A Liposome Encapsulated Ruthenium Polypyridine Complex as a Theranostic Platform for Triple-Negative Breast Cancer.

Ruthenium coordination complexes have the potential to serve as novel theranostic agents for cancer. However, a major limitation in their clinical implementation is effective tumor accumulation. In this study, we have developed a liposome-based theranostic nanodelivery system for [Ru(phen) 2 dppz](ClO 4 ) 2 (Lipo-Ru). This ruthenium polypyridine complex emits a strong fluorescent signal when incorporated in the hydrophobic lipid bilayer of the delivery vehicle or in the DNA helix, enabling visualization of the therapeutic agent in tumor tissues. Incubation of MDA-MB-231 breast cancer cells with Lipo-Ru induced double-strand DNA breaks and triggered apoptosis. In a mouse model of triple-negative breast cancer, treatment with Lipo-Ru dramatically reduced tumor growth. Biodistribution studies of Lipo-Ru revealed that more than 20% of the injected dose accumulated in the tumor. These results suggest that Lipo-Ru could serve as a promising theranostic platform for cancer. ## Results and Discussion Results and Discussion Design and synthesis of Lipo-Ru The preparation process for Lipo-Ru is illustrated in Scheme 1 . Briefly, dppz and cis-[Ru(phen) 2 Cl 2 ] · 2H 2 O were synthesized as previously reported. 13 , 21 , 22 These two compounds were then mixed together to form [Ru(phen) 2 dppz](ClO 4 ) 2 . Results from proton nuclear magnetic resonance ( 1 H-NMR) and electrospray ionization mass spectrometry (ESI-MS) revealed that the synthesis process was successfully carried out ( Figure S1 and S2 ). The Ru-complex was then encapsulated in liposomes consisting of DPPC, cholesterol, and distearoyl phosphatidylethanolamine (DSPE)-PEG. Lipo-Ru was purified by centrifugation and washed in water to remove unencapsulated Ru. Inductively coupled plasma atomic emission spectrometer (ICP-AES) measurements demonstrated that the loading capacity was 4% (1 mg Ru/25 mg liposomes). Characterization of Lipo-Ru The ability of Ru to emit fluorescence when intercalated with DNA was assessed by mixing Ru with plasmid DNA. The results demonstrated that a dose-dependent increase in fluorescence intensity occurred ( Figure 1a ). The addition of DNase to the mixture exposed Ru to a hydrophilic environment that caused the fluorescence signal to be quenched ( Figure 2b ), indicating that DNA binding was essential for light emission. The morphology, size, and zeta potential of Lipo-Ru was evaluated with transmission electron microscopy (TEM) and a Nano-ZS Zetasizer. TEM image and dynamic light scattering revealed a bilayer conformation ( Figure 1c ) with an average nanoparticle size of 82.53±2.66 nm ( Figure 1d and Figure S3c ). Compared to empty liposomes, Lipo-Ru was marginally smaller in size and the zeta potential was slightly elevated ( Figure S3 ). Absorbance measurements of Lipo-Ru did not detect a wavelength shift and the metal-to-ligand charge transfer (MLCT) peak was preserved upon liposomal encapsulation ( Figure 1e ). The non-fluorescent Ru-complex became fluorescent when encapsulated in the lipid bilayer ( Figure 1f ) and the fluorescence intensity decreased in a time-dependent manner when Lipo-Ru was incubated with Triton X-100, which destroys the bilayer structure ( Figure 1g ). Previously, kinetic studies of liposome exposure to Triton X-100 have demonstrated that lipid bilayer solubilization can occur gradually over time. 23 Confocal microscopy demonstrated the presence of distinct fluorescent spots corresponding to Lipo-Ru nanoparticles ( Figure 1h ), while a fluorescence signal was not observed with Ru or empty liposomes ( Figure S4 ). Additionally, the spectral properties of free Ru were determined in the presence and absence of serum ( Figure S5 ). Serum incubation caused a red shift of the ~280 nm peak (305 nm) and hypochromism was observed with increasing concentrations of fetal bovine serum (FBS) ( Figure S5a ). Moreover, the emission spectrum revealed an FBS-induced increase in fluorescence ( Figure S5b ). Furthermore, Lipo-Ru was stable under physiological conditions, as the liposome size remained unchanged for 100 h upon incubation with 50% FBS in phosphate buffered saline (PBS) ( Figure 1i ). The release profile of Ru from Lipo-Ru was also assessed ( Figure S6 ). In the first 72 h, a slow release was observed with approximately 20% of Ru released, indicating that the pegylated bilayer protects Lipo-Ru from biomolecular interactions that reduce nanoparticle stability. Cellular internalization of Lipo-Ru Numerous biological applications have been explored for transition metal complexes, and these agents have shown promise for therapeutic and diagnostic use. 24 – 26 A key factor for achieving therapeutic success is effective delivery of metal-based drugs to target tissues. Since these compounds display therapeutic efficacy through interactions with DNA, they must cross the cellular membrane. Previously, low levels of Ru internalization in cancer cells has been reported. 27 Here, confocal microscopy was used to monitor cellular uptake of Lipo-Ru in MDA-MB-231 human breast cancer cells. A stronger fluorescent signal was detected in the nuclei upon exposure to Lipo-Ru than free Ru at the 6 h time point ( Figure 2a ). These uptake results were confirmed with fluorescence intensity measurements ( Figure 2b ). In contrast, there was no difference between the cellular uptake of free Ru and Lipo-Ru at the 0.5 h time point ( Figure 2b ). It is likely that the time required to activate the endocytic machinery causes the nanodelivery system to display similar uptake levels as the free drug after short incubation periods. Furthermore, the initial delay in liposome uptake could be due to the pegylation layer that causes reduced interactions with cells. Flow cytometry was also performed to analyze cellular uptake levels. After 6 h, cells treated with Lipo-Ru and Ru were 65.6% and 9.15% positive for the fluorescent dye, respectively ( Figure 2c and Table S1 ). Likewise, ICP-AES results demonstrated that the intracellular accumulation of Lipo-Ru after 6 h was 15-fold higher than that of Ru ( Figure 2d ). Taken together, these results demonstrate that cellular internalization is substantially improved as a result of liposomal delivery. Cancer cell death and acute immunotoxicity from Lipo-Ru in vitro A major consideration in the clinical implementation of novel therapeutics is biocompatibility. 28 – 31 Here, the cytotoxicity of free liposomes in MDA-MB-231 cells was evaluated using an MTT assay. After 72 h of incubation, the cell viability was > 95% and > 90% at liposome concentrations of 0–500 μg/mL and 625 μg/mL, respectively ( Figure 3a ). These results are supported by previous studies, which demonstrate that liposomes of similar composition do not cause toxicity. 32 – 34 The in vitro anticancer activity of Lipo-Ru in MDA-MB-231 cells after 24 h, 48 h, and 72 h was also determined with an MTT assay. The results reveal a dramatic difference in the ability of Ru and Lipo-Ru to reduce cell viability. At all three time points, the half maximal inhibitory concentration (IC 50 ) was > 200 μM and < 4 μM for Ru and Lipo-Ru, respectively ( Figure 3b , Figure S7 and Table S2 ). A similar Lipo-Ru-induced reduction in cell viability was observed in three other TNBC cell lines, including SUM 159, MDA-MB-468, and BT-549 ( Figure S8 ). Nanoparticles and small molecule drugs can in certain cases evoke adverse immune reactions that are accompanied by the production of key inflammatory cytokines, such as interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α). 28 , 31 , 35 In this study, the levels of IL-6 and TNF-α were measured in Raw 264.7 murine macrophages and DC2.4 murine dendritic cells exposed to empty liposomes, Ru, or Lipo-Ru. Lipopolysaccharide (LPS) served as a positive control, as its ability to induce an immune response has been well documented. 31 Treatment with LPS or Ru triggered a surge in both TNF-α and IL-6 levels; in contrast, treatment with empty liposomes or Lipo-Ru did not cause any changes in the levels of these cytokines compared to untreated cells ( Figure 3c , 3d and Figure S9 ). These results demonstrate that liposomal encapsulation of Ru prevents immunotoxicity. Evaluation of DNA damage, cell cycle arrest, and apoptosis from Lipo-Ru To evaluate the anticancer activity of Lipo-Ru, MDA-MB-231 cells were co-incubated with Lipo-Ru and cell lysates were analyzed by Western blot to detect proteins and phospho-proteins associated with DNA damage and apoptosis. MDA-MB-231 orthotopic breast cancer tumors from athymic nude mice treated with Lipo-Ru were also analyzed to assess treatment-induced DNA damage in vivo . The results demonstrated that Lipo-Ru increased the levels of γ-H2AX ( Figure 4a and 4b ), a marker for DNA double-strand breaks. 36 , 37 Confocal microscopy analysis also revealed increased levels of γ-H2AX in response to Lipo-Ru treatment ( Figure 4c and 4d ). In addition, Lipo-Ru induced degradation and dephosphorylation of ataxia telangiectasia mutated (ATM) ( Figure 4a and 4b ), a key protein in DNA repair. 38 , 39 It has previously been shown that ATM inactivation is triggered by cellular apoptosis. 40 , 41 Accordingly, DNA damage caused by metal-based drugs typically disrupts normal DNA function, consequently inducing cell cycle arrest and triggering apoptosis. 4 , 42 In this study, the effect of Ru and Lipo-Ru (5 μM) on the cell cycle and apoptosis was assessed in MDA-MB-231 cells with flow cytometry. Ru caused a negligible increase in cell cycle arrest, while 54% of cells treated with Lipo-Ru were in the G2/M phase ( Figure 5a and 5c ). In addition, 75.9% of cells were apoptotic following exposure to Lipo-Ru for 12 h, while less than 5% displayed signs of apoptosis in Ru-treated cells and control cells ( Figure 5b and 5d ). To further assess cell apoptosis, protein levels of key regulators in intrinsic and extrinsic apoptotic pathways 43 were analyzed by Western blot. Cells treated with Lipo-Ru displayed cleaved forms of poly (ADP-ribose) polymerase (PARP), caspase-3, caspase-8, and caspase-9 ( Figure 5e ). Taken together, these results indicate that DNA damage caused by Lipo-Ru can trigger cell cycle arrest and induce apoptosis. Biodistribution of Lipo-Ru in vivo An orthotopic murine model of MDA-MB-231 human breast cancer was applied to evaluate whether Lipo-Ru could effectively accumulate in tumor tissue upon intravenous administration. Whole body images of mice acquired with the IVIS-200 system revealed that the tumor signal was dramatically enhanced in the Lipo-Ru group compared to the Ru group ( Figure S10 ). Additionally, the tumor vasculature was delineated using a FITC-labeled dextran dye and intravital microscopy analysis was used to monitor the tumor accumulation of Lipo-Ru ( Figure 6a ). The results revealed that most of the particles had entered the tumor interstitium and many of them were stationary within the tumor after 0.5 h ( Figure 6a , Video S1 ). Fluorescent spots were clearly visible in tumor tissue sections obtained 2 h after administration of Lipo-Ru ( Figure 6b , Figure S11 , Video S2 and S3 ). The IVIS-200 imaging system was also applied to visualize Lipo-Ru accumulation in the heart, spleen, kidneys, liver, lungs, and tumor 2 h post-injection. The Lipo-Ru particles accumulated primarily in the liver (34% of injected dose) and in the tumor (30% of injected dose) ( Figure 6c and 6e ). Additionally, ICP-AES was used to compare the biodistribution of free Ru and Lipo-Ru. As shown in figure S12 , intratumoral deposition of Ru was substantially increased in the liposome group. Enhanced accumulation of Lipo-Ru particles in tumor tissue is likely due to the enhanced permeability and retention (EPR) effect, which is a consequence of the unique characteristics of tumor vasculature. Namely, tumor blood vessels are highly disorganized, proliferate rapidly, and form a discontinuous barrier, resulting in the formation of fenestrations (<600 nm) that enable nanoparticle entry. 44 – 46 In addition, the PEG chains of Lipo-Ru reduce uptake by macrophages, thereby decreasing liposome accumulation in the mononuclear phagocyte system. Therapeutic efficacy evaluation of Lipo-Ru in vivo The orthotopic MDA-MB-231 tumor model was also applied to evaluate the therapeutic efficacy of Lipo-Ru. Tumor growth was dramatically suppressed in the Lipo-Ru treatment group compared to the PBS or Ru-treatment control groups ( Figure 7a ). Notably, the average tumor weights were 0.992 g, 0.981 g, and 0.342 g in the PBS, Ru, and Lipo-Ru groups, respectively ( Figure 7b ). Immunofluorescent imaging of tumor tissues revealed that the Ki-67 staining index was 58.6%, 50.2%, and 4.8% in mice treated with PBS, Ru, or Lipo-Ru, respectively ( Figure 7c and 7d ), indicating that cancer cell proliferation was dramatically suppressed in response to Lipo-Ru. The TUNEL assay demonstrated that tumors from the Lipo-Ru group displayed much higher levels of apoptosis than the control groups ( Figure 7e and f ). Likewise, Western blot analysis of tumor samples revealed that Lipo-Ru treatment altered the expression levels of proteins involved in intrinsic and extrinsic apoptotic pathways ( Figure 7g ). The potential damage to normal organs from Lipo-Ru exposure was also evaluated. Histological examination demonstrated that repeated injections of Lipo-Ru in tumor-free mice did not cause any apparent morphological changes ( Figure S13 ), as was evident from hematoxylin and eosin (H&E) staining. ## Design and synthesis of Lipo-Ru Design and synthesis of Lipo-Ru The preparation process for Lipo-Ru is illustrated in Scheme 1 . Briefly, dppz and cis-[Ru(phen) 2 Cl 2 ] · 2H 2 O were synthesized as previously reported. 13 , 21 , 22 These two compounds were then mixed together to form [Ru(phen) 2 dppz](ClO 4 ) 2 . Results from proton nuclear magnetic resonance ( 1 H-NMR) and electrospray ionization mass spectrometry (ESI-MS) revealed that the synthesis process was successfully carried out ( Figure S1 and S2 ). The Ru-complex was then encapsulated in liposomes consisting of DPPC, cholesterol, and distearoyl phosphatidylethanolamine (DSPE)-PEG. Lipo-Ru was purified by centrifugation and washed in water to remove unencapsulated Ru. Inductively coupled plasma atomic emission spectrometer (ICP-AES) measurements demonstrated that the loading capacity was 4% (1 mg Ru/25 mg liposomes). ## Characterization of Lipo-Ru Characterization of Lipo-Ru The ability of Ru to emit fluorescence when intercalated with DNA was assessed by mixing Ru with plasmid DNA. The results demonstrated that a dose-dependent increase in fluorescence intensity occurred ( Figure 1a ). The addition of DNase to the mixture exposed Ru to a hydrophilic environment that caused the fluorescence signal to be quenched ( Figure 2b ), indicating that DNA binding was essential for light emission. The morphology, size, and zeta potential of Lipo-Ru was evaluated with transmission electron microscopy (TEM) and a Nano-ZS Zetasizer. TEM image and dynamic light scattering revealed a bilayer conformation ( Figure 1c ) with an average nanoparticle size of 82.53±2.66 nm ( Figure 1d and Figure S3c ). Compared to empty liposomes, Lipo-Ru was marginally smaller in size and the zeta potential was slightly elevated ( Figure S3 ). Absorbance measurements of Lipo-Ru did not detect a wavelength shift and the metal-to-ligand charge transfer (MLCT) peak was preserved upon liposomal encapsulation ( Figure 1e ). The non-fluorescent Ru-complex became fluorescent when encapsulated in the lipid bilayer ( Figure 1f ) and the fluorescence intensity decreased in a time-dependent manner when Lipo-Ru was incubated with Triton X-100, which destroys the bilayer structure ( Figure 1g ). Previously, kinetic studies of liposome exposure to Triton X-100 have demonstrated that lipid bilayer solubilization can occur gradually over time. 23 Confocal microscopy demonstrated the presence of distinct fluorescent spots corresponding to Lipo-Ru nanoparticles ( Figure 1h ), while a fluorescence signal was not observed with Ru or empty liposomes ( Figure S4 ). Additionally, the spectral properties of free Ru were determined in the presence and absence of serum ( Figure S5 ). Serum incubation caused a red shift of the ~280 nm peak (305 nm) and hypochromism was observed with increasing concentrations of fetal bovine serum (FBS) ( Figure S5a ). Moreover, the emission spectrum revealed an FBS-induced increase in fluorescence ( Figure S5b ). Furthermore, Lipo-Ru was stable under physiological conditions, as the liposome size remained unchanged for 100 h upon incubation with 50% FBS in phosphate buffered saline (PBS) ( Figure 1i ). The release profile of Ru from Lipo-Ru was also assessed ( Figure S6 ). In the first 72 h, a slow release was observed with approximately 20% of Ru released, indicating that the pegylated bilayer protects Lipo-Ru from biomolecular interactions that reduce nanoparticle stability. ## Cellular internalization of Lipo-Ru Cellular internalization of Lipo-Ru Numerous biological applications have been explored for transition metal complexes, and these agents have shown promise for therapeutic and diagnostic use. 24 – 26 A key factor for achieving therapeutic success is effective delivery of metal-based drugs to target tissues. Since these compounds display therapeutic efficacy through interactions with DNA, they must cross the cellular membrane. Previously, low levels of Ru internalization in cancer cells has been reported. 27 Here, confocal microscopy was used to monitor cellular uptake of Lipo-Ru in MDA-MB-231 human breast cancer cells. A stronger fluorescent signal was detected in the nuclei upon exposure to Lipo-Ru than free Ru at the 6 h time point ( Figure 2a ). These uptake results were confirmed with fluorescence intensity measurements ( Figure 2b ). In contrast, there was no difference between the cellular uptake of free Ru and Lipo-Ru at the 0.5 h time point ( Figure 2b ). It is likely that the time required to activate the endocytic machinery causes the nanodelivery system to display similar uptake levels as the free drug after short incubation periods. Furthermore, the initial delay in liposome uptake could be due to the pegylation layer that causes reduced interactions with cells. Flow cytometry was also performed to analyze cellular uptake levels. After 6 h, cells treated with Lipo-Ru and Ru were 65.6% and 9.15% positive for the fluorescent dye, respectively ( Figure 2c and Table S1 ). Likewise, ICP-AES results demonstrated that the intracellular accumulation of Lipo-Ru after 6 h was 15-fold higher than that of Ru ( Figure 2d ). Taken together, these results demonstrate that cellular internalization is substantially improved as a result of liposomal delivery. ## Cancer cell death and acute immunotoxicity from Lipo-Ru Cancer cell death and acute immunotoxicity from Lipo-Ru in vitro A major consideration in the clinical implementation of novel therapeutics is biocompatibility. 28 – 31 Here, the cytotoxicity of free liposomes in MDA-MB-231 cells was evaluated using an MTT assay. After 72 h of incubation, the cell viability was > 95% and > 90% at liposome concentrations of 0–500 μg/mL and 625 μg/mL, respectively ( Figure 3a ). These results are supported by previous studies, which demonstrate that liposomes of similar composition do not cause toxicity. 32 – 34 The in vitro anticancer activity of Lipo-Ru in MDA-MB-231 cells after 24 h, 48 h, and 72 h was also determined with an MTT assay. The results reveal a dramatic difference in the ability of Ru and Lipo-Ru to reduce cell viability. At all three time points, the half maximal inhibitory concentration (IC 50 ) was > 200 μM and < 4 μM for Ru and Lipo-Ru, respectively ( Figure 3b , Figure S7 and Table S2 ). A similar Lipo-Ru-induced reduction in cell viability was observed in three other TNBC cell lines, including SUM 159, MDA-MB-468, and BT-549 ( Figure S8 ). Nanoparticles and small molecule drugs can in certain cases evoke adverse immune reactions that are accompanied by the production of key inflammatory cytokines, such as interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α). 28 , 31 , 35 In this study, the levels of IL-6 and TNF-α were measured in Raw 264.7 murine macrophages and DC2.4 murine dendritic cells exposed to empty liposomes, Ru, or Lipo-Ru. Lipopolysaccharide (LPS) served as a positive control, as its ability to induce an immune response has been well documented. 31 Treatment with LPS or Ru triggered a surge in both TNF-α and IL-6 levels; in contrast, treatment with empty liposomes or Lipo-Ru did not cause any changes in the levels of these cytokines compared to untreated cells ( Figure 3c , 3d and Figure S9 ). These results demonstrate that liposomal encapsulation of Ru prevents immunotoxicity. ## Evaluation of DNA damage, cell cycle arrest, and apoptosis from Lipo-Ru Evaluation of DNA damage, cell cycle arrest, and apoptosis from Lipo-Ru To evaluate the anticancer activity of Lipo-Ru, MDA-MB-231 cells were co-incubated with Lipo-Ru and cell lysates were analyzed by Western blot to detect proteins and phospho-proteins associated with DNA damage and apoptosis. MDA-MB-231 orthotopic breast cancer tumors from athymic nude mice treated with Lipo-Ru were also analyzed to assess treatment-induced DNA damage in vivo . The results demonstrated that Lipo-Ru increased the levels of γ-H2AX ( Figure 4a and 4b ), a marker for DNA double-strand breaks. 36 , 37 Confocal microscopy analysis also revealed increased levels of γ-H2AX in response to Lipo-Ru treatment ( Figure 4c and 4d ). In addition, Lipo-Ru induced degradation and dephosphorylation of ataxia telangiectasia mutated (ATM) ( Figure 4a and 4b ), a key protein in DNA repair. 38 , 39 It has previously been shown that ATM inactivation is triggered by cellular apoptosis. 40 , 41 Accordingly, DNA damage caused by metal-based drugs typically disrupts normal DNA function, consequently inducing cell cycle arrest and triggering apoptosis. 4 , 42 In this study, the effect of Ru and Lipo-Ru (5 μM) on the cell cycle and apoptosis was assessed in MDA-MB-231 cells with flow cytometry. Ru caused a negligible increase in cell cycle arrest, while 54% of cells treated with Lipo-Ru were in the G2/M phase ( Figure 5a and 5c ). In addition, 75.9% of cells were apoptotic following exposure to Lipo-Ru for 12 h, while less than 5% displayed signs of apoptosis in Ru-treated cells and control cells ( Figure 5b and 5d ). To further assess cell apoptosis, protein levels of key regulators in intrinsic and extrinsic apoptotic pathways 43 were analyzed by Western blot. Cells treated with Lipo-Ru displayed cleaved forms of poly (ADP-ribose) polymerase (PARP), caspase-3, caspase-8, and caspase-9 ( Figure 5e ). Taken together, these results indicate that DNA damage caused by Lipo-Ru can trigger cell cycle arrest and induce apoptosis. ## Biodistribution of Lipo-Ru Biodistribution of Lipo-Ru in vivo An orthotopic murine model of MDA-MB-231 human breast cancer was applied to evaluate whether Lipo-Ru could effectively accumulate in tumor tissue upon intravenous administration. Whole body images of mice acquired with the IVIS-200 system revealed that the tumor signal was dramatically enhanced in the Lipo-Ru group compared to the Ru group ( Figure S10 ). Additionally, the tumor vasculature was delineated using a FITC-labeled dextran dye and intravital microscopy analysis was used to monitor the tumor accumulation of Lipo-Ru ( Figure 6a ). The results revealed that most of the particles had entered the tumor interstitium and many of them were stationary within the tumor after 0.5 h ( Figure 6a , Video S1 ). Fluorescent spots were clearly visible in tumor tissue sections obtained 2 h after administration of Lipo-Ru ( Figure 6b , Figure S11 , Video S2 and S3 ). The IVIS-200 imaging system was also applied to visualize Lipo-Ru accumulation in the heart, spleen, kidneys, liver, lungs, and tumor 2 h post-injection. The Lipo-Ru particles accumulated primarily in the liver (34% of injected dose) and in the tumor (30% of injected dose) ( Figure 6c and 6e ). Additionally, ICP-AES was used to compare the biodistribution of free Ru and Lipo-Ru. As shown in figure S12 , intratumoral deposition of Ru was substantially increased in the liposome group. Enhanced accumulation of Lipo-Ru particles in tumor tissue is likely due to the enhanced permeability and retention (EPR) effect, which is a consequence of the unique characteristics of tumor vasculature. Namely, tumor blood vessels are highly disorganized, proliferate rapidly, and form a discontinuous barrier, resulting in the formation of fenestrations (<600 nm) that enable nanoparticle entry. 44 – 46 In addition, the PEG chains of Lipo-Ru reduce uptake by macrophages, thereby decreasing liposome accumulation in the mononuclear phagocyte system. ## Therapeutic efficacy evaluation of Lipo-Ru Therapeutic efficacy evaluation of Lipo-Ru in vivo The orthotopic MDA-MB-231 tumor model was also applied to evaluate the therapeutic efficacy of Lipo-Ru. Tumor growth was dramatically suppressed in the Lipo-Ru treatment group compared to the PBS or Ru-treatment control groups ( Figure 7a ). Notably, the average tumor weights were 0.992 g, 0.981 g, and 0.342 g in the PBS, Ru, and Lipo-Ru groups, respectively ( Figure 7b ). Immunofluorescent imaging of tumor tissues revealed that the Ki-67 staining index was 58.6%, 50.2%, and 4.8% in mice treated with PBS, Ru, or Lipo-Ru, respectively ( Figure 7c and 7d ), indicating that cancer cell proliferation was dramatically suppressed in response to Lipo-Ru. The TUNEL assay demonstrated that tumors from the Lipo-Ru group displayed much higher levels of apoptosis than the control groups ( Figure 7e and f ). Likewise, Western blot analysis of tumor samples revealed that Lipo-Ru treatment altered the expression levels of proteins involved in intrinsic and extrinsic apoptotic pathways ( Figure 7g ). The potential damage to normal organs from Lipo-Ru exposure was also evaluated. Histological examination demonstrated that repeated injections of Lipo-Ru in tumor-free mice did not cause any apparent morphological changes ( Figure S13 ), as was evident from hematoxylin and eosin (H&E) staining. ## Conclusion Conclusion In conclusion, the data presented herein indicates that the proposed strategy for liposomal encapsulation of a polypyridine Ru complex is a promising theranostic approach for cancer. The Ru complex is capable of causing DNA damage, which arrests cells in the G2/M phase and triggers pathways that ultimately induce cancer cell apoptosis. Lipo-Ru displayed high levels of uptake in MDA-MB-231 cancer cells and accumulated in high amounts in tumor tissue upon intravenous administration, which enabled efficient tumor imaging and cancer therapy. In particular, this study demonstrates effective delivery and therapeutic efficacy of systemically injected Lipo-Ru in a TNBC model. Approximately 15% of breast cancers in the United States are classified as triple-negative, which is a highly aggressive form of breast cancer that lacks expression of the estrogen receptor (ER), progesterone receptor (PR), and Her2/neu. 47 – 51 Therapeutic options are limited for TNBC patients, as hormonal and targeted therapies cannot be exploited. Although it is common for TNBC patients to initially respond to chemotherapy, most patients relapse with highly metastatic and drug-resistant tumors, leading to high mortality rates. 50 , 52 Accordingly, there is an urgent need to develop new therapeutic agents against TNBC. Here, administration of Lipo-Ru resulted in a dramatic reduction in the growth of TNBC tumors. Liposomal encapsulation of the active compound [Ru(phen) 2 dppz](ClO 4 ) 2 in the lipid bilayer also enabled fluorescence imaging of cancer cells and tumor tissue. In particular, confocal microscopy, intravital microscopy, and an in vivo preclinical imaging system (IVIS) were used to visualize Lipo-Ru in cells and organs. Moreover, Lipo-Ru did not cause any signs of acute immunotoxicity or morphological changes in major organs, suggesting that the platform could be used as a biocompatible theranostic tool for cancer. ## Supplementary Material Supplementary Material