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Mesenchymal stem cells transporting black phosphorus-based biocompatible nanospheres: Active trojan horse for enhanced photothermal cancer therapy
Journal Pre-proofs Mesenchymal stem cells transporting black phosphorus-based biocompatible nanospheres: active trojan horse for enhanced photothermal cancer therapy Miaomiao Luo, Yun Zhou, Nansha Gao, Wei Cheng, Xusheng Wang, Jinxiu Cao, Xiaowei Zeng, Gan Liu, Lin Mei PII: DOI: Reference:
S1385-8947(19)33357-1 https://doi.org/10.1016/j.cej.2019.123942 CEJ 123942
To appear in:
Chemical Engineering Journal
Received Date: Revised Date: Accepted Date:
9 October 2019 21 December 2019 24 December 2019
Please cite this article as: M. Luo, Y. Zhou, N. Gao, W. Cheng, X. Wang, J. Cao, X. Zeng, G. Liu, L. Mei, Mesenchymal stem cells transporting black phosphorus-based biocompatible nanospheres: active trojan horse for enhanced photothermal cancer therapy, Chemical Engineering Journal (2019), doi: https://doi.org/10.1016/j.cej. 2019.123942
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© 2019 Published by Elsevier B.V.
Mesenchymal
stem
cells
transporting
black
phosphorus-based biocompatible nanospheres: active trojan horse for enhanced photothermal cancer therapy Miaomiao Luoa,1, Yun Zhou a,1, Nansha Gaoa, Wei Chenga, Xusheng Wanga, Jinxiu Caoa, Xiaowei Zenga,, Gan Liua,*, Lin Meia,*
a School
of Pharmaceutical Sciences (Shenzhen), Sun Yat-sen University, Guangzhou
510275, P.R. China
1
These authors contributed equally to this work.
*Corresponding
author
E-mail address: [email protected] (L. Mei), [email protected] (G. Liu).
HIGHLIGHTS
MSC possess excellent tumor tropism and low immunogenicity.
PLGA/BPQDs show prominent biocompatibility.
PLGA/BPQDs–loaded MSC exhibited changeless high tumor-tropic ability.
MSC@PLGA/BPQDs would exert tumor ablation in vivo.
GRAPHICAL ABSTRACT
ABSTRACT As potential carriers for specifically transporting photothermal agents to tumors, mesenchymal stem cells (MSC) would enhance their photothermal therapeutic effects. However, a biocompatible and biodegradable nanoscale photothermal agent, which hardly affects the tumor tropism of MSC, is still urgently needed. This study reports an effective therapeutic platform based on MSC loaded with poly (lactic-co-glycolic acid)/black phosphorus quantum dots (PLGA/BPQDs) for targeted photothermal
therapy of U251 glioma tumor cells. PLGA/BPQDs could not only be effectively uptaken by the MSC, but also stably maintained in the MSC for as long as 3 d. PLGA/BPQDs–loaded MSC exhibited changeless high tumor-tropic ability due to the exceptional biocompatibility of PLGA/BPQDs. In vitro results showed that PLGA/BPQDs can be transported from MSC to U251 cells, where U251 cells are killed after irradiation. In vivo results demonstrated that MSC@PLGA/BPQDs can efficiently tend the U251 glioma tumor and showed much longer retention times at the tumor site than PLGA/BPQDs alone. Finally, MSC@PLGA/BPQDs, as an active trojan horse, demonstrated enhanced photothermal effectivity on the U251 glioma tumor in vivo.
Keywords: Mesenchymal stem cells; Black phosphorus; PLGA; Photothermal therapy;
Targeted delivery.
1. Introduction As a promising substitute for traditional cancer therapies, photothermal therapy (PTT) has attracted enormous attention over the past few years due to its minimal invasiveness, less side effects, and high therapeutic efficiency [1-8]. Nanomaterials such as polymer-based semiconductor nanoparticles, metal-based nanomaterials, and carbon nanomaterials have been widely used as PTT agents due to their excellent near-infrared (NIR) photothermal performance [9-12]. Nanoscale photothermal agents can improve the tumor targeting ability through the enhanced permeability
and retention effect (EPR) and modification of targeting groups. However, their targeting efficiency is still very low (below 0.5%), which greatly hampers their anti-tumor effect. In addition, most inorganic nanometer PPT agents can easily accumulate and block blood vessels after injection into the blood, which greatly limits their clinic application [13]. Therefore, novel and safe methods to enhance the targeting efficiency of PPT agents are urgently needed. Recently, mesenchymal stem cells (MSC) have been widely developed for targeted tumor therapy due to their excellent tumor tropism and low immunogenicity [14, 15]. Studies showed that CXCR4 expressed in MSC was predominant in determining MSCs tumor tropism. This behavior was navigated by multifarious chemokine such as chemokine stromal derived factor 1 (SDF-1) released by the tumor site, whose receptor is CXCR4 [15-17]. MSC have been used as carriers to deliver PPT agents (such as gold nanoparticles) to tumor sites for PTT [17-19]. However, these PPT agents have non-negligible toxicity and poor metabolism, which not only affects the tumor tropic ability of MSC, but their long-term accumulation could also cause potential health risks [13]. Therefore, the exploration of novel PTT agents with ideal biocompatibility and biodegradability for efficient MSC transporting is vital. As a new member of the two-dimensional (2D) materials family, black phosphorus (BP) has recently aroused significant attention for biomedical applications due to its excellent properties [20-23]. BP shows layer-dependent bandgaps, which can be adjusted from 0.3 eV (bulk) to 2.0 eV (single layer), leading
to wide absorption ranging from ultraviolet (UV) to infrared regions [24, 25]. The zero-dimensional BP, black phosphorus quantum dots (BPQDs), show interesting photothermal conversion behavior and large extinction coefficients, and were used for PPT [25-29]. More importantly, BP showed outstanding biocompatibility, because P is an essential element in the human body [30-32]. It has been reported that BP is degraded into nontoxic phosphate and phosphonate in aqueous solution, both of which are widely distributed throughout the human body [33]. Therefore, BPQDs can be used as candidate PPT agents that can be loaded into MSC. Despite their promising potential, BPQDs are quite unstable under physiological conditions due to their high reactivity with oxygen and water [34-36], especially after loading into cells. Very recently, a number of strategies have been reported to improve the stability of BP, including surface protective layer protection, surface chemical modification, and doping [37]. Shao et al. [26] encapsulated BPQDs
into
the
Food
and
Drug
Administration
(FDA)-approved
poly
(lactic-co-glycolic acid) (PLGA) to obtain PLGA/BPQDs nanoparticles. This improved the photothermal stability of BPQDs by isolating internal BPQDs from water and oxygen. Thus, loading PLGA/BPQDs into MSC to obtain an active trojan horse, is reasonably anticipated to neither suffer the tumor tropism of MSC, nor rapidly decay the photothermal efficacy of BPQDs. Herein, a therapeutic platform was designed by loading PLGA/BPQDs into MSC (MSC@PLGA/BPQDs) to achieve tumor-tropic migration and enhanced photothermal therapy against U251 glioma cells (Fig. 1). PLGA/BPQDs were
prepared by encapsulating BPQDs with PLGA and were chosen as PTT agent due to their excellent biocompatibility, ideal biodegradability, and high photothermal efficiency. After intravenously injection, PLGA/BPQDs proved to be efficiently transported to the tumor site, released from MSC, and uptaken by U251. This led to enhanced photothermal therapy under NIR irradiation.
Fig. 1. Schematic design of PLGA/BPQD-loaded mesenchymal stem cells (MSC) to tumors for enhanced photothermal therapy.
2. Materials and methods 2.1. Materials Bulk BP was purchased from Nanjing muke nano technology Co., Ltd. (Nanjing, China). PLGA (75:25, MW: 10,000–20,000) was purchased from the Shandong
academy
of
pharmaceutical
N-methyl-2-pyrrolidone
(NMP),
sciences
pilot
polyvinyl
alcohol
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium
plant
(Jinan,
(MW:
China).
9,000–10,000),
bromide
(MTT),
dichloromethane (DCM), 4',6-diamidino-2-phenylindole (DAPI), imethyl sulfoxide (DMSO), Coumarin 6, and IR-780 were purchased from Sigma–Aldrich (St. Louis, MO, USA). Foetal bovine serum, H-DMEM, penicillin-streptomycin, trypsin-EDTA, and PBS (pH 7.4) were obtained from Gibco Life Technologies (AG, witzerland). All chemicals used in this study were of analytical reagent grade and were used without further purification. 2.2. Preparation of BPQDs 10 mg of bulk BP crystal powder was first added to 20 mL of NM Then, the mixture was sonicated with a sonic tip for 14 h (amplifier: 25%, on/off cycle: 5 s/5 s) in an ice bath at 700 W. After that, the mixture solution was sonicated in the water bath for 10 h at 300 W. Subsequently, the dispersion was centrifuged at a speed of 7000 rpm for 20 min and the supernatant was centrifuged at 12000 rpm for 20 min to obtain the precipitate (BPQDs). 2.3. Synthesis of PLGA/BPQDs nanoparticles PLGA/BPQDs NSs were synthesized by a modified oil-in-water emulsion solvent evaporation method. Briefly, 10 mg of BPQDs were added to 5 mL DCM and then mixed with 50 mg PLGA. The mixture was subjected to probe sonication for 10 min and was then dispersed in 50 mL of 0.5% (w/v) polyvinyl alcohol aqueous solution, followed by sonication for another 10 min. Then, the mixture was stirred for
12 h to evaporate residual DCM, then centrifuged at 7,000 rpm for 20 min, and washed with deionized water to remove dissociated BPQDs. The product was designated as PLGA/BPQDs. PLGA/Coumarin 6 and PLGA/IR780 were obtained with the same method. 2.4. Characterization of PLGA/BPQDs Transmission electron microscopy (TEM) was performed on the FEI Tecnai G2 F30 transmission electron microscope (FEI Company, Hillsborough, OR, USA) at an acceleration voltage of 300 Kv. Atomic force microscopy (AFM) Bruker Dimension® Icon™) was used to characterize both the morphology and height of samples. Scanning electron microscopic (SEM) images were obtained on a field-emission SEM. The zeta potential of nanoparticles was acquired by NanoBrook 90Plus PALS. X-ray photoelectron spectroscopy (XPS) was conducted on an X-ray photoelectron spectroscope (Axis HSi, Kratos Ltd., Manchester, UK) with Al Kα radiation (1486.6 eV photons, 150 W) as X-ray source for excitation. Raman spectra of BPQDs and PLGA/BPQDs were obtained with a high-resolution confocal Raman microscope (HORIBA LabRAM HR800). X-ray diffraction (XRD) were performed on a Philips X’Pert Pro Super diffractometer with Cu Kα radiation (λ= 1.54178 Å). 2.5. Cell Culture MSC, isolated from human umbilical cords, were obtained from Dr. Xusheng Wang from the Sun Yat-sen University, China, and were incubated in low glucose Dulbecco's Modified Eagle Medium (DMEM), containing 1% enicillin-streptomycin and 10% fetal bovine serum (FBS). U251 and NIH3T3 cells were also cultured in
DMEM supplemented with 1% penicillin-streptomycin and 10% FBS. All cell lines were cultured in a humid atmosphere with 5% CO2 at 37 °C. 2.6. Cellular uptake of particles MSC were plated in glass-bottomed dishes. When cells reached 80% confluence, the MSCs were cultivated with 40 µg/mL particles for 12 h. To observe the uptake of nanoparticles by MSC cells, the MSCs were treated with PLGA/Coumarin 6 for 12 h, washed with PBS three times, fixed with 4% paraformaldehyde, and finally dyed with DAPI. Samples were observed with a confocal microscope (FV1000, Olympus, Tokyo, Japan). The uptake rate of PLGA/BPQDs by MSC was measured by a multi-function board reader (Perkin Elmer). Firstly, PLGA/BPQDs were suspended by DMEM medium, containing 1% penicillin-streptomycin and 10% FBS. Then, the standard curve of PLGA/BPQDs was made with a multi-function board reader (Perkin Elmer). The MSC were incubated on 6-well plates with 10, 20, and 30 μg/mL of PLGA/BPQDs for 12 h. The absorbance value of PLGA/BPQDs in the supernatant was measured by the multi-function board reader. The concentration of PLGA/BPQDs was calculated by the PLGA/BPQDs standard curve to obtain the amount of PLGA/BPQDs in the supernatant, and to finally calculate the uptake rate. Cellular uptake was quantitatively analyzed by flow cytometry (FACS). In brief, MSCs were incubated with PLGA/Coumarin 6 for 1 h, 6 h, 12 h, and 18 h, respectively. The cells were wished three times and then collected. The fluorescence intensity was detected by FACS.
Quantitative analysis of cellular retention was also conducted by FACS. Briefly, PLGA/Coumarin 6 was incubated with MSCs for 12 h and subsequently washed with PBS three times. After that, the cells continued to be incubated by adding fresh medium. At 12 h, 1 day, 2 days, and 3 days, the cells were collected and the fluorescence intensity was obtained by FACS. 2.7. Cell migration The migration capability of PLGA/BPQDs loaded MSCs was investigated by the transwell assay. MSCs or loaded MSCs were suspended in 200 µL serum-free medium and were seeded in the upper well of 24-well transwell chambers (8 μm pore size, Corning Incorporated) at a density of 105cell/well. The same numbers of U251 glioma cells or NIH3T3 cells were plated in the lower chamber. After culture at 37 °C for 6 h, cells that still remained at the surface of the upper membrane were gently wiped off. Cells that had migrated to the surface of the lower membrane were fixed with 4% paraformaldehyde and stained with DAPI for 15 min. All samples were observed using a fluorescence microscope and the number of migrated cells per optic field was counted and averaged. 2.8. In vitro cytotoxicity assays MSC were seeded on 96-well plates at a density of 8000 cells per well and were cultured for 12 h. Afterward, the medium was replaced with fresh culture medium containing PLGA/BPQDs or BPQDs at various concentrations (2–100 µg BPQDs/mL). After cultivation for 24 h at 37 °C, 20 µL of MTT solution was added to each well and the mixture was cultured for further 4 h. Then, the medium was
removed, which was followed by addition of 150 µL DMSO to each well. The absorption was measured by using a microplate reader at 570 nm. Cells without any treatment were set as 100% cell viability. 2.9. Laser irradiation and photothermal imaging To measure the intracellular photothermal effect of MSC@PLGA/BPQDs, MSCs were seeded on six-well plates and then co-cultured with PLGA/BPQDs at two concentrations (20 and 40 µg BPQDs/mL). After 12 h, the loaded cells were collected and resuspended in 20 μL PBS. The samples (2 × 105 cells in 20 μL of PBS) were placed in 200 μL Eppendorf tubes and were then subjected to 808 nm NIR laser treatment (at 1 W cm-2 for 10 min). The temperature variations of the MSC@PLGA/BPQDs were recorded by an infrared thermal imaging camera (Ti450, Fluke, Everett, WA, USA). 2.10. In vitro inhibition of tumor growth MSC were incubated with PLGA/Coumarin 6 particles for 12 h, washed three times, and collected. MSC@PLGA/Coumarin 6 and GFP labeled U251 cells (at a ratio of 2:1) were co-cultured for 24 h, washed three times with PBS, fixed with 4% paraformaldehyde, and dyed with DAPI. Samples were observed under a confocal microscope (FV1000; Olympus, Tokyo, Japan). The in vitro anti-tumor effect of MSC@PLGA/BPQDs on U251 glioma cells was evaluated by using MTT. Briefly, a co-culture system was constructed by mixing MSC@PLGA/BPQDs with U251 glioma cells at different ratios ranging from 1:4 to 4:1. The plate was illuminated with an 808 nm laser for 10 min at a power density of
1 W/cm2. The cell viability was normalized to the control group, which consisted of U251 glioma cells only. After 12 h of incubation, the viability of cells was evaluated in an MTT assay. 2.11. Xenograft Tumor Models Female balb/c nude mice were bought from Sun Yat-Sen University. U251 cells (107 cells in 100 μL) were subcutaneously injected into the right axillary region of female nude mice. The tumor size was measured with a digital Vernier caliper every two days and the tumor volume (V) was calculated via (tumor length) × (tumor width)2 / 2. Mice were randomly divided into different groups when the tumor volume was approximately 100 mm3. 2.12. In vivo fluorescence imaging When the tumor size reached about 500 mm3, U251 tumor cell bearing mice were randomly divided into two groups (n = 3 per group): a PLGA/IR780 group and an MSC@PLGA/IR780 group (1 mg/kg equivalent IR780 for per group). Mice were intravenously injected with 200 μL dye-loaded NSs via tails. An in-vivo imaging system was employed to take in vivo images of mice at 1 h, 3 h, 24 h, 2 days, 3 days, and 4 days post-injection. NIR light with a peak wavelength of 780 nm was used as excitation source. Nude mice were euthanized at 2 day post-injection and the major organs (heart, liver, spleen, lung, kidney, and tumor tissues) of mice were collected and their fluorescence intensities were measured. 2.13. In vivo PTT and photothermal images When the volume of the tumor reached about 100 mm3, each group (n = 5) was
administered with (A) phosphate buffered saline (PBS, 200 μL), (B) BPQDs (100 μg, 200 μL), (C) PLGA/BPQDs (100 µg BPQDs, 200 μL), (D) MSC + PLGA/BPQDs (100 μg BPQDs, 200 μL), and (E) MSC@PLGA/BPQDs (100 μg of BPQDs in 5 × 105 MSCs, 200 μL) via the tail vein. At 2 days post-injection, all mice were anaesthetized and irradiated with an 808 nm NIR laser (1 W/cm2 for 5 min). At the same time, the tumor temperature change and infrared thermographic maps were recorded by the already mentioned infrared thermal imaging camera. Tumor size and weight of all mice were measured every other day until the end of treatment. The relative tumor volume was calculated as follows: V/V0, where V0 represents the initial tumor volume at the beginning of treatment. After about three weeks of treatment, mice were euthanized and the main organs and tumor tissues were harvested for further study. 2.14. Histological examination and TUNEL assay At day 16 post-transplantation, mice were humanely killed and the organs (including heart, liver, spleen, lung, and kidney) and tumor tissues were harvested. In brief, all samples were fixed in a 10% formalin solution, and then paraffin-embedded. All organs of mice were tested using hematoxylin and eosin (HE) staining for histological examination and all tumors were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay to label apoptotic cells in tumor tissues. 2.15. Statistical analysis All experiments were conducted at least three times. The experimental data are
showed as mean ± standard deviation (SD). Statistical analyses were performed by one-way analysis of variance (ANOVA) followed by Bonferroni test using SPSS 22.0 software. * P < 0.05 was identified as statistically significant and ** P < 0.01 was identified as extremely statistically significant.
3. Results and Discussion 3.1. Synthesis and characterization of PLGA/BPQDs TEM and AFM were used to observe the morphology of BPQDs. As shown in the TEM images (Fig. 2A and B), the average size of BPQDs was 3.5 ± 0.9 nm according to the statistical TEM analysis of 200 BPQDs. The AFM images in Fig. 2C show the topographic morphology of BPQDs. The measured heights of three randomly selected BPQDs were 4.48, 3.6, and 3.11 nm (Fig. 2D), respectively. As shown in fig. S1, all diffraction peaks of BPQDs can be readily indexed to the orthorhombic BP consistent with JCPDS No. 74-1878. PLGA/BPQDs nanospheres were prepared by an oil-in-water emulsion solvent evaporation method. TEM images (Fig. 2E) and SEM images (Fig. 2F) showed that all PLGA/BPQDs nanospheres have a uniform spherical shape. The average size of the PLGA nanospheres was 165.6 ± 55 mm according to the statistical SEM analysis of 200 PLGA/BPQDs (Fig. 2G). Moreover, energy dispersive X-ray spectroscopy (Table 1, see Supporting Information) showed the elemental content of C, O, and P in PLGA nanoparticles. C and O were mainly present due to the introduction of PLGA, which confirmed the successful preparation of PLGA/BPQDs. The zeta potentials of different samples were measured (Fig. S2,
see Supporting Information). The zeta potential increased from -38.47 mV (BPQDs) to -28.18 mV (PLGA/BPQDs), indicating successful fabrication of PLGA/BPQDs. Raman scattering was used to characterize BPQDs and PLGA/BPQDs NPs (Fig. 2H). BPQDs showed three obvious peaks, which could be attributed to one out-of-plane phonon mode (A1g) at 357.6 cm−1 and two in-plane modes at 432.5 cm−1 (B2g) and 460.3 cm−1 (A2g), respectively. Compared with BPQD, the Raman spectra of PLGA/BPQDs NS showed a red shift, proving successful introduction of PLGA into the PLGA/BPQDs NS. PLGA, BPQDs and PLGA/BPQDs were further characterized by XPS (Fig. 2I and Fig. S3A-C) to measure their chemical compositions. The peak intensity of P2p (129.6 eV) (Fig. S3A and Figure 2I) in PLGA/BPQDs and PLGA was significantly weaker compared with BPQDs. At the same time, the peak intensities of C1s (Fig. S3B) and O1s (Fig. S3C) in PLGA/BPQDs and PLGA increased compared with BPQDs. The reason might be that PLGA has no P element and possesses both C and O elements in its chemical structure. As a consequence, these changes demonstrated the successful coating of PLGA. The encapsulation efficiency and loading efficiency were 50.5% and 9.7%, respectively, measured by inductively coupled plasma mass spectrometry (ICP-MS, Thermoscientific, USA). Then, the photothermal property and stability of PLGA/BPQDs were tested by using NIR laser irradiation (808 nm for 10 min). As shown in Fig. 2J and K, both BPQDs and PLGA/BPQDs exhibited a concentration-dependent photothermal effect. The temperature of both samples could increase by nearly 40 °C at a concentration of 60 ppm, and by nearly 10 °C at 7.5 ppm. Furthermore, PLGA coating on BPQD did
not affect the photothermal characteristics of BPQD. As shown in Fig. 2L, after being irradiated by an 808 nm NIR laser at a power density of 1 W/cm2 for 10 min, the temperature of the BPQD solution increased by 20.8 °C; however, the temperature of the BPQD solution increased by only 0.4 °C after 4 days, indicating relatively poor photothermal stability. In contrast, PLGA/BPQD showed better photothermal stability. After 4 days, the temperature increase of the PLGA/BPQDs solution was 17.8 °C, which was close to the initial value of 22.5 °C. This result demonstrated that PLGA can protect BP from degradation for almost three days when exposed to air and water.
Fig. 2. Morphology and characterization (A) transmission electron microscopic (TEM) pictures of BPQDs. (B) Statistical assessment of the size of 200 BPQDs according to the TEM images. (C) Atomic force microscopic (AFM) image of the BPQDs. (D) Height profiles along the red lines in d. (E) TEM and (F) scanning
electron microscopic (SEM) images of the PLGA/BPQDs. (G) Statistical analysis of the size of PLGA/BPQDs based on the SEM images. (H) Raman spectra and (I) X-ray photoelectron spectroscopic (XPS) spectrum of PLGA, BPQDs and PLGA/BPQDs. (J) Photothermal curves of BPQDs and PLGA/BPQDs at different concentrations. (L) Temperature change of BPQDs and PLGA/BPQDs dispersed in water for different durations under NIR laser illumination (1.0 W cm-2) for 10 min.
3.2. PLGA/BPQD cellular uptake and retention in MSC To analyze the PLGA/BPQD uptake capacity of MSC, the green fluorescent probe Coumarin 6 was utilized to replace BPQDs in the NPs. Fig. 3A shows a confocal laser scanning microscopic (CLSM) image of MSC after 12 h of incubation with Coumarin 6-loaded PLGA NPs (PLGA/Coumarin 6). It is clear that PLGA/Coumarin 6 (green) were closely located around the cell nuclei (blue), suggesting that MSC cells possess a high uptake capacity of PLGA NPs. The uptake rate of PLGA/BPQDs by MSC was 50.36% ± 0.012. FACS was also utilized to analyze the uptake of PLGA/Coumarin 6 by MSCs. As shown in Fig. 3B and C, the cellular uptake of NPs was time dependent. The intracellular fluorescence intensity after 12 h of incubation was almost the same as that of 18 h. Therefore, 12 h was chosen as uptake peroid. The retention ability of particles in MSC was a further important index, which was tested by FACS (Fig. 3D, E). With increasing retention time, the fluorescence intensity in the MSC decreased slightly. After three days, the fluorescence intensity of
the cells was still very high, suggesting that PLGA NPs taken by MSC could remain in the MSC cells for a relatively long time.
Fig. 3. (A) Confocal images of MSC incubated with PLGA/Coumarin 6. (B) Flow cytometry histogram profiles of the cellular Coumarin 6 fluorescence intensity in MSC after different incubation durations. (C) Quantification analysis of the Coumarin 6 fluorescence intensity in U251 cells after different incubation times. (D) Flow cytometry histogram profiles of the cellular Coumarin 6 fluorescence intensity in MSC after different retention times. (E) Quantification analysis of Coumarin 6 fluorescence intensity in U251 cells after different retention times.
3.3. Tumor tropism of the MSC-platform Loading of PLGA/BPQDs into MSC without weakening their migratory ability is significant for MSC-based targeting delivery systems. The effect of the tumor tropism capacity of NP loaded MSC and unloaded MSC were obtained by using a 24-well transwell chamber. Compared with the lower chamber with DEME cell medium or NIH3T3 cells, MSC could strongly respond to the chamber with U251 and easily migrated to the lower membrane surface (Fig. 4A, B), indicating a strong tumor-tropic migration ability of MSC. This also showed that the NPs-loaded MSC possessed nearly the same tumor tropism ability as unloaded MSC, suggesting that the NPs inside MSC did not significantly affect the tumor tendency of MSC. Moreover, the biocompatibility of BPQDs and PLGA/BPQDs was measured (Figure 4C). After 24 h of incubation, neither BPQDs nor PLGA/BPQDs exhibited significant cytotoxicity to MSC even at relatively high concentration (100 µg/mL), indicating that BPQDs and PLGA/BPQDs are biocompatible.
Fig. 4. (A) MSC, BPQD-loaded MSC, and PLGA/BPQD loaded MSC migration toward U251 cells, NIH3T3 cells, and Dulbecco's Modified Eagle Medium (DMEM). (B) Quantitative analysis of cell amount of the migrated MSC. (C) Cell viability with different concentrations of PLGA/BPQDs or BPQDs.
3.4. In vitro PTT effect and transport mechanism The photothermal ability of the BPQDs for killing U251 cells was first investigated. After incubated with BPQDs (0, 6.25, 12.5, 25 and 50 ppm) for 4h, the U251 cells were exposure with a NIR laser (808 nm, 1.0 W/cm2) for 10min (Fig. S4). BPQDs exhibited a dose-dependent PPT effect for U251. When concentration of BPQDs is 50 ppm, almost all U251 cells were killed by PPT. The in vitro photothermal effect of MSC@PLGA/BPQDs was then measured by a real-time infrared thermal imaging system during 808 nm laser irradiation (1 W/cm2, 10 min) (Fig. 5A). The temperatures of the MSC treated with PLGA/BPQDs at a
concentration of 40 ppm and 20 ppm increased by 42.61, and 25.87 °C, respectively, after 10 min of irradiation. This demonstrates that PLGA/BPQD loaded MSC possess a good photothermal effect. To evaluate the in vitro antitumor effect of MSCs@PLGA/BPQDs, MSCs@PLGA/BPQDs and U251 cells were co-cultured for PTT. Then, the mechanism of how PLGA/BPQDs inside MSC take effect was studied to test whether PLGA/BPQDs are transported from MSC to U251 cells. U251 (GFP-labeled) cells and MSC loaded with Cy-3-labeled PLGA NPs were co-cultured for 36 h and then observed by confocal imaging. As shown in Fig. 5B, red fluorescence was observed in green fluorescence GPF-labeled U251 cells, which showed that PLGA/BPQDs loaded in MSC were transported from MSC to U251. That might be because MSC releases microvesicles containing PLGA/ Cy3 and then the microvesicles were uptaken by U251, resulting in transportation of Cy-3-labeled PLGA NPs from MSC to U251. This result indicated that MSC could be used as vehicle to deliver NPs into cancer cells and then generated heat to kill U251 cell under NIR irradiation. Subsequently, the in vitro PTT effect of U251 cells was assessed by co-culture with PLGA/BPQD loaded MSC at different ratios. The MSC were incubated with PLGA/BPQDs for 12 h and then mixed with U251 cells at a series of ratios, ranging from 1:4 to 4:1 (MSC/U251). Cell viability was measured by MTT assay after irradiation (808 nm, 1 W cm−2 for 10 min). The cell viability exhibited a MSCs@PLGA/BPQD dose-dependent property (Fig. 5C). With increased ratios of MSCs@PLGA/BPQDs to U251 cells, the cell viability decreased significantly after NIR irradiation. The cell toxicities of MSCs@PLGA/BPQDs to
U251 cells at ratios of 4:1, 2:1, 1:1, 1:2, and 1:4 were 22.42 ± 8.39%, 68.47 ± 8.67%, 94.59 ± 2.11%, 98.45 ± 2.54%, and 99.05 ± 1.34%, respectively. These results show that PLGA/BPQD-loaded MSCs could effectively kill cancer cells via PTT. Therefore, due to the excellent tumor targeting ability, prominent PTT effect, and remarkable biocompatibility, PLGA/BPQDs-loaded MSC have a great prospect for the clinic application in cancer therapy.
Fig. 5. (A) Temperature profiles of PLGA/BPQD-loaded MSC under laser irradiation. (B) Confocal images of GFP-labeled U251 cells co-cultured with MSC@PLGA/Cy3 (green florescence represents U251 cells, red florescence represents Cy3 coated in PLGA/Cy3, and blue florescence represents cell nuclei). (C) Cell viability of MSC@PLGA/BPQDs and U251 cells with different ratios with irradiation tested by MTT after 24 h.
3.5. In vivo tumor-targeting effect and biodistributions of MSC@PLGA/BPQDs
To monitor the in vivo targeting ability of the NP-loaded MSC delivery system, the NIR fluorescence imaging agent IR780 was utilized to replace BPQDs in the NP formulation. For the PLGA/IR780 group, the fluorescence intensity at the tumor site reached its peak at day 1, while for the MSC@PLGA/IR780 group, the fluorescence intensity at the tumor site reached its peak 2 days post injection (Fig. 6A), which was because MSC possessed low immunogenicity that made it difficult for the immune system to recognize and so MSC had a longer in vivo blood circulation to reach its peak fluorescence intensity. Moreover, the peak intensity of the MSC group was clearly higher than that of the NP group (Fig. S5, see Supporting Information), due to intrinsic tumor-tropic ability of MSC was stronger than tumor targeting caused by EPR of NPs and low immunogenicity of MSC. This result suggested that MSC had a longer in vivo blood circulation time and far better tumor targeting effect compared with NPs [38, 39]. Subsequently, the biodistributions of MSC@PLGA/IR780 and PLGA/IR780 were measured after intravenous injection into tumor-bearing mice. Two days after administration, the mice were euthanized, tumor tissues and major organs (spleen, liver, kidney, lung, heart, and tumor) were extracted, and the fluorescence intensity was measured by using an in vivo imaging system. As shown in Fig. 6B and C, the signal intensity of tumors of the MSC@PLGA/IR780 group was stronger than that of the PLGA/IR780 group, which was attributed to the tumor-tropic properties of MSC. The signal intensity in lungs of the MSC@PLGA/IR780 group was also stronger than that of the PLGA/IR780 group. The reason might be that MSC tends to be trapped in the lung [40]. Overall, the IR780 signal distribution for two
drug delivery systems, especially for the MSC@PLGA/IR780, revealed a predominant accumulation in tumor. As shown in Fig. 6D and E, after NIR laser irradiation (808 nm for 5 min), the temperatures of tumors treated with the PLGA/BPQDs + NIR, BPQDs + NIR, and PBS increased to 54.29, 48.43, and 41.23 °C, respectively. In contrast, the temperature of tumors treated with MSC@PLGA/BPQDs + NIR increased to 58.84 °C, which is the maximum temperature in all four groups after the same exposure conditions. This demonstrates that MSC possessed strong tumor tropism ability and long blood circulation time. These results showed that MSC possessed great potential for the in vivo delivery of drugs to tumor sites.
Fig. 6. (A) (a) Fluorescence imaging of U251 tumor-bearing nude mice at different times. (B) Fluorescence imaging of tumors and major organs after intravenous injection at 2 d. Abbreviations: H: Heart; LI: Liver; S: Spleen; LU; Lung; K: Kidney; T: Tumor. (C) Biodistribution of PLGA/Cy3 in H, LI, S, LU, K, and T after
intravenous injection at 2 d. (D) In vivo thermal images and (E) corresponding temperature increase curves of U251 tumor-bearing nude mice under illumination (808 nm and 1 W cm−2 for 5 min) after injection of PBS, BPQDs, PLGA/BPQDs, MSC + PLGA/BPQDs, and MSC@PLGA/BPQDs.
3.6. In vivo antitumor effect of MSC@PLGA/BPQDs Inspired by the excellent results of in vitro and in vivo biodistributions, in vivo PTT was further implemented by using MSC@PLGA/BPQDs on mice bearing U251 tumors. As shown in Fig. 7A, 7B and S6, BPQDs + NIR treatment group exhibited moderate tumor growth inhibition. This could be attributed to the fact that BPQDs rapidly degradedafter two days in the body. Compared with the BPQDs + NIR treatment group, the PLGA/BPQDs + NIR treatment group and the MSC + PLGA/BPQDs + NIR treatment group showed better anti-tumor effects, due to the EPR effect of NPs. The MSC@PLGA/BPQDs + NIR treatment group exhibited the best anti-tumor effect, which was because MSC possessed a high tumor targeting ability and BPQDs had an excellent photothermal effect. These results demonstrated that MSC@PLGA/BPQDs could effectively kill U251 tumor cells after NIR irradiation. TUNEL assay (Fig. 7C) was also used to evaluate the anticancer efficiency by detecting the intratumoral apoptosis levels. Few TUNEL-positive cells (exhibited in green) were found in the PBS-treated group. After BPQDs + NIR treatment, a slight increase of the number of TUNEL-positive cells was observed, which could be
attributed to cell death caused by PPT. PLGA/BPQDs + NIR treatment group and MSC + PLGA/BPQDs + NIR treatment group exhibited more TUNEL-positive cells, which could be attributed to PLGA protection of BPQDs from degradation, resulting in enhanced photothermal effect. Compared with the above-mentioned groups, the MSC@PLGA/BPQDs + NIR treatment group showed the most obvious colocalization of TUNEL-positive apoptotic cells and nuclei, indicating that the MSC@PLGA/BPQDs + NIR treatment group possessed the best antitumor effect according to the result shown in Fig. 7A and B. H&E staining was used to assess the in vivo toxicity in all experimental groups (Fig. 8). No obvious pathologic changes were found in major organs (heart, liver, spleen, lung, and kidney) in any of the experimental groups, suggesting excellent biocompatibility in all therapeutic systems, which is essential for clinic applications. Notably, the body weight of mice bearing U251 cells did not change significantly hroughout the whole treatment process (Fig. S7), indicating that there were no severe side effects in all therapeutic systems.
Fig. 7. (A) Relative tumor growth curves after different treatments. (B) Representative images of ultimate tumors after 16 days of treatment. (C) Fluorescence microscopic images of apoptotic cells in tumor tissues irradiated by the 808 nm laser (1 W cm-2) in response to different treatments. (n=5, * P < 0.05, ** P < 0.01).
Fig. 8. Hematoxylin and eosin (H&E) staining of major organ slices from U251 tumor-bearing nude mice after 16 days of treatment with different groups.
4. Conclusions In summary, this study reported a unique stem-cell-based delivery platform for targeted PTT of U251 cancer by utilizing MSCs as cell carrier and PLGA/BPQDs as cargo. PLGA/BPQD NPs can be easily uptaken into MSC and exert a negligible influence on tumor tropism of MSC. Furthermore, PLGA NPs loaded in MSC can be transported to U251 cancer cells. Due to the high tumor-targeting efficiency of MSC and the excellent photothermal performance of PLGA/BPQDs, this delivery system of MSC@PLGA/BPQDs realized an outstanding antitumor therapeutic effect both in vitro and in vivo. Given the exceptional tumor targeting ability and low
immunogenicity, PLGA/BPQDs-engineered MSC with unique biocompatibility and eminent photothermal effect possess great potential for future clinic applications.
Acknowledgements We are grateful for the financial support from the National Natural Science Foundation of China (31922042, 81971081, 81772449, 81771966 and 51703258), Guangzhou science technology and innovation commission (201804010309, 201803010090), Science, Technology & Innovation Commission of Shenzhen Municipality
(JCYJ20170811160129498,
JCYJ20170818162637217
and
JCYJ20180307154606793,
JCYJ20180507181654186),
the
Fundamental
Research Funds for the Central Universities (19ykzd31).
Author contributions L.M., G.L. and X.Z. conceived the idea and whole experimental project. M.L., N.G., W.C. and J.C. carried out the synthesis, characterization, data analysis and interpretation. M.L., Y.Z., W.C. and X.W. assisted with data analysis. M.L. and G.L. wrote the manuscript. L.M., G.L., Y.Z. and X.Z. provided revisions. Notes The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. The authors have no conflicts of interest to disclose.
Appendix A. Supplementary data Supplementary data related to this article can be found at:
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The authors declare no competing financial interest.
HIGHTINGS
MSC possess excellent tumor tropism and low immunogenicity.
PLGA/BPQDs show prominent biocompatibility and enhanced photothermal effect.
PLGA/BPQDs–loaded MSC exhibited changeless high tumor-tropic ability.
MSC@PLGA/BPQDs would exert tumor ablation in vivo.
S1385-8947(19)33357-1 https://doi.org/10.1016/j.cej.2019.123942 CEJ 123942
To appear in:
Chemical Engineering Journal
Received Date: Revised Date: Accepted Date:
9 October 2019 21 December 2019 24 December 2019
Please cite this article as: M. Luo, Y. Zhou, N. Gao, W. Cheng, X. Wang, J. Cao, X. Zeng, G. Liu, L. Mei, Mesenchymal stem cells transporting black phosphorus-based biocompatible nanospheres: active trojan horse for enhanced photothermal cancer therapy, Chemical Engineering Journal (2019), doi: https://doi.org/10.1016/j.cej. 2019.123942
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Mesenchymal
stem
cells
transporting
black
phosphorus-based biocompatible nanospheres: active trojan horse for enhanced photothermal cancer therapy Miaomiao Luoa,1, Yun Zhou a,1, Nansha Gaoa, Wei Chenga, Xusheng Wanga, Jinxiu Caoa, Xiaowei Zenga,, Gan Liua,*, Lin Meia,*
a School
of Pharmaceutical Sciences (Shenzhen), Sun Yat-sen University, Guangzhou
510275, P.R. China
1
These authors contributed equally to this work.
*Corresponding
author
E-mail address: [email protected] (L. Mei), [email protected] (G. Liu).
HIGHLIGHTS
MSC possess excellent tumor tropism and low immunogenicity.
PLGA/BPQDs show prominent biocompatibility.
PLGA/BPQDs–loaded MSC exhibited changeless high tumor-tropic ability.
MSC@PLGA/BPQDs would exert tumor ablation in vivo.
GRAPHICAL ABSTRACT
ABSTRACT As potential carriers for specifically transporting photothermal agents to tumors, mesenchymal stem cells (MSC) would enhance their photothermal therapeutic effects. However, a biocompatible and biodegradable nanoscale photothermal agent, which hardly affects the tumor tropism of MSC, is still urgently needed. This study reports an effective therapeutic platform based on MSC loaded with poly (lactic-co-glycolic acid)/black phosphorus quantum dots (PLGA/BPQDs) for targeted photothermal
therapy of U251 glioma tumor cells. PLGA/BPQDs could not only be effectively uptaken by the MSC, but also stably maintained in the MSC for as long as 3 d. PLGA/BPQDs–loaded MSC exhibited changeless high tumor-tropic ability due to the exceptional biocompatibility of PLGA/BPQDs. In vitro results showed that PLGA/BPQDs can be transported from MSC to U251 cells, where U251 cells are killed after irradiation. In vivo results demonstrated that MSC@PLGA/BPQDs can efficiently tend the U251 glioma tumor and showed much longer retention times at the tumor site than PLGA/BPQDs alone. Finally, MSC@PLGA/BPQDs, as an active trojan horse, demonstrated enhanced photothermal effectivity on the U251 glioma tumor in vivo.
Keywords: Mesenchymal stem cells; Black phosphorus; PLGA; Photothermal therapy;
Targeted delivery.
1. Introduction As a promising substitute for traditional cancer therapies, photothermal therapy (PTT) has attracted enormous attention over the past few years due to its minimal invasiveness, less side effects, and high therapeutic efficiency [1-8]. Nanomaterials such as polymer-based semiconductor nanoparticles, metal-based nanomaterials, and carbon nanomaterials have been widely used as PTT agents due to their excellent near-infrared (NIR) photothermal performance [9-12]. Nanoscale photothermal agents can improve the tumor targeting ability through the enhanced permeability
and retention effect (EPR) and modification of targeting groups. However, their targeting efficiency is still very low (below 0.5%), which greatly hampers their anti-tumor effect. In addition, most inorganic nanometer PPT agents can easily accumulate and block blood vessels after injection into the blood, which greatly limits their clinic application [13]. Therefore, novel and safe methods to enhance the targeting efficiency of PPT agents are urgently needed. Recently, mesenchymal stem cells (MSC) have been widely developed for targeted tumor therapy due to their excellent tumor tropism and low immunogenicity [14, 15]. Studies showed that CXCR4 expressed in MSC was predominant in determining MSCs tumor tropism. This behavior was navigated by multifarious chemokine such as chemokine stromal derived factor 1 (SDF-1) released by the tumor site, whose receptor is CXCR4 [15-17]. MSC have been used as carriers to deliver PPT agents (such as gold nanoparticles) to tumor sites for PTT [17-19]. However, these PPT agents have non-negligible toxicity and poor metabolism, which not only affects the tumor tropic ability of MSC, but their long-term accumulation could also cause potential health risks [13]. Therefore, the exploration of novel PTT agents with ideal biocompatibility and biodegradability for efficient MSC transporting is vital. As a new member of the two-dimensional (2D) materials family, black phosphorus (BP) has recently aroused significant attention for biomedical applications due to its excellent properties [20-23]. BP shows layer-dependent bandgaps, which can be adjusted from 0.3 eV (bulk) to 2.0 eV (single layer), leading
to wide absorption ranging from ultraviolet (UV) to infrared regions [24, 25]. The zero-dimensional BP, black phosphorus quantum dots (BPQDs), show interesting photothermal conversion behavior and large extinction coefficients, and were used for PPT [25-29]. More importantly, BP showed outstanding biocompatibility, because P is an essential element in the human body [30-32]. It has been reported that BP is degraded into nontoxic phosphate and phosphonate in aqueous solution, both of which are widely distributed throughout the human body [33]. Therefore, BPQDs can be used as candidate PPT agents that can be loaded into MSC. Despite their promising potential, BPQDs are quite unstable under physiological conditions due to their high reactivity with oxygen and water [34-36], especially after loading into cells. Very recently, a number of strategies have been reported to improve the stability of BP, including surface protective layer protection, surface chemical modification, and doping [37]. Shao et al. [26] encapsulated BPQDs
into
the
Food
and
Drug
Administration
(FDA)-approved
poly
(lactic-co-glycolic acid) (PLGA) to obtain PLGA/BPQDs nanoparticles. This improved the photothermal stability of BPQDs by isolating internal BPQDs from water and oxygen. Thus, loading PLGA/BPQDs into MSC to obtain an active trojan horse, is reasonably anticipated to neither suffer the tumor tropism of MSC, nor rapidly decay the photothermal efficacy of BPQDs. Herein, a therapeutic platform was designed by loading PLGA/BPQDs into MSC (MSC@PLGA/BPQDs) to achieve tumor-tropic migration and enhanced photothermal therapy against U251 glioma cells (Fig. 1). PLGA/BPQDs were
prepared by encapsulating BPQDs with PLGA and were chosen as PTT agent due to their excellent biocompatibility, ideal biodegradability, and high photothermal efficiency. After intravenously injection, PLGA/BPQDs proved to be efficiently transported to the tumor site, released from MSC, and uptaken by U251. This led to enhanced photothermal therapy under NIR irradiation.
Fig. 1. Schematic design of PLGA/BPQD-loaded mesenchymal stem cells (MSC) to tumors for enhanced photothermal therapy.
2. Materials and methods 2.1. Materials Bulk BP was purchased from Nanjing muke nano technology Co., Ltd. (Nanjing, China). PLGA (75:25, MW: 10,000–20,000) was purchased from the Shandong
academy
of
pharmaceutical
N-methyl-2-pyrrolidone
(NMP),
sciences
pilot
polyvinyl
alcohol
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium
plant
(Jinan,
(MW:
China).
9,000–10,000),
bromide
(MTT),
dichloromethane (DCM), 4',6-diamidino-2-phenylindole (DAPI), imethyl sulfoxide (DMSO), Coumarin 6, and IR-780 were purchased from Sigma–Aldrich (St. Louis, MO, USA). Foetal bovine serum, H-DMEM, penicillin-streptomycin, trypsin-EDTA, and PBS (pH 7.4) were obtained from Gibco Life Technologies (AG, witzerland). All chemicals used in this study were of analytical reagent grade and were used without further purification. 2.2. Preparation of BPQDs 10 mg of bulk BP crystal powder was first added to 20 mL of NM Then, the mixture was sonicated with a sonic tip for 14 h (amplifier: 25%, on/off cycle: 5 s/5 s) in an ice bath at 700 W. After that, the mixture solution was sonicated in the water bath for 10 h at 300 W. Subsequently, the dispersion was centrifuged at a speed of 7000 rpm for 20 min and the supernatant was centrifuged at 12000 rpm for 20 min to obtain the precipitate (BPQDs). 2.3. Synthesis of PLGA/BPQDs nanoparticles PLGA/BPQDs NSs were synthesized by a modified oil-in-water emulsion solvent evaporation method. Briefly, 10 mg of BPQDs were added to 5 mL DCM and then mixed with 50 mg PLGA. The mixture was subjected to probe sonication for 10 min and was then dispersed in 50 mL of 0.5% (w/v) polyvinyl alcohol aqueous solution, followed by sonication for another 10 min. Then, the mixture was stirred for
12 h to evaporate residual DCM, then centrifuged at 7,000 rpm for 20 min, and washed with deionized water to remove dissociated BPQDs. The product was designated as PLGA/BPQDs. PLGA/Coumarin 6 and PLGA/IR780 were obtained with the same method. 2.4. Characterization of PLGA/BPQDs Transmission electron microscopy (TEM) was performed on the FEI Tecnai G2 F30 transmission electron microscope (FEI Company, Hillsborough, OR, USA) at an acceleration voltage of 300 Kv. Atomic force microscopy (AFM) Bruker Dimension® Icon™) was used to characterize both the morphology and height of samples. Scanning electron microscopic (SEM) images were obtained on a field-emission SEM. The zeta potential of nanoparticles was acquired by NanoBrook 90Plus PALS. X-ray photoelectron spectroscopy (XPS) was conducted on an X-ray photoelectron spectroscope (Axis HSi, Kratos Ltd., Manchester, UK) with Al Kα radiation (1486.6 eV photons, 150 W) as X-ray source for excitation. Raman spectra of BPQDs and PLGA/BPQDs were obtained with a high-resolution confocal Raman microscope (HORIBA LabRAM HR800). X-ray diffraction (XRD) were performed on a Philips X’Pert Pro Super diffractometer with Cu Kα radiation (λ= 1.54178 Å). 2.5. Cell Culture MSC, isolated from human umbilical cords, were obtained from Dr. Xusheng Wang from the Sun Yat-sen University, China, and were incubated in low glucose Dulbecco's Modified Eagle Medium (DMEM), containing 1% enicillin-streptomycin and 10% fetal bovine serum (FBS). U251 and NIH3T3 cells were also cultured in
DMEM supplemented with 1% penicillin-streptomycin and 10% FBS. All cell lines were cultured in a humid atmosphere with 5% CO2 at 37 °C. 2.6. Cellular uptake of particles MSC were plated in glass-bottomed dishes. When cells reached 80% confluence, the MSCs were cultivated with 40 µg/mL particles for 12 h. To observe the uptake of nanoparticles by MSC cells, the MSCs were treated with PLGA/Coumarin 6 for 12 h, washed with PBS three times, fixed with 4% paraformaldehyde, and finally dyed with DAPI. Samples were observed with a confocal microscope (FV1000, Olympus, Tokyo, Japan). The uptake rate of PLGA/BPQDs by MSC was measured by a multi-function board reader (Perkin Elmer). Firstly, PLGA/BPQDs were suspended by DMEM medium, containing 1% penicillin-streptomycin and 10% FBS. Then, the standard curve of PLGA/BPQDs was made with a multi-function board reader (Perkin Elmer). The MSC were incubated on 6-well plates with 10, 20, and 30 μg/mL of PLGA/BPQDs for 12 h. The absorbance value of PLGA/BPQDs in the supernatant was measured by the multi-function board reader. The concentration of PLGA/BPQDs was calculated by the PLGA/BPQDs standard curve to obtain the amount of PLGA/BPQDs in the supernatant, and to finally calculate the uptake rate. Cellular uptake was quantitatively analyzed by flow cytometry (FACS). In brief, MSCs were incubated with PLGA/Coumarin 6 for 1 h, 6 h, 12 h, and 18 h, respectively. The cells were wished three times and then collected. The fluorescence intensity was detected by FACS.
Quantitative analysis of cellular retention was also conducted by FACS. Briefly, PLGA/Coumarin 6 was incubated with MSCs for 12 h and subsequently washed with PBS three times. After that, the cells continued to be incubated by adding fresh medium. At 12 h, 1 day, 2 days, and 3 days, the cells were collected and the fluorescence intensity was obtained by FACS. 2.7. Cell migration The migration capability of PLGA/BPQDs loaded MSCs was investigated by the transwell assay. MSCs or loaded MSCs were suspended in 200 µL serum-free medium and were seeded in the upper well of 24-well transwell chambers (8 μm pore size, Corning Incorporated) at a density of 105cell/well. The same numbers of U251 glioma cells or NIH3T3 cells were plated in the lower chamber. After culture at 37 °C for 6 h, cells that still remained at the surface of the upper membrane were gently wiped off. Cells that had migrated to the surface of the lower membrane were fixed with 4% paraformaldehyde and stained with DAPI for 15 min. All samples were observed using a fluorescence microscope and the number of migrated cells per optic field was counted and averaged. 2.8. In vitro cytotoxicity assays MSC were seeded on 96-well plates at a density of 8000 cells per well and were cultured for 12 h. Afterward, the medium was replaced with fresh culture medium containing PLGA/BPQDs or BPQDs at various concentrations (2–100 µg BPQDs/mL). After cultivation for 24 h at 37 °C, 20 µL of MTT solution was added to each well and the mixture was cultured for further 4 h. Then, the medium was
removed, which was followed by addition of 150 µL DMSO to each well. The absorption was measured by using a microplate reader at 570 nm. Cells without any treatment were set as 100% cell viability. 2.9. Laser irradiation and photothermal imaging To measure the intracellular photothermal effect of MSC@PLGA/BPQDs, MSCs were seeded on six-well plates and then co-cultured with PLGA/BPQDs at two concentrations (20 and 40 µg BPQDs/mL). After 12 h, the loaded cells were collected and resuspended in 20 μL PBS. The samples (2 × 105 cells in 20 μL of PBS) were placed in 200 μL Eppendorf tubes and were then subjected to 808 nm NIR laser treatment (at 1 W cm-2 for 10 min). The temperature variations of the MSC@PLGA/BPQDs were recorded by an infrared thermal imaging camera (Ti450, Fluke, Everett, WA, USA). 2.10. In vitro inhibition of tumor growth MSC were incubated with PLGA/Coumarin 6 particles for 12 h, washed three times, and collected. MSC@PLGA/Coumarin 6 and GFP labeled U251 cells (at a ratio of 2:1) were co-cultured for 24 h, washed three times with PBS, fixed with 4% paraformaldehyde, and dyed with DAPI. Samples were observed under a confocal microscope (FV1000; Olympus, Tokyo, Japan). The in vitro anti-tumor effect of MSC@PLGA/BPQDs on U251 glioma cells was evaluated by using MTT. Briefly, a co-culture system was constructed by mixing MSC@PLGA/BPQDs with U251 glioma cells at different ratios ranging from 1:4 to 4:1. The plate was illuminated with an 808 nm laser for 10 min at a power density of
1 W/cm2. The cell viability was normalized to the control group, which consisted of U251 glioma cells only. After 12 h of incubation, the viability of cells was evaluated in an MTT assay. 2.11. Xenograft Tumor Models Female balb/c nude mice were bought from Sun Yat-Sen University. U251 cells (107 cells in 100 μL) were subcutaneously injected into the right axillary region of female nude mice. The tumor size was measured with a digital Vernier caliper every two days and the tumor volume (V) was calculated via (tumor length) × (tumor width)2 / 2. Mice were randomly divided into different groups when the tumor volume was approximately 100 mm3. 2.12. In vivo fluorescence imaging When the tumor size reached about 500 mm3, U251 tumor cell bearing mice were randomly divided into two groups (n = 3 per group): a PLGA/IR780 group and an MSC@PLGA/IR780 group (1 mg/kg equivalent IR780 for per group). Mice were intravenously injected with 200 μL dye-loaded NSs via tails. An in-vivo imaging system was employed to take in vivo images of mice at 1 h, 3 h, 24 h, 2 days, 3 days, and 4 days post-injection. NIR light with a peak wavelength of 780 nm was used as excitation source. Nude mice were euthanized at 2 day post-injection and the major organs (heart, liver, spleen, lung, kidney, and tumor tissues) of mice were collected and their fluorescence intensities were measured. 2.13. In vivo PTT and photothermal images When the volume of the tumor reached about 100 mm3, each group (n = 5) was
administered with (A) phosphate buffered saline (PBS, 200 μL), (B) BPQDs (100 μg, 200 μL), (C) PLGA/BPQDs (100 µg BPQDs, 200 μL), (D) MSC + PLGA/BPQDs (100 μg BPQDs, 200 μL), and (E) MSC@PLGA/BPQDs (100 μg of BPQDs in 5 × 105 MSCs, 200 μL) via the tail vein. At 2 days post-injection, all mice were anaesthetized and irradiated with an 808 nm NIR laser (1 W/cm2 for 5 min). At the same time, the tumor temperature change and infrared thermographic maps were recorded by the already mentioned infrared thermal imaging camera. Tumor size and weight of all mice were measured every other day until the end of treatment. The relative tumor volume was calculated as follows: V/V0, where V0 represents the initial tumor volume at the beginning of treatment. After about three weeks of treatment, mice were euthanized and the main organs and tumor tissues were harvested for further study. 2.14. Histological examination and TUNEL assay At day 16 post-transplantation, mice were humanely killed and the organs (including heart, liver, spleen, lung, and kidney) and tumor tissues were harvested. In brief, all samples were fixed in a 10% formalin solution, and then paraffin-embedded. All organs of mice were tested using hematoxylin and eosin (HE) staining for histological examination and all tumors were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay to label apoptotic cells in tumor tissues. 2.15. Statistical analysis All experiments were conducted at least three times. The experimental data are
showed as mean ± standard deviation (SD). Statistical analyses were performed by one-way analysis of variance (ANOVA) followed by Bonferroni test using SPSS 22.0 software. * P < 0.05 was identified as statistically significant and ** P < 0.01 was identified as extremely statistically significant.
3. Results and Discussion 3.1. Synthesis and characterization of PLGA/BPQDs TEM and AFM were used to observe the morphology of BPQDs. As shown in the TEM images (Fig. 2A and B), the average size of BPQDs was 3.5 ± 0.9 nm according to the statistical TEM analysis of 200 BPQDs. The AFM images in Fig. 2C show the topographic morphology of BPQDs. The measured heights of three randomly selected BPQDs were 4.48, 3.6, and 3.11 nm (Fig. 2D), respectively. As shown in fig. S1, all diffraction peaks of BPQDs can be readily indexed to the orthorhombic BP consistent with JCPDS No. 74-1878. PLGA/BPQDs nanospheres were prepared by an oil-in-water emulsion solvent evaporation method. TEM images (Fig. 2E) and SEM images (Fig. 2F) showed that all PLGA/BPQDs nanospheres have a uniform spherical shape. The average size of the PLGA nanospheres was 165.6 ± 55 mm according to the statistical SEM analysis of 200 PLGA/BPQDs (Fig. 2G). Moreover, energy dispersive X-ray spectroscopy (Table 1, see Supporting Information) showed the elemental content of C, O, and P in PLGA nanoparticles. C and O were mainly present due to the introduction of PLGA, which confirmed the successful preparation of PLGA/BPQDs. The zeta potentials of different samples were measured (Fig. S2,
see Supporting Information). The zeta potential increased from -38.47 mV (BPQDs) to -28.18 mV (PLGA/BPQDs), indicating successful fabrication of PLGA/BPQDs. Raman scattering was used to characterize BPQDs and PLGA/BPQDs NPs (Fig. 2H). BPQDs showed three obvious peaks, which could be attributed to one out-of-plane phonon mode (A1g) at 357.6 cm−1 and two in-plane modes at 432.5 cm−1 (B2g) and 460.3 cm−1 (A2g), respectively. Compared with BPQD, the Raman spectra of PLGA/BPQDs NS showed a red shift, proving successful introduction of PLGA into the PLGA/BPQDs NS. PLGA, BPQDs and PLGA/BPQDs were further characterized by XPS (Fig. 2I and Fig. S3A-C) to measure their chemical compositions. The peak intensity of P2p (129.6 eV) (Fig. S3A and Figure 2I) in PLGA/BPQDs and PLGA was significantly weaker compared with BPQDs. At the same time, the peak intensities of C1s (Fig. S3B) and O1s (Fig. S3C) in PLGA/BPQDs and PLGA increased compared with BPQDs. The reason might be that PLGA has no P element and possesses both C and O elements in its chemical structure. As a consequence, these changes demonstrated the successful coating of PLGA. The encapsulation efficiency and loading efficiency were 50.5% and 9.7%, respectively, measured by inductively coupled plasma mass spectrometry (ICP-MS, Thermoscientific, USA). Then, the photothermal property and stability of PLGA/BPQDs were tested by using NIR laser irradiation (808 nm for 10 min). As shown in Fig. 2J and K, both BPQDs and PLGA/BPQDs exhibited a concentration-dependent photothermal effect. The temperature of both samples could increase by nearly 40 °C at a concentration of 60 ppm, and by nearly 10 °C at 7.5 ppm. Furthermore, PLGA coating on BPQD did
not affect the photothermal characteristics of BPQD. As shown in Fig. 2L, after being irradiated by an 808 nm NIR laser at a power density of 1 W/cm2 for 10 min, the temperature of the BPQD solution increased by 20.8 °C; however, the temperature of the BPQD solution increased by only 0.4 °C after 4 days, indicating relatively poor photothermal stability. In contrast, PLGA/BPQD showed better photothermal stability. After 4 days, the temperature increase of the PLGA/BPQDs solution was 17.8 °C, which was close to the initial value of 22.5 °C. This result demonstrated that PLGA can protect BP from degradation for almost three days when exposed to air and water.
Fig. 2. Morphology and characterization (A) transmission electron microscopic (TEM) pictures of BPQDs. (B) Statistical assessment of the size of 200 BPQDs according to the TEM images. (C) Atomic force microscopic (AFM) image of the BPQDs. (D) Height profiles along the red lines in d. (E) TEM and (F) scanning
electron microscopic (SEM) images of the PLGA/BPQDs. (G) Statistical analysis of the size of PLGA/BPQDs based on the SEM images. (H) Raman spectra and (I) X-ray photoelectron spectroscopic (XPS) spectrum of PLGA, BPQDs and PLGA/BPQDs. (J) Photothermal curves of BPQDs and PLGA/BPQDs at different concentrations. (L) Temperature change of BPQDs and PLGA/BPQDs dispersed in water for different durations under NIR laser illumination (1.0 W cm-2) for 10 min.
3.2. PLGA/BPQD cellular uptake and retention in MSC To analyze the PLGA/BPQD uptake capacity of MSC, the green fluorescent probe Coumarin 6 was utilized to replace BPQDs in the NPs. Fig. 3A shows a confocal laser scanning microscopic (CLSM) image of MSC after 12 h of incubation with Coumarin 6-loaded PLGA NPs (PLGA/Coumarin 6). It is clear that PLGA/Coumarin 6 (green) were closely located around the cell nuclei (blue), suggesting that MSC cells possess a high uptake capacity of PLGA NPs. The uptake rate of PLGA/BPQDs by MSC was 50.36% ± 0.012. FACS was also utilized to analyze the uptake of PLGA/Coumarin 6 by MSCs. As shown in Fig. 3B and C, the cellular uptake of NPs was time dependent. The intracellular fluorescence intensity after 12 h of incubation was almost the same as that of 18 h. Therefore, 12 h was chosen as uptake peroid. The retention ability of particles in MSC was a further important index, which was tested by FACS (Fig. 3D, E). With increasing retention time, the fluorescence intensity in the MSC decreased slightly. After three days, the fluorescence intensity of
the cells was still very high, suggesting that PLGA NPs taken by MSC could remain in the MSC cells for a relatively long time.
Fig. 3. (A) Confocal images of MSC incubated with PLGA/Coumarin 6. (B) Flow cytometry histogram profiles of the cellular Coumarin 6 fluorescence intensity in MSC after different incubation durations. (C) Quantification analysis of the Coumarin 6 fluorescence intensity in U251 cells after different incubation times. (D) Flow cytometry histogram profiles of the cellular Coumarin 6 fluorescence intensity in MSC after different retention times. (E) Quantification analysis of Coumarin 6 fluorescence intensity in U251 cells after different retention times.
3.3. Tumor tropism of the MSC-platform Loading of PLGA/BPQDs into MSC without weakening their migratory ability is significant for MSC-based targeting delivery systems. The effect of the tumor tropism capacity of NP loaded MSC and unloaded MSC were obtained by using a 24-well transwell chamber. Compared with the lower chamber with DEME cell medium or NIH3T3 cells, MSC could strongly respond to the chamber with U251 and easily migrated to the lower membrane surface (Fig. 4A, B), indicating a strong tumor-tropic migration ability of MSC. This also showed that the NPs-loaded MSC possessed nearly the same tumor tropism ability as unloaded MSC, suggesting that the NPs inside MSC did not significantly affect the tumor tendency of MSC. Moreover, the biocompatibility of BPQDs and PLGA/BPQDs was measured (Figure 4C). After 24 h of incubation, neither BPQDs nor PLGA/BPQDs exhibited significant cytotoxicity to MSC even at relatively high concentration (100 µg/mL), indicating that BPQDs and PLGA/BPQDs are biocompatible.
Fig. 4. (A) MSC, BPQD-loaded MSC, and PLGA/BPQD loaded MSC migration toward U251 cells, NIH3T3 cells, and Dulbecco's Modified Eagle Medium (DMEM). (B) Quantitative analysis of cell amount of the migrated MSC. (C) Cell viability with different concentrations of PLGA/BPQDs or BPQDs.
3.4. In vitro PTT effect and transport mechanism The photothermal ability of the BPQDs for killing U251 cells was first investigated. After incubated with BPQDs (0, 6.25, 12.5, 25 and 50 ppm) for 4h, the U251 cells were exposure with a NIR laser (808 nm, 1.0 W/cm2) for 10min (Fig. S4). BPQDs exhibited a dose-dependent PPT effect for U251. When concentration of BPQDs is 50 ppm, almost all U251 cells were killed by PPT. The in vitro photothermal effect of MSC@PLGA/BPQDs was then measured by a real-time infrared thermal imaging system during 808 nm laser irradiation (1 W/cm2, 10 min) (Fig. 5A). The temperatures of the MSC treated with PLGA/BPQDs at a
concentration of 40 ppm and 20 ppm increased by 42.61, and 25.87 °C, respectively, after 10 min of irradiation. This demonstrates that PLGA/BPQD loaded MSC possess a good photothermal effect. To evaluate the in vitro antitumor effect of MSCs@PLGA/BPQDs, MSCs@PLGA/BPQDs and U251 cells were co-cultured for PTT. Then, the mechanism of how PLGA/BPQDs inside MSC take effect was studied to test whether PLGA/BPQDs are transported from MSC to U251 cells. U251 (GFP-labeled) cells and MSC loaded with Cy-3-labeled PLGA NPs were co-cultured for 36 h and then observed by confocal imaging. As shown in Fig. 5B, red fluorescence was observed in green fluorescence GPF-labeled U251 cells, which showed that PLGA/BPQDs loaded in MSC were transported from MSC to U251. That might be because MSC releases microvesicles containing PLGA/ Cy3 and then the microvesicles were uptaken by U251, resulting in transportation of Cy-3-labeled PLGA NPs from MSC to U251. This result indicated that MSC could be used as vehicle to deliver NPs into cancer cells and then generated heat to kill U251 cell under NIR irradiation. Subsequently, the in vitro PTT effect of U251 cells was assessed by co-culture with PLGA/BPQD loaded MSC at different ratios. The MSC were incubated with PLGA/BPQDs for 12 h and then mixed with U251 cells at a series of ratios, ranging from 1:4 to 4:1 (MSC/U251). Cell viability was measured by MTT assay after irradiation (808 nm, 1 W cm−2 for 10 min). The cell viability exhibited a MSCs@PLGA/BPQD dose-dependent property (Fig. 5C). With increased ratios of MSCs@PLGA/BPQDs to U251 cells, the cell viability decreased significantly after NIR irradiation. The cell toxicities of MSCs@PLGA/BPQDs to
U251 cells at ratios of 4:1, 2:1, 1:1, 1:2, and 1:4 were 22.42 ± 8.39%, 68.47 ± 8.67%, 94.59 ± 2.11%, 98.45 ± 2.54%, and 99.05 ± 1.34%, respectively. These results show that PLGA/BPQD-loaded MSCs could effectively kill cancer cells via PTT. Therefore, due to the excellent tumor targeting ability, prominent PTT effect, and remarkable biocompatibility, PLGA/BPQDs-loaded MSC have a great prospect for the clinic application in cancer therapy.
Fig. 5. (A) Temperature profiles of PLGA/BPQD-loaded MSC under laser irradiation. (B) Confocal images of GFP-labeled U251 cells co-cultured with MSC@PLGA/Cy3 (green florescence represents U251 cells, red florescence represents Cy3 coated in PLGA/Cy3, and blue florescence represents cell nuclei). (C) Cell viability of MSC@PLGA/BPQDs and U251 cells with different ratios with irradiation tested by MTT after 24 h.
3.5. In vivo tumor-targeting effect and biodistributions of MSC@PLGA/BPQDs
To monitor the in vivo targeting ability of the NP-loaded MSC delivery system, the NIR fluorescence imaging agent IR780 was utilized to replace BPQDs in the NP formulation. For the PLGA/IR780 group, the fluorescence intensity at the tumor site reached its peak at day 1, while for the MSC@PLGA/IR780 group, the fluorescence intensity at the tumor site reached its peak 2 days post injection (Fig. 6A), which was because MSC possessed low immunogenicity that made it difficult for the immune system to recognize and so MSC had a longer in vivo blood circulation to reach its peak fluorescence intensity. Moreover, the peak intensity of the MSC group was clearly higher than that of the NP group (Fig. S5, see Supporting Information), due to intrinsic tumor-tropic ability of MSC was stronger than tumor targeting caused by EPR of NPs and low immunogenicity of MSC. This result suggested that MSC had a longer in vivo blood circulation time and far better tumor targeting effect compared with NPs [38, 39]. Subsequently, the biodistributions of MSC@PLGA/IR780 and PLGA/IR780 were measured after intravenous injection into tumor-bearing mice. Two days after administration, the mice were euthanized, tumor tissues and major organs (spleen, liver, kidney, lung, heart, and tumor) were extracted, and the fluorescence intensity was measured by using an in vivo imaging system. As shown in Fig. 6B and C, the signal intensity of tumors of the MSC@PLGA/IR780 group was stronger than that of the PLGA/IR780 group, which was attributed to the tumor-tropic properties of MSC. The signal intensity in lungs of the MSC@PLGA/IR780 group was also stronger than that of the PLGA/IR780 group. The reason might be that MSC tends to be trapped in the lung [40]. Overall, the IR780 signal distribution for two
drug delivery systems, especially for the MSC@PLGA/IR780, revealed a predominant accumulation in tumor. As shown in Fig. 6D and E, after NIR laser irradiation (808 nm for 5 min), the temperatures of tumors treated with the PLGA/BPQDs + NIR, BPQDs + NIR, and PBS increased to 54.29, 48.43, and 41.23 °C, respectively. In contrast, the temperature of tumors treated with MSC@PLGA/BPQDs + NIR increased to 58.84 °C, which is the maximum temperature in all four groups after the same exposure conditions. This demonstrates that MSC possessed strong tumor tropism ability and long blood circulation time. These results showed that MSC possessed great potential for the in vivo delivery of drugs to tumor sites.
Fig. 6. (A) (a) Fluorescence imaging of U251 tumor-bearing nude mice at different times. (B) Fluorescence imaging of tumors and major organs after intravenous injection at 2 d. Abbreviations: H: Heart; LI: Liver; S: Spleen; LU; Lung; K: Kidney; T: Tumor. (C) Biodistribution of PLGA/Cy3 in H, LI, S, LU, K, and T after
intravenous injection at 2 d. (D) In vivo thermal images and (E) corresponding temperature increase curves of U251 tumor-bearing nude mice under illumination (808 nm and 1 W cm−2 for 5 min) after injection of PBS, BPQDs, PLGA/BPQDs, MSC + PLGA/BPQDs, and MSC@PLGA/BPQDs.
3.6. In vivo antitumor effect of MSC@PLGA/BPQDs Inspired by the excellent results of in vitro and in vivo biodistributions, in vivo PTT was further implemented by using MSC@PLGA/BPQDs on mice bearing U251 tumors. As shown in Fig. 7A, 7B and S6, BPQDs + NIR treatment group exhibited moderate tumor growth inhibition. This could be attributed to the fact that BPQDs rapidly degradedafter two days in the body. Compared with the BPQDs + NIR treatment group, the PLGA/BPQDs + NIR treatment group and the MSC + PLGA/BPQDs + NIR treatment group showed better anti-tumor effects, due to the EPR effect of NPs. The MSC@PLGA/BPQDs + NIR treatment group exhibited the best anti-tumor effect, which was because MSC possessed a high tumor targeting ability and BPQDs had an excellent photothermal effect. These results demonstrated that MSC@PLGA/BPQDs could effectively kill U251 tumor cells after NIR irradiation. TUNEL assay (Fig. 7C) was also used to evaluate the anticancer efficiency by detecting the intratumoral apoptosis levels. Few TUNEL-positive cells (exhibited in green) were found in the PBS-treated group. After BPQDs + NIR treatment, a slight increase of the number of TUNEL-positive cells was observed, which could be
attributed to cell death caused by PPT. PLGA/BPQDs + NIR treatment group and MSC + PLGA/BPQDs + NIR treatment group exhibited more TUNEL-positive cells, which could be attributed to PLGA protection of BPQDs from degradation, resulting in enhanced photothermal effect. Compared with the above-mentioned groups, the MSC@PLGA/BPQDs + NIR treatment group showed the most obvious colocalization of TUNEL-positive apoptotic cells and nuclei, indicating that the MSC@PLGA/BPQDs + NIR treatment group possessed the best antitumor effect according to the result shown in Fig. 7A and B. H&E staining was used to assess the in vivo toxicity in all experimental groups (Fig. 8). No obvious pathologic changes were found in major organs (heart, liver, spleen, lung, and kidney) in any of the experimental groups, suggesting excellent biocompatibility in all therapeutic systems, which is essential for clinic applications. Notably, the body weight of mice bearing U251 cells did not change significantly hroughout the whole treatment process (Fig. S7), indicating that there were no severe side effects in all therapeutic systems.
Fig. 7. (A) Relative tumor growth curves after different treatments. (B) Representative images of ultimate tumors after 16 days of treatment. (C) Fluorescence microscopic images of apoptotic cells in tumor tissues irradiated by the 808 nm laser (1 W cm-2) in response to different treatments. (n=5, * P < 0.05, ** P < 0.01).
Fig. 8. Hematoxylin and eosin (H&E) staining of major organ slices from U251 tumor-bearing nude mice after 16 days of treatment with different groups.
4. Conclusions In summary, this study reported a unique stem-cell-based delivery platform for targeted PTT of U251 cancer by utilizing MSCs as cell carrier and PLGA/BPQDs as cargo. PLGA/BPQD NPs can be easily uptaken into MSC and exert a negligible influence on tumor tropism of MSC. Furthermore, PLGA NPs loaded in MSC can be transported to U251 cancer cells. Due to the high tumor-targeting efficiency of MSC and the excellent photothermal performance of PLGA/BPQDs, this delivery system of MSC@PLGA/BPQDs realized an outstanding antitumor therapeutic effect both in vitro and in vivo. Given the exceptional tumor targeting ability and low
immunogenicity, PLGA/BPQDs-engineered MSC with unique biocompatibility and eminent photothermal effect possess great potential for future clinic applications.
Acknowledgements We are grateful for the financial support from the National Natural Science Foundation of China (31922042, 81971081, 81772449, 81771966 and 51703258), Guangzhou science technology and innovation commission (201804010309, 201803010090), Science, Technology & Innovation Commission of Shenzhen Municipality
(JCYJ20170811160129498,
JCYJ20170818162637217
and
JCYJ20180307154606793,
JCYJ20180507181654186),
the
Fundamental
Research Funds for the Central Universities (19ykzd31).
Author contributions L.M., G.L. and X.Z. conceived the idea and whole experimental project. M.L., N.G., W.C. and J.C. carried out the synthesis, characterization, data analysis and interpretation. M.L., Y.Z., W.C. and X.W. assisted with data analysis. M.L. and G.L. wrote the manuscript. L.M., G.L., Y.Z. and X.Z. provided revisions. Notes The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. The authors have no conflicts of interest to disclose.
Appendix A. Supplementary data Supplementary data related to this article can be found at:
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The authors declare no competing financial interest.
HIGHTINGS
MSC possess excellent tumor tropism and low immunogenicity.
PLGA/BPQDs show prominent biocompatibility and enhanced photothermal effect.
PLGA/BPQDs–loaded MSC exhibited changeless high tumor-tropic ability.
MSC@PLGA/BPQDs would exert tumor ablation in vivo.