| Objective:Bufalin is the main effective component of Chinese traditional medicine Chan’su,which exhibits significant activity against a wide variety of malignancies. However, ithas adverse effects, such as poor water solubility, high toxicity, short half-life and narrowtherapeutic window, which limit its clinical application. In this study, we adoptedliposomes as a new drug delivery system, not only improve the difficult solubilityproperties of bufalin, but also have controlled release. We designed citrus pectin-bufalinliposomes (CPL) by coated commercially available citrus pectin (CP) on bufalinliposomes (BFL), provided a new thought of medicine for intestinal mucosa cavity. Inaddition, we preparated the other kind of immunoliposome, anti-CD40-BFL, which wasconjugated BFL with anti-CD40antibody. It used to enhance anti-tumor effects viasynergetic systemic immunity while blocking systemic toxicity.Methods:A high performance liquid chromatography（HPLC）method was developed toevaluate the concentration of bufalin in BFL. The optimized preparation condition of BFLwas evaluated by orthogonal test. The CPL was prepared by forming an ion-complexbetween pectin and cationic lipid in the liposomal formulations. And the characterization, stability, mucoadhesion, anticancer effect and pharmacokinetics were estimated.Anti-CD40-BFL was preparated through the maleimide-thiol reaction, which conjugatedanti-CD40monoclonal antibody to maleimide-functionalized liposomes, and theanti-effection of melanoma was estimated in vivo and in vitro. The levels ofapoptosis-associated protein, such as Caspase-3, Caspase-9and cytochrome c werequantified by Western blot analysis. The tumor cell apoptosis was observed by TUNELassay and hematoxylin-eosin staining (H&E staining). The serum levels of tumornecrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), interleukin-6(IL-6) andinterferon-γ (IFN-γ) were quantifed by ELISA detection kits, and the biodistribution wasinvestigated by immunofluorescence histological analysis on cryosections of tumors,spleens and lymph nodes.Results:The regression equation of concentration of bufalin is A=17865C-2116.7,(r=1),with good linearity in concentration range of0.8~40μg/mL. The specificity, precision andrecovery conformed to the requirements of the measurement.The liposomes preparated by ethanol injection method have high encapsulationefficiency. The optimized preparation condition was: ratio of drug to lipid is1∶5; ratio ofcholesterol to EPC was1∶5, and the volume of phosphate buffer saline （PBS） was10.0mL. The above compositions were dissolved in ethanol, and rapidly injected into PBS,magnetic stirred at50℃until volatilized the residual ethanol. The liposomes suspensionwas supplemented by PBS to10.0mL and extrusted with a Lipex extruder immediately.CPL had an almost spherical shape and well disperse; average particle size was296.3±77.4nm (PI=0.171); surface charge was18.1±5.33mV; encapsulation efficiencywas72.31±5.84%. The liposomes stored as the freeze-dried powder had good stability.The in vitro release of liposomes was prolonged and mucoadhesive increased three times.The cellular uptake had not been affected after coated with CP. In the meantime, theinhibition effect of CPL on SW480colon cancer cells was enhanced due to a block of cellcycle at S phase.The plasma AUC and elimination time of liposomes were greater than that of freebufalin. The pharmacokinetic parameters of bufalin, BFL, CPL were: CLz/F=0.101±0.006,0.049±0.007,0.062±0.02L/min/kg; AUC(0-t)=8.718±1.14,16.531±2.842,12.186±1.88mg/L min. The average particle size of anti-CD40-BFL was205.4±68.4nm（PI=0.062);surface charge was-15.68mV and encapsulation efficiency was73.59±3.14%. Theanti-CD40density on the liposomes was estimated to be5mg/mL by ELISA method.The IC50results of bufalin, BFL and anti-CD40-BFL on B16melanoma cells were224.12±3.14,69.91±0.93and61.73±1.21nmol/mL. After treated on a C57mice B16melanoma model, the average tumor volumes of physiological saline, bufalin solution,anti-CD40solution, BFL or anti-CD40-BFL were726.354±102.53，477.96±76.89，284.35±84.05，254.53±44.99and176.34±43.86mm3. The Integral Optical Density(IOD) of anti-CD40-BFL was strongest in caspase-3(0.92±0.026), caspase-9(1.58±0.013) and cytochrome c (1.069±0.016). The serum cytokine levels of anti-CD40-BFLwere lower than anti-CD40, while enhanced retention in tumor, spleen and lymph nodes.Conclusion:CPL exhibited an excellent stability, mucoadhesive, controlled release properties andanticancer effect. It can slow down the metabolic rate; increase the half-life andbioavailability of drugs effectively. It is concluded that CPL is a potentially promisingdrug carrier system treatment for colon cancer. The anti-CD40-BFL could enhanceanti-cancer therapeutic effcacy while reducing systemic toxicity, due to the prolongedrelease of drug. |
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