**3.2 DOX release from the complexes**

The most important property of the complex is to response to PH and gelatinase. PHA-Cgelatin-polyAT-Hoechst complex was used in vivo instead of CPX2 to test the drug release property of CPX2 in response to tumor acidic microenvironments. Results shown in Figure 3A demonstrated that the three kinds of fluorescence were completely overlapped in liver sections. In liver tumor sections, blue "drug" could be seen in a separated area from the green and red fluorescence.

The ability of gelatinase and Dnase I (DA1) to free DOX from CPX1 and CPX2 in PBS buffers with different pH values was tested. The results in Figure 3B demonstrated that DOX in CPX1 could be released under both PH values of 7.2 and 6.5 while CPX2 could only be digested when PH was 6.5. CPX1 and CPX2 were incubated with tumor tissue homogenate supernatant (THS) and then were examined for the released DOX. Serum and normal liver tissue homogenate supernatant (LHS) was used as the control. The results were shown in Figure 3C and Figure 3D. DOX in CPX1 or CPX2 was not released in serum while THS could efficiently release free DOX from CPX1 and CPX2. LHS could only release free DOX from CPX1. CPX2 remained stable in LHS. Figure 3E demonstrated that more than 60% DOX was released from both CPX1 and CPX2 within 2 hours when incubated with THS. When incubated with LHS, CPX1 was also destructed and released DOX very quickly while CPX2 remained stable and kept the DOX from release for more than 20 hours.

Fig. 3. Release of DOX from CPX1 and CPX2. A) Tumor-specific drug release of CPX2. 0.2 mg Hoechst 33258 contained in multi-fluorescence-labeled CPX2 (polyGC was substituted by polyAT) was injected into the tumor or animal liver after the animals were anesthetized 1 hour before the tissues were separated, sectioned and analyzed by microscope. B) Fluorescent intensity of CPX1 and CPX2 digested by 5 U/ml gelatinase (GA) and 0.5 U/ml Dnase I (DA1) in PBS buffer with PH value of 7.2 or 6.5 for 2 hours at 37 C at 590nm; C) Fluorescent intensity of CPX1 digested by plasma, THS or LHS for 2 hours at 37 C at 590nm; D) Fluorescent intensity of CPX2 by plasma, THS or LHS for 2 hours at 37 C at 590nm; E) DOX release rates from the complexes in THS or LHS. In all experiments, the concentration of DOX contained in CPX1 and CPX2 in the solution is 0.2mg/ml.

### **3.3 Bio-distribution of doxorubicin**

Free DOX, CPX1 and CPX2 were administrated into mice via tail vein at a dose of 20 mg DOX per kg body weight. Different tissues and organs were harvested 48 hours later. The

Fig. 3. Release of DOX from CPX1 and CPX2. A) Tumor-specific drug release of CPX2. 0.2 mg Hoechst 33258 contained in multi-fluorescence-labeled CPX2 (polyGC was

anesthetized 1 hour before the tissues were separated, sectioned and analyzed by

the concentration of DOX contained in CPX1 and CPX2 in the solution is 0.2mg/ml.

**3.3 Bio-distribution of doxorubicin** 

substituted by polyAT) was injected into the tumor or animal liver after the animals were

microscope. B) Fluorescent intensity of CPX1 and CPX2 digested by 5 U/ml gelatinase (GA) and 0.5 U/ml Dnase I (DA1) in PBS buffer with PH value of 7.2 or 6.5 for 2 hours at 37 C at 590nm; C) Fluorescent intensity of CPX1 digested by plasma, THS or LHS for 2 hours at 37 C at 590nm; D) Fluorescent intensity of CPX2 by plasma, THS or LHS for 2 hours at 37 C at 590nm; E) DOX release rates from the complexes in THS or LHS. In all experiments,

Free DOX, CPX1 and CPX2 were administrated into mice via tail vein at a dose of 20 mg DOX per kg body weight. Different tissues and organs were harvested 48 hours later. The concentration of DOX in these tissues was measured. Data in Figure 4A indicated that CPX2 efficiently enhanced the accumulation of DOX in tumor tissue and reduced the deposition of the drug in heart and the other organs. As a control, CPX1 not only enhanced the drug concentration in tumor but also in liver. The concentration of DOX delivered by CPX1 was 2 times higher than free DOX. Figure 4B and Figure 4C described the variation of DOX concentrations in tumor and liver after CPX1, CPX2 or free DOX was administrated. CPX2 dramatically increased the deposition of DOX in tumor and decreased the liver concentration of the drug. CPX1 could increase the concentration of DOX in both tumor and liver.

Fig. 4. A) Bio-distribution of free DOX or delivered by CPX1 and CPX2 given i.v. at a dose of 20 mg/kg body weight; B) DOX concentrations in tumor after free DOX, CPX1 or CPX2 were injected i.v. at the dose of 20 mg/kg body weight; C) DOX concentrations in liver after free DOX, CPX1 or CPX2 were injected i.v. at the dose of 20 mg/kg body weight.

### **3.4 Toxicology investigation**

Daily injection of doxorubicin exhibited serious toxicity which resulted in loss of weight, cardiac toxicity and death. CPX1 and CPX2 remarkably reduced the toxicity of doxorubicin, which caused less weight loss than free DOX (Figure 5A). 30 and 20 mg/kg body weight of DOX caused 100% and 60% death in 5 days. The same dose of DOX in the form of CPX1 caused 50% and 20% death in up to 8 days while 20 mg/kg body weight DOX in the form of CPX2 cause no death and the extreme high dose of 30 mg/kg body weight only caused 10% death (Figure 5B).

Fig. 5. Toxicology analysis. A) Body weight changes of animals received i.v. injections of saline, free DOX, CPX1 or CPX2 at a dose of 20 mg/kg body weight; B) Mortality caused by free DOX, CPX1 or CPX2 at different doses; C) H & E stained heart, liver and lung tissue sections of animal treated by 20 mg/kg body weight free DOX or delivered by CPX1 and CPX2.

Treatment with free doxorubicin caused massive myocardial degeneration (Figure 5C), which resulted in the increase of the plasma level of CK and LDH (Table 1) and heart failure which induced significant pulmonary congestion (Figure 5C). This toxicity was greatly reduced when DOX was delivered by CPX1 and CPX2 (Figure 5C and Table 1). CPX1 showed more hepatotoxicity than free doxorubicin at the same dose, which resulted in the necrosis of hepatocytes (Figure 5C) and increase in plasma levels of ALT (Table 1) while CPX2 did not cause this toxicity. CPX2 also reduced the nephrotoxicity of doxorubicin which was demonstrated by the decrease of BUN level (Table 1).


Table 1. Serological analysis of mice treated with free DOX, CPX1 and CPX2.
