594
J.-Q. Zhang et al. / Journal of Molecular Liquids 219 (2016) 592–598
was employed as the nebulizing gas, as well as the drying gas, to aid des-
olation. After optimization of the MS parameters, the spray voltage was
set to 4.0 kV in the positive mode and the heated metal capillary tem-
perature was set at 300 °C. The fragmentor and skimmer voltages
were set at 250 and 75 V, respectively. The mass scale was calibrated
by using the standard calibration procedure and compounds provided
by the manufacturer.
from the complexation phenomena was calculated by applying Eq. (1)
as follows:
Δδ ¼ δcomplex−δfree
ð1Þ
1H NMR spectra (Fig. 2A) of the complexes showed the proton peaks
of both CDs and PD. Furthermore, several 1H chemical shifts of CDs were
changed. This confirmed that the complexes were formed. Table 1
showed that the chemical shift of outside protons H-1 and H-6 had a lit-
tle variation before and after forming the complex. While significant
chemical shift changes were exhibited by H-5 and H-3 protons in the
inner surface of CD with an up-field shift from −0.110 ppm to
−0.150 ppm. It is noteworthy that the chemical shift variation for H-3
was smaller than H-5 after resulting inclusion complex. Since both H-
3 and H-5 protons from each sugar unit are located in the internal cavity
of CD, we can propose from the 1H NMR data that PD was included in
the CD cavity.
Two-dimensional (2D) NMR [34] provides the most direct evidence
for the spatial proximity between the host and guest atoms following
the observation of intermolecular dipolar cross-correlations. Two pro-
tons closely located in space can produce a Nuclear Overhauser Effect
(NOE) cross-correlation in NOE spectroscopy (NOESY) or rotating-
frame NOE spectroscopy (ROESY) [35]. These NOE cross-peaks between
the protons of the host and guest molecules point to spatial contacts
within 0.4 nm. 2D ROESY off resonance was used to study the inclusion
of PD with β-CD and γ-CD.
Fig. 2B shows a section of the contour plot of the ROESY spectrum of
the PD/γ-CD complex. First of all, it should be noted that the ROESY
spectrum showed a correlation between H-4, H-6, H-8, H-9, H-11, H-
12, H-14 and H-15 of PD and H-3 and H-5 of γ-CD, indicating that the
phenyl group (a and b rings) of PD were incorporated inside the γ-CD
cavity. These observations were not surprising since the most probable
mode of binding in the CD inclusion complexes involved the incorpora-
tion of the less polar moiety of the guest inside the cavity. We also found
NOE cross peaks with the inner CD protons, and to a larger extent with
H-6, demonstrating the PD binding with outside contribution. 2D NMR
results were in good agreement with results obtained from the 1H
chemical shifts analysis. It was also shown that PD should be included
in the β-CD cavity from the wide side (see Supplementary Data).
2.5. Cell growth inhibition assay
The cell growth inhibition on human lung adenocarcinoma cell
(A549), human cervical cancer (Hela), human ovarian cancer cell
(SKOV-3) and human breast adenocarcinoma (HepG2) cell lines
were determined by an MTT assay [30] as described previously. In
this assay, the increase or decrease in the number of viable cells is
linearly correlated with the mitochondrial activity, highlighted by
the conversion of the tetrazolium salt (MTT) into formazan crystals,
which can be solubilized and spectrophotometrically quantified.
First, cells were grown in 96-well plates at 5000 cells per well in a
final volume of 200 μL of culture medium per well. Then, the cells
were cultured in an incubator (5% CO2, 37 °C) until the cells reached
70–80% confluency. After culturing the cells in an incubator for 48 h,
20 μL of MTT solution was added to each well and incubated for 2 h.
The culture medium was discarded, and 200 μL of DMSO were added
to each well. The solution was then swirled gently and left in the dark
for 10 min. The absorbance in each well was measured at 570 nm in a
microtiter plate reader. The cell viability in treated cells was
expressed as the amount of dye reduction relative to that of the un-
treated control cells.
3. Results and discussion
3.1. NMR spectra analysis
1H NMR spectra are some of the most direct evidence for the forma-
tion of the inclusion complex [31–33]. If a guest molecule is incorporat-
ed into the CDs cavity, the screening constants of the CDs protons inside
the cavity (H-3 and H-5) should be sensitive to the changed environ-
ment, but that of the outside protons (H-1, H-2, and H-4) should not.
This should result in chemical shift changes of the inside protons [20].
The 1H chemical shifts of CDs were determined through the 1H NMR
spectra of CDs and PD, as well as their complexes. These results are
shown in Table 1.
3.2. Scanning electron microscopy
Scanning electron microscopy (SEM) [36,37] is a qualitative method
used to visualize the surface structure of raw materials or the prepared
products. The SEM images of the PD, β-CD, and PD/β-CD inclusion com-
plex, as well as their physical mixtures, were illustrated in Fig. 3A. Pure
PD existed in a needle-like crystal with many different sizes (Fig. 3A
(a)), whereas β-CD was observed as rod-like crystals (Fig. 3A (b)). In
the physical mixture, the characteristic β-CD microspheres, which
were mixed with PD crystals or adhered to their surface, were clearly
observed (Fig. 3A (d)). However, the PD/β-CD inclusion complex ap-
peared in the form of irregular block-like particles (Fig. 3A (c)) in
which the original morphology of both components disappeared,
confirming the formation of the inclusion complex of PD and β-CD.
These changes can be taken as proof of the formation of new inclusion
complexes by molecular encapsulation (SEM of γ-CD and inclusion
complex, see the Supplementary Data).
The chemical shifts for the PD (Fig. 2A(a)) are as follows: 1H NMR
(500 MHz, DMSO-d6) δ 9.60 (s, H-16 of PD), 9.47 (s, H-28 of PD),
7.45–7.39 (m, H-11 and H-15 of PD), 7.05 (d, J = 16.3 Hz, H-8 of PD),
6.89 (d, J = 16.3 Hz, H-9 of PD), 6.81–6.73 (m, H-2, H-12 and H-14 of
PD), 6.59 (t, J = 1.7 Hz, H-6 of PD), 6.36 (t, J = 2.2 Hz, H-4 of PD),
5.31 (d, J = 5.1 Hz, H-17 of PD), 5.12 (d, J = 4.8 Hz, H-24 of PD), 5.05
(d, J = 5.3 Hz, H-23 of PD), 4.82 (d, J = 7.6 Hz, H-27 of PD), 4.66 (t,
J = 5.7 Hz, H-25 of PD), 3.75 (ddd, J = 11.8, 5.3, 2.1 Hz, H-22 of PD),
3.51 (dt, J = 11.9, 6.1 Hz, H-19 of PD), 3.38–3.14 (m, H-20, H-21 and
H-26 of PD). The Δδ in the 1H chemical shift for PD and CDs originating
Table 1
Chemical shifts of 1H NMR of CD protons in the presence and absence of PD.
Protons
Chemical shift (ppm)
3.3. Stoichiometry determination: Job's method
δβ-CD
δPD/β-CD
Δδγ
δγ-CD
δPD/γ-CD
Δδγ
H-1
H-2
H-3
H-4
H-5
H-6
4.916
3.495
3.815
3.434
3.707
3.720
4.865
3.440
3.705
3.395
3.565
3.632
−0.051
−0.055
−0.110
−0.039
−0.142
−0.088
4.977
3.526
3.864
3.462
3.765
3.786
4.925
3.475
3.715
3.426
3.615
3.652
−0.052
−0.051
−0.149
−0.036
−0.150
−0.134
The standard curve was performed using a UV–vis spectrophotome-
ter at 317 nm, which was prepared using the concentration (C, mM) as
the x-coordinate and the absorbance (A) as the y-coordinate. The stan-
dard curve of PD can be expressed as A = 29.1199C + 3.8 × 10−3 (R2 =
0.9947).