Journal of Pharmaceutical Sciences 109 (2020) 3262-3281

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Journal of Pharmaceutical Sciences

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Review

Perspectives About Self-Immolative Drug Delivery Systems

Rodrigo Vieira Gonzaga, Lucas Adriano do Nascimento, Soraya Silva Santos,
Bruna Araujo Machado Sanches, Jeanine Giarolla, Elizabeth Igne Ferreira*
~o
Department of Pharmacy, Faculty of Pharmaceutical Sciences, University of Sao Paulo, Av. Prof. Lineu Prestes, 580 e Bl 13 e ZC 05580-000, Sa
Paulo, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Self-immolative drug delivery system is one of the delivery systems, which have drawn attention, in
Received 1 April 2020 recent research, highlighting the improvement they generate in drug selectivity and efficacy. Self-
Revised 27 July 2020 immolative linkers, or spacers, are covalent groups, which have the role of cleavaging two bonds be-
Accepted 17 August 2020
tween a protector group and a drug, in the case of drug delivery systems, after a stimuli.The cascade of
Available online 27 August 2020
reactions allows to control the release of the drug. The choice of the adequate self-immolative linker is
essential and depend on many variables and goals as well. Many approaches can be explored when
Keywords:
Drug delivery systems designing a system adequate for achieving these goals, especially prodrugs. Some of the most used
Prodrug design stimuli-responses for self-immolative drugs e enzyme triggers, chemical triggers, as pH, redox system,
Trigger classes 1,4-, 1,6-, 1,8-eliminations, photodegradable triggers, multiple triggers, among others e are described in
Self-immolative linkers
this ten-year review, along with their application as theranostic agents. We intend that the examples
presented in this review inspire researchers working on drug delivery systems to further explore their
application.
© 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Introduction compounds that respond to many changes in the environment such

Self-immolative systems (SIS), including polymer systems, have
been employed in diverse fields, such as drug delivery, supramo-
lecular chemistry, signal amplification, chemical materials, and
diagnostic probe design. When activated by a specific stimulus,
these molecules undergo spontaneous intramolecular disassem-
bling, in which they are broken down to their building blocks, and
the compound of interest attached to them is released. Self-
immolative linker displays an important role in the cascade
mechanism of release of the compound linked.1 It is defined as a
covalent groups, which have the role of cleavaging two bonds be-
tween a protector group and a drug, in the case of drug delivery
systems, after a stimuli.Then, this allows to control the release of
the drug and the adequate choice of its nature is very important for
the success of the system.
SIS draw attention as potential drug delivery systems (SIDD) as
they can be tailored to provide programmable drug release by
exploiting particular features found in diseased tissues, such as a
different pH, reductive conditions, or even enzyme expression.2e5
These SIS are considered stimuli-responsive polymeric bioma-
terial, also known as smart polymers,6,7 which are defined as
* Corresponding author.
E-mail address: hajudan@usp.br (E.I. Ferreira).
as pH, temperature, light, electrical or magnetic changes and me-
chanical forces. Both internal and external stimuli can be used to
trigger the self-immolation.8,9
Fig. 1 shows a general structure of SIDD and itys mechanism of
drug release.
Increasing research of SIS in the last few decades highlights their
potential in the drug delivery field, especially in noncommunicable
chronic diseases. Their features, such as changed chemical envi-
ronment and modified metabolism, pose an interesting means for
drug delivery as they can potentially be exploited to achieve higher
drug activity and lower systemic toxicity by preferential accumu-
lation at the drug's active site.2e5
As stated, SIS are compounds which undergo cascade reactions
for disintegrating via end-to-end decomposition or cyclization
mechanisms by a stimulus that is able to trigger the sequential
release of their small constituent molecules. These cascade re-
actions are based on carbamate fragments, elimination reaction,
cyclization of an amine-terminated spacer to a five membered urea
ring, or cyclization of a trimethyl lock linker to the lactone
portion.10 In the presence of a specific stimulus, the trigger is
broken, creating an unstable intermediate that self-immolates to
release the output derivative.2e5 Upon stimulation, these systems
suffer head-to-tail disassembly, triggered by a single cleavage at a
focal point, which initiates a sequential fragmentation into the
substance's building blocks, releasing multiple end-groups from

https://doi.org/10.1016/j.xphs.2020.08.014
0022-3549/© 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.
 R.V. Gonzaga et al. / Journal of Pharmaceutical Sciences 109 (2020) 3262-3281 3263

Fig. 1. General structure of a self-immolative systems as drug delivery system and its mechanism of drug release.1

the periphery in a domino-like way.11,12 In general, self-immolative
linkers can be divided into two main groups, depending on the
fragmentation process: either 1,4-,1,6-, or 1,8-elimination or
cyclization.1
Three different research groups started the synthesis of self-
immolative dendrimers constituted by a focal trigger, where re-
actions on it initiate the fragmentation into their building blocks
providing dendrimer disassembly.13 These self-disassembling
multiple-release compounds were named “Cascade-release” by
Groot et al.14 (2003), “Self-immolative” by Amir et al.15 (2003), or
“Dendritic amplification” by Szalai et al.16 (2003). In the dendrimer
field, poly(amidoamine) - PAMAM and poly(propylene imine) - PPI
dendrimers are not biodegradable, and thus, these self-immolative
that are responsive to enzyme triggers have been researched as a
promising approach in drug delivery systems.15e19
This 10 year-review encompasses many examples of smart self-
immolative drug delivery systems, according to each of the stimuli
mentioned above. Since most of the SIS presented here were
designed as antitumoral drug delivery systems, doxorubicin (DOX),
paclitaxel (PTX) and camptothecin (CPT) are the usual model drugs
for them.
Table 1 shows some more recent examples of self-immolative
linkers for SIS in general, including SIDD.
Disulfide linkers are by far the most used for self-immolative
purposes.
It is worth to note that the use of self-immolative linkers,
particularly with drugs, provide a versatile process for the moni-
toring of their delivery1 while decreasing the toxicity of the drugs
involved. Sometimes, it is also essential to control the kinetic of
delivery to dimish the instability of the bond between a carrier and
the drug, as in prodrug design. In this design, it is important to
choose the most adequate linker, not only because of the its sta-
bility and the controlled disassembly, but also considering its
toxicity. Besides, addressing the aspects of kinetics in the process of
self-immolation is important in the choice for the best self-
immolative linker.
Despite the novelty that the self-immolative drug delivery sys-
tems can bring into the therapeutics, there is no clinical trials
developed with these systems, nothwithstanding the potentiality
of its use in clinics, specially in cancer, must be considered.
Enzymatic Triggers
Enzyme expression is controlled by their biological role in the
cell. These catalysts are found in different concentrations among
human tissues depending on their function. It is worth noting that
diseased tissues produce very different enzyme concentrations
when compared to healthy tissues. Thus, diseased tissues’ enzy-
matic activity can be exploited when designing SIDD, as their target
substrate can be incorporated in their activatable linker, causing

Table 1
Some More Recent Examples of Self-Immolative Linkers (2019e2020).

Linker Type Application Reference

20
Disulfide Cancer
21,22
Leukemia
23
Drug delivery systems, theranostic prodrugs, biological sensors
24
25
Hetheroaminebifuncional disulfide Stability of self-immolative systems
26
Thiol-based pirydazinediones Cancer
27
p-Aminebenzyloxycarbonyl Cancer
Dipeptides
27
Gly-Pro Cancer
28
L-Phe-Sar HIV
29
Trans-cyclooctene tetrazine Anti-inflammatory
30
Alcohols
31
Ortho Hydroxy-protected Aryl sulfate Cancer
32
Phosphoramidate-based Drug delivery systems, smart materials, advanced tools for chemical biology
33
Hydroxybenzyl Small molecules ligands, therapeutic agents, peptides, proteins, oligonucleotides
34
Trimethyl carbamate Detection of enzyme reactions
35
Quinone methide-based Drug delivery, theranostic
3264 R.V. Gonzaga et al. / Journal of Pharmaceutical Sciences 109 (2020) 3262-3281
drug release to occur locally, and thus, reducing their adverse
effects.36
Penicillin-G-Amidase (PGA)
Penicillin-G-amidase (PGA), also known as penicillin-G-acylase,
is an enzyme from a microorganism origin, generally used in order
to prove a SIDD is efficiently disassembled when challenged by a
specific stimulus. For such assays, these delivery systems are built
with a PGA-substrate moiety, as it is readily metabolized by the
enzyme. This enzyme has many different uses,37 as in the synthesis
of the precursor of semisynthetic penicillins, 6-APA, 6-
aminopenicillanic acid, besides being applied to trigger drugs in
SIDD. It can be used conjugated to antibody in ADEPT (Antibody-
Directed Enzyme Prodrug Therapy), and also in the case of SIDD.
The substrate for PGA can be amide or ester. Gopin et al.37 (2006)
designed a dendrimer with poly(ethylene glycol) (PEG) in order to
decrease the aggregation resulting from its hydrophobic features,
and also to enhance its water solubility. This dendrimer was con-
jugated to four CPT molecules and to a substrate, as phenyl-
acetamide, for PGA activation under physiological conditions.
Amir and Shabat38 (2004) reported self-immolative dendrimers
able to disassemble by multi-enzymatic triggers, in which phe-
nylacetamide (PAA) is used as a trigger for PGA coupled to reporter
unity. The cleavage assays in a PBS buffer (pH 7.4) caused the
generation of a free amine group responsible for initiating an intra-
cyclization, which lead to the dendrimer fragmentation. The same
research group described a receiver-amplifier self-immolative
dendritic system capable of transferring a cleavage signal in a
convergent way to the core and amplify it divergently to the den-
drimer surface.39 The dendritic molecule initiates signal trans-
duction through enzymatic cleavage of the PAA trigger by PGA and
6-aminoquinoline is used as the reporter unity (Fig. 2). In addition,
they designed dendrimer prodrugs containing two different bio-
logical substrates (PAA for PGA, and a retro-aldol-retro-Michael
substrate recognized by catalytic antibody 38C2) and with DOX
attached to the system in a self-immolative manner. The cleavage of
both triggers leads to dendrimer disintegration and consequent
release of DOX.40
Jin et al.41 (2012) reported the synthesis of a new controlled
enzyme-responsive self-immolative spacer conjugate. This drug
delivery system was designed with L-cysteine as a central core
linked to PAA, and 7-amino-3-trifluoromethylcoumarin as the
model drug attached to the probe by a self-immolative linker
triggered by PGA. A tris-triethylene glycol tail was used to increase
its water solubility. According to the authors, this delivery system is
a versatile platform that can be bonded to different compounds
through generic linkages. This probe also showed that it is stable in
physiological conditions, as it can only be activated by PGA
enzymatic.
The dendritic platform for theranostic prodrugs was based on
self-immolative units containing fluorescence. Thereby, a self-
immolative prodrug was designed with PAA and CPT, as the
spacer built from two aniline compounds attached to a couple of
fluorescein dyes. PAA metabolization by PGA is initiated by the
trigger, thus releasing the anilines, which undergo sequential 1,6-
eliminations. These hybrid systems composed of theranostic
agents and drug delivery compounds through nanomedicine is an
advanced approach that integrates treatment and diagnosis. The
Fo€rster resonance energy transfer (FRET) method is widely used for
the detection or imaging of a specific reactivity, resulting from self-
quenching fluorophore, as a convenient approach to the develop-
ment of new prodrugs for cancer therapy. The results of fluores-
cence assays, monitored using a fluorescence spectrophotometer,
correlate with those of the release. Only in the presence of the
enzyme did the prodrug show fluorescence, increasing progres-
sively over 2 h.42
A comparative study described the self-immolation capacity of
p-aminobenzylalcohol (PABA) versus hemithioaminal-based
linkers connected to trigger moiety and evaluated their stability
under physiological conditions and self-immolation mediated by a
peptide substrate. The general system structure is based on PAA
moiety, which is cleaved by protease PGA, and a 7-
hydroxycoumarin unity, which exhibits fluorescence when free.
In general, a conjugation with PABA derivatives has a faster
disassembly-behavior than a hemithioaminal core with N,N-
dimethylethylendiamine as a stabilizing spacer, which modulates
the release of the fluorescent unity and functions as a model for
drug delivery.43
b-Glucuronidase/b-Galactosidase
b-glucuronidase is a major target in cancer treatment, as it is
overexpressed in a wide range of malignant tumors. It is an enzyme
that catalyzes the hydrolysis of a glucuronide from various from a
variety of substrates. This metabolic feature enables specific b-
glucuronidase substrate-bearing antitumoral SIDD design.44
Grinda et al.44 (2012) proposed a self-immolative prodrug
containing DOX and a glucuronide substrate. Not only did this SIDD
exhibit higher cytoxicity against lung cancer cells when compared
to free DOX, but it also showed controlled release since it depends
on the presence of the cancerous cell's highly expressed b-glucu-
ronidase. Previously, the same research group45 analyzed this same
SIDD, focusing on the importance of the amplifier portion in its
antitumoral activity. DOX and MS-275, a histone deacetylase in-
hibitor, were both attached to this probe and its cytotoxicity was
compared to both parental drugs and to a monomeric self-
immolative prodrug containing either drug but without the
amplifier moiety. Aside from higher activity when compared to the
free drugs, the absence of the amplifier moiety led to a decreased
cytotoxicity, as this portion creates a cytotoxic agent after both
drugs are released. As such, this versatile SIDD exhibits both pro-
grammable b-glucuronidase-dependent activation and innate
antitumoral proprieties attributable to its structure.
A heterodimeric AB2 (A ¼ focal head and B ¼ branched tail
group) self-immolative dendrimer composed of two CPT and DOX,
and a retro-aldol-retro-Michael substrate of 38C2 antibodies as a
trigger moiety, was investigated for synergic cytotoxic effect. Its
bioactivation was led by a cyclization step, followed by triple
quinone methide eliminations for the release of the drug.17
Glycosyl-bearing linkers were widely used for prodrug design.
Schuster et al.46 (2010) synthesized a duocarmycin prodrug
employing a b-D-galactose trigger coupled to 4-hydroxy-3-nitro-
benzaldehyde with ethylenediamine as spacer group connected to
duocarmycin via carbamate linkages. In vitro assays showed high
prodrug stability under physiological conditions in specific cell
culture medium over 24 h. Cytotoxicity assay results reflects its
selectivity.
A similar self-immolative prodrug was developed for DOX de-
livery, which was created using a self-immolative spacer linked to
two molecules of DOX, a central core and a chemical amplifier
coupled to a glucuronide moiety. Comparatively, fast DOX release of
two molecules of this antibiotics occurred in the presence of b-
glucuronidase, producing a higher anti-proliferative effect and a
decreased toxicity.47
Thomas et al.48 (2011) designed a glucuronide prodrug con-
taining DOX, MS-275 and a self-immolative spacer, named prodrug
39 B (Fig. 3). It was 8-fold more cytotoxic than the DOX glucuronide
prodrug on a lung mesothelioma cell line. The authors reported
that the toxicity increase was caused by azaquinone, produced from
 R.V. Gonzaga et al. / Journal of Pharmaceutical Sciences 109 (2020) 3262-3281 3265

Fig. 2. Self-immolative dendrimers containing phenylacetamide, triggered by PGA cleavage, and 6-aminoquinoline as the reporter group.39

the self-immolation of the linker. These results are in agreement
with those found by Grinda et al.45 (2011), as both SIDD were
designed similarly.
An abnormal activation of the Hedgehog protein (Hh) was
identified in several types of cancers such as breast, prostate,
gastric, lung and brain tumors. Cyclopamine was the first potent Hh
inhibitor, which is a poorly water-soluble natural alkaloid, isolated
from Veratrum californicum. Renoux et al.49 (2011) reported the
synthesis of a new cyclopamine glucuronide prodrugs with
improved pharmacokinetics and specific drug release by b-glucu-
ronidase. Two prodrugs were designed, both constituted by b-
glucuronidase-responsive triggers and cyclopamine, and a central
3266 R.V. Gonzaga et al. / Journal of Pharmaceutical Sciences 109 (2020) 3262-3281
Fig. 3. Glucuronide prodrug containing DOX, MS-275 and the self-immolative trigger, p-aminobenzyl alcohol able to lead 1,4- and 1,6-eliminations.32
self-immolative spacer; to one of them, a hydrophilic side chain
was introduced via “click chemistry”. This prodrug demonstrated a
better kinetic profile than the one without the hydrophilic portion.
Moreover, in the presence of b-glucuronidase, cyclopamine anti-
proliferative activity is reestablished in U87 gliblastoma cells. This
is a promising strategy for tumor targeted drug delivery, but further
in vivo assays are needed in order to validate the in vitro results.
Monomethyl auristatin E (MMAE) is a potent tubulin inhibitor,
about 100e1000 times more potent than DOX. First generation
galactoside-bearing prodrugs were developed combining a galac-
toside trigger coupled to a central self-immolative linker, MMAE,
and a targeting ligand (folic acid) to be recognized by cancer cells
providing selective treatment for solids. This strategy consists of
exploiting the high expression of folate receptors (FR) on the sur-
face of some cancerous tissues. When recognized by the FR, the
prodrug is endocytized and, in the presence of lysosomal b-galac-
tosidase, MMAE is released inside the lysosome. Overall this pro-
drug demonstrated, through in vivo assays, the ability for selective
activation by lysosomal b-galactosidase and excellent antitumor
activity, better than MMAE, when tested against FR-expressing KB
xenograft, with no significant toxicity.47
Another interesting approach is the use of glycosidic linkages for
specific release when they come into contact with metallic im-
plants, as they can be designed to degrad in the presence of iron
and release the drug. Glucuronide prodrugs were developed
exploiting their drug releasing feature in drugs used for orthopedic
and vascular procedures that are considered critical and susceptible
to infection and inflammation. A glucuronic acid was coupled to
fluoroquinolone antibacterial agents using 4-(hydroxymethyl)
phenol as a linker and these compounds were evaluated against
bacterial cultures of Escherichia coli and Staphylococcus aureus,
pathogens of great clinical importance. The higher growth inhibi-
tion opens new opportunities for engineering, especially when it
comes to implantable biomaterials.50
Cathepsin B/Antibody Triggers
The approval of antibodyedrug conjugates (ADC) (Adcetris® - a
brentuximab vedotin; Kadcyla® - ado-trastuzimab emtansine) by
regulatory organizations, such as the FDA, in the last year empha-
size how promising this technology is and why it has attracted
attention over the last few years. Seattle Genetics developed
 R.V. Gonzaga et al. / Journal of Pharmaceutical Sciences 109 (2020) 3262-3281
Adcetris® 15 (SGN-35 or brentuximab vedotin), a targeted drug for
Hodgkin and anaplastic large cell lymphoma treatment. It is an
antibody-drug (anti-CD30 mAb) bioconjugated through a cleavable
heterobifunctional linker, containing a maleimide and a cathepsin
B-sensitive trigger (mc-vc-PAB 14 or maleimidocaproyl-valine-
citrulline-p-aminobenzyloxycarbonyl spacer).51e54 Based on this
research, a new approach, which consists in connecting alcohol-
containing payloads to antibodies by methylenealkoxycarbamate
(MAC) self-immolative spacer, was developed by Kolakowski
et al.55 (2016). The authors, inspired by the work reported by Jeffrey
et al.56 (2006), synthesized a series of b-glucuronidase-sensitive
compounds containing MMAE to evaluate MAC technology for ADC.
The results showed that MAC can aid in the development of new
ADCs.
ADCs for targeted delivery of potent CPT analogues attached to
monoclonal antibodies (mAbs) using a valine-citrulline-PABA
dipeptide linker (val-cit) or glucuronide linkers were designed
and synthesized. The ADC, after recognition and internalization
into antigen-positive cell lines, suffers lysosomal degradation,
releasing the drug from its conjugate. These conjugates, on a renal
cell carcinoma xenograft model, effectively impaired tumor
growth, but the glucuronide conjugate outperformed the val-cit
conjugate, as the val-cit derivatives delayed tumor growth while
the glucuronide conjugate led to complete regression.57
Sulfate-containing ADCs using trastuzumab58 were designed to
take advantage of lysosomal sulfatases. After recognition and
incorporation, opposing to ADCs which linkers were design to be
Fig. 4. A e DOX prodrug conjugating the apoptotic cell-binding moiety to caspase-3 activated by concurrent chemoradiotherapy composed by DEVD tetrapeptide (Asp-Glu-Val-
Asp), caspase-3 substrate (green), self-immolative linker p-aminobenzyloxycarbonyl (PABC (black), ApoPep-1 (AP1) apoptotic cell-binding moiety (red); B e Tumoral tissue under
RITAC effect.60
3267
triggered by cathepsins, this prodrug is metabolized by lysosomal
sulfatases: a sulfate-bearing p-aminobenzyloxycarbonyl (PABC) is
enzymatically activated and then releases the drug attached to it.
They showed to be highly cytoroxic.
A MMAE prodrug, activated by caspase-3, was synthesized
following a strategy termed “radiation-induced apoptosis-targeted
chemotherapy” (RIATC). This technique is based on the expression
of caspases triggered by radiation exposure, as these proteolytic
proteins will not only induce apoptosis in tumoral cells, but also
activate the prodrug. The prodrug MPD02 is composed of mal-
eimidocaproyl, an human serum albumin (HAS)-binding moiety,
caspase-3-cleavable linker (Ac-Lys-Gly-Asp-Glu-Val-Asp), p-ami-
nobenzyl (PAB) as the self-immolative spacer, and the cytotoxic
payload MMAE. MPD02 demonstrated to be a potent RIATC,
enhancing antitumor activity when associated with radiotherapy,
indicating the metabolization into MMAE in a tumor specific
manner by caspase-3 hydrolysis. Then, RIATC proved to be a
promising strategy for drug delivery, as radiotherapy is almost al-
ways followed by chemotherapy.59
Cho et al.60 (2019) also explored RIATC when designing prodrug
AP1-DEVD-S-DOX (Fig. 4). It was synthesized based on a DEVD
tetrapeptide Asp-Glu-Val-Asp, used to release free DOX after
enzymatic hydrolysis by caspase-3 followed by the spontaneous
release of the self-immolative linker PABC. Besides that, the
ApoPep-1 (AP1), an apoptotic cell-binding moiety, which binds to
histone H1 translocated on the surface of apoptotic cells, was also
included in this SIS. The results showed effective tumor regression
3268 R.V. Gonzaga et al. / Journal of Pharmaceutical Sciences 109 (2020) 3262-3281
with no significant toxicity, as the prodrug connected to the AP1
proved to be more efficient in the delivery of DOX.
The self-immolative spacer PAB has been widely investigated in
conjugation to different dipeptides to increase selectivity towards
cathepsin, a cysteine protease overexpressed in human cancers.
Dipeptides Phe-Lys and Lys-Lys have been included in the design of
selective fluorogenic peptide prodrugs. Phe-Lys-bearing prodrugs
displayed efficacy in vitro and in vivo with low toxicity in healthy
tissues.61,62 An albumin-binding DOX prodrug (Fig. 5) was engi-
neered to contain a maleimide moiety and a PABC, as self-
immolative linker, conjugated to a cathepsin B dipeptide Phe-Lys
substrate.63
Ajaj and Kratz64 (2009) synthesized a cathepsin B-responsive
dual-acting prodrug for circumventing multidrug resistance. This
nanosystem contains a dual-acting maleimide-bearing moiety as
its central core connected to trigger dipeptides (Phe-Lys), PABC,
organized into two branches. DOX was introduced in one of those
branches and a P-glycoprotein efflux pump inhibitor in the other
one, as this protein is overexpressed in multidrug resistant tumor
cells. In preliminary in vitro tests, this prodrug demonstrated tar-
geted protein-binding due to its maleimide moiety and it was
cleavable in the presence of cathepsin B; preliminary toxicity
studies in mice also demonstrated that this SIDD exhibits low
toxicity even when high doses were administered.
It has been previously reported in the literature that the com-
bination of DOX and PTX enhances antitumoral treatment effi-
ciency, which has led to some examples in preclinical and clinical
Fig. 5. A e Chemical structure of single or dual-acting albumin-binding self-disassemble prodrug for drug delivery composed by a PABC linker and dipeptide Phe-Lys. B e Human
serum albumin-binding (HSA) ability.63,64
studies. Ajaj et al.65 (2009b) designed another prodrug based on the
same strategy. In vitro assays demonstrated a synergic effect in
neoplastic cell lines such as androgen-independent prostate cancer,
advanced breast cancer, and endometrial cancer. This dual-prodrug
was also able to bind to HSA, releasing almost all the drug content
within 20 h.
Sunitinib (Sutent®) belongs to the tyrosine kinase inhibitors
(TKIs) family, used for the treatment of gastrointestinal stromal
tumors (GIST), advanced renal cell carcinoma (RCC), and unre-
sectable or metastatic pancreatic neuroendocrine tumors, when
conventional treatments have no effect. The TKIs presented severe
adverse effects, which have limited their therapeutic use. In this
context, cathepsin B dipeptide cleavable prodrugs targeting
vascular endothelial growth factor receptors (VEGFR) were inves-
tigated. This prodrug showed a great potential in antitumoral
therapy.66
A smart prodrug named Ac-Phe-Lys-PABC-ADM was designed to
reduce the adverse effects of DOX, in which Phe-Lys is used as a
cathepsin B substrate, and PABC acts as the self-immolative linker.
This prodrug provided excellent performance in vitro assays, and
the results of in vivo tests demonstrated that the group treated with
the smart prodrug did not present toxicity, which was comparable
to the control group, while the DOX treated group showed a
marked decrease in body weight, and, in addition, one of the mice
died due to severe toxicity.61
Erez et al.67 (2009) designed a dendritic prodrug through tar-
geting approaches to selectively release chemotherapeutic drugs.
 R.V. Gonzaga et al. / Journal of Pharmaceutical Sciences 109 (2020) 3262-3281
Initially, it was composed of three PTX molecules coupled to 2,4,6-
tris(hydroxymethyl)aniline AB3 linked to a dipeptide Phe-Lys
coupled to a PABC self-immolative linker. In order to increase sol-
ubility, HPMA (N-(2-hydroxypropyl)-methacrylamide) was intro-
duced through a peptide linker via carbamate linkage to PABC-
dendrimer. HPMA copolymers are water soluble, non-toxic and
biocompatible, and they have been used for tumor targeting, as
they accumulate selectively in the tumor due to the enhanced
permeability and retention (EPR) effect (Fig. 6). The polymer-
conjugate with three branches of PTX demonstrated better cyto-
toxicity than the polymer-conjugate with just one PTX substitution
in a prostate adenocarcinoma (TRAMP C2) cell line.
Cryptophycin, a potent tubulin polymerization inhibitor, has
incredible activity in in vitro assays and excellent activity against
multidrug-resistant cancer cells; however, it has limited activity
in vivo and high neurological toxicity, thus necessitating the use of a
targeted drug delivery system to minimize adverse effects. Borbe ly
et al.27 (2019) developed conjugates containing some derivatives of
cryptophycin, which were conjugated to RGDfK cyclopeptide C,
targeting integrin v3 through the protease-cleavable Val-Cit linker,
and two different self-immolative spacers, PABC or Gly-Pro. The
conjugate with the PABC linker exhibited high stability in human
plasma and satisfactory stability in mice plasma, and efficient drug
release in the presence of cathepsin B, except for the Gly-Pro
bearing conjugate.
Neutrophil-Secreted Elastase
Neutrophil-secreted elastase (NSE) is an enzyme related to
oncogenesis signaling and inhibition of tumor suppressors, being
associated with bad prognosis. The neutrophil elastase, matrix
metalloprotease (MMP-9) and cathepsin G are proteases, which
have been correlated with tumor hyperproliferation.68 Integrin
Fig. 6. HPMA copolymer AB3 dendritic-prodrug conjugate with triple PTX payload.51
3269
avb3 receptor has been reported in the literature as being able to
bind to cyclic peptides bearing the Arg-Gly-Asp (RGD) sequence.
Linker cleavage assays and cell antiproliferative experiments per-
formed in vitro demonstrated the efficacy of this tumor-targeting
conjugate compared to the free drug in the presence of elastase,
suggesting a potential for therapeutic applications.
Chemical Triggers
The most used chemical triggers are those related to pH, 1,4-,
1,6- and 1,8-eliminations, and redox conversions. These latter are
most applied, considering disulfide self-immolative linkers are
frequently used as a spacer that needs this type of conversion to
give rise to the cascade of reactions and the delivery of drugs.
pH-Sensitive Nanocarriers
pH changes are an option for controlling drug delivery in
different body regions and have been explored in many examples
presented here.
The slightly acidic pH of lysosomes has been studied for the
development of pH-targeted nanocarriers able to release their
loading inside the cell. Acidic extracellular pH is a major feature of
tumor tissue, and this has been employed as an approach to design
pH-sensitive prodrugs in cancer therapy, such as DOX-thioureas
prodrugs. In these conjugates, DOX and the biologically active 2-
thiohydantoin are released in the acidic environment of cancer
tissues. The thiourea-modified prodrug presented higher accumu-
lation in tumor cells than free DOX.69
Additionally, Gisbert-Garzaran et al.70 (2017) described acidic
pH-sensitive nanocarriers based on mesoporous silica nano-
particles (MSN) bearing self-immolative polymers as promising
candidates for drug delivery in cancer treatment. They employed
3270 R.V. Gonzaga et al. / Journal of Pharmaceutical Sciences 109 (2020) 3262-3281

Fig. 7. pH-responsive prodrug containing biotin, as tumor targeting group, nitrobenzene to measure the fluorescence intensity in situ, and DOX.71

MSN as the main carrier and tert-butyl carbamate as a trigger
sensitive to acidic pH, which hinders pore entrance and prevents
premature drug release. On the other hand, an acid-triggered
prodrug containing biotin (tumor targeting group), nitrobenzene
(fluorescence quencher moiety to measure the fluorescence in-
tensity in situ) and DOX was designed to target particular cellular
uptake in tumor cells. DOX was coupled to a nitrobenzene-biotin
carrier via an acid-cleavable hydrazone bond, which functions as
the pH-responsive trigger (Fig. 7).71
In another example, a pH-activatable self-immolative L-Phe-Sar
dipeptide prodrug, containing atazanavir (HIV-1 protease inhibitor)
(Fig. 8) improved oral bioavailability when compared to the free
drug. Herein, the drug is released by a cyclization process of the
diketopiperazine induced at neutral pH.72
An activatable anticancer polymeredrug conjugate designed for
DOX delivery (Fig. 9) was built with polyacrylate linked to a
azobenzene-bearing polyethylene glycol segment through a
carbamate bond. The polymer-drug conjugate is neutral and after
contact with neoplastic cells was cleaved due to different cell pH,
generally acidic. This activates the azobenzene linkage, initiating a
1,6-self-elimination cascade reaction. Consequently, a cationic
portion is formed, which interacts with the negative surface of the
neoplastic cell membrane phospholipids, promoting cellular up-
take and subsequent DOX intracellular releasing. In vitro assays
with the HT-29 cell line showed no uptake and the prodrug was
only able to enter the cells after activation with sodium dithionite e
which cleaves the azobenzene linkage e, indicating that cell entry
is specific and dependent of ammonium cation formation. This
approach can be used in the future for targeted drug delivery ap-
plications reducing toxicity effects.73
benzylic substituents, resulting in first-generation self-immolative
dendrons. The disassembly happens via quinone methide elimi-
nation provided by 4-hydroxy-benzylalcohol, which is an example
of triggering via this elimination.75,76 The AB2 dendron suffers a
single cleavage in the trigger, releasing two branches from the
benzylic substituents through two subsequent quinone-methide
eliminations.77 Trigger activation of agents 1 or 2 makes up phe-
nolates 1a or 2a, respectively (Fig. 10). Thus, phenolate 1a un-
dergoes 1,6-elimination, releasing a reporter group and para-
quinone-methide 1b, while phenolate 2a undergoes 1,4-
elimination to release a reporter group and ortho-quinone-
methide 2b.13
Another example using dendrimers is that of Szalai et al.16
(2003). They described a self-immolative dendrimer based on an
AB2 unit with benzyl ether linkages, where removal of the pro-
tecting group triggers a cascade of both 1,4- and 1,6-eliminations,
resulting in dendrimer fragmentation and release of the 4-
nitrophenol, the end unit.
Concerning 1,8-elimination, first and second-generation den-
drimers containing vinylbenzyl moieties were synthesized to
disassemble through this reaction. Focal-point aniline was masked
in an oxidized form, as a nitro; in addition, PTX was coupled to the
dendrimer (Fig. 10). The activation occurs after nitro group

1,4-, 1,6- and 1,8-Eliminations

Shabat's research group described a linear polymer based on

polyurethane with a protecting group on the terminal amine acting
as a trigger. Upon trigger activation and eventual removal,
sequential 1,6-elimination and decarboxylation reactions take
place, which stimulate the releasing of the original precursors.74
Another example gives rise to the introduction of dendrimers, or
dendrons, in the scenario of self-immolative drugs. Self-immolative
AB2 dendrimers were designed based on phenol or aniline de-
rivatives with two hydroxymethyl substituents at the ortho and/or Fig. 8. pH-Activatable prodrug containing atazanavir (in blue) named L-Phe-Sar
para positions. These compounds present tail units attached to trigger (in red).72
 R.V. Gonzaga et al. / Journal of Pharmaceutical Sciences 109 (2020) 3262-3281 3271

Fig. 9. Chemical structure of an activatable polymeredrug conjugate based on the self-immolative azobenzene motif and pH-sensitive release of DOX.73

reduction takes place, leading to double 1,8-eliminations, followed
by a decarboxylation reaction, releasing PTX from the dendrimer.
G2 self-immolative dendrimers with double-release linkers were
attached through a carbamate bond to a nitrodiol (Fig. 11). Again,
the nitro group works as a trigger and, when reduced to an amine
group, leads to a cascade of self-eliminations, resulting in den-
drimer disassembly and PTX release.14
On the other hand, the reduction of azide has also been used as a
trigger in the design of self-immolative dendrimer prodrugs. They
undergo nucleophilic addition or self-elimination and,

Fig. 10. AB2 dendrons based on hydroxybenzyl alcohol, resulting in p-quinone-methide elimination or o-quinone-methide elimination.75,78
3272 R.V. Gonzaga et al. / Journal of Pharmaceutical Sciences 109 (2020) 3262-3281

Fig. 11. First and second-generation dendrimers containing PTX, as drug and, upon nitro reduction, vinylbenzyl moieties are responsible for double 1,8-eliminations, releasing
PTX.14

consequently, release the latent amine group. This strategy was acids.81,82 Some disulfide self-immolative linkers have been
carried out by Azoulay et al.78 (2006) to synthesize a self- recently reported (Table 1).
immolative DOX prodrug. They developed a Staudinger linker, Based on the tumoral tissue microenvironment, drug carriers
which involves intramolecular cyclization, followed by a 1,6- presenting a disulfide bond have been proposed, as they can be
elimination, releasing quinone-methide species and DOX. cleaved easily in cancer cells by highly concentrated GSH.83 In this
Ciprofloxacin (CIP) is a fluoroquinolone antibacterial used for context, H2S donors have been widely employed as self-immolative
the treatment of various infections, though its use is restricted due spacers in prodrug design. An example of a H2S donor investigated
to toxicity. Recently, a prodrug was synthesized constituting of a is a class of dual carbonyl sulfide/H2S donors based on a benzyl
self-immolative linker, as substrate for nitroreductase (NTR) thiocarbamate structure, named “caged thiocarbamates” (Fig. 13).
bacteria-specific cleavage, CIP, and a fluorescence moiety which is These structures release carbonyl sulfide and are quickly converted
quenched until linker activation. This CIP-prodrug showed evi-
dence of specific bacteria activation, while the prodrug was not
activated in mammalian cells. The conjugate presented similar ac-
tivity to free CIP, reducing the bacterial burden in a neutropenic
mouse thigh infection model, proving to be an interesting model
for delivery of bacteria-specific fluoroquinolone.79
Fumagalli et al. (2019)80 developed a new self-assembling drug
conjugate, composed of a self-immolative linker coupled to 4-(1,2-
diphenylbut-1-en-1-yl)aniline, which is used as a self-assembling
drug, and amino-thiocolchicine, forming nanoparticles (NPs).
These NPs are released in vitro by lipases. NP-1 and 2 consist of
different spacers - the first has only carbonic bonds and the second
one a disulfide bond (Fig. 12) - and both exhibited an anti-
proliferative profile on two human cancer cell lines (HeLa and MCF-
7). Therefore, it is possible to modulate the nanocarrier responses
through changes in the self-assembling linker.

Redox Conversion

Disulfide chemistry, especially those involving glutathione

(GSH)-responsive compounds (endogenous reducing agent), has
provided some versatile and multifunctional drug delivery systems,
which overcome intracellular and extracellular barriers. For this
reason, GSH-sensitives have been evaluated in the search for anti- Fig. 12. Chemical structure of self-immolative thiocolchicine diphenylbutenylaniline
cancer drugs, antioxidants, peptides, proteins, and nucleic conjugates with self-assembling ability.80
 R.V. Gonzaga et al. / Journal of Pharmaceutical Sciences 109 (2020) 3262-3281
Fig. 13. Dual carbonyl sulfide/H2S donors based on a benzyl thiocarbamate structure, named “caged thiocarbamates”, releasing carbonyl sulfide.5
into H2S in the presence of the ubiquitous enzyme carbonic
anhydrase.5
In addition, reshuffling disulfides have been employed as a
trigger for disassembly of drug delivery systems. The thiol group is
a functionality rarely found in the structure of most available drugs.
In order to overcome this chemical limitation, when there is a need
to introduce a temporary coupling, allowing a specific intracellular
release, disulfide spacers can be introduced in practically any
drug.84e86 In this context, a disulfide trigger for drug releasing has
been engineered for several classes of substances that are thiol free,
including nucleoside analogues,87,88 histone deacetylase inhibitors,
CPT (anticancer),89 and receptor antagonist.90
Reduction-triggered self-immolative based prodrugs containing
both cytotoxic drugs and director group are an interesting approach
taking in consideration their enhanced specificity towards tumor
cells and consequent positive results.53,91,92
Zhao et al.92 (2019) developed a “one pot” method for facile
synthesis of GSH-responsive polyesters (S-PEG-400 and S-PEG-
800) for encapsulated DOX release. When functionalized with a
disulfide bond, polyesters showed to be biocompatible as they did
not present cytotoxicity in vitro cell viability assays. DOX-loaded
functionalized copolymer demonstrated effective drug release in
the presence of GSH, proving to be a fast and practical technique of
preparation for polyester or polyamide that can be used for drug
delivery.93,94
Curcumin (CUR) is a natural compound with various pharmaco-
logical activities. It was reported that it could treat several types of
cancers, including gastrointestinal, melanoma, leukemia, breast,
lung, hematological, head and neck, neurological and sarcoma.
Furthermore, it can be part of cancer prevention. However, it is an
exceedingly hydrophobic compound, which has low stability at
physiological pH, undergoing fast elimination. To work around the
pharmacokinetic problems of CUR, GSH-responsive, PEGylated pro-
drug nano-micelles (PPNMs) were developed with self-assembly
proprieties. Altogether, the data demonstrates the potential of
micellar prodrugs and their future application for drug delivery,
improving the antitumor activity of natural compounds.95,96
3273
Protein-function control in response to peculiar physiological
characteristics can be achieved by attachment of redox-responsive
trigger structures. Wang et al.95 (2013) reported a redox-sensitive
nanocarrier to RNase A, using a RNase A lysine with a disulfide
bond, and 3-((2(((4-nitrophenoxy) carbonyl)-oxy)ethyl) disul-
faneyl)propanoic acid, as the reduction-responsive trigger, which
could be activated by intracellular GSH levels, with potential ap-
plications for targeted cancer therapy.
Another approach involving reduction-sensitive linkers is the
usage of the diselenide bond, as it shows similar chemical proper-
ties to disulfide bonds, being also cleaved by GSH; it is, however,
more easily cleaved since it shows a lower bond energy. Hu et al.96
(2018) proposed a theranostic nanoparticle composed of a modified
PEG containing diselenide bonds attached to a diester linker, to
which CPT was also attached. This SIDD follows the same self-
immolation mechanism proposed for disulfide bonds, whereby
free selenide can perform a nucleophilic addition on the ester next
to it, releasing the cytotoxic agent from the latent form.
Gut microbiome reductive environment can be exploited when
treating gut inflammatory diseases. Qiao et al.97 (2017) proposed an
amphiphilic polymer composed of PEG and polyCUR, both linked by
a disulfide bond, for the treatment of inflammatory bowel disease.
Both in vitro and in vivo assays showed this SIDD has not only good
compatibility and adequate pharmacokinetic potential and phar-
macodynamics, but also effective linker reduction when in the
presence of a normal gut microbiome.
Theranostic prodrugs able to be activated by specific stimuli that
trigger the release of the drug and image agents used to diagnose is
an interesting approach that has attracted attention due to the
possibility for its use to treat a disease and monitor its evolution at
the same time. Wang et al.98 (2014) designed a near infrared (NIR)
dye bearing theranostic, which the trigger e DNS e also being used
for its fluorescence-quenching effect over the NIR dye due to its
electron-withdrawing effect. Fluorescence is fully restored by
trigger activation and further drug and dye are released from the
theranostic system, making this probe efficient for both
applications.
3274 R.V. Gonzaga et al. / Journal of Pharmaceutical Sciences 109 (2020) 3262-3281
Yang et al.99 (2019) designed a gadolinium-complex, a self-
immolative prodrug (Fig. 14), which could be used as theranostic
for clinical magnetic resonance imaging (MRI), conjugate to biotin, a
selective group used for tumor targeting. This theranostic prodrug is
composed by a modified gadolinium (III)-1,4,7,10-
tetraazacyclododecane-1,4,7,10-tetraacetate (Gd-DOTA) complex, a
contrast agent used for clinical MRI, biotin, and CPT bound to a di-
sulfide GSH-responsive self-immolative linker. Assays performed
with it showed its targeting ability to a biotin-receptor, which is
overexpressed in cancer cell lines. Another important fact is that this
prodrug presented excellent diagnostic performance coupled with
MRI, high anticancer efficacy, and a reduction of adverse effects.
A disulfide-reduction model designed by Yang et al.100 (2012)
allowed the releasing drug to be monitored in vivo by two photon
FRET imaging methods. This system is composed by folic acid, as the
targeting group, a fluorescent agent, boron-dipyrromethene, BOD-
IPY, which serves as a FRET donor and emits green fluorescence upon
disulfide bond reduction, and a red fluorescent agent, rhodamine
(FRET acceptor), which is linked via a reducible disulfide bond to
folate. Folate receptors (FR) are abundant in cells of malignant cancer
and in normal kidney proximal tubule (PT) cells, so that the folate
targeting and releasing in the kidney was investigated in vivo. The
results demonstrated that low disulfide reduction occurred in PT
cells even during prolonged monitoring of folateFRET. In mice, a
modest progression of reduction in PT cells over1 time was shown.
Furthermore, in the same folateFRET colchicine prodrug, cytosolic
accumulation did not occur, indicating low kidney toxicity.
Vlahov et al.101 (2020) also used a similar approach when
designing a folate-targeting prodrug, which is proposed to be acti-
vated by biothiols to release a sequence selective DNA-crosslinking
alkylating agent. This SIDD showed sub-nanomolar activity and
good specificity towards its target, and high cure rates with minimal
toxicity.
Self-assembling nanostructures bearing reduction-triggered
bonds are commonly designed for cancer treatment. Disulfide
bonds show good versatility since they can be used on the nano-
particle's main structure, linking its building blocks,94,96,102e109 or
employed in self-immolative linkers, attaching drugs to the nano-
particle backbone.105,110e114 Both applications show highly efficient
drug release profiles when in in vivo or in vitro reductive environ-
ments, in addition to an adequate EPR effect, which make these SIS
an outstanding new technology.
Polymersomes are artificial polymeric vesicles with spherical
shapes composed of a hydrophilic core, which can encapsulate
Fig. 14. Chemical structure of the gadolinium-complex based self-immolative prodrug.99
water-soluble molecules and a hydrophobic membrane, which may
incorporate hydrophobic molecules.115 Self-assembled polymer-
somes have been designed from amphiphilic copolymers to obtain
hollow structures surrounded by a polymeric bilayer membrane.116
Considering that, few oxidation-responsive polymersomes have
been developed, the first being that reported by Napoli117 et al.
(2004), employing oxidative conversion to destabilize them. Poly-
mersomes containing aryl boronic esters have been developed to
be hydrogen peroxide responsive.118e120 These oxidation-sensitive
polymersomes displayed enhanced imaging/drug release features.
Thus, mitochondria-targeted H2O2 reactive polymersomes were
synthesized composed of self-assembling of amphiphilic block
copolymers and arylboronate ester-capped self-immolative bonds
in the hydrophobic block, followed by surface functionalization
with targeted peptides.121
Oxidative stress caused by high concentrations of Reactive Ox-
ygen Species (ROS) makes for an interesting approach, not only for
cancer treatment, but also for the treatment of other diseases like
Alzheimer's Disease (AD) and cardiovascular injuries, in which ROS
play a significant role in pathogenesis and pathology progres-
sion.122 When compared to reduction-triggered self-immolative
drug delivery systems e mainly used for cancer treatment -
oxidation-triggered prodrugs can be designed for a wider disease
spectrum.123,124 Lu et al.125 (2019) designed a boronic acid-bearing
self-immolative prodrug micelle aimed for AD treatment,
composed of a RAGE-targeting peptide, an amphiphilic polymer
PEG-lysine B and CUR. This probe exhibited efficient neuro-
protection and microglia modulation and, consequently, enhanced
cognitive activity in AD model mice.ROS-responsive self-immola-
tive prodrug nanoparticles were also designed for hormone
dependent cancer therapy. In this approach, diethylstilbestrol
(synthetic non-steroidal estrogen) was used, leading to the
respective prodrug, which showed to be more active in vitro against
prostate and breast cancer cells, presenting lower action in healthy
cells due to its higher selectivity for ROS-rich tumor
environments.126,127
Photodegradable Triggers
Light is an attractive pathway for stimulating SIS degradation as
it can be remotely employed for a short period of time with high
spatial and temporal precision.128 Light-sensitive SIDD can release
their loaded drug after external light-activation, in order to
improve treatment efficacy and safety. Thus, light-induced self-
 R.V. Gonzaga et al. / Journal of Pharmaceutical Sciences 109 (2020) 3262-3281 3275

Fig. 15. Polymersome consisted of hydrophilic poly(acrylic acid) and hydrophobic ONB-substituted poly(g-methyl-ε-caprolactone) (PMCL-ONB) blocks and bearing a photode-
gradable spacer, o-nitrobenzyl, for self-disassembly upon UV irradiation.131,132

immolative polymers have been shown to be promising in imaging
and drug delivery investigations.129,130 In this context, Anderski
et al.116 (2019) developed light-responsive polycarbonates and their
respective PEGylated derivatives loaded with the photosensitizer
5,10,15,20-tetrakis(m-hydroxyphenyl)chlorin for photodynamic
therapy. These polymers degrade after a UV light stimulus in a
specific wavelength triggers their breakdown. Moreover, they
observed that light-sensitive compounds had higher drug release
than non-light-sensitive polymers.
Polymersomes able to disintegrate upon UV irradiation were
designed containing a photodegradable spacer, o-nitrobenzyl, as a
junction point between hydrophilic poly(acrylic acid) and hydro-
phobic ONB-substituted poly(g-methyl-ε-caprolactone) (PMCL-
ONB) blocks. These copolymers may self-disassemble when
exposed to UV irradiation, resulting in small micellar-like struc-
tures, which controls the release of the payload through the in-
tensity of UV light131,132 (Fig. 15).
Photo-cleavable polymersomes employing 2-
nitrophenylalanine were shown to obtain UV-responsive com-
pounds, leading to the vesicle destabilization and releasing loaded
contents upon irradiation.133 Liu et al.134 (2014) reported self-
immolative polymersomes composed of amphiphilic block
copolymers that are activated by different triggers which lead to
their disintegration, such as visible light (420 nm), UV light
(365 nm), or a reductive medium. They obtained block copolymers
constituted of a trigger photodegradable poly(benzylcarbamate)
block and a hydrophilic poly(N,N-dimethylacrylamide), which is a
self-immolative moiety caged with perylen-3-yl,2-nitrobenzyl, or
disulfide portions sensitive to the stimuli described above.
Carbamate-based self-immolative dendrimers composed of an
AB2 dendrimer branch unit and N,N0 -dimethylethylenediamine, as
a cyclization spacer, were developed to be cleaved at the 4,5-
dimethoxy-2-nitrobenzyl photolabile trigger (Fig. 16). Drugs can
be conjugated to the two hydroxybenzyl groups through a carba-
mate linkage, while the phenol group can be attached to the N,N0 -
dimethylethylenediamine linker. The light-stimulus triggers the
self-disassembly, forming the derivatives 1a and 1b, which suffer
double 1,4-eliminations followed by decarboxylation reactions,
thus through a process of self-immolative sequential reactions, four
aminomethylpyrene molecules are released from the second-
generation dendrimer.15
Tan et al.135 (2015) reported a UV responsive self-immolative
dendrimer consisting of a phenol-based self-immolative AB3 sys-
tem through carbonate bonds conjugated to three CPT molecules at

Fig. 16. Carbamate-based self-immolative dendrimers composed of N,N0 -dimethylethylenediamine, as a cyclization spacer and, 4,5-dimethoxy-2-nitrobenzyl, as photolabile
trigger.15
3276 R.V. Gonzaga et al. / Journal of Pharmaceutical Sciences 109 (2020) 3262-3281
its periphery. In addition, the phenol group is capped with a pho-
tolabile 2-nitrobenzyl ether moiety coupled to a DNA strand. UV
irradiation releases the nucleic acid shell, while the core, which
consists of prodrug molecules, disintegrates via an irreversible self-
immolative process, releasing three free CPT. Other photodegrad-
able dendrimers were developed to be water-dispersible and visible
light-responsive self-immolative agents through masking in the core
of a perylen-3-yl methanol, visible light-cleavable group.136
Hydrogels based on hyaluronic acid, a polysaccharide of extra-
cellular matrix, were investigated for drug-controlled releasing. In
this context, a UV light-triggered hydrogel prodrug was designed
containing dopamine coupled to a photo-labile spacer (o-nitro-
benzyl) and to hyaluronic acid modified with thiol and hydrazide
groups, which lead the disassembly.137
Exploring viruses as macromolecular vehicles for controlled
releasing, Brasch et al.138 (2013) used the capsid of a plant virus
named Cowpea chlorotic mottle virus for loading a self-immolative
polyurethane with a photolabile trigger, p-nitrobenzyl alcohol.
The disassembly is led by UV irradiation, triggering the depoly-
merization in monomeric units. Therefore, this is another approach
that could be utilized in drug delivery systems.
As a light-stimulus, the NIR light can penetrate deeper into
tissues and presents promising in vivo uses. Taking this into ac-
count, a NIR-responsive polymer was designed to encapsulate a
pharmaceutical model Nile Red, which contains triggering groups,
such as quinone-methide, that initiate a domino effect where a
single event leads to multiple light-sensitive reactions. After the
breakdown induced by light, the cyclization of the diamine spacer
occurs, unmasking an unstable quinone-methide trigger. Thereby,
upon NIR irradiation, the nanoparticle degradation occurs, and the
payload is released.128
Multiple-Stimuli Release
There are some examples that use a multiple stimuli approach,
which represents an even more controlled release of the conju-
gated drugs and an enhancement of cascade release. The most
common combination is between pH and redox triggers. Some of
them are presented as follows.
pH/redox Triggers
pH and redox responsive chitosan nanogels were engineered as
nanocarriers for intracellular delivery in cancer cells. These nano-
gels are obtained from ionic gelation between o-carboxymethyl-
chitosan and thiolated chitosan, which act as pH sensitive triggers
able to achieve endo/lysosomal escape via the proton sponge effect.
Thereby, nanogels are oxidized forming disulfide bonds, which are
trigger activated by high intratumoral GSH levels. In one of the
examples, DOX was loaded into nanogels and thus, there was an
increase in the drug's release caused by the synergistic action of
acidic pH and reductive potential.139
Another example of dual or multi-stimuli responsive nanogels
was reported by Cheng et al.81 to provide tumor-targeted and
reduction-triggered intracellular release of DOX into human glio-
blastoma, with nanogels consisted of a poly(vinyl alcohol) con-
taining cyclo(Arg-Gly-Asp) peptide, and a targeting group that
favours the intracellular release in integrin overexpressing human
glioblastoma.
In a similar approach, Wang et al.140 (2017) designed acid- and
redox-dual responsive (bearing reduction-sensitive disulfide
bonds) polysaccharide-based (dextran) nanohydrogels to improve
the drug delivery ability of anti-cancer DOX delivery systems. The
findings showed higher inhibition of tumor growth, as well as,
quick drug release behavior in acidic and reducing environments,
enhancing DOX controlled release at tumor sites and decreasing its
toxicity in comparison to the free drug.
Acidic pH and reduction-sensitive nanomicelles were designed
to encapsulate DOX to achieve passive targeting and control drug
release at tumor sites,141 for leukemia treatment. The authors found
that the GSH and pH dual-stimulation prodrug significantly
enhanced tumor cell-selective uptake.142
Works with CPT prodrug,143 and chondroitin sulfate micelles144
dual stimulation were also developed.
A polyphosphoester prodrug of DOX containing a diselenide
bond was developed to be pH/reduction-responsive. The DOX de-
rivative presents an azide group connected to a polyphosphoester
chain by click chemistry to yield a pH/reduction-responsive poly-
meric prodrug.9 The SeeSe bond can be oxidized to selenic acid
through oxidative agents and reduced to selenol via reducers, as
well as, its g-ray-responsiveness, which results in it being cleaved
when exposed to g radiation. After internalization into tumor cells,
the diselenide bonds could be cleaved and the hydrazone bonds
could also be hydrolyzed in low a pH medium, finally resulting in
prodrug disintegration and freeing of the DOX derivative to inhibit
tumor growth.98,145
Other Approaches and Multiple Triggers
Some other approaches have also been used in addition to
multiple trigger approaches, which enrich the versatility of SIDD,
making them useful in attaining the highest selectivity, especially
for diseases as cancer.
Wang et al.146 (2013) developed a glutathione and ROS-
responsive drug nanocarrier targeted to tumor cells, employing a
CPT-based topoisomerase I inhibitor 7-ethyl-10-
hydroxylcamptothecin (SN38), which is the active metabolite of
irinotecan. Considering that SN38 has limited therapeutic efficacy
due to its low water solubility, they designed an ester spacer joined
to the hydrophilic oligomer of ethylene oxide to couple with SN38
through a thioether chain, which could be easily oxidized by ROS to
sulfone or sulfoxide and thus become hydrophilic, triggering hy-
drolysis of the phenol ester and subsequent release of SN38. This
compound is an amphiphilic prodrug and its disassembly is trig-
gered by thiolysis in the presence of GSH or hydrolysis of the linker
caused by ROS-oxidation (Fig. 17). Tang et al.147 (2019) reported a
redox-sensitive nanocarrier to RNase A, using a RNase A lysine with
a disulfide bond, 3-((2(((4-nitrophenoxy)carbonyl)-oxy)ethyl)
disulfaneyl) propanoic acid, a responsive trigger which could be
activated by intracellular levels of GSH with potential application
for targeted cancer therapy.
Ester prodrugs of platinumeacridine (anticancer drugs) were
obtained to break through two different pathways: a platinum-
assisted pathway via self-immolative ester cleavage in a low-
chloride environment, and other enzymatic cleavage by human
carboxylesterase-2 (Fig. 18). These derivatives present a hydroxyl
group masked with a cleavable lipophilic acyl moiety (butyric and
valproic esters), in order to improve the log D properties and
decrease the drug's systemic toxicity. In lung adenocarcinoma cell
lines, the hybrids displayed lower systemic toxicity and higher
chemical stability.148
Cytotoxic quinones, which act on the NQO1, an enzyme widely
found in several cancer, displayed interesting anticancer activity.
Additionally, these quinones can be used as a trigger to activate
fluorescent probes. Three hybrid Pt(IV) complexes and quinone
propionic acid derivative were engineered to present dual stimuli-
responsive disassembly by action of both NQO1 and ascorbic acid
(cellular biomolecular reducing agent), thus releasing the cytotoxic
Pt(II) agent. Among them, the complex with coumarin were also a
theranostic agent.149
 R.V. Gonzaga et al. / Journal of Pharmaceutical Sciences 109 (2020) 3262-3281
Fig. 17. Nanocarrier GSH-sensitive and ROS-responsive targeted to tumor cells, employing 7-ethyl-10-hydroxycamptothecin (SN38).146
DT-diaphorase is a cytosolic flavoprotein overexpressed in
various malignant tumors and responsible for reducing some bio-
logical substances like quinones, by co-factors like nicotinamide
adenine dinucleotide (NADH)/nicotinamide adenine dinucleotide
phosphate (NADPH). The use of DT-diaphorase represents an
excellent opportunity for the development of a prodrug with the
ability to trigger the release of the drug and the image agent used to
monitor treatment, as well.150 Liu et al.136 (2015) also developed a
DT-diaphorese-responsive prodrug, in which a drug model CPT is
linked by a cleavable linker to a quinone propionic acid moiety
acting as a self-immolative group, which is rapidly and specifically
reduced by DT-diaphorese, releasing CPT and emitting fluores-
cence. The results demonstrated higher cytotoxicity upon DT-
diaphorase overexpressed cancer cells. According to the authors,
these elements will promote the development of a theranostic
enzyme catalyzed for antineoplastic delivery.
In diabetes research, poly(cyclic phenylboronic ester) was
employed as a self-immolative trigger for the design of glucose and
H2O2-sensitive dual-responsive polymeric nanogels, in order to
carry and increase glucose-responsive insulin delivery. These
nanogels were obtained via one-pot thiol-ene click chemistry. In this
context, glucose oxidase oxidizes glucose from the nanogel, leading
to H2O2 generation, which is responsible for oxidizing and hydro-
lyzing the phenylboronic ester. In subcutaneous injections for the
treatment of diabetic mice, these compounds revealed an effective
hypoglycemic effect.151
Testosterone was used as a model for the design of glyceride-
mimetic prodrugs employing self-immolative spacers that promote
lymphatic transport, avoiding first-pass metabolism, and enhancing
oral bioavailability. The transport of lipids can be an important strategy
for drug delivery, exploring the lymphatic system, as it plays an
3277
important role for the transportation of dietary lipids such as triglyc-
eride (TG), which is converted in the intestine to diglyceride and, ul-
timately, to monoglyceride (MG) and fatty acid (FA). Such lipids were
used as lymphatic targets to design prodrugs with a self-immolative
unit responsive to enzymatic action and promoting a cascade for
releasing the drug. The in vivo assays showed the prodrugs were able to
improve systemic exposure of testosterone, which has limited
bioavailability due to first-pass metabolism, by about 10 to 90-fold.10
An interesting system using PSA-antigen for prostate cancer
though a selective activation of peptide carrier, via a carbamate
bond to a self-immolative linker, was design by Alonysius and Hu152
(2020). They exploited prostate-specific antigen (SPA) to create
prostate cancer targeting peptide-linked DOX conjugates. One of
the proposed prodrugs, after peptidic bond cleavage, produces a
self-immolative portion attached to the cytotoxic agent, which
undergoes spontaneous self-cyclization, releasing its payload. Tu-
mor specificity will de further studied.
Thermal-responsive SIDD can be found in literature, but most of
their applications are on polymeric and not necessarily in thera-
peutic or theranostic agents,153e156 as they are only activated under
mild to high temperatures. Even so, they finely controllable payload
release is a remarkably interesting feature to be incorporated in
biomedicinal materials,157e160 as mild temperatures can be ach-
ieved using magnetic xyz linked to the SIS backbone.161
Future Perspectives
Drug delivery systems using stimuli-response polymers as car-
riers have been intensively studied in the past decade and the
perspectives point out to an increase in their importance in the next
few decades.
3278 R.V. Gonzaga et al. / Journal of Pharmaceutical Sciences 109 (2020) 3262-3281
Fig. 18. Ester prodrugs of platinumeacridine designed with the aim at disintegration through a platinum-assisted via self-immolative ester cleavage in a low-chloride environment
and also enzymatic cleavage by human carboxylesterase-2.148
This 10-year review provides several interesting examples of
compounds under study using many triggers as stimuli-responses,
such as enzymes, pH, redox systems, light, in addition to multiple
stimuli. Enzymes and chemical triggers are the current focus of
most of the interest in this field.
Although many classes of drugs or bioactive compounds can be
conjugated to those smart systems, it is worth noting that most of
the studies focus on cancer treatment, as expected. This is
completely understandable, since selectivity is a property most
searched for in this area, with the aim of achieving a high cytotoxic
effect in the tumor cells with minimum damage to the health cells.
Despite this, there is rising interest in other therapeutic classes,
such as Alzheimer's disease and diabetes.
This is a very complex technology and needs a deep under-
standing of the biochemistry of the cell systems and, also, the
chemical reactions that can, selectively, occur through the immo-
lative effect in cascade. Despite this, the versatility of this approach
and the advanced tools currently used to achieve these goals have
allowed us a glimpse of the evolution possible in this area.
Finding novel self-immolative linkers is one of the challenges in
this area, which could be bypassed by the knowledge of self-
immolative drug delivery kinetics. This would open up many pos-
sibilities for adequately controlling the process [12].Notwith-
standing, the main problem for this approach is that most of the
studies have been developed in vitro. In vivo studies should be
implemented as this is the way for clinical translational step [24].
Some stimuli, as light, wich lead us to photonic systems for
trigger the disassembly of the systems, have raised much interest
nowadays. It is worth to note that this has happened also to
theranostic compounds using this approach.We can foresee much
more examples in the near future, taking into account the advan-
tages of this kind of design, mainly in the case of cancer.
We have been studyed these systems in our laboratories in order
to design the more effective and selective drugs for neglected dis-
eases such as Chagas disease, using carbamate and disulfide groups
as triggers and dendrimers or dendrons as carriers (data not pub-
lished). They have been designed for responding to the enzyme
trypanothione reductase, specific to Trypanosoma cruzi, through the
reduction of the disulfide group.
We hope that the examples presented herein inspire re-
searchers working on selective drug delivery systems and increase
the perspectives for their application in therapeutics.
Acknowledgements
We are grateful to FAPESP, CNPq, and CAPES for their financial
support and scholarships.
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