To encapsulate both drugs, they first prepared liposomes before active loading of CPT-11 by a pH gradient method, with the protonated CPT-11 retained in liposomes after complex formation with FOA. Mice treated with coloaded liposomes had increased survival compared to the combination with separate liposomes. However, the therapeutic efficacy was lower than with liposomes loaded with FOA only, probably because the FOA content had to be lowered for CPT-11 coloading, further demonstrating the difficulty of reproducing a synergy with liposomes relative to free drugs.
When tested in phase I trial with acute leukemia patients, the 5:1 ratio was maintained Inhibitors,research,lifescience,medical in plasma for 24h, and CPX-351 induced complete responses in 9 out of 43 patients . The same group developed irinotecan: floxuridine liposomes (CPX-1,
1:1 molar ratio). In phase I clinical trial they demonstrated that the drug ratio was maintained in plasma up to 12h after Inhibitors,research,lifescience,medical infusion and showed positive clinical responses in patients with colorectal cancer . It is noteworthy that the high therapeutic efficacy of liposomes encapsulating two anticancer drugs was always correlated with the maintenance of their synergistic molar ratio in plasma, in animal models  as well Inhibitors,research,lifescience,medical as in cancer patients [24, 25, 264] indicating optimization of drug loading and liposomal stability as primary concerns for effective combination therapy. Ko et al. codelivered the proapoptotic peptide D-(KLAKKLAK)2 and the Bcl-2 antisense oligodeoxynucleotide G3139 . The authors took the advantage of the electrostatic properties of Inhibitors,research,lifescience,medical these therapeutic molecules to codeliver them by formation of a negatively charged complex between the peptide and G3139 before mixing with positively charged liposomes. Inhibitors,research,lifescience,medical Intratumoral injection of coloaded liposomes led to an enhanced tumor growth suppression. Finally, the combined liposomal delivery of magnetic fluid hyperthermia and photodynamic therapy using magnetic fluid and zinc phthalocyanine as the photosensitizer demonstrated superior
toxicity in vitro of combined light and magnetic stimuli over their separate applications suggesting a new treatment modality for enhanced tumor therapy . 5. Tumor Stimuli-Triggered and PEG Release The addition of PEG to the liposome surface was reported to find more decrease the interaction of the ligand-targeted liposomes with their ligand, either when small molecules were conjugated to the liposome surface  or with antibody-targeted liposomes [48, 118] by steric hindrance of the surface ligand. Moreover, PEGylation decreases targeted liposomal accumulation and drug release . Finally, for gene delivery, PEGylation has been shown to decrease intracellular trafficking of DNA .