Photodegradable coumarin-derived amphiphilic dendrons for DNA binding: Self-assembly and phototriggered disassembly in water and air-water interface

Article abstract of DOI:10.1016/j.colsurfb.2018.12.018

In this article, we demonstrate the self-assembly and photoresponive behavior of a novel coumarin-based amphiphilic dendron in both aqueous solution and air-water interface. The dendritic structure, namely C-IG₁, was composed of a lipophilic cholesterol and hydrophilic poly(amido amine) (PAMAM) dendron, and the amphiphilic counterpart is interconnected by a photolabile coumarin carbonate ester, enabling the photoinduced degradation of the amphiphiles in protic solvents via S_N1-like mechanism. A Nile red solubilization fluorescence assay suggests a low critical aggregation concentration for the micelle formation of C-IG₁ in aqueous solutions (3.9 × 10^?5 M); the Langmuir analysis further indicates that C-IG₁ possesses significant compressibility in air-water interface, eventually forming homogeneous monolayers with a final molecular area (A₀) of 36 ?². Notably, the micelles and Langmuir monolayer are quite stable until photo-triggered dissociation based on the photocleavage of C-IG₁ amphiphile activated by 365-nm incident light. Moreover, the transition in interfacial morphology of the Langmuir monolayer during the assembly and photodegradation processes also can be visually analyzed by incorporating Nile red probes with in situ monitoring through fluorescence microscopy. The thin film deposited on a glass substrate by the Langmuir-Blodgett technique also shows a photoresponsive behavior based on the change in the contact angles of a water droplet on the surface upon light stimulation. The binding affinity of C-IG₁ and cyclic DNA determined by the fluorescence quenching analysis of the coumarin reporter suggests a ground-state macromolecular complexation process occurring through polyvalent interactions between the pseudodendrimers and biomacromolecules. The ethidium bromide displacement assay further indicates thus dendriplex formation at low nitrogen-to-phosphorous value (N/P < 1) and confirms that the decomplexation accompanied by DNA release can be achieved through an active phototriggered route under spatiotemporal control.

Full text of DOI:10.1016/j.colsurfb.2018.12.018

ColloidsandSurfacesB:Biointerfaces175(2019)428–435
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces
journal homepage: www.elsevier.com/locate/colsurfb
Photodegradable coumarin-derived amphiphilic dendrons for DNA binding:
Self-assembly and phototriggered disassembly in water and air-water
interface
⁎
⁎
Jia-Yu Oua, Yu-Chan Shiha, Bing-Yen Wangb,c,d,e,f,g, , Chih-Chien Chua,h,
a Department of Medical Applied Chemistry, Chung Shan Medical University, Taichung City, Taiwan
b Division of Thoracic Surgery, Department of Surgery, Changhua Christian Hospital, Changhua County, Taiwan
c School of Medicine, Chung Shan Medical University, Taichung City, Taiwan
d Institute of Genomics and Bioinformatics, National Chung Hsing University, Taichung City, Taiwan
e School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung City, Taiwan
f Ph.D. Program in Translational Medicine, National Chung Hsing University, Taichung City, Taiwan
g Center of General Education, Ming Dao University, Changhua County, Taiwan
h Department of Medical Education, Chung Shan Medical University Hospital, Taichung City, Taiwan
A R T I C L E I N F O
A B S T R A C T
Keywords:
In this article, we demonstrate the self-assembly and photoresponive behavior of a novel coumarin-based am-
phiphilic dendron in both aqueous solution and air-water interface. The dendritic structure, namely C-IG1, was
composed of a lipophilic cholesterol and hydrophilic poly(amido amine) (PAMAM) dendron, and the amphi-
philic counterpart is interconnected by a photolabile coumarin carbonate ester, enabling the photoinduced
degradation of the amphiphiles in protic solvents via SN1-like mechanism. A Nile red solubilization fluorescence
assay suggests a low critical aggregation concentration for the micelle formation of C-IG1 in aqueous solutions
(3.9 × 10−5 M); the Langmuir analysis further indicates that C-IG1 possesses significant compressibility in air-
water interface, eventually forming homogeneous monolayers with a final molecular area (A0) of 36 Å2. Notably,
the micelles and Langmuir monolayer are quite stable until photo-triggered dissociation based on the photo-
cleavage of C-IG1 amphiphile activated by 365-nm incident light. Moreover, the transition in interfacial mor-
phology of the Langmuir monolayer during the assembly and photodegradation processes also can be visually
analyzed by incorporating Nile red probes with in situ monitoring through fluorescence microscopy. The thin
film deposited on a glass substrate by the Langmuir-Blodgett technique also shows a photoresponsive behavior
based on the change in the contact angles of a water droplet on the surface upon light stimulation. The binding
affinity of C-IG1 and cyclic DNA determined by the fluorescence quenching analysis of the coumarin reporter
suggests a ground-state macromolecular complexation process occurring through polyvalent interactions be-
tween the pseudodendrimers and biomacromolecules. The ethidium bromide displacement assay further in-
dicates thus dendriplex formation at low nitrogen-to-phosphorous value (N/P < 1) and confirms that the de-
complexation accompanied by DNA release can be achieved through an active phototriggered route under
spatiotemporal control.
Dendrimers
Amphiphilic dendrons
Langmuir analysis
Photocleavage
Gene delivery
1. Introduction
structural degradation combined with the release of bioactive payloads
[2–9]. One of the most well-known light-absorbing photocages is the
Phototriggers provide a useful strategy for the photocontrolled drug
delivery system (PDDS) because they enable rapid and accurate spatial
and temporal control with external light stimulation [1]. The biological
relevant materials containing photocaged building blocks can undergo
efficient photolysis through active phototriggers, thereby leading to
coumarin family, which enables one- or two-photon–regulated drug
release [1,10–13]. Because coumarin derivatives undergo photo-
solvolysis through a photo SN1 mechanism, the microenvironment for
achieving effective bond cleavage requires access to a nucleophile
in a protic media (typically water) [14]. Furthermore, the intrinsic
⁎ Corresponding authors at: Department of Medical Applied Chemistry, Chung Shan Medical University, No. 110, Sec. 1, Jianguo N. Rd., Taichung 40201, Taiwan
(C.-C. Chu); Division of Thoracic Surgery, Department of Surgery, Changhua Christian Hospital, No. 135, Nanxiao St., Changhua City 500, Taiwan
E-mail addresses: 156283@cch.org.tw (B.-Y. Wang), jrchu@csmu.edu.tw (C.-C. Chu).
https://doi.org/10.1016/j.colsurfb.2018.12.018
Received 6 August 2018; Received in revised form 28 November 2018; Accepted 8 December 2018
Availableonline11December2018
0927-7765/©2018ElsevierB.V.Allrightsreserved.

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ColloidsandSurfacesB:Biointerfaces175(2019)428–435
fluorescent nature of the coumarin ring makes these photocages robust
fluorescent probes for simultaneously imaging the biodistribution of the
PDDS in living tissues [11].
2. Results and discussion
As shown in Fig. 1b, the azide-functionalized cholesterol and cou-
marin conjugate 1 were synthesized from a commercially available 7-
hydroxy-4-methylcoumarin in four steps. On the basis of our design, the
cholesterol not only acts as the lipophilic head but also assists cellular
uptake by enhancing penetration across a cell membrane. The inter-
connecting coumarin-derived carbonate ester is highly sensitive to ul-
traviolet (UV) light, and the photolabile C–O bond (marked in bold) is
readily cleaved to recover compound 2 and release a free cholesterol
molecule. Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC)
between the alkyl-functionalized PAMAM dendrons and 1 affords the
amphiphilic dendrons, namely C-IG1 (Fig. 1) [18]. Notably, compared
with the conventional PAMAM dendron, this dendritic analogue has
two additional primary amine terminals but possesses an inverse ter-
tiary amine branch and amide linkage [30]. Here, the amphiphilic
dendron was well-characterized through nuclear magnetic resonance
(NMR) and mass spectrometry (MS) analysis. The click conjugation was
confirmed according to the appearance of triazole proton resonance,
and the observed mass values were consistent with the calculated va-
lues of the protonated adducts of C-IG1.
The use of amphiphilic dendron architectures, in which hydro-
phobic groups at the focal point form pseudodendrimers through a self-
assembly process, has facilitated a new type of dendrimer-mediated
delivery in vitro and in vivo [15–22]. Notably, this supramolecular
strategy, which enables combining the characteristics of polymers and
lipids, can give lead to a synergistic effect, particularly during nucleic
acid delivery [23]. Principally, when used as gene vectors, pseudo-
dendrimers apply dynamic and responsive association and dissociation
toward nucleic acids [24]. Moreover, complete dendron degradation
may be required for effective decomplexation to release nucleic acids.
However, experimental and computer-aided simulation data have re-
vealed that the structural degradation of dendrons when bound to nu-
cleic acids becomes ineffective on the transfection timescale, even at
lower pH associated with endosomes [25]. This key problem makes
gene delivery a challenging task, particularly for the in vivo system.
In this study, we developed amphiphilic dendritic scaffolds with
photocages for creating a photoresponsive pseudodendrimer that can
achieve controlled release under an active light trigger [15]. The am-
phiphilic structure composed of a hydrophilic poly(amido amine)
(PAMAM) dendron and lipophilic cholesterol molecule combines the
advantageous gene delivery features of both lipids and polymer vectors.
As shown in Fig. 1a, the amphiphilic counterpart is further inter-
connected by a photolabile coumarin carbonate ester, enabling the
photoinduced degradation of the amphiphilic structure (C-IG1). The
arrow indicates an electrophilic allylic carbon, and the C–O bond can be
readily cleaved in the presence of weak nucleophiles under light illu-
mination [26]. Consequently, this strategy provides an active route for
accelerating nucleic acid release and enhancing gene transfection effi-
ciency. Moreover, to understand the self-assembly process of the am-
phiphiles and their photoresponsive behavior, the Langmuir technique
was introduced for investigating the interfacial phenomenon at the
air–water interface [27–29]. We believe that the thus-formed Langmuir
monolayers are more fluid and therefore provide a more realistic model
for studying DNA binding and phototriggered release.
The time-dependent photolytic reaction of 1 was monitored by
UV–Vis absorption and fluorescence spectra. The maximum absorption
and emission wavelengths of 1 were found to be 320 and 390 nm, re-
spectively. As shown in Fig. 2a, the absorbance at 320 nm decreases
slightly as the sample solution is exposed to a 365-nm light emitting
diode (LED); however, the fluorescence intensity at 390 nm gradually
increases with the irradiation time (Fig. 2b). The fluorescence en-
hancement is evidently attributable to the SN1-like photolytic reaction
of the coumarin ester with an increasing fluorescence quantum yield
[14]. Moreover, high-performance liquid chromatography (HPLC)
analysis showed the depletion of the retention peak of 1 after light
exposure (Fig. 2c). Gas chromatography (GC)–MS analysis further
confirmed the released cholesterol through photocleavage as the char-
acteristic elution peak (Fig. 2d). All results suggested a successful
photoinduced structural degradation of the coumarin derivative.
Because of the amphiphilic structure being composed of
a
Fig. 1. (a) Chemical structure of a coumarin-
based amphiphilic dendron C-IG1. (b)
Synthetic condition for compound 1: (i) 1,2-
dibromoethane, K2CO3, 18-crown-6, THF,
40 °C; (ii) 1. SeO2, dry toluene, reflux and 2.
NaBH4, MeOH, 0 °C to room temperature; (iii)
NaN3, DMF, 50 °C; (iv) cholesteryl chlor-
oformate, pyridine, THF, 50 °C.
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Fig. 2. (a) UV–vis absorption and (b) fluorescence spectra of compound 1 recorded upon 365-nm light irradiation at fixed time intervals. (c) HPLC analysis shows the
decrease in the retention peak of 1 and (d) GC analysis shows the retention peak of free cholesterol released from 1 after light excitation.
Fig. 3. The Nile red solubilization fluorescence assay for amphiphilic dendron C-IG1. (a) Fluorescence intensity for the Nile-red probe (2.5 μM) gradually increases as
the concentration of dendron increases. (b) The critical aggregation concentration (CAC) was extracted from the intersection of two linear regressions based on lower
and higher concentration of dendrons against the fluorescence intensity at 635 nm.
hydrophilic PAMAM dendron and a lipophilic cholesterol, C-IG1 could
self-assemble into Percec-type pseudodendrimers with micelle-like
structures in an aqueous medium [31]. This self-assembly process was
confirmed using a Nile red solubilization fluorescence assay to detect
the formation of a hydrophobic domain of cholesterol within an as-
sembled nanostructure [32]. In this assay, the hydrophobic stain Nile
red was gradually solubilized in the micelles and emitted red fluores-
cence as the concentration of C-IG1 increases (Fig. 3a). Fig. 3b shows
self-assembly with discontinuity in the fluorescence intensity of Nile red
at 635 nm, plotted against the increasing dendron concentration at the
critical aggregation concentration (CAC). The CAC for C-IG1 was ap-
proximately 39 μM, much lower than that for a coumarin-derived sur-
factant [33]. Moreover, dynamic light scattering analysis shows that the
particle size distribution for C-IG1 at 100 μM above the CAC value was
117.5
21.4 nm, confirming the amphiphilic dendrons possess effec-
tive self-assembly process.
To understand the self-assembly behavior of the amphiphile, the
Langmuir technique was introduced to investigate the interfacial phe-
nomenon at the air–water interface. As shown in Fig. 4a, π-A isotherms
have three distinguishable phase transitions from a gas (i)-like state to
liquid (ii)- and finally solid (iii)-like states, suggesting that C-IG1 pos-
sesses significant compressibility, eventually forming homogeneous
Langmuir monolayers upon compression, until the surface pressure of
collapse is approximately 50 mN/m. This result evidently suggests that
the amphiphilic structure also favors the molecular assembly at the
air–water interface. The final molecular area (A0) in a compressed film
determined by extrapolation to zero surface pressure is approximately
36 Å2 for C-IG1, consistent with the A0 of the amphiphilic dendrons with
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Fig. 4. (a) The Langmuir π-A isotherm for amphiphilic dendron C-IG1 monolayer at air-water interface with subphase of pure water. Fluorescence micrographs of the
Langmuir monolayers doped with the Nile red probes at various surface pressure: (a) 0, (b) 5, and (c) 40 mN/m.
small dendritic branches [28]. Moreover, the interfacial morphology of
the Langmuir film under increasing surface pressures was analyzed by
incorporating Nile red probes with in situ monitoring through fluores-
cence microscopy [29]. Fig. 4b–d denotes the fluorescence distributions
of the probe molecules under different molecular packings of the am-
phiphiles in the three states. Fig. 4d illustrates the closely packed and
highly fluorescent domains under the surface pressure of 40 mN/m,
suggesting the successful formation of a condensed Langmuir mono-
layer at the interface. This result corroborates with that of the solubi-
lization assay analysis that the emission intensity considerably in-
creases in the presence of the micelle-like assembled structure of the
amphiphiles in water. This result also confirms that the C-IG1 possesses
a strong self-assembly capability in both aqueous phases and at the
air–water interface.
immediately after photostimulation was activated (t = 0). The surface
pressure of the monolayer composed of C-IG1 gradually decreases as the
light is turned on (red line); by contrast, this monolayer remains rela-
tively stable in darkness (black line). Notably, the nonlinear decay of
the surface pressure following light excitation may be attributable to
the first-order kinetics of the SN1-like photolysis mechanism for cou-
marin derivatives, because the reaction rate is correlated only with the
initial C-IG1 concentration, and the photoinduced dissociation of C-IG1
to yield a carbocation intermediate is the rate-determining step for the
overall photolytic reaction [14]. In addition, in situ monitoring of the
interface morphology by using Nile red probes also indicated that the
fluorescence intensity for the assembled pattern decreased on light
excitation (Fig. 5c). The visualized images clearly confirm the efficient
photodegradation of the Langmuir monolayer.
After confirming that the amphiphilic dendron can process mole-
cular assembly, we examined the photoresponsive behavior of the as-
sembled structures. The photocleavable building block of coumarin
ester aids the self-aggregated micelles in readily dissociating under UV
irradiation. First, this deformation can be analyzed using micelle-en-
capsulated Nile red probes. In contrast to the controlled experiment,
where the solution stored in darkness only exhibited a slight decrease in
fluorescence intensity, 365-nm light irradiation can cause a consider-
able fluorescence decrement within 5 min, until reaching a plateau
(Fig. 5a). This result suggests that the micelles are stable until active
light stimulation degrades these self-assembled structures. Second, a
Langmuir monolayer at a certain surface pressure (approximately
40 mN/m) and molecular area, maintained by keeping the barriers
constant, was prepared. Subsequently, the equilibrium was con-
tinuously disturbed through 365-nm light irradiation. Before analysis,
control experiment showed that the surface pressure of the monolayer
of stearic acids remains constant on light irradiation, suggesting that if
the amphiphile is insensitive to UV light, light stimulation does not
influence the integrity of the assembled structure. Fig. 5b shows the
correlation of elapsed time (t) versus surface pressure, recorded
As shown in Fig. 6, the monolayer at the interface partially col-
lapsed on light exposure because it could be reconstructed through
continuous compression in darkness. The surface pressure initially de-
creased upon light excitation (black arrows) but increased again after
barrier compression in darkness. The photoinduced collapse and re-
construction of the monolayer can be alternatively manipulated until
the maximum pressure of approximately 50 mN/m is reached. Taken
together, in the Langmuir technique, the assembled structure composed
of coumarin-based amphiphilic dendrons is consistently highly sensitive
to UV light. The bond cleavage between the coumarin ester and cho-
lesterol at the interface results in the collapse of the monolayer under
light stimulation. The current Langmuir analysis also confirmed that the
photostimulation strategy provides accurate spatial and temporal con-
trol over the self-assembled system integrity.
To further reveal the thin-film properties of the amphiphilic C-IG1,
three-layer monolayers were transferred to a glass substrate by using
up–down–up strokes based on the Langmuir–Blodgett (LB) technique.
Because the coverglass possesses a hydrophilic surface, the bottom layer
of the sandwich structure comprises the hydrophilic PAMAM dendron
and the upper layer should comprise lipophilic cholesterols (Fig. 7a).
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Fig. 5. The change in (a) fluorescence intensity of the Nile-red incorporated micelles ([C-IG1] = 100 μM) and in (b) surface pressure of the Langmuir monolayer
composed of the amphiphilic C-IG1 upon 365-nm light irradiation at fixed time intervals. (c) Fluorescence micrographs of the Langmuir monolayer (40 mN/m) doped
with the Nile-red probes upon light irradiation.
binding ability of C-IG1 toward cyclic DNA, a pEGFP-C1 reporter gene
(approximately 4700 bp), was first studied through fluorescence titra-
tion experiments. In Stern–Volmer analysis, the intrinsic fluorescence of
the coumarin moiety enables direct resolution of the binding affinity of
the vector and DNA [33]. As shown in Fig. 8, the emission intensity of
coumarin at λ = 390 nm gradually decreased with the continuous ad-
dition of DNA as the fluorescence quencher, indicating successful
binding between the pseudodendrimers and giant DNA through elec-
trostatic association. The Stern–Volmer plot (inset in Fig. 8a) further
illustrates a linear correlation between the change in fluorescence in-
tensity and concentration of the quencher, suggesting
a static
quenching mode for both species. Assuming that the molecular mass for
cyclic DNA is approximately 3 × 106 Da, the quenching constant re-
trieved from the slope is 0.998 μg−1, corresponding to 5.9 × 109 M−1
.
The value is close to the magnitude of the binding affinity between
polymers and proteins rather than that of the groove binding of small
molecules (e.g., drug) and DNA [33,34]. Thus, the pseudodendrimer
assembled from amphiphilic C-IG1 is an excellent biomacromolecular
binder and the dendriplex formation is a ground-state macromolecular
complexation process occurring through polyvalent interactions.
The binding and photoinduced release were analyzed using an
ethidium bromide (EtBr) displacement assay [15]. Fig. 8b shows the
fluorescence titration experiment by increasing C-IG1 concentration at
constant amounts of DNA and EtBr. Initially, the EtBr undergoes a large
increase in fluorescence intensity on intercalation with stacks of nucleic
acid base pairs. However, the fluorescence quenching of the EtBr/DNA
complexes occurs in the presence of amine-based vectors, engendered
by the competitive displacement of EtBr by the vectors. The emission
intensity at 590 nm gradually decreases as the nitrogen-to-phosphorus
(N/P) ratio increases, suggesting increasing dendriplex formation with
increasing C-IG1 concentration. When the vectors degrade under UV
light exposure, DNA should be released and EtBr should reintercalate
into the double helix, thus reactivating its fluorescence. Accordingly,
Fig. 8c shows the fluorescence increment from the minimum intensity
Fig. 6. A stepped control for the collapse and reconstruction of the Langmuir
monolayer by alternating photoexcitation and barrier compression processes.
The monolayer was first fabricated at 15 mN/m and then repetitively irradiated
with 365-nm LED light for 5 min (arrows) and recompressed in dark until de-
signated surface pressure.
From the measurement of static contact angles (Fig. 7b–e), the angle for
a water drop on the surface considerably increases from approximately
18° to 90° after three-layer film deposition. This result confirms that the
surface becomes a lipophilic domain because of a tidy arrangement of
the amphiphiles. Notably, the contact angles gradually decrease when
the film is exposed to UV light, implying that the ordering LB film is
disturbed by an incident light. The photolabile coumarins undergoing a
photolytic reaction cause the structural degradation of the amphiphiles.
Accordingly, the LB film of C-IG1 also exhibits a photoresponsive be-
havior in a solid state.
After the coumarin-based amphiphilic dendrons self-aggregate into
the assembled pseudodendrimers with positively charged peripherals,
they can effectively interact with polyanionic bioactive targets, such as
DNA, to form dendriplexes through electrostatic interaction. The
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ColloidsandSurfacesB:Biointerfaces175(2019)428–435
Fig. 7. (a) Three-layer monolayers transferred to a glass substrate by the Langmuir-Blodgett (LB) technique. A contact angle measurement for a water droplet on (b) a
clean substrate and (c–e) a LB film-deposited substrate: (c) dark and 365-nm light irradiation for (d) 15 and (e) 30 min.
Fig. 8. (a) Fluorescence titration analysis of C-IG1 (1.5 μM) upon addition of pEGF-C1 (0–2.5 μg). Inset: Stern-Volmer plot for the change in fluorescence intensity at
390 nm as a function of the amount of quencher. (b) Fluorescence titration data of ethidium bromide (EtBr) displacement assay for addition of C-IG1 to pEGFP-C1 at
increasing nitrogen-to-phosphorous (N/P) ratios. The inset shows the correlation of relative fluorescence intensity of EtBr at 590 nm versus N/P values. (c) Recovery
ratio of the fluorescence intensity for EtBr reintercalating into pEGFP-C1 after dissociation of the C-IG1/DNA complexes (N/P = 2) upon 365-nm light excitation.
of the DNA/C-IG1 complexes at N/P ratio = 2 after LED light excitation.
By contrast, the fluorescence intensity remained static when the com-
plex solution was maintained in darkness. This result confirms that the
photolytic reaction of the vectors induces decomplexation accompanied
by DNA release through an active phototriggered route.
from the dendriplexes upon light excitation. Thus, the assembled den-
drons may be more resistant to the photoinduced disruption than a
single dendron is. The Langmuir analysis results also revealed that the
assembled structure would soon be recovered under external com-
pression after a partial collapse. Therefore, these electrostatic com-
plexes may be relatively resistant to environmental fluctuation.
Although UV light is insufficient to induce complete dissociation of the
DNA complexes using a coumarin-derived amphiphilic dendron as the
vector, it does assist nucleic acid release.
After light exposure for 50 min, the fluorescence intensity was
partially recovered to approximately 60% of the original magnitude for
the emission of the DNA/EtBr complex in the absence of the dendritic
vectors (N/P = 0). This result implies that only 60% of DNA is released
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3. Conclusions
A dimethylformamide solution of 3 (83.9 mg, 0.28 mmol) and NaN3
(20.1 mg, 0.3 mmol) was stirred at 50 °C for 1 h under N2. The solvent
was removed under vacuum, and the mixture was extracted by ethyl
acetate/brine. Combined organic phase was dried over magnesium
sulfate, and rotatory evaporation to dryness afforded compound 2
(97%, Rf = 0.25, ethyl acetate/hexane = 1:1). A THF solution of 2
(100 mg, 0.38 mmol), cholesteryl chloroformate (249 mg, 1.4 mmol),
and pyridine (75 μL, 0.93 mmol) was stirred at 50 °C for overnight
under N2. The solvent was removed under vacuum, and the crude
product was purified by flash column chromatography (SiO2) to give
compound 1 (76%, Rf = 0.8, ethyl acetate/hexane = 1:1). The overall
yield for the four-step reaction is 12%, and this lower yield is mainly
attributed to the heterogeneous allylic oxidation of compound 4 and
SeO2. 1H-NMR (400 MHz, CDCl3) for 1: δ = 7.44 (d, J = 8.8 Hz, 1 H),
6.91 (dd, J = 8.8, 2.5 Hz, 1 H), 6.86 (d, J = 2.5 Hz, 1 H), 6.40 (t,
J = 1.4 Hz, 1 H), 5.41 (d, J = 5.1 Hz, 1 H), 5.30 (d, J = 1.4 Hz, 2 H),
4.50–4.58 (m, 1 H), 4.21 (t, J = 5.1 Hz, 2 H), 3.67 (t, J = 5.1 Hz, 2 H),
2.43 (m, 2 H), 2.06 – 0.82 (m, 38 H), 0.68 (s, 3 H); 13C-NMR (75 MHz,
CDCl3): δ = 161.6, 160.8, 155.6, 154.1, 148.9, 139.3, 124.9, 123.5,
113.2, 111.2, 110.8, 102.0, 79.3, 67.7, 64.3, 56.9, 56.3, 50.1, 42.5,
39.9, 39.7, 38.2, 37.0, 36.8, 36.4, 36.01, 32.1, 32.0, 28.5, 28.3, 27.9,
24.5, 24.0, 23.1, 22.8, 21.3, 19.5, 18.9, 12.1.
In summary, we successfully synthesized photoresponsive amphi-
philic PAMAM dendrons bearing
photolabile coumarin ester building blocks as DNA carriers. The
amphiphilic dendrons C-IG1 exhibit the capability for self-assembly
during micelle-like pseudodendrimer formation in an aqueous solution,
which was confirmed by spectroscopy and Langmuir isotherm analysis.
Based on the bipolar functionality, C-IG1 also demonstrates substantial
binding affinity with cyclic DNA at low N/P ratios. Notably, the thus-
formed DNA complexes readily dissociate under UV light irradiation
because the coumarin ester group in the dendritic structure undergoes
efficient photolytic cleavage, causing effective dendron degradation
accompanied by DNA release.
4. Experimental sections
4.1. Materials and instruments
The chemical reagents and organic solvents for materials synthesis
were obtained as high-purity reagent-grade from commercial suppliers
and used without further purification. Carbamate-protected dendron
(Boc-IG1) was synthesized following the published procedure [15]. 1H
(400 MHz) and 13C (75 MHz) NMR spectra were recorded on a Varian
Mercury Plus 400 MHz spectrometer at room temperature using CDCl3,
DMSO-d6, methanol-d4, or D2O as the solvents. Spectral processing
(Fourier transform, peak assignment, and integration) was performed
using MestReNova 6.2.1 software. Matrix-assisted laser desorption io-
nization/time-of-flight (MALDI–TOF) MS was performed on a Bruker
AutoFlex III TOF/TOF system in positive ion mode using either 2,5-
dihydroxybenzoic acid or α-cyano-4-hydroxycinnamic acid as the des-
orption matrix. UV–vis absorption spectra was performed on a Thermo
Genesys 10S UV–Vis spectrometer. Fluorescence emission spectra was
recorded on a Hitachi F-2500 spectrometer. GC–MS was performed on a
Hewlett-Packard 5890 series II gas chromatography equipped with
5972 series mass selective detector. HPLC was performed on a JASCO
instrument equipped with a MD-2015 PLUS photodiode array detector,
covering the wavelengths from 200 to 900 nm with an interval of
1.5 nm. The HPLC analysis was conducted at 25 °C using Dr. Maisch
Reprosil 100-Si column (250 × 4.6 mm, normal phase with 5 um
porous spherical silica). A solvent mixture of hexane (97%) and iso-
propanol (3%) was used as a mobile phase and the flow rate was
maintained at 1.0 cm3/min. Photolysis of the ester conjugates were
carried out by using light-emitting diodes (LED) at 365 nm and an
output power of 10-watt.
4.3. Synthesis of the click cluster C-IG1
An anhydrous CH2Cl2 solution of compound
1 (87.7 mg,
0.13 mmol), Boc-protected IG1 (76.2 mg, 0.16 mmol), and CuBr
(22.8 mg, 0.16 mmol) was vigorously stirred at room temperature until
the complete disappearance of compound 1. The resulting solution was
then extracted with CH2Cl2; the organic phase was washed with aqu-
eous ammonia solution to remove copper catalyst and then dried over
magnesium sulfate. Rotatory evaporating to dryness yields the Boc-
protected click clusters C-IG1 without further purification (96%). Boc-
deprotection is readily carried out by acid-promoted hydrolysis.
Trifluoroacetic acid (0.14 mL, 1.9 mmol) was added dropwise into an
anhydrous CH2Cl2 solution of Boc-protected C-IG1 (51.6 mg, 45 μmol).
The mixture was then stirred under room temperature for 3 days, and
the volatiles were removed under reduced pressure. The mixture was
rinsed with hexane repetitively to remove excess acid, and then freeze-
drying afforded amphiphilic dendron C-IG1 as yellowish fluffy powders
(97%). For Boc-protected C-IG1, 1H-NMR (400 MHz, CDCl3): δ = 8.20
(bs, 1 H), 7.78 (s, 1 H), 7.43 (d, 1 H), 6.86 (d, 1 H), 6.81 (s, 1 H), 6.40 (s,
1 H), 5.42 (m, 1 H), 5.29 (s, 2 H), 5.01 (bs, 2 H), 4.79 (t, 2 H), 4.54–4.58
(m, 1 H), 4.51 (d, 2 H), 4.43 (t, 2 H), 3.05–3.10 (m, 4 H), 2.64 (t, 2 H),
2.38–2.44 (m, 7 H), 2.32 (t, 2 H), 0.85–2.04 (m, 60 H), 0.68 (s, 3 H). For
C-IG1, 1H-NMR (400 MHz, CD3OD): δ = 8.17 (s, 1 H), 7.60 (d, 1 H),
6.98 (d, 1 H), 6.91 (s, 1 H), 6.28 (s, 1 H), 5.42 (s, 1 H), 5.37 (s, 2 H),
4.39–4.57 (m, 5 H), 3.52 (s, 2 H), 3.06 (s, 6 H), 2.80 (s, 2 H), 2.41 (s,
2 H), 2.15 (s, 4 H), 0.86–2.07 (m, 38 H), 0.70 (s, 3 H); MALDI-TOF-MS:
Cacld. For (M+H)+ C52H80N7O7: 914.61 Da; Found: 914.28 Da.
4.2. Synthesis of compound 1
To an anhydrous tetrahydrofuran (THF) solution of 7-hydroxy-4-
methylcoumarin (528 mg, 1 mmol), K2CO3 (1.46 g, 3.5 mmol), and 18-
crown-6 (4.36 g, 5.5 mmol), 1,2-diboromoethane (5.69 g, 10 mmol) was
added dropwise under N2. After stirred at 40 °C for 2 days, the volatile
was removed by rotatory evaporation, and then the mixture was ex-
tracted with CH2Cl2 and brine. Combed organic phase was dried over
anhydrous magnesium sulfate, and rotatory evaporation to dryness af-
forded crude product. Flash column chromatography (SiO2) yields
compound 4 (76%, Rf = 0.55, ethyl acetate/hexane = 1:1).
4.4. Nile-red solubilization assay
Nile red stock solution (2.5 mM) was prepared in ethanol, and a
dendron stock solution was prepared in PBS buffer at various con-
centrations depending on the starting concentration for the assay.
Aliquots of the stock solution were taken and diluted with PBS to the
desired concentration in a 1 mL assay volume. Nile red (1 μL) was
added and the fluorescence emission was measured on a spectro-
fluorometer using an excitation wavelength of 550 nm. Fluorescence
intensity was recorded at 635 nm. The photolytic reaction for the single
molecule and the micelles in the solutions was performed by irradiating
the sample solutions prepared in a quartz cuvette (1 cm x 1 cm) under a
365-nm LED. The distance between the sample and light source was
kept at approximately 3 cm, and then the exposed solutions were ana-
lyzed by UV–vis and fluorescence spectroscopy.
A mixture of 4 (609 mg, 2.2 mmol) and SeO2 (715 mg, 6.6 mmol)
dissolved in anhydrous toluene was heated under reflux for 2 days, and
then the volatile was removed by rotatory evaporation. The mixture
dissolved in anhydrous methanol was added dropwise into a methanol
solution of NaBH4 (83.9 mg, 0.28 mmol) under ice bath, and the reac-
tion was further stirred at room temperature for 1 day. The reaction was
quenched by dilute HCl solution and extracted with ethyl acetate. Flash
column chromatography (SiO2) yields compound 3 (21%, Rf = 0.28,
ethyl acetate/hexane = 1:1).
434

J.-Y. Ou et al.
ColloidsandSurfacesB:Biointerfaces175(2019)428–435
4.5. Preparation of the Langmuir monolayers
Acknowledgments
A computer-controlled Langmuir trough (364 mm x 75 mm, KN2002
KSVNIMA) equipped with a Wilhelmy plate for surface pressure mea-
surement was used obtain the surface pressure-area per molecule (π-A)
isotherms. A monolayer at the air/water interface in the trough can be
symmetrically compressed with two Teflon barriers at a given rate.
Before each run, the trough was first filled with Millipore (18.2 MΩ cm)
water as the subphase, and the surface pressure of the air/water in-
terface was then zeroed. A chloroform solution of the compounds was
then spread at the interface, and a period was allowed for solvent
evaporation and for the system equilibrium. The monolayer was con-
tinuously compressed at a rate of 5 mm2/min to record π-A isotherms
with in situ fluorescence microscope observation (Axioskop, Carl Zeiss).
The Nile red dye was selected the fluorescence probe for monitoring the
assembly process at the interface. The excitation/emission wavelengths
were selected by an appropriate beam splitter/filter combination for the
probes, and the monolayer was observed by using 50-fold magnification
of an objective lens. For the photolytic reaction occurred at the air/
water interface, the monolayer formed at a certain surface pressure and
molecular area, maintained by keeping the barriers constant, was ex-
posed to 365-nm LED irradiation. The change of the surface pressure
with irradiation time was then recorded.
The authors would like to thank Changhua Christian Hospital (105-
CCH-IST-124), Chung Shan Medical University, and Ministry of Science
and Technology (MOST-105-2119-M-040-001) of Taiwan for finan-
cially supporting this research. The authors also grateful to Prof. Chien-
Hsiang Chang at National Cheng Kung University (Taiwan) for support
with fluorescence microscopy analysis on the Langmuir films.
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