Rapid release from near-infrared polymer loaded liposomes for photothermal and chemo-combined therapy

Article abstract of DOI:10.1039/c8nj04778b

PEGylated liposomal doxorubicin is a polymeric antitumor drug approved clinically by the Food and Drug Administration, but it has no essentially increasing efficacy compared to free doxorubicin (DOX) because of the slow release rate. In this paper, a near-infrared polymer was designed and encapsulated in liposomes to photothermally accelerate the release of DOX. This polymer loaded liposome provides a maximum absorption that covers the ideal phototherapeutic window between 800 and 850 nm, which ensures an efficient photothermal release of DOX as a nano drug for the delivery of photothermal and chemo-combined therapy in a highly efficient cancer treatment.

Full text of DOI:10.1039/c8nj04778b

NJC
View Article Online
View Journal
PAPER
Rapid release from near-infrared polymer
loaded liposomes for photothermal and
Cite this:DOI: 10.1039/c8nj04778b
chemo-combined therapy†
Dehua Li, *a Meiduo Zhang,a Jingke Yaob and Zhe Zhang*b
PEGylated liposomal doxorubicin is a polymeric antitumor drug approved clinically by the Food and
Drug Administration, but it has no essentially increasing efficacy compared to free doxorubicin (DOX)
because of the slow release rate. In this paper, a near-infrared polymer was designed and encapsulated
in liposomes to photothermally accelerate the release of DOX. This polymer loaded liposome provides a
maximum absorption that covers the ideal phototherapeutic window between 800 and 850 nm, which
ensures an efficient photothermal release of DOX as a nano drug for the delivery of photothermal and
chemo-combined therapy in a highly efficient cancer treatment.
Received 20th September 2018,
Accepted 23rd December 2018
DOI: 10.1039/c8nj04778b
rsc.li/njc
Introduction
PEGylated liposomal doxorubicin (DOX) (Doxils/Caelyxs) was the
first polymeric antitumor drug to be used clinically by the Food and
Drug Administration (FDA).1 Its valid enhancement in clinical
therapy could be achieved by accelerating the release rate of DOX
and raising the bioavailability (o50%) of liposomes.2–6 Extensive
attention has been focused on accelerating the release of liposomes
from specific stimuli, such as pH,7,8 temperature,9–13 and ultra-
sound,14,15 etc. Of these stimuli, NIR light16–21 has provided a
promising approach to accelerate the release of liposomes for
photothermal and chemo-combination therapy. Due to the high
photothermal efficiency and photostability of polymers,22–25 poly-
meric nanoparticles became promising photothermal agents com-
pared to inorganic nanoparticles and organic small molecules.
Taking the above description into account, a NIR conjugated
polymer could be ideal material for accelerating the release rate
of DOX and raising the bioavailability of liposomes. In this paper, a
novel NIR conjugated polymer, PDPPT, was designed to be encap-
sulated in PEG-protected 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
(DPPC) liposomes to facilitate the release of DOX (Scheme 1). Thanks
to the adjustable absorption wavelength of conjugated polymers, the
maximum absorption of liposomes can cover the ideal photo-
therapeutic window between 800 and 850 nm, which enabled light
to perfectly reach a high penetration depth.
Scheme 1 Illustration of NIR irradiation responding to coencapsulated
liposomes for cancer therapy.
Results and discussion
Characterization of liposomes and coencapsulated liposomes
a Engineering Research Center of Forensic Sciences, Department of Forensic
Sciences, Jilin Police College, Changchun 130117, Jilin, P. R. China.
E-mail: lidh785@126.com
b State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of
Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China.
E-mail: zhangzhe@ciac.ac.cn
The synthesis of PDPPT and preparation of liposomes (PDPPT-Lip)
are shown in Schemes S1–S5 and Fig. S1–S5 (ESI†). The color
of the liposome solution changes from colorless to dark green
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c8nj04778b after PDPPT is loaded, which turns to brown after DOX is
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2019
New J. Chem.

View Article Online
Paper
NJC
Fig. 2 (A) Temperature evolution of DOX/PDPPT-Lip with 1.0 mg mLÀ1
under an 808 nm laser irradiation at various power densities; (B) temperature
evolution of DOX/PDPPT-Lip under an 808 nm laser irradiation of 3 W cmÀ2
with various concentrations; (C) thermal images of 1.0 mg mLÀ1 DOX/
PDPPT-Lip solution under an 808 nm laser irradiation of 3.0 W cmÀ2 at
different times; (D) the photothermal response of DOX/PDPPT-Lip with a
NIR laser (808 nm, 3.0 W cmÀ2, 1.0 mg mLÀ1) and then the laser shut off.
(E) The time constant for heat transfer from the system is determined by
applying linear time data from the cooling period versus the negative
natural logarithm of the driving force temperature, which is obtained from
the cooling stage of panel (D).
Fig. 1 (A) Photographs of liposomal solution (a), PDPPT-Lip solution (b) and
DOX/PDPPT-Lip (c); (B) UV-Vis absorption of (a) liposomal, (b) PDPPT-Lip,
and (c) DOX/PDPPT-Lip in aqueous solution; (C) TEM image of blank lipo-
somes; (D) hydrodynamic Dh distribution of liposomes; (E) hydrodynamic
Dh of liposomes at different temperatures; (F) time-dependent release of
DOX from DOX/PDPPT-Lip under 808 nm laser irradiation at various power
densities. The data are represented as the mean Æ S.D. (n = 3).
loaded (Fig. 1A). Before and after DOX loading, the absorption
peaks do not show a significant change and can cover well the
ideal phototherapeutic window between 800 and 850 nm (Fig. 1B)
with an absorption coefficient of 54.1 L gÀ1 cmÀ1 at 808 nm.
In vitro cancer cell ablation of liposomes
The diameter (Dh) of liposomes at pH 7.4 is around 170 nm As shown in Fig. 3A and B, under laser irradiation, the percen-
(Fig. 1C and D), which rapidly steps down to 120 nm when the tage of total apoptotic cells (including early and late apoptosis)
temperature goes above 40 1C (Fig. 1E), indicating the disinte- treated with the PDPPT-Lip rises up to B64.9%, which is much
gration of the liposomes. The loading of DOX and PDPPT in the more than the other three groups. This indicates that PDPPT-
liposomes is 5.3% and 5.7%, respectively. As shown in Fig. 1F Lip with laser irradiation is efficient for PTT. A live/dead assay
and Fig. S6 (ESI†), under NIR irradiation of 3.0 W cmÀ2, the was performed to further demonstrate the highly effective
release of DOX is up to 89.9% within 4 min, while DOX release in vitro cytotoxicity of the DOX/PDPPT-Lip. After treatment,
without NIR irradiation does not rise at 37 1C, demonstrating HepG2 cells are measured by co-staining the cells with calcein
that the release of DOX can be triggered by NIR irradiation.
acetoxymethylester (calcein AM) and propidium iodide (PI) to
differentiate live (green) and dead (red) cells (Fig. 3C). The cells
incubated with PBS do not show an observable cytotoxicity upon
Photothermal effect of coencapsulated liposomes
As the laser intensity increases, the temperature elevation indicates irradiation, while the cells incubated with DOX/PDPPT-Lip are
that DOX/PDPPT-Lip can convert the light into sufficient heat. In mostly killed upon the combined action of chemotherapy and
particular, the temperature of a 1.0 mg mLÀ1 DOX/PDPPT-Lip PTT. The result is consistent with those of MTT.
solution significantly increases from 27 1C to 47 1C (Fig. 2A). On
Cytotoxicity of the PDPPT-Lip and DOX/PDPPT-Lip with
the contrary, the temperature of a water solution merely increased laser irradiation or not against HepG2 was evaluated by MTT
from 27 1C to 30 1C upon laser irradiation of 10 min (Fig. 2B). With assay. As depicted in Fig. 4A, PDPPT-Lip without laser irradiation
exposure time continuing, the temperature of the DOX/PDPPT-Lip exhibits no detectable cytotoxicity against HepG2 cells. Both
solution gradually increased as shown in the thermal images in PDPPT-Lip with laser irradiation and DOX/PDPPT-Lip without
Fig. 2C. The photothermal conversion efficiency of DOX/PDPPT-Lip laser irradiation show obvious cytotoxicity against HepG2 cells.
was calculated to be 38.9% (Fig. 2D, E and see ESI†).
Furthermore, DOX/PDPPT-Lip presents a higher cytotoxicity,
New J. Chem.
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2019

View Article Online
NJC
Paper
Fig. 5 Antitumor study in vivo via intravenous injection. (A and B) Relative
tumor volume and body weight change of different groups during treat-
ment with PBS (a), PDPPT-Lip (b), PBS + laser (c), DOX (d), DOX/PDPPT-Lip
(e), PDPPT-Lip + laser (f) and DOX/PDPPT-Lip + laser (g).
indicating an excellent photothermal and chemo- combination
therapy effect.
Obviously, a higher DOX fluorescence intensity can be observed
in the cells incubated with DOX/PDPPT-Lip with an 808 nm laser
compared to other groups without laser irradiation. This clearly indi-
cates a faster intracellular drug release process of DOX/PDPPT-Lip in
the tumor micro-environment by external irradiation (Fig. 4B and C).
The therapeutic effects were assessed by monitoring the change in
tumor volume (Fig. 5A) and body weights of mice (Fig. 5B). No tumor
growth inhibition or tumor tissue necrosis was observed in the
group of mice with NIR only, which indicated that irradiation with
such a low power intensity NIR had little photothermal effect. The
DOX/PDPPT-Lip group displayed a better therapeutic effect than
pure DOX resulting from an enhanced EPR-induced intratumoral
accumulation in the tumor site. After tumors were treated with
PDPPT-Lip and DOX/PDPPT-Lip plus laser irradiation, the complete
remission of tumors was apparent. Black scars were left in the
original tumor site, which fell off about 10 days later. Four mice in
the PDPPT-Lip + laser group presented tumor recurrence over a
course of 20 days. Surprisingly, no tumor recurrence was observed in
the DOX/PDPPT-Lip + laser group. Overall, the combination treat-
ment of DOX/PDPPT-Lip with laser irradiation demonstrated a
remarkable anticancer efficacy in contrast to single treatment alone.
Fig. 3 (A) Flow cytometry analyses of apoptosis to HepG2 cells stained
with Annexin V-FITC and PI with various formulas. (B) The percentage of
apoptotic cells of (A). The data are represented as a mean Æ S.D. (n = 3,
***p o 0.001). (C) FL images of HepG2 cells after different treatments.
Viable cells were stained green with calcein AM, and dead/later apoptosis
cells were floating and eluted, or stained red with PI.
Conclusions
A conjugated polymer with an ideal NIR absorption in the
phototherapeutic window was designed and encapsulated in
liposomes to accelerate the DOX release in a tumor microenviron-
ment. The efficient photothermal and chemo-combined therapy
demonstrates that this polymer has great potential to be used for
highly efficient cancer treatments.
Conflicts of interest
Fig. 4 (A) In vitro evaluation of HepG2 cell viabilities with different treat-
ments. The data are represented as a mean Æ S.D. (n = 3, **p o 0.01);
(B) flow cytometric profiles of HepG2 cells incubated with DOX/PDPPT-
Lip after 2 h; (C) representative CLSM images of HepG2 cells incubated
with DOX/PDPPT-Lip after 2 h. For each panel, the images from left to
right show a differential interference contrast (DIC) image, cell nuclei
stained by DAPI (blue), DOX fluorescence in cells (red), and overlays of
the three images.
There are no conflicts to declare.
Acknowledgements
This work was supported by the 13th Five-Year Plan in Science
and Technology of the Education Department of Jilin Province
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2019
New J. Chem.

View Article Online
Paper
NJC
12 A. Agarwal, M. A. Mackey, M. A. El-Sayed and R. V. Bellamkonda,
ACS Nano, 2011, 5, 4919–4926.
(JJKH20181295KJ and JJKH20181294KJ) and the National Natural
Science Foundation of China (21504088).
13 G. Kong, G. Anyarambhatla, W. P. Petros and R. D. Braun,
Cancer Res., 2000, 60, 6950–6957.
14 X. L. Liang, J. Gao, L. D. Jiang, J. W. Luo, L. J. Jing, X. D. Li,
Y. S. Jin and Z. F. Dai, ACS Nano, 2015, 9, 1280–1293.
15 H. G. Moussa, A. M. Martins and G. A. Husseini, Curr. Cancer
Drug Targets, 2015, 15, 282–313.
16 D. D. Luo, K. A. Carter, A. Razi, J. M. Geng, S. Shao, D. Giraldo,
U. Sunar, J. Ortega and J. F. Lovell, Biomaterials, 2016, 75,
193–202.
17 V. Shanmugam, S. Selvakumar and C. S. Yeh, Chem. Soc.
Rev., 2014, 43, 6254–6287.
18 Y. Lyu, C. Xie, S. A. Chechetka, E. Miyako and K. Y. Pu, J. Am.
Chem. Soc., 2016, 138, 9049–9052.
19 C. Zhang, W. B. Bu, D. L. Ni, C. J. Zuo, C. Cheng, Q. Li,
L. L. Zhang, Z. Wang and J. L. Shi, J. Am. Chem. Soc., 2016,
138, 8156–8164.
Notes and references
1 T. Tagami, M. J. Ernsting and S. D. Li, J. Controlled Release,
2011, 154, 290–297.
2 Z. Cheng, A. Al Zaki, J. Z. Hui, V. R. Muzykantov and
A. Tsourkas, Science, 2012, 338, 903–910.
3 M. E. R. O’Brien, N. Wigler, M. Inbar, R. Rosso, E. Grischke,
A. Santoro, R. Catane, D. G. Kieback, P. Tomczak,
S. P. Ackland, F. Orlandi, L. Mellars, L. Alland, C. Tendler
and C. B. C. S. Grp, Ann. Oncol., 2004, 15, 440–449.
4 T. M. Allen and P. R. Cullis, Science, 2004, 303, 1818–1822.
5 K. M. Laginha, S. Verwoert and G. J. R. T. M. Allen Charrois,
Clin. Cancer Res., 2005, 11, 6944–6949.
6 A. Gabizon, H. Shmeeda and Y. Barenholz, Clin. Pharmaco-
kinet., 2003, 42, 419–436.
20 S. W. Lv, Y. Liu, M. Xie, J. Wang, X. W. Yan, Z. Li, W. G. Dong
and W. H. Huang, ACS Nano, 2016, 10, 6201–6210.
21 C. X. Sun, L. Wen, J. F. Zeng, Y. Wang, Q. Sun, L. J. Deng,
C. J. Zhao and Z. Li, Biomaterials, 2016, 91, 81–89.
22 J. L. Geng, C. Y. Sun, J. Liu, L. D. Liao, Y. Y. Yuan, N. Thakor,
J. Wang and B. Liu, Small, 2015, 11, 1603–1610.
23 L. G. Xu, L. Cheng, C. Wang, R. Peng and Z. Liu, Polym.
Chem., 2014, 5, 1573–1580.
7 E. Yuba, A. Harada, Y. Sakanishi, S. Watarai and K. Kono,
Biomaterials, 2013, 34, 3042–3052.
8 R. M. Reja, M. Khan, S. K. Singh, R. Misra, A. Shiras and
H. N. Gopi, Nanoscale, 2016, 8, 5139–5145.
9 T. Tagami, W. D. Foltz, M. J. Ernsting, C. M. Lee, I. F.
Tannock, J. P. May and S. D. Li, Biomaterials, 2011, 32,
6570–6578.
10 K. J. Chen, E. Y. Chaung, S. P. Wey, K. J. Lin, F. Cheng, C. C.
Lin, H. L. Liu, H. W. Tseng, C. P. Liu, M. C. Wei, C. M. Liu
and H. W. Sung, ACS Nano, 2014, 8, 5105–5115.
24 D. D. Liu, L. Y. Ma, Y. X. An, Y. Li, Y. X. Liu, L. Wang, J. Guo,
J. H. Wang and J. Zhou, Adv. Funct. Mater., 2016, 26, 4749–4759.
11 M. B. Yatvin, J. N. Weinstein, W. H. Dennis and R. Blumenthal, 25 D. Luo, K. A. Carter, D. Miranda and J. F. Lovell, Adv. Sci.,
Science, 1978, 202, 1290–1293.
2017, 4, 1600106.
New J. Chem.
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2019