*3.4. Cytotoxicity of N-AI PAI-2 Liposomes against Breast Cancer Spheroids*

MCF-7 and MDA-MB-231 cells are reported to form spheroids under low-attachment growth conditions [35]. EMP PAI-2 and *N*-AI PAI-2 liposomes at equivalent phospholipid concentrations were incubated with the preformed spheroids and imaged every 24 h (Figure 3). Spheroids treated with EMP PAI-2 liposomes showed continued growth and an increase in spheroid diameter, over time. In contrast, treatment with *N*-AI PAI-2 liposomes showed a time- and concentration-dependent disassembly of the spheroid structure, at concentrations above 62 µM for both cell lines (Figure 3a). However, MDA-MB-231 spheroids appeared to be more sensitive to *N*-AI PAI-2 liposome treatment, which showed clear evidence of spheroid dissociation, as early as 24 h, compared to the MCF-7 spheroids (Figure 3b). By 48 h, the MDA-MB-231 spheroids were almost completely dissociated in contrast to MCF-7 spheroids. A comparison of the Calcein AM stained spheroids after 96 h found MDA-MB-231 spheroids treated with *N*-AI and *N*-AI PAI-2 to be fully dissociated, while the MCF-7 spheroids, although smaller than the control (EMP and EMP PAI-2), remained largely intact (Figure 3c). *Pharmaceutics* **2020**, *12*, x FOR PEER REVIEW 9 of 16 *3.5. Pharmacokinetics and Biodistribution of N-AI PAI-2 Liposomes* To determine the pharmacokinetic and organ distribution profiles of *N*-AI liposomes and *N*-AI PAI-2 liposomes in tumor-bearing mice, liposomes were labelled with tritiated cholesteryl hexadecyl ether (3H-CHE), to enable their detection in plasma and tissues, through liquid scintillation counting. Liposomes were monodispersed with average diameters of 115 ± 34 nm and 117 ± 39 nm, for *N*-AI and *N*-AI PAI-2 liposomes, respectively. Scintillation counts of the two liposome stock preparations were 319,698 CPM/ml and 312,163 CPM/ml for *N*-AI and *N*-AI PAI-2 liposomes, respectively. The plasma half-life was determined to be 5.63 h and 5.82 h for the *N*-AI and *N*-AI PAI-2 liposomes, respectively (Figure 4a). The plasma clearance profiles of the two liposomes and the pharmacokinetic parameters from the curve-fitting analysis were not significantly different (*p* > 0.05) (Table 2).

**Figure 3.** Cytotoxic effect of *N*-AI PAI-2 liposomes on breast cancer spheroids. (**a**) Spheroid diameter was measured after incubation with EMP PAI-2 liposomes or *N*-AI PAI-2 liposomes with MCF-7 and MDA-MB-231 multicellular tumor spheroids, over a period of 96 h. (**b**) Representative bright-field images and (**c**) fluorescent images, following the addition of calcein-AM to visualize the viable cells were captured at the same magnification (*n* = 3). Spheroids in (**b**, **c**) were treated with 25 µM *N*-AI or the equivalent concentration of phospholipid in liposomal formulation. Scale bars are 100 µm. Data are the mean ± s.d. (*n* = 3). **Figure 3.** Cytotoxic effect of *N*-AI PAI-2 liposomes on breast cancer spheroids. (**a**) Spheroid diameter was measured after incubation with EMP PAI-2 liposomes or *N*-AI PAI-2 liposomes with MCF-7 and MDA-MB-231 multicellular tumor spheroids, over a period of 96 h. (**b**) Representative bright-field images and (**c**) fluorescent images, following the addition of calcein-AM to visualize the viable cells were captured at the same magnification (*n* = 3). Spheroids in (**b**, **c**) were treated with 25 µM *N*-AI or the equivalent concentration of phospholipid in liposomal formulation. Scale bars are 100 µm. Data are the mean ± s.d. (*n* = 3).

lungs was minimal for both liposome formulations (Figure 4g–h).

At 24 h, 48 h, and 96 h, tumor uptake of the *N*-AI and *N*-AI PAI-2 liposomes was not significantly different (*p* > 0.05). Liposome accumulation in the kidneys, liver, spleen, and lungs at each time-point

4b).

#### *3.5. Pharmacokinetics and Biodistribution of N-AI PAI-2 Liposomes* intravenously as a single bolus dose. 3H-CHE signal was measured in plasma at each time-point to

To determine the pharmacokinetic and organ distribution profiles of *N*-AI liposomes and *N*-AI PAI-2 liposomes in tumor-bearing mice, liposomes were labelled with tritiated cholesteryl hexadecyl ether (3H-CHE), to enable their detection in plasma and tissues, through liquid scintillation counting. Liposomes were monodispersed with average diameters of 115 ± 34 nm and 117 ± 39 nm, for *N*-AI and *N*-AI PAI-2 liposomes, respectively. Scintillation counts of the two liposome stock preparations were 319,698 CPM/ml and 312,163 CPM/ml for *N*-AI and *N*-AI PAI-2 liposomes, respectively. The plasma half-life was determined to be 5.63 h and 5.82 h for the *N*-AI and *N*-AI PAI-2 liposomes, respectively (Figure 4a). The plasma clearance profiles of the two liposomes and the pharmacokinetic parameters from the curve-fitting analysis were not significantly different (*p* > 0.05) (Table 2). determine the following parameters. **PK Parameter** *N***-AI** *N***-AI PAI-2**  Cmax (% ID/mL) 84.66 (± 9.79) 83.76 (± 9.25) Kelim α (fast) min−1 0.061 0.058 Kelim β (slow) min−1 0.002 0.002 T1/2 α (fast) min 11.419 12.050 T1/2 β (slow) min 408.152 410.843 Correlation coefficient (R2) 0.9629 0.9836 AUC (% ID/min/mL) 860.3 (± 66.89) 873.4 (± 50.79)

*Pharmaceutics* **2020**, *12*, x FOR PEER REVIEW 10 of 16

liposomes were labeled with tritiated cholesteryl hexadecyl ether (3H-CHE) and administered

**Figure 4.** Biodistribution and pharmacokinetics of the radiolabeled liposomes in mice. *N*-AI and *N*-AI PAI-2 liposomes were labeled with tritiated cholesteryl hexadecyl ether (3H-CHE) and administered intravenously as a single bolus dose. Tritiated signal was measured in the (**a**) plasma, (**b**) tumor, (**c**) kidneys, (**d**) liver, (**e**) spleen, and (**f**) lungs at each time-point. Kidney, liver, spleen, and lung data are also presented on the same graph for the (**g**) *N*-AI liposomes and (**h**) *N*-AI PAI-2 liposomes, for biodistribution comparison. Results are expressed as the percentage of injected dose (ID) per gram of tissue or milliliter of plasma, and as the percentage of the injected dose (ID) in the whole analyzed primary tumor. Values are the mean ± s.e.m. (*n* = 4). **Figure 4.** Biodistribution and pharmacokinetics of the radiolabeled liposomes in mice. *N*-AI and *N*-AI PAI-2 liposomes were labeled with tritiated cholesteryl hexadecyl ether (3H-CHE) and administered intravenously as a single bolus dose. Tritiated signal was measured in the (**a**) plasma, (**b**) tumor, (**c**) kidneys, (**d**) liver, (**e**) spleen, and (**f**) lungs at each time-point. Kidney, liver, spleen, and lung data are also presented on the same graph for the (**g**) *N*-AI liposomes and (**h**) *N*-AI PAI-2 liposomes, for biodistribution comparison. Results are expressed as the percentage of injected dose (ID) per gram of tissue or milliliter of plasma, and as the percentage of the injected dose (ID) in the whole analyzed primary tumor. Values are the mean ± s.e.m. (*n* = 4).

Tumors were removed from mice and analyzed for tritiated liposome signal. The results showed rapid accumulation of *N*-AI PAI-2 liposome signal in tumors, compared to *N*-AI liposomes, as indicated by the significantly increased %ID at 10 min, 3 h, and 6 h post-injection (*p* < 0.001; Figure


**Table 2.** Pharmacokinetic parameters of *N*-AI and *N*-AI PAI-2 liposomes. *N*-AI and *N*-AI PAI-2 liposomes were labeled with tritiated cholesteryl hexadecyl ether (3H-CHE) and administered intravenously as a single bolus dose. <sup>3</sup>H-CHE signal was measured in plasma at each time-point to determine the following parameters.

At 24 h, 48 h, and 96 h, tumor uptake of the *N*-AI and *N*-AI PAI-2 liposomes was not significantly different (*p* > 0.05). Liposome accumulation in the kidneys, liver, spleen, and lungs at each time-point was similar between the *N*-AI and *N*-AI PAI-2 liposomes (Figure 4c–f). The trends indicated increased clearance via the liver and spleen, over time, with clearance via the kidneys and accumulation in the lungs was minimal for both liposome formulations (Figure 4g–h).

Tumors were removed from mice and analyzed for tritiated liposome signal. The results showed rapid accumulation of *N*-AI PAI-2 liposome signal in tumors, compared to *N*-AI liposomes, as indicated by the significantly increased %ID at 10 min, 3 h, and 6 h post-injection (*p* < 0.001; Figure 4b).
