그래핀의약물전달시스템및암치료 연구동향 성균관대학교나노소재기반휴먼인터페이스융합연구센터선임연구원윤옥자

그래핀의약물전달시스템및암치료 연구동향 성균관대학교나노소재기반휴먼인터페이스융합연구센터선임연구원윤옥자

1. 서론 Ø 나노물질은높은생체친화력, 넓은표면적, 표면의기능화에따라질병의진단과맞춤형표적치료에다양하게적용되고생체분자영상, 바이오센서등의연구에활발히응용되고있다. 1 Ø 약물전달시스템은약물을시간에따라방출 흡수하도록제어, 특정한곳에약물을전달하는표적치료, 약물의부작용을최소화한맞춤형치료및조기진단을목적으로한다. Ø 그래핀을이용한약물전달시스템으로의응용가능성은그래핀의낮은독성, biocompatibility, stability, 두께와크기제어, p-p 결합에의한표면기능화등의특성들을가지고있기때문이다. 2 Ø 카본나노물질인탄소나노튜브나그래핀은암치료의화학요법에서 multi-drug resistance 를위한항암치료제로의연구와 gene co- delivered 하거나 photothermal, photoacoustic, RF ablation 등을이용하여최선의암치료방법을찾기위해활발히연구하고있다. 3 Ø 본정보는그래핀의약물전달시스템및암치료연구에대한최근연구동향에대해기술하고자한다

2. 그래핀의약물전달시스템연구 Ø A novel graphene oxide-para amino benzoic acid nanosheet as effective drug delivery system to treat drug resistant bacteria 4 - Graphene oxide-para amino benzoic acid nanosheet(go-paba) 의평균크기는 100nm 이였고 zeta potential 은 34.9 (±7.03) mv 임을보여줌. - 효과적으로 tetracycline 약물이탑재됨을확인하였고약물방출이효과가있음을연구하였음. - Go-PABA 에탑재된 tetracycline (GO-PABA tet) 의항균활동 (antimicrobial activity) 이 tetracycline 에의한내성박테리아 Escherichia coli XL-1 에대한최소억제농도 (minimal inhibitory concentration ) 와 putative mode 를결정되는것을보고함. - 이 GO-PABA-tet 의최소억제농도는 110 μg/ml 이였고 drug resistant bacteria 를치료하기위한훌륭한약물전달시스템임을보여줌. - 또한 GO-PABA 는 biocompatibility, 효과적인물리적약물탑재, drug resistant bacteria 를치료하기위한약물전달시스템등을포함한복합기능화의기능을가지고있어생물학적, 치료분야에서응용이기대됨.

Figure 1. Evaluation of MIC of GO-PABA-tet nanosheet on tetracycline resistant bacteria Escherichia coli XL-1. Uninoculated represents the only autoclaved nutrient broth (negative control); Inoculated ( GO-PABA) represents the autoclaved nutrient with tetracycline resistant bacteria Escherichia coli XL-1 without GO-PABA nanosheet; Inoculated +GO-PABA designates inoculated medium with GO-PABA nanosheet but without tetracycline loading (positive control). Figure 2. Evaluation of a putative mode of action of GO-PABA-tet nanosheet on tetracycline resistant bacteria Escherichia coli XL-1. (A) Untreated E. coli XL-1; (B) treated E. coli XL-1; (C) treated after 12 hrs; (D) treated after 24 hours under 20,000 X magnification. In (C) scar was appeared in middle region of bacteria; in (D) the scar become bigger and spreading toward the Polar Regions (Considering the same dilution in each FE-SEM analysis of GO-PABA-tet treated bacterial sample preparations before gold coating).

Ø Engineered redox-responsive PEG retachment mechanism in PEGylated nanographene oxide for Intracellular drug delivery 5 - 이연구는 redox-responsive PEG detachment mechanism 을이용하여중요한세포내약물전달 문제를해결하고자함. - Disulfide linkage 를매개로결함한 PEGylated nano-graphene oxide (NGO-SS-mPEG) 는세포내 약물전달하기위하여개발하였으며약물전달시세포내에서 diffusion barrier 라는문제가있어 tumor-relevant glutathione (GSH) levels 에서빠르게방출을할수있도록개발하였음. - NGO-SS-mPEG 는생리학적환경에서높은 solubility, stability, circulation 의성질를가지는복합 체이고이것은세포내 GSH 자극에따라선택적으로 NGO 로부터약물을분리할수있음을증명. - Redox-sensitive NGO-SS-mPEG nanocarriers 는세포내 GHS 의높은농도를가진표적암에빠른 doxorubicin hydrochloride (DXR) 방출에의하여세포증식억제가세포내 GSH 농도의증가와직 접적인연관이있음을증명하였고 NGO-SS-mPEG 는 HeLa cell 안에흡수되어 cytoplasm 안에주 로모여있음을입증함. - 이 nanocarriers 는 stability, circulation time, 표적암에서의약물전달, 약물방출제어방법등의 결정적임문제들을다루고있어 photodynamic therapy 와 gene therapy 와같은 biomedical applications 의접근을제시함. Figure 3. Schematic diagram showing antitumor activity of redoxsensitive DXR-loaded NGO-SS-mPEG: (a) PEG- shielded NGO with disulfi de linkage for prolonged blood circulation; (b) endocytosis of NGO-SS-mPEG in tumor cells via the EPR effect; (c) GSH trigger (GSH > fourfold relative to normal cells) resulting in PEG detachment; and d) rapid drug release on the tumor site.

Figure 5. (a) Cell proliferation of HeLa cells incubated with free DXR, NGO-SS-mPEG, and DXR loaded NGO-SS-mPEG for 24 h. Data are presented as the mean ± standard deviation (SD; n = 5). (b) Cell proliferation of pretreated HeLa cells with either 0 or 10 m M GSH-OEt incubated with DXR-loaded NGO-SS-mPEG (0.25, 0.125, 0.0625 mg ml 1 ) for the time periods indicated. Figure 4. Representative CLSM images of (a) 10 m M GSH-OEt and (b) 0 m M GSH-OEt pretreated HeLa cells after 6 h of incubation with DXR-loaded NGO-SS-mPEG (0.25 mg ml 1 ). The red channel visualizes DXR fl uorescence. Figure 6 Flow cytometric analyses of nontreated and 10 m M GSH-Oet pretreated HeLa cells incubated with DXR-loaded NGO-SS-mPEG at the concentrations indicated for 2 h. The equivalent DXR dose is 46.5, 23.3, and 11.6 mg L 1, respectively. HeLa cells without any treatment are used as control. Fluorescence intensity is denoted as FL3-H.

Ø Synthesis and drug-delivery behavior of chitosan-functionalized graphene oxide hybrid nanosheets 6 - 합성된 chitosan-functionalized graphene oxides (FGOCs) 는산성수용액에서 GO 의 solubility 와 분산성이향상되었으며두가지약물인 ibuprofen (IBU) 와 5-fluorouracil (5-FU) 를탑재하여약물 방출제어와 biocompatibility 를입증하였음. - 약물의분자구조의크기에따라 FGOCs 와결합및 release behavior 의차이가있어작은분자구조를가지는두가지약물중 IBU 는 benzene ring (presumably higher p- stacking) 을가진 소수성이기때문에 FGOCs 와강한결합을보였고, 5-FU 는 benzenoid (presumably less p-stacking) 의 resonance 원인으로 diamide group 과친수성성질을가지고있어약한결합력을 가짐을확인함. -IBU 와 5-FU 약물의성공적인 controlled release behavior 를보고하였고약물의구조와물리적특 징에의존함을증명함. - FGOCs 의 Biocompatible 와 drug release behavior 를증명하였고생물학적응용물질임을보고함. Figure 7. Synthesis of the FGOCs and the dispersion of (a) GO and (b) the FGOCs in an aqueous acetic acid solution (CH3COOH/H2O 0.2/1). More details on the synthesis and characterization of FGOCs are given in the Supporting Information. Figure 8. In vitro cell viability assay. a) Relative cell viability of CEM cancer cell line and b) MCF-7 cancer cell line incubated with GO, FGOCs, and drug loaded FGOCs graphene sheets at different concentrations for 5 d (SD3%).

3. 그래핀의암치료연구 Ø Functional graphene oxide as a nanocarrier for controlled loading and targeted delivery of mixed anticancer drugs 7 - 항암제의탑제제어 (controlled loading) 나표적전달 (targeted delivery) 하기위한나노운반체로서 nanoscale graphene oxide (NGO) 를 sulfonic acid groups 으로기능화하였고 folic acid (FA) 분자와공유결합한후에 FA receptors 를가지고있는 human breast cancer cells (MCF-7 cells) 를표적화하고자했음. - p p stacking 과 hydrophobic interactions 에의하여 FA-conjugated NGO (FA NGO) 위에두가지항암제 doxorubicin (DOX) 와 camptothecin (CPT) 를복합화하여탑재함으로써정확히 MCF-7 cells를표적화했고 DOX 이나 CPT 중한가지항암제만탑재한것에비교하여높은세포독성을보였음. - 임상시험에서한가지항암제의치료효과보다두가지나그이상의복합항암제의치료효과가좋았음을고려하여그래핀기반나노운반체를이용하여복합항암제의탑제제어나표적전달를이용한치료효과를입증하였음. - 이연구는임상에서환자들의치료효과를극대화시키고 biomedicine 응용이기대됨.

Figure 9. Schematic illustration of the preparation of FA NGO conjugates. EDC¼1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; NHS¼N-hydroxysuccinimide Figure 11. Relative cell viability of MCF-7 treated with FA NGO/CPT and FA NGO/DOX/CPT. The asterisks indicate P<0.05 versus normal cells. When the P value was less than 0.05, differences were considered statistically significant. Figure 10. a)uv/visand b)fluorescence spectra of Rho B before and after adsorptionon FA NGO (suspended in water and 50% ethyl alcohol, respectively). c) Bright-field (left), fluorescence (center),and merge (right) imagesof MCF-7 cells incubated withfa NGO/RhoBfor2 h; d) same as (c) except A549 cells were used.

Ø Multi-functionalized graphene oxide based anticancer drug-carrier with dualtargeting function and ph-sensitivity 8 - 이연구는암세포를외부자기장에의한자기표적 (magnetic targeting) 하기위한 superparamagnetic iron oxide nanoparticles 와분자표적리간드 (molecular targeting ligand) 를결합한 multi-functionalized graphene oxide (GO) 를기반으로암세포에대한항암제의 dual-targeting drug delivery 와 ph-sensitive controlled release system 을준비하여향상된효과를보고함. -Chemical precipitation method에의해 GO Fe3O4 nanohybrid준비à amino groups 를형성하기위하여 3-aminopropyl triethoxysilane (APS) 의가수분해에의한표면개질 à targeting agent 인 folic acid (FA) 를결합하여자기적성질과 tumor dualtargeting properties를가진 multi-functionalized GO를준비à p-p 결합에의해항암제Doxorubicin hydrochloride (Dox) 를탑재하거나현광물질인 fluorescein isothiocyanate labeled 하였음. -Multi-functionalized GO 는상자성성질 (superparamagnetic property) 에의한자기표적화하였고약물방출이 ph 값에의존함을보고함. -Cell uptake 연구결과는현광이미지를통해암세포의향상된 dual-targeted delivery property 와 Dox 약물에의한독성효과를입증함으로써 multi-functionalized GO 가 tumor combination therapy 를위한항암제표적전달과 controlled release 의훌륭한응용성을입증함.

Figure13. Confocal fluorescence images of GO-Fe3O4-FA-FITC (A) and GO-Fe3O4-FITC (B) after incubation with SK3 at 37 C for 1 h. Figure 12. The preparation of the multi-functionalized GO based anticancer drug-carrier with dual-targeting function and ph-sensitivity. Figure 14. Relative cellular viability of Hela after treatment with GO, GOFe3O4, Dox, GO-Fe3O4-Dox and GO-Fe3O4-FA-Dox.

Ø In vitro comparison of the photothermal anticancer activity of graphene nanoparticles and carbon nanotubes 9 - 악성종양치료시외과적치료가어려울경우고열요법으로열치료 (thermal ablation) 를선택 하는데이미이에대한연구로 carbon nanotubes (CNT), gold nanoparticle 에 near-infrared (NIR) radiation 의발열을일으키는파장대 700-1100 nm range 에노출시켜나노입자들의진동에너지 (vibrational energy) 가발생되고이러한에너지가종양세포를사멸시키기에충분한열로 에너지전환되어치료하는 photothermal therapy 를보고함. -Laser, ultrasound, microwave 또는 radio frequency 에의한열치료는악성종양에대한고유 특이성이없어적합하지않으며열치료를위해서는독성을줄이고효율성을증가시켜야함. - 이연구에서는그래핀나노입자와 CNT 의 Near-infrared (NIR) 에의한 photothermal anticancer activity 를비교하였음. -낮은 NIR-absorbing capacity (808 nm, 2 W/cm2) 임에도불구하고 DNA 또는 sodium dodecylbenzene sulfonate-solubilized single-wall CNT (CNT DNA 또는 CNT SDBS ) 보다 polyvinyl pyrrolidone-coated graphene sheets (Gpvp) 가열이더많이발생하였고결과적으로 in vitro에서 U251 human glioma cells 의 photothermal 사멸이증가함. -그래핀과 CNT는 thermodynamic, optical, geometrical properties등을가지고있으나그래핀이 CNT보다분산성이더좋고더작은크기제어에의하여효율성이증가함을보고하고있음. 그결과그래핀을매개로한종양세포의 photothermal killing 은종양세포의 oxidative stress와 mitochondrial damage를일으킨다는것을입증하여확인함.

Figure 15. The type of graphene-induced photothermal cell death. (AeD) U251 cells were exposed for 3 min to NIR laser (808 nm, 2 W/cm2) in the absence or presence of GPVP (10 mg/ml). After 24 h, the cell morphology was examined by inverted microscopy (A), while flow cytometry was used to assess the cell size (FSC) and granularity (SSC) (B), DNA fragmentation (C) or externalization of phosphatidylserine and membrane permeability (Ann/PI-staining) (D). The representative photomicrographs, histograms and dot plots from one of three experiments are presented (no significant changes were observed in cell cultures exposed to NIR or GPVP alone - not shown). Figure 16. The intracellular mechanisms of graphene-induced photothermal cell death. (AeD) U251 cells were exposed for 3 min to NIR laser (808 nm, 2 W/cm2) in the absence or presence of GPVP (10 mg/ml). After 24 h, flow cytometry was used to assess caspase activation (ApoStat staining; A), while the mitochondrial membrane potential (DJ) (DePsipher staining; B), production of ROS (DHR staining; C) and superoxide (DHE staining; D) were measured after 4 h.

4. 결론 Ø 그래핀의낮은독성, biocompatibility, stability, 두께와크기제어, p-p 결합에의한표면기능화등우수한물성을이용하여시간에따라약물의제어방출, 표적치료, 맞춤형치료진단이가능한약물전달시스템개발이활발히보고되고있다. 또한암치료를위한 mixed anticancer drugs 7, tumor combination Therapy 8, photothermal therapy 9 등의연구보고가진행하고있다. Ø 그래핀의약물전달시스템및암치료연구동향및보고는기존에보고된탄소나노튜브와비교하여 thermodynamic, optical, geometrical properties 등유사한결과를보이고있으나그래핀이 CNT 보다분산성이더좋고더작은크기제어에의한효율성, 저비용등에의하여약물전달시스템및암치료연구에더좋은효과를보고하고있다. Ø 이러한결과는 in vitro 에서의연구뿐만아니라 in vivo 적용에의한연구결과를기대할수있으며임상에서환자들의맞춤형치료효과에의한약물의부작용을최소화하고 biomedicine 에널리적용될것을기대됨.

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