Intelligent Photosensitive Mesenchymal Stem Cells and Cell-Derived Microvesicles for Photothermal Therapy of Prostate Cancer

Abstract

Targeted delivery of nanomedicines into the tumor site and improving the intratumoral distribution remain challenging in cancer treatment. Here, we report an effective transportation system utilizing both of mesenchymal stem cells (MSCs) and their secreted microvesicles containing assembled gold nanostars (GNS) for targeted photothermal therapy of prostate cancer. The stem cells act as a cell carrier to actively load and assemble GNS into the lysosomes. Accumulation of GNS in the lysosomes facilitates the close interaction of nanoparticles, which could result in a 20 nm red-shift of surface plasmon resonance of GNS with a broad absorption in the near infrared region. Moreover, the MSCs can behave like an engineering factory to pack and release the GNS clusters into microvesicles. The secretion of GNS can be stimulated via light irradiation, providing an external trigger-assisted approach to encapsulate nanoparticles into cell derived microvesicles. In vivo studies demonstrate that GNS-loaded MSCs have an extensive intratumoral distribution, as monitored via photoacoustic imaging, and efficient antitumor effect under light exposure in a prostate-cancer subcutaneous model by intratumoral and intravenous injection. Our work presents a light-responsive transportation approach for GNS in combination of MSCs and their extracellular microvesicles and holds the promise as an effective strategy for targeted cancer therapy including prostate cancer.

Keywords: gold nanostars; mesenchymal stem cells; microvesicles; photothermal therapy; targeted transportation.

Scheme 1

Schematic design of MSCs-mediated delivery of GNS for photothermal ablation of cancer cells.

Figure 1

Characterization of TAT-GNS. A. The scheme of TAT-GNS synthesis. B. The TEM image of GNS; C. The TEM image of the spikes of GNS. D. UV-vis-NIR absorption of TAT-GNS (The insert shows the TAT-GNS resuspension solution.); E. DLS size distribution of the bare GNS (grey column), HOOC-PEG-GNS (red column) and TAT-GNS (green column). F. Zeta potential of GNS, PEG-GNS and TAT-GNS; G. △T-t curve of TAT-GNS with different concentrations (rang from 0-160 pM) by laser irradiation with an intensity of 2 W/cm².

Figure 2

Cellular uptake and the tropic migration of TAT-GNS loaded MSCs. A. MSCs cellular uptake of PEG-GNS and TAT-GNS (ranging from 0-160 pM); B. ICP-MS of Au content in MSCs incubated with 160 pM TAT-GNS during 4 h and 24 h. C. Cell viability after labeling with TAT-GNS at different concentrations ranging from 20 to 160 pM. D. FACS analyses of the surface markers of labeled and unlabeled MSCs after 24 h incubation. E. Light microscopy images showing TAT-GNS loaded MSCs migration towards PC-3 cells. F. Western blot analysis of CXCR4 expression in GNS-labeled MSCs, β-actin as control.

Figure 3

Cellular localization of TAT-GNS in MSCs and the photothermal property of TAT-GNS in MSC lysosomes. A. Laser scanning confocal microscopy images of the FITC-TAT-GNS loaded MSCs. B. UV-Vis-NIR spectrum of GNS-loaded MSCs (3×10⁵ cells) suspension in PBS (1 mL). C. representative TEM images of MSCs incubated with TAT-GNS for 24 hours. The aggregation state of GNS in the lysosomes of MSCs. And the distance between the sharps of GNS inside the MCSs were determined by TEM. D. the △T-t curve of GNS-loaded MSCs (3×10⁵ cells) suspension in PBS (1 mL) under the optical density of 2 W/cm².

Figure 4

Microvesicles containing GNS clusters released from GNS-loaded MSCs and their transportation to cancer cells. A. Representative images showing an intracellular GNS cluster (blue arrow) and GNS clusters exocytosis (red arrow) by GNS-loaded MSCs. B. GNS clusters produced by exocytosis from GNS-loaded MSCs in the extracellular medium. C. Size distribution of the extracellular microvesicles containing GNS clusters by DLS. D. UV-Vis-NIR spectrum of microvesicles containing GNS clusters in PBS. E. SDS-PAGE protein analysis of microvesicles containing GNS clusters. Samples were stained with Coomassie brilliant blue. I, markers. II, MSCs extracellular vesicles as control. III, GNS-loaded MSCs extracellular vesicles. F. Protein quantification of supernatant in GNS loaded MSCs by BCA assay 4 h after NIR exposure (1.5 W/cm², 3 min). The concentration of TAT-GNS was 160 pM. G. Confocal laser scanning microscopy of FITC-labeled GNS clusters released from MSCs to PC-3 (labeled by RFP). The red arrow shows GNS clusters labeled by FITC and taken up by a PC-3 cell (labeled by RFP).

Figure 5

In vitro PTT effect of GNS-loaded MSCs. A. PTT effects on GNS-loaded MSCs. B. Photothermal therapy effects on co-cultured GNS-loaded MSCs and PC-3 with different ratios (ranging from 1:4 to 4:1). Representative 10× images obtained 4 hours after laser exposure (Live-dead staining with PI and calcein-AM); C. Cell viability of GNS-loaded MSCs post light irradiation; D. Cell viability of co-cultured GNS-loaded MSCs and PC-3 post PTT. Error bars indicate s.d. (n=4). P < 0.05(*), P < 0.01(**), P < 0.001 (***) compared with the control group.

Figure 6

Photothermal ablation of prostate tumors via GNS-loaded MSCs. A. PAI of GNS distribution after intratumoral injection. Fusion of ultrasound and PAI images of the tumors after the injection of TAT-GNS and GNS-loaded MSCs. Scale bar is 0.5 cm. B. Representative H&E and silver staining sections of the tumor after PA imaging. The yellow circles and yellow arrows noted the GNS signals (brown spots) in the sections. All scale bars are 200 μm. C. Infrared microscopic imaging: NIR laser irradiation of the tumor bearing mice after injections of PBS, TAT-GNS and GNS-loaded MSCs after 3 days. D. Temperature rise profiles at the tumor site under the 808 nm NIR laser irradiation with an intensity of 1 W/cm² for 10 min. E. Relative tumor volume from mice intratumorally injected with PBS, GNS and GNS-loaded MSCs (n = 5) post PTT; F. Representative images of PC-3 tumors harvested from the three different groups post 16 days of laser irradiation. G. Representative H&E staining section of the mice organs after treatment for 16 days. All scale bars are 200 μm.

Figure 7

Photothermal ablation of prostate tumors via intravenous injection. A. Infrared microscopic imaging, NIR laser irradiation (808 nm, 1.5 W/cm², 10 min) of the tumor bearing mice after intravenous injections of PBS, TAT-GNS and GNS-loaded MSCs (5 × 10⁵ cells) after 3 days. B. Temperature rise profiles at the tumor site under the 808 nm NIR laser irradiation with an intensity of 1.5 W/cm² for 10 min. C. Relative tumor volume from mice intravenously injected with PBS, TAT-GNS and GNS-loaded MSCs (n = 5) post PTT in 14 days; D. Representative images of PC-3 tumors harvested from the three different groups post 14 days of laser irradiation.