We report for the first time that the copper-doxorubicin complex, when loaded within a liposome, has substantial advantages in reducing systemic toxicity. Further, we exploit this reduced toxicity to create an efficacious therapy through repeated administration over a multi-week regimen and augment the therapy with rapamycin and/or ultrasound.
Copper-doxorubicin complex quenches fluorescence and demonstrates enhanced stability at neutral pH
Doxorubicin loading increased with the ratio of copper to doxorubicin, reaching a maximum at a 1:2 molar ratio with 100% loading efficiency (Supplementary Methods and Supplementary Figure S1
). Loading also increased linearly with the intra-liposomal copper concentration, up to 0.6 mg doxorubicin per mg lipid (Supplementary Figure S1c
). In all following studies, particles were loaded using 100 mM copper-gluconate and 270 mM TEA, achieving a final ratio of 0.2 mg-doxorubicin per mg-lipid in order to facilitate a comparison with Doxil.
Although doxorubicin loading increased in proportion to the TEA gradient, intra-liposomal doxorubicin fluorescence was quenched (). For copper-doxorubicin liposomes, full restoration of doxorubicin fluorescence was observed only with the combination of Triton X-100, trans-chelation with EDTA and incubation at 55°C for 1 hour, indicating that doxorubicin was associated with copper (). Even in the presence of serum albumin, EDTA and elevated temperature were required to achieve trans-chelation and restore fluorescence (). Alternatively, for liposomes that did not contain copper, the fluorescence intensity of released doxorubicin was not affected by the addition of EDTA (). Long term in vitro stability of doxorubicin encapsulation was also assessed; free doxorubicin was not detected during a 30-day in vitro incubation of either copper-doxorubicin liposomes or Doxil at 37°C.
Loading optimization and in vitro characterization of copper-doxorubicin in long-circulating liposomes (LCLs)
Fluorescence of both Doxil and copper-doxorubicin liposomes in plasma remained quenched after 24 hours of circulation (). Upon addition of Triton X-100, fluorescence of doxorubicin was fully restored for Doxil, whereas, a combination of Triton X-100 and EDTA at elevated temperatures was required to restore the fluorescence of doxorubicin for copper-doxorubicin liposomes as shown previously in the in vitro stability assay (). Thus, the copper-doxorubicin complex circulates stably within liposomes and remains associated in plasma after release from liposomes. Trans-chelation kinetic of copper from copper-doxorubicin complex in the presence of albumin, one of the major trans-chelating component of blood, was strongly dependent on pH and exhibited a significantly lower dissociation rate (≤ 20%) at pH values of 7 and higher over a period of 48h, p<0.001 (). In contrast, copper trans-chelation increased as the pH decreased below 7 with a rapid dissociation of copper (≥75%) observed at pH≤ 5 ().
Cryo-electron microscopy verified the presence of precipitation as a dotted and diffuse structure of the copper- doxorubicin complex uniformly distributed inside the liposomes ( left), which was substantially different than the needle-like precipitate formed by ammonium sulfate loading of doxorubicin31, 32
or the subtle precipitate of copper alone ( right). Given the molar ratio of 1:2, the hypothesized structure for the liposomal copper: doxorubicin complex is schematically depicted ().
Pharmacokinetics of copper-doxorubicin liposomes and comparison with control vehicles
In vitro cytotoxicity of copper-doxorubicin liposomes is enhanced
When evaluated with the Met-1 cell line, the cytotoxicity of the copper-doxorubicin liposomes (IC50 of 0.33±0.16 μM, n=12) was greater than that of Doxil (IC50 of 1.72±0.85 μM, n=6), p
<0.001, whereas free doxorubicin exhibited the lowest IC50 value (0.02±0.01 μM, n=15) (, Supplementary Figure S1d
). Empty liposomes and copper liposomes (each tested with an equal lipid concentration) had no effect on cell viability. Delivery of copper and doxorubicin in two separate liposomal formulations (copper liposomes and Doxil) did not change the IC50 value of Doxil.
Copper-doxorubicin liposome stability is associated with reduced systemic toxicity
In vivo stability was assessed by serial imaging of fluorescent doxorubicin, PET labeling of the liposomal shell, and ICP-MS measurements of copper accumulation. At 24 hours after the injection of copper-doxorubicin liposomes, the concentration of doxorubicin and copper in plasma were 43.3±3.8% ID/cc (n=6) and 39.1±6% ID/cc (n=6) of the initial dose, indicating a stable association of doxorubicin with copper in circulation. The concentration of doxorubicin in Doxil in the blood pool was higher 24 hours after injection with ~50% of the initial dose continuing to circulate (p<0.01).
Following organ perfusion with saline and excision at 24 hours after injection, fluorescence was similar for Doxil and copper-doxorubicin liposomes in organs associated with drug clearance (spleen, liver, kidney). However, in the heart, fluorescence resulting from copper-doxorubicin liposomes was one-fifth that resulting from Doxil administration, p
<0.01 (). Skin fluorescence increased to a greater extent following Doxil administration than with Cu-doxorubicin liposomes or free doxorubicin, increasing with time for Doxil and decreasing with time for free drug and Cu-doxorubicin liposomes, p
<0.05 (). A unique spectrum, associated with intact Doxil liposomes, was detected within the skin by multi-spectral optical imaging (Supplementary Figure S2a–b
), indicating that the increased fluorescence resulted at least partially from intact liposomes.
Doxorubicin-associated toxicity was then assessed with an aggressive four-week dose schedule of 6 mg/kg of doxorubicin liposomes twice per week, which is equivalent to 66.7 mg/m2 per week. Animals receiving Doxil at this relatively high dose demonstrated fur loss and a skin rash as early as 7–10 days post-treatment; such effects were not observed with the equivalent dose copper-doxorubicin liposomal therapy throughout a 28-day course of treatment (). Doxil-treated animals showed a significant increase in heart weight (p<0.001), circulating albumin (p<0.05) and total protein (p<0.001) and a significant weight loss (p<0.001), as compared to control mice and mice treated with copper-doxorubicin liposomes (). Leucopenia was observed with each liposomal doxorubicin group; however, the effect was greater with Doxil. Red and white blood cell counts following the administration of a control diluent, copper-doxorubicin liposomes or Doxil were 7.6±0.7 (n=5), 4.5±0.3 (n=7), 3.1±0.1 M/μL (n=5) for red blood cells and 5.7±2.1, 3.6±1.1, 1.9±0.6 K/μL for white blood cells, respectively.
Toxicity of copper-doxorubicin (CuDox) liposomes and Doxil assessed over 28 day administration of 6 mg/kg (33.4 mg/m2) twice per week (total of 266.7 mg/m2)
Efficacy of copper-doxorubicin liposomes demonstrated in multi-component regime
In initial studies with copper doxorubicin liposomes or Doxil with 3 mg/kg (~17 mg/m2) biweekly dosing, growth of the Met-1 tumor continued with only a small extension of survival (data not shown). Thus, the dose was increased to 6 mg/kg (~33 mg/m2) and the treatments were incorporated into a multi-treatment regime with rapamycin, which is also known to be efficacious in the Met-1 line. Also, we recognize that the penetration of liposomal particles within solid tumors is problematic and, thus we added ultrasound to improve the accumulation and diffusion of the particles and drug within the tumor. In our study, ultrasound was applied immediately after injection, with a goal of increasing tumor accumulation (). With the mechanical index (MI) of 0.9 applied here, changes in vascular permeability are not produced when short (1–2 cycle) imaging pulses are applied; however, in this study, long pulses were employed and controlled such that an increase in tumor temperature to 42°C was achieved and maintained for two minutes. Immediately after insonation, the tumor blood vessel diameter increased to 25.5 ± 25.3 μm (n=4, , upper left panel) as compared to 11.6 ± 6.9 μm for untreated tumors (n=4, , upper right panel), p<0.001. In the absence of drug, insonifying one of two bilateral tumors did not change the tumor growth rate (, lower panels).
Enhanced accumulation of copper-doxorubicin liposomes using therapeutic ultrasound (US)
As assessed with PET and optical imaging, the concentration of liposomes within the tumors peaks between 18 and 20 hours after injection (, Supplementary Figure S2c
). Copper (assessed by ICP-MS) accumulated in tumors similarly over time, reaching a maximum of 10% ID/g at 18–20h post-injection. The concentration of liposomes within the insonified tumor increased by approximately 2-fold compared with the contralateral tumor, as quantified using PET () and optical imaging (Supplementary Figure S2c
). Further, the concentration of copper also increased by approximately two-fold in the insonified tumor, reaching a maximum of 15% ID/g which translates to 20 μg-doxorubicin/g-tumor, p
<0.05. Doxorubicin fluorescence was also evident within tumors, increasing as a function of time following the injection of both Doxil and copper-doxorubicin formulations.
A ten day course of therapy was next applied to compare the efficacy of free doxorubicin, copper-doxorubicin liposomes (with and without ultrasound), ultrasound only, and systemic injection of a saline control therapy (). The treatment with free doxorubicin showed a therapeutic effect when compared with the saline control (p<0.001). Tumor growth suppression was greater for copper-doxorubicin liposomes than free doxorubicin (p<0.001).
In vivo treatment efficiency including ultrasound (US), free doxorubicin (Free Dox), rapamycin and copper-doxorubicin liposomes (CuDox-lipo) in Met-1 tumor mice
The therapeutic effect of copper-doxorubicin liposomes was then tested in an aggressive multi-dose 28-day treatment, in combination with rapamycin and ultrasound and appropriate single therapy control groups. Tumor longitudinal diameter ranged from 4–6 mm prior to treatment and was similar in all groups. Animals treated with diluent (control group) or rapamycin survived only 18 and 24 days post-treatment, respectively, whereas all animals receiving sole or combination therapy with liposomal doxorubicin or Doxil survived the entire 28-day course of treatment (). All therapies suppressed the tumor growth as compared to control after 18 days of treatment (p<0.001). The in vivo efficacy of Doxil was similar to the efficacy of copper-doxorubicin liposomes in the two sub-groups that were evaluated, which were liposomal doxorubicin alone (not shown) and combined therapy with liposomal doxorubicin and rapamycin (). Suppression of tumor growth was observed in all copper-doxorubicin liposome-treated animals (, p<0.001); however, average tumor growth was ~700% with copper-doxorubicin liposomes and was reduced to <200%, 85% or −11% with the addition of rapamycin (p<0.05), ultrasound (p<0.001) or rapamycin plus ultrasound (p < 0.01), respectively ().
Histological measurements confirm efficacy with liposomal doxorubicin and enhancement with rapamycin and ultrasound
Histological sections obtained from tumors confirmed the efficacy of therapy; however viable tumor assessed by histology was smaller than the diameter measured by ultrasound at the end of the treatment due to the presence of cysts (). As a result of copper-doxorubicin liposomes and rapamycin or ultrasound therapy, a cystic, epithelial phenotype with reduced proliferation (as compared with control tumors) was observed (–). Mammary lymph nodes within the sections provided a control for proliferation. With the combination of copper-doxorubicin liposomes and therapeutic ultrasound, viable tumor was not detected in a subset of tumors (). Total tumor nuclei were reduced in all treatments with copper-doxorubicin liposomes as compared to diluent injection, p<0.01 (). As compared to control tumors, CD31 and Ki67 were reduced and apoptosis increased with copper-doxorubicin liposomes alone or in combination with rapamycin and therapeutic ultrasound, p<0.05 (). Contrast ultrasound imaging confirmed the reduced vascularity observed in treated as compared with control tumors (, ), where functional vasculature was not detected in the copper-doxorubicin liposomes plus rapamycin plus therapeutic ultrasound-treated tumors. Gold indicates the presence of contrast agent within the control tumor in the middle panels and yellow indicates fast flow within the control tumors in the right upper panel ().
Histology and immunohistochemistry of tumors treated with sole or combined therapies using copper-doxorubicin liposomes