A light-responsive, self-immolative linker for controlled drug delivery: Via peptide- and protein-drug conjugates

Article abstract of DOI:10.1039/c9sc03016f

When designing prodrugs, choosing an appropriate linker is the key to achieving efficient, controlled drug delivery. Herein, we report the use of a photocaged C4′-oxidized abasic site (PC4AP) as a light-responsive, self-immolative linker. Any amine- or hydroxyl-bearing drug can be loaded onto the linker via a carbamate or carbonate bond, and the linker is then conjugated to a carrier peptide or protein via an alkyl chain. The PC4AP linker is stable under physiologically relevant conditions. However, photodecaging of the linker generates an active intermediate that reacts intramolecularly with a primary amine (the ?-amine of a lysine residue and the N-terminal amine) on the carrier, leading to rapid and efficient release of the drug via an addition-elimination cascade, without generating any toxic side products. We demonstrated that the use of this self-immolative linker to conjugate the anticancer drug doxorubicin to a cell-penetrating peptide or an antibody enabled targeted, controlled delivery of the drug to cells. Our results suggest that the linker can be used with a broad range of carriers, such as cell-penetrating peptides, proteins, antibodies, and amine-functionalized polymers, and thus will find a wide range of practical applications.

Full text of DOI:10.1039/c9sc03016f

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A light-responsive, self-immolative linker for
controlled drug delivery via peptide- and protein-
drug conjugates†‡
Cite this: DOI: 10.1039/c9sc03016f
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
a
a
b
a
a
Chuanlong Zang, Huawei Wang, Tiantian Li, Yingqian Zhang, Jiahui Li,
a
b
a
a
Mengdi Shang, Juanjuan Du, Zhen Xi and Chuanzheng Zhou
*
When designing prodrugs, choosing an appropriate linker is the key to achieving efficient, controlled drug
0
delivery. Herein, we report the use of a photocaged C4 -oxidized abasic site (PC4AP) as a light-responsive,
self-immolative linker. Any amine- or hydroxyl-bearing drug can be loaded onto the linker via a carbamate
or carbonate bond, and the linker is then conjugated to a carrier peptide or protein via an alkyl chain. The
PC4AP linker is stable under physiologically relevant conditions. However, photodecaging of the linker
generates an active intermediate that reacts intramolecularly with a primary amine (the 3-amine of
a lysine residue and the N-terminal amine) on the carrier, leading to rapid and efficient release of the
drug via an addition–elimination cascade, without generating any toxic side products. We demonstrated
that the use of this self-immolative linker to conjugate the anticancer drug doxorubicin to a cell-
penetrating peptide or an antibody enabled targeted, controlled delivery of the drug to cells. Our results
suggest that the linker can be used with a broad range of carriers, such as cell-penetrating peptides,
proteins, antibodies, and amine-functionalized polymers, and thus will find a wide range of practical
applications.
Received 19th June 2019
Accepted 8th August 2019
DOI: 10.1039/c9sc03016f
rsc.li/chemical-science
induces a cascade of disassembly reactions that ultimately lead
to drug release. So far, only two types of self-immolative linkers
Introduction
In drug design and development, prodrug strategies are widely have gained wide acceptance, and both types undergo self-
used to improve the pharmacokinetic properties of drugs, immolative elimination, cyclization, or both to release the
1,2
16,17
especially targeted delivery. A prodrug is generally constructed conjugated drug.
However, disassembly of these self-
by conjugation of a drug molecule to a carrier via a linker immolative linkers can generate toxic side products such as
18
containing a trigger moiety. Aer delivery of the prodrug to the quinone methides, which can have unwanted side effects.
target cells or tissues, the active drug is released via cleavage of Therefore, the development of biocompatible self-immolative
1
1,19,20
the linker, either by an endogenous stimulus such as a pH linkers has attracted considerable attention.
3,4
5,6
7,8
change, a redox reaction, or an enzyme, or by an exoge-
We and others have shown that primary amines can catalyze
DNA cleavage at C4 -oxidized abasic sites (C4APs, which are
The key to achieving efficient, controlled drug release is to formed by abstraction of the C4 –H from 2 -deoxyribose;
9–11
2,12
0
nous stimulus such as light
or a small-molecule trigger.
0
0
13,14
21–26
choose an appropriate linker.
Fig. 1A).
Specically, addition of a primary amine to the C1
In some cases, the close proximity of the drug and the carrier of a C4AP via Schiff base formation leads to sequential elimi-
impairs linker cleavage by the stimulus. This problem can be nation of the C3 and C5 phosphates, generating the 5-methy-
23
overcome by introducing an additional linker, referred to as lene pyrrolone (5MP) derivative of the primary amine. On the
15,16
a self-immolative linker, between the trigger and the drug.
basis of these addition–elimination cascade reactions, we
Removal or cleavage of the trigger by an appropriate stimulus designed a photocaged C4AP (PC4AP, Fig. 1B) as a novel light-
responsive, self-immolative linker for controlled drug delivery
a
via peptide- and protein-drug conjugates.
State Key Laboratory of Elemento-Organic Chemistry, Department of Chemical
Biology, College of Chemistry, Nankai University, Tianjin 300071, China. E-mail:
chuanzheng.zhou@nankai.edu.cn
Results and discussion
Design of the PC4AP linker
b
School of Pharmaceutical Sciences, Tsinghua University, 30 Shuangqing Rd., Beijing
1
†
‡
00084, China
Dedicated to the 100th anniversary of Nankai University.
Electronic supplementary information (ESI) available: General experimental
The PC4AP linker is constructed by protection of the C1–OH
and C4–OH of the C4AP with photolabile O-nitrobenzyl groups
methods, spectroscopic data and supplementary gures (PDF). See DOI:
0.1039/c9sc03016f
27,28
22,29
1
(–ONv),
which make the system light-responsive.
The C3–
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carbamate or carbonate bond and concomitant release of the
active drug, along with nontoxic CO . Because C5–OH is linked
2
to the carrier via a stable O–C bond, cleavage of the linker via
ꢀ
departure of C5–O is disfavored. Therefore, the linker remains
attached to the carrier, and a cyclic species is generated.
Synthesis of a doxorubicin prodrug based on the PC4AP linker
Doxorubicin (DOX) is a cytotoxic anthracycline antibiotic and
anticancer drug. Because of its natural uorescence, DOX is
widely used as
a
model cytotoxin for cellular delivery
30–32
studies.
Here, in a proof-of-principle experiment, DOX was
employed to examine the efficacy of controlled drug delivery by
means of the PC4AP linker. To this end, we designed and
synthesized Mal-PC4AP-DOX (9, Scheme 1), in which DOX and
a maleimide (Mal) moiety are bridged by the PC4AP linker. The
purpose of the maleimide moiety was to enable site-specic
bioconjugation of 9 to a Cys residue in carrier peptides and
proteins.
The synthesis of 9 started from acetal 1, in which the C5–OH
is protected with a benzoyl group. Aer protection of the C3–OH
as a t-butyldiphenylsilyl ether, the benzoyl group was selectively
removed by treatment with NaOMe, and the C5–OH was then
allowed to react with propargyl bromide to afford alkyne 3,
0
Fig. 1 Design of a photocaged C4 -oxidized abasic site (PC4AP) as
a light-responsive, self-immolative linker for controlled drug delivery
via peptide- and protein-drug conjugates. (A) Primary-amine-cata-
0
lyzed DNA cleavage at the C4 -oxidized abasic site (C4AP). (B) Principle
of PC4AP-based drug delivery via peptide- and protein-drug which was transformed into dithiane 4 in 85% yield. Aer
conjugates.
oxidation of the C4–OH to a ketone, the dithiane moiety of 5 was
oxidatively cleaved in the presence of 4,5-dimethoxy-2-
nitrobenzyl alcohol (NvOH) to afford protected PC4AP 6 as
29
OH and C5–OH are used to load the drug and the carrier, a mixture of four stereoisomers. The C3–OH was then depro-
respectively (Fig. 1B). Specically, an amine- or hydroxyl-bearing tected by treatment with tetra-n-butylammonium uoride, and
drug payload is attached to the C3–OH via a carbamate or three isomers of 7 were isolated and characterized by NMR
carbonate bond, and the C5–OH of the PC4AP linker is conju- spectroscopy. The predominant isomer, 7a, was subjected to
gated to a carrier peptide or protein via an alkyl chain. Upon further transformations. Specically, treatment of 7a with tri-
photodecaging, the C4AP moiety undergoes an intramolecular phosgene converted the C3–OH to the corresponding carbon-
0
addition reaction with any nearby amine on the carrier, and ochloridate (8 ), which, without purication, was allowed to
a subsequent elimination reaction leads to cleavage of the react selectively with the amino group of DOX to give 8 in 65%
Scheme 1 Synthesis of Mal-PC4AP-DOX (9).
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yield. Finally, target product 9 was obtained by coupling 8 with of 8 under the same conditions furnished the decaged inter-
Mal-PEG -N via click chemistry. It is worth noting that various mediate 11 quantitatively (Fig. 2 and S3A‡). Compound 11 was
3
3
functional groups can be introduced into the PC4AP linker by stable in a neutral buffer, but upon addition of 20 equiv. of
means of click reactions between 8 and azide-containing benzyl amine (BnNH ), 11 decomposed to DOX and 12, the
2
molecules. The linker can thereby be site-specically attached structure of which was determined unambiguously by means of
to a broad range of residues in carrier peptides and proteins.
Although the carbonochloridate intermediate reacts readily was complete in 5 min, and the yield was essentially quantita-
NMR spectroscopy (Fig. S3A and S25–S28‡). The release of DOX
3
3–35
with amines, it can also react with alcohols and thiols.
For tive. In contrast, no reaction occurred upon incubation of 8 and
in the absence of light (Fig. 2C). These results indicate
0
example, when 8 was treated in situ with 1-pyrenemethanol BnNH
2
(
PyrCH OH), carbonate 10 was obtained in 50% yield.
that the PC4AP was a stable linker and that both light and
a primary amine were required to trigger release of the drug.
We propose the following mechanism for the primary-
amine-catalyzed release of DOX (Fig. 2A). Nucleophilic attack
2
Analysis of PC4AP linker stability and disassembly
To evaluate the stability of the PC4AP linker under physiologi- at the anomeric carbon (C1) of 11 by the primary amine
cally relevant conditions, we incubated 8 in pH 7.5 and 5.2 produces Schiff base 13. The presence of the C1]N bond
buffers, which simulate intercellular and lysosomal environ- increases the acidity of the C2–H, which in turn promotes the
ments, respectively. Ultra performance liquid chromatography departure of the C3-carbamate via b-elimination to furnish 14.
(
UPLC) revealed that under both conditions, 8 was completely Concomitant cleavage of the carbamate bond releases CO
and
2
stable aer 24 h of incubation (Fig. S1‡).
DOX. Subsequently, hydrolysis of the C1]N bond of 14 regen-
Next, we examined the kinetics and mechanism of the pho- erates a nucleophilic amine, which intramolecularly attacks the
todecaging process by using model compounds 8 and 9. Anal- C4-ketone, generating cyclic intermediate 15. Finally, elimina-
ysis of the UV-vis absorption spectra of 9 as a function of tion of a H
irradiation time (365 nm, 7.0 mw cm ) revealed a rapid
2
O molecule followed by isomerization produces 12.
To conrm this proposed mechanism, we carried out pho-
decrease in absorbance at 350 nm along with a concomitant todecaging of 8 in the presence of both BnNH and NaBH CN.
ꢀ
2
2
3
31
+
increase at 270 nm, and irradiation for 3 min was sufficient to Aer 0.5 h, a single product with a [MH] value of 817.3194 was
completely remove the photolabile O-nitrobenzyl groups obtained (Fig. 2D and S3B‡). We assumed that this product was
(
Fig. S2‡). Similarly, UPLC-MS analysis showed that irradiation compound 16§ and that it had been generated via intermediates
Fig. 2 Mechanism of primary-amine-catalyzed disassembly of the PC4AP linker. (A) Proposed mechanism of disassembly of the PC4AP linker in
by irradiation and subsequent reaction with BnNH . (B) Disassembly of the PC4AP linker in 10 by irradiation and subsequent reaction with
BnNH . (C) Ultra performance liquid chromatography-mass spectrometry (UPLC-MS) analysis of light-triggered decomposition of 8 in the
presence of BnNH . (D) UPLC-MS analysis of light-triggered decomposition of 8 in the presence of BnNH and NaBH CN. DOX was reduced by
NaBH CN to give DOX , which contains two more hydrogen atoms than DOX. (E) UPLC-MS analysis of decomposition of 10 by irradiation and
subsequent reaction with BnNH
8
2
2
2
2
3
R
3
2
.
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7 and 18 (Fig. 2A). It is known that NaBH
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25
1
3
CN is unreactive a Lys residue, followed by incubation in phosphate buffer (pH
ꢁ
toward aldehydes and ketones but can reduce C]N to C–N. 7.5) at 37 C for 5 min led to the formation of intermediate 30
Therefore, NaBH CN would reduce intermediate 13 to give 17, and release of approximately 50% of the DOX (Fig. 3B). The
3
the C2–H of which is not acidic enough for rapid elimination of amount of intermediate 30 decreased with increasing incuba-
the C3-carbamate. Instead, 17 would undergo rapid cyclization tion time, and complete release of DOX was achieved aer
followed by another reduction to afford observed product 16.
Intermediates 13–15, 17, and 18 were not observed either in transformed into a cyclic species (Fig. S5A‡). In contrast, irra-
the presence or in the absence of NaBH CN, suggesting that diation of conjugate 26, which lacks the N-terminal amine and
incubation for 1 h. Simultaneously, the peptide moiety was
3
once the C]N bond was formed by the reaction of the C4AP has no Lys residues, followed by incubation in phosphate buffer
with a primary amine, the drug was rapidly released via intra- for 0.5 h produced intermediate 31 quantitatively, and none of
molecular reactions.
Similarly, photoirradiation of 10 generated intermediate 19, irradiation of conjugates 27 and 28, which contain either an N-
which decomposed to release PyrCH OH quantitatively upon terminal amine or a Lys residue, respectively (but not both),
treatment with BnNH for 5 min (Fig. 2B, E and S4‡). This result resulted in efficient release of DOX (Fig. S5C and D‡). Taken
the DOX was released (Fig. 3C). We also found that photo-
2
2
indicates that a payload attached to the PC4AP linker via together, these results conrm that only a primary amine in the
a carbonate bond could be released as efficiently as a payload peptides (i.e., the 3-amine of Lys and the N-terminal amine)
attached via a carbamate bond.
could intramolecularly induce drug release from the peptide-
PC4AP-drug conjugates.
We also demonstrated that DOX release from peptide-
PC4AP-DOX conjugates was not noticeably affected by the
distance between the Cys anchor and the Lys or the N-terminal
amine. For instance, even though the Lys and the N-terminal
Gly of peptide-PC4AP-DOX conjugate 29 are four and six resi-
dues, respectively, away from the Cys anchor, photoirradiation
of 29 followed by incubation for 0.5 h led to release of 70% of
the DOX (Fig. 3D), suggesting that the drug-release efficiency of
this conjugate was similar to that of 25. Given that the PC4AP
linker is anchored to the side chain of Cys via a exible 25-atom-
long chain, distance-independent drug release is reasonable.
Requirement of either a lysine residue or an N-terminal amine
for drug release from peptide-PC4AP-drug conjugates
The above-described results conrmed that the intermolecular
reaction with BnNH could trigger rapid disassembly of the
2
PC4AP linker. To determine whether a primary amine in
a peptide, such as the 3-amine of a Lys residue or the N-terminal
amine, could induce efficient drug release from a peptide-
PC4AP-drug conjugate via an intramolecular process, we
prepared peptide-PC4AP-DOX conjugates 25–29 by attaching 9
to the Cys residues of peptides 20–24 (Fig. 3A). Irradiation of 25,
the Cys residue of which is anked by the N-terminal Gly and
Fig. 3 Light-triggered release of DOX from peptide-PC4AP-DOX conjugates. (A) Overview of DOX release from peptide-PC4AP-DOX conju-
gates 25–29 upon photoirradiation. (B) UPLC-MS analyses of light-triggered decomposition of conjugate 25. (C) UPLC-MS analyses of light-
triggered decomposition of conjugate 26. (D) UPLC-MS analyses of light-triggered decomposition of conjugate 29. Reaction conditions:
a solution of each conjugate (1 mM) in phosphate buffer (20 mM, pH 7.5) was photoirradiated at 365 nm for 3 min and then incubated in the dark.
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Efficient cellular delivery of DOX by means of an H3-PC4AP-
NaBH
3
CN, and analyzed by means of 15% SDS-PAGE (Fig. S8‡).
DOX conjugate
Quantication of the uorescence intensity of each band
revealed that 75% of the DOX was released aer 5 min of
incubation, and the amount reached 85% at 1 h (Fig. 4C). In
contrast, little, if any, DOX was released when the irradiation
step was omitted. Taken together, these results conrm that the
PC4AP linker was stable under neutral conditions but aer
photodecaging, the self-immolative process occurred sponta-
neously, releasing the cargo from the carrier protein rapidly and
efficiently.
Cell-penetrating peptides (CPPs), which are short peptides that
can translocate across cell membranes, have been extensively
used for intracellular cargo transport both in vitro and in vivo.
36
38
We therefore conjugated 9 to a small CPP, histone H3, and
demonstrated the utility of the resulting conjugate for
controlled cellular uptake of DOX. Specically, the H3 mutant
H3-V35C, which contains 13 Lys residues but only one Cys (at
37
position 35), was treated with 1.1 equiv. of 9 in HEPES buffer
pH 7.5) to afford H3-PC4AP-DOX (36) aer purication by gel
ltration (Fig. 4A and S6‡). A solution of 36 in phosphate buffer
Any intermediate 37 present in the aliquots removed from
(

the reaction would have been quenched by the NaBH CN to
3
afford a stabilized product (as 16 in Fig. 2A), which would have
prevented release of DOX from H3 during SDS-PAGE analysis.
Thus, rapid release of DOX observed by SDS-PAGE analysis can
be attributed solely to intramolecular Lys-catalyzed reactions.
DOX release from H3-PC4AP-DOX (36) upon photoirradiation
was even faster than release from peptide-PC4AP-DOX conju-
gate 25. This result led us to speculate that in addition to the Lys
residue and the N-terminal amine group, other residues in the
carrier protein may also have promoted linker disassembly. For
instance, acidic residues may have catalyzed Schiff base
formation between the linker and a primary amine, and basic
residues may have catalyzed the elimination step (see Fig. 2A).
To demonstrate the utility of our strategy for cellular delivery
of drugs, we treated HeLa cells with 10 mM H3-PC4AP-DOX (36)
(
20 mM, pH 7.5) was irradiated at 365 nm for 3 min and then
ꢁ
incubated at 37 C for 2 h. UPLC-MS analysis of the reaction
mixture showed that more than 70% of the DOX was released
(
of 15 746, which is consistent with the proposed cyclic structure
3
intermediate 37 ([MH] , calcd 16 351, found 16 351) was
observed as well (Fig. S7‡).
We also assessed the kinetics of DOX release. Specically,
a solution of 36 in phosphate buffer (20 mM, pH 7.5) was irra-
+
Fig. 4B). The remaining carrier protein exhibited a [MH] value
+
8 ([MH] , calcd 15 746). A very small amount of photodecaged
+
diated at 365 nm for 5 min and then incubated in the dark at
3
ꢁ
7
C. Aliquots were periodically removed, quenched with
ꢁ
or free DOX for 3 h at 37 C. Fluorescence confocal microscopy
38
analysis of the treated cells showed that, as expected, free DOX
was efficiently transported to the nucleus, where it bound to
genomic DNA (Fig. 5A, le panel); in contrast, in cells treated
with 36, the red uorescence of DOX was observed predomi-
nantly in the cytoplasm, indicating that the protein-drug
conjugate successfully transported DOX to the cytoplasm,
where it was not released in the absence of irradiation (Fig. S9‡).
Aer medium removal and addition of fresh DMEM (Dulbecco's
Modied Eagle Medium), the 36-treated cells were irradiated at
ꢁ
3
65 nm for 5 min and then incubated at 37 C for another 3 h;
under these conditions, DOX was transported to the nucleus
Fig. 5A, middle panel). Given that DOX remained in the cyto-
(
plasm of unirradiated cells (Fig. 5A, right panel), the DOX
transport to the nucleus of the irradiated cells can be attributed
to photo triggered release of free DOX from the H3 carrier
protein.
Cell counting kit-8 assays showed that incubation of HeLa
cells with H3-PC4AP-DOX (36) followed by photoirradiation
inhibited cell growth by up to 20%. In contrast, no cytotoxicity
was observed in the absence of light, even at high concentra-
tions of the conjugate (Fig. 5B). These results once again
conrm that 36 was stable inside cells but that it released active
DOX upon photoirradiation.
Targeted cellular delivery of DOX by an antibody-PC4AP-DOX
Fig. 4 Preparation and light-triggered disassembly of H3-PC4AP-
DOX (36). (A) Structures of 36 and its decomposition products. (B) conjugate
UPLC-MS analyses of light-triggered disassembly of 36 in phosphate
ꢁ
The above-described results inspired us to determine whether
the PC4AP linker could be used for targeted drug delivery with
buffer (20 mM, pH 7.5) at 37 C. (C) Kinetics and photoirradiation-
dependence of DOX release from 36 in phosphate buffer (20 mM, pH
ꢁ
39,40
7
.5) at 37 C.
an antibody as the carrier.
As the antibody, we chose
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Fig. 5 Controlled delivery of DOX into HeLa cells by H3-PC4AP-DOX
(36). (A) Fluorescence confocal microscopy analyses showing the
cellular location of DOX after treatment of HeLa cells with DOX and 36.
The images were obtained by overlaying the DAPI channel (excitation
at 408 nm and emission at 425–475 nm), the DOX channel (excitation
at 561 nm and emission at 570–620 nm), and the DIC channel. (B)
Light-dependence of 36 cytotoxicity to HeLa cells.
Fig. 6 Targeted delivery of DOX to breast cancer cell lines via a tras-
tuzumab-PC4AP-DOX conjugate (39). (A) Preparation of 39. (B) DOX
fluorescence quantification revealed the kinetics of DOX release from
trastuzumab, a clinical monoclonal IgG that recognizes and
induces internalization of HER2, a receptor that is highly
expressed in some breast cancer cell lines.
the heavy and light chains of 39 upon incubation in human serum. (C)
41,42
To prepare DOX fluorescence quantification revealed the kinetics of DOX release
trastuzumab-DOX conjugates with the PC4AP linker, we treated from the heavy and light chains of 39 upon photoirradiation and
subsequent incubation in human serum. (D) Light- and concentration-
dependent cytotoxicities of 9, 39, and DOX against SK-BR-3 cells
trastuzumab with 4 equiv. of tris(2-carboxyethyl)phosphine
(TCEP) in phosphate-buffered saline for 2 h to reduce the
(HER2 positive). (E) Comparison of the cytotoxicities of 39 (200 nM)
interchain disulde bonds, and then incubated it with 8 equiv.
against SK-BR-3 (HER2 positive) and MCF7 (HER2 negative) cell lines.
ꢁ
of Mal-PC4AP-DOX (9) at 37 C for 1 h to give antibody-drug
conjugate (ADC) 39, which was puried by gel ltration. LC-
MS analysis indicated the drug/antibody ratio to be 8 (Fig. 6A
and S10‡).
Photoirradiation of 39 at 365 nm for 5 min and subsequent
incubation in human serum led to release of 80% of the free
DOX from the antibody aer incubation for 10 min (Fig. 6C and
S12‡), conrming that once the photocage was removed, self-
immolative disassembly of the C4AP linker in the ADC was
rapid and efficient.
The linker of an ADC must be stable enough to prevent off-
target effects. To test the stability of the PC4AP linker in ADC
ꢁ
3
9, we incubated it in human serum at 37 C. Aliquots were
removed periodically, quenched with NaBH CN, and analyzed
3
by means of 12% reducing glycine-SDS-PAGE (Fig. S11‡). The
heavy and light chains appeared as two separated bands in the
gel, and quantication of the uorescence intensities of the
bands revealed that DOX was released very slowly from both
chains (Fig. 6B and S11‡); approximately 85% of the DOX
remained attached to the antibody aer 72 h of incubation.
Note, however, that DOX release from the antibody was not
attributed to cleavage of the PC4AP linker, given that no
released free DOX was observed. Instead, we detected some
slower-migrating uorescent bands (Fig. S11‡), which we
attributed to transfer of the conjugated moiety (Mal-PC4AP-
DOX, 9) from the antibody to other serum proteins (e.g.,
Zhang et al. conjugated DOX to trastuzumab via an acid-
labile MMCCH linker. At pH 7.4, both linker degradation
42
and drug release were rather slow (the release rate was 25% per
day), but in a lysosomal environment (pH 5.4), 80% of the DOX
was released from the ADC aer 24 h. Our trastuzumab-PC4AP-
DOX conjugate was much more stable than the trastuzumab-
MMCCH-DOX conjugate under physiologically relevant condi-
tions. In addition, aer being triggered by a biocompatible
stimulus, drug release was more efficient from the former than
from the latter. Thus, the PC4AP linker can be expected to be
useful for ADC development.
Next, we evaluated the cytotoxicity of the trastuzumab-
PC4AP-DOX conjugate (39). Aer incubation of two xed
breast cancer cell lines, SK-BR-3 (HER2 positive) and MCF7
43,44
human serum albumin) via thiol exchange reactions.
That
is, the PC4AP linker itself was stable in human serum.
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(HER2 negative), with ADC 39, an immunouorescence assay shi the activation wavelength, indicating the versatility of this
showed that 39 bound specically to the antigen HER2 on the linker. Fourth, Lys is the most abundant amino acid residue in
surface of the SK-BR-3 cells (Fig. S13‡). Having conrmed the typical proteins; it is widely distributed in CPPs and on the outer
antigen-specic binding, we determined the cytotoxicity of 39 to surface of proteins. In addition to Lys, any other primary amine,
the SK-BR-3 cells over a wide concentration range. ADC 39 including the N-terminal amine, can catalyze disassembly of the
showed marginal cytotoxicity in the absence of light (Fig. 6D), PC4AP linker. Therefore, this linker could be used with a broad
whereas incubation of the cells with 39 and subsequent irradi- range of carriers, including CPPs, proteins, antibodies, amine-
ation led to decreased cell viability in a dose-dependent bearing polymers such as polyethyleneimine, and amine-
manner; the potency of 39 (IC50 ¼ 203 nM) was comparable to functionalized nanoparticles. These advantages make the
that of free DOX (IC50 ¼ 186 nM). Furthermore, we found that PC4AP linker invaluable for controlled cargo delivery, and it can
Mal-PC4AP-DOX (9) was efficiently taken up by the SK-BR-3 cells be expected to nd a wide range of practical applications.
but showed no obvious cytotoxicity either in the presence or in
the absence of light. Given that the free amino group of DOX is
45
Conflicts of interest
vital for its cytotoxicity, this result indicates that irradiation
did not release active DOX from 9. That is, photodecaging was
not sufficient to release DOX from the PC4AP linker in the
absence of intramolecular catalysis by a primary amine inside
the cells. In other words, light-triggered cellular release of DOX
from the antibody-PC4AP-DOX conjugate (39) occurred by
means of intramolecular catalysis, as we proposed.
There are no conicts to declare.
Acknowledgements
This work was supported by the NSFC (21877064 and 21572109),
Finally, we evaluated the cell line specicity of ADC 39 the National Key Research and Development Program of China
(Fig. 6E). SK-BR-3 (HER2 positive) and MCF7 (HER2 negative) (2017YFD0200501 and 2017YFA0207900) and the Fundamental
cells were preincubated with 200 nM 39 for 6 h, the medium was Research Funds for the Central Universities, Nankai University
changed to fresh DMEM, and the cells were photoirradiated and (63191523).
then incubated for an additional 48 h. Cell viability assays
showed that the irradiated SK-BR-3 cells displayed remarkably
decreased cell viability, whereas little, if any, effect on the MCF7
cells was observed, whether they were irradiated or not. This
result conrms that conjugation of the drug to the antibody via
Notes and references
R
§
In compound 16, the DOX moiety was reduced to DOX by NaBH
3
CN. Treatment
of free DOX with NaBH CN gave the same result (Fig. 2D). UPLC-MS analysis
3
R
the PC4AP linker enabled targeted drug delivery in a controlled showed that DOX contains two more hydrogen atoms than DOX (Fig. S3‡).
manner.
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In summary, we have described the use of a PC4AP as a light-
responsive, self-immolative linker for controlled drug delivery.
Amine- or hydroxyl-bearing drugs can be easily introduced at C3
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