Blood Interactions, Pharmacokinetics, and Depth-Dependent Ablation of Rat Mammary Tumors with Photoactivatable, Liposomal Doxorubicin

The delivery of anticancer agents at therapeutically relevant concentrations is a major challenge in treating solid tumors (1–4). Nanoparticles, particularly liposomes, have been widely used clinically to address this concern (5–8). However, nanoparticles do not necessarily provide enhanced therapeutic effects over the free drug; rather, they provide a reduction in toxicity and alteration of pharmacokinetics (9–13). Doxil, a PEGylated liposomal form of doxorubicin (Dox), for example, is clinically approved for the treatment of Kaposi sarcoma, ovarian cancer, and multiple melanoma (14), and is widely used for the treatment of metastatic breast cancer (15). Yet it does not offer clearly superior efficacy over non-liposomal doxorubicin (9, 16–19). Rather, its use is driven by a reduction in side-effects compared with free Dox, including cardiotoxicity (9, 17). This lack of greater efficacy, despite enhanced delivery to the tumor, is attributed to poor bioavailability of liposome-encapsulated drugs due to their slow release (9, 20). Numerous molecular-targeting strategies have been explored for liposomes (21–24). Alternatively, several stimulus controlled systems have been developed to address this problem, including systems that use heat, light, and pH to trigger drug release (25–31). The most advanced of which, Thermodox, a heat-triggered, liposome-encapsulated Dox, has been tested clinically for the treatment multiple forms of cancer, including liver cancer and breast cancer (32–34). The combination of light therapy and chemotherapy, chemophototherapy (CPT), is an emerging treatment paradigm for solid tumors (35). Photodynamic therapy (PDT) itself is a clinically used ablative modality that has been shown to treat a variety of tumor types (36, 37).

We previously described long-circulating Dox in porphyrin–phospholipid (LC-Dox-PoP) liposomes, a PEGylated liposomal form of Dox, co-encapsulated with a bilayer-confined and lipid-like porphyrin–phospholipid (PoP) conjugate incorporated at 2 mole % (total lipid). In murine studies, LC-Dox-PoP liposomes were shown to have long circulating properties similar to that of sterically stabilized stealth liposomal doxorubicin, and enhanced antitumor efficacy when combined with laser treatment, at doses significantly lower than Doxil-like liposomes (38–40). Here, we report in vitro/in vivo biological evaluation of the LC-Dox-PoP liposomes, including in vitro immunological characterization, blood partitioning, in vivo pharmacokinetics, and antitumor efficacy in an orthotopic rat tumor model with specific emphasis on determining the suitability of LC-Dox-PoP for use in humans.

LC-Dox-PoP liposomes were prepared as previously described (38). Briefly, 200 mg of lipids were dissolved in 2 mL of ethanol in the molar ratio [53:2:5:40; 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, Corden LP-R4-076):pyro-lipid (synthesized as previously described; ref. 38)]: 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG-2000, Corden, LP-R4-039): cholesterol (Chol, PhytoChol; Wilshire Technologies, CAS 57-88-5)]. The solution of dissolved lipids was heated to 60°C and 8 mL of 250 mmol/L ammonium sulfate (60°C) was added to the ethanol. The liposomes were then extruded 10 times using a 10 mL nitrogen pressurized extruder (Northern Lipids) using stacked (80, 100, and 200 nm) polycarbonate membranes at 60°C. Excess ammonium sulfate and ethanol were removed by dialysis (MWCO 12,000-14,000, Fisher # 21-152-16) in a buffer of 10 mmol/L histidine (pH 6.5), 10% sucrose solution with three buffer changes. Dox (LC Laboratories # D-4000) was encapsulated into the liposomes by mixing the liposome and Dox solution, and incubating for 60 minutes at 60°C. Following Dox loading, liposomes were sterile filtered using a 0.2-μm Supor membrane filter (Pall # 28143-300). Doxil was obtained from the NIH pharmacy.

A Malvern Zetasizer Nano ZS instrument (Southborough, MA) was used for measuring the hydrodynamic size (diameter) and zeta potential in accordance to National Institute of Standards and Technology (NIST)—National Cancer Institute Nanocharacterization laboratory (NCL) joint protocol PCC-1 and NCL protocol PCC-2, respectively. Hydrodynamic size was measured by diluting the liposomes 100- and 1,000-fold in PBS or 10 mmol/L NaCl (zeta potential measurement conditions). Samples were measured at 25°C in a quartz microcuvette with a back scattering detector (173°) in batch mode. Zeta potential was measured by diluting the liposomes 100-fold in 10 mmol/L NaCl. Sample pH was measured before loading into a pre-rinsed folded capillary cell. Measurements were made at both native pH and after adjustment to neutral pH using 1 N standardized NaOH. An applied voltage of 150 V was used.

Cryo-transmission electron microscopy (cryo-TEM) was performed to assess the morphology of the liposomes. To prepare the sample for cryo-TEM, 4 μL of stock solution or solution diluted ×20 in ultrapure water was applied to a quantifoil holey film 200 mesh copper grid (Electron Microscopy Sciences) and blotted either for 3 or 2.5 sec, respectively, before plunge freezing in liquid ethane using a Vitrobot (FEI Company) to form a thin film of vitreous ice. Imaging was performed using a Tecnai T-20 equipped with a tungsten thermoionic gun using low-dose mode.

Encapsulation efficiency of doxorubicin was assessed using UV-Vis spectroscopy and centrifugal filtration to measure free doxorubicin. Samples (250 μL) were centrifuged at 14,000 rpm at 24°C to dryness using Microcon DNA Fast Flow centrifugal devices (Millipore, MRCFOR100, RC). The filtrate, containing any free doxorubicin, was analyzed by UV-Vis spectroscopy. UV-Vis spectra were recorded using a PerkinElmer Lambda 35 spectrophotometer. All samples were measured in quartz microcuvettes (path length, b = 10 mm, QS105.250). Spectra were collected from 200 to 800 nm at 480 nm/min in 1-nm steps against water as the reference. Calibration standards of doxorubicin were prepared from a 2,000 μg/mL doxorubicin stock solution in 25 mmol/L NaCl. Calibration standards were prepared by diluting this calibration stock with 25 mmol/L NaCl to final concentrations ranging from 5 to 50 μg/mL. Doxorubicin calibration standards were subjected to the same centrifugation process as the liposome samples.

Hemolysis tests were conducted in vitro in accordance to the NCL protocol “ITA-1, Analysis of Hemolytic Properties of Nanoparticles.” In brief, freshly drawn human blood anticoagulated with lithium heparin was diluted in PBS to a concentration of 10 mg/mL total blood hemoglobin. The diluted whole blood was then incubated with test samples for 3 hours at 37°C. Following incubation, cell-free supernatants were prepared and analyzed for the presence of plasma-free hemoglobin by converting hemoglobin and its metabolites into cyanmethemoglobin (CMH) using Drabkin's reagent. CMH was then quantified against a hemoglobin standard by measuring the absorbance of the samples at 540 nm.

Platelet aggregation tests were conducted in vitro in accordance to NCL protocol “ITA-2, Analysis of Platelet Aggregation.” In brief, platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were prepared from freshly drawn human blood. PPP was used as the background control. PRP was incubated with the ChronoLum reagent and test samples, and sample turbidity was measured using the ChronoLog aggregometer. The ChronoLum reagent was used to monitor ATP release from platelets. The latter correlates with platelet aggregation and is used as an additional control to verify that any changes observed in the light transmission were due to platelet aggregation.

Plasma coagulation tests were conducted in vitro in accordance with NCL protocol “ITA-12, Coagulation Assay.” In brief, three plasma coagulation tests were performed, prothrombin time (PT), activated partial thromboplastin time (APTT), and thrombin time (TT). Freshly drawn human blood was used to prepare plasma. Control N (normal plasma standard) and control P (abnormal plasma standard) were obtained as citrated human plasma from a commercial source specializing in the clinical diagnostic blood coagulation products (Diagnostica Stago). The controls are verified by the manufacturer against relevant standards so that coagulation time of the Control N is below and that of the Control P is above the threshold normal coagulation time for the given plasma coagulation assay. Plasma was incubated with test samples for 30 minutes at 37°C. Following incubation, plasma coagulation initiation reagents (neoplastin, CaCl₂, or thrombin, respectively) were added to the mixture and the coagulation times measured using the STArt4 coagulometer (Diagnostica Stago).

Complement activation tests were conducted in vitro in accordance with NCL protocol “ITA-5.2, Analysis of Complement Activation by EIA.” Briefly, plasma was prepared from freshly drawn human blood and incubated with test samples and veronal buffer for 30 minutes at 37°C. Following incubation, the samples were analyzed for the presence of the iC3b component of complement using a commercial enzyme immunoassay kit. LC-Dox-PoP liposomes were analyzed at four concentrations ranging 0.002 to 0.66 mg/mL Dox. Doxil was used as an additional, commercially relevant nanoparticle control. Doxil was tested at equivalent Dox concentrations.

A previously described blood partitioning method (41) was used to determine the percentage of plasma fraction for both PoP and Dox, using the equation F_P% = (C_P/C_B)(1-HCT) × 100, where C_P = concentration in plasma; C_B = concentration in the blood; and HCT = hematocrit. Fresh human blood was collected in K2-EDTA tubes (Thermo Fischer Scientific, Waltham, MA, Cat. # 02-689-4) for these studies. For analysis in whole blood, 3 mL blood was spiked with LC-Dox-PoP liposomes, in triplicate, to yield final concentrations of 0.5, 2, and 10 μg PoP/mL, corresponding to 2.5, 10.2, and 51.2 μg Dox/mL, in glass vials. Samples were incubated at 37°C with agitation. At specified time points (time zero, 30 minutes, 2, 4, and 24 hours), 50 μL blood was taken for PoP and Dox analysis. A capillary sample was also taken for hematocrit measurement. For plasma analysis, 500 μL of blood was also taken at each time point above, transferred to an Eppendorf tube and spun at 2,500 x g for 10 minutes to collect plasma. Plasma (50 μL each) was sampled for PoP and Dox analysis using LC-MS. See Supplementary Information for detailed LC-MS methods.

Before injection, LC-Dox-PoP liposomes were diluted in PBS to a concentration of 1 mg Dox/mL. Double jugular catheterized 10-week-old male Fischer 344 rats (approximately weight of 250 grams) were purchased from Charles River Laboratories. Six rats were treated intravenously by left catheter with 5 mg Dox/2.4 mg PoP/5 mL/kg LC-Dox-PoP liposomes. Blood samples (250 μL) were collected in K2EDTA tubes by the right jugular catheter at 0.25, 0.5, 1, 2, 4, 8, 24, and 48 hours after injection, and spun to collect plasma and stored at −80°C. Plasma from the untreated control animals was also collected and used to prepare plasma matrix standard curve, and quality control standards. Frozen plasma samples were prepared for PoP and Dox analysis by LC-MS. See Supplementary Information for detailed LC-MS methods.

Noncompartmental pharmacokinetic parameters were determined using Phoenix WinNonlin Version 6.3 software (Pharsight Corporation): The area under the time concentration curve (AUC_inf) was calculated using the linear trapezoidal rule with extrapolation to time infinity; total clearance (CL) was calculated from dose/AUC_inf; terminal half-life (t_1/2) was calculated from 0.693/slope of the terminal elimination phase; the C_max term is the maximum concentration; volume of distribution steady-state (V_ss) was calculated from (dose/AUC_inf) × [area under the first moment curve (AUMC)/AUC_inf]; mean residence time (MRT) was calculated as (AUMC)/AUC_inf).

Female Fischer 344 rats were inoculated with orthotopic R3230 mammary adenocarcinoma tumors on both the left and right sides skin immediately adjacent to the base of the most caudal pair of nipples. R3230 cells were provided as a kind gift from Dr. Nahum Goldberg (Beth Israel Deaconess Medical Center) and were maintained for tumor studies without authentication. Once the tumors were approximately 20 mm (2 cm) in diameter, rats were injected intravenously with 2 mg/kg LC-Dox-PoP liposomes and 1 hour after injection, tumors irradiated with a 665 nm laser diode (RPMC laser, LDX-3115-665) at a fluence rate of 150 mW/cm² (total fluence: 250 J/cm²). Twenty-four hours after laser treatment rats were sacrificed, and tumors and key organs surgically excised. PoP and Dox tissue concentrations were analyzed using previously described fluorescence methods (38). To measure the drug concentration as a function of tumor depth tumors were sliced into 2-mm segments starting from the side adjacent to skin.

Female Fischer 344 rats were inoculated with 5 × 10⁶ R3230 mammary adenocarcinoma cells in a volume of 100 μL injected through the skin immediately adjacent to the base of the most caudal pair of nipples. Once the tumors were large enough (8–12 mm or 12–16 mm depending on the treatment group) the rats were injected intravenously with 2 mg/kg LC-Dox-PoP liposomes, or an equivalent dose of empty PoP liposomes, and tumors irradiated with a 665 nm laser diode (RPMC laser, LDX-3115-665) at a fluence rate of 150 mW/cm² (250 J/cm²) 1 hour after injection. Tumor size was monitored 2 to 3 times per week and tumor volumes calculated using caliper measurements and the formula: V = π/6·L·W², where V, L, and W are the volume, length, and width of the tumor, respectively. Rats were sacrificed when the volume exceeded 10 times the initial volume or ulceration was observed.

The skin response to laser treatment was assessed visually and rated using the following scale: 0, no observable reaction; 1 Eschar covers less than 50% treated surface area; 2 Eschar covers more than 50% treated surface area; 3 Eschar covers >50% of treated area and is >1 mm thick.

Kaplan–Meier curves were used to analyze tumor growth inhibition efficacy with GraphPad prism (Version 5.01). Each pair of groups were compared by the Log-rank (Mantel–Cox) test. Differences were considered significant at P < 0.05. Median survival was defined as the time at which the staircase survival curve crosses the 50% survival point.

Human blood used for in vitro studies was collected in accordance with Protocol OH99-C-N046. All animal studies in this work were approved by either the University at Buffalo Institutional Animal Care and Use Committee, or by the NCI at Fredrick Institutional Animal Care and Use Committee.

LC-Dox-PoP liposomes, were formed with the same composition ([DSPC:DSPE-PEG2000:Pyro-lipid:Chol] in a mol. ratio of [53:5:2:40]) as previously described (38). To conduct immunological characterization, liposomes were prepared using a protocol to minimize endotoxins to prevent contaminant interference of the results. This batch of liposomes was characterized for its endotoxin content (Supplementary Table S1 in the Supplementary Information), size, zeta potential, and morphology. The liposome diameter ranged from 90 to 100 nm, depending on the measurement conditions and the zeta potential showed a mild negative charge (Supplementary Table S2 in the Supplementary Information). Cryo-TEM images showed the liposomes had a somewhat elongated shape due to the presence of the prominent doxorubicin bundles contained entirely within the liposome (Fig. 1). The overall morphology is similar to the observed structure of the FDA approved liposomal doxorubicin, Doxil (14). Dox entrapment efficiency for this batch of liposomes was found to be approximately 93% as assessed using a UV-Vis micro-centrifugal assay, consistent with the reported loading efficacy of >90% for Doxil (14).

The general compatibility of LC-Dox-PoP liposomes with human blood was tested in vitro, using an in vivo relevant concentration range if the liposomes were administered intravenously at a human dose level of 0.8 mg/kg, which is analogous to a 10 mg/kg mouse dose used in previous efficacy studies (39). The analysis included analysis of the effects of LC-Dox-PoP liposomes on erythrocytes, platelets, plasma coagulation cascade and the complement system.

LC-Dox-PoP liposomes were evaluated for potential particle effects on the integrity of red blood cells (Fig. 2A). Three independent samples were prepared for each test concentration and analyzed in duplicate (%coefficient of variation, CV < 20). Triton X-100 was used as a positive control (PC) and PBS as the negative control (NC). The LC-Dox-PoP liposomes were found to interfere with the hemolysis assay at all tested concentrations and the data therefore had to be normalized to account for this interference. At 0.1 mg/mL, LC-Dox-PoP liposomes appeared slightly hemolytic (hemolysis between 2% and 5%); however, at 0.2 mg/mL less hemolysis was observed.

Potential effects of LC-Dox-PoP liposomes on the cellular component of the blood coagulation cascade was evaluated at Dox concentrations ranging from 0.0004 to 0.1 mg/mL (Fig. 2B) with collagen used as the positive control. Light transmission was used to determine the presence of platelet aggregation. An increase in light transmission indicated by an increase in area under the curve (AUC) was used to indicate platelet aggregation. LC-Dox-PoP was also tested for the ability to interfere with platelet aggregation induced by collagen alone. LC-Dox-PoP liposomes added to plasma before addition of assay's positive control did not induce platelet aggregation and did not alter collagen-induced platelet aggregation at the tested concentrations (Fig. 2C).

The effects of LC-Dox-PoP liposomes on the biochemical component of the blood coagulation cascade prothrombin time, thrombin time and activated partial thromboplastin time were tested at different concentrations (Fig. 2D). Three independent samples were prepared and analyzed in duplicate (%CV <5). Normal plasma standard (Control N) and abnormal plasma standard (Control P) were used for instrument controls. LC-Dox-PoP did not alter plasma coagulation times in vitro for the prothrombin or activated partial thromboplastin time assays. A statistically significant prolongation of thrombin time was noted at the highest test concentration. This suggests that LC-Dox-PoP may affect either fibrinogen or thrombin or both of these proteins.

Complement activation of LC-Dox-PoP was tested at Dox concentrations ranging 0.002 to 0.66 mg/mL. PBS was used as a negative control and cobra venom factor (CVF) as a positive control. Doxil was used as an additional, clinically relevant nanoparticle for comparison. For unknown reasons, LC-Dox-PoP at all tested concentrations appeared to interfere with measurement of iC3b (detected using an inhibition/enhancement control), resulting in a diminished apparent iC3b concentration by approximately 50% (Supplementary Table S3 in the Supplementary Information). Despite this observed degree of inhibition in its detection, the amount of iC3b induced by LC-Dox-PoP in plasma were elevated, and comparable, to that observed in the Doxil-treated samples at equivalent Dox concentrations (Fig. 2E). Therefore, similar to clinical and preclinical observations with Doxil (42–45), LC-Dox-PoP could be expected to induce complement activation-related pseudoallergy (CARPA) in sensitive individuals (46, 47).

LC-Dox-PoP liposomes contain two active components, PoP (bilayer loaded) and Dox (actively loaded in the aqueous core), entrapped in different locations of the liposomes. Therefore, blood partitioning of each active drug component was measured to determine the release from LC-Dox-PoP liposomes and the partitioning of each in blood and plasma. LC-Dox-PoP was incubated in blood at three concentrations, and PoP (Fig. 2F) and Dox (Fig. 2G) plasma concentration measured at specified time points. A correlation plot (Fig. 2H) was used to compare the mean PoP and Dox percent fraction in plasma (%Fp). The data for Dox and PoP are highly correlated, falling within 30% of either side of the perfect correlation 45-degree line. Most notably, in the 0.5 μg/mL PoP concentration samples, both drugs were found in both blood and plasma equally, with a %Fp of approximately 50%, whereas in the higher 2 and 10 μg/mL PoP concentration samples, both drugs were primarily found in the plasma, with a %Fp of approximately 100%. This may be due to uptake of liposomes into the mononuclear blood cell fraction at lower liposome concentrations, which then saturates at higher concentrations resulting in liposome remaining in the plasma fraction. These data suggest that the LC-Dox-PoP liposomes remains intact, with both the PoP and Dox remaining associated with the liposome at the same ratios as the initial formulation.

Pharmacokinetic studies were conducted in Fischer 344 rats. Rats were intravenously administered LC-Dox-PoP liposomes at 5 mg/kg Dox. Both the PoP and Dox pharmacokinetic profiles displayed similar monophasic decays (Fig. 3A), suggesting the absence of a rapid tissue distribution phase. When normalized to injected dose, with concentrations expressed as %ID*kg/mL, the PoP and Dox profiles were superimposable (Fig. 3B), suggesting that LC-Dox-PoP liposomes are stable in vivo and retain both the bilayer loaded PoP and aqueous-core loaded Dox drugs in the same ratio as initially formulated. The pharmacokinetic parameters, determined by non-compartmental analysis (Supplementary Table S4 in the Supplementary Information) show as expected, the dose normalized parameters, C_L, V_ss, t_1/2, and MRT were very similar for both PoP and Dox, at 1.33 ± 0.10 and 1.16 ± 0.10 mL/h/kg, 45 ± 5 and 41 ± 4 mL/kg, 25 ± 1 and 26 ± 1 hour, 32 ± 2 and 33 ± 2 hour, respectively. These values are similar to those previously reported for Doxil in rats (48), indicating LC-Dox-PoP is a long circulating formulation and further evidence of stability.

Tissue distribution studies were conducted in female Fischer 344 rats bearing orthotopic R3230 mammary adenocarcinoma tumors. The rats were injected with 2 mg/kg LC-Dox-PoP and treated with a 665 nm laser at a fluence rate of 150 mW/cm² for a total fluence of 250 J/cm². Similar distribution of PoP (Fig. 4A) and Dox (Fig. 4B) was observed in key organs with the majority of each accumulating in the spleen and liver, indicating that both drugs remain within the liposomes during circulation and experience clearance mechanisms. An enhancement of both PoP and Dox was also observed in the skin adjacent to light treated tumors.

Tumor uptake of PoP and Dox was studied as a function of tumor depth. Laser-treated and -untreated tumors from rats injected with 2 mg/kg LC-Dox-PoP were removed and cut into 2 mm segments with distance 0 representing the side to the tumor adjacent to the skin (the location that receives the greatest amount of light). Both PoP and Dox in the laser treated tumors exhibited the same trend in which drug concentration decreased as the tumor depth increased. The untreated tumors exhibited no significant change in drug concentration throughout the tumor depth. Enhancement of drug deposition was observed at tumor tissue depths to 12 mm for Dox and 10 mm for PoP, with statistically significant differences (P < 0.05, student T test) compared with non-irradiated tumors observed up to 6 mm for Dox (Fig. 4C) and 4 mm for PoP (Fig. 4D). Within laser-treated tumors, when compared with the amount of Dox found at 18-mm depth, significantly more Dox was found at all depths up to 12 mm (P < 0.05, student T test). The greater deposition range of Dox over PoP may be due to diffusion of released Dox from the liposomes. The general trend was consistent with calculated light propagation in tumor tissue, which exhibits exponential decay over tumor depth using parameters for light attenuation in breast (Supplementary Fig. S1 in the Supplementary Information; ref. 49).

Female Fischer 344 rats inoculated with R3230 mammary adenocarcinoma cells orthotopically in the mammary fat pad were used in all tumor studies. In initial studies rats were injected with 2 mg/kg LC-Dox-PoP and treated with a 665 nm laser at a fluence rate of 150 mW/cm² for a total light dose of 250 J/cm² (Fig. 5A). A second group received only LC-Dox-PoP without laser (Fig. 5B) and a third an equivalent dose of empty-PoP liposomes with laser treatment (Fig. 5C). The empty-PoP liposomes (median survival 31 days) and LC-Dox-PoP liposomes without laser treatment (median survival 30 days) groups showed no significant improvement over the untreated control (median survival 26 days; Fig. 5D), whereas the laser-treated LC-Dox-PoP (median survival >90 days) showed significant improvement in tumor suppression (P < 0.05) over all three groups. Tumors were effectively cured in three of six rats over 90 days, whereas one tumor grew slowly over the same time. Two tumors grew at a faster rate, though slower than the tumors in the other groups. Post treatment analysis found that both of these tumors had an initial starting diameter greater than 11.5 mm.

To test whether or not the tumor size was an important factor in the therapeutic outcome of LC-Dox-PoP liposome treatment two additional groups were tested. Ten rats were split into two groups, one with tumor diameters <11.5 mm, and a second with tumor diameters >11.5 mm. Both groups were injected with 2 mg/kg LC-Dox-PoP and treated with a 665 nm laser at a fluence rate of 150 mW/cm² for a total light dose of 250 J/cm². In this study, no significant differences were found between the >11.5 mm and <11.5 mm groups with 2 of 5, and 3 of 5 rats displaying complete tumor suppression, respectively (Supplementary Fig. S2 in the Supplementary Information). However, when analyzed with the previous data, a statistically significant difference (P < 0.05) was observed between the two groups with median survival of 40 days and >90 days in the >11.5 mm and <11.5 mm groups, respectively (Fig. 5E). In the combined data, with treated tumors <11.5 mm, 7/9 rats survived until 90 days with 6/9 rats having cures. In comparison, for treated tumors >11.5 mm, only 2/7 survived until 90 days, with a single cure. The difference in efficacy based on tumor size may be due to light propagation limitations in the larger tumors. The light received by deeper parts of the tumor may not be therapeutically sufficient. These results are similar to clinical observation with photodynamic therapy where the greatest efficacy is observed in tumors with diameters <1 cm (50–52).

The effects of laser treatment on skin of LC-Dox-PoP and empty-PoP laser–treated rats were monitored visually immediately following laser treatment for 30 days. Eschar formation was rated on a 1 to 3 scale (See Supplementary Fig. S3 in the Supplementary Information for a description). Immediately following laser treatment mild swelling at the treatment site was observed. The following day eschar formation could be observed visually in all animals. The eschars were most intense in the first week following treatment, gradually and completely healing over the following weeks (Fig. 6).

The purpose of these studies was to estimate general compatibility of LC-Dox-PoP with human blood at concentrations relevant to those which may occur in vivo when this material is intravenously administered at a human-relevant dose level (0.8 mg/kg), determine the pharmacokinetic profile of LC-Dox-PoP with emphasis on the stability of the liposome entrapped components, and test antitumor efficacy at human relevant doses at varying depths.

The effects on erythrocytes, platelets, plasma coagulation cascade, and the complement system were unremarkable in all assays with a few exceptions. The most notable of which is the activation of complement in vitro similar to that observed with the PEGylated liposomal doxorubicin formulation, Doxil, which is known to cause CARPA in sensitive individuals. In vitro blood partitioning and in vivo pharmacokinetics showed the liposomes to be stable retaining both the PoP and Dox at similar levels.

Laser treatment enhanced drug delivery to 1.2-cm depth in tumor tissue, and tumors with initial diameters of less than 1.2 cm had better treatment outcomes. As light diffusion through tissues is subject to rapid attenuation due to scattering and absorption, the depth of photoablation is an important parameter for planning the treatment of larger tumors using approach such as the use of multiple and interstitial optical fibers for light delivery.

A single human relevant dose of 2 mg/kg was able to potently suppress tumor growth, with antitumor synergy observed between chemo- and photocomponents. CPT using LC-Dox-PoP liposomes had a markedly improved effect compared with PDT using the same liposomes (containing the PoP photosensitizer) that lacked Dox, and to LC-Dox-PoP without the laser treatment. Despite this potent ablation efficacy, skin healing with CPT was similar to PDT.

In summary, LC-Dox-PoP liposomes appear to have similar blood behavior and immunological properties to Doxil, are stable and long circulating, and exhibit potent antitumor efficacy in a single treatment at a human relevant drug dose, lower than typically used in rats. Together, these data show that additional studies of LC-Dox-PoP are warranted, including assessing additional treatment efficacy in different tumor types, assessing impact of varying drug and light doses, and testing CPT in larger animals.

K.A. Carter is a scientist and has ownership interest (including stock, patents, etc.) in POP Biotechnologies. J.F. Lovell has ownership interest (including stock, patents, etc.) in POP Biotechnologies. No potential conflicts of interest were disclosed by the other authors.

The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. government.

Conception and design: K.A. Carter, S.T. Stern, J.F. Lovell

Development of methodology: K.A. Carter, S.T. Stern, J.F. Lovell

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): K.A. Carter, D. Luo, J. Geng

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): K.A. Carter, D. Luo, S.T. Stern, J.F. Lovell

Writing, review, and/or revision of the manuscript: K.A. Carter, S.T. Stern, J.F. Lovell

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J. Geng, J.F. Lovell

Study supervision: J.F. Lovell

This work was supported by the National Institutes of Health (R01EB017270 and DP5OD017898; to J.F. Lovell) and the National Science Foundation (1555220; to J.F. Lovell).The formulation described herein was characterized by the Nanotechnology Characterization Laboratory as part of its free Assay Cascade characterization service for cancer nanomedicines, which is supported by the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E (to S.T. Stern).