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Article
Biomimetic Nanovesicles for Enhanced Antitumor Activity of Combinational Photothermal and Chemotherapy Tingting Wu, Dan Zhang, Qi Qiao, Xianya Qin, Conglian Yang, Miao Kong, Huan Deng, and Zhiping Zhang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b01142 • Publication Date (Web): 04 Feb 2018 Downloaded from http://pubs.acs.org on February 4, 2018
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Molecular Pharmaceutics
1
Biomimetic Nanovesicles for Enhanced Antitumor
2
Activity of Combinational Photothermal and
3
Chemotherapy
4 1
1
1
1
1
1
5
Tingting Wu , Dan Zhang , Qi Qiao , Xianya Qin , Conglian Yang , Miao Kong ,
6
Huan Deng , Zhiping Zhang
1
1,2,3*
7 8 9 10 11
1
Tongji School of Pharmacy
2
National Engineering Research Center for Nanomedicine
3
Hubei Engineering Research Center for Novel Drug Delivery System
HuaZhong University of Science and Technology, Wuhan, China 430030
12 13 14 15 16 17 18 19 20 21 22
ABSTRACT Combination of multiple modalities has shown great potential in cancer treatment 1
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with improved therapeutic effects and minimized side effects. Here we fabricated a
24
kind of doxorubicin encapsulated biomimetic nanovesicles (NVs) by a facile method
25
with near-infrared dye insertion in the membrane for combinatorial photothermal and
26
chemotherapy. With innate biomimetic properties, NVs enhanced the uptake by tumor
27
cells while reduced the phagocytosis of macrophages. Upon laser irradiation, NVs can
28
convert the absorbed fluorescent energy into heat for effective tumor killing. The
29
hyperthermia can further induce membrane ablation of NVs to accelerate the release
30
of chemotherapeutic drug for potent cytotoxicity to tumor cells. The NVs improved
31
the drug accumulation and showed more efficient in vivo photothermal effect with
32
rapid temperature increase in tumor. Moreover, the NVs based combinational
33
photothermal and chemotherapy exhibited significant tumor growth suppression with
34
a high inhibitory rate of 91.6% and negligible systemic toxicity. The results indicate
35
that NVs could be an appealing vehicle for combinational cancer treatment.
36 37
KEY WORDS: Nanovesicles, photothermal therapy, chemotherapy, near-infrared,
38
cancer
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1. INTRODUCTION
46
With increasing incidence and mortality, cancer keeps as the major cause of death
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all over the world.1 Chemotherapy, which can cause direct cytotoxicity to tumor cells,
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is the commonly used treatment regimen in clinic cancer therapy. However, the low
49
selectivity of cytotoxic drugs always causes severe toxicity to normal tissues, which
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would greatly limit the successful application of chemotherapy. Combinational
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therapy with multiple modalities can improve the therapeutic efficiency as well as
52
minimize the side effects, which has shown great potential in cancer treatment.2-3 In
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the past few decades, the combination of chemotherapy with other therapeutic
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modalities has attracted much attention and achieved satisfied outcomes for tumor
55
killing.4-6 Photothermal therapy (PTT) is a well-developed technique which can utilize
56
the comparable heat generated by near-infrared (NIR) laser to directly destroy the
57
malignant tissues.7-8 Owing to the highly specific spatial-temporal selectivity and the
58
minimal toxicity to normal tissues, PTT has emerged as an appealing cancer
59
therapeutic modality.9 However, as heat distribution cannot be so uniform in whole
60
tumor, especially in the areas near large blood vessels where circulating blood can
61
rapidly dissipate the heat, PTT alone is unable to clear tumor cells.10 The combination
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of PTT with chemotherapy can utilize multiple antitumor mechanisms and may work
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cooperatively to suppress cancer development. On one hand, the hyperthermia
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induced by PTT can disrupt extracellular matrix, relieve the tumor interstitial fluid
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pressure and trigger the rapid release of cytotoxic drugs in tumor site.11-12 On the other
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hand, the cytotoxic drugs can kill tumor cells which are unlikely to be cleared by PTT 3
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owing to the nonuniform heat distribution. Such a multimodal approach may possess
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the potential to eradicate tumor cells.
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With rapid development of nanotechnology, nanosized drug delivery systems
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have been extensively applied in cancer treatment. Encapsulating free drugs into
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nanoformulations can protect the drug from degradation during blood circulation,
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improve pharmacokinetics and pharmacodynamics as well as reduce side effects.13
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The nanoformulations are usually prepared with synthesized materials. However,
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there are still several factors which impede the therapeutic potential of these
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synthesized material based delivery systems. Firstly, as intruders to the host, foreign
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materials can be easily detected by the immune system, which may cause some
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immune disorder.14 Besides, the opsonization and nonspecific clearance by
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reticuloendothelial system (RES) remain as a major challenge.15 Even the
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modification with poly(ethylene glycol) (PEG) cannot completely prevent this
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nonspecific clearance,16 which would then activate the complement system to induce
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undesired immune response.17 Furthermore, the potential toxicity of foreign
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nanomaterials with artificial nature has kept as a considerable concern for biomedical
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applications.18 Given the limitations of synthetic materials, it is gaining popularity to
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develop biomimetic strategies for drug delivery. Cell based nanovesicles (NVs) are
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spherical membrane vesicles consisting of various proteins, lipids and nucleic acids,19
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which can mimic the natural properties of parent cells.20 As a top-down approach, the
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biomimetic carriers can get rid of laborious and costly groups-modified engineering.21
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With good biocompatibility, reduced uptake by macrophages, decreased blood 4
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clearance and specific targeting ability for enhanced tumor accumulation, NVs
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showed great potential for drug delivery.22-23 Generally, NVs are spontaneously
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secreted by cells and used as drug carriers. However, NVs generated in this way are
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always suffered from low production yield, heterogeneity in size and composition,
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and inefficient drug package.24 An alternative and facile method for generating
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homogenous drug loaded-NVs is to subject cells and therapeutic agents to serial
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extrusions using filters with diminishing pore sizes. Moreover, this approach can
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promote the production of biomimetic vesicles, which has been reported to be more
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than a 100-fold higher production yield as comparison to spontaneously generated
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extracellular vesicles.25-26
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In this work, we prepared a kind of biomimetic nanovesicles co-loading
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near-infrared dye DIR and chemotherapeutic drug doxorubicin (DOX) for
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combinational photothermal-chemotherapy. DIR, a lipophilic fluorescent probe, can
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be embedded in the lipid bilayer of cell membrane with negligible influence on its
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physiological properties and commonly used for in vivo fluorescence imaging and
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tracking.12, 27 Notably, DIR has an absorption in NIR region and can convert absorbed
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fluorescent energy into heat under NIR irradiation.28-29 Moreover, it can also work as
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a photosensitizer to generate reactive oxygen species (ROS) upon light exposure.
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Here we inserted DIR in the membrane of DOX loaded NVs (NV-DOX) for
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combinatorial photothermal and chemotherapy. This NV based combinational
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platform (NV-DOX-DIR) can accumulate in tumor owing to the enhanced permeation
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and retention (EPR) effect of nanoformulations and the specific targeting ability of 5
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cell derived membrane vesicles. Upon NIR irradiation, DIR inserted in the membrane
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of NVs can convert near-infrared fluorescence into thermal energy to kill tumor cells.
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The induced hyperthermia can further destroy the membrane of NVs and release the
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encapsulated DOX for direct cytotoxicity to tumor cells (Scheme 1). The biomimetic
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NVs can realize synergistic antitumor efficiency through the combination of
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photothermal and chemotherapy.
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Scheme 1. Schematic illustration for biomimetic NVs preparation and in vivo
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synergistic antitumor effect by combination of photothermal and chemotherapy. To
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prepare NV-DOX-DIR, DC2.4 cells were firstly mixed with DOX and subjected to
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serial extrusions by polycarbonate membranes with diminishing core size. After
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removing the cells, debris and free DOX, NVs were then incubated with DIR to
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obtain NV-DOX-DIR. When intravenously injected to the mice, NVs can accumulate
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in tumor site owing to EPR effect and specific targeting ability of NVs. Upon
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irradiation by NIR laser, NV-DOX-DIR can effectively convert the absorbed 6
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fluorescent energy into heat for tumor killing. The induced hyperthermia can further
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destroy the NVs to release the encapsulated DOX for enhanced cytotoxicity.
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2. EXPERIMENTAL SECTION
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2.1. Materials and reagents
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Doxorubicin hydrochloride was purchased from Beijing Huafeng United
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Technology Co., China. 1, 1′-Dioctadecyl-3, 3, 3′, 3′- tetramethylindotricarbocyanine
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iodide
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5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was acquired from
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Biosharp, South Korea. 4', 6-Diamidino-2-phenylindole (DAPI) was obtained from
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Nanjing KeyGen Biotech. Inc., China. Radioimmunoprecipitation assay (RIPA) buffer,
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dichlorodihydrofluorescein diacetate (DCFH-DA) and bicinchoninic acid (BCA)
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protein quantitation kit were acquired from Beyotime Institute of Biotechnology,
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China. 1, 1'-Dioctadecyl-3, 3, 3', 3'-tetramethylindocarbocyanine perchlorate (DIL)
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was purchased from Invitrogen Corporation, USA. Phosphatidylcholine was obtained
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from Avanti Polar Lipids, Inc., USA. Cholesterol was purchased from Lipoid GmbH,
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Germany. DSPE-PEG 2000 was purchased from Corden Pharma Switzerland LLC,
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Switzerland. Pentobarbital sodium was obtained from Sigma-Aldrich, USA.
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2.2. Cell lines and animals
(DIR)
was
acquired
from
AAT
Bioquest
Inc.,
USA.
3-(4,
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Murine DC2.4 cells were maintained in RPMI-1640 containing 10% fetal bovine
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serum (FBS), 100 µg/mL of streptomycin and 100 IU/mL of penicillin in a humidified
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atmosphere incubator with 5% CO2 at 37 °C. Murine B16F10 melanoma cells were 7
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cultured in Dulbecco's modified eagle medium (DMEM) with the same supplements
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and culture condition as DC2.4 cells. Female C57BL/6 mice (6-8 week) were
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obtained from the Experimental Animal Center of Hunan SJA, China. All mice were
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maintained under specific pathogen-free (SPF) condition which keeps at 25 ± 1°C and
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60% ± 10% humidity under a 12 h light/dark cycle in the Animal Center of Huazhong
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University of Science and Technology (HUST), China. All animals were treated
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according to the regulations of Chinese law, and the study was approved by the
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Institutional Animal Care and Use Committee at Tongji Medical College, HUST,
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China.
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2.3. Preparation and characterization of NVs
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NV-DOX were prepared as reported previously with minor modification.25
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Briefly, DC2.4 cells were collected and resuspended in phosphate buffered saline
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(PBS) at a concentration of 2.5×106 cells/mL. Then DOX was added to cell
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suspensions and the resulted mixture were sequentially extruded through 10, 5, and 1
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µm polycarbonate membrane (Whatman International Ltd, Maidstone, England) using
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a mini-extruder (Avanti Polar Lipids, USA). To isolate the NVs, the suspension was
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firstly centrifuged at 1500g for 15 min to get rid of the cells and debris. Then the
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supernatant was centrifuged at 17, 000g for 45 min and NVs were obtained after
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washing twice with PBS. To prepare DIR inserted NV-DOX, DIR was dissolved in
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ethanol and added dropwise to the aqueous phase of NV-DOX. The mixture was then
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incubated at 37 oC for 1 h and centrifuged at 17, 000 g for 45 min to remove the
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unincorporated DIR. The resulting pellets were resuspended in PBS for further use. 8
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The hydrodynamic diameter of NVs was monitored by dynamic light scattering (DLS)
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(Zeta Plus, Brookhaven Instruments, USA). To observe the morphology, the NVs
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were fixed with 2% paraformaldehyde for 30 min, dropped on EM grids and stained
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with 0.5% phosphotungstic acid for 2 min. The morphology of NVs was then
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observed by TEM (JEM-1230, Japan). To monitor the successful package of DOX,
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UV-Vis spectra of NVs were detected using a microplate reader (Multiskan MK3,
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Thermo, USA).
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2.4. Determination of drug loading efficiency in NVs
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To detect the loading efficiency of DOX by NVs, DC2.4 cells were collected and
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mixed with DOX in PBS. The drug loaded NVs were obtained as described above.
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The NVs were lysed with RIPA buffer containing phenylmethanesulfonyl fluoride
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(PMSF). The concentration of DOX was measured using a fluorescent microplate
182
reader and the total content of protein was detected by BCA protein quantitation kit.
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To further determine the loading efficiency of DIR by NVs, different amounts of DIR
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(w/w, DIR/total protein) were incubated with the NVs. After removing the free drug,
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NVs were resuspended in DMSO to entirely dissolve the NVs and DIR. Then the
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UV-Vis spectra were detected by the microplate reader. The amount of DIR insertion
187
in the membrane was calculated according to the linear standard curve of DIR in
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DMSO.
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2.5. In vitro cumulative release of DOX
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The cumulative release behavior of DOX from NVs was performed in pH 7.4
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PBS using an orbital shaker (37 °C, 100 rpm). To monitor the impact of NIR laser on 9
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drug release, NV-DOX-DIR was pre-treated with or without 808 nm laser at a power
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density of 2.5 W/cm2 for 2 min. The released drug was collected by centrifuge at
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various time points and fresh medium were supplemented for continuous release. The
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DOX content was then determined using fluorescent microplate reader.
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2.6. In vitro photothermal profiles
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The photothermal profiles of free DIR, DIR inserted blank NVs (NV-DIR) and
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NV-DOX-DIR with equivalent DIR concentration of 100 µg/mL were treated with
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808 nm laser at a power density of 2.5 W/cm2. At different time points, the images of
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each group upon irradiation were photographed and corresponding temperature was
201
recorded using the infrared thermal imaging camera (FLIR System E40, Boston, MA,
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USA). The thermal profiles of NV-DOX-DIR with different concentrations of DIR
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were also evaluated.
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2.7. The cell uptake of NVs by tumor cells
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Confocal microscope and flow cytometer were used to determine the cell uptake
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of NVs by tumor cells. Briefly, B16F10 cells were seeded into 6-well plate and
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incubated overnight in the incubator. Afterward, all the medium were removed and
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replaced with fresh medium containing DOX, NV-DOX and NV-DOX-DIR,
209
respectively. The cells were further incubated at 37 oC for different time intervals and
210
detected by flow cytometer (Accuri C6, BD, USA). To visualize the cell uptake, cells
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were further monitored by confocal microscope at 4 h. After removing the supernatant,
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cells were washed by cold PBS, fixed with 4% paraformaldehyde at room temperature
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for 15 min and stained with DAPI. The cells were then rinsed with PBS and 10
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monitored by confocal laser scanning microscopy (710META, Zeiss, Germany).
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2.8. Evasion of NVs from the uptake by macrophages
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To investigate the evasion of NVs from phagocytosis by macrophages, DIL
217
labeled NVs were incubated with Raw 264.7 macrophages for 2 h. Instead of DIR,
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DIL was used here owing to the visualized fluorescence of DIL for detection. A lipid
219
structure based liposome was prepared by filming-hydration method and used as
220
control. Briefly, phosphatidylcholine, cholesterol and DSPE-PEG 2000 (w/w, 4:4:1)
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were dissolved in chloroform and evaporated by rotary evaporation to form a uniform
222
film. The liposome was obtained after hydration, labeled with DIL and incubated with
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Raw 264.7 macrophages. Then the cells were rinsed with cold PBS, stained with
224
DAPI (5 µg/mL) for 5 min and imaged by confocal microscope. To quantitatively
225
determine the cellular uptake, the cells were resuspended in PBS and detected by flow
226
cytometer.
227
2.9. Intracellular temperature measurement and ROS generation
228
To measure the intracellular temperature, the photothermal profiles of B16F10
229
cells were evaluated after treating with free DIR, NV-DIR and NV-DOX-DIR.
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B16F10 cells (1×104 cells/well) were seeded in 24-well plates and cultured overnight.
231
Then the medium were replaced with fresh medium containing 100 µg/mL free DIR,
232
NV-DIR or NV-DOX-DIR and incubated for 4 h. Then the cells were irradiated with
233
808 nm. The temperature of each time point was recorded using infrared thermal
234
imaging camera. To further detect the intracellular ROS generation, the cells treated
235
with various formulations were incubated with DCFH-DA at 37 °C for 20 min. Then 11
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the cells were rinsed with PBS for three times, irradiated with NIR laser and collected
237
for detection by flow cytometer.
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2.10. In vitro combinatorial effects of photothermal and chemotherapy
239
To investigate the enhanced antitumor effect in vitro, B16F10 cells were seeded
240
into 96-well plate and allowed for attachment overnight. Then the cells were treated
241
with different formulations. The concentrations of DOX and DIR range from 0.01 to 5
242
µg/mL and 0.008 to 4 µg/mL, respectively. After incubation for 4 h, the supernatant
243
was removed and fresh medium were added. Then the cells were irradiated with 808
244
nm laser for 2 min and cultured for another 20 h. The cell viability was determined by
245
MTT assay. The synergistic cytotoxicity of photothermal-chemotherapy was also
246
evaluated by Calcein AM/PI live/dead staining (Molecular Probes-Invitrogen).
247
B16F10 cells were seeded into 24-well plates and incubated overnight. The original
248
medium were replaced with the medium containing different formulations. After 4 h
249
incubation, the cells were irradiated with NIR laser. Twenty hours later, the cells were
250
stained with Calcein-AM/PI according to the manufacture’s instruction and then
251
monitored using the fluorescent microscope (CKX53, Olympus, Japan).
252
2.11. In vivo imaging and biodistribution of NVs
253
To study the biodistribution of NVs after systematic administration, free DIR and
254
NV-DIR were given to melanoma bearing mice by tail vein. The biodistribution of
255
injected formulations at various time points was monitored in live mice under
256
isoflurane anesthesia using the Pearl Impulse Small Animal Imager (LI-COR
257
Biosciences, Lincoln, NE). At 48 h post injection, all the mice were sacrificed. The 12
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major organs were collected, rinsed with PBS and imaged by imaging system.
259
2.12. In vivo temperature measurement and photothermal profiles
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A well-established murine melanoma model was used to determine the in vivo
261
photothermal effect after systematic injection of NVs. PBS, free DIR, NV-DIR and
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NV-DOX-DIR were intravenously injected to mice, respectively. After 24 h, the mice
263
were anesthetized by intraperitoneally injecting 50 µl 1% pentobarbital sodium. And
264
tumors were exposed to 808 nm laser at a power intensity of 2.5 W/cm2 and the
265
temperature was simultaneously monitored by infrared thermal imaging camera.
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2.13. In vivo synergistic antitumor efficacy of NVs
267
To
investigate
the
in
vivo
synergistic
antitumor
effect
of
268
photothermal-chemotherapy, murine melanoma model was used for tumor inhibition
269
experiment. B16F10 cells were implanted on the left hind flank of female C57BL/6
270
mice on day 0. From day 11, the mice were intravenously injected with PBS, free
271
DOX (2.5 mg/kg), free DOX+DIR (2 mg/kg DIR), NV-DOX, NV-DIR and
272
NV-DOX-DIR every three days for 3 times, respectively. Twenty four hours after each
273
injection, the mice were irradiated with NIR laser. Tumor volume of each group,
274
calculated by (width)2 × length × 1/2, and body weight were recorded every other day.
275
All mice were sacrificed at day 21 and tumors were excised for imaging and
276
hematoxylin and eosin (HE) staining. Terminal-deoxynucleoitidyl transferase
277
mediated nick-end labeling (TUNEL) assay was conducted to evaluate the apoptosis
278
and necrosis of tumor after various treatments.
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2.14. Systemic toxicity of NVs 13
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To investigate the systemic toxicity of NVs, C57BL/6 mice were treated with
281
PBS,
PBS+Laser,
DOX,
DOX+DIR+Laser,
NV-DOX,
NV-DIR+Laser
and
282
NV-DOX-DIR+Laser, respectively. Then the serum was sampled to measure the
283
blood biochemistry parameters. According to the manufacture's protocol (Nanjing
284
Jiangcheng Bioengineering Institute, China), the levels of alanine aminotransferase
285
(ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), creatinine (Cr)
286
and lactate dehydrogenase (LDH) were measured using a microplate reader. The
287
major organs were excised, stained with HE and observed under a microscope to
288
monitor the histopathological changes after systemic administration of NVs.
289
2.15. Statistical analysis
290
Data were represented as mean ± standard deviation (SD). The difference of the
291
mean was calculated by one-way ANOVA using SPSS software (version 19.0). There
292
was significantly statistical difference when the p value was less than 0.05.
293 294
3. RESULTS AND DISCUSSION
295
3.1. Preparation and characterization of NVs
296
NVs are commonly generated by spontaneous secretion from the cells under both
297
pathological and physiological conditions. However, this process is usually faced with
298
many challenges including low production yield, heterogeneity in size and
299
compositions and inefficient drug packaging. Here we introduced an alternative
300
method to generate a population of homogenous drug loaded-NVs for combinational
301
photothermal and chemotherapy. DC2.4 cells were detached from the culture plate 14
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and resuspended in PBS. The cells were then mixed with DOX and conducted with
303
serial extrusion through polycarbonate membranes (10 µm, 5 µm, and 1 µm). After
304
removing the cells and debris, the supernatant were centrifuged to collect DOX
305
loaded NVs (NV-DOX). To further prepare DIR incorporated NVs, NV-DOX were
306
incubated with DIR at 37 ℃ for 1 h following with centrifugation to remove the
307
unincorporated DIR. The morphology of NVs were observed by transmission electron
308
microscope (TEM). As shown in Figure 1A, NV-DOX showed regular morphology
309
with a near-spheric shape. As DIR showed negligible interference on the
310
physiological properties of cell membrane, no obvious morphology change was
311
monitored after inserting DIR in the membrane of NVs. After NIR irradiation, the
312
membrane structure of NVs was destroyed, which was mainly attributed to
313
photothermal ablation on cell membrane induced by NIR laser.30-32 To verify the
314
successful package of DOX and incorporation of DIR in NVs, the UV-Vis absorption
315
spectra of NV-DOX, free DIR, NV-DIR and NV-DOX-DIR were measured using a
316
microplate reader (Figure 1B). Free DIR in PBS showed very weak absorption while
317
NV-DIR and NV-DOX-DIR exhibited a peak absorption at 760 nm and remarkably
318
increased absorption in NIR region. It ensured the potential of NV-DIR and
319
NV-DOX-DIR for photothermal therapy induced by NIR laser. NV-DOX showed a
320
peak absorption at 490 nm of DOX, verifying the successful encapsulation of DOX by
321
NVs. After NIR irradiation, the absorption of DIR decreased owing to the consumed
322
DIR during the process of heat converting. The absorption of DOX was not
323
influenced by the irradiation, indicating DOX was in good stability during laser 15
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324
illumination (Figure 1C). Then the quantitative DOX loading by NVs was determined.
325
As Figure 1D showed, the amount of DOX loaded in NVs was dependent on the
326
initial DOX concentration in cell suspensions before extrusion. With the increasing
327
concentration of DOX, the encapsulation of DOX by NVs was increased. To further
328
determine the incorporated amount of DIR into NVs, UV-Vis spectra of NVs with
329
different amounts of DIR insertion were recorded (Figure 1E). The absorption of DIR
330
in NVs increased with the elevating amount of DIR and became saturated when the
331
weight ratio of DIR/total protein raised to 11%. It seems that no more DIR could be
332
incorporated into the membrane of NVs. In contrast, free DIR with similar amount as
333
NVs in PBS showed very weak absorption (Figure 1F). According to DOX dosage in
334
our previous combinational therapy33 and effective DIR amount for PTT,29 200 μg/mL
335
initial DOX concentration was used to prepare NV-DOX and 11% DIR was
336
incorporated in the membrane to obtain NV-DIR in the following experiments. The
337
stability of drug carriers in physiological condition is critical for further biomedical
338
application. The stability of NVs was then monitored in commonly used physiological
339
media. As shown in Figure 1G and Figure S1, both NV-DOX and NV-DOX-DIR
340
showed good stability in PBS and FBS with negligible change of hydrodynamic size
341
during storage for 5 days at 4 oC. To further detect the drug retention during storage,
342
the leakage of DOX in the supernatant was detected using the fluorescent microplate
343
reader. As shown in Figure S2, 84.1 ± 1.1% and 83.0 ± 1.0% of DOX retained in
344
NV-DOX and NV-DOX-DIR after 24 h respectively. This may be attributed to the
345
good hydrophilicity of doxorubicin hydrochloride and the slight permeability of cell 16
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346
membrane.34 The cumulative release of DOX from NVs was also performed in PBS
347
(Figure 1H). There was no obvious difference in the release profiles of NV-DOX and
348
NV-DOX-DIR, which further confirmed the negligible interference on membrane of
349
NVs after DIR insertion. After laser irradiation, the release of DOX from
350
NV-DOX-DIR was accelerated and can reach to 88.6 ± 0.2% after 24 h while that of
351
NV-DOX and NV-DOX-DIR was 61.2 ± 0.7% and 62.3 ± 1.2%, respectively. This
352
may be caused by the NIR laser irradiation which can generate comparable localized
353
heat to destroy the membrane of NVs and thus trigger the accelerated DOX release
354
from the NVs.29
355 356
Figure 1. .Preparation and characterization of NVs. (A). TEM images of (a) NV-DOX
357
(b) NV-DOX-DIR and (c) NV-DOX-DIR with NIR laser (Scale bar, 500 nm). (B-C).
358
The UV-Vis spectra of NV-DOX, free DIR, NV-DIR and NV-DOX-DIR (B) before
359
and (C) after NIR irradiation. (D). DOX loading efficiency in NVs with the increasing 17
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360
initial concentration of DOX (n = 3). (E). The UV-Vis absorption spectra of NVs after
361
different amount of DIR inserting into membrane. (F), The UV-Vis spectra of free
362
DIR with similar amount as NVs in PBS. (G). the physiologic stability of NV-DOX
363
and NV-DOX-DIR in PBS (n = 3). (H) In vitro cumulative DOX release of NV-DOX
364
and NV-DOX-DIR with or without NIR (n = 3).
365 366
3.2. In vitro photothermal effects of NVs
367
As described above, we have demonstrated that DIR incorporated in NVs showed
368
a remarkably increased absorbance in NIR region. To investigate the photothermal
369
effect of NVs, the real-time temperature changes with 808 nm NIR laser irradiation
370
were recorded by an infrared thermal imaging camera. As Figure 2A-B displayed,
371
upon laser illumination for 5 min, no obvious temperature change was observed in
372
PBS control. The temperature of free DIR raised slowly and reached maximum as
373
44.9 oC. However, the temperature of NV-DIR and NV-DOX-DIR could increase over
374
50 °C within a short period of irradiation for 1 min. After 2 min irradiation, the
375
temperature of NV-DIR and NV-DOX-DIR was raised up to 61.4 °C and 62.8 °C,
376
respectively. When the irradiation time was up to 5 min, the temperature of NVs can
377
reach above 70.0 °C with a dramatic temperature increase (△T) over 45 °C. This
378
could be resulted from the more concentrated status of DIR in NVs, which would lead
379
to higher energy converting efficiency under laser illumination.29 Besides, free DIR
380
exhibited very quick photodegradation with a rapid fluorescence quenching under
381
NIR laser irradiation while NV-DIR showed a much higher photostability (data not 18
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382
shown).
The
photothermal
profiles
of
NV-DOX-DIR
exhibited
a
383
concentration-dependent pattern upon NIR laser irradiation (Figure 2C). These results
384
elucidated that DIR inserted in NVs could increase the photostability and meanwhile
385
enhance the heat converting efficiency compared with free DIR. Therefore, DIR
386
incorporated NVs could serve as a potent candidate for photothermal therapy.
387 388
Figure 2. The in vitro temperature profiles after NIR laser irradiation. (A). The
389
real-time photothermal images of PBS, free DIR in PBS (100 µg/mL), NV-DIR and
390
NV-DOX-DIR under NIR irradiation. (B). The corresponding photothermal profiles
391
of PBS, free DIR, NV-DIR and NV-DOX-DIR upon NIR laser illumination. (C). The
392
temperature profiles of NV-DOX-DIR at different DIR concentrations (808 nm, 2.5
393
W/cm2). 19
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394 395
3.3. The uptake by tumor cells and evasion from macrophages
396
The effective internalization of therapeutic agents by tumor cells plays an
397
essential role in the subsequent tumor killing. Here, the intracellular uptake of NVs by
398
tumor cells was determined after incubation with free DOX, NV-DOX or
399
NV-DOX-DIR, respectively. For quantification of the cellular uptake, cells were
400
harvested for analysis by flow cytometer. As Figure 3A showed, the uptake behavior
401
of all the formulations displayed a time-dependent manner. Both NV-DOX and
402
NV-DOX-DIR showed enhanced cellular uptake with improved florescence intensity
403
compared to free DOX. This may be due to that the internalization of NVs was
404
mediated by the fusion of membrane components of NVs with the similar cell
405
membrane of tumor cells, which would promote the drug accumulation in the
406
cells.35-36 NV-DOX and NV-DOX-DIR exhibited comparable cellular uptake as the
407
DIR insertion did not influence the physiological properties on the membrane of NVs.
408
Furthermore, after incubation with different formulations for 4 h, the cells were
409
observed under the confocal microscope to visualize the internalization of NVs. It
410
showed the similar tendency as the results displayed in flow cytometer (Figure 3B).
411
RES clearance keeps as one of the major obstacles in the application of
412
nanomedicines as nanocarriers can be easily recognized as intruders to the host and
413
readily cleared by RES, which would greatly reduce the in vivo circulation time and
414
drug delivery efficiency.37 Surface modification by PEG is a commonly used strategy
415
to reduce the opsonization by RES but cannot effectively prevent this kind of 20
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416
nonspecific clearance.16 With innate biomimetic nature, membrane vesicles may
417
evade the opsonization and clearance by RES and achieve prolonged in vivo
418
circulation time. To determine the evasion function of NVs from RES, we examined
419
the cellular uptake in Raw 264.7 macrophages. A liposome consisting of
420
phosphatidylcholine, cholesterol and DSPE-PEG 2000 was used as the control. As
421
shown in Figure 3C, DIL labeled NVs showed significantly decreased internalization
422
by RAW264.7 macrophages compared with the liposome, indicating the effective
423
evasion of NVs from RES clearance. The quantitative uptake of NVs by macrophages
424
detected by flow cytometer also showed a reduced mean fluorescence intensity (MFI)
425
in comparison to the liposome (Figure 3D). It may be attributed to that the
426
nanovesicles with similar functionality as cell membrane were not “non-self”
427
components after injection, which could effectively evade non-specific clearance from
428
the body.38 Taken together, NVs can effectively promote the internalization by tumor
429
cells while decrease the clearance by RES, which may be an ideal drug carrier in
430
cancer therapy.
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431 432
Figure 3. The intracellular uptake of NVs by tumor cells and evasion from
433
macrophages. (A). The quantitative uptake of NVs by B16F10 cells detected by flow
434
cytometer (n = 3, *p < 0.05). (B). Confocal images of B16F10 cells after incubation
435
with free DOX, NV-DOX and NV-DOX-DIR for 4 h, respectively. Cell nucleus was
436
stained with DAPI (scale bar, 50 µm). (C). The cell uptake of DC2.4 derived NVs and
437
liposome by Raw 264.7 macrophages by confocal microscope (scale bar, 50 µm). (D).
438
The determination of quantitative cellular uptake by Raw 264.7 macrophages using
439
flow cytometer (n = 3, *p < 0.05).
440 441
3.4. In vitro combinational photothermal-chemotherapy
442
To investigate the potential photothermal effect on tumor cells, the intracellular
443
temperature of B16F10 cells upon irradiation was recorded using infrared thermal
444
camera after treating with different formulations. As Figure 4A showed, the 22
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445
temperature change of PBS treated cells was only 5.5 ± 0.6 oC after NIR irradiation
446
for 3 min. Free DIR pretreated cells showed a maximum temperature increase as 10.5
447
± 1.1 oC and reduced to 8.2 ± 1.4 oC after 3 min illumination. However, after
448
treatment with NV-DIR or NV-DOX-DIR, the temperature of tumor cells was
449
dramatically increased within 30 s and then remained a relatively steady state during
450
the irradiation. To further investigate the impact of DIR inserted NVs on ROS
451
generation, the cells were stained with DCFH-DA and irradiated with NIR laser. As
452
shown in Figure 4B and Figure S3, NV-DIR and NV-DOX-DIR can remarkably
453
increase the level of intracellular ROS, which would induce irreversible damage or
454
apoptosis of cells. Therefore, incorporating DIR into NVs showed higher efficiency to
455
induce NIR triggered hyperthermia in B16F10 cells than free DIR, facilitating the
456
photothermal ablation of tumor cells. Then the cytotoxicity against tumor cells was
457
determined by MTT assay to evaluate the in vitro combinatorial antitumor effect of
458
photothermal-chemotherapy. B16F10 cells were treated with free DOX, free DOX
459
and
460
NV-DOX-DIR+Laser, respectively. The cytotoxicity induced by all the formulations
461
showed a concentration-dependent manner. Compared with free drugs (DOX or
462
DOX+DIR+Laser), the drug loaded NVs exhibited much lower cell viability, which
463
could be resulted from the enhanced drug accumulation and increased heat converting
464
efficiency by NVs. With a combinational effect of photothermal and chemotherapy,
465
NV-DOX-DIR showed the highest cytotoxicity to tumor cells compared with that of
466
single treatment with NV-DOX or NV-DIR+Laser (Figure 4C). To further visualize
DIR
with
laser
(DOX+DIR+Laser),
NV-DOX,
23
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NV-DIR+Laser
and
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467
the cytotoxic effect, B16F10 cells were incubated with different formulations and
468
treated with or without NIR laser. Afterward, the cells were stained with Calcein
469
AM/PI for visualizing the live/dead cells. Figure 4D showed that the NIR laser
470
irradiation alone did not significantly affect the cell viability, suggesting the safety of
471
laser illumination at 2.5 W/cm2. DOX and DOX+DIR+Laser showed higher
472
cytotoxicity to B16F10 cells than control, which was demonstrated by the reduced
473
cell density and increased PI staining. Monotherapy with NV-DIR+Laser or NV-DOX
474
also caused cytotoxicity to B16F10 cells. Notably, after treatment with
475
NV-DOX-DIR+Laser, the combinational therapy demonstrated higher tumor killing
476
effect as the presented much stronger PI signal for dead cells and rarely found Calcein
477
AM signal for live cells. Collectively, these results indicated that NVs could enhance
478
the cytotoxicity against tumor cells compared with free drugs and the
479
photothermal-chemotherapy based on NVs may possess great potential to realize
480
synergistic antitumor effect.
481 24
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482
Figure 4. In vitro combinatorial photothermal-chemotherapy against tumor cells. (A).
483
The photothermal profiles of B16F10 cells after treating with free DIR, NV-DIR and
484
NV-DOX-DIR (n = 3, *p < 0,05). (B).The intracellular ROS generation after treating
485
with different formulations. The orange, black, green, blue and red line represented
486
CTRL, PBS, free DIR, NV-DIR and NV-DOX-DIR, respectively. (C) The cell
487
viability of B16F10 cells after treating with DOX, DOX+DIR+Laser, NV-DOX,
488
NV-DIR+Laser and NV-DOX-DIR+Laser, respectively. (D). Fluorescent images of
489
B16F10 cells after various treatments. The live cells were stained with Calcein AM
490
(green) and the dead cells were stained with PI (red). The scale bar is 50 µm.
491 492
3.5. In vivo imaging and biodistribution
493
We then observed the in vivo biodistribution of NVs after intravenous
494
administration on melanoma bearing mice to determine the feasibility of NVs as
495
effective drug carriers. Taking advantage of the intrinsic absorption of DIR in NIR
496
region, the distribution of free DIR and NV-DIR after administration can be easily
497
visualized.39 As shown in Figure 5A, the in vivo NIR fluorescence images were
498
monitored at various time points after intravenous injection of free DIR and NV-DIR.
499
It was found that NV-DIR showed gradually increased fluorescence of DIR with time
500
prolonging and reached the maximum after 24 h. However, free DIR showed much
501
weak fluorescence in tumor site, indicating the poor accumulation of free DIR in
502
tumor. The corresponding average fluorescence intensity also showed the similar
503
tendency (Figure 5B). The better distribution and accumulation of NV-DIR in tumor 25
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504
may be attributed to the EPR effect of nano-sized drug carriers40 and the specific
505
targeting ability of cell derived NVs.22 To further investigate the distribution of free
506
DIR and NV-DIR, the mice were sacrificed, tumors and major organs were excised
507
for ex vivo imaging and fluorescence quantification at 48 h post injection. As shown
508
in Figure 5C, free DIR mainly distributed in liver, lung and spleen while showed a
509
weak accumulation in tumor. NV-DIR also showed a high accumulation in liver, lung
510
and spleen. However, the accumulation of NV-DIR in tumor site was much higher
511
than that of free DIR. The corresponding quantitative analysis also exhibited the
512
higher fluorescence intensity in tumor of NV-DIR than that of free DIR (Figure S4).
513
These results indicated that NVs showed an improved accumulation in tumor and may
514
realize the satisfied antitumor efficiency.
515 516
Figure 5. The in vivo biodistribution of free DIR and NV-DIR after systematic
517
administration. (A). The NIR fluorescent images of mice. (B). The quantitation of
518
fluorescence intensity of tumors (n = 3, *p < 0.05). (C). The ex vivo images of excised
519
tumor and major organs of C57BL/6 mice at 48 h after systematic injection of free 26
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520
DIR and NV-DIR, respectively.
521 522
3.6. In vivo combinational photothermal and chemotherapy
523
Accoording to the results above, it has been demonstrated that incorporating DIR
524
into NVs could induce efficient hyperthermia in vitro. To further monitor the
525
photothermal effect of NVs in vivo, tumor bearing mice were injected with PBS, free
526
DIR, NV-DIR and NV-DOX-DIR, respectively. On the basis of accumulation of NVs
527
in tumor presented above, the mice were anesthetized and exposed to NIR laser after
528
24 h. As Figure 6A-B displayed, within 2 min irradiation, free DIR showed slightly
529
higher but no significant temperature increase compared to PBS, which may be
530
resulted from the restricted tumor accumulation and weak photothermal converting
531
efficiency of free DIR. However, for NV-DIR and NV-DOX-DIR treated mice, the
532
temperature of tumor was rapidly elevated over 50 °C, which would induce an
533
irreversible photothermal ablation of tumor cells.41 The results demonstrated the great
534
potential of incorporating DIR into NVs to realize effective PTT.
535
To further investigate the synergistic antitumor effect of combinational
536
photothermal and chemotherapy, C57BL/6 mice were inoculated with B16F10 cells
537
(5×104) at the left flank of back. At day 11, the mice were treated with PBS,
538
PBS+Laser, DOX, DOX+Laser, NV-DOX, NV-DIR+Laser and NV-DOX-DIR+Laser
539
at an interval of 2 days for 3 times, respectively. The NIR irradiation was conducted at
540
24 h after injection. As Figure 6C showed, there is no significant difference in tumor
541
growth after treatment with laser alone compared with PBS, which further 27
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542
demonstrated the in vivo safety of laser illumination at 2.5 W/cm2. Comapred with
543
free DOX and DOX+DIR+Laser, the drug loaded NVs showed much better tumor
544
growth inhibition, suggesting the promising efficiency for NVs as drug carrier. As a
545
combination for photothermal and chemotherapy, NV-DOX-DIR achieved the best
546
tumor growth inhibition with the minimum tumor volume as 189.2 ± 53.4 mm3 at day
547
21. Moreover, NV-DOX-DIR showed the highest tumor inhibitory rate as 91.6%
548
compared with DOX, DOX+Laser, NV-DOX, NV-DIR+Laser of 38.7%, 45.6%, 76.8%
549
and 72.7%, respectively (Figure S5). During the treatment period, the body weight of
550
mice treating with different formulations all showed an increasing tendency (Figure
551
S6). Then all the mice were sacrificed, the direct image (Figure 6D) and weight of
552
excised tumors (Figure S7) showed the similar tendency as tumor growth curves.
553
Besides, the tumors were stained with HE and observed using a microscope. In PBS
554
and PBS+Laser treated groups, the tumor tissue showed compact and disordered cell
555
arrangement, indicating negligible tumor necrosis. DOX and DOX+Laser treated
556
tumors showed partial nucleus cracking while the necrosis region in NV-DOX and
557
NV-DIR+Laser
558
NV-DOX-DIR+Laser treated tumor, it showed a much larger area of tumor necrosis
559
compared with other groups (Figure 6E). To further indicate cell apoptosis at tumor
560
site, the tumors were then conducted with TUNEL assay. As Figure 6F showed,
561
NV-DOX-DIR+Laser displayed the highest expression of apoptosis fluorescence
562
compared with other groups. All in all, the combination of photothermal and
563
chemotherapy based on NVs may be a promising strategy for tumor therapy.
treated
groups
was
further
28
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enlarged.
Notably,
for
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Molecular Pharmaceutics
564 565
Figure 6. In vivo synergistic antitumor efficacy of NVs against murine melanoma. (A).
566
Infrared photothermal images of tumor-bearing mice upon exposure to NIR laser. (B).
567
In vivo photothermal profiles of the tumor tissues after treating with PBS, free DIR,
568
NV-DIR and NV-DOX-DIR, respectively (n = 3). (C). The tumor growth curves after
569
various treatments during a therapeutic period for 21 days (n = 7, *p < 0.05). (D).
570
Images of excised tumor at day 21 after treating with PBS, PBS+Laser, DOX,
571
DOX+Laser, NV-DOX, NV-DIR+Laser and NV-DOX-DIR+Laser, respectively (n = 7,
572
*p < 0.05). (E). Representative HE staining images of tumor after various treatments
573
(scale bar, 250 µm). (F). TUNEL fluorescence examination images of tumor sections
574
after treating with PBS, PBS+Laser, DOX, DOX+Laser, NV-DOX, NV-DIR+Laser
575
and NV-DOX-DIR+Laser, respectively (scale bar, 50 µm). 29
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576 577
3.7. Evaluation of systemic toxicity of NVs
578
To evaluate the systemic toxicity, the mice were treated with PBS, PBS+Laser,
579
DOX, DOX+Laser, NV-DOX, NV-DIR+Laser and NV-DOX-DIR+Laser, respectively.
580
Afterward, the mice were sacrificed and the serum was collected to measure a series
581
of blood biochemistry parameters. ALT and AST have been considered as indicators
582
for liver pathology. After different treatment, the levels of ALT and AST were all
583
within normal range (Figure 7A-B). BUN and Cr are the commonly used indices to
584
assess the function of renal. As Figure 7C-D showed, no apparent difference was
585
observed in both BUN and Cr among all the groups. LDH is a kind of cytoplasmic
586
enzyme which can be found in all tissues. The increase of serum LDH is closely
587
related with cell damage, which directly reflects the tissue injury. In this experiment
588
setting, all the groups showed comparable level of LDH, indicating negligible damage
589
to major tissues. To further investigate the systematic toxicity, the major organs were
590
excised and stained with HE. As Figure 7F showed, there was no obvious
591
histopathological changes in all tissues after systematic treatment of various
592
formulations, respectively. The results suggested that NVs can be used as a kind of
593
safe vehicle for systematic drug delivery.
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Molecular Pharmaceutics
594 595
Figure 7. Systemic evaluation after intravenous administration of NVs. The
596
determination of blood biochemistry parameters including (A). ALT, (B). AST, (C).
597
BUN, (D). Cr and (E). LDH after treating with PBS, PBS+Laser, DOX, DOX+Laser,
598
NV-DOX, NV-DIR+Laser and NV-DOX-DIR+Laser, respectively (n = 3). (F). The
599
representative HE staining images of the major organs after various treatments (scale
600
bar, 250 µm).
601 31
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602
4. CONCLUSION
603
In this work, we fabricated a kind of DOX loaded and NIR dye inserted
604
biomimetic nanovesicles by a facile method for combinational photothermal and
605
chemotherapy. As a kind of membrane vesicles, NVs could effectively evade the
606
clearance by RES. Owing to the specific targeting ability of membrane vesicles and
607
EPR effect of nanosized vehicles, NVs can accumulate in tumor site after intravenous
608
injection. Upon NIR laser irradiation, the NVs with DIR insertion can convert
609
absorbed fluorescent energy into comparable heat with high efficiency to eradicate
610
tumor cells. Meanwhile, the NIR triggered hyperthermia can destroy the membrane of
611
NVs and thus induce the accelerated release of DOX to further cause direct
612
cytotoxicity to tumor cells. The combination of photothermal and chemotherapy could
613
achieve efficient in vitro tumor killing and satisfied in vivo tumor growth inhibition
614
against murine melanoma. The NVs demonstrated the great potential in the
615
combinatorial antitumor therapy with good biomimetic properties and feasibility for
616
clinic translation.
617 618
ASSOCIATED CONTENT
619
Supporting Information
620
The Supporting Information is available free of charge on the ACS Publications
621
website.
622
Supplementary Figures S1−S7
623
Figure S1. The relative diameter change of NV-DOX and NV-DOX-DIR in FBS for 5 32
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624
days (n = 3).
625
Figure S2. The relative retained DOX in NVs for 5 days at 4 oC (n = 3).
626
Figure S3. Quantitative detection of ROS generation after treating with NIR
627
irradiation (n = 3).
628
Figure S4. The quantitation of fluorescent intensity of tumors and the major organs at
629
48 h after intravenous injection of free DIR and NV-DIR (n = 3, *p < 0.05).
630
Figure S5. The tumor inhibitory rate after treating with PBS+Laser, DOX,
631
DOX+Laser, NV-DOX, NV-DIR+Laser and NV-DOX-DIR+Laser, respectively (n = 7,
632
*p < 0.05).
633
Figure S6. The relative body weight change after various treatments (n = 7)
634
Figure S7. The weight of the excised tumors after different treatments (n = 7, *p