Studies On Heparin-modified Reduced Graphene Oxide Nanoparticles

Reduced graphene oxide(rGO),developed rapidly in the past ten years,is a kind of graphene oxide with some characteristics,such as,low oxygen content,high drug loading,photothermal effect and wonderful modification,which has been applied on many aspects of biological medicinefield,including drug delivery system,gene transfection,DNA manipulation,tumor imaging,photothermal therapy,bone tissue engineering.The thesis consists three parts.In the first part,hemolysis assay served as evaluation method,and hemolytical index(HI)used as evaluation index in order to find suitable physicochemical feature of GO derivatives with high biocompatibility.In the second part,two preparations were studied.Based on curcumin(Cur)as a model drug,unfractionated heparin(UFH)as hydrophilic macromolecule,and folic acid as the active targeting modification,GO-2 as a starting material,and two Cur nanoparticles(Cur-rGO-UFH and Cur-rGO-UFH-FA)were synthesized and then evaluated by release test,cell assay,pharmacokinetics,biodistribution and immunohistochemistry.The molecular simulations of rGO were detailed in the third part.In order to reasonably explain mechanism that(1)the unmodified rGO was thermodynamically unstable,and modification of hydrophilic macromolecule was needed.(2)the drug-loaded process of rGO.The interaction between rGO and protein was simulated as well as,in which Lysozyme(Lyso)or Myoglobin(Myo)as model protein,rGO as typical nanomaterial,and GRO as reference.These simulations indicated that nanoparticles was biocompatible after i.v.administration.For example,rGO was biocompatible with Lyso-rich liver,spleen,lung and kidney,and with Myo-rich heart.The main research methods and results were as follows:In the first chapter,nine kind of 25 GO derivatives were synthesized.In the second chapter,a novel method to calculate HI was established and incubation condition of hemolysis assay was optimized.The calculation method,a typical three-point absorbance difference method,was 540-576-600 method in which absorption values at 540nm,576nm and 600nm were used.In order to illustrate the new method with high accuracy,two HP methods including 540 method as a typical one-point absorbance method and 540-655 method as the representative two-point absorbance difference method were compared.Additionally,576-600 method as the representativetwo-point absorbance method,a new HI method,is established.Finally,the noval 540-576-600 method had features with high accuracy and low errors after compare of three examples,while other methods had larger false-positive errors because the HP results were even more than 100%sometimes when used the two methods.The optimal condition was chosen,such as 5%glucose solution as medium,rabbit-originated,1%final concentration,fresh and healthy RBCs as ideal RBCs.3000rpm as centrifugal speed,3-6h as incubation time,and 37? as incubation temperature.We quantitatively explained the interaction between GO and RBCs in two tests of RBCs concentration and centrifugal speed.This study has not been reported in the literature.In the third chapter,25 GO derivatives were evaluated by hemolytic assay.The physicochemical features were found of GO derivatives with high biocompatibility including the decrease of particle size,modification of hydrophilic macromolecule,reduction from GO to rGO,and diminishment of surface charge.In the fourth chapter,two UFH-modified materials were prepared and characterized.The result of photothermal effect showed that photothermal effect was dependent of concentration and time.After irradiation at 808nm for 5min,the low limit of temperature of 50? required for photothermal treatment was achieved for two materials with 10 ?g·mL-1.rGO-UFH-FA was non-toxic for MCF-7 and A549 cells.When exposed to irradiation at 808 nm,toxicity of two materials was in a dose-dependent manner and enhanced for rGO-UFH-FA on MCF-7 cells,which exhibited rGO-UFH-FA entered MCF-7 cells by active targeting mediated by FR and probable EPR effect,while rGO-UFH probably internalized into MCF-7 and A549 cells only through EPR effect.In the fifth chapter,two Cur nanoparticles were evaluated in vitro.The method to calculate drug loading and entrapment efficiency was set up according to UV-vis characteristics of Cur and GO.The preparation process of Cur-rGO-UFH-FA was optimized by CCD-RSM.Release test in vitro showed that temperature effect was larger,while pH effect was less,which indicated that the adsorption of Cur on surface of rGO-UFH-FA was mainly affected by the ?-? stacking interaction,and slightly by hydrogen bonding.The assay of MCF-7 and A549 cell shows that irradiation is significant for cell inhibition.The cytotoxicity of Cur significantly was enhanced after irradiation due to photothermal effect of rGO when was exposed to 808 nm for MCF-7 and A549 cell,which was explained by a synergistic effect of toxicity of Cur and photothermal effect of rGO.However,the cytotoxicity of Cur substantially was enhanced for Cur-rGO-UFH-FA after irradiation.These phenomena show Cur-rGO-UFH-FA entered MCF-7 cells by FR and probable EPR effect,while Cur-rGO-UFH internalized into MCF-7 and A549 cells only through probable EPR effect.Cell uptake tests showed that uptake is dependent on rGO concentration and exposure time for MCF-7 and A549 cells.In the sixth chapter,two Cur nanoparticles were evaluated in vivo,free Cur has short half-life after administration into rats,while half-life of Cur-rGO-UFH or Cur-rGO-UFH-FA nanoparticles was greatly prolonged.There existed liver-targeting effect for the two preparations in mice biodistribution tests,and lung targeting effect for Cur-rGO-UFH-FA.The tissues examined by immunohistochemistry and showed that some mild damage of tissues were gradually eliminated and tissues restored after 48h.In the seventh chapter,self-aggregation and drug-loading process of rGO were simulated by Gromacs.The results showed self-aggregation of rGO occurred in manner of point-line-plane adsorption.Low concentration of rGO solution was thermodynamically unstable.To decrease particle size of GO and to modify with hydrophilic macromolecule were vital to enhance stability.The adsorption process of Cur to GO or graphene(GRO)was simulated as well.The results showed that GO could adsorb Cur at 5.01ns,and that GRO couldn't adsorb Cur during 20ns,which meant adsorption process was not only concerned with the ?-? stacking interaction,but also related to Van der Waals' force,electrostatic force,hydrogen bonding and collision from water.From these facts,long time stirring was beneficial for enhancement of drug loading via increase of molecular collision probability between molecules.In the eighth chapter,the interaction between GRO or rGO and Lyso or Myo was simulated.Even in the condition of adsorption between rGO and protein,the effect of acidic and alkaline amino was uncertain,and the binding process was complicated and changed with the environment.Although rGO could adsorb protein,but its binding energy decreased significantly when compared with GRO,indicating that the rGO is biocompatible as a drug carrier due to low binding energy.