Dually Enzyme- And Acid-Triggered Self-Immolative Ketal Glycoside Nanoparticles for Effective Cancer Prodrug Monotherapy

Article abstract of DOI:10.1021/acs.nanolett.0c01973

The use of glycoside prodrugs is a promising strategy for developing new targeted medicines for chemotherapy. However, the in vivo utility of such prodrugs is hindered by insufficient activation and the lack of convenient synthetic methods. We have developed an innovative strategy for synthesizing ketal glycoside prodrugs that are unique in being activated by a dual enzyme- and acid-triggered self-immolative mechanism. Amphiphilic glucosyl acetone-based ketal-linked etoposide glycoside prodrug isomers were synthesized and fabricated into excipient-free nanoparticles for effective cancer prodrug monotherapy. Hydrolysis of the glycosidic linkage or the ketal linkage triggered hydrolysis of the other linkage, which resulted in spontaneous self-immolative hydrolysis of the prodrugs. Nanoparticles of the prodrug isomer that was the most labile in a lysosome-mimicking environment displayed high intratumoral accumulation and strong antitumor activity in an A549 xenograft mouse model. Our strategy may be useful for the development of stimulus-responsive self-immolative prodrugs and their nanomedicines.

Full text of DOI:10.1021/acs.nanolett.0c01973

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Letter
Dually Enzyme- and Acid-Triggered Self-Immolative Ketal Glycoside
Nanoparticles for Effective Cancer Prodrug Monotherapy
Na Yu, Tao Liu, Xi Zhang, Ningqiang Gong, Tianjiao Ji, Jing Chen, Xing-Jie Liang, Daniel S. Kohane,
and Shutao Guo*
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ABSTRACT: The use of glycoside prodrugs is a promising strategy for
developing new targeted medicines for chemotherapy. However, the in vivo
utility of such prodrugs is hindered by insufficient activation and the lack of
convenient synthetic methods. We have developed an innovative strategy for
synthesizing ketal glycoside prodrugs that are unique in being activated by a
dual enzyme- and acid-triggered self-immolative mechanism. Amphiphilic
glucosyl acetone-based ketal-linked etoposide glycoside prodrug isomers
were synthesized and fabricated into excipient-free nanoparticles for effective
cancer prodrug monotherapy. Hydrolysis of the glycosidic linkage or the
ketal linkage triggered hydrolysis of the other linkage, which resulted in
spontaneous self-immolative hydrolysis of the prodrugs. Nanoparticles of the
prodrug isomer that was the most labile in a lysosome-mimicking
environment displayed high intratumoral accumulation and strong antitumor
activity in an A549 xenograft mouse model. Our strategy may be useful for
the development of stimulus-responsive self-immolative prodrugs and their nanomedicines.
KEYWORDS: Nanomedicines, Prodrugs, Glycosides, Cancer Therapy, pH-Sensitive
glycoside prodrugs is complex and difficult, their practical
utility is limited. Recently, however their synthesis has been
simplified by incorporation of self-immolative linkers, which
have facilitated the development of glycosidase-activatable
prodrugs.21−26 The glycosidic linkages of these prodrugs serve
as the sole trigger for activation, which is thus predominantly
under enzymatic control. Although the elevated glycosidase
levels in necrotic tumors can activate glycoside prodrugs,27,28
in vivo activation of most of these prodrugs remains insufficient
when they are used as monotherapies;29−32 thus exogenous
stimulus (e.g., immunoconjugates of enzyme and tumor-
targeted antibody) is often combined to achieve efficient
prodrug activation. In light of these findings, the development
of glycoside prodrugs that can be readily synthesized and
undergo efficient in vivo transformation would be highly
desirable.
INTRODUCTION
■
Nanomedicines are broadly employed in pharmaceutical
development to treat different diseases and have achieved
great success indubitably.1−3 However, only few anticancer
nanomedicines succeed so far, by solving the solubility issue
for poorly soluble drugs (e.g., paclitaxel) and/or reducing
adverse effects through modulating pharmacokinetics of drugs
(e.g., doxorubicin).4 The successful development of new
anticancer nanomedicines has been significantly hindered by
poor compatibility between nanocarriers and drugs, resulting
in low drug loading, poor colloidal stability and/or premature
burst release after administration, and so forth.
Prodrug strategies can improve drug performance and
facilitate the design and development of new medicines,
including nanomedicines.5−15 Because of the good biocompat-
ibility and tumor-targeting capability of various small
molecules that occur naturally in the human body (e.g.,
monosaccharides, cholesterol, and fatty acids), they are ideal
components for constructing anticancer prodrugs.16,17 In fact,
glycoside prodrugs in which monosaccharides are directly
linked to the hydroxyl group of drugs via glycosidic linkages
that can be activated by glycosidases have been synthesized for
selective chemotherapy against malignant tumors.18,19 One
example is glufosfamide, a glycoside isophosphoramide
mustard prodrug in a phase 3 clinical trial for the treatment
of pancreatic cancer.20 However, because the synthesis of
Herein, we report a simple yet novel strategy for
synthesizing glycoside prodrugs by conjugating hydroxyl-
group-containing monosaccharides with hydroxyl-group-con-
Received: May 8, 2020
Revised: June 19, 2020
https://dx.doi.org/10.1021/acs.nanolett.0c01973
© XXXX American Chemical Society
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a
Scheme 1. Schematic Illustration
a
Schematic of self-assembly of glucosyl prodrug NPs, their glucose transporter−mediated uptake by an A549 tumor cell in vivo, and subsequent
self-immolative activation of the prodrug by β-glucosidase and acid in a lysosome. EPR = enhanced permeability and retention.
Figure 1. Syntheses of isomeric ETP glycoside prodrugs.
taining drugs via pH-sensitive asymmetrical acetone-based
ketal linkages. The resulting amphiphilic ketal glycoside
prodrugs self-assembled into glucose-decorated nanoparticles
(NPs) that could be internalized into enzyme-rich acidic
organelles of tumor cells, where they can be activated by a
unique mechanism involving the dual action of glycosidase and
B
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Figure 2. (a) Transmission electron microscopy images of (I) prodrug 1, (II) prodrug 2, (III) prodrug 3, and (IV) prodrug 4 NPs. Hydrolysis
curves of NPs of (b) α-anomeric and (c) β-anomeric prodrugs in the presence or absence of β-glucosidase (10 U/mL) at 37 °C at pH 5.0. Data are
means SD, n = 4. (d) Prodrug t1/2 values were derived from the data in panels (b,c). The numbers denote fold increases in hydrolysis rate in the
presence of β-glucosidase. (e) Dual glycosidase- and acid-catalyzed self-immolative hydrolysis of ketal glycoside prodrugs; hydrolysis of the
glycosidic or ketal linkage triggers hydrolysis of the other linkage, resulting in spontaneous self-immolation.
acid; that is, hydrolysis of either the glycosidic linkage or the
ketal linkage triggered spontaneous self-immolative hydrolysis
of the other linkage, efficiently releasing the drug, along with
acetone and glucose.
by acid) are novel features that have rarely been reported
previously.
Synthesis of Ketal Glycoside Prodrugs. Given acetone-
based ketal-linked prodrugs can be obtained from acid-
catalyzed reactions of alcohols and isopropenyl ethers34−36
and that substitution at the 1-position of glucose does not
affect the affinity of glucose for its transporters,37,38 we began
by carrying out reactions of ETP with isopropenyl 2,3,4,6-tetra-
O-benzyl-α- or β-D-glucopyranoside and subsequent hydro-
genolysis to remove the benzyl protecting groups (Figure 1,
Schemes S1−S4). For most ETP prodrugs, nucleophilic
substitution under basic conditions proceeds at the phenol
group of the ETP molecule. However, in our case, the reaction
between isopropenyl 2,3,4,6-tetra-O-benzyl-D-glucopyranoside
and the ETP phenol group was inhibited, even when an excess
of the isopropenyl ether was used and the reaction time was
prolonged. We speculate that previously reported syntheses of
ETP glycoside prodrugs at the phenol position succeeded
owing to the use of a longer spacer and/or the use of a less
bulky protecting group (e.g., acetyl). The reactions occurred
instead at the 2″- and 3″-hydroxyl groups. The similar
polarities of the two resulting isomers impaired their
separation, however they could be isolated by using preparative
high-performance liquid chromatography. Prodrugs 1 and 2
were synthesized from the α-anomeric isopropenyl ethers, and
prodrugs 3 and 4 were synthesized from the β-anomeric ethers.
The 2″ and 3″ isomers were unambiguously confirmed on the
RESULTS AND DISCUSSION
■
As a proof of concept, glucosyl acetone-based ketal-linked
glycoside prodrugs of etoposide (ETP, a topoisomerase II
inhibitor for the treatment of various cancers33) were
synthesized and fabricated into prodrug NPs (Scheme 1).
These NPs exhibited glucose transporter-mediated uptake by
tumor cells, where they might undergo dual β-glucosidase- and
acid-triggered self-immolative hydrolysis in lysosomes or
undergo acid-triggered ketal hydrolysis in endosomes. In an
A549 xenograft mouse model, they displayed high intratumoral
accumulation and strong antitumor activity. The use of
excipient-free NPs of amphiphilic prodrugs that can be
internalized into enzyme-rich acidic organelles represents a
novel strategy for overcoming the problems caused by
insufficient acidity and low enzyme levels outside tumor
cells. This feature is particularly important in situations in
which the extracellular acidity and enzyme levels in tumors are
too low for glycoside prodrug activation in traditional glycoside
prodrug monotherapies. The structure of ketal glycoside
prodrugs reported herein, the method by which they were
synthesized, and the unique mechanism by which they are
released (self-immolation dually triggered by an enzyme and
C
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Figure 3. (a) IC50 of ETP formulations in A549 cells. (b) Cell cycle arrest by flow cytometry. Cells were treated with 20 μM ETP formulations for
12 h. Data are means SD; n = 3. (c,e) Cellular uptake and (d,f) intracellular hydrolysis (%) of prodrugs. In c−f, cells were incubated with ETP
formulations at 20 μM; data are means SD; n = 4; * indicates p < 0.05, and ** indicates p < 0.01; ns = not significant. Ten micromolar conduritol
B epoxide (CBE) and 50 μM cytochalasin B were used. “Prodrug NPs (Free)” means free etoposide hydrolyzed from prodrugs, and “Prodrug NPs
(Total)” means total etoposide in prodrugs including those hydrolyzed and unhydrolyzed forms.
1
basis of their H- and 13C NMR (including DEPT-135) and
2D-NMR (HSQC and HMBC) spectroscopy (Figures S1−
S24). The overall yield of each prodrug was approximately
35%, and HPLC confirmed them to be highly pure (Figure
S25). Note that there has been only one report of the synthesis
of a ketal glycoside prodrug by Tietze et al., who used a
multistep approach involving a TMSOTf-catalyzed nucleo-
philic substitution reaction.39 However, this approach is
limited in that it releases carbonyl-group-containing drug
derivatives rather than native drugs. In contrast, our simple yet
novel approach, which features mild reaction conditions and
high yields, can release native drugs rather than derivatives and
may boost the development of ketal glycosides.
of 20−30 nm (Figure 2a), a result that was confirmed by
dynamic light scattering (Figure S26, Table S1). The zeta
potentials of the glucose-decorated NPs were approximately
−14 mV and their ETP content was 72.8 wt %, which is much
higher than that of previously reported nanoparticulate ETP
formulations.40,41 Moreover, we quantified the number
concentrations of prodrug NPs (1 mg/mL) by the NP
tracking analysis (NTA) system and calculated the average
number of prodrug compound molecules in each NP (Figure
S27). As a result, the number concentrations of NPs ranged
from 5.68 × 108 to 6.65 × 108 particles/mL, and each prodrug
NP contained approximately 1.12 × 109 to 1.31 × 109
molecules.
Preparation and Characterizations of Ketal Glycoside
Prodrug NPs. To elucidate the relationship between prodrug
structure and properties, we carried out various experiments
with the above-mentioned isomeric ETP prodrugs, which had
differing anomeric and positional configurations. Given that
prodrug-containing NPs can be internalized by tumor cells into
enzyme-rich acidic organelles and then efficiently activated by
enzymes and acid, we fabricated excipient-free NPs from the
amphiphilic ETP glycoside prodrugs by means of a nano-
precipitation method. Transmission electron microscopy
showed that the NPs were uniform spheres with diameters
For practical use, the prodrug in the NPs must undergo acid-
and enzyme-catalyzed transformation in lysosomal environ-
ments. Therefore, we investigated the kinetics of hydrolysis of
the prodrug NPs at lysosomal pH (5.0)42 and physiological pH
(7.4) by using HPLC. Note that the acetal linkage in the
glucose moiety is stable at weakly acidic pH,43 and thus any
pH sensitivity exhibited by the prodrugs can be attributed to
the acetone-based ketal linkages. In all cases, prodrug
hydrolysis followed pseudo-first-order kinetics (Figures 2b,c,
S28, and S29 and Table S2). Calculation of the half-lives (t1/2
)
of the prodrugs at pH 5.0, ln 2/kobs, where kobs is the hydrolytic
D
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Figure 4. (a) In vivo biodistribution of ETP formulations in an A549 xenograft tumor model and (b) hydrolysis (%) of prodrug 3 NPs. Data are
means SD, n = 4; * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001; ns = not significant. (c) Temporal dependence of
tumor volume after IV injection of ETP formulations (15 mg/kg ETP equivalent) on days 0, 3, and 6. Data are means
Representative H&E staining and Ki67 staining of tumor sections.
SD, n = 7. (d)
constant, showed that the α-anomeric prodrugs hydrolyzed
faster than the β-anomeric prodrugs; the 3″-isomer of each
anomer hydrolyzed faster than the 2″-isomer. The t1/2 values
for NPs of prodrugs 1−4 at pH 5.0 were 1.56, 1.83, 1.76, and
2.88 h, respectively. However, after 24 h at pH 7.4, less than
5% of the prodrugs had hydrolyzed (Figure S29).
biocompatible) (Figure S30). These results indicate that our
rationally designed ketal-linked β-anomeric glycoside ETP
prodrugs exhibited β-glucosidase- and acid-catalyzed self-
immolative hydrolysis by the mechanism depicted in Figure
2e, which is different from that for previously reported self-
immolative glycoside prodrugs.
We also evaluated enzymatic hydrolysis by incubating the
prodrug NPs with 10 U/mL β-glucosidase at pH 5.0 and pH
7.4 (Figures 2b,c, S28, and S29 and Table S2). At pH 5.0, the
hydrolysis rates for β-anomeric prodrugs 3 and 4 in the
presence of the enzyme were 1.6 and 1.18 times the values in
its absence, respectively (Figure 2d); whereas, the enzyme had
a negligible effect on the rate of hydrolysis of the α-anomeric
prodrugs. At pH 7.4, no significant difference in prodrug
hydrolysis in the absence and presence of β-glycosidase was
observed (Figure S29), which may have been due to the low
activity of the enzyme at pH 7.4. However, it must be noted
the effect of β-glycosidase on prodrug activation may be
different in vivo than in buffers. HPLC analysis revealed that
the only degradation products of the prodrugs were native
ETP, acetone (a metabolite), and glucose (which is
In Vitro Cell Studies. Modification of NPs with glucose
has been demonstrated to enhance their intratumoral
accumulation by glucose transporters present in endothelial
cells and tumor cells.44,45 Therefore, we used A549 cells, which
highly express glucose transporters,46,47 to assess the
cytotoxicity and therapeutic efficacy of the ETP prodrug
NPs. Compared with free ETP (IC50 = 7.7 μM), prodrug 2
(IC50 = 4.4 μM) and prodrug 3 (IC50 = 3.66 μM) were more
toxic, whereas prodrugs 1 and 4 were less toxic (IC50 = 14.2
and 7.95 μM, respectively) (Figures 3a and S31). The presence
of conduritol B epoxide (a β-glucosidase inhibitor) decreased
the cytotoxicities of β-anomeric prodrugs 3 and 4 (IC50 = 9.56
and 12.1 μM, respectively), but the toxicities were still
comparable to that of free ETP. This result demonstrates the
stereoselectivity of the interaction between β-glucosidase and
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the glycoside prodrugs and strong acid-activation of the
glycoside prodrugs in cells. A cell cycle arrest assay
demonstrated that the arrest ability of prodrug 3 in the S
phase and the early G2/M phase was higher than the arrest
abilities of the other drugs in these two phases (Figures 3b and
S32), in accordance with the cytotoxicity results. Among the
prodrug NPs, the prodrug 3 NPs showed the highest cellular
uptake, which exceeded that of free ETP at 2 h (Figure 3c).
However, the uptake of ETP was similar to or better than other
prodrug NPs, indicating that the glucose transporter-mediated
endocytosis for prodrug NPs was markedly affected by the
prodrug structure and other pathways might also be involved
in cellular uptake of prodrug NPs. The intracellular hydrolysis
percentages of the prodrugs exceeded 50%, and intracellular
hydrolysis of prodrug 3 reached 94% at 2 h (Figure 3d).
However, in the presence of conduritol B epoxide, the
intracellular hydrolysis percentage of prodrug 3 at 2 h was
only 51%. In contrast, the presence of the β-glucosidase
inhibitor had no statistically significant effect on the hydrolysis
percentage for the other prodrugs. We also found that the
presence of cytochalasin B, an inhibitor of glucose transporter
type I, reduced the cellular uptake of all four prodrugs (Figure
3e) without affecting their intracellular hydrolysis (Figure 3f),
indicating that this transporter facilitated the cellular uptake of
the glucose-decorated NPs.
In Vivo Ketal Glycoside Prodrug NPs Monotherapy.
Because prodrug 3 had the lowest IC50, the highest cellular
uptake, and the most efficient hydrolysis in A549 cells, we used
it for in vivo studies. Specifically, we evaluated the
biodistributions and antitumor efficacies of ETP formulations
in an A549 xenograft tumor model (Figure 4). Free ETP
injection prepared according to the method used for the
commercial formulation Toposar (which is more efficacious
than Etopophos25) was used for comparison. Like other kinds
of NPs, prodrug 3 NPs were cleared mainly by the liver and
spleen. Four hours after administration, the accumulations
(μg/g) of prodrug 3 NPs in the liver, spleen, lungs, and
kidneys were higher than the accumulation in tumor tissue;
whereas 24 h after administration, the accumulations in these
organs were significantly decreased (Figure 4a). Note that the
ratio of intratumoral prodrug accumulation to prodrug
accumulation in organs increased over time, indicating the
enhanced permeability and retention of NPs48−50 and GLUT-
mediated transport of the glucose-decorated prodrug NPs.
Compared with free ETP injection, prodrug 3 NPs exhibited
higher accumulation in tumor tissue (Figure 4a). Furthermore,
prodrug 3 was efficiently transformed to native ETP to a
greater extent in tumor tissue than in the organs (Figure 4b);
intratumoral hydrolysis of prodrug 3 was about 85% at 24 h.
The efficient transformation of prodrug 3 in tumor tissue was
probably due to enhanced cellular uptake mediated by the high
levels of the GLUT1 transporter on tumor cells, the high
abundance of β-glucosidase in the tumor cells, and the acidic
tumor microenvironment. Prodrug 3 NPs exhibited much
better inhibitory effects on A549 tumor than free ETP
injection (Figures 4c and S33), and there was no obvious
change in body weight in mice treated with the NPs (Figure
S34). Hematoxylin and eosin (H&E) and Ki67 immunohis-
tochemical staining confirmed that prodrug 3 NPs significantly
inhibited the growth of tumor cells (Figure 4d). In addition,
H&E staining of organs and blood biochemistry analysis
showed no obvious abnormalities in the treatment groups
(Figures S35 and S36).
Although many glycoside prodrugs have been synthesized,
the mechanism of their activation in cells and animals has not
been investigated in detail, and the resulting lack of
information is partially responsible for the fact that glycoside
prodrugs have not advanced into practical use. Here, we
comprehensively investigated the characteristics of the ketal
glycoside prodrugs and their NPs in cells and animal models.
Glucosyl prodrug NPs were internalized by tumor cells into
enzyme-rich acidic organelles, where they can be efficiently
activated by enzymes and acid. Our findings provide important
insight into the mechanism of action of ketal glycoside prodrug
formulations and should facilitate the design of more-efficient
glucoside prodrugs.
The development of more-efficacious glycoside prodrugs
and prodrug formulations is an important goal of work that is
ongoing in our group and the groups of others in the field. The
strategy reported herein, which combines rational chemical
design and nanotechnology for the development of efficacious
glycoside prodrug monotherapy, is an important one.
CONCLUSIONS
■
In summary, we have developed a simple yet novel method for
synthesizing ketal glycoside prodrugs in which a mono-
saccharide moiety is directly conjugated to the drug via an
acetone-based ketal linkage, and we evaluated the activities of
self-assembled NPs of isomeric ETP glycoside prodrugs in an
A549 xenograft tumor model. The pH sensitivity and β-
glucosidase sensitivity of the prodrug NPs depended on the
anomer of the prodrug: specifically, NPs of α-anomeric
prodrugs underwent only acid-activated hydrolysis, whereas
hydrolysis of β-anomeric prodrug NPs was activated by both β-
glycosidase and acid. Of all the prodrugs, the 3″-positional β-
anomeric prodrug (i.e., prodrug 3) was the most labile toward
both acid and β-glucosidase, and the prodrug 3 NPs exhibited
the highest cytotoxicity against A549 cells. The presence of a
GLUT1 inhibitor reduced uptake of the NPs by tumor cells,
and the presence of a glucosidase inhibitor downregulated the
extent of β-anomeric prodrug hydrolysis. In a biodistribution
study, prodrug 3 NPs showed notably higher accumulation
than free ETP injection; the NPs were effectively transformed
in tumor tissue and showed much better efficacy in reducing
tumor volume than did free ETP injection. Thus, they have the
potential to be effective as a prodrug monotherapy, which has
been difficult to achieve with previously reported glycoside
prodrugs. Our strategy of combining rational chemical design
and nanotechnology for the development of efficacious
glycoside prodrug monotherapy may be useful for the synthesis
of stimulus-responsive self-immolative prodrugs and may
facilitate the development of targeted chemotherapeutics.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.nanolett.0c01973.
Detailed methods, synthesis procedures of ketal glyco-
side prodrugs, NMR spectra, hydrolysis parameters,
DLS, NanoSight characterization, HPLC chromato-
grams, cell toxicities, cell cycle arrest by flow cytometry,
H&E staining of organs, weight of resected tumors,
blood biochemistry analysis and change in body weight
of mice treated with the NPs (PDF)
F
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AUTHOR INFORMATION
REFERENCES
■
■
(1) Ventola, C. L. Progress in Nanomedicine: Approved and
Investigational Nanodrugs. P&T 2017, 42 (12), 742−755.
(2) Bobo, D.; Robinson, K. J.; Islam, J.; Thurecht, K. J.; Corrie, S. R.
Nanoparticle-Based Medicines: A Review of Fda-Approved Materials
and Clinical Trials to Date. Pharm. Res. 2016, 33 (10), 2373−2387.
(3) Qi, B.; Wang, C.; Ding, J.; Tao, W. Editorial: Applications of
Nanobiotechnology in Pharmacology. Front. Pharmacol. 2019, 10,
1451.
Corresponding Author
Shutao Guo − Key Laboratory of Functional Polymer Materials
of Ministry of Education, State Key Laboratory of Medicinal
Chemical Biology, Institute of Polymer Chemistry, College of
Chemistry, Nankai University, Tianjin 300071, China;
orcid.org/0000-0002-0916-7451; Email: stguo@
nankai.edu.cn
(4) Dawidczyk, C. M.; Kim, C.; Park, J. H.; Russell, L. M.; Lee, K.
H.; Pomper, M. G.; Searson, P. C. State-of-the-Art in Design Rules for
Drug Delivery Platforms: Lessons Learned from Fda-Approved
Nanomedicines. J. Controlled Release 2014, 187, 133−144.
(5) Rautio, J.; Meanwell, N. A.; Di, L.; Hageman, M. J. The
Expanding Role of Prodrugs in Contemporary Drug Design and
Development. Nat. Rev. Drug Discovery 2018, 17 (8), 559−587.
(6) van der Meel, R.; Sulheim, E.; Shi, Y.; Kiessling, F.; Mulder, W.
J.; Lammers, T. Smart Cancer Nanomedicine. Nat. Nanotechnol. 2019,
14 (11), 1007−1017.
Authors
Na Yu − Key Laboratory of Functional Polymer Materials of
Ministry of Education, State Key Laboratory of Medicinal
Chemical Biology, Institute of Polymer Chemistry, College of
Chemistry, Nankai University, Tianjin 300071, China
Tao Liu − Key Laboratory of Functional Polymer Materials of
Ministry of Education, State Key Laboratory of Medicinal
Chemical Biology, Institute of Polymer Chemistry, College of
Chemistry, Nankai University, Tianjin 300071, China
Xi Zhang − Key Laboratory of Functional Polymer Materials of
Ministry of Education, State Key Laboratory of Medicinal
Chemical Biology, Institute of Polymer Chemistry, College of
Chemistry, Nankai University, Tianjin 300071, China
Ningqiang Gong − Laboratory of Controllable
Nanopharmaceuticals, Chinese Academy of Sciences (CAS)
Center for Excellence in Nanoscience and CAS Key Laboratory
for Biomedical Effects of Nanomaterials and Nanosafety,
National Center for Nanoscience and Technology of China,
Beijing 100190, China
Tianjiao Ji − Laboratory for Biomaterials and Drug Delivery,
Division of Critical Care Medicine, Children’s Hospital Boston,
Harvard Medical School, Boston, Massachusetts 02115, United
States
Jing Chen − Laboratory of Controllable Nanopharmaceuticals,
Chinese Academy of Sciences (CAS) Center for Excellence in
Nanoscience and CAS Key Laboratory for Biomedical Effects of
Nanomaterials and Nanosafety, National Center for
Nanoscience and Technology of China, Beijing 100190, China
Xing-Jie Liang − Laboratory of Controllable
Nanopharmaceuticals, Chinese Academy of Sciences (CAS)
Center for Excellence in Nanoscience and CAS Key Laboratory
for Biomedical Effects of Nanomaterials and Nanosafety,
National Center for Nanoscience and Technology of China,
Beijing 100190, China; University of Chinese Academy of
Sciences, Beijing 100049, China; orcid.org/0000-0002-
4793-1705
̈
(7) Desmaele, D.; Gref, R.; Couvreur, P. Squalenoylation: A Generic
Platform for Nanoparticular Drug Delivery. J. Controlled Release 2012,
161 (2), 609−618.
(8) Couvreur, P.; Stella, B.; Reddy, L. H.; Hillaireau, H.; Dubernet,
̂
̈
C.; Desmaele, D.; Lepetre-Mouelhi, S.; Rocco, F.; Dereuddre-
Bosquet, N.; Clayette, P.; Rosilio, V.; Marsaud, V.; Renoir, J.-M.;
Cattel, L. Squalenoyl Nanomedicines as Potential Therapeutics. Nano
Lett. 2006, 6 (11), 2544−2548.
(9) Ling, X.; Chen, X.; Riddell, I. A.; Tao, W.; Wang, J.; Hollett, G.;
Lippard, S. J.; Farokhzad, O. C.; Shi, J.; Wu, J. Glutathione-
Scavenging Poly(Disulfide Amide) Nanoparticles for the Effective
Delivery of Pt(Iv) Prodrugs and Reversal of Cisplatin Resistance.
Nano Lett. 2018, 18 (7), 4618−4625.
(10) Wang, Y.; Liu, D.; Zheng, Q.; Zhao, Q.; Zhang, H.; Ma, Y.;
Fallon, J. K.; Fu, Q.; Haynes, M. T.; Lin, G.; Zhang, R.; Wang, D.;
Yang, X.; Zhao, L.; He, Z.; Liu, F. Disulfide Bond Bridge Insertion
Turns Hydrophobic Anticancer Prodrugs into Self-Assembled Nano-
medicines. Nano Lett. 2014, 14 (10), 5577−5583.
(11) Li, J.; Li, Y.; Wang, Y.; Ke, W.; Chen, W.; Wang, W.; Ge, Z.
Polymer Prodrug-Based Nanoreactors Activated by Tumor Acidity for
Orchestrated Oxidation/Chemotherapy. Nano Lett. 2017, 17 (11),
6983−6990.
(12) Ling, X.; Tu, J.; Wang, J.; Shajii, A.; Kong, N.; Feng, C.; Zhang,
Y.; Yu, M.; Xie, T.; Bharwani, Z.; Aljaeid, B. M.; Shi, B.; Tao, W.;
Farokhzad, O. C. Glutathione-Responsive Prodrug Nanoparticles for
Effective Drug Delivery and Cancer Therapy. ACS Nano 2019, 13
(1), 357−370.
(13) Lv, W.; Li, Y.; Li, F.; Lan, X.; Zhang, Y.; Du, L.; Zhao, Q.;
Phillips, D. L.; Wang, W. Upconversion-Like Photolysis of Bodipy-
Based Prodrugs Via a One-Photon Process. J. Am. Chem. Soc. 2019,
141 (44), 17482−17486.
(14) Zhang, W.; Ji, T.; Li, Y.; Zheng, Y.; Mehta, M.; Zhao, C.; Liu,
A.; Kohane, D. S. Light-Triggered Release of Conventional Local
Anesthetics from a Macromolecular Prodrug for on-Demand Local
Anesthesia. Nat. Commun. 2020, 11 (1), 2323.
Daniel S. Kohane − Laboratory for Biomaterials and Drug
Delivery, Division of Critical Care Medicine, Children’s Hospital
Boston, Harvard Medical School, Boston, Massachusetts 02115,
United States; orcid.org/0000-0001-5369-5932
(15) Zhao, C.; Liu, A.; Santamaria, C. M.; Shomorony, A.; Ji, T.;
Wei, T.; Gordon, A.; Elofsson, H.; Mehta, M.; Yang, R.; Kohane, D. S.
Polymer-Tetrodotoxin Conjugates to Induce Prolonged Duration
Local Anesthesia with Minimal Toxicity. Nat. Commun. 2019, 10 (1),
2566.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.nanolett.0c01973
Notes
(16) Chen, F.; Huang, G. Application of Glycosylation in Targeted
Drug Delivery. Eur. J. Med. Chem. 2019, 182, 111612.
(17) Irby, D.; Du, C.; Li, F. Lipid-Drug Conjugate for Enhancing
Drug Delivery. Mol. Pharmaceutics 2017, 14 (5), 1325−1338.
(18) Tietze, L. F.; Major, F.; Schuberth, I. Antitumor Agents:
Development of Highly Potent Glycosidic Duocarmycin Analogues
for Selective Cancer Therapy. Angew. Chem., Int. Ed. 2006, 45 (39),
6574−6577.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors acknowledge financial support from the National
Natural Science Foundation of China (NSFC, No. 51773098),
the Natural Science Foundation of Tianjin of China (No.
18JCYBJC28300), and a joint NSFC−German Research
Foundation project (No. 31761133013).
(19) Tietze, L. F.; Herzig, T.; Fecher, A.; Haunert, F.; Schuberth, I.
Highly Selective Glycosylated Prodrugs of Cytostatic Cc-1065
G
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Nano Lett. XXXX, XXX, XXX−XXX

Nano Letters
pubs.acs.org/NanoLett
Letter
Analogues for Antibody-Directed Enzyme Tumor Therapy. Chem-
BioChem 2001, 2 (10), 758−765.
(20) Ciuleanu, T. E.; Pavlovsky, A. V.; Bodoky, G.; Garin, A. M.;
Langmuir, V. K.; Kroll, S.; Tidmarsh, G. T. A Randomised Phase Iii
Trial of Glufosfamide Compared with Best Supportive Care in
Metastatic Pancreatic Adenocarcinoma Previously Treated with
Gemcitabine. Eur. J. Cancer 2009, 45 (9), 1589−1596.
(35) Zhong, H.; Mu, J.; Du, Y.; Xu, Z.; Xu, Y.; Yu, N.; Zhang, S.;
Guo, S. Acid-Triggered Release of Native Gemcitabine Conjugated in
Polyketal Nanoparticles for Enhanced Anticancer Therapy. Biomacro-
molecules 2020, 21 (2), 803−814.
(36) Guo, S.; Nakagawa, Y.; Barhoumi, A.; Wang, W.; Zhan, C.;
Tong, R.; Santamaria, C.; Kohane, D. S. Extended Release of Native
Drug Conjugated in Polyketal Microparticles. J. Am. Chem. Soc. 2016,
138 (19), 6127−6130.
(37) Calvaresi, E. C.; Hergenrother, P. J. Glucose Conjugation for
the Specific Targeting and Treatment of Cancer. Chem. Sci. 2013, 4
(6), 2319−2333.
(21) Andrianomenjanahary, S.; Dong, X.; Florent, J. C.; Gaudel, G.;
Gesson, J. P.; Jacquesy, J. C.; Koch, M.; Michel, S.; Mondon, M.;
Monneret, C.; Petit, P.; Renoux, B.; Tillequin, F. Synthesis of Novel
Targeted Pro-Prodrugs of Anthracyclines Potentially Activated by a
Monoclonal Antibody Galactosidase Conjugate (Part 1). Bioorg. Med.
Chem. Lett. 1992, 2 (9), 1093−1096.
(38) Patra, M.; Awuah, S. G.; Lippard, S. J. Chemical Approach to
Positional Isomers of Glucose-Platinum Conjugates Reveals Specific
Cancer Targeting through Glucose-Transporter-Mediated Uptake in
Vitro and in Vivo. J. Am. Chem. Soc. 2016, 138 (38), 12541−12551.
(22) Bouvier, E.; Thirot, S.; Schmidt, F.; Monneret, C. A New
Paclitaxel Prodrug for Use in Adept Strategy. Org. Biomol. Chem.
2003, 1 (19), 3343−3352.
̈
̈
(39) Tietze, L. F.; Beller, M.; Fischer, R.; Logers, M.; Jahde, E.;
Glusenkamp, K.-H.; Rajewsky, M. F. Development of Tailor-Made
̈
̀
(23) Alouane, A.; Labruere, R.; Le Saux, T.; Schmidt, F.; Jullien, L.
Cytostatics Activable by Acid-Catalyzed Hydrolysis for Selective
Tumor Therapy. Angew. Chem., Int. Ed. Engl. 1990, 29 (7), 782−783.
(40) Wang, J.; Zhu, R.; Sun, X.; Zhu, Y.; Liu, H.; Wang, S.-L.
Intracellular Uptake of Etoposide-Loaded Solid Lipid Nanoparticles
Induces an Enhancing Inhibitory Effect on Gastric Cancer through
Mitochondria-Mediated Apoptosis Pathway. Int. J. Nanomed. 2014, 9,
3987−3998.
Self-Immolative Spacers: Kinetic Aspects, Structure-Property Rela-
tionships, and Applications. Angew. Chem., Int. Ed. 2015, 54 (26),
7492−7509.
(24) Fernandes, A.; Viterisi, A.; Coutrot, F.; Potok, S.; Leigh, D. A.;
Aucagne, V.; Papot, S. Rotaxane-Based Propeptides: Protection and
Enzymatic Release of a Bioactive Pentapeptide. Angew. Chem., Int. Ed.
2009, 48 (35), 6443−6447.
(41) Pooja, D.; Kulhari, H.; Tunki, L.; Chinde, S.; Kuncha, M.;
Grover, P.; Rachamalla, S. S.; Sistla, R. Nanomedicines for Targeted
Delivery of Etoposide to Non-Small Cell Lung Cancer Using
Transferrin Functionalized Nanoparticles. RSC Adv. 2015, 5 (61),
49122−49131.
(42) Tao, W.; Wang, J.; Parak, W. J.; Farokhzad, O. C.; Shi, J.
Nanobuffering of Ph-Responsive Polymers: A Known but Sometimes
Overlooked Phenomenon and Its Biological Applications. ACS Nano
2019, 13 (5), 4876−4882.
(43) Bemiller, J. N. Acid-Catalyzed Hydrolysis of Glycosides. In
Advances in Carbohydrate Chemistry; Wolfrom, M. L., Tipson, R. S.,
Eds.; Academic Press, 1967; Vol. 22, pp 25−108.
(44) Min, H. S.; Kim, H. J.; Naito, M.; Ogura, S.; Toh, K.; Hayashi,
K.; Kim, B. S.; Fukushima, S.; Anraku, Y.; Miyata, K.; Kataoka, K.
Systemic Brain Delivery of Antisense Oligonucleotides across the
Blood-Brain Barrier with a Glucose-Installed Polymeric Nanocarrier.
Angew. Chem., Int. Ed. 2020, 59 (21), 8173−8180.
(45) Patra, M.; Johnstone, T. C.; Suntharalingam, K.; Lippard, S. J. A
Potent Glucose-Platinum Conjugate Exploits Glucose Transporters
and Preferentially Accumulates in Cancer Cells. Angew. Chem., Int. Ed.
2016, 55 (7), 2550−2554.
(46) Zhou, X.; Chen, R.; Xie, W.; Ni, Y.; Liu, J.; Huang, G.
Relationship between 18f-Fdg Accumulation and Lactate Dehydro-
genase a Expression in Lung Adenocarcinomas. J. Nucl. Med. 2014, 55
(11), 1766−1771.
(47) Kaira, K.; Serizawa, M.; Koh, Y.; Takahashi, T.; Yamaguchi, A.;
Hanaoka, H.; Oriuchi, N.; Endo, M.; Ohde, Y.; Nakajima, T.;
Yamamoto, N. Biological Significance of 18f-Fdg Uptake on Pet in
Patients with Non-Small-Cell Lung Cancer. Lung Cancer 2014, 83
(2), 197−204.
(25) Schmidt, F.; Monneret, C. Prodrug Mono Therapy: Synthesis
and Biological Evaluation of an Etoposide Glucuronide-Prodrug.
Bioorg. Med. Chem. 2003, 11 (10), 2277−2283.
(26) Florent, J.-C.; Dong, X.; Gaudel, G.; Mitaku, S.; Monneret, C.;
Gesson, J.-P.; Jacquesy, J.-C.; Mondon, M.; Renoux, B.;
Andrianomenjanahary, S.; Michel, S.; Koch, M.; Tillequin, F.;
Gerken, M.; Czech, J.; Straub, R.; Bosslet, K. Prodrugs of
Anthracyclines for Use in Antibody-Directed Enzyme Prodrug
Therapy. J. Med. Chem. 1998, 41 (19), 3572−3581.
(27) Sperker, B.; Backman, J. T.; Kroemer, H. K. The Role of B-
Glucuronidase in Drug Disposition and Drug Targeting in Humans.
Clin. Pharmacokinet. 1997, 33 (1), 18−31.
(28) Groot, F. M. d.; Damen, E. W.; Scheeren, H. W. Anticancer
Prodrugs for Application in Monotherapy Targeting Hypoxia, Tumor-
Associated Enzymes, and Receptors. Curr. Med. Chem. 2001, 8 (9),
1093−1122.
(29) Murdter, T. E.; Sperker, B.; Kivisto, K. T.; McClellan, M.; Fritz,
̈
̈
P.; Friedel, G.; Linder, A.; Bosslet, K.; Toomes, H.; Dierkesmann, R.;
Kroemer, H. K. Enhanced Uptake of Doxorubicin into Bronchial
Carcinoma: B-Glucuronidase Mediates Release of Doxorubicin from a
Glucuronide Prodrug (Hmr 1826) at the Tumor Site. Cancer Res.
1997, 57 (12), 2440−2445.
(30) Bosslet, K.; Straub, R.; Blumrich, M.; Czech, J.; Gerken, M.;
Sperker, B.; Kroemer, H. K.; Gesson, J.-P.; Koch, M.; Monneret, C.
Elucidation of the Mechanism Enabling Tumor Selective Prodrug
Monotherapy. Cancer Res. 1998, 58 (6), 1195−1201.
(31) Murdter, T. E.; Friedel, G.; Backman, J. T.; McClellan, M.;
̈
Schick, M.; Gerken, M.; Bosslet, K.; Fritz, P.; Toomes, H.; Kroemer,
H. K.; Sperker, B. Dose Optimization of a Doxorubicin Prodrug (Hmr
1826) in Isolated Perfused Human Lungs: Low Tumor Ph Promotes
Prodrug Activation by B-Glucuronidase. J. Pharmacol. Exp. Ther.
2002, 301 (1), 223−228.
(48) Golombek, S. K.; May, J.-N.; Theek, B.; Appold, L.; Drude, N.;
Kiessling, F.; Lammers, T. Tumor Targeting Via Epr: Strategies to
Enhance Patient Responses. Adv. Drug Delivery Rev. 2018, 130, 17−
38.
(32) Houba, P. H. J.; Boven, E.; Meulen-Muileman, I. H. v. d.;
Leenders, R. G. G.; Scheeren, J. W.; Pinedo, H. M.; Haisma, H. J. A
Novel Doxorubicin-Glucuronide Prodrug Dox-Ga3 for Tumour-
Selective Chemotherapy: Distribution and Efficacy in Experimental
Human Ovarian Cancer. Br. J. Cancer 2001, 84 (4), 550−557.
(33) Hande, K. R. Etoposide: Four Decades of Development of a
Topoisomerase Ii Inhibitor. Eur. J. Cancer 1998, 34 (10), 1514−1521.
(34) Xu, Y.; Mu, J.; Xu, Z.; Zhong, H.; Chen, Z.; Ni, Q.; Liang, X.-J.;
Guo, S. Modular Acid-Activatable Acetone-Based Ketal-Linked
Nanomedicine by Dexamethasone Prodrugs for Enhanced Anti-
Rheumatoid Arthritis with Low Side Effects. Nano Lett. 2020, 20 (4),
2558−2568.
(49) Lee, H.; Shields, A. F.; Siegel, B. A.; Miller, K. D.; Krop, I.; Ma,
C. X.; LoRusso, P. M.; Munster, P. N.; Campbell, K.; Gaddy, D. F.;
Leonard, S. C.; Geretti, E.; Blocker, S. J.; Kirpotin, D. B.; Moyo, V.;
Wickham, T. J.; Hendriks, B. S. 64Cu-Mm-302 Positron Emission
Tomography Quantifies Variability of Enhanced Permeability and
Retention of Nanoparticles in Relation to Treatment Response in
Patients with Metastatic Breast Cancer. Clin. Cancer Res. 2017, 23
(15), 4190−4202.
(50) Maeda, H.; Wu, J.; Sawa, T.; Matsumura, Y.; Hori, K. Tumor
Vascular Permeability and the Epr Effect in Macromolecular
Therapeutics: A Review. J. Controlled Release 2000, 65 (1), 271−284.
H
https://dx.doi.org/10.1021/acs.nanolett.0c01973
Nano Lett. XXXX, XXX, XXX−XXX