The Study Of Preparation Of Bufalin Multilevel Targeted Nanoparticles With RGD Modification And Its Anti-cancer Mechanism On Colorectal Cancer

ObjectiveThis study is aimed to establish the preparation and quality standard for bufalin-loadedmPEG-PLGA-PLL-cRGD nanoparticles and to investigate their pharmacologicalcharacters such as drug release, in vivo and in vitro drug targeting distribution, cytostaticeffects on human colon cancer cells and therapeutic effects in colon cancer-bearing nudemice.MethodPreparation of bufalin-mPEG-PLGA-PLL-cRGD NPs:Methoxy polyethylene glycols (mPEG), polylactic-co-glycolic acid (PLGA), poly-L-lysine(PLL) were used as carrier material, then co-modified with cyclic arginine-glycine-asparticacid (cRGD) and loaded with bufalin.(i.e. bufalin-loaded mPEG-PLGA-PLL-cRGDnanoparticles, bufalin-mPEG-PLGA-PLL-cRGD NPs).The bufalin-mPEG-PLGA-PLL-cRGD NPs were prepared via single oil-in-water (O/W)emulsion/solvent evaporation method. Seven factors affecting nanoparticle formationwere observed, the preparation process of BNP was further optimized by orthogonalexperiment.Study of the relevant physicochemical properties: The surface morphology of NPs wasobserved via scanning electron microscopy. Particle size distribution of NPs was assessedby optical microscopy. Entrapment rate and drug loading rate were tested. The law of invitro drug release was also observed.Drug targeting: Distribution of nanoparticles taken in by cells was observed by confocallaser scanning microscope, with encapsulated RB as fluorescent probe. Bio-distribution ofdrug-loaded nanoparticles in SW620cancer-bearing mice was observed via in vivotargeting fluorescence imaging. Anti-tumor experiment in vitro:CCK-8assay was used to assess the cytotoxicity and cytostatic effect of BNP in thehuman colon cancer cell line (SW620SMMMC7721).In vivo study of the therapeutic efficacy in mice: The mice were divided into6groupsaccording to the substance administered (Route: intra-vena caudalis): Normal saline group(NS,13.5mL/kg), mPEG-PLGA-PLL NP group (mPEG-PLGA-PLL NPs,24.5mg/kg),bufalin solution group (bufalin,1mg/kg), mPEG-PLGA-PLL-cRGD NP group(mPEG-PLGA-PLL-cRGD NPs24.5mg/kg), bufalin-mPEG-PLGA-PLL NP group(bufalin-mPEG-PLGA-PLL,1mg/kg) and bufalin-mPEG-PLGA-PLL-cRGD NP group(bufalin-mPEG-PLGA-PLL-cRGD,1mg/kg). The size and necrosis of the tumor, as wellas the survival time of mice in each group were documented and compared.ResultsCharacterization of bufalin-mPEG-PLGA-PLL-cRGD NPs: ThemPEG-PLGA-PLL-cRGD NPs were spherical and well-dispersed particles, with adiameter of164±84nm. Zeta potential was2.77mV. Entrapment rate and drug-loadingrate of the bufalin-mPEG-PLGA-PLL-cRGD NPs was81.7±0.89%and3.92±0.16%. Thepreparation method was proved duplicable.Drug release in vitro: Over time, bufalin in NPs has a much slower releasing ratecomparing with unbound drug. At24h, the cumulative releasing rate of free drug was90%,while in NPs it was as low as50%. The90%cumulative releasing rate in NPs was reachedat192h.Distribution of drug: The fluorescence intensity of HUVECs cultivated with Rb-bufalin-mPEG-PLGA-PLL NPs was significantly lower than that in Rb-bufalin-mPEG-PLGA-PLL-cRGD NPs group. At four hours after i.v administration, thefluorescence intensity in tumor site of cancer-bearing nude mice was slightly stronger inRb-bufalin-mPEG-PLGA-PLL-cRGD group. At24hours, the intensity has graduallystrengthened and was much higher than Rb-bufalin-mPEG-PLGA-PLL group. Tumor targeting: This finding shows that the cellular uptake ofRb-mPEG-PLGA-PLL-cRGD NPs by HUVEC is more effective than that ofRb-mPEG-PLGA-PLL NPs. The possible explanation was thatRb-mPEG-PLGA-PLL-cRGD NPs could be internalized into the cells via their ability topreferentially bind with αvβ3integrins, which were overexpressed on HUVECs.The present experimental results further showed that, compared to theRb-mPEG-PLGA-PLL NPs, Rb-mPEG-PLGA-PLL-cRGD NPs have ideal αvβ3integrin-targeting properties and better therapeutic potential.In vitro anti-tumor effect of NPs: The survival rate of SW620cell line was calculatedusing the CCK-8assay. The survival rate in mPEG-PLGA-PLL andmPEG-PLGA-PLL-cRGD NPs remained in the level of90-96%despite increasedconcentrations. Comparing with unbound bufalin, bufalin-loaded NPs showed a greatercytotoxicity and stronger growth inhibition to SW260, which was proportional to dosageand action time.cRGD modified mPEG-PLGA-PLL NPs could target tumor cells and inhibit angiogenesisand proliferation of tumor blood vessels. Additionally, bufalin could directly kill theSW620colon cancer cells. The growth inhibitory effect was concentration-andtime-dependent.Therapeutic efficacy in colon cancer-bearing mice: After administration, we measuredthe tumor volume at predetermined intervals and calculated the inhibitory rate. Comparedto the NS treatment, mPEG-PLGA-PLL NP treatment had no effect on the tumor in mice(P>0.05). When same dose of drugs were administered to the mice, the inhibition oftumor was more remarkable in the bufalin-mPEG-PLGA-PLL NP group andbufalin-mPEG-PLGA-PLL-cRGD NP group than that in the unbounded bufalin solutiongroup. The tumor size was significantly smaller in the bufalin-mPEG-PLGA-PLL-cRGDNP group comparing with other groups.Conclusions1. In this study, preparative method and quality standard ofBufalin-mPEG-PLGA-PLL-cRGD NPs were established using emulsion-solventevaporation method. 2. The bufalin-mPEG-PLGA-PLL-cRGD NPs are characterized by slow release of the drugwith a cumulative release rate of (31.77±2.08)%at384h.3. The significantly higher uptake rate of Rb-bufalin-mPEG-PLGA-PLL-cRGD and agreater targeting efficacy showed in targeting fluorescence imaging indicated a satisfyingtumor targeting property of cRGD moiety.4. Bufalin-unloaded mPEG-PLGA-PLL and mPEG-PLGA-PLL-cRGD NPs showedextremely low cytotoxicity to cells. Comparing with unbounded bufalin, cytotoxicity wasgreatly increased in bufalin-loaded group, indicating a great enhancement of thetherapeutic effect in the form of bufalin-loaded NPs.5. The anti-cancer effect is greatly enhanced when given inbufalin-mPEG-PLGA-PLL-cRGD NPs form, which is dose-effect dependent. Thismulti-targeted drug was proved with a good therapeutic effect on colon cancer-bearingnude mice and could greatly prolong the survival of the mice.