Overcoming Ovarian Cancer Drug Resistance with a Cold Responsive Nanomaterial

Abstract

Drug resistance due to overexpression of membrane transporters in cancer cells and the existence of cancer stem cells (CSCs) is a major hurdle to effective and safe cancer chemotherapy. Nanoparticles have been explored to overcome cancer drug resistance. However, drug slowly released from nanoparticles can still be efficiently pumped out of drug-resistant cells. Here, a hybrid nanoparticle of phospholipid and polymers is developed to achieve cold-triggered burst release of encapsulated drug. With ice cooling to below ∼12 °C for both burst drug release and reduced membrane transporter activity, binding of the drug with its target in drug-resistant cells is evident, while it is minimal in the cells kept at 37 °C. Moreover, targeted drug delivery with the cold-responsive nanoparticles in combination with ice cooling not only can effectively kill drug-resistant ovarian cancer cells and their CSCs in vitro but also destroy both subcutaneous and orthotopic ovarian tumors in vivo with no evident systemic toxicity.

Figure 1

Synthesis and characterization of cold-responsive nanoparticle. (a) Hyaluronic acid (HA or H), lipid (dipalmitoylphosphatidylcholine or DPPC in this study, L), Pluronic F127 (PF127, P), poly(N-isopropylacrylamide-co-butyl acrylate) (PNIPAM-B or N), and chitosan (C)-modified Pluronic F127 (PF127-chitosan) were used to prepare the doxorubicin (DOX, D) laden HCLPN-D nanoparticles using the double-emulsion method. (b) TEM images showing the HCLPN-D nanoparticles are spherical with a multicore–shell configuration. (c) The thermally induced phase transition behavior of PNIPAM-B from being water-insoluble to highly water-soluble, which can cause disassembly of the HCLPN-D nanoparticles upon cooling to below room temperature. This can result in burst release of the encapsulated drug. (d) TEM images showing the HCLPN-D nanoparticles become completely disassembled after 3 min incubation at 10 °C. (e) An extensive network of polymer fibers rather than nanoparticles is observable after warming back to 22 °C. (f) Photographs of the aqueous samples of HCLPN-D nanoparticles at various temperatures before and after shining a red laser beam through them in the dark. As a result of the Tyndall effect (i.e., scattering of laser beam by nanoparticles in solution), a bright white track of light is visible in the dark in the solutions of HCLPN-D nanoparticles above 10 °C. However, it is not clearly observable at or below 10 °C and after warming back to 22 or 37 °C, indicating the HCLPN-D nanoparticles disassemble upon cooling to 10 °C (or a lower temperature), and the disassembling process is not reversible. (g) Size distribution of HCLPN-D nanoparticles measured by dynamic light scattering (DLS) at different temperatures. The results show a narrow size distribution of the HCLPN-D nanoparticles at 37, 22, and 15 °C. An additional peak of large particles is seen at 12 °C, probably due to aggregation of polymers. No stable peak of nanoparticles can be detected when the temperature is decreased to 10 and 6 °C.

Figure 2

Cold-triggered burst drug release from HCLPN-D nanoparticles. (a) UV–vis absorbance of HCLPN-D nanoparticles at different temperatures showing the cold-responsiveness of HCLPN-D nanoparticles. Arrows indicate the absorbance peaks of DOX. (b) A comparison of the release of DOX from HCLPN-D nanoparticles under pH 7.4, acidic pH (5.0, 5 min), and ice cooling (5 min), showing the cold temperature is much more effective than low pH in triggering drug release from the HCLPN-D nanoparticles. Error bars represent ± standard deviation (SD, n = 3).

Figure 3

Overcoming cancer drug resistance with cold-triggered burst drug release form HCLPN-D nanoparticles. (a) Confocal micrographs of 2D cultured NCI/RES-ADR multidrug-resistant cancer cells after incubating them with either free DOX or HCLPN-D nanoparticles for 3 h at 37 °C, followed by either continued culturing in incubator (37 °C) or ice cooling (+I) for 5 or 10 min. (b) Confocal images of CSC-enriched spheres derived from the multidrug-resistant cancer cells after incubating them with HCLPN-D nanoparticles for 3 h at 37 °C, followed by either continued culturing in incubator (37 °C) or ice cooling (+I) for 5 or 10 min. DOX could enter the cell nuclei only when treated with both HCLPN-D nanoparticles and ice cooling, indicating the cold-triggered burst drug release from the HCLPN-D nanoparticles could be used to overcome the drug resistance of the 2D cultured cancer cells and their CSCs.

Figure 4

Enhanced in vitro anticancer capacity by HCLPN-D nanoparticles with ice cooling for overcoming drug resistance. Viability of (a) 2D cultured NCI/RES-ADR multidrug-resistant cancer cells and (b) CSC-enriched spheres derived from the multidrug-resistant cancer cells after treating them with blank nanoparticles (HCLPN), free DOX, and HCLPN-D nanoparticles without or with ice cooling for 5 or 10 min. The viability of control cells cultured in pure medium is 100%. Error bars represent SD (n = 3). *: p < 0.05 (Kruskal–Wallis H test), which indicates cells treated with HCLPN-D nanoparticles and ice cooling for 10 min is significantly lower than other treatments with the same drug concentration. (c) TEM images of the NCI/RES-ADR cancer cells treated with saline, HCLPN-D nanoparticles with or without ice cooling for 10 min. The endo-/lysosomes in HCLPN-D treated cells light up due to the existence of intact (without ice cooling) or disassembled (with ice cooling) HCLPN-D nanoparticles. The insets are the endo-/lysosomes indicated by the arrows with either intact or disassembled HCPN-CG nanoparticles. (d) A schematic illustration of the combination of the HCLPN-D nanoparticle and ice cooling for overcoming the multidrug resistance to enhance cancer destruction, in comparison to the HCLPN-D nanoparticle alone and free drug. The combination can overcome the drug resistance in cancer cells by (1) cold-triggered burst drug release from the HCLPN-D nanoparticles and (2) the cold-induced low activity of the membrane transporters to pump out the released drug.

Figure 5

In vivo tumor targeting capacity of HCLPN-D nanoparticles. (a) In vivo whole animal imaging of ICG fluorescence at different times after intravenous injection of free ICG and ICG-laden HCLPN-G nanoparticles via the tail vein. Arrows indicate the locations of tumors in mice. (b) Ex vivo imaging of ICG fluorescence in tumor and five critical organs collected after sacrificing the mice at 9 h. (c) Imaging of total ICG fluorescence of free ICG and ICG-laden HCLPN-G nanoparticles in three samples prepared in the same way as the solutions used for injection into mice. The images were taken under the same condition as that for both the in vivo and ex vivo imaging. (d) Quantitative analysis of the distribution of HCLPN-G and free ICG in tumor and five critical organs collected from free ICG and HCLPN-D nanoparticles treated mice. The data show that the HCLPN-G nanoparticles could accumulate in tumor much more efficiently than free ICG. NCI/RES-ADR cells detached (with trypsin) from CSC-enriched spheres were used to obtain xenografts of multidrug-resistant tumors for imaging.

Figure 6

In vivo antitumor capacity of HCLPN-D nanoparticles with ice cooling studied using subcutaneous tumor model. (a) Near infrared thermographs of whole animal and human hand before and after ice cooling for 10 min (+I), showing temperature in the region with cooling can be effectively decreased to ∼0 °C. (b) Typical photographs showing the size of tumors (indicated by arrows) on day 59 in mice with six different treatments. (c) Tumor growth curves for the six different treatments. Error bars represent SD (n = 5). The red arrow heads indicate the times of conducting injections. *: p < 0.05, **: p < 0.01 (Kruskal–Wallis H test). (d) Weight of the tumors collected after sacrificing the mice on day 59. Error bars represent SD (n = 5). **: p < 0.01 (Kruskal–Wallis H test). (e) Representative histology (H&E) images of the tumors collected on day 59. The HCLPN-D+I treated tumors are more necrotic than tumors with the other five treatments. (f) Immunofluorescent staining of CD44 and CD133 in tumor showing diminished expression of both CD44 and CD133 after the treatment with HCLPN-D+I. (g) Body weight and (h) representative micrographs of H&E staining of four important organs with various treatments showing the minimized systemic toxicity of HCLPN-D+I compared to treatments with free DOX (DOX and DOX+I).

Figure 7

In vivo antitumor capacity of HCLPN-D nanoparticles with ice cooling studied using orthotopic metastasis model of ovarian cancer. (a) Near infrared thermographs of whole animal on the ventral side before and after ice cooling for 10 min. The data show that temperature on the skin with cooling on the ventral side of the peritoneal cavity can be effectively decreased to ∼6–10 °C. (b) Photographs showing the typical in situ locations of tumors (indicated by arrows and circles) from mice treated with saline and free DOX. (c) Photograph showing the size of tumors collected after sacrificing the mice on day 32 with three different treatments. (d) Weight of the tumors collected on day 32. Error bars represent SD (n = 3). (e) Representative histology (H&E) images of the tumors collected on day 32. (f–g) Body weight (f) and representative micrographs of H&E staining of five critical organs (g) with various treatments. The data show reduced systemic toxicity of the treatment of HCLPN-D nanoparticles with ice cooling for 10 min (HCLPN-D+I) compared with the free DOX+I treatment. *: p < 0.05 (Kruskal–Wallis H test).

Figure 8

A Schematic illustration of overcoming drug resistance with HCLPN-D nanoparticles and ice cooling for enhanced cancer therapy. In vivo accumulation of HCLPN-D nanoparticles in tumor through the enhanced permeability and retention (EPR) effect of tumor vasculature could minimize the side effects associated with free DOX. Moreover, the HCLPN-D nanoparticles can specifically target cancer stem cells (CSCs) via the HA-CD44 interaction to facilitate their uptake by the CSCs. Although drug slowly released from nanoparticles at 37 °C (or mild hyperthermic temperatures) could be still pumped out of the multidrug-resistant cancer cells, the cold-triggered burst drug release together with the compromised pumping activity of membrane transporters in the multidrug-resistant cancer cells under cold temperature could efficiently overcome their drug-resistant capacity. As a result, the cold-responsive nanoparticle in combination with ice cooling could efficiently inhibit the growth of multidrug-resistant tumor in vivo.