| Betulinic acid (BA) is a plant-derived pentacyclic lupine-type triterpene, and has been recognized to possess potent pharmacological properties. Since 1995, cancer cells derived from many tumor types were also discovered to be sensitive to BA. Although BA has demonstrated an anticancer activity, it still has some drawbacks such as low water solubility and relatively short half-life, which has limited its drug efficacy. The dissertation work is directly aimed to address the problems and further to promote sustained, controlled, and targeted delivery of BA. The properties of a series of well-defined BA-prodrugs and nanoparticles are studied in detail. Moreover, the potential application of BA delivery system is evaluated as well. Synthesizing BA prodrugs by multiarm-polyethylene glycol and carboxymethylcellulose linkers, and preparing the nanoparticles by introducing target molecules, we evaluated their physico-chemical and pharmaceutical property. Moreover, cellulose-graft-poly(L-lactic acid), bovine serum albumin-poly(L-lactic acid), and ginsenoside Rbl nanoparticles have been developed and evaluated.The main contents of this work are as follows:1. Synthesizing BA prodrugs by multiarm-polyethylene glycol linkers, and preparing the nanoparticles by introducing target molecules (folate), we evaluated their physico-chemical and pharmaceutical property. The effect of molecular weight and the number of functional groups to pharmaceutical property of the prodrugs has been investigated. For better targeting, folate-conjugated eight-arm-polyethylene glycol-betulinic acid (F-8arm-PEG-BA) was synthesized by introducing folate. Then another anticancer drug hydroxycamptothecine (HCPT) was encapsulated into the self-assembled nanoparticles from the conjugate by a simple nanoprecipitation method.8arm-PEG40K-BA conjugate shows antitumor activity that is more potent than those other BA prodrugs. Compared to the prodrugs, the drug-loaded nanoparticles significantly improved the cellular cytotoxicity and exhibited the obviously synergistic effect by the co-delivery of two different anticancer drugs. Pharmacokinetics study revealed the nanoparticles could prolong the circulation of BA and HCPT in the blood. In conclusion, F-8arm-PEG-BA/HCPT NPs have great potential for targeted chemotherapy for cancer.2. On the basis of aforementioned investigations, we evaluated the pharmaceutical property of the novel targeted nanoparticle platform based on carboxymethylcellulose co-delivery. A conjugate based on carboxymethylcellulose (CMC) was first synthesized by introducing hydrophilic molecules (PEG), target molecules (folate), and drug molecules (betulinic acid) to CMC. Then another anticancer drug HCPT was encapsulated into the nanoparticles from the conjugate using a simple nanoprecipitation method. The obtained nanoparticles possessed appropriate size (～180 nm), high drug loading efficiency (～23 wt%BA,～21.15wt% HCPT), slowly drug release rate, higher blood circulation half-time of free BA (6.4-fold) and HCPT (6.0-fold), and high synergetic activity of BA and HCPT toward cancer cells. Furthermore, the targeted nanoparticles showed rapid cellular uptake by tumor cells. The antitumor effect of the nanoparticles in a mouse tumor xenograft model exhibited much better tumor inhibition efficacy and fewer side effects than that of BA and HCPT, strongly supporting their application as efficient carriers for anticancer therapy.3. Developing novel BA delivery material cellulose-graft-poly(L-lactic acid) (CE-g-PLLA), we evaluated the anticancer effect of the blank and drug-loaded nanoparticles. As far as the high-value utilization of cellulose and biocompatibility of biomedical polymer material is concerned, poly(L-lactic acid) (PLLA) has been choice to graft onto the backbones of cellulose via ring-opening polymerization. The degree of polymerization of poly(L-lactic acid) (DPPLLA) can be well-controlled, and different DPPLLA of the polymer showed different degradability and particle size. Then, BA-loaded cellulose-graft-poly(L-lactic acid) nanoparticles (CE-g-PLLA/BA NPs) were fabricated. Both drug-free and BA-loaded nanoparticles were spherical in shape with a uniform size of 100-170 nm. The release of BA from CE-g-PLLA/BA NPs was relatively slow. In vitro cytotoxicity studies with A549 and LLC cell lines suggested that CE-g-PLLA/BA NPs was slight superior to BA in antitumor activity and CE-g-PLLA NPs were non-toxic. The antitumor effect of the CE-g-PLLA/BA NPs in a mouse tumor xenograft model exhibited much better tumor inhibition efficacy and fewer side effects, strongly supporting their use as efficient carriers for anti-cancer therapy.4. On the basis of aforementioned investigations, we evaluated the anticancer effect of the BA delivery material bovine serum albumin-poly(L-lactic acid) polymer conjugate. Here, we developed a new self-assembled bovine serum albumin-poly(L-lactic acid) nanoparticle platform for anticancer drug delivery made of bovine serum albumin-poly(L-lactic acid) polymer conjugate. Depending on the ratio of bovine serum albumin (BSA) to poly(L-lactic acid) (PLLA), these conjugates self-assemble into uniform spherical nanoparticles with different size. In vitro cytotoxicity studies suggested that the BSA-PLLA/BA NPs were significantly superior to the model drug BA in antitumor activity and the BSA-PLLA NPs were non-toxic. Compared to free BA, the BSA-PLLA/BA NPs provided significantly higher the blood circulation half-time of free BA (5.02-fold). The antitumor effect of the BSA-PLLA/BA NPs in a mouse tumor xenograft model showed much better tumor inhibition efficacy and fewer side effects than that of free BA. It may be attributed to the preferential tumor accumulation and increase the solubility of the drug in water, strongly supporting their use as high-performance carriers for anti-cancer therapy.5. Using ginsenoside Rbl self-assemble with anticancer drugs directly to form stable nanoparticles, we successfully explored their pharmaceutical properties. The use of non-toxic materials in pharmaceutical formulations could minimize the adverse effects of pharmaceutical residues entering the body and environment. Here, we firstly find that ginsenoside Rbl can self-assemble with anticancer drugs to form stable nanoparticles, which have greater anticancer effects in vitro and in vivo than the free drugs. The obtained nanoparticles possessed an appropriate size (～100 nm), better tumor selectivity, high drug loading capacity (～35 wt%BA,～32 wt% dihydroartemisinin, and ～21 wt% HCPT), and a higher blood circulation half-time. Furthermore, the antitumor effect of the nanoparticles in a mouse tumor xenograft model exhibited a much better tumor inhibition efficacy and fewer side effects than those of the free drugs, strongly supporting their application as a novel efficient nanocarrier for anticancer therapy. Moreover, ginsenoside nanoparticles with a simple, green preparation method and easy large-scale production are promising for the delivery of various insoluble drugs.In summary, a series of well-defined BA-prodrugs and nanoparticles were fabricated. And these drug carriers showed good promise for the potential BA therapy. Actually, this strategy provided a facile method to prepare well-defined drug carriers with various drugs and a versatile method for the design of nanostructures. These self-assembled nanostructures may find attractive applications in the fields of drug and gene delivery. |
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