Preparation of Betulinic Acid Light-responsive Drug Delivery Systems and Study on Their Antitumor Effects

Author:Liu Yan Ping

Supervisor:gao da wei


Degree Year:2017





Cancer has become the main disease that endangers human health,however,there is still no obvious breakthrough in treatment of cancer.The nanoscale drug delivery systems(NDDSs)combining nanotechnology and drug research have brought new hope for the treatment of cancer.Owning to excellent tissue penetration capacity and noninvasive nature of near-infrared(NIR)-light,NIR responsive NDDSs have become promising and vital in the current research of nanodrug.Herein,based on the natural plant compounds of betulinic acid(BA),NIR-light responsive noble metal nanomaterials coated drug-carrying liposomes have been rationally designed and synthesized.Moreover,the antitumor effects of the NDDSs were further studied,and their possible synthetic mechanism and antitumor mechanism were deeply explored.Thereinto,PEGylated BA liposomes were firstly prepared by ethanol injection method,and the optimal preparation process was obtained by orthogonal experiment.Noble metal nanomaterials coated BA-liposomes were formed on the surface of liposomes functionalized with glutathione(GSH)by seed-mediated growth,which could achieve efficient NIR light controlled drug release,and synergistic effect of chemo-photothermal treatment.First,the preparation formulation of PEGylated BA liposomes was optimized by orthogonal experiment.The encapsulating efficiency of PEGylated BA liposomes was93.5%.Transmission electron microscopy(TEM)image showed that PEGylated BA liposomes were spherical shapes with a smooth surface,and the mean diameter was 142nm as determined by laser particle size analyzer.As shown in atomic force microscopy(AFM)image,PEGylated BA liposomes exhibited good rigidity,which enhanced their stability and blood circulation time.Moreover,the BA released from PEGylated BA liposomes was up to 78%within 72 h,which showed a slow drug release over a prolonged period of time.The antitumor effects of all formulations against HepG2 cells and HeLa cells were evaluated by MTT assay.The results proved that both BA liposomal formulations showed preferable antitumor effects.Meanwhile,PEGylated BA liposomes can significantly inhibit tumor cells growth over a long period of time,showing good long circulation and sustained drug release effect.In addition,PEGylated BA liposomes exhibited a good antitumor effect through intratumoral injection in U14 tumor-bearing mice.Second,based on surface plasmon resonance of metal nanoparticles,three types of NIR-light responsive BA-liposomes were successfully designed and prepared,gold nanoshells coated BA liposomes(AuNS-BA-Lips),branched gold nanoshells coated BA liposomes(BGNs-BA-Lips)and Au-Pd nanoflowers coated BA liposomes(BA-Lips@Pd@Au NFs).The nanocarriers were modified by GSH for promoting the interaction between metal nanoparticles and thiol/amino groups,which provided nucleation sites for the growth of Au or Pd nanoparticles;the preparation of nanocarriers through surfactant-free synthesis could guarantee biological safety.The obtained nanocarriers exhibited high entrapment efficiency,ideal size distribution and excellent stability.The energy-dispersive X-ray spectroscopy(EDS)and fourier transform infrared spectroscopy(FTIR)spectrum revealed the presence of Au on the liposomal surface.The absorption peak of three types of noble metal nanocarriers had significant red shift entering into the NIR region,which also exhibited obvious light response potential.In particular,after the NIR laser irradiation(2 W/cm~2)for 10 min,the temperature changes(?T)of AuNS-BA-Lips,BGNs-BA-Lips and BA-Lips@Pd@Au NFs solution were up to18°C,50.2°C and 47.3°C,respectively.Thereinto,the photothermal conversion efficiency of BA-Lips@Pd@Au NFs was as high as 64.6%.Meanwhile,the hyperthermia derived from these nanocarriers under NIR-light irradiation can effectively control on-demand drug release at tumor site,resulting in enhancing the effect of chemotherapy and reducing the toxic side effects on normal tissues.Finally,Coumarin-6(Cou6),which was used as a model drug with a green fluorescence signal,was encapsulated into the liposomes to monitor the cellular uptake process.The results showed that the laser irradiation obviously promoted the cellular uptake of drug-loading liposomes,resulting in even higher intercellular drug accumulation.The cell viabilities of these nanocarriers against tumor cells were detected by MTT assay.Upon NIR laser irradiation,the tumor cells treated with AuNS-BA-Lips,BGNs-BA-Lips and Au@Pd@BA-Lips NFs showed the cell viabilities of 9.8%,11.9%and 6.3%,respectively,which achieved synergistic effect of chemo-photothermal therapy and efficient treatment of tumor.Besides,U14 bearing mice treated with nanocarriers plus laser irradiation exhibited an enhanced antitumor efficiency.Obviously,NIR laser irradiation not only induced PTT for cancer therapy but also could accelerate the BA release from nanocarriers leading to an enhanced chemotherapy.In this study,NIR-light responsive nanocarriers encapsulated BA were prepared and achieved the synergistic effect of chemo-photothermal treatment.Moreover,the nanocarriers effectively controlled the release of drugs in response to NIR irradiation,which resulted in decreased the dose of drugs,thus achieving the accurate treatment of tumor.Therefore,the drug delivery system provided a promising prospect for future development of antitumor drugs.