| Background: Nasopharyngeal carcinoma(NPC)is a unique squamous cell carcinoma frequently found in head and neck,which is one of the most common and lethal cancers of the respiratory system.At present,the therapeutic effect of NPC is not satisfactory.Novel anti-nasopharyngeal carcinoma drugs with high efficiency and low toxicity have become a focused issue and trend in the treatment of NPC.Guang Chenpi,derived from the dried peel of Citrus reticulata ’Chachi’,is widely used in medicine and health food.Polymethoxyflavonoids(PMFs)are the bioactive compounds extracted from Guang Chenpi.Studies have shown that PMFs are potential anticancer agents for various cancers and have a variety of biological activities,especially in respiratory diseases.However,owing to the high degree of methoxylation on the flavonoid skeleton of PMFs,the poor aqueous solubility and low bioavailability are hindered its further application.Liposome refers to the micro-capsule formed by the encapsulation of drugs in the lipid bilayer,which can encapsulate drugs,especially hydrophobic drugs.Liposome had potential enhancing the bioavailability of drugs,prolonging drug circulation time in vivo,improving drug stability and have good safety.Bioactive components with poor solubility and dispersity could not be well absorbed in vivo,resulting in poor pharmacological activity.In this paper,therefore,PMFs extract was enriched from Guang Chenpi,and PMFs encapsulated liposomes(Lip-PMFs)were prepared.The physicochemical properties of Lip-PMFs were characterized by transmission electron microscopy(TEM),particle size,polydispersion index(PDI),zeta potential(ZP)and encapsulation efficiency(EE).In addition,combined with in vivo pharmacokinetics and in vitro pharmacokinetics experiments,the potential and efficacy of Lip-PMFs on bioavailability and anti-nasopharyngeal carcinoma activity were investigated.This paper provided a new idea for the treatment of nasopharyngeal carcinoma,which might broaden the clinical applicability of PMFs and Guang Chenpi for respiratory diseases in functional supplement.Aims:(1)The effective parts of PMFs were enriched and isolated from Guang Chenpi,which might provide valuable information for the valorization and sustainability of Guang Chenpi,as well as the applicability of PMFs in subsequent biological activity study.(2)The liposomes loaded with PMFs(Lip-PMFs)were prepared,and the physicochemical characterization and stability of Lip-PMFs were investigated.(3)The inhibitory effect of constructed Lip-PMFs on nasopharyngeal carcinoma(NPC)cells was investigated in vitro.(4)The pharmacokinetics and antiNPC efficacy of Lip-PMFs was carried out in vivo.Methods:(1)The PMFs extract from Guang Chenpi was enriched via reflux extraction method after solvent optimization.The PMFs extract was analyzed by the UPLC-Q-Exactive Orbitrap/MS method and determined by the UV-visible and HPLC quantification.(2)Lip-PMFs was prepared by thin film hydration-high pressure homogenization method.The physicochemical characteristics of liposomal structure were characterized by transmission electron microscope(TEM),particle size,polydispersity index(PDI),zeta potential(ZP)and encapsulation efficiency(EE).The release rule of PLS was studied by in vitro simulated gastrointestinal model study and the release kinetics model was established.The stability investigation for 15 days and anti-lipase activity were studied to confirm the potential application on NPC.(3)The in vitro cell experiments including cell counting kit 8(CCK8)assay,colony formation assay,hoechst assay,flow cytometry assay,scratch-wound assay and transwell assay were used to investigate the effects of Lip-PMFs on proliferation,apoptosis,migration and invasion of NPC cells.(4)To investigate the bioavailability,the pharmacokinetics of PMFs liposome were evaluated on the healthy Sprague-Dawley(SD)rats via a single intragastric administration,compared with free PMFs.To confirm the in vivo antitumor efficacy,the antitumor study of PMFs liposome were performed on the CNE-2 xenograft nude mice mode.Results:(1)The purity of PMFs have been tested to be 85.86 ± 6.24% with0.65% productivity by UV assay.Eight compounds in PMFs were confirmed by the UPLC-Q-Exactive Orbitrap/MS technique,including 6-demethoxytangeritin,eupatilin,nobiletin,chrysosplenetin B,3,5,6,7,8,3′,4′-heptamethoxyflavone,tangeretin,5-hydroxy-6,7,8,3′,4′-pentamethoxyflavone and gardenin B.Furthermore,five PMFs components were successfully separated and quantified according to the reference standards,including nobiletin,tangeretin,5-HPMF,6-demethoxytangeritin and HMF.The HPLC analysis showed that the content of the five PMFs components above in the CRP extracts was 38.85% ± 1.41%、30.35% ± 0.52%、2.68% ±0.52%、1.98% ± 0.34%和0.68% ± 0.01%,respectively.(2)The Lip-PMFs prepared is a milky white liquid in appearance and found to be spherical vesicles with a uniform size of approximate 50 nm under TEM observation.The Lip-PMFs fabricated possessed small particle size(approximately 70 nm),high EE(>87%)in physical characteristics.The in vitro release studies showed that PLS had an obvious sustainedrelease effect in simulated gastric fluids(SGF)and simulated intestinal fluid(SIF).Moreover,Lip-PMFs still exhibited superior physical stability during 15 days storage by maintaining nanometric size(particles size < 130 nm),narrow distribution(PDI <0.3),negative surface charge(approximately-1 m V)and high encapsulation efficiency(EE>87%).(3)The pharmacological experiments in vitro revealed that PMFs significantly repressed CNE-2 cell proliferation,migration,invasion,and enhanced apoptosis in a concentration-dependent mode,and Lip-PMFs and DMSOPMFs were superior to the AQ-PMFs group.(4)Compared with free PMFs,the oral absorption and relative oral bioavailability of Lip-PMFs were significantly increased.The relative oral bioavailability of nobiletin in Lip-PMFs was 1.78 times higher than that of free PMFs,and the relative oral bioavailability of tangerine in Lip-PMFs was5.73 times higher than that of free PMFs.The plasma concentration of Lip-PMFs was significantly higher than that of AQ-PMFs at the same time point after treatment.Both nobiletin and tangeretin in liposomes could last for 12 h in plasma,while the detection of nobiletin in free aqueous solution is lower than the LOQ after 10 h,and the tangeretin concentration were out of LOQ after 5 h as well.Meanwhile,the CNE-2tumor-bearing animal model in vivo was successful established,and PMFs both in loaded liposome and free aqueous solution exerted significant inhibitory effect on nasopharyngeal carcinoma.The results of H&E staining and Ki-67 immunohistochemistry assay indicated that saline-administrated nude mice had more severe necrosis in tumor tissues than those of PMFs group,and free PMFs and LipPMFs treatment clearly inhibited tumor cell proliferation in vivo.Conclusion:(1)Petroleum ether is a good solvent for PMFs enrichment with high selectivity.The extract from Guang Chenpi had a high concentration of PMFs via the optimal refluxing extraction with petroleum ether.PMFs extract were enriched with high purity and the PMFs enriched might be applied further.(2)The Lip-PMFs fabricated possessed small particle size(approximately 70 nm),high EE(>87%),sustained release and good stability in physical characteristics.(3)Hydrophobic PMFs could be effectively encapsulated in liposomes.Compared with free PMFs,Lip-PMFs had stronger inhibitory effects on NPC cell proliferation,migration and invasion,and stronger induction of cell apoptosis.(4)Lip-PMFs was successfully constructed which exhibited prolonged oral bioavailability improvement and exerted good antitumor efficacy on NPC. |
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