**3.1 Preparation of CPX1 and CPX2**

The anthracycline class of drugs, including doxorubicin, has fluorescence properties that become quenched after intercalation into DNA. Fluorescence spectroscopy was used to examine the binding of doxorubicin to DNA and the drug encapsulation efficiency of CPX1 and CPX2. A series of weight ratio of DOX/polyGC was tested. DOX could be completely combined by polyGC at the weight ratio of 2:1. Cationic gelatin could combine with DOX-polyGC intercalation at a weight ratio of polyGC/C-gelatin = 1:2. In CPX1, weight ratio of DOX/polyGC/C-gelatin = 2:1:2. Its zeta potential was +18 mV. PEG-Hisalginate was added to the solution of CPX1 at the C-gelatin/PEG-His-alginate ratio of 1:1

CPX1 and CPX2 solutions and free DOX solution were separately injected into liver tumor bearing mice via tail vain at a dose of 20 mg/kg body weight. Different organs were harvested from the experimental mice bearing implanted tumors. Doxorubicin in the organ was extracted according a reported method and quantified by the examination of its

Healthy mice were given CPX1, CPX2 or free DOX to examine their toxicity. CPX1, CPX2 and DOX were intravenously injected into animals at the doses of 20 mg DOX/kg body weight and 30 mg DOX/kg body weight. The changes of their body weights were examined every day for one week. The mortality of the animals was also calculated. For histological examination, different tissues were harvested at the 4th day after the drugs were given and sectioned and analyzed by hematoxylin and eosin staining. Animal plasma activity of alanine transaminase (ALT) and creatine kinase (CK) were determined for the evaluation of

Animals bearing implanted liver tumors were intravenously given CPX1, CPX2 and free DOX at a dose of 10 mg DOX/kg body weight every two days from day 5 after the allograft model establishment. All tumors were separated, weighed and sectioned for pathological analyze on day 21. For the calculation of survives, testing animals bearing tumors were divided into 4 group, 10 animals each. CPX1 and CPX2 at a dose of 10 mg DOX/kg body weight were given intravenously every two days with free doxorubicin and saline as

Results are expressed as the mean ± standard error of the mean (S.E.M). The differences between groups were analyzed by Mann-Whitney U test and, if appropriate, by Kruskal-Wallis ANOVA test. Survival curves were analyzed by the Kaplan-Meyer log-rank test. Changes in body weight were compared by use of the Wilcoxon matched-pair signed-rank

The anthracycline class of drugs, including doxorubicin, has fluorescence properties that become quenched after intercalation into DNA. Fluorescence spectroscopy was used to examine the binding of doxorubicin to DNA and the drug encapsulation efficiency of CPX1 and CPX2. A series of weight ratio of DOX/polyGC was tested. DOX could be completely combined by polyGC at the weight ratio of 2:1. Cationic gelatin could combine with DOX-polyGC intercalation at a weight ratio of polyGC/C-gelatin = 1:2. In CPX1, weight ratio of DOX/polyGC/C-gelatin = 2:1:2. Its zeta potential was +18 mV. PEG-Hisalginate was added to the solution of CPX1 at the C-gelatin/PEG-His-alginate ratio of 1:1

**2.5 Bio-distribution of doxorubicin** 

**2.6 Toxicology investigation** 

the functions of livers and hearts.

**2.7 Anti-cancer activity** 

**2.8 Statistical analysis** 

test. A value of P <0.05 was considered significant.

**3.1 Preparation of CPX1 and CPX2** 

controls.

**3. Results** 

fluorescence intensity at 590 nm (Bigotte, 1985).

to form CPX2. The zeta potential of CPX2 was -1.2 mV. Figure 2A show the fluorescence intensity of doxorubicin, DOX-polyGC, CPX1 and CPX2. Figure 2B show the particle sizes of CPX2.

Fig. 2. Preparation of CPX1 and CPX2. A) Fluorescent intensity of free DOX, DOX-polyGC intercalation (weight ratio : DOX:polyGC = 2:1), CPX1 (weight ratio: C-gelatin : polyGC : DOX = 2:1:2) and CPX2 (weight ratio: PEG-His-alginate : C-gelatin : polyGC : DOX = 2:2:1:2) at 590nm; B) Size distribution of CPX2 in saline.
