Steric Protected and illumination-activated tumor targeting accessory for endowing drug-delivery systems with tumor selectivity

Article abstract of DOI:10.1002/adfm.201301309

Here, an ABA-typed polymer, octadecyl-polyethylene glycol (biotin)-(o-nitrobenzyl)-octadecyl ester (CPB-p-C) with an o-nitrobenzyl group inserted between polyethylene glycol (PEG) and octadecyl ester is synthesized as an illumination-activated tumor targeting accessory for micelle-based drug carriers. The functional accessory can form a flower-like structure with folded PEG segments in aqueous solution to hide targeting biotin ligands in the core of the mixed micelle. Thus the specific binding between biotin and avidin can be suppressed by the steric hindrance of PEG shell. Upon illumination, the flower-like structure of CPB-p-C is destroyed due to the cleavage of the o-nitrobenzyl group and the biotin moieties are exposed on the surface of the micelle through the stretching process of PEG segments, generating ligand-receptor-mediated targeted delivery. By confocal laser scanning microscopy and flow cytometry, the illumination-activated, tumor-targeting delivery is studied. The influence of the amount of functional accessory in the mixed micelle on the targeting property is investigated and the optimal amount of CPB-p-C to achieve less side effects and better illumination activated tumor targeting activity is identified. The observed properties of CPB-p-C qualify it as a promising functional accessory to endow traditional drug-delivery systems with tumor selectivity. An ABA-typed polymeric accessory is synthesized to endow traditional micelle-based drug carriers with an illumination-activated tumor specific intracellular drug-delivery capacity. The targeting function of this accessory can be effectively suppressed due to the hiding of ligands. After photo-irradiation, the cleavage of o-nitrobenzyl groups in the functional accessory leads to the stretch of PEG chains, resulting in activated tumor-targeting functions.

Full text of DOI:10.1002/adfm.201301309

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Steric Protected and Illumination-Activated Tumor
Targeting Accessory for Endowing Drug-Delivery Systems
with Tumor Selectivity
Zhefan Yuan, Dan Zhao, Xiaoqing Yi, Renxi Zhuo, and Feng Li*
develop tumor-targeting drug carriers
Here, an ABA-typed polymer, octadecyl-polyethylene glycol (biotin)-(o-
nitrobenzyl)-octadecyl ester (CPB-p-C) with an o-nitrobenzyl group inserted
between polyethylene glycol (PEG) and octadecyl ester is synthesized as an
illumination-activated tumor targeting accessory for micelle-based drug car-
riers. The functional accessory can form a flower-like structure with folded
PEG segments in aqueous solution to hide targeting biotin ligands in the core
of the mixed micelle. Thus the specific binding between biotin and avidin
can be suppressed by the steric hindrance of PEG shell. Upon illumination,
the flower-like structure of CPB-p-C is destroyed due to the cleavage of the
o-nitrobenzyl group and the biotin moieties are exposed on the surface of the
micelle through the stretching process of PEG segments, generating ligand-
receptor-mediated targeted delivery. By confocal laser scanning microscopy
and flow cytometry, the illumination-activated, tumor-targeting delivery is
studied. The influence of the amount of functional accessory in the mixed
micelle on the targeting property is investigated and the optimal amount of
CPB-p-C to achieve less side effects and better illumination activated tumor
targeting activity is identified. The observed properties of CPB-p-C qualify it as
a promising functional accessory to endow traditional drug-delivery systems
with tumor selectivity.
anchored with targeting ligands, such as
antibodies,[1] vitamins,[2] peptides[3] and
aptamers.[4] These targeting molecules
are capable of binding specific cell mem-
brane receptors and benefit the drug car-
riers across cell barriers. Although direct
anchoring targeting ligands on the sur-
face of drug carriers could significantly
enhance the drug accumulation in tumor
cells status is more complicated and
delivery systems face severe challenges,
such as aggregation, rapid clearance and
immunogenicity in physiological environ-
ment, before arriving at pathological tis-
sues.[5] The targeting ligands possibly not
only fail to accomplish their missions but
also become burdens due to the specific or
nonspecific contact. In our opinion, ideal
target-mediated delivery systems should
have such properties as follows: the tar-
geting ligands of drug carriers should
be protected or their targeting functions
should be shut down during the trans-
porting process in systematic circulation,
and then be activated at pathological sites.
1. Introduction
In other words, the delivery systems should have ’targeting pro-
tection and stimuli activation’ properties. In previous studies
on intelligent drug carriers,[6–9] charge-conversion polymers
attracted intensive research interest.[6,10] Amine-rich polymers
were reacted with some derivatives of maleic anhydride to form
serum-stable anionic vehicles. When these vehicles located in
slightly acidic environment of tumor tissues, the surface charge
turned from negative to positive, resulting in an enhanced
intracellular uptake via electrostatic interaction. Different from
direct chemical modification, another approach is to physi-
cally block the contact opportunities between targeting ligands
and receptors. Zhang’s group has anchored a targeting drug
carrier with acid-cleavable polyethylene glycol (PEG) shells to
inactivate the targeting functions.[11] Due to the detachment of
PEG shells in acidic environment of tumor tissue, the exposed
targeting ligands could facilitate cellular uptake. It is worth
noting that these strategies all focused on the specific pH value
in tumor tissues, which is slightly acidic (6.8 < pH < 7.4) in
comparison with normal tissues. Since the operable pH scale is
relatively narrow, it is hard to keep a balance between pH sen-
sitivity and physiological stability, which limits their potential
Chemotherapy, as an important treatment modality, has been
widely used in cancer therapy along with radiotherapy and
surgery. However, systemic administration of anti-tumor
drugs usually leads to undesirable biodistribution and severe
side effects. A promising approach to solve this problem is to
Dr. Z. Yuan, D. Zhao, X. Yi, Prof. R. Zhuo, Prof. F. Li
Key Laboratory of Biomedical Polymers
of Ministry of Education
College of Chemistry and Molecular Science
Wuhan University
Wuhan, 430072, China
E-mail: lfsj2004@hotmail.com
Dr. Z. Yuan
State Key Laboratory of Chemical Engineering
Department of Chemical and Biological Engineering
Zhejiang University
Hangzhou, 310027, China
DOI: 10.1002/adfm.201301309
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Recently, Bae’s group has reported a new
approach to inactivate and activate the tar-
geting function.[20,21] In their studies, pLLA-
b-PEG-b-polyHis-ligand tri-block polymers
were prepared. The targeting ligands were
shielded by folded PEG chains due to the
hydrophobicity of polyHis block in physi-
ological pH value. When the drug carrier
Figure 1. The structure of CPB-p-C.
exposed to weak acidic environment, the
applications.[12] Therefore, there is an urgent need for addi-
tional breakthrough of tumor-specific targeting drug carriers.
Light, different from tumor extracellular pH gradient, is a
controllable external stimulus. Photochemical reaction and
light-induced structural transformation can be completed
within seconds or minutes by adjusting the parameters of
external light source such as wavelength and intensity.[13–15]
Recently, Kohane’ group reported an example of photo-targeted
nanoparticles.[16] This intelligent system was based on the
photocaging method.[17] The targeting peptide YIGSR on nano-
particles was firstly modified with 4,5-dimethoxy-2-nitrobenzyl
(DMNB) group to inactivate its targeting function. Then DMNB
was removed by UV-irradiation, leading to selective uptake of
nanoparticles at illuminated sites. Similar strategies were also
used to cage folic acid and the minimum sequence of cell pen-
etrating peptide (RRMKWKK) by Yeh’s[18] and Ashkqenasy’s[19]
groups, respectively. However, this photocaging method needs
to cage every targeting ligand with more than one photo-cleav-
able moiety for effective inactivation, which may be helpless for
those targeting ligands without (such as biotin) reaction site or
with many reaction sites (such as long chain cell penetrating
peptides). Furthermore, the caged ligands are still on the carrier
surface and have to face complicated physiological environment
to undergo unexpected changes.
ligands were pushed to the micellar surface by the protonated
polyHis block and then mediated the drug carriers across cell
membranes. Inspired by this strategy, we present here a steric
protected and illumination activated tumor targeting drug car-
rier based on accessory-functionalized Pluronic P123 micelles.
The accessory is an ABA-typed amphiphilic polymer, which is
shown in Figure 1. Biotin moiety, chosen as a model ligand,
is hidden between hydrophobic core and hydrophilic shell of
the micelles and the steric hindrance of the hydrophilic PEG
shell can effectively restrict specific and non-specific contacts.
Moreover, an o-nitrobenzyl group is located adjacently to biotin
as a photo-cleavable junction. Upon illumination, this junc-
tion will disconnect and biotin moiety will be popped up to
the micellar surface to facilitate the binding with the recep-
tors on cell membrane (Scheme 1). Different from the strategy
of directly removing PEG shells, the shielding effect in our
system owing to the steric hindrance may disappear immedi-
ately without relying on the degree of degradation. More impor-
tantly, by utilizing the polymer to participate into the formation
of nanoparticles with functionalized surface layer, this strategy
could endow conventional nano-sized drug carriers with acute
photo-triggered targeting function.
2. Results and Discussion
2.1. Synthesis and Characterization of Functional Accessory
The functional accessory, C18-PEG-biotin-photo site-C18 (CPB-p-
C), was prepared by a similar procedure based on click chem-
istry according to our previous report.[7] C18-PEG-biotin-N3
(CPBN3) was synthesized by introducing azide-modified biotin
moiety (Biotin-lys-N3) into carbonyldiimidazole activated Brij
100. 1-(5-Methoxy-2-nitro-4-(octadecyloxy) phenyl) ethyl prop-
2-ynylcarbamate (Alkyne-photo-C18) was prepared based on
the synthetic protocols reported by Holmes.[22] Cu-catalyzed
azide-alkyne cycloaddition (CuAAC) click chemistry was uti-
lized to connect CPBN3 and Alkyne-photo-C18. As indicated by
1
the H NMR spectrum of CPB-p-C (Figure S1), nearly 100% of
CPBN3 was transformed into CPB-p-C according to the inte-
gration ratio of H atoms on the amide bond of biotin to that
on the benzene ring. The successful synthesis of CPB-p-C was
also proved by its gel permeation chromatography (GPC) trace
and the change of FT-IR spectra before and after click chem-
istry (Figure S2 and Figure S3). The micelles prepared by Plu-
ronic P123 (PEG20-PPG70-PEG20) were chosen as model drug
carriers, which were extensively investigated as a type of long-
circulating drug vehicle.[23] To incorporate the ’targeting pro-
tection and stimuli activation’ property to the delivery system,
Scheme 1. The mechanism of illumination-activated, tumor-targeting
accessory for micelle-based drug carrier.
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2.2. Steric Protection and Phototriggered
Exposure of Targeting Ligands
In our study, the phototriggered cleavage
of CPB-p-C was based on the property of
o-nitrobenzyl group with photo-triggered
rapid cleavage property.[22] UV-vis spectrum
of CPB-p-C showed two absorption peaks
at 300 nm and at 345 nm (Figure 3A). After
5 min of UV-irradiation (λ = 365 nm, I =
30 mW/cm2), o-nitrobenzyl group was trans-
formed to o-nitrosophenyl ethanone moiety.
Thus the absorption maximum was red
shifted to λ = 382 nm. The change of UV-vis
spectrum of CPB-p-C was consistent with
previous literatures.[27] Detailed changes of
UV-vis spectrum of CPB-p-C were shown in
Figure S6. We further investigated the influ-
ence of steric hindrance on specific protein
binding and whether the biotin ligands could
be popped up onto the micellar surface
after the cleavage of CPB-p-C. It is known
that the complex of 4-hydroxyazobenzene-
2′-carboxylic acid (HABA) and avidin has
a strong absorption at 500 nm. Since the
affinity between HABA and avidin is weaker
than that between biotin and avidin, HABA
can be easily replaced by biotin from HABA/
avidin complex. Therefore, the contact
between biotin and avidin could be quantita-
tively detected by the absorption decrease at
500 nm (see Experimental Section). By the
Figure 2. Transmission electron microscopy (TEM) images of a) P123, b) 10p, and c) 20p
micelles. The scale bar is 100 nm. d) the size and distribution of the micelles obtained by
dynamic light scattering (DLS).
HABA/avidin assays, we simulated the pro-
CPB-p-C was used as a functional accessory to decorate the
P123 micelles. CPB-p-C with a hydrophobic octadecyl chain and
P123 with a hydrophobic PPG chain could self-assembled in
aqueous solution to form mixed micelles. The critical micelle
concentrations (CMC) of CPB-p-C and P123 were 32.3 mg/L
and 33.8 mg/L, respectively (Figure S4). Two types of mixed
micelles, 10p with CPB-p-C/P123 weight ratio of 1/10 and 20p
with CPB-p-C/P123 weight ratio of 2/10, were prepared in this
study. The sizes of both mixed micelles (10p: 23.4 0.2 nm,
20p: 35.8 0.3 nm) decreased in comparison with single P123
micelles (75.6 1.2 nm) due to the increase in the ratio of
hydrophilic part to hydrophobic part (Figure 2). A smaller par-
ticle size would lead to more accumulation in tumor based on
the enhanced permeability and retention (EPR) effect.[24,25] The
DLS experiments showed that the proportion of CPB-p-C also
affected the size distribution of the mixed micelles. A small
amount of large aggregations were formed in 20p, which was
not found in 10p. With the increase of CPB-p-C amount, PEG
chain tended to expand and form bridges among neighboring
micelles, resulting in the formation of large aggregations.[26] We
also tested size and distribution change of the mixed micelles
after UV-irradiation (Figure S5). 10p showed no obvious
change. In contrast, the polydispersity index (PDI) value of 20p
became much smaller, and some of large aggregations disap-
peared at the same time, which implied the damage of bridge-
linked structure in 20p after UV-irradiation.
cess of inactivating and activating targeting function. As shown
in Figure 3B, only 5.2% of biotin was available to avidin in 10p,
but nearly 11.6% of biotin could not be protected from avidin in
20p. It indicated that the outer PEG shell could sterically inac-
tivate the targeting function, and the shield capability became
weaker with the increase of CPB-p-C in the mixed micelles.
After irradiation for 5 min, there was about 7-fold increase of
available biotin found in 10p as compared to the sample before
irradiation, while the change in 20p was relatively smaller and
a 2.8-fold increase was observed after irradiation. The change
of binding capacity indicated that the shielding effect of PEG
shell was destroyed due to the photo-triggered cleavage of CPB-
p-C. The biotin moieties were popped up out of PEG shell to
bind avidin freely. It was worth noting that the photo-activated
binding capacity was still lower than that of the positive con-
trols. The introduction of triazole ring by click chemistry might
increase the hydrophobic contact with the micellar core, which
restrained the chain unfolding of PEG.
2.3. Phototriggered Cellular Uptake
Phototriggered cellular uptake of the mixed micelles was
examined in Human cervical cancer cells (HeLa cells) with
over-expression of biotin receptors[28] by confocal laser scan-
ning microscopy (CLSM). P123 was labeled with fluorescein
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in the other two samples. The shielded biotin moieties were
re-exposed on the surface of micelles due to the photo-trig-
gered cleavage of CPB-p-C. The enhanced cellular internaliza-
tion of the mixed micelles of P123-FITC was due to the biotin
receptor-mediated endocytosis.
Quantitative characterization of cellular uptake was per-
formed by flow cytometry (Figure 4E). There was no sig-
nificant difference in intracellular fluorescence intensities
between the cells treated with 10p and P123-FITC micelle.
A 2.2-fold increase of fluorescence could be found in cells
treated with 20p. Upon light stimulation, the sample of P123
micelle did not show significant difference but 10p and 20p
with the functional accessory CPB-p-C exhibited 5.2- and 6.5-
fold increase in cellular uptake, respectively. Obviously, the
increase of fluorescence intensities was correlated with the
amount of free biotin moieties on the micelle surface. It was
noted that fluorescence intensities of both samples after irra-
diation was close to that of the positive controls although there
were only about 50% of biotin moieties were free to receptors
in 10p and 20p after UV-irradiation. It is known that not all
ligands on micelles are required for effective cell targeting.[29]
The excessive addition of functional accessory did not further
improve phototriggered uptake of fundamental micelles but
led to un-specific intracellular accumulation. So 10p seems to
be a better formula than 20p.
2.4. Determination of Endocytosis Pathways
It is well known that biotinylated conjugates could cross cancer
cell membranes by a ligand receptor-mediated process,[28,30]
which was known as a clathrin-mediated endocytosis. In
our system, biotin moieties underwent a process of position
change from micellar inner core to micellar outer layer upon
light stimulation. Correspondingly, cellular uptake of the
mixed micelles increased as the exposure of biotin ligands.
To further understand the role of biotins, we investigated the
mechanism involved in the endocytosis of the mixed micelles
before and after UV-irradiation. Three inhibitors of endocy-
tosis, chlorpromazine, amiloride and genistein, were used to
inhibit clathrin-mediated endocytosis, macropinocytosis and
caveolin-mediated endocytosis, respectively (Figure 5). Gen-
istein and amiloride showed much weaker influence on cellular
uptake of these mixed micelles than chlorpromazine, implying
that the clathrin-mediated endocytosis was the primary endo-
cytosis pathway for P123 based mixed micelles. Although the
cellular internalization of 10p was mainly inhibited by chlor-
promazine regardless of illumination, the cellular uptake of
10p with activated targeting function was more dependent
on clathrin-mediated endocytosis than the one without illu-
mination. Before UV-irradiation, about 25% of cellular inter-
nalization of 10p was inhibited by chlorpromazine and this
value increased to 53% after UV-irradiation. It indicated that
biotin-receptor mediated endocytosis was the most important
endocytic pathway in this photo-activated targeting delivery. In
contrast, chlorpromazine decreased the cellular internalization
of 20p by 57% and 48% with or without UV-irradiation, respec-
tively. It implied that more unprotected biotin ligands on 20p
resulted in more unexpected clathrin-mediated endocytosis
Figure 3. A) The UV-vis absorption spectra of CPB-p-C in PBS before
and after UV-irradiation (5 min, I = 30 mw/cm2). B) Results of HABA/
avidin assay of 10p and 20p before and after UV-irradiation (5 min, I =
30 mw/cm2). Positive controls were 10+ and 20+ (WP123/WCPBN3 = 10/1
and 10/2), respectively. The amounts of biotin available in 10p and 20p
were normalized to their theoretical amount of biotin in CPB-p-C, respec-
tively; and the amounts of biotin available in 10+ and 20+ were normal-
ized to their theoretical amount of biotin in CPBN3, respectively. The
amounts of biotin in CPB-p-C and CPBN3 were calculated by 1H NMR.
isothiocyanate (FITC) to obtain P123-FITC, and the mixed
micelles were prepared by mixing P123-FITC solution and
CPB-p-C solution. Then the HeLa cells were treated with these
mixed micelles for 30 min with or without UV-irradiation (30 s
of irradiation with 30 s of nonirradiation interval between
light irradiations for total 5 min). As shown in Figure 4, 10p
and P123-FITC micelles exhibited similar cellular uptake,
which implied that introducing a small amount of functional
accessories did not lead to additional unexpected cellular
uptake. It is noted that a side effect was observed when the
amount of CPB-p-C increased. As compared to negative con-
trol, the cells treated with 20p showed enhanced green fluo-
rescence although there was no light stimulus. According to
the results of HABA/Avidin assay, we believed that the unex-
pected cellular uptake was related with the amount of unpro-
tected biotin moieties in 20p. Upon UV-irradiation, no change
could be observed in the HeLa cells treated with P123 micelles
but obviously enhanced green fluorescence could be found
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Figure 4. The CLSM images of HeLa cells treated with mixed micelles before and after UV-irradiation. The HeLa cells were incubated with FITC-
labeled P123, 10p and 20p for 30 min at 37 °C. Then the cell nucleuses were stained with Hochest 33258. For photo-triggered experiments, the cells
were exposed to UV-irradiation (30 s of irradiation with 30 s of nonirradiation interval between light irradiations) for total 5 min. A1-A6 were the green
fluorescence channel and B1-B6 were the overlap of green and blue channel. The scale bar is 20 μm. Flow cytometry analysis of FITC-labeled 10p
(C) and 20p (D) exposed to HeLa cells before (black line) and after UV-irradiation (gray filled). Quantitative comparison of photo-triggered cell uptake
of FITC-labeled mixed micelles (*p < 0.05). The negative control was P123 and the positive controls were 10+ and 20+.
hydrophilic channels[31] for drug release were shut down,
before UV-irradiation, which also confirmed our previous
resulting in the decreased release of DOX. Similar trend was
observed in the drug release profile of 20p and the amount of
released DOX from 20p was decreased from 56.1% to 48.8%
upon irradiation after 24 h. We noted that the total amount
of released DOX from 20p was more than that of 10p. It also
could be explained by the increase of hydrophilic channels in
20p.
Finally, in vitro photocontrolled targeted drug delivery was
evaluated by MTT assay in HeLa cells. As shown in Figure 7,
before UV-irradiation, the cells treated with 10p showed sim-
ilar cytotoxicity to that of negative control, which meant effec-
tive protection of PEG shells in 10p. After UV-irradiation, there
was a dramatic cell viability decrease and IC50 of 10p dropped
from 0.261 to 0.121 μg/mL. As shown in Figure 7B, compared
to 10p, 20p showed a stronger tumor cell inhibition due to the
increased amount of CPB-p-C, which caused a higher cytotox-
icity in HeLa cells even without UV-irradiation. After UV-irra-
diation, the IC50 value of 20p dropped to 0.099 μg/mL, which
was close to that of positive control (IC50: 0.072 μg/mL). It was
worth noting that the improvement on cytotoxicity of 10p or
20p after UV-irradiation was less obvious than the results of cel-
lular uptake shown in Figure 4E. According to the results in
Figure 6, a fast drug release from P123/CPB-p-C based micelles
without irradiation was found, which might override the effect
speculation.
2.5. In Vitro Photocontrolled Drug Release and Cytotoxicity
Next these mixed micelles were used to encapsulate anti-
tumor drug doxorubicin (DOX) to study the photocontrolled
targeted drug delivery. The amount of DOX loaded in these
P123 based micelles was shown in Table S1. We first evalu-
ated the drug release behavior of the mixed micelles with
or without UV irradiation. As shown in Figure 6, all sam-
ples showed relatively fast release of DOX at the initial stage
and 10p exhibited slightly quicker release of DOX than P123
/
brij100 (10/1) in 24 h. Upon UV irradiation, the drug release
behavior of 10p was suppressed and about 45.8% of DOX was
finally released after 24 h. This value was about 50.3% in the
same formulation but without UV irradiation. Considering
the negligible change of drug release behavior of P123/brij100
(10/1) after irradiation (Figure 6A), we could conclude that
the bended structure of CPB-p-C in the mixed micelles might
increase drug release from the mixed micelles before irradia-
tion. After UV irradiation, the photo-cleavage of o-nitrobenzyl
group in CPB-p-C decreased the number of PEG chains on
the surface of the micelles (two became one). Thus some
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Figure 5. Relative uptake efficiency of 10p (A) and 20p (B) in HeLa cells in
the presence of various endocytosis inhibitors with and without UV-irradi-
ation. Chlorpromazine, amiloride, and genistein were used as inhibitors
for clathrin-mediated endocytosis, macropinocytosis, and caveolin-medi-
ated endocytosis, respectively. The results obtained by by flow cytometry.
*p < 0.05, **p < 0.01.
Figure 6. The cumulative DOX release from (A) 10p and (B) 20p in
PBS (0.1 M) at 37 °C. The samples were exposed to UV-irradiation (I =
30 mW/cm2) for 5 min after 1 h of shaking.
of cellular uptake, and finally reduced the phototriggered
cytotoxcity.
The facile strategy to achieve ’targeting protection and
stimuli activation’ property in this study is applicable to other
micelle based drug carriers by using the functional accesso-
ries with similar structures. To further improve the compat-
ibility of the functional accessories for other drug carriers, we
can change the type or the molecular weights of the hydro-
phobic parts. We may also introduce different types of targeting
ligands for various cancerous cell lines. All these changes can
be easily achieved by the ’click chemistry’ based synthesis route.
Moreover, o-nitrobenzyl groups used here can respond to near
infrared light sources based on two-photon absorption,[34,35]
which will enhance the in vivo applications of our ’steric hin-
drance protection and illumination activated’ tumor targeting
drug delivery systems.
We further evaluated the cytotoxicity of blank micelles
and UV irradiation on HeLa cells. It was reported that Plu-
ronic P123 micelles displayed certain cytotoxicity at high
concentration due to the cytostatic action of Pluronic
micelles.[32] However, a low dose of P123 might limit its drug
loading content (DOX < 0.3% w/w, Table S1) and affect its
micellar formation (CMCP123 = 33.8 mg/L). Therefore, we
set the maximum dose of P123 at 200 μg/mL based on the
balance between its concentration and toxicity. HeLa cells
showed nearly 70% of cell viability at this concentration for
24 h (Figure S7). It should be noted that the concentration
of mixed micelles at DOX concentration of 0.05 μg/mL was
little lower than the CMC of P123. However, we still observed
the photo-triggered cell apoptosis. We speculated that the
mixing of P123 and CPB-p-C and the encapsulation of hydro-
phobic drugs might improve the stability of micelle.[33] Except
3. Conclusion
P
123, the other materials and UV irradiation were less toxic at
the dose used in this work. It proved again that it would safe
to endow conventional nano-sized drug carriers with acute
photo-triggered targeting function by adding small amount of
functional accessories.
We report here an accessory to endow traditional micelle-based
drug carriers with illumination-activated tumor specific intra-
cellular drug delivery capacity. Due to the folded PEG structure
in the mixed micelles, the targeting function of CPB-p-C was
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PEG100-OH, Mn: 4670), Pluronic P123 (PEG20-PPG70-PEG20, Mn: 5800),
Avidin, chlorpromazine, amiloride and genistein were purchased from
Sigma-Aldrich and used as received. Hydroxyazobenzene-2-carboxylic
acid (HABA) and Fluorescein isothiocyanate isomer I (FITC) were
purchased from Alfa aesar and used as received. Merrifield Resin
(1.0∼1.1 mmol/g) was purchased from GL Biochem (Shanghai) Ltd.
Doxorubicin hydrochloride (Dox·HCl) was purchased from Zhejiang
Hisun Pharmaceutical Co., Ltd. (China). Bovine serum albumin (BSA),
Dubelcco’s Modified Eagle’s Medium (DMEM), penicillin–streptomycin,
trypsin, and phosphate-buffered saline (PBS), and 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from
GIBCO Invitrogen Corporation. All the organic solvent used here was
dried and re-distilled by common method.
1-(3-Methoxy-4-(octadecyloxy)phenyl) Ethanone (1):
A slurry of
acetovanillone (5.12 g, 30.8 mmol), 1-bromooctadecane (11.31
g, 33.9 mmol), and K2CO3 (6.38 g, 46.2 mmol) in 50 mL of
dimethylformamide (DMF) and acetone (v/v = 1/1) was stirred at 60 °C
overnight. Then the mixture was poured into excess distilled water to
remove salts. The precipitate was collected and dissolved in 200 mL
of DCM. The solution was washed with distilled water, brine and
dried over anhydrous Na2SO4, then concentrated. The crude product
was recrystallized with CH3OH to yield white solid (1). Yield: 12.46 g
(96.7%). 1H NMR (300 MHz, CDCl3) δ: 7.56∼7.50 (m, 2H), 6.88 (s, 1H),
4.07 (t, 2H), 3.92 (s, 3H), 2.56 (s, 3H), 1.88 (m, 2H), 1.46 (m, 2H),
1.26 (m, 28H), 0.88 (d, 3H). 13C NMR (75 MHz, CDCl3) δ: 197.0, 153.3,
149.5, 130.5, 123.5, 111.3, 110.7, 69.3, 56.3, 32.2, 30.4, 30.0, 29.7, 29.3,
26.4, 26.2, 23.0, 14.4.
1-(5-Methoxy-2-nitro-4-(octadecyloxy)phenyl) Ethanone (2): A mixture
of (1) (6.28 g, 15.0 mmol) and sodium nitrite (0.14 g, 2.0 mmol) in
30 mL of DCM was stirred at room temperature. Nitric acid (65–68%,
4.36, 45.0 mmol) in 10 mL of DCM was added dropwise to the mixture
and the reaction was chilled in an ice-water bath. After addition, the
emulsion was kept stirring at room temperature for 6 h. Then the
mixture was poured into water and extracted with CH2Cl2. The organic
layer was combined and washed with saturated NaHCO3, distilled water,
brine and dried over anhydrous Na2SO4. The solvent was concentrated
and the residue was purified by chromatography on silica gel by eluting
petroleum ether with 15% of ethyl acetate. The product was obtained as
yellow crystal (2). Yield: 4.41 g (63.5%). 1H NMR (300 MHz, CDCl3) δ
7.59 (s, 1H), 6.77 (s, 1H), 4.09 (t, 2H), 3.96 (s, 3H), 2.49 (s, 3H), 1.88
(m, 2H), 1.47 (m, 2H), 1.26 (m, 28H), 0.87 (d, 3H). 13C NMR (75 MHz,
CDCl3) δ 200.2, 154.3, 149.3, 138.5, 132.6, 108.8, 107.8, 69.9, 56.8, 32.1,
30.5, 29.9, 29.7, 29.5, 28.9, 26.0, 22.9, 14.3.
Figure 7. Cell viability studies of HeLa cells incubated individually with
DOX-loaded 10p (A) and 20p (B) for 24 h. Negative controls were DOX
loaded P123/Brij100 mixed micelles and positive controls were DOX
loaded P123/CPBN3 mixed micelles (w/w = 10/1 for 10p and 10/2 for
20p, respectively). *p < 0.05, **p < 0.01. The maximum amount of DOX
loaded micelles (at CDOX = 0.6 μg/mL) used in MTT assays were 215.8,
226.4, 212.0, 229.0, 236.2 and 232.6 μg/mL, which corresponded to DOX
loaded 10p, 10+, P123/Brij 100 (10/1), 20p, 20+, and P123/Brij 100 (10/2)
micelles, respectively.
1-(5-Methoxy-2-nitro-4-(octadecyloxy)phenyl) Ethanol (3): A solution
of (2) (1.50 g, 3.23 mmol) in 50 mL of THF was mixed with NaBH4
(263 mg, 6.95 mmol) at room temperature. The reaction mixture was
stirred for 24 h and then partitioned between EtOAc and saturated
NH4Cl. The combined organic phase was dried over anhydrous
Na2SO4 and concentrated under vacuum. The residue was purified by
chromatography on silica gel by eluting (20% ethyl acetate/petroleum
ether to 30% ethyl acetate/petroleum ether) to give (3) as yellow solid.
effectively suppressed by PEG shells before UV-irradiation.
After UV-irradiation, the cleavage of o-nitrobenzyl groups in
CPB-p-C led to the stretch of PEG chains, resulting in the expo-
sure of biotin moieties. In vitro studies showed that CPB-p-C
indeed improved the intracellular uptake of mixed micelles by
photo stimulus and further enhanced the cytotoxicity of DOX-
loaded micelles in HeLa cells. The strategy developed in this
study is applicable to other micelle based drug carriers to
achieve ’targeting protection and stimuli activation’ property.
1
Yield 1.34 g (88.9%). H NMR (300 MHz, CDCl3) δ: 7.88 (dd, 1H), 7.71
(d, 1H), 6.88 (d, 1H), 4.08 (t, 2H), 3.92 (s, 3H), 1.86 (m, 2H), 1.45 (m,
2H), 1.23 (m, 28H), 0.85 (d, 3H). 13C NMR (75 MHz, CDCl3) δ: 154.5,
149.3, 141.4, 118.0, 110.9, 106.8, 69.7, 56.5, 32.2, 29.9, 29.8, 29.8, 29.6,
29.1, 26.1, 22.9, 14.4.
1-(5-Methoxy-2-nitro-4-(octadecyloxy)phenyl) Ethanol (Alkyne-p-C, 4): A
solution of (3) (1.0 g, 2.38 mmol) in 20 mL of DCM was mixed with
1,1′-carbonyldiimidazole (CDI, 0.77 g, 4.75 mmol) at room temperature.
The reaction was stirred for 4 h and then partitioned between DCM
and distilled water. The combined organic phase was washed twice
with distilled water, dried over anhydrous Na2SO4 and concentrated
under vacuum. The residue was directly used in the next step without
further purification. The aforementioned residue was resolved in 15
mL of DCM and added dropwise with a solution of 2-propynylamine
(0.26 g, 4.72 mmol) in 5 mL of DCM. The reaction was stirred overnight
at room temperature and then washed twice with distilled water, dried
over anhydrous Na2SO4 and concentrated under vacuum. The residue
4. Experimental Section
Materials:
Acetovanillone,
1-bromooctadecane,
sodium
nitrite, nitric acid (65%∼68%), sodium borohydride (NaBH4),
1,1′-carbonyldiimidazole (CDI), 2-propynylamine and N,N,N′,N″,N″–
pentamethyl diethylenetriamine (PMDETA) were purchased from
Aladdin-reagent (China) and used as received. CuBr, Brij 100 (C18-
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was purified by column chromatography (SiO2, eluent: petroleum ether
with 15% of ethyl acetate). The (4) was obtained as yellow powder. Yield:
0.85 g (72.6%). 1H NMR (300 MHz, CDCl3) δ: 7.57 (s, 1H), 7.00 (s, 1H),
6.41 (m, 1H), 5.34∼5.17 (m, 1H), 4.04 (t, 2H), 3.96 (m, 5H), 2.23 (s,
1H), 1.85 (m, 2H), 1.60 (d, 3H), 1.45 (m, 2H), 1.26 (m, 28H), 0.88 (t,
3H). 13C NMR (75 MHz, CDCl3) δ: 155.1, 154.2, 147.7, 139.8, 133.6,
108.9, 108.2, 79.8, 71.9. 69.7, 56.5, 32.1, 31.0, 29.9, 29.8, 29.8, 29.6, 29.1,
27.4, 26.1, 24.6, 22.9, 22.4, 14.3.
(aqueous phase). At the fixed time intervals, the UV spectrum of cpb-p-C
solution was recorded on UV-Vis spectrophotometer.
HABA/Avidin Assay: As described in a previous publication,[7] the
HABA/avidin assay was performed according to protocols reported by
Wooly.[36] The HABA solution was prepared by dissolving 24.2 mg of
4-hydroxyazobenzene-2-carboxylic acid (0.1 mmol) in 10 mL of aqueous
sodium hydroxide solution (10 mM). Then the HABA/avidin solution
was made by dissolving 5.0 mg of avidin in 9.7 mL of 50 mM PBS with
50 mM NaCl (pH = 7.2), followed by adding HABA solution (300 μL).
10p or 20p (CP123 was fixed at 10 mg/mL) was added into a sealed quartz
cuvette and placed under the UV lamp at an intensity of I = 30 mW/cm2
for 5 min. Aliquots (22.2 μL) were taken out at the regular time intervals
and mixed with 200 μL of HABA/avidin solution. The final mixtures
were kept at 37 °C for 6 h to reach equilibrium. 200 μL of the mixtures
were transferred into a 96-well plate and read by UV/Vis microplate
spectrophotometer (Multiskan GO, Thermo Fisher).
Biotin-lys(PEG-C18)-N3 (5): According to a previous publication,[7]
biotin-lys(PEG-C18)-N3 was synthesized as follows. Briefly,
H-lys(Boc)-OH was reacted with biotin N-hydroxy succinimide
ester (biotin-NHS) in the presence of triethylamine to form
biotin-lys(Boc)-OH. After the carboxyl group was reacted with
aminopropylazide, the Boc group was removed by adding excess TFA
(90% in DCM) to obtain free amino group. Finally, biotin-lys(PEG-
C18)-N3 was obtained by the reaction between biotin-lys-N3 and
carbonyl imidazole activated Brij 100.
Cellular Uptake by CLSM: Cell internalization was observed on
confocal laser scanning microscopy (CLSM, Nikon C1-si). All the
mixed micelles used in this measurement were based on fluorescein
isothiocyanate (FITC) modified P123 (concentration: 0.1 mg/mL). Hela
cells were seeded in a 35 mm cell culture dish with glass bottom and
incubated at 37 °C for 24 h. The samples in DMEM with 10% fetal
bovine serum (FBS) were added to replace the medium. After 30 min of
incubation, the medium was removed and the cells were washed three
times with PBS. The nuclear was stained with Hochest 33258 and the
cells were fixed with 4 wt% formaldehyde in PBS for 20 min at room
temperature. A drop of mounting media (10% PBS, 90% glycerol) were
added to mount the cells. For UV irradiation measurement, the cell
culture dish was exposure to the UV light in an alternate way (30 s of
irradiation with 30 s of nonirradiation interval between light irradiations)
for total 5 min of irradiation at an intensity of I = 30 mW/cm2 and
incubated for another 20 min. The fluorescence was examined under
excitation at 405 nm for Hochest 33258 and 488 nm for FITC.
Flow Cytometry: HeLa cells were seeded in 6-well plates and
incubated for 24 h at 37 °C with 5% CO2. The samples in DMEM with
10% fetal bovine serum (FBS) were added to replace the medium and
incubated for 30 min. For UV irradiation measurement, the 6-well plate
was exposed to UV light in an alternate way (30 s of irradiation with 30 s
of nonirradiation interval between light irradiations) for total 5 min of
irradiation at an intensity of I = 30 mW/cm2 and incubated for another
20 min. Then the medium was removed and the cells were washed with
PBS (4 °C) for three times, harvested by 0.25% (w/v) trypsin-0.03%
(w/v) EDTA solution. The cells were pelleted by centrifuge (1000 rpm
for 5 min), resuspended in fresh PBS and analyzed on a flow cytometer
(FACSAriaTMIII). Flow cytometry analysis was used to evaluate the
endocytosis pathways of 10p and 20p mixed micelles with and without
UV irradiation. The HeLa cells were pre-incubated with endocytosis
inhibitors (inhibitor of clathrin-mediated endocytosis: chlorpromazine
hydrochloride (30 μg/mL); inhibitor of caveolin-mediated endocytosis:
genistein (15 μg/mL); inhibitor of macropinocytosis: amiloride
(133 μg/mL) for 30 min at 37 °C. Then the mixed micelles in complete
medium with corresponding concentration of inhibitors were added to
replace the medium. After the same procedures as cell uptake, the cells
were analyzed on a flow cytometer.
Alkyne-resin: Merifield’s resin (1 g, 1.0∼1.1 mmol), K2CO3 (6.9 g,
0.050 mol), and propargyl alcohol (2.8 g, 0.050 mol) were added in 40
mL of DMF and the resulting mixture was stirred at 110 °C for 48 h.
The obtained resin was washed several times with water and THF,
respectively. The alkyne-resin was collected by filtration and dried under
vaccum. FTIR (cm−1): 2330 (HCϵC−CH2) and 3300 (HC ϵC−CH2).
CPB-p-C: Biotin-lys(PEG-C18)-N3 (0.5 g, 0.10 mmol) and 4 (0.16 g,
0.29 mmol) were dissolved in 2 mL of DMF. After three freeze-pump-thaw
cycles, PMDETA (5.2 mg, 0.03 mmol) and CuBr (4.3 mg, 0.03 mmol)
were added and the solution was stirred under Ar atmosphere at 35 °C
for 48 h. Then the solution was frozen under liquid nitrogen and alkyne-
resin (0.2 g, ∼0.2 mmol), fresh PMDETA (17.3 mg, 0.1 mmol) and CuBr
(14.3 mg, 0.1 mmol) were added. After three freeze-pump-thaw cycles
again, the solution was stirred for another 48 h. Finally, the reaction was
stopped and the solution was diluted with THF. After passing through
a short alumina column to eliminate the copper and resin, the product
was obtained by three times of precipitation in excess ice-cooled ethyl
ether and dried under vacuum.
1
Polymer Characterization: H NMR spectra were recorded at 300 MHz
on a Mercury VX-300 spectrometer by using tetramethylsilane (TMS) as
the internal reference. 13C-NMR spectra were recorded at 75 MHz with
the solvent carbon signal as reference. Infrared spectra were recorded on a
Nicolet Avator 360 FT-IR spectrometer. The molecular weight and molecular
weight distribution of the polymer were determined by gel permeation
chromatography equipped with a Waters 2690 separation module and a
Waters 2410 refractive index detector. DMF was used as eluent at a flow
rate of 0.5 mL min−1 with the temperature maintained at 30 °C and the
results were calibrated against with polyethylene glycol standards.
Micelle Preparation and Characterization: In this work, all the micelle
solutions were prepared by diluting pre-made high concentration
micelle stocks and kept in 37 °C for 24 h to reach equilibrium before
use. For example, 10p (P123: 1 mg/mL) was prepared as follows:
equal volumes of P123 micelle (10 mg/mL) and CPB-p-C (1 mg/mL)
were mixed and then diluted to 5-fold volume. Size and distribution
measurements of the micelles were performed on a Nano-ZSZEN3600
(Malvern) instrument. Critical micelle concentration (CMC) of CPB-p-C
and P123 were collected by the pendant drop method for measuring
surface tension for a series of sample solutions. An optical contact
angle measuring device (OCA 30, Dataphysics) was used to measure
the surface tension. The CMC was estimated as the intersection when
extrapolating the surface tension at low and high concentration regions.
Transmission electron microscopy images (TEM) were obtained using
a JEM-100CXII transmission electron microscope. A drop of micelle
solution was placed onto a copper grid with carbon film and then
stained with phosphotungstic acid. The TEM images were observed at
an acceleration voltage of 200 keV.
Drug Encapsulation: DOX was loaded into the micelles as a model anti-
tumor drug by o/w emulsion method. Typically, 0.1 mg of doxorubicin
hydrochloride was added into CHCl3 (3 mL) and solubilized by adding
two equivalents of triethylamine under sonication. The above solution
was added into a stirred mixed micelle solution (CP123: 0.5 mg/mL,
volume: 60 mL), to form o/w emulsion. The emulsion was kept stirring
overnight to evaporate CHCl3 and then dialyzed against with deionized
water to get rid of free DOX and triethylamine salt. Then the mixture was
passed through a 0.45 μm filter and lyophilized (2 mL per one tube).
The loaded content of DOX was determined by fluorescence emission
spectroscopy. The excitation wavelength was set as 480 nm and the
emission wavelength was 560 nm with 5 nm of slit width. The Loading
content (LC) and loading efficacy (LE) were calculated by the following
equations.
Photodegradation: The photo-triggered detachment of CPB-p-C was
performed in aqueous phase (phósphate buffered saline (PBS), 0.1 M)
and organic phase (1,4-dioxane). CPB-p-C solution was added into a
sealed quartz cuvette and placed under UV lamp (FC-100/F, Spectronics)
at an intensity of I = 10 mW/cm2 (organic phase) or 30 mW/cm2
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LC% = (weight of the drug in micelles)/
×(weight of the drug loaded micelles) × 100%
LE% = (weight of the drug in micelles)/
×(weight of the feeding drug) × 100%
In vitro Release of DOX-Loaded Micelles: Typically, the lyophilized
DOX-loaded micelles were resolved in PBS (2 mL, 0.5 mg/mL) and
transferred into a dialysis bag (Mw cutoff: 3500). The dialysis bag was
immersed into 20 mL of PBS dialysate (0.1 M, pH 7.4) and then shaken
at 37 °C. At the preset intervals, 2 ml of solution was withdrawn from
the release medium and replaced with equal volume of fresh PBS. As to
the photo-triggered experiments, the dialysis bag was exposed to 5 min
of UV irradiation after 1 h of shaking. The DOX content released from
micelles was determined by fluorescence emission spectroscopy.
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cells by using MTT assay. 100 μL of cell suspension containing 5×103
cells were seeded into each well of a 96-well plate and incubated at
37 °C with 5% CO2 for 24 h. Then the cells were treated with samples
at various concentrations and carried out a further incubation for 24 h.
For UV irradiation measurement, the cells were exposed to UV light
in an alternate way (30 s with and 30 s without irradiation) for total
5 min of irradiation at an intensity of I = 30 mW/cm2 and incubated
for 24 h in incubator. Then the medium in each well was replaced with
200 μL of fresh mediums and 20 μL of MTT solution in PBS (5 mg/
mL). After 4 h of incubation, the medium was carefully removed and
replaced by 100 μL DMSO to solve the purple crystals. When the purple
solution was homogeneous, the absorbance at 570 nm was recorded by
a microplate reader (Multiskan GO, Thermo Fisher). Cell viability was
calculated as follows.
Cell viability(%) = (Atreated− Ablank)/
× (Acontrol − Ablank) × 100%
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Acknowledgements
This work was financially supported by National Basic Research Program
of China (2011CB606202), the Natural Science Foundation of Hubei
Province (2012FFB04327) and National Natural Science Foundation of
China (20704032 and 21374082).
Received: April 18, 2013
Revised: September 14, 2013
Published online: December 2, 2013
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wileyonlinelibrary.com
Adv. Funct. Mater. 2014, 24, 1799–1807
2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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