Self-assembly Of Bovine Serum Albumin And Polysacchride And Its Application On Anti-tumor Drug Delivery

Recently, nanocarriers are extensively investigated as drug delivery systems in the pharmaceutical research. These carriers can protect the drug from degradation, enhance drug absorption at targeting tissue, and promote drug concentration in the cells. Furthermore, by modulating the surface properties and composition, desired drug release profile and biodistribution can be achieved. Biomacromolecules such as albumin and polysaccharides with good biocompatibility and biodegradability are extensively used in pharmaceutical fields, due to their natural source, low cost, non-toxic, as well as binding abilities with drugs. Therefore, developing self-assembly technique to prepare superior biomacromolecular drug carriers is very important. Paclitaxel/human serum albumin (HSA) nanoparticles (Abraxane(?)) has been proved by FDA in 2005, however, the high cost and limited sources of HSA is a drawback of this paclitaxel/HSA formulation. Thus, it is very practical to produce functional carriers using bovine serum albumin (BSA) that is abundant and has a low price.In this thesis, we used Maillard reaction and heat treatment to form nanocarriers by preparing BSA-dextran conjugates and inducing BSA gelation. We focus on preparing nanoparticles and nanoemulsion based on biomacromolecular self-assembly for delivering anti-cancer drugs. The carrier preparation and drug encapsulation need no organic solvents, surfactants, nor any other toxic reagents. The drug loaded nanocarriers are biocompatible, biodegradable, stable against long term storage in physiological conditions, as well as having a long circulating nature. In addition, non-covalent interactions between BSA and drug are introduced into our work to obtain high drug encapsulation efficiency and sustained release. This thesis contains the following four parts.The first part is preparation of nanoparticles by self-assembly of BSA-dextran conjugate and its application on doxorubicin (DOX) delivery. This part follows previous work in our lab. Firstly, Maillard reaction was used to prepare BSA-dextran conjugate; and secondly, nanoparticles were produced by heat-gelation process. We managed to prepare nanoparticles with a higher BSA concentration to increase interactions between drug and carrier, thus to enhance encapsulation efficiency. On the other hand, DOX has a pH dependent nature with pKa 8.2. We developed a simple and effective method to load DOX:DOX and nanoparticles were mixed at certain pH to form electrostatic complex, then higher pH value was achieved to enhance their hydrophobic interaction. In this way, more than 90% of the DOX was encapsulated. In vitro release study showed a sustained and relatively faster release of DOX from the nanoparticles at pH 5.0 compared with the release at pH 7.4. In vitro cytotoxicity and in vivo tumor bearing mice treatment indicate that the blank nanoparticles have no toxicity while DOX loaded nanoparticles are less effective than free drug because of the sustained release of DOX possibly.In the second part we prepared pH sensitive BSA-dextran/chitosan/DOX nanoparticles. Based on foregoing section, in this part, we introduced positively charged chitosan to realize pH sensitive targeting delivery. At first, chitosan and BSA-dextran was mixed at pH 5.6 to form electrostatic complex. Then, after a heat treatment, BSA was denatured; stable dual-polysaccharide shell and protein core nanoparticles formed. The pKa of chitosan is about 6.2-7.0, so chitosan will aggregate at physiological condition. In that condition, chitosan will collapse on the nanoparticle surface to avoid macrophage uptake. At tumor site where the pH is slightly lower, chitosan is protonated. The positive polymer chains can interact with negatively charged cell membrane, therefore benefit for cancer cell uptake while reduce damages of normal tissue. Additionally, dextran can not only enhance nanoparticles circulation time in blood, but also prevent chitosan from aggregation at pH 7.4. Similarly, DOX was encapsulated in the nanoparticles, and the encapsulation efficiency is about 72%. Blank nanoparticles are non-toxic both in vitro and in vivo. Compare to DOX loaded BSA-dextran nanoparticles, BSA-dextran/chitosan/DOX nanoparticles have significant improvement on prolonging the life of murine ascites hepatoma H22 tumor bearing mice.In the third part, we prepared BSA-dextran paclitaxel emulsions by ultrasound sonication. In the previous systems we found that the anti-tumor effectof DOX loaded nanoparticles in vivo is not so efficiency as free DOX. The nanoparticles are solid spheres, so that it is not very easy to be degraded that may result in the sustained release profile of DOX from the nanoparticles. In this part, we developed paclitaxel loaded BSA-dextran nanoemulsions by ultrasound sonication. As an emulsifier, BSA forms a thin interfacial film on the droplet, which is much easier to be degraded. Dextran on the droplet surface can protect emulsion from aggregation against pH changes, heat treatment, ionic strength change, as well as long term storage. Blank emulsion shows no toxicity while drug loaded emulsion has the same anti-tumor effect as free paclitaxel solution in vitro. It means that this interfacial protein film is easily degraded, thus the drug can be released fast.In the fourth part, we further improved BSA-dextran paclitaxel emulsion preparation by high pressure homogenization, the most efficient and high-throughput comminution system to produce nanoemulsions. Emulsion produced by sonication is not stable; the droplets inosculate forming cannulations that are not suitable for parenteral delivery. After emulsification via high pressure homogenization, the emulsions were immediately heated at 90 ℃ for 1 h to induce permanent crosslinking of BSA that can produce irreversible interfacial films and also can decrease possible epitopes on the BSA surface. Dextrans with different molecular weight were conjugated on BSA surface. We found that the anti-tumor efficiency of drug loaded emulsion with short dextran chains is better than that with long dextran chains and commercial product both in vitro and in vivo. The reasons may be that the emulsion with the short dextran chains has smaller droplets and larger surface charges. We further proved that the BSA-dextran nanoemulsion is suitable for other hydrophobic drug loading.In the last part, folic acid was conjugated to dextran so that the drug loaded emulsion would have a targeting property. It is comfirmed that the targeting paclitaxel loaded emulsion has a better anti-tumor activity in vitro.