Glutathione-responsive nanoscale MOFs for effective intracellular delivery of the anticancer drug 6-mercaptopurine

Article abstract of DOI:10.1039/d0cc02872j

A glutathione-triggered drug delivery system (DDS) based on nanoscale metal-organic frameworks (NMOFs) is developed, which features an intracellular redox-responsive release of an anticancer drug. Compared to normal cells, the current NMOF-based DDS has 3-fold higher cytotoxicity to cancer cells, prefiguring its great potential for selective cancer therapy.

Full text of DOI:10.1039/d0cc02872j

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Glutathione-responsive nanoscale MOFs for effective intracellular
delivery of anticancer drug of 6-mercaptopurine
Ming Gong,a Jian Yang, a Yongsheng Li a and Jinlou Gu*a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
A glutathione-triggered drug delivery system (DDS) based on premature drug release in normal tissues could be effectively
nanoscale metal-organic framework (NMOF) is developed, which avoided using GSH-responsive NMOFs. We suppose this suite
features an intracellular redox-responsive release of anticancer of characteristics would render the reducible NMOFs valuable
drug. Compared to normal cells, current NMOF-based DDS for tumor-specific drug delivery.
performs a 3-fold higher cytotoxicity to cancer cells, prefiguring its
great potentials for selective cancer therapy.
In this work, a thiol-functionalized NMOF, UiO-66-(SH)2 is
employed as a new type of redox-sensitive DDS, which is
composed of zirconium ion (Zr4+, metal node), 2,5-
disulfanylterephthalic acid (BDC-(SH)2, organic ligand) and
benzoic acid (BA, modulator). During the MOFs synthesis, BA
would compete with BDC-(SH)2 to coordinate on Zr6 clusters,
which effectively controls the crystal growth of NMOFs and
leads to the formation of small particle size. Meanwhile, the
internal pore volume could be increased after the removal of
the BA modulator. In this case, a thiol-containing anticancer
drug, 6-mercaptopurine (6-MP), could be stably loaded into
the NMOF via the formation of disulfide bonds between the
thiol groups on BDC-(SH)2 and 6-MP (Scheme 1). This smart
NMOF-based DDS has several advantages: (1) the small
particle size could be elaborated through the modulation of
the BA contents, which provides the rapid and effective
internalization for tumor cells. (2) The hierarchical pores inside
the NMOF could be produced after the removal of BA, offering
sufficient space for the diffusion of drug and GSH. (3) The
disulfide bonds inside the NMOFs could only be cleaved in the
presence of GSH, resulting in a GSH-responsive release of 6-
MP. As expected, the drug-loaded NMOF showed an
interesting intracellular redox-responsive drug release
capability. A 3-fold higher cytotoxicity towards cancer cells
Since cancer has become a serious worldwide threat to human
health, various drug delivery systems (DDSs) towards cancer
treatment have been explored.1 Although the chemotherapy
based on traditional DDSs is serving as a principal method in
clinical therapy, its undesirable side effects remain a major
problem due to the premature drug release.2 To overcome this
limitation and improve the therapeutic efficacy, advanced
DDSs with stimulus-response are gradually designed, which
could release the encapsulated drugs upon certain exogenous
(i.e., light and temperature) or endogenous (i.e., pH and
glutathione (GSH)) triggers.3 To date, several carriers such as
metals and quantum dots have joined into the class of
advanced DDSs to meet the requirements of specific drug
delivery.4 However, the drawbacks of low loading capabilities
significantly prevent their further application. Therefore, a
stimulus-responsive nanoplatform with high cargo capacity is
highly desirable for more efficient cancer therapy.
Thanks to the large pore volume, easy functionalization
and excellent biocompatibility, nanoscale metal-organic
frameworks (NMOFs) have attracted great interest in
biological fields.5 NMOFs-based advanced DDSs are emerging
as the superior candidates since they could achieve spatially
and temporally controllable drug release. Considering the
intrinsically reductive microenvironment inside solid tumors, in
which the GSH concentration (0.5-10 mM) is significantly
higher than extracellular matrices and normal cells,6 the
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than normal ones were revealed from the IC50 (half-maximal to remove the BA modulator coordinated on the Zr6 clusters,
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inhibitory concentration) values, which displays the excellent further increasing the volume of tDhOeI: 1p0o.1r0e39/Dch0aCnCn0e28ls7.21J2
tumor-selective chemotherapy. Such smart DDS would provide Obviously, two hierarchical pores of 1.5 and 2.7 nm (Fig. 2B
)
great convenience of MOFs for more biological applications. are created, which are larger than the dimension of the MP-SS-
To endow the MOF with redox-responsive property, the MP dimer (1.29 nm × 0.17 nm × 0.89 nm).13 As a result, the
thiol-containing organic ligand BDC-(SH)2 was prepared from drug molecule 6-MP could be effectively linked to the ligands
2,5-dihydroxyterephthalic acid diethyl ester (Fig. S1) and of NMOF. The surface area significantly decreases from 517 to
confirmed by 1H NMR measurement (Table S1).7 Then, UiO-66- 250 m2 g-1, indicating the partial occupation of 6-MP to the
(SH)2 was synthesized solvothermally. Powder XRD technique pore space. The retained high pore volume of 0.70 cm3 g-1
was utilized to track the structural evolvements under ensures the accessibility of 6-MP to the intracellular GSH,
different amounts of the modulator (Fig. 1A). All the as- which is beneficial to its redox-responsive drug release.
synthesized MOFs exhibit similar Bragg diffraction peaks
The successful loading of 6-MP was validated by FT-IR
present at 2θ = 7.3 and 8.4o in comparison to the simulated measurement (Fig. 2C). The presence of thiol groups on 6-MP
UiO-66 structure, which correspond to the octahedral and and UiO-66-(SH)2 were affirmed by the weak characteristic
tetrahedral cages in the frameworks of UiO-66.8 As the feed peaks at the wavenumber of 2570 cm-1.14 After the reaction of
ratio of BA/BDC-(SH)2 increases from 40 : 1 to 70 : 1, the peak 6-MP with NMOF, this peak completely disappears. At the
intensity at 2θ = 7.3o shows a 17-fold enhancement as shown same time, a new characteristic peak appears in the spectrum
in the amplified XRD spectra, which indicates the increased of UiO-66-SS-MP at the wavenumber of 1336 cm-1,
crystallinity of the synthesized MOFs. Furthermore, since the corresponding to the C-N stretching vibration on 6-MP.15
full-width at half maximum (FWHM) is inversely proportional These results indicate that the drug has been successfully
to the crystallite size according to the Scherrer equation,9 the grafted in the frameworks of UiO-66-(SH)2 through the
gradual growth of the particle diameter could be inferred from formation of a disulfide bond between the thiol groups on the
the reduced FWHM listed in Table S2. Among all, the uniform drug and the ligand of NMOF. Furthermore, the zeta potential
MOF particles with the small size of 40 nm are visible from the of the drug-loaded NMOF increases from -47.2 to -30.4 mV
SEM images (Fig. 1B) when the feed ratio of BA/BDC-(SH)2
(Fig. 2D), which is explained by the consumption of thiol
reaches 50 : 1. Such monodispersed nanoparticles are fully groups.10
instrumental in rapid cellular endocytosis. However, both
To explore the interaction of 6-MP and the framework, the
inadequate and excess dosages of BA would yield negative unfunctionalized and defective UiO-66 was synthesized (Fig.
impacts on the morphology evolvement such as severe S2) as a control group. After the same HCl-activated process,
aggregation and inhomogeneous diameter. Therefore, the the pore with diameter of 2.6 nm was created (Fig. S3) which is
optimal feed ratio of BA/BDC-(SH)2 is chosen as 50 : 1 to large enough for the drug loading. Afterwards, the drug
balance the good crystallinity and small size.
loading efficiency of the NMOFs were measured to be 0.8 mg
The drug loading process was conducted by
a
g-1 for UiO-66 and 35 mg g-1 for UiO-66-(SH)2 based on the
mercaptopurine oxidation and a disulfide bond exchange.10 6- standard curve (Fig. S4).16 Such negligible drug loading in
MP was firstly oxidized to a dimer form as MP-SS-MP (1,2- unfunctionalized of UiO-66 demonstrates the weak adsorption
di(purin-6-yl)disufane), which would further react with the
thiol groups on the ligand of UiO-66-(SH)2. Thus, the adequate
porosity of the NMOF is of great importance. The typical type I
isotherm shows the presence of massive micropores in the
UiO-66-(SH)2 framework (Fig. 2A).11 However, this narrow pore
width (< 1 nm) strongly prohibits the inner loading of the drug
molecules. Therefore, a HCl-activating procedure is introduced
2 | J. Name., 2012, 00, 1-3
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capability of NMOFs towards 6-MP or its dimer MP-SS-MP.
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Considering the more acid milieu in cancer cells than
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Therefore, the formation of the disulfide bond plays a key role normal cells, the 6-MP release in differeDnOt Ip: 1H0.b10u3f9fe/Dr0sCoClu02ti8o7n2Js
in the drug loading process. The drug-loaded UiO-66-SS-MP was also investigated (Fig. 3C). Upon the addition of the same
remain its structural integrity, small particle size and excellent GSH concentration of 5 mM, the similar release behaviors are
stability (Fig. S5 and S6), which are the prerequisite for NMOF found in the first 4 h. However, compared to the maximum
in biomedical applications.
release of 6-MP at pH = 5.5 (90%), the value at pH = 7.4 is
Developing the redox-triggered DDS is meaningful since the obviously lower (72%). This might be caused by the diverse
GSH concentration varies significantly between the reducing activity of GSH under different pH values.18 When the
intracellular milieus of normal and cancer cells. As illustrated in pH value increases from 5.5 to 7.4, the reduced form of GSH
Fig. 3A, the anticancer drug 6-MP is pre-installed in the pore of (G-SH) trends to be converted to the deprotonated form (GS-),
UiO-66-SS-MP via the disulfide bond. When GSH appears in the which owns higher reductive activity. The released 6-MP could
system, it would replace the encapsulated 6-MP through the react to GS- in the neutral solution easily, resulting in the GS-
formation of a new disulfide bond between the thiol groups on MP compound. On the other hand, the dimer form of GS- itself
GSH molecule and the ligand of NMOF, simultaneously (GSSG) would also be generated, due to the relatively higher
releasing 6-MP.17 The drug release profiles could be obtained concentration of GS- in the solution. The large consumption of
through the standard curves in different releasing GSH in the neutral environment decreases the probability of
environments(Fig. S7-S9), thanks to the separated UV-vis the reaction to the encapsulated 6-MP, resulting in a reduced
spectra peaks of GSH and 6-MP (Fig. S10). Additionally, drug release that this work (Fig. 3C, curve iii) has shown. At
although there is a small amount of spectra overlap between the same time, the drug release from UiO-66-MP was also
the ligand and drug (Fig. S11), the high stability of the NMOFs measured. No obvious absorbance at 324 nm assigned to the
could avoid the leakage of ligand (Fig. S12-S14) and ensure the 6-MP (Fig. S15) further rules out the possibility of the
reliability of the drug release profiles. To measure the redox- adsorption-based drug loading in thiol-functionalized DDS.
responsiveness, the drug release profiles were tested in pH =
To demonstrate the biocompatibility of UiO-66-(SH)2, both
5.5 MES buffer solution in the presence of different NIH/3T3 normal cells and SMMC-7721 cancer cells were
concentrations of GSH (Fig. 3B). A rapid release could be found treated with different concentrations of NMOF. As shown in
in the first 4 h, which is mainly attributed to the cleavage of Fig. S16, the high cell viabilities (> 90%) verify the good
the surface disulfide bonds. The cumulative release of 6-MP biosafety of the DDS for stimulus-responsive drug delivery.
reaches 90% after 5 d of incubation with 5 mM GSH, but only
Due to the inappreciable luminescence of UiO-66-SS-MP
64% of 6-MP releases with 1 mM GSH. Furthermore, the NPs, FMN (riboflavin sodium phosphate) was selected as a
release profile in the absence of GSH was also determined and fluorescent molecule which can be stably anchored onto the
a negligible release amount of 2.7% is finally found. Such a surface of NMOF through a strong Zr-O-P bond as our previous
significant difference in the releasing behavior demonstrates work shown.19 A strong fluorescent peak at the wavelength of
an excellent GSH-responsive ability of UiO-66-SS-MP.
537 nm could be emitted from FMN@UiO-66-SS-MP under the
excitation of 405 nm. After its soaking in water for 24 h, only
negligible fluorescent change could be monitored (Fig. S17),
demonstrating the excellent optical stability of the FMN-
labelled NMOFs. Then, the flow cytometry was conducted to
evaluate the endocytosis behavior of FMN@UiO-66-SS-MP by
SMMC-7721 cancer cells. The significant increase of the FMN
fluorescence in the first 2 h (Fig. S18) reflects the rapid
internalization of such smart DDS thanks to the small particle
size. Through a further comparison of the mean fluorescent
intensity (MFI) values (Fig. S19), a time-dependent uptake
property could be revealed from the much higher fluorescent
intensity (ca. 5400) with the incubation time of 24 h than the
value with the time of 2 h (ca. 3300).
A similar phenomenon could be also visible from laser
scanning confocal microscopy (LSCM) as shown in Fig. 4A. The
strong blue fluorescence is emitted from the DAPI-stained
nucleus, and the green fluorescence is detected from the
NMOF regions in cancer cells. With the prolonging of the
incubation time from 2 to 24 h, the green fluorescence is
noticeably enhanced (Fig. 4A, iii and vii). Such a long-time
accumulation of nanocarriers offers enough opportunity for
effective contact between GSH and NMOF as well as the
subsequent release of 6-MP in cancer cells.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
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Key Research and Development Program (2016VYieFwCA1r1tic0le2O1n0li0ne)
DOI: 10.1039/D0CC02872J
and 111 Project (B14018).
Conflicts of interest
There are no conflicts to declare.
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In summary, a GSH-responsive drug-delivery platform
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This work was financially supported by the Natural Science
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DOI: 10.1039/D0CC02872J
Glutathione-responsive nanoscale MOFs for effective
intracellular delivery of anticancer drug of 6-mercaptopurine
Ming Gong a, Jian Yang a, Yongsheng Li a and Jinlou Gu*a
A glutathione-responsive drug-delivery platform based on nanoMOF was developed
for the selective cancer therapy through the introduction of disulfide bonds.