GE11chitosan Nanoparticles' Cellular Uptake Mechanism And Targeting Ability Study

Purpose:Synthesize and modify GE11-chitosan,prepare GE11-chitosan-doxrubicin nanoparticles to evaluate the quality and investigate in vitro release degree,as well as study the cellular uptake mechanism and targeting ability in vitro and in vivo.Methods:1.Synthesize GE11-chitosan through a two-step reaction from starting materials glutathione and chitosan.By amidation reaction to produce thiolated chitosan intermediate,followed by a disulfide bond formation between two thiol groups from thiolated chitosan and the end of polypeptide respectively to give GE11 modified chitosan.Product is characterized by IR,NMR spectroscopy,and the coupling rate of peptide is tested by fluorescence spectrophotometry to provide evidence for the modification of starting materials' ratio to get optimized GE11 modified chitosan.2.Synthesize GE11-chitosan-doxrubicin nanoparticles through ionic gelation method.Then characterize the modified nanoparticles by particle size,zeta potential and transmission electron microscopy(TEM)examinations.MTT assay to exam blank carrier's cytotoxicity.Dialysis bag method to test nanoparticles release in vitro,investigate the drug release profile of GE11-chitosan-doxrubicin nanoparticles in pH values of 5.5,6.5 and 7.4.HPLC is used to detect drug concentration in each group and calculate their cumulative release degree.3.Investigate factors affecting nanoparticles' cellular uptake.Treat EGFR-high expressing cell line A431 and EGFR-low expressing cell line HepG2 with GE11-chitosan doxrubicin nanoparticles(GE11-DOX-NP),chitosandoxrubicin nanoparticles(DOX-NP)and doxorubicin solution.Investigate the effect of time,concentration and energy on cellular uptake.HPLC is used to measure drug concentration in cells,BCA protein assay kit is used to exam cellular protein concentration.The cellular uptake drug concentration is presented as the amount of doxorubicin contained per microgram protein.4.Explore the nanoparticles' cellular uptake pathway.Label GE11-CS-NP nanoparticles with FITC,and screen non-toxic dose concentrations of various inhibitors.Treat A431 and HepG2 with FITC-labeled nanoparticles,the one with the intervention of inhibitors as experimental groups,and the one without inhibitors as control groups.Using flow cytometry to study each group's inhibitory effects.Confocal laser scanning microscopy(CLSM)is applied to observe the nanoparticles' intracellular distribution.5.Investigate GE11-chitosan-doxrubicin nanoparticles' in vivo targeting ability.A431 and HepG2 cells are selected,and treated with different concentration of GE11-DOX-NP,DOX-NP and doxorubicin solution respectively.Using MTT assay to exam the inhibition effect of those three preparations on tumor cells.A431 tumor multicellular spheroids model are treated with those three preparations mentioned above,another one group without preparation treatment as control group.Administrate once every two days and measure tumor sizes.Administration last for 6 days to exam those preparations' inhibitory ability towards tumor multicellular spheroids.Establish A431 tumor multicellular spheroids model,treat with active-targeting nanoparticles and passive-targeting nanoparticles.The multicellular spheroids' s uptake of nanoparticles are examed by CLSM.6.In vivo targeting inspection of GE11-chitosan-doxrubicin nanoparticles.Establish mice s180 tumor model and randomly divide them into four groups,namely: GE11-DOX-NP group,DOX-NP group,doxorubicin solution group and control saline group.All groups are administrated once every two days,continuously of 4 times for 7 days.Each dose for experimental groups contain doxrubicin 5 mg/kg.Weight mice every two days,as well as measure tumorspheres.Weight the mass of tumor at the end of experiment and calculate tumor inhibition rate for each group.Results:1.Optimize and synthesize GE11 modified chitosan.The IR and NMR spectrum examination of the product showes characteristic peaks from polypeptide,indicating the successful connecting of GE11 polypeptide to chitosan.2.Synthesize GE11-doxorubicin-chitosan nanoparticles(GE11-DOX-NP)with 28.5±1.06% encapsulation efficiency,about 400 nm particle diameter,about +34mv zeta potential,while the average size of DOX-NPs is about 390 nm particle diameter with encapsulation efficiency of(29.23±2.06%),+31mv zeta potential.GE11-DOX-NP's nanoparticle carrier is not cytotoxic at 500 ?g/mL concentration.In vitro study shows that the release degree increase as the decrease of pH value.3.Investigation of factors affecting nanoparticles' cellular uptake.The cellular uptake of nanoparticles increase as the elongation of drug administration time at first 4 h.The cellular uptake of nanoparticles also increase as the higher concentration of drug.It maintains at low lever under low temperature and low energy conditions.4.Investigation of the nanoparticles' cellular uptake pathway.After the addition of CME pathway blocker,the cellular uptake of nanoparticle of A431 is reduced by 54%.Compared with A431,the inhibitory effect of CME pathway blocker and macropinocytosis pathway blocker on Hep G2 is more significant.A431 cell's uptake of nanoparticles is meditated by CME,while the uptake pathway for HepG2 is a result of synergistic effect of clathrin-meditated pathway and micropinocytosis.CLSM demonstrates the concentration of nanoparticles in lysosome after internalization.5.Investigation of in vivo targeting ability.For A431,GE11-DOX-NPs poss a lower IC50 value compared with DOX-NPs and doxrubicin solution.With respect to the tumorsphere inhibition,the sizes of tumorspheres after 6 days treatment are 0.57,0.74,0.50 times related to beginning.CLSM demonstrates that active-targeting nanoparticles internalize into cell more and thus show a stronger fluorescence signal,which indicate a better targeting ability of GE11-DOX-NPs in in vitro experiments.6.Investigation of in vivo targeting ablility.After administration,the mice body weight decrease obviously in doxrubicin solution group,whereas in other experimental groups the mice body weight increase to deferent degrees.The tumor inhibitory effects for GE11-DOX-NP,DOX-NP and doxorubicin solution group are(50.39±20.22%),(44.15±20.05%)and(75.39±7.21%)respectively.Indicating a better targeting ability towards EGFR-high expressing tumor of GE11-DOX-NP compared with DOX-NP,as well as reduced side effects of doxorubicin for organism compared with doxorubicin solution group.Conclusion:It is demonstrated that the GE11-DOX-NP synthesized from this work shows a more complete in vitro release in low pH conditions.The cellular uptake of nanoparticles is affected by energy significantly and is positively correlated with time and concentration.Cellular uptake mechanism study demonstrates that in A431 cells the uptake is mainly mediated by CME and can be medicated by EGFR receptor.In HepG2 cells,however,the uptake of nanoparticle is a result of synergistic effect of CME and micropinocytosis.The formulation shows a targeting ability towards EGFR receptor high expression A431 cells,as well as good targeting ability towards EGFR receptor high expression S180 tumor model and thus exhibit certain tumor suppression ability.