Linker-determined drug release mechanism of free camptothecin from self-assembling drug amphiphiles

Article abstract of DOI:10.1039/c3cc49453e

We report here that the release mechanism of free camptothecin from self-assembling drug amphiphiles can be regulated by use of different linker groups. Our results highlight the significance of the linker group of drug amphiphiles on the drug release efficiency and their consequent in vitro efficacy. the Partner Organisations 2014.

Full text of DOI:10.1039/c3cc49453e

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Linker-determined drug release mechanism of
free camptothecin from self-assembling drug
amphiphiles†
Cite this: Chem. Commun., 2014,
50, 6039
Received 16th December 2013,
Accepted 16th April 2014
Andrew G. Cheetham, Yu-Chuan Ou, Pengcheng Zhang and Honggang Cui*
DOI: 10.1039/c3cc49453e
www.rsc.org/chemcomm
We report here that the release mechanism of free camptothecin analogue, and attributed this to the lack of endosomal esterases
from self-assembling drug amphiphiles can be regulated by use of that can accelerate hydrolysis of the cleaved thiol. In contrast, a
different linker groups. Our results highlight the significance of the report by Wender and co-workers suggests that intramolecular
linker group of drug amphiphiles on the drug release efficiency and cyclization of the cleaved thiol to form a 5-membered lactone can
their consequent in vitro efficacy.
help spur the release of the free drug in paclitaxel (PTX)-
conjugated octaarginine transporters,2 with this conjugate showing
Covalent linkage of anticancer drugs to a carrier presents an effective greater activity than its carbonate analogue. These contradictory
strategy to improve the drug’s solubility, to enhance the drug results may be due to greater steric hindrance around the 31
targeting efficiency,1 and to help circumvent multidrug resistance.2 hydroxyl of CPT versus the 21 hydroxyl of PTX.
Such a prodrug strategy has been used to create peptide–drug
We recently reported the creation of drug amphiphiles
conjugates,3 polymer–drug conjugates,4 dendrimer–drug (DAs)—peptide–drug conjugates with the capacity for self-assembly
conjugates,5 nanoparticle–drug conjugates,6 and most recently into well-defined nanostructures.8 Our initial design8a connected
drug-based molecular hydrogelators7 and self-assembling drug the hydrophobic anticancer drug camptothecin15 to a hydrophilic
amphiphiles.8 Indeed, the linkage of a hydrophilic segment b-sheet forming Tau-derived peptide16 via a reducible disulfanyl
to a hydrophobic anticancer drug to create self-assembling butyrate (buSS) linker. The single CPT-containing buSS-linked
amphiphiles offers the means to create self-delivering drugs drug amphiphile displayed a much reduced activity when com-
that possess a high and fixed drug content, allowing for the pared with free CPT, an observation that may be due to the
minimum use of excipients. These self-assembling prodrugs retarded release of the active drug. In this communication, we
combine the processability of small molecules with the controlled show that the in vitro activity of this buSS-linked drug amphiphile
pharmacokinetics potential offered by larger structures such as is compromised by the nanostructure-promoted formation and
polymer–drug conjugates.4b,9 One characteristic that all these drug protection of a CPT-based disulphide dimer that reduces its ability
conjugates must share is the ability to effectively release their cargo to release the free drug (Fig. 1). The carbonate-based analogue,
in an active form at the target site. A popular method for this is the which undergoes rapid self-immolation upon reduction, was found
use of stimuli-sensitive linkers10 between the drug and carrier that to largely negate this effect.
can selectively release the drug, examples of which include pH-,11
To probe the release of CPT from the buSS-linked drug
reduction-,2,12 or enzyme-sensitive13 linkers. The linker chemistry is amphiphile, CPT-buSS-Tau (1),8a we performed a more mechanistic
thus of critical importance in determining the overall efficacy of the study of its glutathione (GSH)-induced degradation using HPLC
delivery system and, at times can be complicated by the auxiliary and LC-MS analyses at relatively low (5 mM) and high (50 mM)
segment5b,14 and also by drug’s chemical structure. For example, concentrations. This allows us to explore the degradation behaviour
work by Low and co-workers12 reveals that a camptothecin (CPT)– close to and much higher than the critical assembly concentration,
folate conjugate with a disulfanyl butyrate linker shows lower which was previously found to be sub-micromolar.8a Following the
activity against cancer cell lines in comparison to its carbonate initial reduction of the buSS linker, a dynamic mixture of products
is expected to form through a series of disulphide exchange and
hydrolysis reactions (Fig. 2a). It can be seen from the HPLC analysis
that reduction of 1 proceeded to give free CPT via two intermediary
species at 11.7 and 15.7 min (Fig. 2b and c). LC-MS analysis
Department of Chemical and Biomolecular Engineering and Institute for
NanoBioTechnology (INBT), The Johns Hopkins University, Baltimore, MD 21218,
USA. E-mail: hcui6@jhu.edu
(S3.3 and Fig. S11 in ESI†) identified the first of these as the
expected cleavage product, 3, with the second being the symmetrical
† Electronic supplementary information (ESI) available: Experimental methods,
characterization data. See DOI: 10.1039/c3cc49453e
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Fig. 1 Self-assembly of the ester-based CPT-buSS-Tau (1) and carbonate-
based CPT-etcSS-Tau (2) drug amphiphiles into nanofilamentous structures
and the effect of the linker on the release mechanism of the free drug, CPT,
and the subsequent cytotoxicity. Representative TEM images were obtained
from 1 mM samples of 1 and 2 in water, staining with 2 wt% uranyl acetate.
CPT-containing disulphide dimer, 4. To the best of our knowl-
edge, this is the first time that such a disulphide species has been
reported to have been formed through reduction of this linker type.
The efficiency with which free CPT is released is therefore dependent
upon the stability of these two intermediates.
Examining the release profile over a 24 h period shows that the
solution concentration has a significant effect on the amount of
released CPT (Fig. 2d and e). At low concentrations, 3 and 4 make up
around 13% of the total CPT-containing components in solution after
24 h, with approximately 49% free CPT released. In contrast, 3 and 4
make up 34% after 24 h at the 50 mM concentration with only 26%
Fig. 2 GSH-induced release of CPT from 1. CPT-containing reaction products
formed and the exchange reactions that can occur during the GSH-induced
reduction of 1 (a). HPLC analysis of GSH-induced release of CPT from DA 1 at
5 (b) and 50 mM (c) in 10 mM sodium phosphate containing 10 mM GSH at 37 1C
after 1 and 24 h. Peak areas of the 4 main species (expressed as percentages of
the total area; the area for 4 was halved to reflect the increased absorbance due
to two CPT molecules) showing the evolution over time for the 5 (d) and 50 mM
(e) solutions of 1. Possible mechanism for the formation of the disulphide dimer
4 within nanofilaments or smaller b-sheet aggregates (f).
free CPT released. This occurs despite a similar level of degradation of early stages of reaction, the reduction of DA 1 proceeds pre-
the parent conjugate 1 (almost 60% loss at both concentrations). The dominantly via the formation of sulphide 3 and Tau-GSH (as
data also show that at the higher concentration, 4 appears to undergo identified by LC-MS analysis, Fig. S11g in ESI†), because there
little further hydrolysis or reduction, even increasing slightly after 7 h is little evidence of CPT-buSS-GSH and only a small amount of
incubation, whereas 3 depletes at both concentrations. This clearly CPT formed within 1 h. The released 3 would then go on to
indicates that there is some stabilizing influence over the disulphide form the disulphide dimer by further reaction with 1. It is clear
dimer that prevents further degradation at the higher concentration. from these results that the supramolecular nature of the
There is also little evidence to suggest that the degradation of 3 is conjugate is affecting the release process.
undergoing any accelerated release due to intramolecular cyclization,
instead being simply hydrolysed in agreement with the observations the data obtained suggests the mechanism shown in Fig. 2f as a
of Low and co-workers.12
possible explanation for the observed disulphide formation and
Taking into account the supramolecular nature of DA 1,8a
A comparison of the GSH-induced CPT release from solutions of subsequent stability. In a fully assembled state, the disulphide
DA 1 and the non-assembling CPT-buSS-Pyr conjugate (both 25 mM) bond of 1 will only be able to react with GSH at the terminal
found that reduction of 1 generates twice the relative amount of 4 ends of the nanofilaments or, more likely, with those in smaller
than CPT-buSS-Pyr (Fig. S12 in ESI†). Interestingly, this experiment b-sheet aggregates that have dissociated from the larger nano-
also shows that CPT-buSS-GSH is very sensitive to further degrada- structures. The close proximity of the newly released 3 (as the
tion as a significant amount of free CPT has already been generated thiolate) to a neighbouring conjugate molecule would then
after only 1 h of incubation. This observation suggests that in the make disulphide exchange highly favourable, either reforming 1 or
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giving the disulphide dimer 4. This reaction of GSH with nanofibers
or non-micellar b-sheet structures is expected to be enhanced at
relatively higher concentrations, accounting for the greater fraction
of 4 at 50 mM relative to that of 5 mM. Since the CPT segment is
hydrophobic and possesses the potential for p–p interactions,
molecules of 4 formed from reaction of GSH with nanofilaments
or non-micellar b-sheets at higher concentrations may remain
associated with the larger nanostructure or form some other types
of aggregates, rather than escaping into the aqueous environment.
As a result these disulphide dimers would be protected from
hydrolysis that would give free CPT. At low concentrations, the
degradation of the conjugate will lead to eventual disassembly of
the nanofibers or b-sheets as the concentration falls closer to the
critical aggregation concentration with subsequent exposure of the
CPT-products to water—the significant proportion of 4 remaining
after 24 h incubation at 50 mM in comparison to the 5 mM study is in
agreement with this proposition.
Fig. 4 Cytotoxicity study (48 h incubation) comparing free CPT, 1, 2, 3, 4
and ethylene sulphide against the MCF-7 breast cancer cell line. Cell viability
was determined by SRB assay and data are presented as mean Æ s.d. (n = 3).
generating only the free drug. A room temperature study confirmed
To negate the effect of this nanostructure-promoted disulphide no appreciable amount of intermediates 5 and 6 were formed
formation, we incorporated a carbonate-based linker that has a greater (Fig. 3b), indicating that rapid degradation via the expected intra-
propensity for self-immolation. Our rationale is that if the release molecular pathway was occurring rather than direct hydrolysis at the
product can rapidly degrade then it should reduce any opportunity for elevated temperature. A kinetic study also confirmed that the con-
disulphide formation. To this end, we utilized the disulfanyl-ethyl jugate possesses excellent hydrolytic stability (Fig. 3c). It is clear that
carbonate linker2,12,17 to furnish CPT-etcSS-Tau, 2 (Fig. 1 and Scheme the fast and direct conversion to free CPT upon GSH reduction of 2
S2 in ESI†). It has been proposed that, upon reduction, this linker can eliminates any possibility of disulphide formation due to the self-
release the attached cargo via a two-step pathway to produce the free assembled nature of the conjugate.
drug (Fig. 3a), with thiolate 5 undergoing intramolecular displacement
In vitro experiments using the MCF-7 breast cancer cell line were
to give carbonate 6, which then decarboxylates to give free CPT.17 performed to determine the relative cytotoxicity of DAs 1 and 2, and
The observation of the rapid hydrolysis of peptide thioesters in the their degradation products 3, 4 and ethylene sulphide (Fig. 4). It can
presence of 2-mercaptoethanol provides validation for this intra- be seen that the carbonate linker of 2 offers a greater efficacy
molecular displacement step.18
compared to the ester linker of 1, being comparable to free CPT.
Self-assembly of 2 was seen to give similar filamentous This result is consistent with previous observations12 and our release
nanostructures to 1 (Fig. 1), with CD analysis confirming the experiments. Interestingly, intermediate 3 was found to have a
b-sheet structure was present (Fig. S9 in ESI†). Given that the nature similar toxicity to free CPT, whereas disulphide 4 was comparable
of the linker has no significant effect on the nanostructure adopted, to its parent DA, 1. The fact that 3 is apparently more toxic than both
the drug release was studied using the same protocol. We found that 1 and 4 may be due its greater susceptibility to hydrolysis (less
the GSH-induced cleavage of 2 occurs very rapidly (Fig. 3b and c), propensity for aggregation) and differences in cellular uptake. The
proposed release product from the degradation of 2, ethylene
sulphide, was found to be non-toxic over the studied range, indicat-
ing the toxicity of 2 arises solely from the release of CPT.
In summary, our studies have revealed that the filamentous nature
of disulfanyl ester-linked CPT drug amphiphiles can promote the
formation of CPT-based disulphide dimers upon reduction, with higher
concentrations of the conjugate reducing the amount of bioactive CPT
being released through apparent protection of the intermediates from
hydrolysis. Incorporating a carbonate-based linker restored much of the
conjugated drug’s activity by facilitating a more effective release upon
reduction, giving a more potent drug amphiphile.
This work was supported by the National Science Founda-
tion (DMR 1255281) and NIH (A.C.: T-32CA130840). We
acknowledge the use of the JHU Department of Chemistry
Shared Facilities and the Integrated Imaging Center (IIC).
Notes and references
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