmaleic
anhydride (CDM) to produce PCL-CDM (SI Appendix, Fig. S3).
A Pt prodrug c,c,t-[Pt(NH3)2Cl2(OH)(O2CCH2CH2CO2H)] wasconjugated to PAMAM to afford PAMAM/Pt, which was further coupled to PCL-CDM through the reaction of the aminogroups of PAMAM with the CDM anhydride residue (SI Appendix, Scheme S1). The resultant amide bond is acid labile(38) and will be cleaved at pHe to release PAMAM attumor sites.
The Pt-containing pH-instable clustered nanoparticle (iCluster/Pt)
was prepared from coassembly of PCL-CDM-PAMAM/Pt, PEG-

additional incubation focompletely disintegrateS5) and changed to ananoparticles formed b
S6A). Meanwhile, plent
the surroundings, with
dix,research
Fig. S6B),①suggestinreleased upon cleavage
Cluster/Pt maintained
identical conditions (FiNext, high-performan
used to monitor and co

pH-sensi5ve clustered nanopar5cles

pH 〜6.5-7

PCL-CDM-PAMAM/Pt

B
PCL-CDM-PAMAM/Pt
Self-assembly
PCL

Intracellular
Reduction

Tumor acidity

PEG-b-PCL

C

20

D

G
0h

16

4h

Ascorbic acid
5 mM, pH 7.4

100

24 h

12
8
4
0
10

Cumulative release (%)

pH 〜7.4

A

Intensity (%)

model diagram

100 1000

PB
pH 6.880
60
PB
pH 7.4

40

20
0

0

4

PAMAM
Pt drug
812E

Intensity (%)

20
16
12
8
4

F

H

0h

4h

24 h

tive release (%)

Time (h) 15
Li, H. J., Du, J. Z., et al. ACSSize
Nano(nm)2016, 10, 6753−6761.; Li, H., Du, J., et al. PNAS 2016, 113(15), 4164-4169.
100
80
60
40

16

Ascorbic acid
5 mM, pH 7.4
PB
pH 6.8
PB

PAMAM

Fig.
of th
of P
tura
acid
(C a
Clus
(D) a
and
Inse
and
and
whic
ronm
envi

cleavage linker (maleamate deriva,ves)
O
R

R1

N
H
O

O

H

R1
R NH2

R2

O

Rozema et al.

O

O

O

R1

O

R2

Me

Me

O

O

O

OH

O
O

O

t1/2 = 300 min

R2
O

O
H

R1

N
H
O

Me
O

t1/2 = 2 min

R

O
OH

O
O

O

R2
O

research ①

t1/2 = 1.5 min
O
Me
O
H
O

t1/2 = 300 min

Rozema, D. B., et al. Bioconjugate Chemistry 2003, 14(1), 51–57. 16

Figure 3. The cleavage of maleamic acid modifications of theglycinylalanine dipeptide modified with dimethylmaleic (b),CDM (O), citraconic (9), or cis-aconitic anhydride (0).

://www.pnas.org by UNIVERSITY OF TOKYO on January 10, 2023 from I

Downloaded from https://www.pnas.org by UNIVERSITY OF TOKYO on January 10, 2023 from IP address 133.

ther coupled to PCL-CDM throughthereaction of the amino
the surroundThe
Pt-containing
pH-instableclugroups of PAMAM with the CDM anhydride residue (SI Apdix, Fig. S6B
was preparedreleasedof
upopendix, Scheme S1). The resultant
amide bondfrom
is acid coassembly
labile(38) and will be cleaved at pHe to release PAMAM at
Cluster/Pt midentical con
tumor sites.
Next, high
The Pt-containing pH-instable clustered nanoparticle (iCluster/Pt)
used
was prepared from coassembly of PCL-CDM-PAMAM/Pt, PEGresearch① to monS5muli-responsive clustered nanopar5cles

PEG-b-PCL

C

20
16
12
8
4
0
10

E

20

D

100 1000Size (nm)F

B

Intracellular
Reduction

Tumor acidity

PCL-CDM-PAMAM/Pt
Self-assembly
PCL

G

0h

C

4h

PEG-b-PCL

16

H

Size (nm)E

Intensity (%)

24 h

60
40

20
0

PB
pH 7.40

PAMAM
Pt drug0

4

8Time (h)

100
80
PB
12
pH 6.8460
8Retrieved from hJps://www.sigmaaldrich.com/JP/ja/technical-documents/technical-arZcle/materials-science-and-engineering/drug-delivery/dendrimers
40 PB 17
Li, H., Du, J., et al. PNAS 2016,4
0 113(15), 4164-4169. (Fig. modiﬁed)
20 pH 7.4
0
10
100
1000
0
10
100 1000
16

84h

PB
pH 6.880D

12Ascorbic ac
5 mM, pH 7

100

24 h

20

0h

Cumulative release (%)

PCL-CDM-PAMAM/Pt
Self-assembly
PCL

PCL-CDM-PAMAM/Pt

Cumulative release (%)

B

AIntensity (%)

・ highly symmetrical・ can control size andsurface chemistryA

Intensity (%)Ø dendrimer

Size (nm)

20

F

0

4

8

12

Ascorbic a
5 mM, pHTime (h)PAMA
Pt dru12

Downloaded from https://www.pnas.org by UNIVERSITY OF TOKYO on January 10, 2023 from IP address 133

groups of PAMAM with the CDM anhydride residue (SI Appendix, Scheme S1). The resultant amide bond is acid labile(38) and will be cleaved at pHe to release PAMAM attumor sites.
The Pt-containing pH-instable clustered nanoparticle (iCluster/Pt)
was prepared from coassembly of PCL-CDM-PAMAM/Pt, PEG-

S5muli-responsive clustered nanopar5cles

dix, Fig. S6B), suggesting that small PAMAM dereleased upon cleavage of the amide bond at pH
Cluster/Pt maintained its initial morphology aidentical conditions (Fig. 1F).
Next, high-performance liquid chromatographused to monitor and compare the release kineti

research ①

A
PCL-CDM-PAMAM/Pt

B
PCL-CDM-PAMAM/Pt
Self-assembly
PCL

Intracellular
Reduction

Tumor acidity

PEG-b-PCL
20

D

Li, H., Du,GJ., et al. PNAS 2016, 113(15),4164-4169.
Fig. 1. Preparation and physicoch
Ascorbicacid
Cumulative release (%)

C

5 mM, pH 7.4

F

Intensity (%)

E

Li et al.

4h

100

24 h

H

PB
pH 6.8Cumulative release (%)

Intensity (%)

of the clustered nanoparticles. (A) C
of PCL-CDM-PAMAM/Pt. (B) Self-as
80
Developmentof Pt(IV)-Encapsulated,
12
tural change of iCluster/Pt in re60(Pt-NP-Apt).
PLGA-COOH and NH
8
acidity and intracellular reducti
40 PB
PAMAMwere used to prepare a PLGA-b-P(C and E) DLS distributions of iC
pH 7.4
4
Pt drug
20
carboxylicacid(E).groups(PLGA-b
Cluster/Pt(D and F)
TEM ima00
10
100 1000
COOH
wasconverted
to
its
succi
(D)
and
Cluster/Pt(F)treated
in PB
0
4
8
12
16
Size (nm)Time (h)and 24 h, respectively. (Scale bar, 1
dimethylaminopropyl]carbodiimid
Inset images, 50 nm.)(NHS),(G and H)whPA
Ascorbicacid
N-hydroxysuccinimide
20
5
mM,
pH
7.4
and platinum drug (red line) releas
0h 4h
24 h
100
with NH
16
2-PEG-COOH. We used
and Cluster (H) under three diff
80
PB
Dhar, S., Gu, FX., et al. PNAS 2008, 105(45), 17356-17361.
(35) towhich
encapsulate
1 within a PL
12
include PB at pH 7.4 to mim
pH 6.860
havingronment,a terminal
acida gt
Scheme 1. Chemical structure of the hydrophobic platinum(IV) compound8
PB atcarboxylic
pH 6.8 to mimic
40 is
PB
PAMAM
1
and
the
chemistry
by
which
the
activedrug,
cisplatin
released,
after(Fig. 1A).
The properties
ofacidthesoluten
4 WL., et al. JACS 2009, 131(41), 14652–14653.; Maitani, Y., & Nagai, T. Drug Delivery System
environment,
and ascorbic
Dhar, S., Daniel,
Pt drug 2002, 17(4), 314-320. 18
20 pH 7.4
reduction
in
the
cell.
characterized
byintracellular
dynamicredox
lightenvisc
0
to mimic an0
10
100 1000
releasewasquantifiedbyHPLC,
polydispersityof each preparatio
0
4
8
12
16
Size (nm)Time (h)releasewas determined
by ICP-MS.
loading,a seriesof encapsulated
N
0h

16

because of its physico-chemical properties (27, 28). Cisplatin is
insoluble in organic solvents, and its partial solubility in water
makes it difficult to obtain cisplatin-encapsulated-PLGA sus-

weight percentage of 1 to polymer
molecular
In2016
this| way,
| April 12,
PNAS masses.
vol. 113w

Nanoparticles which has cleavable linker or notresearch ①

iCluster(pH-instable)
SchemeS1. Synthetic
procedures of PCL-CDM-PAMAM/Pt and PCL-CDM-PAMAMFlu.

Cluster (pH-stable)

Scheme S1. Synthetic procedures

Scheme S1. Synthetic procedures of PCL-CDM-PAMAM/Pt and PCL-CDM-PAMAMFlu.

Scheme S1. Synthetic procedures of PCL-CDM-PAMA

Li, H., Du, J., et al. PNAS 2016, 113(15), 4164-4169. (Fig. modiﬁed) 19

Scheme S2. Synthetic procedures of PCL-PAMAM/Pt and PCL-PAMAMFlu.

Downloaded from https://www.pnas.org by UNIVERSITY OF TOKYO on Januar

A
PCL-CDM-PAMAM/Pt

Bphysicochemical properties of the clustered nanoparticles
20

PB pH 6.8
0h

16

4h

G

8
4
100 1000Size (nm)

20

F

0h

16
12
8
4
0
10

PB pH 6.8

100

1000

Size (nm)

4h

H
24 h

PB
pH 6.8

80
60
40

20
0

research ①

Ascorbic acid
5 mM, pH 7.4

100

24 h

12

0
10

E

D

Cumulative release (%)

Intensity (%)

C

Intracellular
Reduction

Tumor acidity

Cumulative release (%)

Cluster (pH-stable)

PEG-b-PCL

Intensity (%)

iCluster (pH-instable)

PCL-CDM-PAMAM/Pt
Self-assembly
PCL

PB
pH 7.40

PAMAM
Pt drug

4

8Time (h)10080PBpH 6.860
40 PB
20 pH 7.4
0
0

4

8

12

16

Ascorbic acid
5 mM, pH 7.4

Time (h)PAMAM
Pt drug12

Ø Each nanoparticle showed
Li et al.similar morphology, sizeØ plenty of small nanoparticles (PAMAM) were observed after 4h or 24h incubation
Ø under an intracellular redox environment, both cluster released Pt drugs.
Li, H., Du, J., et al. PNAS 2016, 113(15), 4164-4169. (Fig. modiﬁed) 20

16

Fig. 1.
of the cof PCLtural cacidity(C andCluster/(D) andand 24
Inset imand pla
and Clu
which ironmenenvironto mimireleasereleasePNASDownloaded from https://www.pnas.org by UNIVERSITY OF TOKYO on January 10, 2023 from IP address 13

24 h at pH 6.8, then washed and observed under confocal laser
scanning microscopy (CLSM, Fig. 2A and SI Appendix, Fig. S8).
For RhBiClusterFlu treatment, the red fluorescence from the hydrophobic core attached to the periphery of MCSs, and no noticeable fluorescence was detected in the internal area by 24 h.
Instead, the green signals from PAMAM inside MCSs clearly
increased over time, demonstrating the improved penetration of
PAMAM. By contrast, both red and green fluorescence signals
from nonresponsive RhBClusterFlu were on the peripheral regionof the MCSs by 24 h, revealing limited penetration of larger
particles. Quantitative analysis indicated that more than 10-fold
3D-modeliClusterFlu-treatedhigher green fluorescence was detected in RhB
RhBClusterFlu-treated ones (Fig.
These results
MCSs than inof2B).
cancercell penetra3on and an3tumor ac3vity
iCluster (pH-instable)

Cluster (pH-stable)

bearing BxPC-3 human pancre
a notoriously poorly permeableAs shown in Fig. 3B, free cis
modest therapeutic efficacy, w
versus PBS control, whereas C
hibition. In contrast, iCluster/P
research①of tumor
growth, reaching88
versus Cluster/Pt treatment).
mass excised at the end of trea
trend (SI Appendix, Fig. S11A
hematoxylin and eosin (H&E)(PCNA), and terminal deoxy
deoxyuridine triphosphate nin celllarge nanopar)cles (RhB), PAMAM dendrimers (Flu)Ø The size-changeable iCluster had beOer tumor penetra)onØ Compared with Cluster/Pt, iCluster/Pt exhibited signiﬁcant suppression of tumor growth
Li, H., Du, J., et al. PNAS 2016, 113(15), 4164-4169.; Kamatur, A., et al. Polymers 2020, 12(11), 2506. 21
4166 | www.pnas.org/cgi/doi/10.1073/pnas.1522080113

Fig. 2. In v
of clusteredetration of R
pH 6.8 afte
marked witharea. (Scaletensity (MFI
MCSs. (C) Q
cells after in
pH 6.8 for 4
tent in MCS
tions at pH 6
in DNA of M
for 24 h. (treatments amean ± SD (cell penetra3on and an3tumor ac3vity
iCluster (pH-instable)

research ①

in vivo

Cluster (pH-stable)

Ø The size-changeable iCluster had beOer tumor penetra)onØ Compared with Cluster/Pt, iCluster/Pt exhibited signiﬁcant suppression of tumor growth
Li, H., Du, J., et al. PNAS 2016, 113(15), 4164-4169. 22

nloaded from https://www.pnas.org by UNIVERSITY OF TOKYO on January 10, 2023 from IP address 133.11.4

conjugated to PAMAM to afford PAMAM/Pt, which was further coupled to PCL-CDM through the reaction of the aminogroups of PAMAM with the CDM anhydride residue (SI Appendix, Scheme S1). The resultant amide bond is acid labile(38) and will be cleaved at pHe to release PAMAM attumor sites.
The Pt-containing pH-instable clustered nanoparticle (iCluster/Pt)
was prepared from coassembly of PCL-CDM-PAMAM/Pt, PEG-

Short summary

S6A). Meanwhile, plenty of small nanoparticles
the surroundings, with size comparable to PA
dix, Fig. S6B), suggesting that small PAMAM
released upon cleavage of the amide bond at p
Cluster/Pt maintained its initial morphology
identical conditions (Fig. 1F).
①
Next, high-performanceresearchliquid chromatogra
used to monitor and compare the release kin

A
PCL-CDM-PAMAM/Pt

B
PCL-CDM-PAMAM/Pt
Self-assembly
PCL

Intracellular
Reduction

Tumor acidity

PEG-b-PCL

D

20

G
0h

16

4h

24 h

Fig. 1. Preparation and physicof the clustered nanoparticles. (100
PBof PCL-CDM-PAMAM/Pt. (B) Sel
80
pH 6.8tural change of iCluster/Pt in60
acidity and intracellular redu
40 PB
PAMAM
(C and E) DLS distributions o
pH 7.4Pt drug
20
Cluster/Pt (E). (D and F) TEM
0(D) and Cluster/Pt (F) treated in
0
4
8
12
16
Time (h)and 24 h, respectively. (Scale ba
1/2
Inset images, 50 nm.) (G and H)
Ascorbic acidmM, pH 7.4
andplatinum drug100
23 (red line) rel
Li, H., Du, J., et al.5PNAS
2016, 113(15),4164-4169.
andCluster(H)
under three80
PB
which include PB at pH 7.4 topH 6.860
ronment, PB at pH 6.8 to mimic
40 PB
PAMAM
environment, and ascorbic acid so
Pt drug
20 pH 7.4
to mimic an intracellular redox e0release was quantified by HPL
0
4
8
12
16

Cumulative release (%)

Intensity (%)

C

Ascorbic acid
5 mM, pH 7.4

Intensity (%)

20

0h

16
12
8
4
0
10

100

1000

4h

24 h

mulative release (%)üiCluster12system enables that robust tumor penetra2on achieved through8
pH-triggered
sha?ering of small PAMAM dendrimers at tumor sites
4üexhibited
0 signiﬁcant an2tumor ac2vity
10
100 1000Size (nm)× takeEmuch 2me to
F complete the transi2onH(t = 2min)research ①→②

pressuretake much time to complete the transition

Ø high inters22al ﬂuid pressure typically prevent permea2on of nanopar2clesØ easily pumped back to vessels
Cheng, G., Wu, D., et al. Nano Today 2021, 38, 101208.; Mitchell, M. J., Billingsley, M. M., et al. Nature Reviews Drug Discovery 2021, 20(2), 101–124.;
Wong, C., Stylianopoulos, T., et al. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 2426−2431. Yu, W., Liu, R., Zhou, Y., & Gao, H. ACS Central Science 2020, 6(2), 100–116. 24

ultra-pH-sensi,ve and fast reac,vity siteresearch ①→②

Ø 2-(Hexamethyleneimino)ethyl group was the best.
Zhou, K., Wang, Y., et al. Angew. Chem., Int. Ed. 2011, 50, 6109−6114. 25

Scheme 1. (a) Structure of PEG-b-PAEMA-PAMAM/Pt. (b) Schematic illustration showing the self-assembly of PEG-bPAEMA-PAMAM/Pt into the pH-sensitive cluster nanobombs (SCNs/Pt) at neutral pH and the disintegration of SCNs/Pt into
small particles at tumor acidic pH. (c) Schematic illustration of SCNs/Pt as a robust nanoplatform to overcome biologicalbarriers to in vivo drug delivery in poorly permeably pancreatic tumor models. (I) Large SCNs/Pt superstructures are favorable
for prolonged blood circulation. (II) Prolonged circulation increases the propensity of large nanoparticles to extravasate fromleaky tumor vasculature to accumulate in the vicinity of blood vessels. (III) Once deposited in the acidic tumor research ②
microenvironment (pH ∼6.5−7.0), SCNs/Pt instantaneously switch to small particles with a size less than 10 nm for deep
tumor penetration.

improved nanopar,cle design

improvedwas
cancertreatment eﬃcacy. However, the means that
intrinsically conﬂicting attributes Øof using
nanoparticlesnanopar2cle
for
pH sensi2ve
improved
were employed to trigger size alterations can be practicallycancer treatment.
Li, H.For
J., Du,J. Z., et al.
Nanoconcerns
2016, 10, 6753−6761.
To address the predicament to improve therapeutic eﬃcacy,
problematic.
example,theACSmajor
for UV light26arevarious innovative strategies have been reported.8−12 Since
its poor tissue penetration depth and harmfulness to normal
tissues.29 For the existing enzyme- or pH-triggered sizetumor penetration is hindered by the elevated IFP and dense
matrix, normalization of tumor vessels to restore the pressure
switchable systems, their size alterations involve the cleavage
13of chemical bonds and usually take hours to complete thegradients and degradation of the collagen matrix to reduce
14

26,30

protonation type reacted faster

research ②

iCluster (pH-instable)-assembly of PEG-PCL and PCL, (B) PAMAM

Relative scattering intensity

improved cancer
intrinsically conﬂicting attributes of using nanoparticles
pH 6.7for
1.0
were employedcancer treatment.
pH 6.0To address the predicament to improve therapeutic eﬃcacy,
problematic. For
8−12
0.8
pH
5.6
pH 6.8various innovative strategies have been reported.
Since
its poor tissue ppH
5.1
tissues.29 For thtumor penetration is hindered by the elevated IFP and dense
0.6 of tumor vessels to restore the pH
5.0
matrix, normalizationpressureswitchable system
13
of chemical bon
gradients and degradation of the collagen matrix to reduce
14
0.4transport hindrance
have been promising choices. However,transition,26,30 whthese approaches have limitations. For example, the normalsmall particles an
0.2
To fully explo
ization of tumor vessels needs delicate balance in order not tocancertherapy, thdramatically compromise0 the0.2
EPR0.4eﬀect,
while
uncontrollable
0.6 0.8 1.0 1.2 1.4
small nanoparticl
degradation of the tumor matrix may lead to unexpected risks
5Time(second)for promoting pasuch as promoting tumor progression or even metastasis.
Compared with remodeling of the tumor microenvironment,
and boosting cellLi, H. J., Du, J. Z., et al. ACS Nano 2016, 10, 6753−6761.;
Li,
H.,
Du,
J.,
et
al.
PNAS
2016,
113(15),4164-4169.
(Fig.
modiﬁed)27
rationally regulating the physiochemical properties of nanoestablished a seri
S3. pH-Dependentof SCNs/Pt measured
by stopped
particles suchFigure
as particle
size and shaperesponse
providestime
an alternative
tertiary-amine-co
15−18
It has beenwith another resolution to addressing this problem.
demonstrated that large stealth nanoparticles (∼60−100 nm)
polymers underg
are usually suitable for operating the EPR eﬀect and hold highthe scale of mileﬀective tumor dpropensity of extravasation across tumor vasculature toSCN and ICNSCN (pH-sensi2ve cluster nanostructure)research ②

ICN (pH-insensitive cluster nanostructure)

Li, H. J., Du, J. Z., et al. ACS Nano 2016, 10, 6753−6761. (Fig. modified) 28

Relative scattering intensity

employed
as an inthat
vitrocancer
model to assess pen
Spheroids
have shown
carries
twowerePEG-b-PAEMA
chainson(MCSs).
average.
For gelstudies
which was
N-(ε-maleimidocaproyloxy)less than
10 nm
observed atpH
6.7 (Figure1c).RecentThe
Pt were PAEMA,
self-assembled
frommodiﬁed
PEG-b-withPAMAM
andexhibit
cellof our sy
nanomedicineswith shift
smallerenhanced ininhibitionvivo
we preparedwaspH-insensitive
amphiphilic
block
succinimide
ester.according
The resulting
PEG-b-PAEMA-NHS
ﬁltration chromatography
also showed
a 1.0
distinct
peakfrom sizes
PAMAM/Pt
was prepared
to comparison,pH
6.7 proliferation
16,19,39,40
28
strated,performancethroughgreater
penetration.
The three-dimenspoly(ethylene
methcoupledPEG-b-PAEMA
with PAMAM/Ptpolymers
to6.6obtain
PEG-b-PAEMAminat
pH 7.4 glycol)-b-poly(2-cyclohexylethyl
to 11.2 min
at pH 6.7,
indicating
thetumor
pH
6.0MCSs are versatilen. The further
pH-sensitive
studying
tumor
biologyaswell as screening
supersensitivityof SCNs/Pt
in response
to tumor extracellular(PEG-b-PCHMA)(SchemeS1), which acrylate)wasdissolution
conﬁrmedby theleft shift
ofand PEG-b-PCHMA-PAMAM/Ptofsuperstructure
into small
particles(Figure0.8
pH 5.6
opolymer PAMAM/Pt
was obtainedby reversible
due pH
to5.1
their
in itsmorphology and
pHS2
to andreleasesmallerparticles
us tosimilarity
investigate
following
similarICNs/Pt
procedures
S2).stablethe elution
time
in gel ﬁltration
chromatography(Figure1d).
Bothand(Scheme
SCNs/Pt
wereFigurequite
in motivatedn chain transfer(RAFT)
polymerization38
0.6
to solid humantumors.41 The penet
potential
drug S5).
delivery. In environment
ourpHstudy,
BxPC-3
SCNs/Pt
preparedby (PBS)
self-assemblyof forPEG-b-PAEMAS1c). Throughdetermination
contentwere
in PAMAM/Pt
5.0
phosphate-buﬀeredsalineand serum(Figure. PEG-b-PAEMA
was reducedby 2- of Pt
and
ICNs
a
pancreaticcancercell
derived Cy5-labeled
MCSs were SCNs
incubatedand (denoted
throughdialysis.
Dynamic
light scattering(DLS)
and PEG-b-PAEMA-PAMAM/Pt,
welearnedthatone
research②
Penetration
and
Tumor
Cell
Killingin Multicellularoduce sulfhydryl-terminatedPEG-b- PAMAM/Pt
0.4
were capability
monitored by confoc
employed
an in that
vitro cancer
model toICNs/Cy5)
assess penetration
andSpheroids
transmission
electronRecent
microscopy(TEM)measurements
PAMAM
carries two PEG-b-PAEMA
chains on (MCSs).
average.
For
studieshave asshown
odiﬁed with
N-(ε-maleimidocaproyloxy)(CLSM)(Figure 2). MCSs weshowedthatthe diameterof SCNs/Pt
wasaround80 inhibition
nmvivo microscopy
and exhibit
cellof our system.
As demoncomparison,
we preparedwas
pH-insensitive
amphiphilic
0.2proliferation
nanomedicineswith block
smallersizes
enhancedinhe resulting
PEG-b-PAEMA-NHS
16,19,39,40(Figure1a). ThethroughpH-insensitive
nanostructures
SCNs/Cy5 or ICNs/Cy5
strated,MCSs are(ICNs/
versatilemodels for for 4 h atpolymerspoly(ethylene
glycol)-b-poly(2-cyclohexylethyl
methperformance
greater cluster
tumor
penetration.
The three-dimensional
PAMAM/Pt
to obtain
PEG-b-PAEMA0
0.2 0.4 0.6 0.8 1.0 1.2 1.4
and therapeutics
subjected to CLSM Z-st
studying totumor
biology
as well as respectively,
screening cancer(PEG-b-PCHMA)
and PEG-b-PCHMA-PAMAM/Pt
supersensitivity of SCNs/Pt in response
tumor
extracellularS1), whichacrylate)
was conﬁrmed
by left shift ofTime(second)of theMCSs microwas deﬁned as 0 μm
due to theirsimilarity
in morphology
biologicalfollowing
similar procedures
FigureS2). particles motivatedreleasesmaller
us toinvestigate
its41 surface andgel ﬁltration
chromatography(Figure(SchemepHS2toand
we foundthat ofthe red ﬂuoresc
solid tumors.
The penetration
activities
wereby self-assemblyof PEG-b-PAEMApotential
for drug delivery. Inenvironment
our study, to
BxPC-3
human treatment,
rmination ofSCNs/Pt
Pt content
in prepared
PAMAM/Pt
mostlylocatedontheperipheryof the M
Cy5-labeled
SCNs
and
ICNs
(denotedas
SCNs/Cy5and
PAMAM/Ptthroughdialysis.
Dynamiclightscattering(DLS)
pancreatic cancer cell derived MCSs
incubatedresponse
and time of SCNs/Pt measured by stopped-flow instrument.
-PAMAM/Pt, we learned that one
Figurewere
S3. pH-Dependent
values.
At thelaser
scanning
depth of 25 μm
ICNs/Cy5)
were monitoredconfocalscanning
and transmission
electron For
microscopy(TEM)asmeasurements
employed
an in vitro model toassess penetration
capability by
PEG-b-PAEMA
chains on average.
in theinteriorwith
area of the M(CLSM)MCSs wereincubatedshowed thatamphiphilic
the diameterof SCNs/Pt
was proliferation
around 80 nm
and cellinhibitionmicroscopyof our system.
As (Figure
demon- 2).intensity
ared pH-insensitiveblock
incubated at ne(Figure 1a). The pH-insensitive
nanostructures(ICNs/
SCNs/Cy5 or ICNs/Cy5
4 h at pH SCNs/Cy5
6.7 and 7.4,
strated,MCSs areversatile three-dimensionalmodels forfor considerably.
e glycol)-b-poly(2-cyclohexylethyl
meth- clustercomparable
to ICNs/C
and subjected
to CLSM
Z-stackpenetration
scanning. behavior
The
studying tumor biology as well asrespectively,
screening cancer
therapeutics
HMA) and PEG-b-PCHMA-PAMAM/Ptabas 400 μm.capability
penetrationof SCNs/Cy5 at tum
surface of
MCSs wasdeﬁnedFor ICNs/Cy5
duepHto6.7 their similarity in morphology
andbiologicalmicroedures (Scheme pH
S27.4and Figure S2).
40 the7.4
pH 6.741 treatment, wepH foundimprovedsigniﬁcantly.
that theﬂuorescenceof Cy5The
wasred ﬂuorescenc
environment to solid tumors. The penetration
activities
of redared by self-assembly of PEG-b-PAEMA30
30
observed
evenpHat the scanninglocatedon the peripheryof clearly
the MCSs
at both
Cy5-labeled SCNs and ICNs mostly(denotedas SCNs/Cy5
and wasdialysis. Dynamic light scattering (DLS)20
20
deep penetration and unif
At the laser
scanning
depth demonstratingof 25 μm, thetheﬂuorescence
ICNs/Cy5) were monitored values.
by confocal
scanning
tron microscopy (TEM) measurements
10in the interior area of
10 MCSs
SCNs/Cy5
atacidicpH because of their r
intensitythehasdroppedmicroscopy (CLSM) (Figure 2). MCSs were incubated withmeter of SCNs/Pt was around 80 nm
into small
particles.
contrast, both SCN
incubated
at0 neutral
pH Inshowednsensitive cluster nanostructures (ICNs/
SCNs/Cy5 or ICNs/Cy5 forconsiderably.
4 h at 01pHSCNs/Cy5
6.7
and100 7.4,
10
1000
1
10
100
1000
showed limited
penetration
at pH 7.4, pcomparable
penetration
to ICNs/Cy5.
However,thesize (nm)behavior
size
(nm)
respectively, and subjected to CLSM
Z-stack
scanning.
The Cy5of theirlarge sizes.
penetrationof SCNs/Cy5
at tumor
pH (pH 6.7)
surfaceof the MCSs wassizedeﬁned
asc of0 SCNs/Pt.
μm.capability
For (a)ICNs/Cy5
Figure1. Tumor-pH-triggeredtransition
DLS
The robustinsidepenetrationof SCNs/Cy5 at a
improvedThe
red ﬂuorescencethe MCSs
80signiﬁcantly.
found
the measurements
red ﬂuorescenceof Cy5(lefttreatment,
panel) andwe
TEM(rightthat
panel)of SCNs/Pt
inwas
with adepth
higherofopportunity
wasofclearlyobservedevenat thethemscanning
85 μm, to access m
mostly buﬀer
locatedonat theperiphery
the60MCSs
at bothphosphate(PB)
pH 7.4.
(b) pH-Dependent
size change
of pH
boost
cellular distribution
uptake and ofcell killing eﬃ
thedeep
penetration
and uniform
values.analyzed
At thebyscanning
of 25
μm,
the
ﬂuorescence
40 andSCNs/Pt
DLS. (c)depth
DLS demonstrating(leftpanel)
TEM(rightby ﬂow cytometryat GelacidicpH
becauseuptakeof theirwas
rapidstudied
disintegration
intensityin the ofinterior
areaatSCNs/Cy5
ofpHtheMCSs
has
droppedpanel)
measurements
SCNs/Pt
6.7.20(d)ﬁltration
Cy5-positive
cells after incubatin
into
small
Inshowed
contrast, analyzing
both SCNs/Cy5
and ICNs/
chromatographyof SCNs/Pt
treatedpH 6.7pH
and7.4.
considerably. analysis
SCNs/Cy5
incubated
at atneutral
0 particles.
6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6
Thecomparable
gel ﬁltrationpenetration
chromatographysystemwasequipped
with
a
size
or ICNs/Cy5
at pH 7.4 and 6
showed limited
penetration atSCNs/Cy5
pH 7.4, presumably
becausebehaviorCy5
to ICNs/Cy5.
However,
pH value theexclusioncolumnandevaporative
light-scattering
detector.
respectively(Figure3aandFigure S6). At pH
of
their
large
sizes.
penetration
capabilityof SCNs/Cy5
at J.tumor
pH
Li, H. J., Du,
Z., et al.
ACS(pH
Nano6.7)
2016, 10, 6753−6761. (Fig. modiﬁed) 29
Figure 1. Tumor-pH-triggered size transitionof SCNs/Pt.
(a) DLSCy5 and
ICNs/Cy5
treatments exhibited
The robustinsidepenetrationof SCNs/Cy5
at acidic
pH endowed
improved signiﬁcantly.
the MCSs(left panel) and TEM (right panel) measurementsof SCNs/Pt The
in red ﬂuorescence
Figure S4. DLS measurements of ICNs/Pt in PB at pH 7.4 (a) and 6.7 (b). (c) Sizeinternalization(∼20% cells
positivethemwitha depthhigher ofopportunity
access more cancer
to cells for 4 h
wereobtainedfromeven
self-assemblyof PEG-b-PCHMAwas
clearlyobserved
at thescanning
85 μm, tophosphate buﬀer (PB) at pH 7.4. (b)Pt)
pH-Dependent
size changeofICNs/Pt
analyzedbyshowed
DLSandat a seriesof
pHInvalues
from 6.3
to7.5.
contrast,
atThepH
6.7, the portion of po
boost
uptakecell h).
killingeﬃcacy.
cellular
PAMAM/Pt
under
identical
conditions.
ICNs/Pt
demonstratingthedeeppenetration
andcellular
uniform
distribution
of
SCNs/Pt analyzed by DLS. (c) DLS(left
panel) andTEM(rightwith SCNs/Cy5
was throughsigniﬁcantly higher th
wasrapid
studiedby ﬂow cytometry(FACS)
zeta
potential,
Pt uptakedrug
content
todisintegration
SCNs/Pt
atGel
acidicpH andbecauseof theirpanel) measurements of SCNs/Pt similarat SCNs/Cy5
pH size,
6.7. (d)ﬁltration
ICNs/Cy5
4 hwith(2.9-fold, p < 0.00
analyzingCy5-positive
cells afterwith
incubating
the after
MCSs(TableS1).
keyof SCNs/Pt
theirultrasensitive
chromatography analysis of SCNs/Pt
treatedat ApH
6.7feature
andIn7.4.
into
small
particles.
contrast,
bothisSCNs/Cy5
and ICNs/
Size (nm)Number (percent)Number (percent)Tumor-pH-triggered size transi,on of SCNs/Pt.

penetration of SCNs/Cy5 and ICNs/Cy5
ACS Nanoin cellinteractions[8,9],
increaseddrug resistance[10–12],cell polarity
and nutrie
increaseddrug
resistance[10–12],cell polarity[13], andnutrient[13],di↵usion
grad[14].Studies on MCTS began in the 1970s and 1980s, led by the e↵orts ofStudieson MCTSbegan inthe 1970s
and1980s, led byleads
the efforts
ofocolleagues[15,16].
In metastatic
tumors,lack ofvascularization
to areascolleagues[15,16].
In
metastatic
tumors,lackofvascularization
leads
to
areas
nutrient deficiency, hypoxia, and acidity [16–18]. Tumors also exhibit layers ofop
nutrient deficiency, hypoxia, and acidity [16–18]. Tumors also exhibit la(non-proliferating), and necrotic cells at progressively greater distances fro
quiescent (non-proliferating), and necrotic cells at progressively greater distan
As we demonstrated in Figure 1, it is now well established that MCTS replica[16]. As we demonstrated in Figure 1, it is now well established that MCTS
oxygen and pH gradients and cell layers in those typically above 500 µm in dnutrient, oxygen and pH gradients and cell layers in those typically above 500 μAnother hallmark of metastatic cancer is the epithelial-to-mesenchymal trans1) [19]. Another hallmark of metastatic cancer is the epithelial-to-mesenchym
epithelial cancer cells lose cell–cell adhesions such as E-cadherin, apical-basolawhich epithelial cancer cells lose cell–cell adhesions such as E-cadherin, apica
on a mesenchymal phenotype to promote migration and invasion [13]. MCTS
and take on a mesenchymal phenotype to promote migration and invasion [2D culture, can induce thisArticle
EMT [20] and a↵ect the migratory profiles of MCT
opposed to 2D culture, can induce this EMT [20] and affect the migratory profilein studies attempting to understand and prevent metastasis. In fact, in ovaria
is crucial in studies attempting to understand and prevent metastasis. In fa
proposed to occur through detachment of cells into the peritoneal cavity, whemetastasis is proposed to occur through detachment of cells into the periton
MCTS and create malignant ascites [13], further highlighting the direct relevan
aggregate into MCTS and create malignant ascites [13], further highlighting t
to human cancer pathophysiology.
the MCTS model to human cancer pathophysiology.

research ②

SCNs(pH-sensi2ve)

3D-model of cancer

Figure1.1.Multicellular
Multicellulartumor
tumorspheroids
spheroids (MCTS)(MCTS) biology.
biology. MCTS
MCTS provide
Figureprovide an
an in
in vi
vi
investigationofofcell–cell
cell–celland
andcell–extracellularcell–extracellular matrix
matrix (ECM)(ECM) interactions.
interactions. Additio
investigation
Additiovivosolidsolidtumors
tumorsininterms
termsofofnutrient,nutrient,oxygen
oxygenandandpH
pH gradients
gradients andand zonezone form
form
ininvivo

Therearearetwotwobroadbroadstrategies
strategiesfor
forpreparation
preparation ofof MCTS
MCTS as an in vitro mo
Thereandcancer
cancerbiology
biology
investigation:
matrix-freeformationandmatrix-dependeandinvestigation:
matrix-freeformationandmatrix-dependent
free MCTS
formationtechniques
includecultureliquidoverlay,
overlay,hanging
hangingd
MCTS
formationtechniques
includecultureviavialiquidflasks,andandmagnetic
magneticlevitation
levitation[21,22].
[21,22]. Matrix-free
Matrix-free techniques
techniques haveflasks,have been
been widwi
highthroughput,
highreproducibility,
reproducibility, andand relative
relative ease
ease ofof culture,
culture, but
Figure 2. CLSM images showing in vitro penetration of ﬂuorescence-labeled SCNs/Cy5 and ICNs/Cy5highin
BxPC-3
multicellularspheroids
throughput,high
but they
they(MCSs). The MCSs were incubated with SCNs/Cy5 or ICNs/Cy5 for 4 h at designated pH and measured
by CLSM Z-stack
The initialextracellularmatrixscanning.
(ECM)during
duringinitial assembly,
assembly, which
which provides
provides important
importantextracellularmatrix(ECM)
surface of the MCSs was deﬁned as 0 μm. Scale bar = 100 μm.
intumor
developmentinvivo[23,24].
Matrix-dependent
techniques,in tumor development in vivo [23,24]. Matrix-dependent techniques, inin contr
contr
biomaterials
torecreate
these
in
vivo
cell–ECM
interactions.
Itshouldbebiomaterials to recreate these in vivo cell–ECM interactions. It should noted
be no
also
been
considered
a promisingstrategy
for MCTS
formationand culture,
an
has
also
been
considered
a promisingstrategy
for MCTS
formationand cultu
incorporates
polymers
thatthat
are arediscussed
throughout
thisreview.
often
incorporates
polymers
discussed
throughout
thisreview.Engineeri
Engineemicrofluidic
MCTS2008,
formation
have been
extensively reviewed
elsewhere [25],Li, H. J., Du, J. Z., et al. ACS Nano 2016, 10, 6753−6761.; Lin, R. Z., & Chang, H. Y. Biotechnology
Journal3(9–10),1172–1184.
30

ICNs(pH-insensi2ve)

Ø The robust penetra2on of SCNs/Cy5 at acidic pHtumor penetra,on and an,tumor ac,vity study

research ②

in vivo

SCNs(pH-sensi2ve)

ICNs(pH-insensitive)Ø SCNs/Pt was eﬀec7ve in suppressing tumor growth
Li, H. J., Du, J. Z., et al. ACS Nano 2016, 10, 6753−6761. 31

ContentsØIntroduc)on§ What are Drug Delivery Systems ?
§ What are Nanopar7cles ?
§ Current problems

ØpH-sensi)ve size-changeable nanopar)cles§ pH-sensi7ve linker cleavage type§ pH-sensi7ve protona7on typeØSummary and perspec)ve32

Summary

ACS Nano

Scheme 1. (a) Structure of PEG-b-PAEMA-PAMAM/Pt. (b) Schematic il
PAEMA-PAMAM/Pt into the pH-sensitive cluster nanobombs (SCNs/Pt)
small particles at tumor acidic pH. (c) Schematic illustration of SCNs/P
barriers to in vivo drug delivery in poorly permeably pancreatic tumor mofor prolonged blood circulation. (II) Prolonged circulation increases theleaky tumor vasculature to accumulate in the vicinity of blood vessels. (Imicroenvironment (pH ∼6.5−7.0), SCNs/Pt instantaneously switch to sm
tumor penetration.

ØSummaryüthe problem of nanopar7cles for DDS is size-dilemmalarge size for EPR eﬀect ↔ small size for penetra7on→ size-change strategy

üpH-sensi7ve and rapid reac7ve size-changeable nanopar7cle wasdesignedügreat tumor penetra7on and an7tumor ac7vity
Li, H. J., Du, J. Z., et al. ACS Nano 2016, 10, 6753−6761.
intrinsically conﬂicting attributes of using nanoparticles33 forcancer treatment.
To address the predicament to improve therapeutic eﬃcacy,various innovative strategies have been reported.8−12 Since
tumor penetration is hindered by the elevated IFP and dense
matrix, normalization of tumor vessels to restore the pressure
gradients13 and degradation of the collagen matrix to reduce
14

improve
were emproblemits poortissues.2
switchabof chemProblems and Perspectives
Ø Problems
・oﬀ-target of EPR eﬀect (liver, Spleen, etc.)
・cell penetra7on・complicated tumor microenvironment
・reten7on 7me → size-increasing strategy
・toxicity (an7genicity, slow elimina7on)・protein corona・transla7onal gap
Yu, W., Liu, R., Zhou, Y., & Gao, H. ACS Central Science 2020, 6(2), 100–116.; Blanco, E., Shen, H., & Ferrari, M. Nature Biotechnology 2015, 33(9), 941–951.;
34
Yokoyama, M. Drug Delivery System 2018, 33(2), 89–97.; Zanganeh, S., Spitler, R., et al. InternaLonal Journal of Biochemistry and Cell Biology 2016, 75, 143–147.

can cause NPs to preferentially distribute to certain
tissues. For example, a poly(β- amino- ester) (PBAE)
ter- polymer/PEG lipid conjugate was optimized forlung localization, achieving efficacy two orders ofmagnitude above the pre-optimized form both in vitro
and in vivo179. Other PBAE polymers have been developed that preferentially target glioblastoma cells overhealthy cells in vitro200. Even AuNPs can be optimized
to passively target triple- negative breast cancer cells,

Problems and Perspec,ves

Surface andmaterial propertiesØ Perspec7ves
・combina7on of modiﬁca7on

Self
peptidesCarbohydrates(glucose, galactose,mannose, dextran)
Antibodies

Responsiveness
Endogenous triggers:• Acidic/hypoxic
environment• Enzyme levels• Metabolic shifts
Exogenous triggers:• Light• Sound• Force (physicalor magnetic)

PEGylation

・Precision medicine

TargetingCellcoating

Density andtoughness

Cell surface
receptors
Vitamins(vitamin D,
folate)Aptamers(peptides)

Architecture
Size
Shape
Charge–+Fig. 5 | Commonly engineered NP surface properties that allow for enhanced
Surfaceand material
properties,
architecture,targetingand35
Mitchell, M. J., Billingsley,delivery.
M. M., etal. Nature
Reviews
Drug Discovery
2021,
20(2),moieties
101–124.
responsiveness are all attributes of nanoparticles (NPs) that can be altered in intelligent
designs to tailor the platform to a specific application. Different combinations of thesefour properties allows for seemingly endless permutations of NP features and platforms.
PEG, poly(ethylene glycol).
NATURE REVIEWS | DRUG DISCOVERY

exogenand ththe trig
this reand otOn
stimuldown
biologinitialthe bloNP ex
At thesystemhave imof thecells204
ditionaadmin
by 10%
up to 9commin the kthis sy
triggerhave gtheir dto the tAnopoly(acan beapplicatransdor degplatformodif
cule, por recoa similto deli
respon