Photodegradable self-assembling PAMAM dendrons for gene delivery involving dendriplex formation and phototriggered circular DNA release

Article abstract of DOI:10.1039/c5nj03173g

For effective gene delivery, structural degradation of synthetic carriers is crucial to the release of nucleic acids on the transfection time scale. In this study, we have synthesized the amphiphilic dendritic scaffolds with a photolabile o-nitrobenzyl (o-NB) group that can enable the structural decomposition and controlled release of nucleic acids under active light stimulation. The amphiphilic counterpart composed of a lipophilic cholesterol and a hydrophilic poly(amido amine) (PAMAM) dendron allows the self-assembly into a core-shell-like pseudodendrimer above the critical aggregation concentration (CAC) of approximately 20 μM. On the basis of electrostatic interaction, the polycationic pseudodendrimers are capable of forming stable complexes with polyanionic cyclic reporter genes under low charge excess values, suggesting substantial binding affinity of the dendron assembly toward circular DNA. Because the o-NB group in the dendritic structure undergoes efficient photolytic cleavage, an in vitro test shows that thus-formed "dendriplexes" are readily dissociated under 365 nm light irradiation, causing effective dendron degradation accompanied by DNA release. This photochemical strategy provides an opportunity to control gene binding and release in a spatiotemporal manner.

Full text of DOI:10.1039/c5nj03173g

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Full Paper
Photodegradable Self-Assembling PAMAM Dendrons for Gene Delivery
Involving Dendriplexes Formation and Phototriggered Circular DNA
Release†
YuꢀSen Lai,a ChaiꢀLin Kao,b YaꢀPei Chen,a ChiaꢀChia Fang,a ChaoꢀChin Hua and ChihꢀChien
Chua,c
–––––––––
aDepartment of Medical Applied Chemistry, Chung Shan Medical University, Taichung
40201, Taiwan
bDepartment of Medical and Applied Chemistry, Kaohsiung Medical University, Kaohsiung
80708, Taiwan
cDepartment of Medical Education, Chung Shan Medical University Hospital, Taichung
40201, Taiwan
–––––––––
Correspondence to: ChihꢀChien Chu; email: jrchu@csmu.edu.tw
†Electronic supplementary information (ESI) available: materials synthesis, NMR
characterization, mass spectrometric data, particle size and zeta potential analysis. See DOI:
XXX
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Abstract
For effective gene delivery, structural degradation of synthetic carriers is crucial to nucleic
acids releasing on the transfection time scale. In this study, we have synthesized the
amphiphilic dendritic scaffolds with a photolabile oꢀnitrobenzyl (oꢀNB) group that can enable
the structural decomposition and controlled release of nucleic acids under active light
stimulation. The amphiphilic counterpart composed of a lipophilic cholesterol and hydrophilic
poly(amido amine) (PAMAM) dendron allows the selfꢀassembly into a coreꢀshellꢀlike
pseudodendrimer above the critical aggregation concentration (CAC) of approximately 20 ꢁM.
On the basis of electrostatic interaction, the polycationic pseudodendrimers is capable of
forming stable complexes with polyanionic cyclic reporter gene under low charge excess
value, suggesting substantial binding affinity of the dendron assembly toward circular DNA.
Because the oꢀNB group in the dendritic structure undergoes efficient photolytic cleavage, in
vitro test shows that thusꢀformed “dendriplexes” are readily dissociated under 365ꢀnm light
irradiation, causing effective dendron degradation accompanied by DNA release. This
photochemical strategy provides an opportunity to control over the gene binding and releasing
in a spatiotemporal manner.
Key words: amphiphilic dendrons; pseudodendrimer; photocage; nitrobenzyl; gene
transfection
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Introduction
Dendrimers based on tailorꢀmade surface functional groups and multivalent properties have
been suggested as promising nanoscale synthetic carriers for delivering bioactive materials
into target cells. An alternative to the processes required to prepare giant dendrimers with a
wellꢀdefined hyperbranched structure, which are tedious and timeꢀconsuming, is using small
amphiphilic dendron architectures in which a hydrophobic group at the focal point encourages
selfꢀassembly of the resulting amphiphilic dendrons into large “pseudodendrimers.”1ꢀ6 This
supramolecular strategy, which enables combining polymer and lipid characteristics, can
cause a synergistic effect, particularly in nucleic acid delivery. Recently, several studies have
demonstrated remarkable DNA and small interfering RNA (siRNA) transfection in vitro and
in vivo mediated by these amphiphilic dendrons.7ꢀ10
For effective gene delivery, synthetic carriers must overcome several extracellular and
intracellular barriers including (1) nucleic acid complexation and protection, (2) cell
membrane penetration, (3) endosomal escape, and (4) nucleic acid release for gene expression
or knockdown.11, 12 Principally, using the pseudodendrimers as gene vectors enables taking
advantage of dynamic and responsive association and dissociation toward nucleic acids,
which favors the encapsulation of nucleic acids through the multivalent ligand array
assembled by the dendrons and rapid disassembly of these complexes under external stimuli
(e.g., change in pH or ionic strength).13, 14 To achieve the controlled release of nucleic acid
after it enters the cells, complete dendron degradation has been suggested as mandatory for
effective nucleic acid decomplexation. However, experimental and computerꢀaided simulation
data have revealed that the structural degradation of the dendrons when bound to nucleic acids
becomes ineffective on the transfection time scale, even at the lower pH associated with
endosomes.11 This key problem associated with barrier 4 on the transfection pathway makes
gene delivery a challenging task, particularly for in vivo gene transfection.
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Recently, the concept of phototriggers has provided a useful strategy in photocontrolled
drug delivery systems (PDDSs) because it enables rapid and accurate control over specific
sites and times with external light stimulation.15ꢀ20 Biologically relevant materials containing a
photolabile building block can undergo efficient photolysis through active phototriggers, thus
causing structural degradation and the release of biological targets. Among the numerous
photolabile groups that have been studied, oꢀnitrobenzyl (oꢀNB) alcohol derivatives have
aroused much attention in the PDDS field.21ꢀ24 oꢀNB alcohol derivatives that undergo efficient
photoisomerization are readily cleaved upon irradiation with UV light (approximately 300–
350 nm) and then release a free carboxylic acid (COOH) and oꢀnitrosobenzaldehyde.25
Because the oꢀNB group is highly sensitive to UV light, this photocleavage reaction can be
induced within minutes, even when using a lowꢀintensity light source. Moreover, simple
chemical modifications to an aromatic ring enable tuning the absorption profiles of the oꢀNB
group slightly. For example, an electronꢀdonating substituent (e.g., OCH3) functionalized at
the para position of the NO2 group can bathochromically shift the photocleavage wavelength
to within the 350–400 nm range.
In this paper, we present amphiphilic dendritic scaffolds with a photolabile building block
for creating photoresponsive pseudodendrimers that can enable the controlled release of
nucleic acids under active light triggering. Generally, the amphiphilic structure composed of a
hydrophilic poly(amido amine) (PAMAM) dendron and a lipophilic cholesterol molecule
combines the advantageous gene delivery feature of lipid and polymer vectors. Furthermore,
to overcome barrier 4, the cholesterol and dendron are interconnected by a photolabile oꢀNB
group, enabling photoinduced degradation of the amphiphilic structure. Consequently, this
strategy provides a remotely triggered route for accelerating nucleic acid release and
enhancing gene transfection efficiency.26 Aside from classical PAMAM dendrons, the novel
dendron analogue that has inverse amide linkage to the amine branch are introduced for
constructing the amphiphilic structures (Figure 1).27
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Figure 1. The oꢀnitrobenzyl (oꢀNB)ꢀcontaining amphiphilic PAMAM dendrons. The H denote
to characteristic triazole protons of the click clusters.
Results and discussion
Dendron synthesis and characterization
Figure 1 shows the classical PAMAM dendron and its analogue, namely G1 and IG1
respectively. G1 dendron was synthesized through consecutive 1,4ꢀMichael addition and
amidation, with propargyl amine used as the starting material.28 Moreover, IG1 dendron with a
COOH focal point was prepared using the synthetic method developed by Huang et al.27
Carbodiimideꢀpromoted amidation of IG1 with propargyl amine yields the final propargylꢀ
functionalized inverse dendrons. All amino groups of both dendrons were protected by the
tertꢀbutyloxycarbonyl (Boc) groups to prevent unwanted complexation of copper catalysts
with NH2 groups during the click reaction.
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Scheme 1. Synthetic conditions: (i) Benzyl chloride, K2CO3, DMSO, 90oC; (ii) 65% HNO3,
o
o
o
CH3COOH, ice bath to 25 C; (iii) CF3COOH, 25 C; (iv) NaBH4, EtOH, 25 C; (v) 1,2ꢀ
o
o
diboromoethane, K2CO3, 18ꢀcrownꢀ6, THF, 40 C; (vi) NaN3, DMF, 90 C; (vii) cholesteryl
chloroformate, pyridine, THF, 40 oC; (viii) CuBr, THF, and then CF3COOH, CH2Cl2, 25 oC.
As shown in Scheme 1, the oꢀNB alcohol derivative 1 was synthesized from commercially
available vanillic aldehyde by following a modified procedure.29 A nucleophilic substitution
reaction at the phenolic proton yields compound 2; following this with an azide substitution
yields compound 3. Subsequently, a simple esterification of 3 with cholesteryl chloroformate
yields the precursor 4 with a carbonate linkage between the cholesterol and oꢀNB group. The
terminal azide group enables “click” conjugation with propargylꢀfunctionalized classical and
inverse PAMAM dendrons through [3+2] copperꢀcatalyzed azideꢀalkyne click reaction
(CuAAC) protocols.28 Finally, acidꢀpromoted hydrolysis of the click products to remove the
Boc protection yields the desired amphiphilic dendrons, namely Chol-G1 and Chol-IG1, with
photolabile oꢀNB building blocks.
Because the amphiphilic dendrons could exist in the form of giant aggregates rather than a
single molecule in water or organic solvents, interpreting NMR spectra becomes a difficult
task based on these complex resonance patterns of Chol-G1 and Chol-IG1 (see supporting
information, Figure S3). Fortunately, successful click conjugation was readily confirmed
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according to the appearance of the triazole proton resonances at δ = 8.42 and 8.04 ppm, which
correspond to the characteristic protons in the triazole linkages of Chol-G1 and Chol-IG1,
respectively (Figure 1). Moreover, the 1:1 integration ratio between the triazole proton and
aromatic proton on the oꢀNB ring suggests effective CuAAC reaction of azideꢀterminated
compound 4 with propargylꢀfunctionalized G1 or IG1 dendron. More evidently, the absolute
molecular weights for the amphiphilic dendrons were analyzed by performing MALDIꢀTOFꢀ
MS, with αꢀcyanoꢀ4ꢀhydroxycinnamic acid used as a supporting matrix. The observed mass
values were consistent with the calculated values of the protonated adducts of Chol-G1 and
Chol-IG1 at 964.6 and 921.4 Da, respectively.
Self-assembly and photo-induced disassembly of dendrons
Because of the amphiphilic nature of a structure composed of a hydrophilic PAMAM dendron
and
a lipophilic cholesterol, Chol-G1 and Chol-IG1 assemble into Percecꢀtype
pseudodendrimers in an aqueous solution.30 The structure of the pseudodendrimers is based
on a coreꢀshellꢀlike micelle, in which the cholesterol aggregates are sheltered with PAMAM
dendrons in aqueous media. The hydrophilic and lipophilic balance dominates the size,
integrity, and morphology of the micelleꢀlike pseudodendrimers. This selfꢀassembly process
was further confirmed using a Nileꢀred solubilization fluorescence assay, in which
hydrophobic Nileꢀred dyes are used to observe the formation of a hydrophobic domain within
an assembled nanostructure.3, 31 Nile red is solubilized and emits appreciable orangeꢀred
fluorescence only when the dendron itself selfꢀassembles into a micelle with a hydrophobic
domain. Figure 2a,b shows that the selfꢀassembly process was observed for Chol-G1 and
Chol-IG1, with discontinuity in the fluorescence intensity of Nile red at 635 nm plotted
against increasing dendron concentration at the critical aggregation concentration (CAC).
These results confirm that selfꢀassembly occurs above the CAC. Using Nileꢀred assay, the
CAC for Chol-G1 and Chol-IG1 in a phosphate buffer solution could be identified as
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approximately 20 ꢁM, which is comparable to the CAC of cholesterolꢀbased spermine
dendrons.1 In addition, we used dynamic light scattering (DLS) methods and assumed a
spherical aggregation to further analyze the particle size of the selfꢀassembled aggregates. The
dimensions of Chol-G1 and Chol-IG1 aggregates formed in a phosphate buffer solution were
approximately 114 ± 0.9 and 130 ± 1.4 nm, respectively.
(a)
(b)
Figure 2. Fluorescence titration data of Nileꢀred solubilization assay to determine the critical
aggregation concentration (CAC) of (a) Chol-G1 and (b) Chol-IG1 in a phosphate buffer
solution (PBS, pH = 7.4). The CAC values are calculated by linear extrapolation of the linear
regression fitting data.
Because the photocaged oꢀNB derivatives exhibit moderate absorption in the UV region,
we examined the photoinduced degradation of the amphiphilic dendrons by using ultraviolet–
visible (UV–Vis) spectroscopic analysis. The photocleavage reaction can be conducted under
UV light; therefore, both dendron solutions were exposed to a 365ꢀnm lightꢀemitting diode
(LED) at fixed time intervals (10, 20, and 30 min). Figure 3a,b illustrates the apparent
changes in the absorption profiles of the Chol-G1 and Chol-IG1 solutions, respectively, upon
UV light exposure. Furthermore, the Chol-IG1 dendron possesses observable redꢀshifted
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absorption bands in the UV region (300–400 nm). This result clearly suggests structural
decomposition of the oꢀNB moiety from the original oꢀnitrobenzyl carbonate into oꢀ
nitrosobenzaldehyde after UV light irradiation.32 Moreover, for both solutions, the appearance
of a detectable absorption peak at 280 nm after light exposure indicates the formation of oꢀ
nitrosobenzaldehyde (see Figure S4).
(a)
(b)
Figure 3. UVꢀVis absorption spectra of (a) Chol-G1 and (b) Chol-IG1 aqueous solutions
under 365ꢀnm lightꢀemitting diode (LED) irradiation. The appearance of absorption peak at
280 nm indicates successful photolysis of oꢀnitrobenzyl ester. The molar concentration for
both dendron solutions is equal to 1 x 10ꢀ4 M.
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Because these amphiphilic dendrons undergo efficient photolytic structural degradation,
dissociation of the selfꢀassembly aggregates of the amphiphilic dendrons could also be
activated by UV light trigger. The photoinduced disassembly process of Chol-G1 aggregates
was then analyzed by the Nileꢀred assay. Notably, the concentration of the dendron solution
was kept at 100 ꢁM, which is fairly above the CAC, to ensure the micelle formation. As
shown in Figure 4, the fluorescence intensity of Nileꢀred dramatically decreases and reaches a
plateau after the solution was exposed to UV light for 20 min. This result suggests that the
photocleavage of the oꢀNB groups could decompose the amphiphilic structure and thus causes
the micelle deformation.
Figure 4. The relative fluorescence intensity of Nileꢀred in the presence of Chol-G1 (100 ꢁM)
after 365ꢀnm light irradiation. The sample solutions were exposed to UV light at fixed time
intervals, followed by adding the Nileꢀred probe to analyze the dendron assembly.
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DNA binding and photo-induced releasing
We then used the standard ethidium bromide (EtBr) displacement fluorescence spectroscopic
assay to investigate the “dendriplexes” formation of these dendrons to bind pEGFPꢀC1
reporter DNA (approximately 4700 base pairs).33, 34 This assay method involves using the
competition between the DNA binder and EtBr to determine the concentration at which the
DNA binder takes effect. This concentration can be expressed as a charge excess (CE50) value,
which reflects the concentration of the DNA binder required for half of the EtBr to be
displaced from binding to DNA. This value is also the equivalent of a minimum nitrogenꢀtoꢀ
phosphorus (N/P) ratio that is usually used to determine the amount of amineꢀbased binding
motifs required for effective DNA condensation through electrostatic interactions between the
amine and phosphate groups. Initially, EtBr undergoes a large increase in fluorescence
intensity upon intercalation with stacks of nucleic acid base pairs. The fluorometric titration
experiments illustrated in Figure 5a,b reveal the fluorescence quenching of the EtBr/DNA
complex in the presence of competing Chol-G1 and Chol-IG1. This fluorescence quenching is
due to the competitive displacement of EtBr by the amineꢀbased dendrons.
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(a)
(b)
1.2
0.8
0.4
(c)
(d)
0.0
1.2
0.8
0.4
0.0
0
1
2
3
4
5
Charge excess (CE)
Figure 5. Fluorescence titration data of ethidum bromide (EtBr) displacement assay for
addition of (a) Chol-G1 and (b) Chol-IG1 to pEGFPꢀC1 at increasing nitrogenꢀtoꢀ
phosphorous (N/P) ratios. The control 1 corresponds to 1:1 binding of EtBr and a DNA base
in the absence of dendrons, and the control 2 corresponds to the free EtBr in a phosphate
buffer solution (PBS, pH = 7.4). The correlation of relative fluorescence intensity of EtBr at
590 nm versus the charge excess values of (c) Chol-G1 and (d) Chol-IG1 with DNA.
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Moreover, by maintaining the amounts of DNA (0.5 ꢁg) and EtBr (0.7 ꢁg) constant during
all fluorometric assays, reduced fluorescence intensity was found to depend on the
administered N/P values. As shown in Figure 5c,d, both amphiphilic dendrons possess similar
DNA binding affinity, with calculated CE50 values of 0.92 and 0.97 for Chol-G1 and Chol-
IG1, respectively, indicating that 50% of the EtBr intercalated in the DNA could be
successfully replaced by Chol-G1 and Chol-IG1 in phosphate buffer solutions. Accordingly,
the minimum N/P ratio for effective DNA complexation is approximately equal to the CE50
values. We also performed a control experiment by using both G1 and IG1 dendrons as the
DNA binder and found no fluorescent reduction at N/P ratio of 1. This result suggests that the
amphiphilic structure containing cholesteryl and PAMAM dendritic counterparts is crucial for
DNA complexation. DNA complexation was further analyzed by recording zeta potential
measurements. The pristine pEGFPꢀC1 possesses a negative surface charge of approximately
ꢀ54.7 ± 2.6 mV and was neutralized by adding Chol-G1 at an N/P ratio of 2. The surface
potential shifted positively to +0.47 ± 0.1 mV, clearly confirming effective binding between
DNA and the vectors above the CE50.
The EtBr assay can also be used to study the UVꢀlightꢀinduced disassembly of the DNA
and amphiphilic dendron complexes. We reasoned that as the dendrons degraded under UV
light exposure, the DNA should be released and the EtBr is reintercalated into the double
helix, thus reactivating its fluorescence. Before the test, two control experiments confirmed
that (1) the DNA structure is insensitive to the 365ꢀnm LED because the fluorescence
intensity of the EtBr/DNA complex in the absence of amphiphilic dendrons remained at the
highest value upon light exposure for 30 min, and (2) the fluorescence intensity remained
essentially at the lowest value if the complex solution was maintained in the dark for 24 h in
the presence of amphiphilic dendrons at an N/P ratio of 2. This result suggests that the DNA
cannot be released from the electrostatic complexes because the amphiphilic dendrons possess
an intact structure. The bar chart in Figure 6 indicates successful DNA release upon UVꢀlight
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exposure. First, the fluorescence of EtBr at λmax = 590 nm decreases because the EtBr is
displaced from the DNA double helix by the presence of Chol-G1 and Chol-IG1 at an N/P
ratio of 2. After UV light irradiation within 10 min, the clear increase in fluorescence intensity
indicates disassembly of the DNA complexes. UV–Vis analysis demonstrated that both Chol-
G1 and Chol-IG1 are highly sensitive to UV light, and thus the fluorescence enhancement is
apparently due to EtBr reintercalation into DNA resulting from the photoinduced structural
degradation of the amphiphilic dendrons that associates with DNA release. DLS measurement
also supports the photoinduced dissociation of the DNA complexes. Initially, the dimensions
of DNA–Chol-G1 complexes formed in an aqueous buffer solution were approximately 277 ±
57.3 nm; however, after light irradiation, hydrodynamic size distribution was undetectable by
the DLS method, suggesting the disappearance of the DNA complexes. Moreover, zeta
potential of the complex solution decreases from +0.47 ± 0.1 mV before irradiation to ꢀ44.6 ±
1.5 mV after irradiation. This negative shift of surface potential also confirms the
phototriggered release of polyanionic DNA into surrounding solutions.
Figure 6. Fluorescence intensity of EtBr in the presence of (a) pEFGPꢀC1 and of (b)
dendriplexes composed of pEGFPꢀC1/Chol-G1 or Chol-IG1 at N/P = 2. (c) The emission
intensity was recovered after 365ꢀnm light irradiation for 10 min, suggesting dendrons
degradation and EtBr reintercalation into DNA.
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Barnard et al. argued that complete dendron degradation is necessary for effective DNA
release.11 However, when bound to DNA, dendron degradation becomes ineffective on a
transfectionꢀrelevant time scale (hours). We supposed that is because DNA complexation may
increase the stability of the dendrons against environmental fluctuation (e.g., the change in pH
associated with endosomes). Because a photolabile oꢀNB building block exists in the
dendrons, structural breakdown can be effectively triggered using UV light irradiation within
several minutes, even in the presence of DNA. This photoinduction strategy enables DNA
release on the transfection time scale under an actively triggered route. Moreover, by using
waterꢀsoluble tetrazolium (WST) salt to assess metabolic cytotoxicity, Chol-G1 and Chol-IG1
were found to be nontoxic to the Smulow–Glikcman (SG) gingival cell lines at up to 100
ꢁg/mL (Figure 7). The test indicated that high doses of both vectors do not induce a cytotoxic
response. The efficiency of gene transfection has been suggested as usually being optimized
with increasing N/P ratios, and thus the transfection can be conducted until an approximate
N/P value of 50, which is substantially higher than the value for effective DNA binding. In
vivo gene transfection facilitated by phototriggers toward SG cells by using the
photoresponsive amphiphilic dendrons as synthetic vectors is currently under investigation.
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Figure 7. Smulow–Glikcman (SG) gingival cell viability test in the presence of Chol-G1 and
Chol-IG1.
Conclusion
In summary, we successfully synthesized photoresponsive amphiphilic PAMAM dendrons
bearing photolabile oꢀNB building blocks as DNA carriers. Both amphiphilic dendrons
comprising classical and inverse PAMAM dendritic scaffolds exhibit similar selfꢀassembly
behavior in the formation of micelleꢀlike pseudodendrimers in an aqueous solution. On the
basis of the bipolar functionality, Chol-G1 and Chol-IG1 also demonstrate substantial binding
affinity with cyclic DNA at low N/P values. Most critically, thusꢀformed DNA complexes are
readily dissociated under UV light irradiation because the oꢀNB group in the dendritic
structure undergoes efficient photolytic cleavage, causing effective dendron degradation
accompanied by DNA release. This photochemical strategy provides an opportunity to control
over the gene binding and releasing in a spatiotemporal manner.
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Experimental section
Materials and instruments
All reagents were purchased as highꢀpurity reagentꢀgrade chemicals from either Sigmaꢀ
Aldrich or Acros and used without further purification. The circular reporter DNA (pEGFPꢀ
C1) was a generous gift from Prof. WenꢀWei Chang. Classical poly(amido amine) (PAMAM)
dendron (G1) was synthesized via a divergent pathway,28 and the dendron analogue (IG1) was
prepared by a solidꢀphase synthetic method.27 All organic solvents for organic synthesis were
distilled over suitable drying reagents under N2 atmosphere before use. 1H (400 MHz) and 13C
(75 MHz) NMR spectra were recorded on a Varian Mercury Plus 400 MHz spectrometer at
room temperature using CDCl3, DMSOꢀd6, methanolꢀd4, or D2O as the solvents. Spectral
processing (Fourier transform, peak assignment, and integration) was performed using
MestReNova 6.2.1 software. Matrixꢀassisted laser desorption ionization/timeꢀofꢀflight mass
spectrometry (MALDIꢀTOFꢀMS) was performed on a Bruker AutoFlex III TOF/TOF system
in positive ion mode using either 2,5ꢀdihydroxybenzoic acid or αꢀcyanoꢀ4ꢀhydroxycinnamic
acid as the desorption matrix. The size and zeta potential measurements were recorded on a
Horiba SZꢀ100 nanopartica series instrument using 532 nm laser as the incident light source.
Ultravioletꢀvisible (UVꢀVis) absorption spectra was performed on a Thermo Genesys 10S
UVꢀVis spectrometer. Fluorescence emission spectra was recorded on a Hitachi Fꢀ2500
spectrometer. Photolysis of the ester conjugates were carried out by using lightꢀemitting
diodes (LED) at 365 nm and an output power of 10ꢀwatt.
Synthesis of propargylꢀfunctionalized IG1
A CH2Cl2 solution (2 mL) of dicyclohexylcarbodiimide (DCC, 51 mg, 0.25 mmol) was added
dropwise into a CH2Cl2 solution (3 mL) of IG1 (50 mg, 0.12 mmol), propargylamine (40 ꢁL,
o
0.62 mmol), and 1ꢀhydroxybenzotriazole (5.1 mg, 10 wt % of DCC) under N2 at 0 C. After
15 min, the mixture was then stirred under room temperature for overnight. The reaction
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mixture was cooled to 0 oC to insure complete precipitation of the byproduct dicyclohexylurea,
which is quickly removed by vacuum filtration. After rotatory evaporation to dryness, the
crude product was further purified by flash column chromatography (SiO2, ethyl
acetate/hexane 2:3) to give propargylꢀfunctionalized IG1 as a colorless liquid (36 mg, 66%).
1HꢀNMR (400 MHz, CDCl3): δ = 4.95 (bs, 2H), 4.04 (dd, J = 5.1, 2.5 Hz, 2H), 3.16 (dd, J =
12.3, 6.2 Hz, 4H), 2.71 (t, J = 6.2 Hz, 2H), 2.46 (t, J = 6.6 Hz, 4H), 2.35 (t, J = 6.2 Hz, 2H),
2.22 (t, J = 2.5 Hz, 1H), 1.60ꢀ1.68 (m, 4H), 1.44 (s, 18H). 13CꢀNMR (75 MHz, CDCl3): δ =
172.4, 156.4, 80.2, 79.5, 71.4, 51.6, 50.9, 38.9, 33.9, 28.9, 28.7, 27.5.
Synthesis of (4ꢀ(2ꢀbromoethoxy)ꢀ5ꢀmethoxyꢀ2ꢀnitrophenyl)methanol 2
4ꢀ(hydroxymethyl)ꢀ2ꢀmethoxyꢀ5ꢀnitrophenol 1 was synthesized from commercial available
vanillin in 4 steps following a modified procedure (see supporting information).29 To a
tetrahydrofuran solution (THF, 25 mL) of 1 (1.35 g, 6.8 mmol), K2CO3 (0.88 g, 24 mmol),
18ꢀcrownꢀ6 (2.7 g, 37 mmol), and 1,2ꢀdibromoethane (3.5 g, 18 mmol) was added dropwise
under N2. After stirred at 40 oC for 4 h until the disappearance of the compound 1, the mixture
was extracted by ethyl acetate/brine for 3 times. The combined organic phase was dried over
anhydrous magnesium sulfate, and rotatory evaporation to dryness afforded compound 2 as
1
paleꢀyellow solids (2.0 g, 96%). HꢀNMR (400 MHz, CDCl3): δ = 7.74 (s, 1H), 7.22 (s, 1H),
4.98 (d, J = 5.8 Hz, 2H), 4.52 (bs, 1H), 4.40 (t, J = 6.4 Hz, 2H), 4.00 (s, 3H), 3.70 (t, J = 6.4
Hz, 2H). 13CꢀNMR (75 MHz, CDCl3): δ = 154.8, 146.8, 139.7, 133.4, 111.6, 110.3, 68.6, 63.2,
56.7, 28.8.
Synthesis of (4ꢀ(2ꢀazidoethoxy)ꢀ5ꢀmethoxyꢀ2ꢀnitrophenyl)methanol 3
A dimethylformamide solution (DMF, 20 mL) of 2 (0.53 g, 1.7 mmol) and NaN3 (1.1 g, 17
o
mmol) was stirred at 90 C for overnight under N2. The solvent was removed under vacuum,
and the mixture was extracted by ethyl acetate/brine for 3 times. The combined organic phase
was dried over anhydrous magnesium sulfate, and rotatory evaporation to dryness afforded
1
compound 3 as paleꢀyellow solids (0.32 g, 70%). HꢀNMR (400 MHz, CDCl3): δ = 7.73 (s,
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1H), 7.22 (s, 1H), 4.98 (bs, 2H), 4.52 (bs, 1H), 4.25 (t, J = 5.0 Hz, 2H), 4.00 (s, 3H), 3.69 (t, J
= 5.0 Hz, 2H). 13CꢀNMR (75 MHz, CDCl3): δ = 154.8, 146.9, 139.7, 133.4, 111.6, 110.4, 68.6,
63.0, 56.7, 50.2.
Synthesis of Cholesterolꢀfunctionalized compound 4
A THF solution (10 mL) of 3 (0.10 g, 0.37 mmol), cholesteryl chloroformate (0.2 g, 0.45
o
mmol), and pyridine (46 ꢁL, 0.57 mmol) was stirred at 40 C for overnight under N2. The
solvent was removed under vacuum, and the crude product was purified by flash column
chromatography (SiO2, ethyl acetate/hexane 1:4) to give compound 4 as paleꢀyellow solids
(0.13 g, 52%). 1HꢀNMR (400 MHz, CDCl3): δ = 7.75 (s, 1H), 7.08 (s, 1H), 5.58 (s, 2H), 5.41
(bs, 1H), 4.50ꢀ4.58 (m, 1H), 4.25 (t, J = 4.9 Hz, 2H), 3.98 (s, 3H), 3.70 (t, J = 4.9 Hz, 2H),
2.43 (bs, 2H), 0.85ꢀ2.03 (m, 38H), 0.68 (s, 3H). 13CꢀNMR (75 MHz, CDCl3): δ = 154.5, 154.3,
147.1, 139.5, 139.3, 128.3, 123.4, 110.4, 78.7, 68.6, 66.2, 56.9, 56.7, 56.3, 50.2, 42.5, 39.9,
39.7, 38.2, 37.1, 36.8, 36.4, 36.0, 32.1, 32.0, 28.5, 28.2, 27.9, 24.5, 24.1, 23.1, 22.8, 21.3, 19.5,
18.9, 12.1.
Synthesis of click clusters CholꢀG1 and CholꢀIG1
An anhydrous THF solution of compound 4 (21.0 mg, 30.8 ꢁmol), Bocꢀprotected G1 (18 mg,
37.2 ꢁmol) or IG1 (17 mg, 38.6 ꢁmol), and CuBr (5.3 mg, 36.9 ꢁmol) was vigorously stirred
at room temperature until the complete disappearance of compound 4. The resulting solution
was then extracted with ethyl acetate; the organic phase was washed with aqueous ammonia
solution to remove copper catalyst and then dried over magnesium sulfate. Rotatory
evaporating to dryness yields the Bocꢀprotected click clusters quantitatively. For Bocꢀ
protected Chol-G1, 1HꢀNMR (400 MHz, CDCl3): δ = 7.78 (s, 1H), 7.70 (s, 1H), 7.08 (s, 1H),
5.56 (s, 2H), 5.40 (bs, 1H), 4.85 (bs, 2H), 4.53 (m, 1H), 4.46 (bs, 2H), 3.97 (s, 3H), 3.40 (bs,
2H), 3.32 (bs, 4H), 3.23 (bs, 4H), 2.74 (bs, 4H), 2.42 (m, 6H), 0.85ꢀ2.05 (m, 56H), 0.68 (s,
3H); For Bocꢀprotected Chol-IG1, 1HꢀNMR (400 MHz, CDCl3): δ = 7.86 (s, 1H), 7.69 (s, 1H),
7.07 (s, 1H), 5.56 (s, 2H), 5.39 (bs, 1H), 4.80 (t, J = 5.0 Hz, 2H), 4.50ꢀ4.54 (m, 3H), 4.44 (t, J
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= 5.0 Hz, 2H), 3.97 (s, 3H), 3.08ꢀ3.16 (m, 4H), 2.68 (m, 2H), 2.43 (m, 6H), 2.35 (bs, 2H),
0.85ꢀ2.03 (m, 60H), 0.68 (s, 3H). Bocꢀdeprotection is readily carried out by acidꢀpromoted
hydrolysis. Excess trifluoroacetic acid was added dropwise into an anhydrous CH2Cl2 solution
of Bocꢀprotected click clusters. 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 freezeꢀdrying afforded amphiphilic
dendrons as yellowish fluffy powders (72% for Chol-G1, 63% for Chol-IG1). The NMR
analysis is shown in the supporting information.
General methods for the photolysis and photorelease
The photolytic reaction was performed by irradiating the sample solutions prepared in a
quartz cuvette (1 cm x 1 cm) under a 10ꢀwatt LED (365 nm). The distance between the
sample and light source was kept at approximately 3 cm, and the exposed solutions were
analyzed by either UVꢀVis absorption or fluorescence spectroscopy.
Nileꢀred solubilization assay
A Nile red stock solution (2.5 mM) was prepared in ethanol, and a dendron stock solution was
prepared in PBS buffer at various concentrations 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 ꢁL) was added and the fluorescence
emission was measured on a spectrofluorometer using an excitation wavelength of 550 nm.
Fluorescence intensity was recorded at 635 nm. Experiments were performed in triplicate.
EtBr displacement assay
0.5 mL of pEGFPꢀC1 solution (1 ꢁg/mL) and 7 ꢁL of EtBr solution (0.1 mg/mL) were mixed
thoroughly in PBS buffer, followed by adding 8 ꢁL of the dendron solutions to the desired
N/P values. EtBr in ultrapure water was measured as the background fluorescence of EtBr,
and the solution that only contains pEGFPꢀC1 and EtBr in 1:1 binding ratio corresponds to
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the N/P = 0 with maximum emission intensity. The fluorescence emission was measured on a
spectrofluorometer using an excitation of 540 nm, and the emission spectra were recorded
from 540 nm to 700 nm. Experiments were performed in triplicate.
WST cell proliferation assay
Prior to cytotoxicity measurement, SG cells were grown in 96ꢀwell plate at a density of
3×104 cells per well. After incubation for 24 h, dendron solutions were added into each well
to attain the desired concentrations. After incubation at 37 oC for 48 h, a solution of 2ꢀ(4ꢀ
iodophenyl)ꢀ3ꢀ(4ꢀnitrophenyl)ꢀ5ꢀ(2,4ꢀdisulfophenyl)ꢀ2Hꢀtetrazolium salt (WSTꢀ1) as 10
ꢁL/well was added and the mixture was incubated at 37 oC for another 3 h. The viability of
the cells was determined by visible absorbance at 440 nm after subtracting the reference
absorbance at 650 nm with an ELISA microplate reader (EZ Read 400, Biochrom Ltd.,
Cambridge, UK). Four independent experiments were performed, and each experiment was
done in triplicate.
Acknowledgements
The authors would like to thank Chung Shan Medical University and Ministry of Science and
Technology (MOST) of Taiwan, for financially supporting this research (MOST103ꢀ2113ꢀMꢀ
040ꢀ003). The authors are also grateful to Prof. HsiehꢀChih Tsai for support with size and zeta
potential measurements and to Prof. WenꢀWei Chang for support with cytotoxicity test.
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Photoresponsive amphiphilic dendron bearing a photolabile o-nitrobenzyl group possesses
self-assembly, DNA binding and photo-induced releasing.