Reversible Covalent Assembly of Nanoparticles through On-Surface Diels-Alder Reaction

Article abstract of DOI:10.1021/acs.langmuir.9b02261

We demonstrate here a controlled assembly of individual nanoscale building blocks into defined architectures based on chemospecific covalent bonding interactions. For this purpose, α-Fe₂O₃, γ-Fe₂O₃, and SiO₂ nanoparticles decorated with surface-conjugated organic ligands were used for performing on-surface Diels-Alder reactions. Driven through their chemical affinity and surface-grafted complementary functionalities, nanoparticles underwent click-reactions to produce covalently organized nanostructures. An advantage of using the Diels-Alder reaction is its reversible nature, which was used to click and unclick the nanoparticles on demand. The efficiency and chemical specificity of this approach opens up another synthetic access to unify materials with complementary properties, where the thermoresponsive nature of particle assemblies imparts to them a fully reversible character. The covalent conjugation strategies demonstrated in this work potentially allow the use of a diverse range of particles and ligands for their applications in different disciplines such as medicine, optics, or photonics. The nanoparticles morphology and crystalline nature were investigated by TEM and XRD analysis, while the presence of surface attached groups was verified by NMR, FTIR, UV-vis, and ζ potential measurements.

Full text of DOI:10.1021/acs.langmuir.9b02261

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Interface-Rich Materials and Assemblies
Reversible Covalent Assembly of Nanoparticles
Through On-Surface Diels-Alder Reaction
Markus Schütz, Khan Lê, Shaista Ilyas, and Sanjay Mathur
Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.9b02261 • Publication Date (Web): 22 Jan 2020
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Reversible Covalent Assembly of Nanoparticles Through
On-Surface Diels-Alder Reaction
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Markus B. Schütz,[a] Khan Lê,[a] Shaista Ilyas,[a] and Sanjay Mathur*[a]
[a]Institute of Inorganic Chemistry, University of Cologne, Greinstraße 6, 50939 Cologne, Germany
Correspondence*: E-mail: sanjay.mathur@uni-koeln.de
Abstract
We demonstrate here a controlled assembly of individual nanoscale building blocks into
defined architectures based on chemospecific covalent bonding interactions. For this
purpose, α-Fe2O3, -Fe2O3 and SiO2 nanoparticles decorated with surface-conjugated
organic ligands were used for performing on-surface Diels-Alderreactions. Driven through
their chemical affinity and surface-grafted complementary functionalities, nanoparticles
underwent click-reactions to produce covalently organized nanostructures. An advantage
of using the Diels-Alder reaction is its reversible nature, which was used to click and un-
click the nanoparticles on demand. The efficiency and chemical specificity of this
approach opens up another synthetic access to unify materials with complementary
properties, where the thermoresponsive nature of particle assemblies imparts them a fully
reversible character. The covalent conjugation strategies demonstrated in this work
potentially allow use of a diverse range of particles and ligands for their applications in
different disciplines such as medicine, optics or photonics. The nanoparticles morphology
and crystalline nature was investigated by TEM and XRD analysis, while the presence of
surface attached groups was verified by NMR, FTIR, UV-vis and ζ-Potential
measurements.
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Keywords: iron oxide • silica • Diels-Alder • nanoassembly, maleimide, propionic acid
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Introduction
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Unifying two or more functionalities such as optical, magnetic and catalytic properties at
nanoscale show unique leveraging effects, however controlled synthesis of biphasic
materials against random mixing of constituent phases remains an overarching challenge
of nanochemistry.1–4 New robust synthetic strategies for covalent functionalization of bare
nanoparticles (NPs) with surface-grafted linker molecules having addressable target units
that can be activated at later processing stages to immobilize functional molecules or
secondary nanoparticles are desired to create nano-assemblies.5–8 Stoichiometrically
defined metal and metal oxide nanostructures owe strategic functions in several medical
and biotechnological applications as diagnostic, therapeutic9 or contrast agents10 and are
therefore useful in early diagnosis and management of diseases.11–13
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Combination of different nanomaterials to obtain unique properties has been achieved in
core-shell-NPs14, Janus type NPs15 or nanocrystal heterojunctions,16 however most of
such fabrication strategies are non-reversible due to surface charges and chemical
heterogeneities of the surface. In this context, new link chemistries enabling controlled
self-assembly and dis-assembly of NPs hold promises for the amalgamation of
complementary properties in individually defined ensemble of nanoparticles.17–19 In
covalent assembly of nanoparticles, the efficiency and specificity of “click-chemistry” is
utilized to obtain preconceived particle-particle interactions and arrangements that are
amendable for implementing orthogonal chemistry for various applications.20,21
Reversible covalent assembly and disassembly of nanoparticles, linked for example by
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imines,22 spiroborates,23 boronate esters,24 and [2+2]photocycloaddition/ cycloreversion
reactions25 were already shown in literature.
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We demonstrate here an efficient and reliable approach to build nanoassemblies by direct
conjugation of two different nanoparticles bearing click-compatible surface groups, for
instance, to trigger a Diels-Alder reaction between a conjugated diene and a substituted
alkene.26 Gheneim et al. reported thermoresponsive polymers with maleimido- and furyl-
derivatives that allowed relatively low temperatures for forward and backward reactions.
The facile reversibility of the conjugation chemistry (Retro-Diels-Alder reaction) at
appropriate temperatures is another appealing feature of the reaction to shuttle
therapeutics or other biomolecules for in vivo applications.27,28 Herein, we report the
application of Retro-Diels-Alder for covalently conjugated nanoparticle assemblies
through furyl- and maleimido-functionalized nanoparticles. A quantitative disassembly
was shown by conducting Retro Diels-Alder reaction. The assembly and disassembly of
the particles could be extended to biomedical applications by conjugating fluorescent
molecules (imaging) and targeted drug-delivery (therapy) currently desired for cancer
treatment.
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Experimental
Details on the instrumentation are given in the SI.
1- Chemicals and solvents
All reagents were of analytical grade and used without further purification. Ethylenglycol
(EG, C2H6O2), Heptane (C7H14), Sodium hydroxide (NaOH), N,N-Dimethylformamide
(DMF, C3H7NO), Sodium dodecylsulfate (SDS, C12H25NaO4S) were obtained from Fisher
Chemical. Ethanol (EtOH), Sodium citrate (Na-Citrate, C6H5Na3O7), Iron(III)chloride
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(FeCl3 ∙ 6 H2O), Sodium ascorbate (NaAs, C6H7NaO6), Ammonium hydroxide solution
(28% NH3 in H2O), Dimethyl sulfoxide-d6 ((CD3)2SO), Magnesium sulfate (MgSO4) and
Tetraethyl orthosilicate (TEOS, C8H20O4Si) were purchased from Sigma Aldrich. Sodium
acetate (NaAc, C2H3ONa ∙ 3 H2O), Iron(II)-chloride (FeCl2 ∙ 4 H2O), Iron(III)-nitrate
(Fe(NO)3 ∙ 9 H2O), 3-(2-Furyl)propionic acid (C7H8O3), Polyvinylpyrrolidone average mol
wt 40,000 (PVP, (C6H9NO)n), Poly(ethylene glycol) average Mn 6,000 (PVP,
H(OCH2CH2)nOH), 6-Maleimidohexanoic acid (C10H13NO4) and 11-Maleimidundecanoic
acid (C15H23NO4) were obtained from Alfa Aesar.
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2- Synthesis of nanoparticles
i- Synthesis of -Fe2O3 nanocubes
1.156 g Fe(NO)3 . 9 H2O were dissolved in 24 mL DMF and 2.512 g PVP were added.
The mixture was transferred into Teflon tubes and autoclaved at 180°C for 30 h. The
particles were washed seven times by centrifugation at 11.000 rpm for 30 min and
redispersion in H2O/EtOH. The α-Fe2O3 nanocubes were analysed by XRD, IR, DLS/Zeta
measurements and images were recorded with SEM/TEM. The particles were used for
surface modification with 6-maleimidohexanoic acid and 11-maleimidoundecanoic for the
Diels-Alder cycloaddition as well as Retro-Diels-Alder reaction.
ii- Synthesis of -Fe2O3 nanospheres
0.811 g FeCl3 ∙ 6 H2O was mixed with 2.641 NaAc ∙ 3 H2O, 1.154 g SDS and 0.4 g PEG
6000 in 24 mL Ethylenglycol. The mixture was transferred into a Teflon tube and
autoclaved at 180°C for 6 h. The particles were washed several times by centrifugation
at 11.000 rpm for 30 min and redispersion in H2O/EtOH. The -Fe2O3 nanospheres were
analysed by XRD, IR, DLS/Zeta measurements and images were recorded with
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SEM/TEM. The particles were used for surface modification with 3-(2-furyl)propionic acid
and subsequent nanoassembly by Diels-Alder cycloaddition and Retro-Diels-Alder
reaction.
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iii- Synthesis of SiO2 nanospheres – Stöber Method
180 mL EtOH, 65 mL H2O and 45 mL aqueous 28% NH3 solution were mixed in a 1 L
Erlenmeyer flask. 15 mL TEOS were slowly added with a dropping funnel over 1.5 h. The
suspension was centrifuged at 11.000 rpm for 10 min and redispersed in H2O/EtOH five
times. The SiO2 nanospheres were analysed by XRD, IR, DLS/Zeta measurements and
images were recorded with SEM/TEM. The particles were used for surface modification
with 3-(2-furyl)propionic acid and subsequent nanoassembly by Diels-Alder cycloaddition
as well as Retro-Diels-Alder reaction.
3- Synthesis of organic Ligands
i- Synthesis of Diels-Alder product 11-(4-(2-carboxyethyl)-1,3-dioxo-3a,4,7,7a-
tetrahydro-1H-4,7-epoxyisoindol-2(3H)-yl)undecanoic acid
200 mg
11-maleimidoundecanoic
acid
(0.71 mmol, 1 Eq.)
and
100 mg
3-(2-furyl)propionic acid (0.71 mmol, 1 Eq.) were dissolved in 5 mL H2O. The solution was
stirred for 48 h at 60°C. The white solid product was filtered, washed with 3 x 5 mL H2O
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and dried. The Diels-Alder adduct was analysed by H-NMR. It was used for the Retro-
Diels-Alder reaction in order to prove the reversible formation of a covalent bond.
ii- Retro-Diels-Alder Reaction of 11-(4-(2-carboxyethyl)-1,3-dioxo-3a,4,7,7a-
tetrahydro-1H-4,7-epoxyisoindol-2(3H)-yl)undecanoic acid
200 mg of 11-(4-(2-carboxyethyl)-1,3-dioxo-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindol-
2(3H)-yl)undecanoic acid (0.47 mmol) were dissolved in 2 mL DMSO-d6. The solution
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was stirred at 130°C for 24 h, cooled in an ice bath and H-NMR measurements were
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done immediately. The obtained solution was analysed by 1H-NMR in order to prove the
reversibility of the Diels-Alder cycloaddition.
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4- Surface conjugation chemistry
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i- 11-maleimidoundecanoic acid@-Fe2O3 nanocubes
10 mg of -Fe2O3 nanocubes were dispersed in 5 mL heptane and 100 mg of
11-maleimidoundecanoic acid were added. The mixture was kept in an ultrasonic bath for
30 min and then centrifuged 5 times at 11.000 rpm for 30 min, redispersing the particles
in H2O/EtOH after each centrifugation. The surface modified particles were analysed by
XRD, DLS/Zeta and IR spectroscopy. They were used for nanoassembly by Diels-Alder
cycloaddition with furyl-functionalized NPs.
ii- 3-(2-furyl)propionic acid@-Fe2O3 nanospheres
10 mg of -Fe2O3 nanospheres were dispersed in 5 mL heptane and 100 mg of
3-(2-furyl)propionic acid were added. The mixture was kept in an ultrasonic bath for
30 min and then centrifuged 5 times at 11.000 rpm for 30 min, redispersing the particles
in H2O/EtOH after each centrifugation. The surface modified particles were analysed by
DLS/Zeta measurements and IR spectroscopy. They were used for nanoassembly by
Diels-Alder cycloaddition with maleimido functionalized NPs.
iii- 3-(2-furyl)propionic acid@SiO2 nanospheres
10 mg of SiO2 nanospheres were dispersed in 5 mL H2O and 100 mg of
3-(2-furyl)propionic acid were added. The mixture was kept in an ultrasonic bath for
30 min and then centrifuged 5 times at 11.000 rpm for 30 min, redispersing the particles
in H2O/EtOH after each centrifugation. The surface modified particles were analysed by
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DLS/Zeta measurements and IR spectroscopy. They were used for nanoassembly by
Diels-Alder cycloaddition with maleimido functionalized NPs.
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5- Diels-Alder Cycloaddition Nanoassembly
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i- Assembly of -Fe2O3@SiO2
10 mg of 11-maleimidohexanoic acid@α-Fe2O3 nanocubes and 10 mg of
3-(2-furyl)propionic acid@SiO2 nanospheres were dispersed in 2 mL H2O each. The
mixture was stirred for 48 h at 60°C. The obtained assembly was washed by five cycles
of centrifugation at 5000 rpm for 5 min and re-dispersed in H2O/EtOH.
ii- Assembly of -Fe2O3@-Fe2O3 nanospheres
10 mg of 11-maleimidohexanoic acid@α-Fe2O3 nanocubes 10 mg of 3-(2-furyl)propionic
acid@-Fe2O3 nanospheres were dispersed in 2 mL H2O each. The mixture was stirred
for 48 h at 60°C. The obtained assembly was washed by five cycles of centrifugation at
5000 rpm for 5 min and re-dispersed in H2O/EtOH.
iii- Proof of covalent bond for -Fe2O3@ -Fe2O3 assembly
10 mg of the assembly obtained from 7 (ii) were dissolved in 5 mL of concentrated
hydrochloric acid and diluted with 30 mL water. The mixture was extracted with 3x10 mL
dichloromethane. The organic layers were combined and the solvent removed under
reduced pressure. The residue was analysed by 1H-NMR spectroscopy in DMSO-d6.
6- Retro-Diels-Alder Reaction Disassembly of particles
i- Separation of - Fe2O3@SiO2
10 mg of the α-Fe2O3@SiO2 assembly obtained from Diels-Alder cycloaddition (7.i) were
dispersed in 5 mL DMSO and stirred for 24 h at 130°C. The mixture was cooled with ice
and shortly after centrifuged at 5000 rpm for 5 min. The supernatant was transferred into
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another falcon tube and the pellet re-dispersed in H2O/EtOH. The supernatant as well as
the dispersed pellet were centrifuged again under the same conditions 4 more times. The
disassembled particles were analysed by TEM/SEM imaging.
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ii- Separation of -Fe2O3@-Fe2O3
10 mg of the -Fe2O3@-Fe2O3 assembly obtained from Diels-Alder cycloaddition (7.ii)
were dispersed in 5 mL DMSO and stirred for 24 h at 130°C. The mixture was cooled with
ice and shortly after centrifuged at 5000 rpm for 5 min. The supernatant was transferred
into another falcon tube and the pellet re-dispersed in H2O/EtOH. The supernatant as well
as the dispersed pellet were centrifuged again under the same conditions four more
times. The disassembled particles were analysed by TEM/SEM imaging.
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Results and Discussion:
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Scheme 1. Reaction scheme for the functionalization of different NPs with
11-maleimidoundecanoic acid and 3-(2-furyl) propionic acid and formation of the reversible
nanoassemblies via Diels-Alder reaction.
Scheme 1 shows the pathway of the chemical approach used for the preparation of
nanoassembly from different particles. First, the selected organic starting materials for
the Diels-Alder reaction were used to synthesize the organic ligand 11-(4-(2-
carboxyethyl)-1,3-dioxo-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindol-(3H)yl)undecanoic
acid for NMR analysis first and later conjugated on the surface of respective
nanoparticles, (Scheme 1). The formation of the ligand 11-(4-(2-carboxyethyl)-1,3-dioxo-
3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindol-2(3H)yl)undecanoic acid was clearly visible in
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the H NMR spectra in which the aromatic furyl derivative δ = 7.45 ppm, δ = 6.29 ppm
and δ = 6.05 ppm are no longer detectable and the signal at δ = 5.05 ppm is ascribable
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to the formation of tetrahydro epoxyisoindol ring, showing the successful implementation
of Diels-Alder reaction. The Retro-Diels-Alder reaction was performed at 130°C in DMSO
for 24 hours, followed by cooling in an ice bath to hinder the Diels-Alder reaction followed
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by the immediate analysis of the reaction mixture. The H NMR spectrum showed the
reoccurrence of signals for the aromatic furyl derivative at δ = 7.45 ppm, δ = 6.29 ppm
and δ = 6.05 ppm demonstrating the efficacy of the reverse reaction and their potential in
the synthesis of reversible assemblies of nanoparticles carrying complementary surface
chemistry. The efficiency of the click-reaction between both ligands was also conducted
without particles and characterized via NMR spectroscopy (Figure 1).
Figure 1. 1H NMR spectra: A) a mixture of the starting materials 3-(2-furyl)propionic acid
and 11-maleimidoundecanoic acid for the Diels-Alder reaction utilized in this work, B) the
product
11-(4-(2-carboxyethyl)-1,3-dioxo-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindol-
2(3H)yl)undecanoic acid obtained after heating the mixture for 48 hours to 60 °C and C)
the same sample after Retro-Diels-Alder reaction at 130 °C for 24 hours showing the
formation of 3-(2-furyl)propionic acid and 11-maleimidoundecanoic acid.
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In order to generate an assembly of particles, -Fe2O3, -Fe2O3 and SiO2 NPs were
synthesized via solvothermal decomposition of iron salts and tetraethyl orthosilicate,
respectively.29 Scanning and transmission electron microscope (SEM /TEM) analysis
indicated a uniform distribution of -Fe2O3 nanocubes with an average size of 40 ± 2 nm,
-Fe2O3 spheres, 250 ± 4 nm and monodispersed SiO2 spheres (300 ± 4 nm) were
obtained as evident in Figure 2 A-C.
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Figure 2: TEM image: A) -Fe2O3 nanocubes with an average diameter of 40 nm.
B) -Fe2O3 nanospheres with an average diameter of 250 nm. SEM image: C) SiO2
nanospheres with an average diameter of 400 nm.
The powder X-ray diffraction patterns recorded for as-synthesized -Fe2O3 and -Fe2O3
NPs showed the diffraction peaks corresponding to crystalline hematite (-Fe2O3, JCPDS
C39-1346) and maghemite (--Fe2O3, JCPDS C79-0007) phases, whereas silica particles
were amorphous (Figure 3). In Figure 3A, NPs peaks positions and orientations [012],
[104], [110], [113], [202], [024], [116], [018], [214] and [300] matched well with the
standard values of α-Fe2O3 nanocrystals.
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Figure 3: XRD pattern: A) -Fe2O3 nanocubes. B) -Fe2O3 nanospheres. C) SiO2
nanospheres.
In Figure 3B shows the presence of [111], [220], [311], [400], [511] and [440]
demonstrating the existence of -Fe2O3 NPs. After Diels-Alder reaction, the diffractogram
of -Fe2O3@SiO2 nanoassembly showed the existence of hematite phase, while the
existence of both hematite and maghemite phases in the -Fe2O3@-Fe2O3
Figure 4. XRD pattern: A) 300 nm SiO2 nanospheres, 40 nm -Fe2O3 nanocubes and
250 nm -Fe2O3 nanospheres and B) -Fe2O3@SiO2 nanoassembly obtained by Diels-
Alder reaction.
nanoassembly was verified by XRD data after purification of particles, involving iterations
of dispersion in EtOH/H2O and centrifugation at different speeds (1000-11000 rpm) in
order to separate the different sized particles. We did not observe the presence of any
impurity or other phase of particles after the formation of the assemblies. Nevertheless,
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the data does not demonstrate any changes in the crystallinity after the surface
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The characteristic Fe-O-Fe vibrational bands can be observed at 520-540 cm-1 (Figure 5,
A and B). In addition the Si-O vibrational bands are visible at 790-1100 cm-1(Figure 5, C).
These particles were surface grafted by organic ligands, 3-(2-furyl)propionic acid,
11-maleimidoundecanoic acid and 6-maleimidohexanoic acid. Additional FT-IR spectra
of pure organic compounds used for the study are presented in the supporting information
and show sharp peaks corresponding to relevant bonds existing in the compounds used.
(Figure SI1 and SI2). Diels-Alder reactions between maleimide and furyl-functionalized,
-Fe2O3 and SiO2 NPs, respectively enabled the formation of chemically conjugated
-Fe2O3@SiO2 assembly.
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The reversibility of as-synthesizedFe2O3@SiO2 nanoassemblies was validated by
Retro-Diels-Alder reaction that efficiently released the entangled nanoparticles. The FT-
IR spectra of surface-functionalized nanoparticles showed (Figure 5 A-C) the carbonyl
vibration bands of the ligands in all samples. In case of Furyl@-Fe2O3: the carbonyl
vibration bands can be seen at 1715 cm-1. The Furyl@SiO2 demonstrates the signals for
carbonyl at 1646 cm-1. Nevertheless Maleimido@-Fe2O3 indicated the availability of the
aforementioned bonds at 1692 cm-1). In case of the Maleimido@-Fe2O3 conjugate, the
-CH vibration frequencies are detected around 2984 cm-1.
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Figure 5. IR spectra for surface group analysis before and after functionalization:
A) Maleimide@-Fe2O3 nanocubes and unfunctionalized -Fe2O3 nanocubes B)
Furyl@γ-Fe2O3 nanospheres and unfunctionalized γ-Fe2O3 nanospheres C) Furyl@SiO2
nanospheres and unfunctionalized SiO2 nanospheres. UV-Vis spectra for surface
group analysis before and after functionalization D) Furyl@SiO2 nanospheres, 3-(2-
furyl) propionic acid and SiO2 nanospheres.
In addition, the furyl group on the surface of the SiO2 nanospheres was detected via UV-
Vis measurements (Figure 5 D). The signal of the 3-(2-furyl)propionic acid exhibited a
bathochromic shift (from 214 nm to 264 nm) upon its covalent conjugation to nanoparticles
surface due to transfer of electron density. The zeta potential measurements of
unfunctionalized (17.7 mV) and functionalized (-4.8 mV) NPs demonstrated the significant
change in the surface charge thereby supporting the success of conjugation reactions. In
case of the -Fe2O3 spheres the potential changed from 17.9 mV to -20.5 mV and for the
SiO2 NPs from 1.6 mV to -28.6 mV (Table 1). The average hydrodynamic radius was
measured by DLS in aqueous media. It showed an increased after functionalizing the
nanoparticles with the respective organic molecules. This can be due to the interaction of
organic molecules with water or possible hydrogen bonding forces of water molecules.The
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average values for the as-synthesized and functionalized nanoparticles are given in Table
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Table 1: DLS and -Potential values.
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Name of the sample
Hydrodynamic Radius [nm]
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--Potential [mV]
SiO2 nanospheres
405.20 ± 1.80
176.00 ±7.00
1.63 ± 0.24
17.70± 0.40
-Fe2O3 nanocubes
11-maleimidoundecanoic
870.00 ±8.00
411.00 ± 4.00
-4.80 ± 0.50
acid@-Fe2O3 nanocubes
3-(2-furyl)propionic
SiO2 nanospheres
acid@
-28.60 ± 0.30
Thermogravimetric analysis of bare and particles after functionalization were measured
under nitrogen atmosphere in the temperature range of 30–800 °C and with a 10 °C/min
heating rate and presented in Figure, SI5. The mass loss measured by TGA for
unfunctionalized SiO2 is 13% (Figure SI5, top), compared to a loss of 15% for 3-(2-
furyl)propionic acid@ SiO2 (Figure SI5, bottom).
The Diels-Alder reaction between maleimide and furyl-functionalized particles resulted in
uniform attachment of hematite cubes on silica spheres demonstrating a successful Diels-
Alder reaction. The TEM/SEM image of particles formed after the Diels-Alder reaction
(Figure 6 A-B) showed a defined arrangement of iron oxide nanocubes on -Fe2O3 or SiO2
nanospheres.
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Figure 6. SEM images: A) -Fe2O3@-Fe2O3, TEM image: B) -Fe2O3@SiO2 C) the
same sample disassembled by Retro-Diels-Alder reaction and purification by
centrifugation.
For a thermoresponsive dis-assembly, the Retro-Diels-Alder reaction was performed by
heating the particle conjugates in DMSO at 130°C. In order to prevent the reformation of
the-Fe2O3@SiO2 nanoassemblies, the particles were quickly transferred into a
centrifuge tube and quenched at 0°C. The fractional differentiation of disassembled NPs
was achieved by centrifugation at different rpm values. The TEM image taken after the
reversible reaction (Figure 6 C) showed that most of covalently attached nanocubes
residing on the spheres have been detached. A few -Fe2O3 still present on silica surface
is most likely due to electrostatic interaction or reformation of a covalent bond because of
the forward Diels-Alder reaction occurring at ambient temperature, since the TEM
analysis was performed at room temperature.(23) Overview images for the assembly and
disassembly of particles is shown in Figure SI6 which clearly demonstrate the occurrence
of Diels-Alder and Retro-Diels-Alder reaction on the surface of nanoparticles. The huge
number of particles were detached from the bigger nanospheres after the reaction
besides trivial attachments of smaller particles on surface.
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For an unambiguous verification of covalent bond formation between the two types of
nanoparticles and to rule out the significance of non-covalent interactions, the
nanoassembly was dissolved in hydrochloric acid and the liberated ligands were extracted
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and analysed by H-NMR to demonstrate the successful implementation of On-surface
Diels-Alder reaction (SI 3). The maleimide and furyl functionalized-NPs (Diels-Alder
reaction) and their thermosensitive detachment (Retro-Diels-Alder reaction) can be used to
accumulate complementary functionalities into single nano-particle. The outer surface
of both particles are available for further ligand conjugation thus being significant for dual-
mode imaging-guided combinational cancer early diagnosis and therapeutic applications.
The small size of nanoparticles and attachment of surface ligands with specific chemical
affinities, several strategies can be investigated to conjugate therapeutic units to selectively
use bioconjugated nanoparticles to target cells or tumor sites that hold promises in drug
delivery applications.
Conclusions
The Click-chemistry protocols are finding increasing applications in the synthesis of
bioconjugates containing therapeutic drugs with other biomolecules. The work reported
here demonstrates the potential of on-surface covalent chemistry on nanoparticles by
integrating clickable ligands with unique surface properties of nanoparticles. Appropriate
implementation of Click-reactions carries enormous potential of decorating nanoparticles
and their assemblies with orthogonal bio/chemical functionalities. The maleimide and furyl
functionalized NPs were successfully used in this work and showed covalent attachment
(Diels-Alder reaction) and their thermosensitive detachment (Retro-Diels-Alder reaction).
The outer surface of the particle assemblies should be available for further ligand
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conjugation which is significant for dual-mode imaging-guided combinational cancer early
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diagnosis and therapeutic applications.
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Acknowledgements
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The authors gratefully acknowledge the infrastructure and financial support provided by
the University of Cologne. Authors extend their thanks to Dr. Roitsch and Mrs. Tosun for
TEM and SEM measurements.
Supplementary information
Instrumentation, FTIR spectrum of pure organic compounds, unfunctionalized and
functionalization of -Fe2O3 nanocubes with 11-maleimidoundecanoic acid
6-Maleimidohexanoic). Unfunctionalized and functionalized -Fe2O3 nanospheres (3-(2-
furyl)propionic acid) while unfunctionalized SiO2 and silica particles after functionalization
with 3-(2-furyl)propionic acid. 1H-NMR of -Fe2O3@-Fe2O3 nanoassembly, mechanism
of the furan-maleimide reaction. TGA of SiO2 nanospheres with 3-(2-furyl)propionic acid
modification. SEM images after Retro-Diels-Alder reaction on SiO2 NPs and existing
examples of Diels-Alder reactions on nanoparticles and differences to current work. This
material is available free of charge via the Internet at http://pubs.acs.org.
Corresponding Author
*Correspondence to Sanjay Mathur:
sanjay.mathur@uni-koeln.de, Tel: +49 221 470 5627
Notes
The authors declare no competing financial interest.
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