Effect Of Transferrin Modified Nanoscaled Graphene Oxide Loading Doxorubicin For Treating Glioma: In Vitro And In Vivo Evaluations

Gliomas are the most common primary malignant tumors in the central nervoussystem. Although invasive surgery, radiotherapy, and chemotherapy, prognosis of patientssuffered glioma has increased little during the past decades. It’s impossible to thoroughlyeradicate by surgery for infiltration growth. Furthermore, blood brain barrier (BBB)restricts the penetration of agents given systemically to glioma. Such factors contribute toits poor prognosis. With the development of biomaterial science, the nano-medicine whichis a subject of conjugation biomaterial science and medical science has attracted muchattention of scientists. Graphene, a two-dimensional nanomaterial firstly reported in2004,has been widely investigated for its applications in many fields. Graphene oxide (GO) is animportant derivative of graphene. Its good biocompatibility and lack of obvious toxicitymake it a promising material for drug carrier. Transferrin (Tf) is a monomeric glycoproteinwith its receptor (TfR) overexpressed on the brain capillary endothelium and surface ofglioma cell. While, a low level of TfR is observed on normal tissues. Tf-mediatedtranscytosis has been demonstrated to transport across BBB.Many researches also revealedthat Tf coupled nanocarrier has potential for glioma targeting therapy. In this paper, adramatic nanovector was designed for glioma targeting drug delivery. Tf was conjugatedPEGylated nanoscaled GO covalently (Tf-PEG-nGO). Doxorubicin (Dox), a widely usedchemotherapy agent, without passing through BBB, which limited its clinical utility onglioma, was loaded onto Tf-PEG-nGO via simple π-stacking with high efficiency. Itstherapeutic efficiency to glioma was evaluated both in vitro and in vivo.PartⅠ Tf-PEG-nGO-Dox conjugation and detectionAim Transferrin (Tf), an iron-transporting serum glycoprotein that binds to receptorsoverexpressed at the surface of glioma cells, was chosen as the ligand to develop Tf-conjugated PEGylated nanoscaled graphene oxide (GO) for glioma targeting delivery ofanticancer drug doxorubicin (Tf-PEG-nGO-Dox).Tf-PEG-nGO-Dox was detected for sizeby atomic force microscope, Dox loading ratio was measured by ultravioletspectrophotometer.Methods GO was prepared by modified Hummers method and converted tocarboxylated GO, followed by PEGylation. Tf-PEG-nGO was developed via a two-stepcoupling of Tf to PEG-GO by EDC chemistry. In the first step, Tf was dissolved at1mg/mL in MES buffer by sonicating for5min. Then, NHS aqueous solution was added tothe Tf solution and reacted at room temperature. EDC was added quickly with to thereaction solution and then continually stirred for1h. The suspension was filtered andrinsed thoroughly with distilled water to remove excess agent. In the second step, PEG-GO(1mg/mL) was added to the above suspension and mixed for4h. The final product waswashed thoroughly to remove unbound Tf. Loading of Dox onto the GO sheets was carriedout by adding Dox (dissolved in DMSO) to the Tf-PEG-GO or PEG-GO aqueoussuspension with stirring for12h. The final product was filtered against distilled water toremove the free drug. The loading ratio of Dox was estimated by UV-vis spectra, aftersubtracting the absorbance from GO. UV-vis spectra were collected with ShimadzuUV-2550spectrophotometer. Morphology of Tf-PEG-GO and PEG-GO was characterizedby AFM.Results AFM was employed to characterize morphology of the PEG-nGO andTf-PEG-nGO. AFM images and cross-section analyse along with the line of GO andTf-PEG-GO. Both GO and Tf-PEG-nGO have lateral dimensions of100–400nm, andthickness of1-2nm for GO, which is characteristic of a single or two layer sheets.Afterconjugation with PEG and transferrin, the thickness of GO is increased to4-5nm,confirming the formation of Tf-PEG-GO. The UV-vis spectra were measured to confirmthat loading of Dox on the Tf-PEG-GO. The characteristic absorption peak of Dox (~490nm) appeared in the sample of Tf-PEG-nGO-Dox, indicating successful conjugation of Tf.The amount of loaded Dox was estimated by absorbance at490nm, after subtracting theabsorbance of GO. In addition, the concentration of Tf-PEG-nGO was evaluated by theabsorbance at600nm and800nm, respectively. The concentration of Tf-PEG-nGO andthat of the loaded Dox were estimated to be240μg/mL and277μg/mL, respectively.Therefore, the loading ratio (the weight ratio of loaded drug to carriers) was determined to be115.4%. This value is close to that of PEG-GO (127%), indicating that Tf-PEG-GOcould load Dox efficiently. Dox releasing test show when pH decreased to4.0, Doxreleasing accelerated, while pH at7.4Dox releasing ratio was at low level.Conclusion A dramatic nanoscaled Tf modified peglated nGO loading Dox wasdesigned,with diameter100to400nm and high Dox loading ratio up to115.4%.PartⅡ Effect of Tf-PEG-nGO-Dox for treating glioma: in vitroevaluationsAim On the basis of congjugation of Tf-PEG-nGO-Dox, in this part, severalexperiments were performed to evaluate its cytotoxicity and glioma-targeting characters.The experiments included hemolysis toxicity, cytotoxicity to C6glioma cell, Competitiontest by Tf, intracellular delivery efficiency by HPLC and effect to apoptosis via flowcytometry.Methods C6glioma cells were seeded into96-well culture plates and grown for24h.Free Dox, GO-Dox and Tf-GO-Dox dissolved in100μL culture medium were filled into96-well culture plates, respectively. The final concentration of Dox was in the range of0–80μg/mL. The cytotoxicity was measured24h later by the MTT assay, and theabsorbance was read on a microplate reader at540nm. The survival percentages werecalculated, and concentration–viability curves were made and IC50values were calculated.Similarly, cytotoxicity of PEG-GO and Tf-PEG-GO on C6glioma cells was also evaluated.The final concentration of GO was in the range of0–800μg/mL. In competition assay, C6glioma cells were seeded into96-well culture plates as above. Then, the cells wereincubated with excessive amount of Tf (50μg)in each well in advance, afterward, Dox,GO-Dox and Tf-GO-Dox were added into the culture plates, respectively. MTT assay wasperformed for cytotoxicity as above. Concentration–viability curves were made and IC50values were calculated. To evaluated C6glioma cellular uptake of Dox quantitatively, C6cells were seeded onto24-well plates,24hours later, Free Dox, GO-Dox and Tf-GO-Doxsolution were added into the culture medium to make final Dox concentration at3μg/mL ineach well. The plates were incubated for1,2,4,8, or12h at37oC. At each time point,cells were collected, and dealed with Triton X-100. Total cell protein content was assessedvia BCA protein assay kit. Intracellular Dox content was evaluated by HPLC assay. Theuptake index (UI) was expressed as Dox (μg)/cellular protein (mg). UI of different formulations was compared.Results Cytotoxicity of PEG-nGO and Tf-PEG-nGO was evaluated via MTT assay,no significant toxicity was found. Even at the highest concentration (0.8mg/mL), thecellular viability was always above80%. There were no significant differences in cellularviability between the PEG-nGO and Tf-PEG-nGO (p>0.05). MTT assay on C6cellsviability demonstrated that the cytotoxicity of Tf-PEG-nGO-Dox, PEG-nGO-Dox and Doxwere concentration depended. Viability-concentration curves showed that the IC50of thesethree formulations was13.61,63.01and28.43μg/mL respectively, manifesting a4.63intensification of cell inhibition of Tf-GO-Dox compared to GO-Dox(p<0.01). In thecompetition assay, after adding Tf in advance, the cytotoxicity of Tf-GO-Dox decreasedmarkedly, nevertheless little effect to PEG-nGO-Dox or Dox. The IC50values ofTf-PEG-nGO-Dox with Tf in advance and only Tf-PEG-nGO-Dox to C6glioma cells were40.16and13.61μg/mL, respectively. The targeting effect of different formulations wasquantitatively measured by the uptake of C6glioma cells via HPLC assay. The intracellularDox level of all groups increased with time. Quantitative determination results also provedthat Tf-PEG-nGO-Dox could transferred more Dox into C6glioma cells compared toPEG-nGO-Dox or Dox.Conclusion PEG-GO has little cytotoxicity and Tf-PEG-GO might be used as apotential nanocarrier for drug delivery. With the help of Tf conjugated to PEG-nGO,Tf-PEG-nGO-Dox can transfer more Dox into C6cells, resulting in the cytotoxicity to theC6cells of Tf-PEG-nGO-Dox higher than free Dox or PEG-nGO-Dox.Part Ⅲ Effect of Tf-PEG-nGO-Dox for treating glioma: in vivoevaluationsAim On the basis of above researches, the Brain glioma-bearing rat model wasestablished. On the rat model, free Dox, PEG-nGO-Dox and Tf-PEG-nGO-Dox wereadministered via tail vein,Dox distribution in major tissues including heart, liver, spleen,lung, kidey, normal tissue and tumor were evaluated, and inhibitory ratio to tumor volumeand survival curves were also monitored.Methods The Brain glioma-bearing rat model was established by stereotacticinjecting C6cells into the right striatum of adult male Sprague-Dawley rats weighing between200-250g.10days after tumour implantation,9rats were randomly and equallydivided into3groups.3hours after administrated intravenously with free Dox,PEG-nGO-Dox and Tf-PEG-nGO-Dox (dose of Dox3mg/kg), the rats were sacrificed andthe brain tissues and major organs including liver, spleen, kidney, lung, and heart wereexcised. Left striatum (normal brain tissue) and right striatum (tumour tissue) wereseparated from the brain tissue. HPLC analysis was performed to determine Dox level inbiological samples.52glioma bearing rats were randomly and equally divided into4groups, on the day7thpost intracranial implantation. Animals in the control group wereadministered with physiological saline. Animals in the other3groups were treated withfree Dox, PEG-nGO-Dox and Tf-PEG-nGO-Dox via tail vein at a dose of2mg/kg Dox,respectively. Administration was made every2days with total3doses per rat. On the15thdays post tumour implantation,3rats of each group were sacrificed randomly, and thebrain tissue was collected，tumours were calculated，tumor volume inhibitory ratio wasalso evaluated. The other10rats in each group were maintained to monitor the survivalcurves. Kaplan–Meier survival curves were plotted for each group.Results Tissue distribution assay demonstrated that in the Tf-PEG-nGO-Dox group,Dox concentration in glioma tissue was significantly higher than that in the left striatum.Moreover, the Dox concentration of glioma tissue in Tf-PEG-nGO-Dox group was muchhigher than that of other groups(p<0.01).Dox distribution in other tissues showed that Doxaccumulated most in the spleen, and followed by the liver, but the concentrations ofTf-PEG-nGO-Dox and PEG-nGO-Dox in spleen and liver were significantly lower thanthat of free Dox group (p<0.05). There was no significant difference of drug distribution inother organs such as kidney, heart, and lung between different groups (p>0.05). Aftertreated with physiological saline, free Dox, PEG-nGO-Dox and Tf-PEG-nGO-Dox, tumourvolume at day15thpost intracranial implantation with reagents injection on day7th,9thand11thwere326.6,303.9,259.7and180.8mm3, respectively. This Result showed thatTf-GO-Dox exhibited the strongest inhibitory effect to the tumour growth compared toother groups (p<0.01).After treating with physiological saline, free Dox, GO-Dox andTf-GO-Dox, with a multidose of2mg/kg Dox on days7,9and11after gliomaimplantation the survival ranges were11–21,13–27,15–28and19–34days, respectively.The rank of median survival time was Tf-PEG-nGO-Dox (25days)> PEG-nGO-Dox (21days)> Dox (19days)> physiological saline (17days). The median survival time of Tf-PEG-nGO-Dox was significantly prolonged compared with that of saline control, freeDox or PEG-nGO-Dox (P <0.05), which reached nearly41.7%,31.6%and19.0%life-span extension compared with the saline control, free Dox or GO-Dox grouprespectively.Conclusion With Tf conjugation to nanovectors, Tf-PEG-nGO-Dox was able toconcentrate at the tumour site and transport more Dox to the tumor site and improve itsantitumor effect, manifesting by more effective inhibition of tumor volume and prolongedlife span, in vivo.