Localization of agents at a target site with a composition and an energy source

#Effective strategies for low-toxicity, multiply-administered cancer therapies are uncommonly reported1. Encapsulating doxorubicin into liposomes has increased the total tolerated dose2-4, while cardiac toxicity, mucositis and palmar-plantar erythrodysesthesia restrict the maximum lifetime dose and limit the clinical dosing schedule to 10-12 mg/kg/week at intervals of two to six weeks1, 5, 6. Unexpected synergies between the cardiotoxicities of anthracyclines and growth factors such as anti-ErbB2 antibodies have further increased the need to reduce toxicity7. Given the impact of the dose limitations on efficacy, particles with reduced toxicity would facilitate treatment, particularly in recurrence.

#To enhance stability of doxorubicin within the particle, we create a complex between the drug and a transition metal, as has previously been reported for doxorubicin with manganese (II) and irinotecan with copper (II)8-10. Creation of a copper-doxorubicin complex during the loading process is particularly attractive, since the formation of the copper (II)-doxorubicin complex has been associated with oxygen radical-mediated stimulation of DNA strand scission, the stimulation of lipid peroxidation mechanisms and resultant toxicities11-13. Formation of a drug-metal complex during loading changes the morphology of the liposomes and subsequently improves circulation lifetime and the accumulation of liposomes in tumors14, 15. Further, a 1:2 complex of copper and doxorubicin with a stability constant of 1016 forms when a neutral pH is created within liposomes16, 17. Yet, at a low pH, such as the pH encountered within a lysosome or tumor, the stable copper:doxorubicin ratio has been reported to change to 1:1 and the stability of the complex decreases17. Here, we track the liposome shell using positron emission tomography (PET) and the drug using multi-spectral fluorescence in order to assess the pharmacokinetics.

#Further, the protective coating of liposomes reduces drug diffusion within the tumor, and the impact of liposomal therapy on clinical efficacy has been modest18. We address the dual issues of toxicity and efficacy by applying our stable particle in an aggressive dosing schedule and incorporating two strategies designed to enhance efficacy: mTOR inhibition to slow proliferation19 and therapeutic ultrasound to enhance accumulation and local diffusion20, 21. The aggressive syngeneic Met-1 model is known to be sensitive to rapamycin (which is an mTOR inhibitor); however, rapamycin alone is not curative in this model22.

#These and other features, aspects, and advantages will become better understood with regard to the following description, and accompanying drawings, where:

#FIG. 1. Loading optimization and in vitro characterization of copper-doxorubicin in long-circulating liposomes (LCLs). a, Fluorescence intensity of doxorubicin encapsulated in LCLs with an increasing TEA gradient, released by Triton X-100 in the presence or absence of 10 mM EDTA. b, Effect of EDTA and heat in trans-chelation of doxorubicin from copper, as assessed by fluorescence. Copper-doxorubicin (CuDox) liposomes and Doxil were incubated in complement-preserved human serum in the presence or absence of 0.25% Triton X-100 for 1 hour. c, Fluorescence images of plasma isolated from mice 24 h post injection of either Doxil or CuDox liposomes, before and after the addition of TritonX-100 at 37° C. or TritonX-100 with 10 mM EDTA at 55° C. for 1 h. d, Dissociation of copper from CuDox complex in 0.5 mM BSA solutions as a function of pH at 37° C. Statistical analyses were performed using one-way ANOVA followed by a Tukey Post Hoc test. ***, †p<0.001.

#FIG. 2. Pharmacokinetics of copper-doxorubicin liposomes and comparison with control carriers. a, i, Cryo-EM images of LCLs encapsulating copper-doxorubicin (left panel) and copper only (right panel) under 100 mM copper/270 mM TEA intraliposomal condition. Scale bar represents 100 nm. ii, Schematic presentation of the hypothesized molecular interaction between copper and doxorubicin upon loading into liposomes. b, IC50 values of free and liposomal doxorubicin were calculated using GraphPad. c, Ex vivo hyper-spectral fluorescence intensity of the organs of mice 24 h after injection of copper-doxorubicin (CuDox) liposomes or Doxil. Mice were perfused with saline immediately prior to organ harvesting and imaging. Inset view of heart fluorescence at 48 h after injection (white indicates higher fluorescent intensity). d, In vivo hyper-spectral fluorescence intensity of the skin of mice after injection of CuDox liposomes or Doxil. Statistical significance is compared to the highest value in each time point. Inset images acquired at 24 h after injection. Statistical analyses were performed using one-way ANOVA followed by a Tukey Post Hoc test (b, d) and Student's t-test (c). *p<0.05, **p<0.01, ***p<0.001.

#Terms used in the claims and specification are defined as set forth below unless otherwise specified.

#The term “ameliorating” refers to any therapeutically beneficial result in the treatment of a disease state, e.g., a cancerous disease state, including prophylaxis, lessening in the severity or progression, remission, or cure thereof.

#The term “in situ” refers to processes that occur in a living cell growing separate from a living organism, e.g., growing in tissue culture.