**4. Discussion**

Doxorubicin, as a chemotherapy drug, has been commonly used in the treatment of hepatocellular carcinoma, but is thwarted by a key obstacle-cumulative cardiotoxicity. To improve its anti-cancer activity and reduce its toxicity, a number of doxorubicin formations such as pegylated liposomal doxorubicin, doxorubicin-eluting-bead has been performed in liver cancer clinical trials recently. Among them, anthracycline-DNA complexes as therapeutic reagents have been used for the treatment of liver cancer for a long history, due its easy-fabrication, no change in the structure of the drug molecules and its anti-cancer activity (Trouet, 1979a). Clinical trials were also carried out to evaluate their enhancement in the performances in effectiveness and toxicology compared to free anthracycline (Trouet, 1979b). However, except a few of *in vitro* reports about successfully reducing the toxicity of the anthracycline by using the complexes, there is no convincing results demonstrating the advantages of using the complexes instead of free anthracycline (Dorr et al., 1991). One of the most important reasons underlies the ineffectiveness of the intercalation between DNA and anthracycline is that there is abundant Dnase in circulation and body fluids which digests un-protected DNA molecular with extremely high efficiency (Paik & Kim, 1970). The anthracycline-DNA complexes were destroyed soon after their injection into the body. The anthracycline in complexes was released very quickly and did not change their pharmaceutical properties any more.

Protection of DNA from destroying by Dnase is necessary for gene delivery technology (Tranchant et al., 2004). Non-viral gene delivery practices take the advantage of using cationic polymers to combine DNA, which prevents the exposure of DNA molecules to Dnases (Wagner, 2004). DNA complexed with cationic polymers could totally resist the degradation of Dnase I or serum Dnases in reported *in vivo* and *in vitro* tests (Paleos et al., 2009). These researches instruct us cationic polymers could be used to combine the intercalation of anthracycline-DNA to form an anthracycline-DNA-cationic polymer ternary complex. Such a complex can avoid the unexpected early release of anthracycline into serum by protecting DNA from the digestion of Dnases. In our experimets, cationic gelatin was used to combine DNA-DOX intercalation. This simple process generated regular nanocomplexes that could be observed. It is difficult to release DOX in short time using Dnase I alone to digest the complex, which suggested that DNA is well protected by cationic gelatin. Additionally, in the present study, synthesized GC oligonucleotide was used to substitute the natural DNA fragments, which increased the drug-loading capacity of DNA. Moreover, the combination between DOX and polyGC is more stable than DOX-natural DNA intercalation fragments which may release DOX when combined by some kind of cationic polymer. No DOX release was observed in the study when DOX-polyGC was combined by cationic gelatin.

A lot of cationic polymers are used as gene carriers, such as polyether imide (PEI), polylysine (PLL), cationic polysaccharides and other polycation materials (Bodley et al., 1989). To ensure the sufficient release of anthracycline in tumor sites, the cationic polymer used to form the anthracycline-DNA-cationic polymer ternary complex must be biodegradable, especially in tumor. Cationic gelatin is a derivate of gelatin by conjugating ethylenediamine, spermine or other compounds with multiple amino groups to the gelatin molecules. Cationic gelatin is a proved effective DNA carrier for gene delivery *in vitro* and *in vivo*. It can protect DNA and even RNA from the degradation of Dnases (Kushibiki et al., 2003). More importantly, cationic gelatin can be easily digested by gelatinase (Ganea et al, 2007). Gelatinase belong to the matrix metallopeptidase (MMP) family. It is composed of MMP-2 and MMP-9 (Eliyahu, 2005). Recent investigations suggest that gelatinase is involved in the genesis, development and metastasis of most kinds of tumors, which is highly expressed in solid tumors (Deryugina & Quigley, 2006). That means cationic gelatin could be effectively digested in tumor. To address this, tumor homogenate supernatant was tested for its ability to digest cationic gelatin in CPX1 and release DOX. Compared to plasma, THS exhibited much higher efficiency of releasing the DOX from CPX1. This implies CPX1 can be destroyed and DOX can be released specifically in tumor sites.

However, only gelatinase-response is not sufficient to enable the tumor-specific drug release ability of the delivery system because gelatinase is relatively high-expressed in liver (Emonard & Grimaud, 1990). In our experiments, CPX1 notablely enhanced the liver accumulation of DOX and caused serious hepatotoxicity which weakened the efficacy of the drug. To avoid this, a pegylated PH-responsive alginate was introduced in the system. After the surface of CPX1 was covered with this polymer (Formed CPX2), it could resist the digestion of gelatinase under physiological conditions. The environment intra tumor is acid which could trigger a change of the charge on the polymer from negative to a positive one, which resulted in the dissociation of the polymer form CPX1 and the digestion of CPX1 by gelatinase in the tumor. Because of the pegylation, CPX2 acquired stronger enhanced permeability and retention (EPR) effect that increased the distribution of the drug in tumor than CPX1 and greatly reduced the liver accumulation.

Cardiotoxicity is the main drawback of anthracycline, which restraints more effective application of this drug to liver cancer patients (Ferreira et al., 2008). Myocardial degeneration is the most commonly encountered side-effects of anthracycline (Combs & Acosta, 1990). It can cause serious heart failure and even death (Ferrans, 1978). In our study, CPX2 efficiently reduced the accumulation of DOX in heart, which greatly alleviated the cardiotoxicity of DOX. It completely rescued animals from death caused by high doses of DOX. Additionally, controlled-release and tumor site specifically release of DOX also eased the acute toxicity-induced loss of body weight.
