Toward Drug Release Using Polymer Mechanochemical Disulfide Scission

Zhiyuan Shi, Jingnan Wu, Qingchuan Song, Robert Go

Article

Toward Drug Release Using Polymer Mechanochemical Disulﬁde

Scission

stl,* and Andreas Herrmann*

Cite This: https://dx.doi.org/10.1021/jacs.0c07077

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sı Supporting Information

ABSTRACT: Traditional pharmacotherapy suﬀers from multiple drawbacks that hamper patient treatment, such as the buildup of

antibiotic resistances or low drug selectivity and toxicity during systemic application. To overcome these challenges, drug activity can

be controlled by employing delivery, targeting, or release solutions that mostly rely on the response to external physicochemical

stimuli. Due to various technical limitations, mechanical force as a stimulus in the context of polymer mechanochemistry has so far

not been used for this purpose, yet it has been proven to be a convenient and robust method to site-selectively rearrange or cleave

bonds with submolecular precision in the realm of materials chemistry. Here, we present an unprecedented mechanochemically

responsive system capable of successively releasing small furan-containing molecules, including the furylated ﬂuorophore dansyl and

the drugs furosemide as well as furylated doxorubicin, by ultrasound-induced selective scission of disulﬁde-centered polymers in

solution. We show that mechanochemically generated thiol-terminated polymers undergo a Michael-type addition to Diels−Alder

(

DA) adducts of furylated drugs and acetylenedicarboxylate derivatives, initiating the downstream release of the small molecule drug

by a retro DA reaction. We believe that this method can serve as a blueprint for the activation of many other small molecules.

INTRODUCTION

conjugating a photoswitch to the bioactive agent. However,

limited tissue penetration depth, phototoxicity, poor tumor

selectivity, and the production of reactive oxygen species

damaging biological membranes constitute currently challeng-

■

Drug delivery systems are actively investigated to satisfy

therapeutic needs and overcome the limitations associated with

traditional pharmacotherapy, such as physical and/or chemical

drug degradation, poor drug selectivity, environmental toxicity,

and the emergence of drug resistances.

targeted drug release systems responding to internal or external

stimuli are popular and successful contenders. While internal

stimuli mostly comprise speciﬁc physiological or endogenous

factors, such as the decrease of interstitial pH or an increase of

ing disadvantages of this technology. Concomitantly, US is

used in biomedical applications already with a wide range of

frequencies, power intensities, and modes of application for

tissue ablation (kHz, W·cm ), theranostics, and imaging

(MHz, mW·cm ). Moreover, US is being employed for the

−3

Among these,

−2

−

spatiotemporally controlled release of drugs from carriers, such

14,16

glutathione concentration, external stimuli, such as ultra-

as micelles, liposomes, or microbubbles,

cally increase drug eﬃcacy.

or to synergisti-

However, the currently applied

US modes of operation generally rely on acoustic streaming,

local heating, sonophoresis as well as sonoporation, or the

1,9

17,18

sound (US) or light, are controlled by the operator to gate

or switch drug release. Yet, many of these systems are limited

by low resistance of the carriers to enzymatic attacks, restricted

superﬁcial applicability, low loading capacity, or systemic side

,10

eﬀects. Hence, it is highly desirable to develop technologies

that oﬀer an alternative control over drug dosing while

simultaneously providing high spatiotemporal control in

conjunction with a minimally invasive character.

Received: July 1, 2020

Within the emerging ﬁeld of photopharmacology, bioactive

compounds can be switched “on” and “oﬀ” with unmatched

spatiotemporal resolution, which is generally achieved by

XXXX American Chemical Society

J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society

Article

Scheme 1. Reaction Cascade of Disulﬁde Mechanophore and DA Proﬂuorophores as Well as Prodrugs Subjected to US

Treatment

Shear force is generated by collapsing cavitation bubbles, leading to scission of disulﬁdes within macromolecules but not within small molecules.

Small (drug) molecules employed in this work are furan-dansyl 1, commercial furosemide 2 intrinsically bearing a furan moiety, and commercial

doxorubicin postfunctionalized with a furyl unit 3.

formation of liquid microjets and hence lack the ability to

address functional molecular motifs selectively.

Polymer mechanochemistry exploits US to control molec-

Here, we report the design and synthesis of an

unprecedented mechanochemically responsive system capable

of successively releasing furan-containing small molecules by

US-induced selective scission of a disulﬁde mechanophore

embedded at the center of a polymer chain. For this, we

employed an immersion probe ultrasonic liquid processor at a

frequency of 20 kHz as a model setup. Upon disulﬁde scission,

thiyl radicals were generated, which in turn abstracted

hydrogen from water and yielded thiols and, presumably,

hydroxyl radicals. These, in turn, underwent a Michael-type

addition to DA adducts of furylated drugs and acetylenedi-

carboxylate, which ultimately induced a retro DA reaction,

ular transformations by rearranging or cleaving bonds at

9−21

predetermined breaking sites.

This is achieved by

applying mechanical force to a macromolecular framework

that transduces it to the mechanochemically labile bond of the

mechanophore. Mechanophore breakage can occur in bulk

material but also in solution via the collapse of US-induced

cavitation bubbles. While undesired tissue interactions by

acoustic heating, cavitation, radiation pressure, force and

torque, and acoustic streaming can still occur using US, it is

useful for biomedical applications, as it combines spatial and

temporal dosage with easy regulation of tissue penetration

depth by varying frequency and energy through exposure

43−45

releasing the corresponding small molecule (Scheme 1).

To outline the conceptual scope of this system, we investigated

the activation of three diﬀerent molecules: While furan-dansyl

1 as a “turn-on” ﬂuorescence probe allowed the analytical

time.

description of the release process, furosemide 2 as an

intrinsically furan-bearing drug and postfurylated doxorubicin

3 were investigated as pharmacologically active moieties.

The design of mechanophores has progressed considerably,

providing access to transformations, such as absorbance and

4−26

ﬂuorescence alterations,

cules,

the release of small mole-

7−33

or the activation of latent catalysts, through

RESULTS AND DISCUSSION

various forms of force application. Prominently, the disulﬁde

moiety was shown to be selectively cleavable by mechano-

chemically-induced homolytic bond scission, thus being

■

Our initial eﬀorts were focused on assessing whether thiol-

terminated polymer chains with mechanochemically promising

35−40

rendered an established mechanophore.

Although ﬁrst

molar mass actually can undergo Michael-type addition to

eﬀorts to converge clinically employed US using relevant high

the respective DA adducts, as this technology was up to today

43,45

power intensities, such as HIFU, with mechanochemistry

only used for the bimolecular reaction of small molecules.

have been carried out, the mechanochemically-induced

release of pharmacologically active small molecules remains

unexplored up to today.

For this purpose, we relied on the liberation of furan-dansyl 1

acting as a “turn-on” ﬂuorescent probe upon thiol-initiated

retro DA reaction, allowing the facile quantiﬁcation of released

J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Article

Figure 1. Retro DA reaction condition optimization with PEG-SH. (a) Reaction scheme and conditions. (b) UV−vis absorption spectra of 1 (100

μM) and DA1 (100 μM). (c) Fluorescence spectra and photograph (inset) of 1 (50 μM) and DA1 (50 μM). (d) Fluorescence spectra for various

PEG-SH concentrations reacting with DA1 (50 μM). The peak at 670 nm is a mode of λ_e_x_ccaused by the grating monochromator. (e) Fractional

release of 1. Mean values ± standard deviation, N = 3. Fluorescence spectra were recorded at λ_e_x_c= 335 nm with spectral bandwidths of 3 nm for

excitation and emission. All spectra were recorded in DMSO at room temperature.

Additionally, the release was monitored by ¹H NMR

spectroscopy cross validating the quantiﬁcation experiments

furan derivative (Figure 1a). Consequently, the DA adduct of

an acetylenedicarboxylate derivative and furan-dansyl DA1 was

synthesized (Scheme S1 ) and reacted with commercial

macromolecular thiol-terminated poly(ethylene glycol)

(

Figure S18 ). A four times excess of PEG-SH regarding DA1

was identiﬁed as an optimal stoichiometry yielding release of

5% of 1 and hence was employed in subsequent experiments.

Note that the reaction partners without PEG-SH, particularly

with regard to N,N-diisopropylethylamine (DIPEA), did not

induce any release from DA1 (Figures S19 and S20).

Moreover and with regard to cell compatibility, we investigated

the potential release in dependence of endogenous thiols, such

as those present in Dulbecco’s modiﬁed eagle medium

(

PEG-SH, M = 30 kDa). While the UV−vis absorption

spectra of 1 and DA1 were similar and hence did not allow

an analytical description of the release process (Figure 1b), a

clearly visible green ﬂuorescent band emerged upon transition

from DA1 to 1 (Figure 1c). This diﬀerence in ﬂuorescence

properties of both compounds enabled the determination of

the released amount of 1 from DA1, hence monitoring the

release of 1 at diﬀerent employed concentrations of PEG-SH

(

DMEM) and human serum albumin (HSA). While

subsequent cell experiments were carried out in DMEM that

showed no unspeciﬁc release, HSA at physiological concen-

trations could release 1 from DA1 (Figure S21). This

(

Figure 1d) and ultimately quantiﬁcation of released 1 (Figure

e).

J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Article

Figure 2. Ex situ and in situ retro DA reaction of DA1 (50 μM) with disulﬁde-centered POEGMEA (100 μM). (a) Reaction routes. (b) Ex situ

retro DA reaction over diﬀerent time points of POEGMEA sonication with regard to released 1 and M as determined by GPC. Mean values ±

standard deviation, N = 3. (c) Fluorescence intensity I_e_min relation to I_e_mat t = 0 during in situ sonicated POEGMEA with DA1, DA1 alone, and 1

alone. N = 2. Fluorescence spectra were recorded at λ_e_x_c= 335 nm with spectral bandwidths of 3 nm for excitation and emission. All experiments

were conducted in MeCN/H O = 2/3 (v/v) at room temperature.

constitutes a current limitation of our release system that is

going to be addressed by macromolecular shielding against the

high molar mass HSA in the future.

fore, DA1 was ﬁrst mixed with disulﬁde-centered POEGMEA

in solution and then sonicated for diﬀerent periods of time,

and subsequently, ﬂuorescence intensities of liberated 1 were

measured (Figure 2c, black line). While the successful release

is clearly visible, reference experiments subjecting DA1 as well

as 1 to ultrasonication independently revealed that (i) a

contribution of unspeciﬁc release of 1 from DA1 was observed

(Figure 2c, blue line) and (ii) the ﬂuorescence of 1 was

bleached over the course of sonication, most likely due to the

formation of hydroxyl radicals in the water−solvent mixture

(Figure 2c, red line). These two observations would yield

false-positive as well as false-negative interpretations of the

ﬂuorescence data and hence, while proving beyond doubt that

the mechanochemical in situ release of 1 from DA1 proceeds as

anticipated, quantiﬁcation is carried out best with the ex situ

approach.

Encouraged by these results, we turned toward the

mechanochemical activation of this process by employing a

disulﬁde-centered polymer, which we prepared by Cu -

mediated controlled radical polymerization starting from a

bifunctional initiator toward PEG-analogue poly(oligo-

(

ethylene glycol) methyl ether acrylate) (POEGMEA, M =

8.9 kDa, Đ_M= 1.09, Figure S17 ). The mechanochemically-

induced release was performed both in situ and ex situ, i.e., by

ultrasonication of the disulﬁde-centered POEGMEA before

addition to DA1 or by ultrasonication in the presence of DA1

(

Figure 2a).

The ex situ approach allowed us to avoid possible

nonspeciﬁc eﬀects caused by ultrasonication of the DA adducts

and was hence carried out initially. For this purpose, disulﬁde-

centered POEGMEA was sonicated for diﬀerent periods of

time and then mixed with DA1 and DIPEA, and the mixture

was left stirring at room temperature for 72 h. Hereafter, the

ﬂuorescence intensity was recorded yielding a released fraction

of 70% of 1 from DA1 after 1 h of sonication (Figure 2b).

To further explore the mechanochemically-induced release,

the experiments were subsequently performed in situ. There-

Having established the proof of concept of this release

mechanism using small probe molecule furan-dansyl 1, we

turned toward pharmacologically relevant drug molecules.

Initially, we investigated the release of furosemide 2 as a

representative example for intrinsically furan-bearing drugs.

Furosemide is a loop diuretic and mostly used in pediatric

patients to reduce edema in both acute and chronic

48,49

cardiovascular, pulmonary, and kidney diseases.

Although

J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Article

Figure 3. In situ US-induced retro DA reaction of DA2 in conjunction with disulﬁde-centered POEGMEA. DA2 (50 μM) was mixed with 100 μM

POEGMEA and 50 μM DIPEA and sonicated in MeCN/H O = 2/3 (v/v) for 75 min. The reaction mixture was then dried, and H NMR spectra

were measured directly or the mixture was dialyzed for UPLC−MS measurements, (a) Reaction route. (b) Excerpt from H NMR spectrum (600

MHz, DMSO-d ). Color-coded signals as indicated in a. (c) UPLC diode-array detector elugram at λ = 335 nm. (d) ESI mass spectrum at t =

−

abs

.24 min. M₂_,_c_a_l_c= 330.0 Da; M₂_,_m_e_a_s= 329.0 Da.

furosemide is one of the most frequently prescribed

medications in the United States, high-dose therapy may

produce excessive diuresis, resulting in hypotension and

dehydration and placing susceptible patients at increased risk

analysis of the remaining solid clearly indicated the emergence

of the characteristic furyl protons of 2analogously to the

GSH- and PEG-SH-induced retro DA reaction (Figure 3b).

This analysis was complemented by ultraperformance liquid

chromatography−mass spectrometry (UPLC−MS) measure-

ments, which clearly revealed the presence of free furosemide

2, both regarding retention time (Figure 3c) and measured

mass (Figure 3d) and thus highlighting the successful

mechanochemically induced release of 2.

for vascular clot formation. Hence, exerting remote control

over furosemide release and therefore activity is desirable.

Initially, the DA adduct of furosemide 2 and an

acetylenedicarboxylate derivative DA2 was synthesized as

described in the Supporting Information (Scheme S1). As

the furyl moiety is involved in the metabolization of

furosemide, formation of a DA adduct at this site was

Following up on the successful release of furan-containing

drugs, we then investigated whether the compound scope

could be expanded beyond this comparatively minor class of

molecules by relying on functionalization of other drugs with a

furan unit. For this purpose, doxorubicin (Dox) as the

representative drug (Scheme S1 and Figure 4) was chosen.

Dox is one of the most frequently used chemotherapeutic

drugs to treat acute leukemia, malignant lymphomas, and solid

50,51

identiﬁed as a plausible method to diminish its activity.

then verify the ability of DA2 to undergo the thiol-induced

retro DA reaction toward free 2, we employed thiol-containing

small molecule glutathione (GSH) and PEG-SH in con-

junction with DA2. Due to absence of ﬂuorescence, the release

of 2 was monitored by H NMR spectroscopy focusing on the

indicative up ﬁ eld region of the generated aromatic furyl

tumors. However, it exhibits disadvantages, such as a short

protons (Figures S22 and S23). The furyl protons in 3-, 4-, and

half-life as well as systematic and speciﬁc cardiac toxicity,

-positions were clearly identiﬁed, highlighting the successful

impeding its application. Consequently, as Dox would beneﬁt

from a remote control over its release and therefore activity, we

employed the mechanochemical ex situ release method

established earlier to liberate 3 from DA3, which we

release of 2 from DA2 by GSH and PEG-SH. Subsequently,

the mechanochemical activation by ultrasonication following

the in situ approach described above was carried out.

Therefore, disulﬁde-centered POEGMEA was mixed with

DA2 and DIPEA, subsequently subjected to US for 75 min,

and then stirred at room temperature for 72 h (Figure 3a).

synthesized according to a protocol reported previously.

Ultrasonication of the disulﬁde-centered POEGMEA for 90

min and then mixing with DIPEA and DA3 (Figure 4a)

yielded, after drying, a solid residue that was again subjected to

Hereafter, the solvent was removed in vacuo and H NMR

J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Article

Figure 4. Release of Dox from DA3 employing disulﬁde-centered POEGMEA. (a) Reaction route of Dox release. (b) UPLC total ion current

−

(

TIC) elugram at 667 Da. (c) ESI mass spectrum at t = 6.75 min. M₃_,_c_a_l_c= 667.2 Da; M₃_,_m_e_a_s= 666.2 Da. (d) MTS proliferation assay of Dox,

DA3 , 3 , POEGMEA, and ex situ sonicated (90 min) DA3/POEGMEA with HeLa cells. Mean values ± standard deviations, N ≥ 3, data compiled

in Table S1. To release 3 from DA3, 100 μM POEGMEA was sonicated in MeCN/H O = 2/3 (v/v) for 90 min. Then, 50 μM DIPEA and 50 μM

DA3 were added, and the mixture stirred for 3 d. To release Dox from 3, sonicated samples were dried, dissolved in DMSO/PBS buﬀer at pH 5,

and stirred for 3 d.

UPLC−MS, conﬁrming the release of 3 (Figure 4b,c). Most

notably, dissolving 3 under tumor-like conditions in a mixture

of DMSO and PBS buﬀer at pH 5 yielded free Dox after 3 d at

of the small molecular precursors DA3 and 3 (Figure 4d, green

line). Importantly, DA3 together with disulﬁde-centered

POEGMEA without ultrasound treatment kept for 3 d at pH

5 did not notably decrease the IC50, demonstrating once again

the stability and inertness of the DA adduct against hydrolysis

(Figure 4d, cyan line). These results highlight the successful

mechanochemically-induced proof of principle of drug

activation.

room temperature due to defurylation, as reported earlier.

Analogously to above, free Dox was detected by UPLC−MS

measurements (Figure S24 ). Importantly, adduct DA3 is not

31,44

hydrolyzed to Dox under identical conditions,

highlighting

the potential stability and inertness of DA3 under these

conditions (Figure S25 ).

Hereafter, we applied our system in vitro and investigated

the half maximal inhibitory concentration (IC ) of Dox, DA3,

CONCLUSION

■

We here reported an unprecedented system to mechanochemi-

cally induce the release and activation of ﬂuorophores and

drugs with US. Therefore, we employed a disulﬁde-centered

polymer in conjunction with DA adducts of furylated small

molecules and acetylenedicarboxylate derivatives. Selective

homolytic disulﬁde scission generated free thiols, which, in

turn, reacted in a Michael-type addition with the DA adducts,

thus inducing the retro DA reaction and subsequent liberation

of the drug. Importantly, we showed that our system is suitable

to release both drugs containing furan as part of the

pharmacophore as well as drugs that were functionalized

with an additional furan unit, demonstrating the broad

versatility of our approach. This ﬁrst example of a

mechanochemically released drug molecule is the stepping

stone toward a new research area that will deal with the precise

mechanochemical activation of pharmacologically active

compounds. In the future, multiple currently challenging

aspects have to be addressed: (i) Regarding the employed US

3, disulﬁde-centered POEGMEA, DA3 together with disulﬁde-

centered POEGMEA, and DA3 together with disulﬁde-

centered POEGMEA after ultrasonication for 90 min (Figure

d and Table S1 ). Initially, it was visible that both furylation of

Dox to 3 and DA reaction of 3 toward DA3 led to an increased

^I^C⁵0 of around 1 order of magnitude (Figure 4d, blue and red

lines), highlighting the potential use of these compounds as

prodrugs. We then subjected DA3 and POEGMEA with

DIPEA to sonication via the ex situ approach, which we

estimated would lead to an approximate release of ca. 70% 3

from DA3 according to our calibration using DA1 (Figure 2b).

Hereafter, the solution was stirred at room temperature for 3 d

31,44

at pH 5 at tumor-like conditions to release Dox from 3.

After the solution was freeze-dried and dissolved in cell

culture medium, it was applied to HeLa cells for the MTS

proliferation assay. It was observed that the IC₅₀of the

sonicated mixture was considerably lower compared to those

J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society

Article

technology, the required energy doses and the compatibiliza-

tion with clinically established US techniques as well as

of the analytical investigations were performed at the Center

for Chemical Polymer Technology CPT, which was supported

by the European Commission and the federal state of North

Rhine-Westphalia (No. 300088302). Financial support is

acknowledged from the European Commission (EUSMI, No.

biomimetic ﬂows will be investigated. (ii) Moreover, the

mechanochemical activity of the polymers, expressed through

mechanochemical scission rates and yields, has to be

converged with medically tolerable ultrasonication timeframes,

avoiding US-speciﬁc side eﬀects. (iii) The pharmacological

applicability of our developed method will be improved by

covalent incorporation of latent DA3 into a disulﬁde-bearing

polymer structure to prevent HSA access and allow for the

intramolecular activation of 3. Alongside, this will support

eﬀorts to increase drug deactivation eﬃciency.

731019).

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sı Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/jacs.0c07077.

■

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stl − DWI − Leibniz Institute for Interactive

Andreas Herrmann − DWI − Leibniz Institute for Interactive

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