Silicon Phthalocyanines Axially Disubstituted with Erlotinib toward Small-Molecular-Target-Based Photodynamic Therapy

Article abstract of DOI:10.1002/cmdc.201700384

Small-molecular-target-based photodynamic therapy—a promising targeted anticancer strategy—was developed by conjugating zinc(II) phthalocyanine with a small-molecular-target-based anticancer drug. To prevent self-aggregation and avoid problems of phthalocyanine isomerization, two silicon phthalocyanines di-substituted axially with erlotinib have been synthesized and fully characterized. These conjugates are present in monomeric form in various solvents as well as culture media. Cell-based experiments showed that these conjugates localize in lysosomes and mitochondria, while maintaining high photodynamic activities (IC₅₀ values as low as 8 nm under a light dose of 1.5 J cm^?2). With erlotinib as the targeting moiety, two conjugates were found to exhibit high specificity for EGFR-overexpressing cancer cells. Various poly(ethylene glycol) (PEG) linker lengths were shown to have an effect on the photophysical/photochemical properties and on in vitro phototoxicity.

Full text of DOI:10.1002/cmdc.201700384

Accepted Article
Title: Silicon Phthalocyanines Axially Disubstituted with Erlotinib
towards Small Molecular Target-Based Photodynamic Therapy
Authors: Juan-Juan Chen, Yi-Zhen Huang, Mei-Ru Song, Zhi-Hong
Zhang, and Jin-Ping Xue
This manuscript has been accepted after peer review and appears as an
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To be cited as: ChemMedChem 10.1002/cmdc.201700384
Link to VoR: http://dx.doi.org/10.1002/cmdc.201700384
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Silicon Phthalocyanines Axially Disubstituted with Erlotinib
towards Small Molecular Target-Based Photodynamic Therapy
Juan-Juan Chen,[a] Yi-Zhen Huang,[a] Mei-Ru Song,[a] Zhi-Hong Zhang*[b] and Jin-Ping Xue*[a]
[a]
Dr. J.-J. Chen, Y.-Z. Huang, M.-R. Song, Prof. J.-P. Xue
National & Local Joint Biomedical Engineering Research Center on Photodynamic Technologies, and Fujian Engineering Research Center of Functional
Materials, College of Chemistry, Fuzhou University, Fuzhou 350116, China
Prof. Z.-H. Zhang
[b]
Fuzhou General Hospital of Nanjing Military Command, Fuzhou 350005, China
E-mail: zhangzhh85@126.com
Supporting information for this article is given via a link at the end of the document.
Abstract: Small molecular target-based photodynamic therapy—a
promising targeted anticancer strategy—has been developed by the
conjugation between zinc (II) phthalocyanine and small molecular
target-based anticancer drug. To inhibit the self-aggregation and
avoid the isomeride problem of phthalocyanine, two silicon
phthalocyanines di-substituted axially with erlotinib have been
synthesized and fully characterized. These conjugates exist as a
monomeric form in different solvents and the culture media. Cellular
experiments showed that these conjugates localize in lysosomes
and mitochondria, and keep high photodynamic activities (IC50 is as
low as 8 nM under 1.5 J/cm2 light dose). Having erlotinib as the
targeting moiety, two conjugates exhibited high specificity to the high
EGFR expression cancer cells. The different PEG linker length was
proven to have an effect on photophysical/photochemical properties
and further in vitro phototoxicity.
moiety for conjugation with a photosensitizer to achieve targeted
PDT. In these systems, we chose erlotinib to conjugate with zinc
(II) phthalocyanines (ZnPc) at α- or β-positions via various
lengths of oligoethylene glycol spacers. By introducing the
erlotinib moiety, these conjugates exhibited high targeting ability
to HepG2 cancer cells and A431 tumor tissues, and also
maintained high photodynamic efficacy.[21–23] There are two
challenges in the clinical PDT applications of ZnPc conjugates:
the great tendency to form aggregates,[8,24–33] and low-purity
resulting from isomeride.[34]
As reported, a silicon center allows the introduction of two
appropriate axial ligands to inhibit the π-π stacking tendency of
phthalocyanine (Pc) rings; thus, axially-substituted silicon
phthalocyanines (SiPcs) have emerged to improve the
hydrophilicity and inhibit the self-aggregation of Pcs.[32,35–39] On
the other hand, axially-substituted SiPc can efficiently avoid the
isomeride problem, and its single structure provides the potential
for clinical application.[34] More importantly, SiPcs enable the
introduction of two small molecular target-based moieties at the
axial positions, which may improve the specificity to cancer and
anticancer activity. For these reasons, SiPcs are of great interest
to our group for conjugating with small molecular target-based
drugs for targeted PDT.
1. Introduction
Photodynamic therapy (PDT) has emerged as a promising
minimally-invasive modality for cancer treatment.[1–4] It is based
on a nontoxic photosensitizer (PS) which can produce cytotoxic
reactive oxygen species (ROS) in the presence of oxygen and
light. The overall therapeutic efficacy depends greatly on the
PS.[3,5] As a result, a large number of PS have been developed
to identify desirable photosensitizing systems and to optimize
their photodynamic efficacy.[5–10] One major drawback regarding
PS is the lack of tumor targeting; therefore, various approaches
which can improve tumor targeting have been explored,
including conjugation with tumor-specific bio-targets[11–15] and
encapsulation in nanocarriers.[4,15–18] These two approaches
have achieved promising results and decades of progress, but
have also faced some challenges. For bio-targets, the
complicated structure and low stability of biomacromolecules
always result in their difficult synthesis and purification process;
moreover, their biological activities are often changed or even
lost during the process of modification.[19] For nanocarriers,
accuracy is a basic demand for drug administration; thus, a
complex control procedure over the size, shape, stability, drug
loading, and releasing capacity of nanoparticles is needed.[20]
As part of our ongoing interest in the development of targeted
PS, we recently developed a ―smart‖ tumor targeted strategy in
PDT: PS was conjugated with a small-molecule target-based
anticancer drug, some of which can target the epidermal growth
In this article, we report erlotinib-based SiPc conjugates for
dual molecular targeting and photodynamic therapy. The
parameters of photophysical/photochemical properties, solubility,
tumor-targeting, and anticancer activity were studied to evaluate
whether these SiPc-erlotinib conjugates have good water-
solubility and exhibit high cancer selectivity and anticancer
activity for targeted photodynamic therapy.
2. Results and Discussion
2.1 Synthesis and Characterization
The preparation of erlotinib-based SiPc conjugates and the
control compound is shown in Scheme 1. Oligoethylene was
mono-tosylated by TsCl and then underwent nucleophilic
substitution with erolotinib analog to afford erlotinib-based PEG
compounds (PEG-n-E). Reaction of isoindoline-1,3-diimine and
SiCl4 in quinoline at 220 °C gave SiPc. Further axial substitution
of SiPc with PEG-n-E compounds or triethylene glycol (TEG)
afforded erlotinib-based SiPc (SiPc-n-E), or the control
compound (SiPc-PEG). The detailed synthetic procedures are
shown Scheme 1.
factor receptor (EGFR) overexpressed in cancer cells.[21–23]
A
small-molecule target-based anticancer drug with a simple
chemical structure and high stability would be a more suitable
1
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Scheme 1. Synthesis of erlotinib-based silicon phthalocyanine (SiPc-n-E) and control compound PEGlyation silicon phthalocyanine (SiPc-PEG).
All the prepared compounds were characterized with 1H-NMR, of SiPc-n-E and SiPc-PEG in different organic solvents all
HR-MS, and HPLC (see Supplementary Materials Figures S7–
S18). As expected, the H NMR spectrum of SiPc-n-E showed
exhibit typical electronic spectra of phthalocyanines with sharp
and intense Q bands. Then, taking SiPc-n-E and SiPc-PEG in
DMF as an example, we examined the UV-vis spectra monitored
at different concentrations ranging from 0.5 μM to 10.0 μM in
Figure 1B. There were no new bands (ca. 710 nm for J-
aggregation[41–43]) observed, and their Q-bands strictly followed
the Lambert–Beer law. Similar results can be seen in Figure S2.
All these results indicate that SiPc-n-E and SiPc-PEG are
essentially free from aggregation in these organic solvents.
The aggregation behavior of SiPc-n-E and SiPc-PEG were
also studied in aqueous solution and culture media (Figure 2).
As shown in Figures 2 and S3, the spectra of SiPc-PEG in water
showed a small new band (ca. 710 nm), which is as the same as
that in the previous report[44]. The similar spectra of SiPc-PEG
were observed in Roswell Park Memorial Institute-1640 (RPMI-
1640), and Dulbecco Minimum Essential Medium (DMEM)
culture media (without antibiotics and fetal bovine serum). This
new band can be attributed to the formation of J-aggregates on
the basis of the exciton theory of Kasha et al.,[41-43] and points
toward the formation of aggregation. The Q band of SiPc-n-E is
sharp and strong with no observable new bands, which indicates
that little or no aggregation is occurring in water, RPMI 1640, or
DMEM culture media. The same results can also be acquired in
the fully supplemented RMPI 1640 culture medium (with
antibiotics and fetal bovine serum, Figure S4). Therefore, these
axial erlotinib moieties are effective in decreasing the
aggregation of the phthalocyanine, which is extremely important
for PDT application.
1
peaks belonging to Hα and Hβ protons at between 9.52–9.31 and
8.26–8.20 ppm, respectively. Because of the magnetic
anisotropy of the phthalocyanine ring,[40] the peaks belonging to
aliphatic CH2 protons in Si-O-CH2 bond were shifted towards the
negative region. The H protons of -NH- appeared at 8.70–8.57
ppm, and the peak belonging to -C≡CH appeared at 2.52 ppm
for SiPc-2-E and 3.05 ppm for SiPc-3-E, respectively. The high
resolution mass spectrometer (HRMS) clearly indicated the
formation of the desired products. All HPLC spectra revealed a
single peak, indicating that SiPc-n-E and SiPc-PEG were pure
(>97%).
2.2 Photophysical and Photochemical Properties
Because of their 18 π-electron systems, phthalocyanine
compounds always have a high aggregation tendency, which
often causes weak solubility or insolubility in many solvents,
which then further seriously affects their photophysical and
photochemical properties.[8,24-32] Therefore, solvents effect and
concentrations effect were investigated according to the
absorption spectra of all compounds in organic solvents and
aqueous solutions. Comparing the UV-vis spectra of free
erlotinib (Figure S1) with that of SiPc-n-E and SiPc-PEG (Figure
1) in DMF, we found that the absorption bands of free erlotinib
did not pollute SiPc-n-E conjugates’ Q band, but contributed to a
part of their B bands. As shown in Figure 1A, the UV-vis spectra
2
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As shown in Figure S5, the emission spectra of these
compounds are essentially a mirror image of the absorption
spectra in the Q-band. The photophysical data are summarized
in Table 1. Both erlotinib-based SiPc conjugates and SiPc-PEG
showed
a strong fluorescence emission at 679–684 nm.
Compared to SiPc-PEG (ΦF = 0.42), the fluorescence quantum
yield of SiPc-2-E is slightly lower (ΦF = 0.34), while SiPc-3-E
maintained the similar fluorescence quantum yield (ΦF = 0.43).
The same trend was observed in the results of single oxygen
quantum yield (ΦΔ), which is an important parameter to evaluate
the photosensitizing efficiency of these compounds (SiPc-2-E <
SiPc-3-E ≈ SiPc-PEG, Table 1). These results indicate that
when the PEG linkers were short (n = 2), erlotinib may perturb
the photophysical and photochemical progress of the Pc ring.
Therefore, suitable linker length (n = 3) is very necessary for the
photosensitizers erlotinib-based SiPc conjugates.
Table 1. Photophysical/photochemical data for SiPC-n-E and SiPc-PEG in
DMF.
[a]
[b]
Compound
λabs/nm (log ε)
λex/nm
λem/nm
ΦF
ΦΔ
SiPc-2-E
SiPc-3-E
SiPc-PEG
675 (5.35)
673 (5.35)
674 (5.36)
674
676
673
680
679
684
0.34
0.43
0.42
0.26
0.34
0.39
Figure 1. Absorption spectra of SiPc-n-E and SiPc-PEG in (a, b, c) different
solvents (DCM, DMF, DMSO, EtOH, and THF) and (d, e, f) different
concentrations in DMF (from bottom to top: 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 5.0, 7.0,
10.0 μM).
[a] Relative to ZnPc (ΦF = 0.28 in DMF). [b] Relative to ZnPc (ΦΔ = 0.56 in
DMF).
2.3 In Vitro Cancer Cells Targeting
To demonstrate the cancer targeting ability of these erlotinib-
based SiPc conjugates, we selected two different EGFR
expression and morphological cells: the HepG2 cancer cells (a
carcinoma cell line overexpressing EGFR) and HELF normal
cells (a normal cell line with low EGFR expression). In the
confocal images study, we mixed these two cell lines in a cell
culture dish to investigate their EGFR-dependent competitive
uptake to avoid small differences in different cell culture dishes.
As shown in Figure 3, all SiPc-n-E conjugates exhibited
obviously brighter intracellular fluorescence in HepG2 cancer
cells than those in HELF normal cells. However, there were no
obvious different cellular fluorescence intensities between
HepG2 and HELF cells for the control compound SiPc-PEG. To
accurately express the intracellular concentration, we lysed the
cells which were incubated with conjugates to extract the
conjugates. The intracellular conjugates concentration and the
cell numbers were determined by measuring their fluorescence
emission intensity and the protein concentration on a microplate
reader, respectively. The intracellular uptake was expressed in
nmol SiPc/mg protein, and the results are depicted in Figure 4.
The cellular uptake of SiPc-n-E by HepG2 cells is clearly higher
than that by HELF cells with high significant differences,
whereas for SiPc-PEG, the cellular uptake by both cell lines is
nearly identical.
Figure 2. Absorption spectra of SiPc-n-E and SiPc-PEG in water.
3
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2.4 Subcellular Localization
The subcellular localization in HepG2 cells was further
investigated by confocal microscopy. As shown in Figure 5,
good colocalization of SiPc-2-E or SiPc-3-E with LysoTracker
Green (lysosomes) and MitoTracker Green FM (mitochodria)
was observed. The very similar fluorescence intensity line
profiles of SiPc-n-E (red line) and LysoTracker or MitoTracker
(blue line) traced along the white line in the confocal images also
confirmed that SiPc-2-E and SiPc-3-E can target mitochondria
and lysosomes, which may be due to the water solubility of
SiPc-n-E conjugates, making it more difficult for them to cross
the membrane of lysosomes and mitochondria and remaining in
these two organelles. In addition, it was found that there was no
staining of nucleus with SiPc-n-E conjugates.
Figure 3. (a) Confocal fluorescence images of mixed HepG2 (red line region)
and HElF cells (white line region) after incubation with SiPc-n-E and SiPc-PEG
for 24 h (all at 1 μM). (b) Average fluorescence intensity (AFI) of SiPc-n-E and
SiPc-PEG in HepG2 and HELF cells (mean ± SD, n = 30–50, paired t-test, ** p
< 0.01, # p > 0.05).
To further investigate EGFR-mediated uptake, competitive
binding experiments were performed by co-incubation of HepG2
cells with erlotinib for 24 h. It was shown that the addition of
erlotinib significantly inhibited the cellular uptake of SiPc-n-E
(Figure 4). However, there were no obvious different cellular
uptakes after adding erlotinib for SiPc-PEG, which has no
erlotinib moieties. All of these results indicate that the
introduction of two erlotinib moieties resulted in EGFR-
dependent cancer targeting of SiPc-n-E conjugates.
Figure 5. Subcellular localization of SiPc-n-E (ex = 633 nm, em = 650-750 nm)
in HepG2 cells with 4′,6-diamidino-2-phenylindole (DAPI, ex = 405nm, em
=450-550 nm ), LysoTracker (ex
= 488 nm, em = 510-570 nm), and
MitoTracker (ex = 488 nm, em = 510-570 nm). Scale bar: 20 μm.
Figure 4. Relative cellular uptake (RCU) of SiPc-n-E and SiPc-PEG in HELF
cells（ white bar） and HepG2 cells (red bar and blue bar) co-incubated
without/with erlotinib (mean ± SD, n = 30–50, paired t-test, ** p < 0.01, # p >
0.05).
2.5 In Vitro Photodynamic Activity
The generation efficiency of reactive oxygen species (ROS)—
which have been implicated in the cell death associated with
conventional PDT—was investigated against HepG2 cancer
4
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cells (Figures
6 and S6). Both SiPc-2-E and SiPc-3-E
conjugates showed high ROS generation. However, SiPc-3-E
had higher ROS levels than that of SiPc-2-E, which is in
accordance with their ΦΔ in DMF. The results also reveal that
the length of the PEG linker has an effect on the relationship
between the two erlotinib moieties and Pc ring, and further
influence the ROS generation efficiency.
The photodynamic therapeutic efficacy of SiPc-n-E was
evaluated against HepG2 cancer cells. The dose-dependent
survival curves are shown in Figure 7. All conjugates essentially
had no dark cytotoxicity, and exhibited significantly high
photocytotoxicity upon low-dose light illumination (670 nm, 1.5 J/
cm2, 25 mW/cm2) which is usually used in phthalocyanine-based
PDT.[21-22, 45-47] The low IC50 (8–27 nM) values follow the order
SiPc-3-E > SiPc-PEG > SiPc-2-E, which is in accordance with
their ΦΔ and ROS levels. Their high photodynamic anticancer
activities are attributed to their low aggregation in the culture
medium and specific subcellular localization. The PEG linker
length has an effect on single oxygen and ROS generation, and
ultimately on phototoxicity.
Figure 6. Cellular reactive oxygen species (ROS) generation level of SiPc-n-E
in HepG2 cells. Data are expressed as the mean ± SD of three independent
experiments, each performed in sextuple (paired t-test, *** p < 0.0001).
Figure 7. Cytotoxic effects of SiPc-n-E and SiPc-PEG towards HepG2 cells in (a) dark and (b) 1.5 J/cm2 light (670 nm). Data are expressed as mean ± SD of
three independent experiments, each performed in quadruplicate.
3. Conclusions
4. Experimental Section
In summary, we have designed and synthesized two silicon
4.1 Reagents and Apparatus
phthalocyanine–erlotinib conjugates to inhibit the self-
aggregation and improve the hydrophilicity of Pcs in small
molecular target-based photodynamic cancer therapy. We
demonstrated that these erlotinib-based SiPcs have no
aggregation in different solvents and concentrations in DMF.
The water solubility made them specifically localized in
lysosomes and mitochondria. These two erlotinib-based SiPcs
were demonstrated to have high cancer targeting abilities due to
the introduction of the erlotinib moiety, and maintained the high
phototoxicity of the SiPc core (IC50 is as low as 8 nM). Moreover,
we showed that the length of the linker has an effect on the
fluorescence and single oxygen quantum yield, intracellular ROS
levels, and in vitro phototoxicity. Overall, the developed silicon
phthalocyanine–erlotinib conjugates are highly promising
anticancer agents for small molecular target-based PDT.
MTT (3-[4,5-Dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide) was
purchased from Sigma Chemical Corp. (St. Louis, MO, USA). 2′,7′-
Dichlorofluorescein diacetate (DCFH-DA), 4′,6-diamidino-2-phenylindole
(DAPI), and Mito-Tracker Green were purchased from Beyotime Institute
of Biotechnology (Haimen, China). LysoTracker Green DND-26 was
purchased from Xiamen Bioluminor Bio-Technology Co., Ltd. (Xiamen,
China). Fetal bovine serum (FBS) was obtained from Sijiqing Biologic.
Co., Ltd. (Zhejiang, China). Roswell Park Memorial Institute-1640 (RPMI-
1640) medium and trypsin-EDTA solution were purchased from Gibco
Life Technologies (Carlsbad, CA, USA). HepG2 cell line was provided by
Institute of Life Sciences, Chinese Academy of Sciences (Shanghai,
China). Water used in the experiments was purified through an Ulupure
system (Chengdu, China) with resistivity higher than 18.2 MΩ. All other
reagents were of analytical grade and used as received. 1H NMR spectra
were recorded on an AVANCE III 400 (1H, 400 MHz) spectrometer in
CDCl3 or DMSO-d6. Chemical shifts (δ) were expressed in ppm relative
to tetramethyl silane (TMS) (δ 0). HRMS analyses were carried out on an
5
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ion trap MS DECAX-30000 LCQ Deca XP mass spectrometer. IR spectra
−1.87 to −1.92 ppm (m, 4H, Si-OCH2CH2O); HRMS (ESI) calculated for
were measured on
a
PerkinElmer SP2000 instrument. Electronic
Beijing PuXi Tu-1901
C83H74N14O12Si [M + H]+ 1473.5257, found: 1473.5303.
adsorption spectra were measured on
a
spectrometer. Fluorescence spectra were obtained on a Varian Cary
eclipse spectrometer. The purity of all new compounds was determined
by HPLC analysis, and was found to be ≥95%.
SiPc-3-E: According to the general procedure, SiPcCl2 (0.052 g, 1.00
equiv.), PEG-3-E (0.19 g, 4.00 equiv. ), and NaH (0.033 g, 16.00 equiv.)
were mixed in dried toluene to give SiPc-3-E (21.23 mg, 16%). 1H NMR
(400 MHz, CDCl3) δ = 9.52–9.38 (m, 8H, Pc-1,4-Har), 8.57 (s, 2H, N-H),
8.26–8.24 (m, 8H, Pc-2,3-Har), 7.66–7.59 (m, 4H, Ar-H), 7.47–7.46 (m,
2H, Ar-H), 7.14–7.13 (m, 4H, Ar-H), 6.98 (s, 2H, Ar-H), 6.79–6.73 (m, 2H,
Ar-H), 3.99 (s, 4H, CH2), 3.67–3.65 (m, 6H, CH3), 3.58–3.55 (m, 4H,
CH2), 3.40–3.38 (m, 4H, CH2), 3.19 (t, J = 8.0 Hz, 4H, CH2), 3.15 (s, 4H,
CH2), 3.05 (s, 2H, CH), 2.86 (t, J = 8.0 Hz, 4H, CH2), 2.42 (t, J = 8.0 Hz,
4H, CH2), 1.77 (t, J = 12.0 Hz, 4H, CH2), 0.51 (t, J = 12.0 Hz, 4H, CH2),
−1.88 to −1.91 ppm (m, 4H, Si-OCH2CH2O); HRMS (ESI) calculated for
C83H74N14O12Si [M + H]+ 1561.5781, found:1561.5811.
4.2 Synthesis
4.2.1. Synthesis of SiPc-PEG: TEG (0.13 g, 9.00 equiv.), SiPcCl2 (0.052
g, 1.00 equiv.), and NaH (0.021 g, 10.00 equiv.) were added in dried
toluene (15 mL) and were refluxed for 16 h. After cooling to room
temperature, the mixture was directly loaded onto a basic Al2O3 column
using CH2Cl2/CH3OH (80:1 v/v) as the eluent. A blue solid SiPc-PEG
(12.49 mg, 25%) was acquired. HRMS (ESI) calculated for C46H46N8O8
[M + Na]+ 889.3208, found: 889.3092.
4.3. Photophysical/Photochemical Studies
4.2.2. Synthesis of PEG-n-E: A mixture of compounds erlotinib analog
(1.00 equiv.), tosyl PEG (PEG-OTs) (1.20–1.50 equiv.), and K2CO3
(3.00–5.00 equiv.) in DMF (10–20 mL) were stirred at 80 °C for 7–10 h
under nitrogen. At the end of the reaction, the solvent was removed in
vacuo. Water (90 mL) was added to the residue and extracted with
CH2Cl2 (3 × 100 mL). The combined organic extracts were dried with
saturated NaHCO3 solution, filtered and concentrated. The yellow oil
product was purified over a silica gel column using CH2Cl2/CH3OH (25:1
v/v) as the eluent to give PEG-n-E.
Fluorescence quantum yields (ΦF) were measured according to the
2
equation: ΦF(sample) = (Fsample/Freference) (Areference/Asample) (ηsample2/ηreference
)
ΦF(reference), where F, A, and η are the integrated fluorescence area (ex =
610 nm, area under the emission peak), the absorbance at the excitation
wavelength (610 nm), and the refractive index of the solvent, respectively.
Unsubstituted zinc (II) phthalocyanine (ZnPc) in DMF was used as the
reference with a value for ΦF = 0.28.[49] The ΦF measurements were
performed using diluted solutions (absorbances of PSs at 610 nm are
0.03–0.05). Singlet oxygen quantum yields (ΦΔ) were measured using
1,3-diphenylisobenzofuran (DPBF) as the scavenger. A light laser (670
nm) was used as the light source and ZnPc was selected as the
reference (ΦΔ = 0.56 in DMF).[50]
PEG-2-E: According to the general procedure, 2-OTs (1.73 g, 1.21
equiv.), erlotinib analog (1.58 g, 1.00 equiv.) and anhydrous K2CO3 (2.60
g, 4.00 equiv.) were mixed in DMF to give PEG-2-E (0.99 g, 45%). 1H
NMR (400 MHz, DMSO-d6): δ 3.38 (s, 3H, CH3), 3.45–3.42 (m, 2H, CH2),
3.49 (t, J = 12.0 Hz, 2H, CH2), 3.58–3.55 (m, 2H, CH2), 3.67–3.65 (m, 2H,
CH2), 3.79 (t, J = 8.0 Hz, 2H, CH2), 3.84 (t, J = 12.0 Hz, 2H, CH2), 4.20 (s,
1H, CH), 4.32–4.28 (m, 4H, CH2), 4.60 (t, J = 12.0 Hz, 1H, OH), 7.24–
7.21 (m, 2H, Ar-H), 7.41 (t, J = 16.0 Hz, 1H, Ar-H), 7.87 (s, 1H, Ar-H),
7.92–7.89 (m, 1H, Ar-H), 8.00 (t, J = 3.2 Hz, 1H, Ar-H), 8.51 (s, 1H, Ar-H),
9.48 (s, 1H, NH); HRMS (ESI) calculated for C25H29N3O6 [M + H]+
468.2090, found: 468.2139.
4.4. Cell Targeting Confocal Imaging
HELF and HepG2 cell suspensions were mixed first, and then plated on
a culture dish (50,000:50,000 cells). After overnight incubation at 37 °C
under 5% CO2, the cells were exposed to 1 μM conjugates and
incubated for 24 h. After incubation, the cells were rinsed with PBS three
times, and the intracellular fluorescence caused by phthalocyanines was
recorded and statistically analyzed by confocal laser scanning
microscopy or ImageJ. First, the cell area was selected by using freeform
selection tools, and then the intracellular fluorescence intensity of
phthalocyanines was calculated by measuring the mean gray value of the
selected area.
PEG-3-E: According to the general procedure, 3-OTs (2.11 g, 1.25
equiv.), erlotinib analog (1.63 g, 1.00 equiv.), and anhydrous K2CO3 (3.02
g, 4.50 equiv.) were mixed in DMF to give PEG-3-E (1.19 g, 48%). 1H
NMR (400 MHz, CDCl3): δ 3.43 (s, 3H, CH3), 3.52–3.48 (m, 8H, CH2),
3.57–3.54 (m, 2H, CH2), 3.66–3.64 (m, 2H, CH2), 3.79 (t, J = 8.0 Hz, 2H,
CH2), 3.84 (t, J = 8.0 Hz, 2H, CH2), 4.18 (s, 1H, CH), 4.29–4.69 (m, 4H,
CH2), 4.61 (s, 1H, OH), 7.23–7.21 (m, 2H, Ar-H), 7.40 (t, J = 16.0 Hz, 1H,
Ar-H), 7.85 (s, 1H, Ar-H), 7.92 (d, J = 8.0 Hz, 1H, Ar-H), 8.02 (s, 1H, Ar-
H), 8.51 (s, 1H, Ar-H), 9.47 (s, 1H, NH); HRMS (ESI) calculated for
C27H33N3O7 [M + H]+ 512.2352, found: 512.2391.
4.5. Cellular Uptake Testing
Approximately 30,000 HepG2 or HELF cells in 100 μL culture medium
(per well) were seeded on 96-multiwell plates and incubated overnight at
37 °C under 5% CO2. The old culture medium was removed, and then
cells were incubated with 4 μM conjugate or 4 μM conjugate and 8 μM
free erlotinib in fresh medium for 24 h under the same conditions. The
solution was then removed, and the cells were rinsed with PBS three
times and then lysed by 1% SDS solution to give a homogenous solution.
The fluorescence of phthalocyanine in the cell extract was measured on
a Bio-Tek microplate reader (excitation/emission: 610/680 nm). Cellular
protein content was determined with a BCA Protein Assay Kit (Beyotime
Institute of Biotechnology Co., Ltd., China). Results are expressed as
nmol photosensitizer per mg cell protein.
4.2.3. Synthesis of SiPc-n-E: SiPc-n-E conjugates were prepared
according to the procedure described in the literature.[48] A mixture of
compounds PEG-n-E (3.00–5.00 equiv.), SiPcCl2 (1.00 equiv.), and NaH
(13.00–16.00 equiv.) in dried toluene (15–20 mL) was stirred at 125 °C
for 1.5–2.5 days. The solvent was then removed in vacuo, and the
mixture was directly loaded onto
CH2Cl2/CH3OH (50:1 v/v) as the eluent.
a basic Al2O3 column using
SiPc-2-E: According to the general procedure, SiPcCl2 (0.052 g, 1.00
equiv.), PEG-2-E (0.17 g, 4.00 equiv.), and NaH (0.031 g, 15.00 equiv.)
were mixed in dried toluene to give SiPc-2-E (17.53 mg, 14%). 1H NMR
(400 MHz, CDCl3): δ = 9.51–9.31 (m, 8H, Pc-1,4-Har), 8.69–8.61 (m, 2H,
N-H), 8,28–8.19 (m, 8H, Pc-2,3-Har), 7.61–7.54 (m, 2H, Ar-H), 7.46–7.44
(m, 2H, Ar-H), 7.17–7.14 (m, 4H, Ar-H), 7.07–7.03 (m, 2H, Ar-H), 6.81 (s,
2H, Ar-H), 6.54 (s, 2H, Ar-H), 3.72–3.62 (m, 6H, CH3), 3.50–3.43 (m, 4H,
CH2), 3.31–3.24 (m, 4H, CH2), 3.07–3.04 (m, 8H, CH2), 2.51 (s, 2H, CH),
2.05–1.87 (m, 4H, CH2), 1.82–1.75 (m, 4H, CH2), 0.70–0.57 (m, 4H, CH2),
4.6. Subcellular Localization
Approximately 100,000 HepG2 cells were plated on a cell culture dish
(diameter = 35 mm) and incubated overnight. The medium then was
replaced by fresh medium containing 1 μM phthalocyanine and incubated
for 24 h. After incubation, the cells were rinsed with PBS three times and
incubated with DAPI (0.3 μM for 1 min), LysoTracker@ Green DND-26
(0.25 μM for 30 min), or MitoTracker@ Green FM (2 μM for 60 min). The
6
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10.1002/cmdc.201700384
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Acknowledgements
This work was supported by the National Natural Science
Foundation of China (No. 21471033), the Major Project of the
State Ministry of Science and Technology of China (No.
2011ZX09101-001-04), the Independent Research Project of
State Key Laboratory of Photocatalysis on Energy and
Environment (Nos. 2014A04), the Natural Science Foundation of
Fujian Province (No. 2016J05034) and Foundation of Fujian
Educational Committee (No. JA15084).
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Keywords: targeted photodynamic therapy • antitumor agents •
phthalocyanines • erlotinib • molecular targeting imaging
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7
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Entry for the Table of Contents
Small molecular target-based anticancer drug, which has a simple chemical structure and high stability, is an idea targeted group for
conjugating with photosensitizers to improve cancer targeting. We have developed this ―smart‖ targeted therapeutic strategy and
synthesized two silicon phthalocyanines-erlotinib conjugates with good biocompatibility, high tumor selectivity and anticancer activity
for photodynamic therapy.
8
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