Letter Cite This: ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
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Polyethylenimine Coated Graphene Oxide Nanoparticles for Targeting Mitochondria in Cancer Cells Abhik Mallick,†,§ Aditi Nandi,†,§ and Sudipta Basu*,‡ †
Department of Chemistry, Indian Institute of Science Education and Research (IISER)-Pune, Dr. Homi Bhabha Road, Pashan, Pune, Maharashtra 411008, India ‡ Discipline of Chemistry, Indian Institute of Technology (IIT)-Gandhinagar, Palaj, Gandhinagar, Gujarat 382355, India
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S Supporting Information *
ABSTRACT: Mitochondrion, the powerhouse of the cells, controls bioenergetics, biosynthesis, metabolism, and signaling. Consequently, it has become an unorthodox target for cancer therapeutics. However, specific targeting of mitochondria into subcellular milieu in cancer cells remains a major challenge. To address this, we have engineered polyethylenimine cloaked positively charged self-assembled graphene oxide nanoparticle (PEI-GTC-NP) comprising topotecan and cisplatin concurrently. These PEI-GTC-NPs effectively homed into mitochondria in HeLa cervical cancer cells at 6 h and impaired mitochondria leading to reactive oxygen species generation followed by remarkably improved cancer cell death. This platform can be used for specific subcellular organelle targeting for future cancer therapy.
KEYWORDS: mitochondria, graphene oxide, nanoparticle, cisplatin, DNA damage, cancer
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mitochondrial outer membrane permeabilization (MOMP) through formation of mitochondrial transition pores (MTPs). PEI-GTC-NP-mediated mitochondrial damage induced generation of reactive oxygen species (ROS) leading to improved death of HeLa cells in a dose dependent manner much effectively compared to free drug combination. Topotecan was chosen due to its ability to conjugate with graphene oxide (GO) by π−π stacking, inherent fluorescent property to visualize subcellular mitochondrial homing, and inhibiting mitochondrial Topoisomerase I to augment the therapeutic effect of cisplatin in cervical cancer.22−24 The synthesis of polyethylenimine (PEI) coated graphene oxide nanoparticle (PEI-GTC-NP) is described in Scheme 1. In short, topotecan (2) was first stacked on 2D-GO-sheets by π−π interaction by incubating GO and topotecan in 1:5 weight ratio in water for 24 h (Figure S1). The formation of GOtopotecan complex (GT, 3) was characterized by fieldemission scanning electron microscopy (FESEM), which confirmed that GT also had the similar 2D-sheet like structure like GO (Figure S2a,b). Further, GO-topotecan complex (GT, 3) was reacted with aquated cisplatin (4) in 1:5 weight ratios in water for 24 h at room temperature to obtain GOtopotecan-CDDP conjugate (5, Figure S1). As expected from our previous study, GO-topotecan-CDDP conjugate selfassembled into spherical nanoparticle (GTC-NP) in water.25
itochondrion is a crucial subcellular organelle that not only synthesizes ATP (energy currency of cells), but also plays an important role in biosynthesis, metabolism, retro/ anterograde signaling, cell death (apoptosis)/survival machinery in stressed condition, and protein synthesis for oxidative phosphorylation (OXPHOS).1−5 Consequently, it is no wonder that mitochondria are instrumental in every steps of tumorigenesis from initiation, growth, survival, and metastasis.6 As a result, mitochondrion has gained much attention as a novel target in cancer therapy.7−9 However, because of ubiquitous nature, reaching mitochondria inside cancer cells has remained a major challenge toward developing effective mitochondria targeted anticancer therapeutics.10 Several nanoscale materials (nanoparticles and peptides) have been explored to target mitochondrial DNA to overcome chemotherapeutic drug resistance in cancer treatment.11−14 In this context, graphene-based carbon materials became interesting candidates to impair mitochondria due to their biocompatibility and biodegradability.15−19 However, lately it was realized that cancer cells can repair the damaged mitochondrial DNA through multiple DNA damage repair mechanisms, leading to the inactivation of drug effects eventually.20,21 To address this, in this manuscript, we have engineered a polyethylenimine (PEI) coated, self-assembled, spherical graphene oxide based nanoparticle (PEI-GTC-NP) that can simultaneously contain cisplatin (CDDP, DNA damaging agent) and topotecan (Topoisomerase I inhibitor) (Scheme 1). These cationic PEI-GTC-NPs localized into mitochondria of HeLa cervical cancer cells within 6 h and triggered © XXXX American Chemical Society
Received: September 12, 2018 Accepted: December 25, 2018 Published: December 25, 2018 A
DOI: 10.1021/acsabm.8b00519 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
Letter
ACS Applied Bio Materials
Scheme 1. Engineering of Polyethylenimine Coated Graphene Oxide Nanoparticle Containing Topotecan and Cisplatin (PEIGTC-NP) To Target Mitochondria in Cancer Cells
Figure 1. Characterization of PEI-GTC-NPs. (a) FESEM image. (b) Fluorescence emission quenching of topotecan in PEI-GTC-NPs at λmax = 525 nm. (c) Single particle resonance Raman spectra of PEI-GTC-NP and GO showing the characteristic D and G bands. (d) Zeta potential of PEIGTC-NPs showing high positive surface charge.
biodegradable cationic polyethylenimine (PEI) (Scheme 1 and Figure S1). The size, shape, and morphology of PEI coated GTC-NPs (PEI-GTC-NPs) were visualized by FESEM. Figure 1a clearly showed that PEI-GTC-NPs retained their spherical morphology with increase in diameter to 140−170 nm. This increment in mean diameter also confirmed the successful coating of PEI over GTC-NPs. The presence of topotecan in PEI-GTC-NPs was further confirmed by characteristic significant quenching of fluorescence emission of free topotecan at λmax = 525 nm after stacking with GO surface (Figure 1b). We further established the existence of topotecan in PEIGTC-NPs by UV−vis spectroscopy through characteristic absorbance peak at λmax = 405 nm (Figure S6). The presence of GO moiety in PEI-GTC-NPs was also confirmed by single particle resonance Raman spectroscopy, which clearly showed the characteristic D and G bands (Figure 1c). The presence of cisplatin in PEI-GTC-NPs was also validated by energy-
This unique morphological transformation was visualized by FESEM (Figure S2c), which confirmed the size of the GTCNPs to be 120−140 nm. The presence of topotecan in 2D-GTsheets and spherical GTC-NPs was confirmed by the dramatic quenching of characteristic fluorescence emission spectrum of free topotecan at λmax = 525 nm (Figure S3). Further, we also confirmed the presence of GO-moiety in GT and GTC-NPs by characteristic D and G bands in resonance Raman spectra (Figure S4). For successful mitochondria targeting, the material should possess high positive surface charge. Hence, we evaluated the surface charge of GO, GT, and GTC-NPs by light scattering experiments. It was observed that GO, GT, and GTC-NPs showed −22.6, −15.9, and −10.7 mV zeta potential, respectively (Figure S5), which were not suitable for mitochondria targeting. To achieve mitochondria homing, we further surface coated GTC-NPs with biocompatible and B
DOI: 10.1021/acsabm.8b00519 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
Letter
ACS Applied Bio Materials
confocal imaging. From the confocal microscopy images, we hardly found any trace of green fluorescence signal in the nucleus at 12 and 24 h (shown by arrows in Figure S10). To establish that PEI coating on GTC-NPs localized the nanoparticle into mitochondria, we further treated HeLa cells with GTC-NPs without any PEI coating for 1 and 3 h. Nucleus and lysosomal compartments were costained with DAPI (blue fluorescent) and LysoTracker Red (red fluorescent) and visualized by confocal microscopy. The confocal images in Figure S11 clearly demonstrated that without PEI coating, GTC-NPs localized into lysomes within 1 h followed by localization into nucleus at 3 h. These fluorescence imaging revealed that PEI-GTC-NPs localized selectively into the mitochondria within 6 h in HeLa cells and sustained there for 24 h. However, without PEI coating, the GTC-NPs localized mainly into lysosomes and nucleus within 3 h. After localization into mitochondria, PEI-GTC-NPs should damage mitochondrial DNA and inhibit mitochondrial Topoisomerase I (mt-Topo1) simultaneously leading to mitochondrial membrane perforation.26 We subsequently assessed mitochondrial membrane damage by JC1 dye, which switches fluorescence emission from red (aggregated form inside healthy mitochondria) to green (monomeric form in cytosol). The HeLa cells were treated with PEI-GTC-NPs for 24 h followed by treatment with JC1 dye. The cells were then visualized by confocal microscopy. From Figure 3, it was apparent that PEI-GTC-NPs induced mitochondrial membrane damage leading to the efflux of aggregated red fluorescent JC1 dye to cytosol triggering the formation of monomeric green fluorescent JC1 in much improved quantity. However, the control cells (without any nanoparticle treatment) generated comparable amount of red and green fluorescent signals indicating healthy intact mitochondria. Further quantification from microscopy images showed that PEI-GTC-NPs induced the formation of J-monomer in 2.2 ± 0.1-fold more compared to J-aggregate (Figure S12a). In contrast, control cells contained J-monomer/J-aggregate in 1:1 ratio. Further validation of mitochondrial damage by PEI-GTCNPs was visualized by TMRM assay. Tetra-methyl-rhodamine methyl ester (TMRM) is a cationic red fluorescent dye that binds with healthy undamaged mitochondria due to their high negative membrane potential. Upon damage, mitochondrial membrane potential reduces dramatically leading to the efflux of TMRM from cells. HeLa cells were treated with PEI-GTCNPs for 24 h followed by staining mitochondria by TMRM. The control cells were not treated with any nanoparticle. The cells were observed under fluorescence microscopy which indicated that TMRM dye stained undamaged mitochondria in control cells leading to generation of very high red fluorescence signal (Figure 4a). In contrast, PEI-GTC-NP treated cells showed remarkably reduced red fluorescent intensity representing damaged mitochondria followed by TMRM efflux from cells. Fluorescence intensity quantification from microscopy also validated that PEI-GTC-NP treatment reduced the red fluorescence intensity into the cells by 2.2 ± 0.6-fold compared to control cells (Figure S12b). These JC1 and TMRM assays evidently showed that PEI-GTC-NPs damaged mitochondria in HeLa cells. Mitochondrial membrane damage is triggered by the formation of mitochondrial transition pores (MTPs). We estimated PEI-GTC-NP-mediated MTP formation by Calcein AM assay.27 Inside the cells, Calcein AM (localized in cytosol)
dispersive X-ray spectra (EDAX) from FESEM images on single particle, which showed nearly 9.61 wt % of Pt content in the nanoparticle (Figure S7). We also measured the loading of cisplatin and topotecan in PEI-GTC-NPs by UV−vis spectroscopy from absorbance versus concentration calibration graph at characteristic λmax = 706 and 405 nm, respectively (Figure S8a,b). The mean loading of cisplatin and topotecan in PEI-GTC-NPs was found to be = 511.3 ± 5.9 μg/mL and 607.6 ± 3.9 μg/mL, respectively (Figure S8c). Finally, we quantified the surface charge of PEI-GTC-NPs by zeta potential. As expected, PEI-GTC-NPs demonstrated +26.1 mV zeta potential, which would be suitable for mitochondria targeting (Figure 1d). The dramatic change in zeta potential of GTC-NPs from highly negative value to highly positive value for PEI-GTC-NPs also confirmed the successful surface coating of PEI over GTC-NPs. After engineering of positively charged PEI-GTC-NPs, we evaluated their mitochondria homing capability. Mitochondria of HeLa cervical cancer cells were stained with MitoTracker Deep Red fluorescent dye followed by incubation with green fluorescent PEI-GTC-NPs for 6 h. The cells were then observed under confocal laser scanning microscopy (CLSM). The fluorescence microscopy images in Figure 2 clearly
Figure 2. Confocal laser scanning microscopy (CLSM) images of HeLa cells at 6 h after treatment with PEI-GTC-NPs (green fluorescence). Mitochondria were stained with MitoTracker Deep Red (red fluorescence). The yellow merged regions show the colocalization of PEI-GTC-NPs into mitochondria. Scale bar = 10 μm.
confirmed that green fluorescent PEI-GTC-NPs localized into filamentous mitochondria (red fluorescently stained) to produce yellow overlapping regions at 6 h. We further visualized the time-dependent retaining of PEI-GTC-NPs into mitochondria by confocal microscopy at 12 and 24 h. It was observed from the fluorescence microscopy images (Figure S9) that PEI-GTC-NPs retained into the mitochondria of the HeLa cells even after 24 h. We also further visualized the timedependent localization of PEI-GTC-NPs in nucleus by C
DOI: 10.1021/acsabm.8b00519 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
Letter
ACS Applied Bio Materials
Figure 3. CLSM images of HeLa cells after 24 h of treatment with PEI-GTC-NPs to show mitochondrial membrane permeabilization. The cells were costained with JC1 dye. JC1 dye in monomeric and aggregated form showed green and red fluorescence signals, respectively. Scale bar = 10 μm.
Figure 4. CLSM images of HeLa cells after 24 h of treatment with PEI-GTC-NPs followed by costaining the cells with (a, b) TMRM and Calcein AM to visualize the mitochondrial damage and (c) H2DCFDA to visualize the ROS generation. Scale bar = 10 μm.
AM in cytosol where it was cleaved by esterases to Calcein. Fluorescence microscopy based quantification also supported that PEI-GTC-NPs increased the subcellular green fluorescence intensity by 7.5 ± 0.8-fold compared to control cells (Figure S12c). This Calcein AM assay indicated that PEIGTC-NPs damaged mitochondria by MTP formation. Mitochondrial membrane damage through MTPs would lead to the generation of reactive oxygen species (ROS).5 Hence, we evaluated ROS production by dichloro-dihydrofluorescein diester (H2DCFDA) reagent.28 ROS produced inside the cells would oxidize H2DCFDA into green fluorescent dichloro-fluorescein (DCF). To assess the ROS generation, we treated HeLa cells with PEI-GTC-NPs for 24 h and exposed the cells in H2DCFDA followed by confocal microscopic visualization. Fluorescence microscopy images
is cleaved by esterase leading to the formation of green fluorescent Calcein dye, which quenches in the presence of CoCl2. On the other hand, Calcein AM localized in healthy mitochondria remains intact. After mitochondrial damage by MTP formation, mitochondrial Calcein AM will be sequestered into cytosol leading to the formation of green fluorescent Calcein, which can be visualized by confocal microscopy. Hence, we treated HeLa cells with PEI-GTC-NPs for 24 h and incubated with Calcein AM and CoCl2. We visualized the live HeLa cells through CLSM. Fluorescence images in Figure 4b showed that in control cells green fluorescence signal was quenched due to undamaged mitochondria. However, in PEIGTC-NP treated cells, green fluorescence signal was significantly increased due to the formation of MTPs from damaged mitochondria leading to the sequestration of Calcein D
DOI: 10.1021/acsabm.8b00519 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
Letter
ACS Applied Bio Materials
damage through pore formation and subsequent generation of ROS confirmed by fluorescence confocal microscopy. Remarkably improved cervical cancer cell killing was also observed by these mitochondria-targeted graphene oxide based nanoparticle compared to free drugs. It can be anticipated that this nanoscale platform has future potential for organelle specific targeting of cancer cells toward next generation cancer therapy.
(Figure 4c) showed that non-nanoparticle treated cells produced negligible amount of green fluorescence signal indicating very low level of subcellular ROS. However, on the contrary, PEI-GTC-NP treated cells showed remarkably increased green fluorescence signal, which evidently indicated that PEI-GTC-NPs triggered generation of increased amount of ROS. This observation was also supported by quantification of subcellular green fluorescence intensity from microscopy. It was measured that PEI-GTC-NPs increased the formation of ROS in HeLa cells 4.5-fold compared to untreated control cells (Figure S12d). This microscopy-based experiment demonstrated that PEI-GTC-NP mediated mitochondrial damage led the formation of ROS. Finally, PEI-GTC-NP mediated mitochondrial damage followed by ROS generation would lead to cellular death. To estimate damaged mitochondria triggered cell death, we incubated HeLa cells with PEI-GTC-NPs in different dosages for 48 h. The cell viability was quantified by adding MTT reagent. The control cells were incubated with the combination of free topotecan, cisplatin, and GO in the same ratio present in the nanoparticle. Cell viability assay (Figure 5)
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsabm.8b00519. Experimental details, synthetic scheme, FESEM images, fluorescence emission spectra, resonance Raman spectra, zeta potential, EDX, drug loading quantification, confocal laser scanning microscopy, quantification from CLSM (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Sudipta Basu: 0000-0002-0433-8899 Author Contributions §
A.M. and A.N. contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was financially supported by Department of Biotechnology (DBT) (BT/RLF/Re-entry/13/2011 and BT/ PR14724/NNT/28/831/2015) and DST-Nanomission [SB/ NM/NB-1083/2017 (G)]. A.M. and A.N. are thankful to CSIR-UGC and IISER-Pune, respectively, for doctoral fellowships.
Figure 5. Viability of HeLa cells at 48 h after treatment with PEIGTC-NPs in a concentration-dependent manner determined by MTT assay. A cocktail of GO, topotecan, and cisplatin was used as control.
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showed that free drug combination with GO induced 50% cell death (IC50) at 1.71 μM concentration with only 12% viable cells at 25 μM concentration based on cisplatin. On the other hand, PEI-GTC-NPs triggered 50% cell death (IC50) at only 0.39 μM concentration based on cisplatin, which is nearly 4.4fold less compared to free drug cocktail. To validate the better efficacy of mitochondria targeted PEI-GTC-NP, we further treated the HeLa cells with non-PEI-coated GTC-NPs for 48 h. The MTT assay demonstrated that GTC-NPs (without PEI coating) induced IC50 = 1.2 μM, which is 3.1-fold higher than PEI-GTC-NPs (Figure S13). These cell viability assays indicated that PEI-GTC-NPs induced much improved HeLa cell death by damaging mitochondria compared to free drug cocktail as well as non-mitochondria targeted nanoparticles. In conclusion, in this study, we have engineered polyethylenimine coated self-assembled graphene oxide based spherical nanoparticles (PEI-GTC-NPs) that can incorporate topotecan (Topoisomerase I inhibitor) and cisplatin (DNA damaging agent) simultaneously. This nanoparticle showed high positive surface charge and nearly 170 nm diameter for successful accumulation of mitochondria in cancer cells specifically. These PEI-GTC-NPs localized into mitochondria of HeLa cells within 6 h followed by mitochondrial membrane
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DOI: 10.1021/acsabm.8b00519 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
