RESEARCH ARTICLE www.advhealthmat.de Graphene Oxide Nanoscale Platform Enhances the Anti-Cancer Properties of Bortezomib in Glioblastoma Models Paul S. Sharp, Maria Stylianou, Luis M. Arellano, Juliana C. Neves, Alfredo M. Gravagnuolo, Abbie Dodd, Katharine Barr, Neus Lozano, Thomas Kisby,* and Kostas Kostarelos* Graphene-based 2D nanomaterials possess unique physicochemical 1. Introduction characteristics which can be utilized in various biomedical applications, Glioblastoma multiforme (GBM; WHO including the transport and presentation of chemotherapeutic agents. In grade IV glioma) is the most aggressive glioblastoma multiforme (GBM), intratumorally administered thin graphene and prevalent form of adult primary can- cer of the central nervous system.[1] The oxide (GO) nanosheets demonstrate a widespread distribution throughout standard of care is surgical resection (if tu- the tumor volume without impact on tumor growth, nor spread into normal mor location allows), followed by radiother- brain tissue. Such intratumoral localization and distribution can offer multiple apy with complementary cycles of system- opportunities for treatment and modulation of the GBM microenvironment. ically administered temozolomide (TMZ) Here, the kinetics of GO nanosheet distribution in orthotopic GBM mouse chemotherapy. Despite this combinatory approach, the prognosis of GBM is in- models is described and a novel nano-chemotherapeutic approach utilizing variably a terminal disease with a me- thin GO sheets as platforms to non-covalently complex a proteasome dian survival of 15–18 months, and 5-year inhibitor, bortezomib (BTZ), is rationally designed. Through the survival of less than 10%.[2] The employ- characterization of the GO:BTZ complexes, a high loading capacity of the ment of novel therapeutic approaches in- small molecule on the GO surface with sustained BTZ biological activity in cluding convection-enhanced delivery, tar- vitro is demonstrated. In vivo, a single low-volume intratumoral geted therapies, monoclonal antibodies, and tumor-treating fields has not yet had a administration of GO:BTZ complex shows an enhanced cytotoxic effect major impact on this poor prognosis.[3–6] compared to free drug in two orthotopic GBM mouse models. This study GBM has proved challenging to treat due provides evidence of the potential that thin and small GO sheets hold as flat to the highly aggressive and invasive na- nanoscale platforms for GBM treatment by increasing the bioavailable drug ture of tumor cells and their unique location concentration locally, leading to an enhanced therapeutic effect. in the brain, protected by the blood-brain barrier (BBB).[7,8] Despite numerous efforts P. S. Sharp[+] , M. Stylianou, A. M. Gravagnuolo, A. Dodd, K. Barr, T. Kisby, L. M. Arellano, J. C. Neves, N. Lozano, K. Kostarelos K. Kostarelos Catalan Institute of Nanoscience and Nanotechnology (ICN2) Nanomedicine Lab Campus UAB, Bellaterra, Barcelona 08193, Spain Faculty of Biology, Medicine & Health National Graphene Institute University of Manchester AV Hill Building, Manchester M13 9PT, UK E-mail: thomas.kisby@manchester.ac.uk; kostas.kostarelos@manchester.ac.uk The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/adhm.202201968 [+] Presentaddress: Medicines Discovery Catapult, Alderley Park, Mere- side, Macclesfield SK10 4TG, UK © 2022 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. DOI: 10.1002/adhm.202201968 Adv. Healthcare Mater. 2023, 12, 2201968 2201968 (1 of 13) © 2022 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH 21922659, 2023, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/adhm.202201968 by Readcube (Labtiva Inc.), Wiley Online Library on [24/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.advhealthmat.de to increase the permeability of the BBB to chemotherapy,[9] key In this work, we first investigated the distribution kinetics of limitations such as the pharmacokinetic profile of chemother- intratumorally injected GO in vivo using orthotopic mouse GBM apeutic agents, along with systemic toxicity have compromised models, and then non-covalently complexed and characterized a the efficacy of GBM treatments. These challenges have led to an GO:BTZ nanoscale complex system. The successful complexa- urgent need to develop new strategies that can achieve higher tion of GO with BTZ demonstrated high drug-loading capacity, and more localized therapeutic concentrations at the tumor site. and more importantly, preservation of the drug’s biological activ- One of those strategies is the direct administration of chemother- ity. We evaluated systematically its cytotoxic activity in vitro and apy into the tumor parenchyma or resection cavity. However, the in vivo, utilizing two GBM mouse models (orthotopic xenograft rapid clearance kinetics and diffusion away from the tumor or and orthotopic syngeneic model). A single low-volume intratu- the rapid enzymatic processing and degradation of the therapeu- moral administration of GO:BTZ resulted in significant tumor tic agents, have led to compromised therapeutic efficacies. More- tissue necrosis and enhanced activity compared to free drug. over, such effects pose neurological and systemic safety concerns and have thus far required the application of complicated admin- istration methods through an implanted catheter or dissolvable 2. Results implants, still with varied efficacy reported.[10,11] Alternatively, the use of nanomaterials to deliver drugs directly into the tumor can 2.1. GO Localizes Throughout the GBM Tumor Volume In Vivo increase the therapeutic agent concentration achieved locally, but also prolong localized high concentrations and provide much- Following our previous observations of extensive GO nanosheet needed drug stability.[12–15] distribution throughout U87-MG tumors in vivo,[27] we initially The unrivaled available surface area of small, thin graphene ox- utilized the U87 model for a more dynamic evaluation of GO ide (GO) nanosheets provides a platform for increased loading ca- translocation within the GBM tissue over time. Human U87- pacity of bioactive molecules and can allow excellent colloidal dis- luc cells were implanted intracranially in athymic mice and in- persibility in physiological fluids, with a proven bio- and neuro- jected at the same site with 1 μl GO (1 mg ml−1 ) alone, 12 days compatibility profile.[16–20] Several efforts have been made to uti- following cell inoculation. GO distribution was evaluated histo- lize GO as a delivery platform for anti-cancer agents by either co- logically at 4, 24, 48, and 96 h post intratumoral injection (Fig- valent attachment or non-covalent interactions and in combina- ure 1). First, the optical signature of GO (identified as dark ma- tion with photothermal effects.[20–26] We previously demonstrated terial) within the histological specimen was validated with Ra- that GO nanosheets are able to localize throughout the tumor man spectroscopy showing co-localization of GO Raman sig- volume using a human glioblastoma model, both in vitro (3D nature with the observed black matter (Figure 1A). Between spheroids) and in vivo (orthotopic xenograft model).[27] Taking 4 and 96 h post-intratumoral injection, we observed a signifi- advantage of the translocation of GO exclusively within the GBM cant spread of GO from the well-localized point of the injec- volume and recognizing the limited activity and diffusivity of lo- tion site to a more diffuse pattern throughout the U87 tumor cally administered small molecules, we have hypothesized that area over time (Figure 1B,C). In addition to the observed lat- GO sheets could be a promising flat platform to achieve trans- eral spread, GO was also identified to translocate both rostrally port, localization, and retention of chemotherapeutic agents in- and caudally by 96 h post-administration (Figure 1B (i)–(iii)). Im- tratumorally. portantly, we did not identify any GO outside the tumor bor- The persistently poor prognosis of GBM patients undergo- der within the healthy brain tissue. In agreement with our pre- ing standard-of-care treatment with TMZ chemotherapy high- vious report that described the interaction and internalization lights the necessity to explore non-standard chemotherapeutics. of GO by macrophages and microglia (IBA1+ cells) within the High content screening of patient-derived cell lines revealed that U87 GBM microenvironment,[27,38] we further observed a strong bortezomib (BTZ; Velcade) was a non-standard chemotherapeu- IBA1+ signal at the initial injection site at 4 h which appeared to tic agent that showed potent cytotoxic activity across a panel of undergo redistribution alongside the spread of the nanomaterial GBM cell lines, multiple times more effective than TMZ.[28] BTZ (Figure 1C). is the first proteasome inhibitor approved by the US Food and Drug Administration (FDA) for multiple myeloma and mantle cell lymphoma,[29] while further demonstrating promising anti- 2.2. Preparation and Characterization of Non-Covalent tumor activity in various cancers including colorectal, pancreatic, GO:Bortezomib (BTZ) Complexes and lung cancer.[30] However, in the clinic, intravenous adminis- trations of BTZ have shown limited therapeutic efficacy in GBM Given the spatial distribution of GO nanosheets throughout the with the boric acid groups compromising the blood stability of tumor volume and their retention within the tumor, we reasoned the drug, exhibiting poor BBB translocation, and leading to fre- that complexation of the nanomaterial with a chemotherapeu- quent adverse effects.[31–36] Preclinical investigations have shown tic drug such as BTZ may improve the localization, persistence, modest improvement of therapeutic efficacy with direct admin- and activity of the drug following direct (intratumoral) adminis- istration to GBM, however, this required continual infusion with tration. BTZ molecules were mixed with GO at a 10:6 GO:BTZ a surgically implanted osmotic mini-pump further limiting the (weight ratio) in an aqueous solution (1 mg mL−1 , GO). BTZ was ease of clinical applicability.[37] Based on such reported knowl- expected to be complex onto the GO lattice primarily through 𝜋– edge, we selected BTZ as a suitable potent candidate chemother- 𝜋 stacking interactions between the aromatic rings, with further apeutic agent for intratumoral (GBM) transport via the GO-based potential for hydrogen bonding among the oxygen-containing flat nanomaterial platform. groups on the GO and the polar groups of BTZ (Figure 2A).[39] Adv. Healthcare Mater. 2023, 12, 2201968 2201968 (2 of 13) © 2022 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH 21922659, 2023, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/adhm.202201968 by Readcube (Labtiva Inc.), Wiley Online Library on [24/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.advhealthmat.de Figure 1. Time-course of graphene oxide nanosheet distribution in orthotopic U87 gliomas. Athymic nude mice were implanted with 1 × 105 U87-luc cells into the right striatum. To assess penetrance, 1 μg of GO was delivered intratumorally at 12 days post inoculation (n = 12) and brains were harvested at 4, 24, 48, and 96 h post GO delivery (n = 3 per group). A) Co-localization of the GO Raman signal and black matter observed under bright-field imaging in brain sections. B) Representative images of the whole brain and corresponding 20 μm sections showing the redistribution of intratumoral GO over time (scale bar 500 μm). For the 96 h time-point, i–iii) images show the presence of GO in an anterior-posterior (rostral-caudal) direction. C) Representative epifluorescence images of the whole brain and tumor region showing the distribution of intratumoral GO in relation to DAPI staining and IBA1+ macrophage/microglia cells. Scale bar = 500 μm. GO alone control (GOc ) was also prepared using the same proto- on the GO:BTZ complex was found to be 0.17–0.19 mg BTZ col as per the GO:BTZ complex for the purpose of characteriza- per 1 mg GO (Figure S1B, Supporting Information) as deter- tion. mined by UV–vis using a BTZ concentration standard curve in To quantify the BTZ loaded onto GO sheets, the concentration water (Figure S1C,D, Supporting Information). Complex forma- of BTZ on the complex was calculated indirectly by serial wash- tion was validated by the XPS survey spectrum for the complex ing and centrifugation steps in water (as shown in Figure S1A, which exhibited signals corresponding to carbon, oxygen, nitro- Supporting Information). The final concentration of BTZ loaded gen, and boron (Figure 2B). The strong nitrogen peak at 400 eV Adv. Healthcare Mater. 2023, 12, 2201968 2201968 (3 of 13) © 2022 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH 21922659, 2023, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/adhm.202201968 by Readcube (Labtiva Inc.), Wiley Online Library on [24/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.advhealthmat.de Figure 2. Physicochemical characterization of GOc and GO:BTZ complex. A) Schematic illustrating GO:BTZ complex and predicted chemical interac- tions. B) XPS survey analysis. C,D) AFM height images and cross-section analysis of GO and E,F) GO:BTZ complex. Measurements were performed in triplicate. and the weak boron contribution at 191 eV provided further ev- bilization of BTZ molecules onto the GO basal plane with uni- idence of the absorption of BTZ onto the GO surface. Next, we form distribution and without evidence of structural damages evaluated BTZ release from the GO:BTZ complex (in water) at 4 (Figure 2E,F and Figure S3A,B, Supporting Information). In the and 24 h post-complex formation using UV–vis based quantifica- Raman spectra of GOc and GO:BTZ (Figure S3C, Supporting In- tion. The total concentration of BTZ released over 24 h was found formation) the characteristic D (1327 cm−1 ) and G band (1599 as 1 μg ml−1 (per 1 mg ml−1 , GO) which indicated high stability cm−1 ) of graphitic materials were identified.[41] When BTZ was of the GO:BTZ complex (Figure S2, Supporting Information). loaded on GO, additional Raman peaks attributed to boronic acid GOc displayed the characteristic topology of isolated mono- compounds were clearly detected,[42] indicating the decoration on layers with an average height of 1.2–1.3 nm by AFM (Fig- the GO surface without disturbing the graphitic lattice. In addi- ure 2C,D),[40] whereas for GO:BTZ complexes, the presence of tion, the G-band was down-shifted by 4 cm−1 with respect to GOc several bright areas with heights of 1.9 nm suggested the immo- suggesting the n-doping of the material by the BTZ moiety.[43] Adv. Healthcare Mater. 2023, 12, 2201968 2201968 (4 of 13) © 2022 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH 21922659, 2023, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/adhm.202201968 by Readcube (Labtiva Inc.), Wiley Online Library on [24/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.advhealthmat.de XRD analysis by analyzing the crystalline phase and interlayer that the complexation of BTZ through non-covalent interaction distance for both GOc and GO:BTZ complex, showed a range of with GO does not inhibit its biological (cytotoxic) activity in vitro. 2𝜃 from 5° to 40° (Figure S3D, Supporting Information). The XRD patterns confirmed the chemical oxidation of graphite and complete formation of GO by the appearance of a new diffrac- 2.4. GO:BTZ Complex Retains Cytotoxic Activity in U87 Gliomas tion peak at 2𝜃 = 11.2° and no residual reflection of the graphite In Vivo plane at 2𝜃 = 26.6°. The increase in the interlayer distance was attributed to the uptake of oxygen-containing functional groups To further evaluate the cytotoxic activity of the GO:BTZ com- and water molecules between GO layers.[44] Furthermore, for plexes we initially utilized an orthotopic xenograft model of the GO:BTZ complex the shift of the characteristic peak of GO glioblastoma.[50] U87-luc cells were injected into the striatum of from 11.2° to 9.9° with a slight increase in the basal spacing evi- athymic mice and tumors were allowed to establish for 14 days dencing the intercalation of BTZ molecules between GO sheets. before mice received intratumoral injection of GOc or GO:BTZ The spectrum of GO:BTZ complex by FTIR spectroscopy was al- (at 0.175 μg BTZ) (Figure 4A). Anatomical T2- weighted MRI was most the same as the GOc revealing the existence of OH, C=O, conducted at 13 days post inoculation (pre-treatment) to establish C=C, and C–O–C functional groups, with new contributions at the size and position of the tumor, then again at days 5 and 12 1527 and 1193 cm−1 ascribed to C=C/C=N and C–B stretching following intratumoral injection. No change, in contrast, was ob- respectively,[45] as well as an increase of the peaks at around 2850– served in mice treated with GOc , whereas a clear impact on the 2950 cm−1 related to C–H vibrations (Figure S3E, Supporting In- tumor was observed as a defined hypointensity extending from formation). Interestingly, the shift of the C=O peak in the com- the site of injection in the GO:BTZ treated group, which is sug- plex in comparison with parent BTZ suggests the hydrogen bond- gestive of drug-induced necrosis (Figure 4B). While no signifi- ing between both entities as reported previously in the formation cant change in the tumor volume was calculated (Figure 4C), fur- of GO and doxorubicin complexes.[46] ther histological analysis of this phenomenon present 12 days Further characterization was obtained by dynamic light scat- after treatment, provided consistent evidence across all mice of a tering (DLS) and zeta potential measurements.[47] DLS was ex- GO:BTZ-induced necrotic zone which was largely devoid of nu- clusively used to evaluate the bulk colloidal characteristics of the clei (Figure 4D and Figure S5, Supporting Information). Despite suspension over time (Figure S3F, Supporting Information). The this prominent effect, endpoint tumor growth was not signifi- surface charge of GO nanosheets before and after BTZ complexa- cantly inhibited by GO:BTZ delivery (Figure 4E), potentially due tion remained negative. Generally speaking, there were no signif- to the use of a low-volume approach in a large established tumor. icant changes after 7 days for both size and surface charge of the To understand the kinetics of this GO:BTZ-induced cytotoxic complexes in water suggesting the formation of a colloidally sta- response we further evaluated this effect at earlier time points ble complex. Furthermore, we evaluated the bulk colloidal charac- (4, 24, and 96 h) following administration of either the GO:BTZ teristics of the GOc and GO:BTZ complexes suspended in serum complex or the equivalent dose of BTZ (Figure S6A, Supporting (10% FBS) for 24 h (Figure S4, Supporting Information). The Information). The presence of this cytotoxic core extending from bulk colloidal properties of both GOc and GO:BTZ remained sim- the injection site became apparent by 24 h post-injection and fur- ilar and in suspension, with some larger agglomerates forming ther developed over the next 48 h consistent with the kinetics for by 24 h in serum as observed by AFM, presumably due to the the translocation of the material observed previously (Figure S6B, serum protein coating of the complex. Taken together, these data Supporting Information). Notably, this effect was not visible in confirm the non-covalent complexation of BTZ on the surface of the free BTZ-treated tumors. Taken together, these data provide GO nanosheets and the formation of a stable complex. proof of concept for GO as a delivery system for chemotherapy drugs in an in vivo GBM model and warrant further investiga- tion. 2.3. GO:BTZ Complex Retains Cytotoxic Activity In Vitro To evaluate the cytotoxicity of BTZ complexed with GO in vitro, 2.5. GO:BTZ Complex Exhibits Higher Cytotoxic Activity in an we used a patient-derived GBM cell line (U87) and a mouse- Aggressive Immunocompetent Tumor Model derived GBM cell line (GL261).[28,48,49] Cell counts using Trypan blue exclusion were performed demonstrating the sensitivity of As the xenograft model did not illustrate a complete and sus- both GBM cell lines to BTZ with IC50 = values of 7.2 nM and tained tumor clearance after GO:BTZ intervention, we decided to 12.2 nM for GL261 and U87-MG respectively (Figure 3A). This explore this response further in an immunocompetent syngeneic observation was also supported by the LDH assay (Figure 3B) and model. The murine GL261 glioblastoma model exhibits a more cell morphology analysis (Figure 3C). To ensure that GO com- rapid tumor growth, retains an intact adaptive immune system plexation with BTZ did not inhibit BTZ function, we treated U87- and thus more closely recapitulates the cellular populations MG and GL261 glioma cells with increasing doses of GO:BTZ and invasive nature of clinical GBM[51] (Figure S7, Supporting (based on BTZ molarity) under the same conditions as previously Information). Furthermore, based on our in vitro cytotoxicity described. GO:BTZ and BTZ alone showed comparable viability assays, GL261 cells show increased sensitivity to BTZ compared curves without significant differences (Figure 3D). In contrast, to U87 (Figure 3A–C). We initially sought to confirm that the GOc showed no impact on cell viability, demonstrating that the effects previously observed in U87 xenograft could be repro- cytotoxic activity is attributed to the presence of BTZ on the sur- duced in this syngeneic model. GL261 cells were inoculated in face of the GO:BTZ complex. Overall, these results demonstrate the striatum and on day 9 after implantation, mice were injected Adv. Healthcare Mater. 2023, 12, 2201968 2201968 (5 of 13) © 2022 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH 21922659, 2023, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/adhm.202201968 by Readcube (Labtiva Inc.), Wiley Online Library on [24/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.advhealthmat.de Figure 3. GO:BTZ retains cytotoxic potential in vitro for both GL261 and U87-MG cells. A) Cell viability graphs of U87-MG (left side) and GL261 (right side) 24 h after treatment with free BTZ at 10 pM to 100 μM determined by live cell counting and corroborated by B) LDH assay. Data presented as mean ± SD % cell viability relative to control (n = 3). C) Representative bright field images taken 24 h post-treatment. Scale bar, 100 μm. D) Cell viability of U87-MG (left side) and GL261 (right side) 24 h post-treatment with GO:BTZ complex, free BTZ, GOc , or 15% DMSO. Data presented as mean ± SD % cell viability relative to control (n = 3). No statistical significance between the BTZ and GO:BTZ groups (Two-way ANOVA, Tukey’s multiple comparison test). intratumorally with GOc , free BTZ, or GO:BTZ (at 0.175 μg BTZ) ment significantly reduced the tumor volume compared to these (Figure S8A, Supporting Information). Histological analysis of two control groups (Figure S8D, Supporting Information). the brains on day 6 after treatment illustrated that GO:BTZ treat- We followed this up with a longitudinal measurement of the ment induced a pronounced tumor clearance effect extending response using bioluminescence imaging. Mice were again inoc- outward from the initial injection site (Figure S8B, Supporting ulated with GL261-luc cells followed by intratumoral intervention Information). Staining of nuclei with DAPI confirmed that this on day 7 post-inoculation (Figure 5A). There was a clear and sig- cytotoxic effect was associated with necrosis with the absence nificant reduction in bioluminescence signal in mice treated with of nuclei within the treated tumor area (Figure S8C, Supporting GO:BTZ compared to dextrose vehicle and free BTZ groups on Information). This effect was not detected in the free BTZ or GOc - both day-4 and day-8 post intratumoral injections (Figure 5B,C). treated tumors and this is further corroborated by the measure- In agreement with in vivo imaging, a remarkable area of tu- ment of tumor volumes which demonstrated that GO:BTZ treat- mor necrosis in GO:BTZ treated tumors was further observed Adv. Healthcare Mater. 2023, 12, 2201968 2201968 (6 of 13) © 2022 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH 21922659, 2023, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/adhm.202201968 by Readcube (Labtiva Inc.), Wiley Online Library on [24/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.advhealthmat.de Figure 4. Evaluation of the biological activity of GO:BTZ in vivo in U87 gliomas. (A) Experimental schematic; Athymic nude mice were implanted with 1 × 105 (1 μl) U87-luc cells into the right striatum. MRI was conducted at 13 days post inoculation for a pre-treatment (baseline) and 5 and 12 days following intratumoral delivery of GOc (1 μl, 0.9 mg mL−1 ; n = 5) or GO:BTZ (0.175 mg ml−1 , BTZ, n = 5). B) Coronal T2 -weighted MRI images showing the progression of U87 glioma growth and the effect of intratumoral delivery of GOc (upper panels) and GO:BTZ (lower panels), 5 and 12 days post- treatment. Arrows highlight areas of hypointensity within the tumor following GO:BTZ treatment that is not observed following GO delivery. Scale bar, 2 mm. C) Volumetric quantification of U87 tumors from MRI scans of GOc (n = 5) and GO:BTZ (n = 5) treated mice, after subtracting the hypointensity area. Data are presented as mean tumor volume ± S.E.M. No statistical significance between the GOc and GO:BTZ groups (Two-way analysis of variance- ANOVA, Sidak’s multiple comparisons test). D) Representative H&E images showing the effect of intratumoral delivery of GOc and GO:BTZ on day 12 post-treatment. Scale bars: 500 μm and 100 μm. E) Quantification of tumor volume based on H&E data after correction for the necrotic zone. Data presented as mean ± SD. No statistical significance after the unpaired Student’s t-test. by histological analysis of brains from day 8 post-injection (Fig- hicle control group, to 25 days in the GO:BTZ treated group ure 6A,B). While tumor volume reconstruction (without correc- which was also increased beyond that achieved by free BTZ (21.5 tion for the necrotic area) did not show any significant differ- days) (Figure 6E). Importantly, GO:BTZ-treated animals showed ence between free BTZ and GO:BTZ groups (Figure 6C), us- no signs of toxicity in relation to bodyweight changes during the ing tissue structure-based classification (Figure S9, Supporting acute treatment phase (Figure S10C, Supporting Information) Information)[64] we confirmed that the percentage of necrosis and maintained their weight longer than animals in the con- was significantly higher in the GO:BTZ group compared to free trol treatment groups (Figure S10D, Supporting Information). BTZ (Figure 6D) demonstrating an enhanced cytotoxic effect. Fi- Histological analysis of mice at the time of sacrifice illustrated nally, we conducted a further study to understand the longevity that tumor growth at the treatment site (striatum) remained rel- of this treatment response. As before, treatment with GO:BTZ atively suppressed, however outgrowth of tumor tissue superfi- significantly reduced the bioluminescence signal by day 4 post- cial to this in the uninjected area within the cortex (originating treatment (Figure S10A,B, Supporting Information) which trans- from the needle tract) was able to progress (Figure S10E, Support- lated to an increase in median survival from 17 days in the ve- ing Information). Notably, an amount of material did appear to Adv. Healthcare Mater. 2023, 12, 2201968 2201968 (7 of 13) © 2022 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH 21922659, 2023, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/adhm.202201968 by Readcube (Labtiva Inc.), Wiley Online Library on [24/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.advhealthmat.de Figure 5. Enhanced tumor-suppressing activity of GO:BTZ in GL261 glioma. A) Experimental schematic for the in vivo analysis. C57Bl/6 mice were implanted with 5 × 104 (1 μl) GL261-luc cells into the right striatum. BLI was conducted at 6 days post inoculation (i.c) for a pre-treatment (baseline) and 4 and 8 days following intratumoral (i.t.) delivery of 5% dextrose (n = 5), free BTZ (n = 7) or GO:BTZ complex (7 days post i.c.). B) Representative bioluminescence images of GL261 mice at baseline (6 days post i.c.), and 8 days following intratumoral delivery of 5% dextrose, free BTZ, or GO:BTZ. C) Fold change in bioluminescence relative to the pre-treatment baseline across all treatment groups (dextrose, free BTZ, or GO:BTZ) on 4 and 8 days post-treatment. Two-way ANOVA, Sidak’s multiple comparisons test (* p ≤0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001). Adv. Healthcare Mater. 2023, 12, 2201968 2201968 (8 of 13) © 2022 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH 21922659, 2023, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/adhm.202201968 by Readcube (Labtiva Inc.), Wiley Online Library on [24/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.advhealthmat.de Figure 6. Elevated cytotoxic activity of GO:BTZ in GL261 glioma mice. A) Representative H&E images across treatment groups 8 days post all treatment (5% Dextrose [n = 5], free BTZ [n = 7], and GO-BTZ complex [n = 7]). Scale bar, 500 μm. B) High magnification H&E images from GO-BTZ treated brain tumor (8-day post-treatment). Scale bar, 500 μM. Zoomed images illustrating different areas of GO-BTZ treated tumor. Scale bar, 100 μM. C) Tumor volume at 8 days post-treatment with 5% dextrose (n = 5), GO-BTZ (n = 7), and free BTZ (n = 7). D) Necrotic area as a percentage of GL261 tumor area determined via orbit image analysis. Data presented as mean ± SD. Ordinary one-way ANOVA, Tukey’s multiple comparisons test (****p ≤ 0.0001). E) Survival analysis of GL261-luc bearing mice treated with 5% dextrose, GO, free BTZ, or GO:BTZ complex (n = 10–11). Day of treatment highlighted with red arrow. Statistical differences shown in index. Log rank (Mantel–Cox) test (***p ≤ 0.001, ****p ≤ 0.0001). Adv. Healthcare Mater. 2023, 12, 2201968 2201968 (9 of 13) © 2022 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH 21922659, 2023, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/adhm.202201968 by Readcube (Labtiva Inc.), Wiley Online Library on [24/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.advhealthmat.de persist in the necrotic area induced by GO:BTZ, which may limit which maintained biological activity while allowing for gradual its translocation in comparison to bare GO and restrict the capac- detachment of the drug from GO locally throughout the tumor ity for GO:BTZ to reach tumor areas further from the initial injec- area where GO has translocated. Furthermore, in this study, we tion site (Figure S10E, Supporting Information and Figure 6B). identified differences in sensitivity between U87 and GL261 or- While these results are encouraging, the observation that the an- thotopic models, which highlights the requirement to consider imals still eventually succumb to the tumor burden even after different chemotherapeutics for a heterogeneous patient popula- GO:BTZ treatment highlights limitations associated with single tion, as is the case in human GBM. Indeed, the versatility of ex- intratumoral injection and warrants further optimization of this ploiting non-covalent interactions of drugs with GO nanosheets nanomaterial-enabled treatment strategy. provides significant advantages when considering adaptation in this scenario. We hypothesize that GO could be used as a univer- 3. Discussion sal platform to interact and deliver various drugs or combinations to improve therapeutic responses or rapidly adapt to treatment- In this study, we utilized GO nanosheets non-covalently com- resistant tumors. plexed with BTZ as a novel, single dose, intratumorally admin- Despite the enhanced anti-cancer effects achieved here, the istered therapy, which achieved an enhanced anti-cancer activity eventual overgrowth of residual tumors beyond the injected treat- compared to the free drug in two orthotopic mouse models of ment site with this approach is a limitation that is shared by al- glioblastoma. ternative preclinical and clinical locally applied therapeutic ap- Following the administration of GO nanoparticles into a hu- proaches in GBM. Future investigations may be able to further re- man U87 xenograft orthotopic mouse model we observed an out- solve this through MRI-guided intratumoral injections to ensure standing time-dependent diffusion effect throughout the tumor more effective tumor targeting at different sites. In addition, the area without exiting the tumor border, confirming our previous current nano-chemotherapeutic approach described here could findings.[27] The high penetrance of this small, thin (1–2 nm be further evaluated in combination with other therapies, or with thick) nanomaterial throughout the tumor area highlighted its adaptation for the post-resection environment that could ulti- potential as a drug-delivery system. Several studies have previ- mately provide a more long-lasting therapeutic benefit for pa- ously investigated graphene-based materials for the loading and tients with GBM. delivery of chemotherapeutics including doxorubicin, cisplatin, and BTZ.[39,52-56] The majority of these studies investigated the ef- fects of their graphene-based delivery system in 2D or 3D cell cul- 4. Conclusion tures that do not recapitulate the complex tissue and cellular in- In this study, we demonstrated that thin GO nanosheets can be teractions of these nanomaterials in the in vivo setting. One study effectively non-covalently complexed with the chemotherapeutic did explore intravenous administration of transferrin functional- drug bortezomib which provided a pronounced tumor necrotic ized GO nanoparticles for doxorubicin delivery in a C6 glioma effect in vivo in two distinct orthotopic glioblastoma tumor mod- rat model.[57] However, the administration of multiple doses was els. These findings suggest that GO could be more widely applied necessary to achieve a modest tumor inhibition due to minimal as a delivery platform for chemotherapeutic drugs to achieve a accumulation of drug at the tumor site with the majority of in- higher local drug concentration when administrated locally. Over- jected dose accumulating in off-target organs. This highlights the all, this work highlights a promising therapeutic strategy that can challenges associated with systemic administration for intracra- be used in combination with other therapies. nially located tumors. Indeed, while intracranial administration is not the primary drug administration approach currently used in the clinic it has been identified as a potential alternative ap- 5. Experimental Section proach with clear benefits in regards to avoiding the off-target side effects,[37,58,59] and could be further improved using nanopar- Reagents: Bortezomib (BTZ) was purchased from (Stratech, UK) with a purity of over 99.77%. Cell culture reagents and chemicals for the pro- ticles that distribute widely throughout but are retained within duction of GO were purchased from Sigma-Aldrich (Merck, UK) unless the tumor as we observed here. Thus, we rationally utilized GO otherwise stated. Additional reagents used were obtained from commer- as a delivery platform to introduce a non-classical chemothera- cial suppliers. peutic following simple intratumoral administration. Synthesis of Graphene Oxide (GO): Biological-grade GO was synthe- The dose of bortezomib in the present study is orders of mag- sized using a modified Hummers’ method previously described. Briefly, nitude below that used for systemic administration in other pre- graphite powder was mixed with sodium nitrate, followed by dropwise addition of sulphuric acid. Subsequently, potassium permanganate was clinical investigations (30–40 μg)[37,60] and was able to achieve effi- added to the mixture. Then, water was added to the solution, keeping the cacy at a lower dose than was previously reported for intratumoral temperature at 98 °C. After that, hydrogen peroxide was added to the mix- administration of BTZ in orthotopic GBM models highlighting ture to stop the reaction. The purification of the GO was performed under the advantages of utilizing a nanoscale delivery system.[37,61] BTZ endotoxin-free conditions by several centrifugation and washing steps. GO has previously been encapsulated in other nanoscale systems, sheets with lateral dimensions ranging from 25 nm to 1.9 μm were pre- such as CuS/carbon dot nanocomposites or dendrimers aiming pared by sonicating for 5 min and consequently purification to remove the for targeted delivery following systemic administration into dif- largest debris. (Table S1, Supporting Information). Preparation of GO:BTZ Complexes: The complexation of GO and BTZ ferent types of cancers, including GBM, again requiring rela- was performed through non-covalent interactions between both entities. tively high doses due to limited tumor localization.[61-63] In ad- Initially, bortezomib was dissolved in non-pyrogenic water containing dition to the doses required, one advantage of a GO nanosheet- 0.001% (w/w) acetic acid (Sigma-Aldrich). Then, 1 mg ml−1 GO suspen- based approach is the rapid non-covalent complexation of BTZ sion was mixed with the BTZ solution at a 10:6 mass ratio (w/w) followed Adv. Healthcare Mater. 2023, 12, 2201968 2201968 (10 of 13) © 2022 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH 21922659, 2023, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/adhm.202201968 by Readcube (Labtiva Inc.), Wiley Online Library on [24/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.advhealthmat.de by incubation for 1 h under moderate agitation (1 G). Afterward, the so- tured via an AxioCam ERc5s camera with ZEN light software. Imaging con- lution was centrifuged for 50 min (21 000 g) at room temperature and ditions were kept consistent throughout. washed three times with water to remove any unbound BTZ, and the ob- Animals: All animal experiments were performed at the University of tained product was re-dispersed in non-pyrogenic water to obtain a ho- Manchester (UK), in accordance with the Animals (Scientific Procedures) mogenous and stable suspension. For biological experiments, following Act 1986 (UK), approved by the University of Manchester Ethical Review a single washing step as described above, the GO:BTZ complex was re- Committee and under a UK Home Office Project License P089E2EOA suspended in either water for injection (Fannin, Greece) with 5% dextrose and following the ARRIVE 2.0 guidelines. Animals were housed in groups (Sigma-Aldrich) for intratumoral injections, or water for cell culture (Ther- within ventilated cages with ad libitum access to food and water. Female moFisher, UK) and re-suspended in cell culture medium for the in vitro athymic nude-Fox1nu (Envigo, UK) and C57/BL6 (Envigo, UK) mice were experiments. allowed to acclimatize to the facility for at least one week prior to any pro- Physicochemical Characterization of GO and GO:BTZ Complex: Ab- cedure. sorbance spectra were acquired with a UV–vis–NIR Jasco V-780 spec- Intracranial Inoculation of Glioma Cells: Female athymic nude mice for trophotometer, at the ICMAB Spectroscopy Facility, at room temperature the U87 model (6–8 weeks old) and female C57/BL6 mice (8–9 weeks using samples prepared in water dispersion. The accuracy of the ab- old) were anesthetized using isoflurane (2.5% induction and 1–2% main- sorbance of UV–vis spectrophotometer was tested using the certified tenance in medical oxygen, at a rate of 1.5 L min−1 ) and placed on a stereo- UV–vis Standard 1 (potassium dichromate, 60.06 mg L−1 in sulphuric tactic frame. Prior to incision, animals received 0.1 mg kg−1 of buprenor- acid, 0.01N, Batch HC909064), complying with the European Phar- phine (Buprenex, Reckitt Benckiser, UK). A midline incision was performed macopeia (Ph. Eur.) specifications. X-Ray photoemission spectroscopy to expose the cranium and a 0.7 mm borehole was drilled (Fine Science measurements (XPS) were obtained using a Phoibos 150 (SPECS, GmbH) Tools, Canada) above the right striatum at 0.0 mm anterior and 2.3 mm lat- electron spectrometer equipped with a hemispherical analyzer, at the eral from bregma. A 10 μl Hamilton syringe (SYR10, Hamilton, USA) fitted ICN2 Photoemission Spectroscopy (XPS&UPS) Facility, operating under with a 26-gauge blunt needle (Hamilton, USA) was lowered to 3 mm below ultrahigh-vacuum conditions, and with an Al K𝛼 (h𝜈 = 1486.74 eV) X-ray the cortical surface and slowly withdrawn 0.6 mm such that the injection source. Samples were prepared using the drop-casting method. The took place at 2.4 mm depth. 1 × 105 U87-luc cells or 5 × 104 GL261-luc morphology of the materials deposited on freshly cleaved mica (Ted Pella) cells in 1 μl of PBS were injected slowly over 5 min at a rate of 0.2 μl min−1 . was determined by atomic force microscopy (AFM, Asylum MFP-3D, Post-injection the needle was kept in place for 3 min to minimize reflux Oxford instruments) in tapping mode and equipped with silicon probes and slowly withdrawn over 1 min to minimize any injury. The skin incision (Ted Pella) with a resonance frequency of 300 kHz and a nominal force of was closed with 5-0 coated vicryl sutures (Ethicon, UK) and animals were 40 N m−1 . allowed to recover in a heated environment. Cell Lines and Cell Culture Conditions: Human glioma cell line U87- Intratumoral Injection: Mice underwent intratumoral injection with MG and U87-luc was obtained from the American type culture collec- 1 μl vehicle (5% dextrose), GO (0.9–1 μg), BTZ (0.175 μg), or GO:BTZ tion (ATCC) and were tested free from mycoplasma contamination. Cells (containing 0.175 μg BTZ) in 5% dextrose at the time specified in the fig- were cultured in normal minimum essential medium eagle (MEM), with ure legend for each experiment. Mice were anesthetized and prepared for L-glutamine, Earle’s salts, (2.2 g L−1 ) sodium bicarbonate (added from the stereotaxic surgery as described above. The original incision was reopened manufacturer), and 10% fetal bovine serum (FBS; Gibco, Thermo Fisher and a 33-gauge needle connected to a 10 μl Hamilton Neuros syringe was Scientific, UK) and 1% antibiotics (penicillin and streptomycin, PenStrep) passed through the original borehole to a depth of 2.4 mm. 1 μl of material at 37 °C, 5% CO2 . The murine GL261-luc cell line was kindly provided or vehicle control was injected over 5 min (0.2 μl min−1 ). Post-injection the by Prof. Brian Bigger (The University of Manchester, UK) and was cul- needle was kept in place for 3 min to minimize reflux and slowly withdrawn tured in RPMI-1640 medium supplemented with 10% FBS and 1% peni- over 1 min. The skin incision was closed with 5-0 coated vicryl sutures and cillin/streptomycin. animals were allowed to recover in a heated environment. LDH Cytotoxicity Assay: The release of lactase dehydrogenase (LDH) In Vivo Bioluminescence Imaging (BLI): Tumor-bearing mice were was measured by using a CytoTox96 nonradioactive cytotoxicity assay kit anesthetized with 1.5% isoflurane followed by intraperitoneal injection of following the manufacturer’s guidelines (Promega, G1780). Cells were 150 mg kg−1 mouse D-luciferin (15 mg ml−1 ; Promega, UK) in PBS. Af- seeded in 12-well or 24-well plates (Costar, UK) and allowed to reach 75% ter 8 min, bioluminescence signals were detected using sequential imag- confluence before the medium was aspirated and replaced with treatments ing (10 measurements at 2 min intervals) with an in vivo imaging system followed by incubation for 24 h at 37 °C with 5% CO2 . Following incuba- (IVIS Lumina II, PerkinElmer, UK). Images were analyzed with Living Im- tion, cell supernatant was transferred to a 96-well plate and centrifuged at age software (version 4.7) (PerkinElmer, UK). 4000 rpm for 20 min. 50 μl of each conditioned media-supernatant was MRI Acquisition and Analysis: MRI was conducted using a 7 Tesla mag- transferred to a separate well in a 96-well-plate and an equal volume of net connected to a Bruker Advance III console (Bruker Biospin Ltd, UK). substrate solution was added. Samples were incubated at room tempera- Mice were placed in a magnet capsule with a cylindrical surface coil and im- ture for 20 min before measurement of the absorbance at 𝜆 = 492 nm us- aged under isoflurane anesthesia (3% induction and 1–2% maintenance ing a Fluostar Omega microplate reader (BMG Labtech). Cytotoxicity was in medical oxygen at a rate of 1.5 L min−1 ). The respiratory rate was moni- measured by the release of LDH and was expressed as the percentage via- tored throughout imaging and anesthetic level was controlled based on the bility relative to control based on n = 3 biological replicates per condition. respiratory parameters (50–70 bpm). After localizing imaging on three or- Control conditions: 15% DMSO –positive control, untreated cells- nega- thogonal axes, the whole brain was imaged using T2 weighted MRI: Rapid tive control. Data are reported as a percentage release of LDH compared Acquisition with Relaxation Enhancement (RARE) pulse sequence, repe- to the untreated control. tition time = 2200 ms, echo time 33 ms, RARE factor 8, the field of view Cell Counting Cytotoxicity Assay: As above, cells were seeded 1 day be- (FOV) 30 × 30 mm matrix size 512 × 512, 17 contiguous slices, thickness fore treatment and allowed to reach 75% confluence. Following incuba- 0.8 mm, averages = 5. Tumor volumes were determined using MRIcron tion with the different treatments, for 24 h, at 37 °C with 5% CO2, cells software (National Institutes of Health, NIH, Bethesda, USA). The tumor were detached using 0.05% trypsin-EDTA for 5 min, followed by the addi- area was delineated for each coronal slice and the total tumor volume was tion of complete media containing 10% of FBS. The cell solution was then generated from the acquired measurements. combined at a 1:1 ratio with 0.4% Trypan blue, and single live cells were Tissue Processing and Staining: At the end of each experiment, tumor- counted using a haemocytometer- twice per sample with three biological bearing mice were anesthetized with 2.5% isoflurane and culled by cardiac replicates per condition. As controls, 15% DMSO and untreated cell sam- perfusion with 2 mM EDTA in PBS, followed by 4% PFA in PBS. Brains were ples were used. Data are reported as a percentage of live cells compared removed and fixed overnight at 4 °C and later placed in 30% sucrose in PBS with untreated cells. for at least 24 h. The brains were snap-frozen in cold isopentane (−40 °C) Imaging: Cells were imaged using a PrimoVert microscope (ZEISS) and coronal sections (20 μm thickness) were taken using a cryostat (Leica using a Primo Plan-ACHTOMAT 10X/0.25 Ph1 lens and images were cap- CM1950, Leica Biosystems, Germany). Adv. Healthcare Mater. 2023, 12, 2201968 2201968 (11 of 13) © 2022 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH 21922659, 2023, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/adhm.202201968 by Readcube (Labtiva Inc.), Wiley Online Library on [24/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.advhealthmat.de Sections were stained with haematoxylin and eosin (H&E) staining to (EP/S030719/1). The European Union Horizon 2020 Research and Inno- observe the histological characteristics of the tumor sections and deter- vation Programme under Grant Agreement no. 881603 (Graphene Flag- mine the tumor volume. Cryosections were left for 15 min to air dry before ship Core 3) also funded this work. The ICN2 is funded by the CERCA fixation with pure ethanol for 2 min. Slides were washed once with PBS programme, Generalitat de Catalunya, and is supported by the Severo for 5 min, and were placed in haematoxylin for 1 min. Slides were washed Ochoa Centres of Excellence programme by the Spanish Research Agency twice with water for 3 min each and were placed in 70% EtOH for 3 min. (AEI, grant no. SEV-2017-0706). The authors would like to acknowledge Following dehydration, slides were placed in eosin solution (1% eosin in the ICN2 Advanced Electronic Materials and Devices Group (Prof. Jose A. 95% alcohol) for approximately 40 s. This was followed by three washes Garrido) for access to the AFM and Raman instrumentation. The authors in 100% alcohol for 3 min each, and slides were placed in two changes of would also like to thank Dr. Cyrill Bussy for assistance with mapping Ra- Xylene for 2 min each. Finally, DPX mount was used to mount coverslips man imaging, the Bioimaging core facility, Biological Services Facility, His- and slides were then left to dry overnight at room temperature. Slides were tology core facility, and Preclinical MRI facility at the University of Manch- scanned using a 3D Histech Panoramic 250 slide scanner. ester for technical support and access to equipment. Histological Evaluation of Tumor Growth: H&E stained sections were imaged using a Panoramic 250 slide scanner (3D Histech, Hungary) and analyzed using 3DHISTECH Case Viewer software version 2.6. Initially, tu- mor diameter was measured in each section so as to identify the maximal Conflict of Interest tumor area. Subsequently, the height and width of the tumor area were The authors declare no conflict of interest. measured and the volume was calculated using the following formula: V = (W2 × H)∕2 (1) Author Contributions *The above formula was chosen after the comparison with V = (W × H × P.S.S. and M.S. contributed equally to this work. K.K. and P.S. initiated and L)/2 where length indicates the tumor thickness based on the slices cut designed the study. K.K. and T.K. coordinated the study. P.S. conducted and slice thickness. most of the in vivo experiments and in vivo imaging. M.S. assisted in all Raman Mapping: U87 brain cryosections were used for Raman Spec- the in vivo experiments and performed the in vitro experiments. T.K. per- troscopy imaging in which GO was identified by a Raman spectrum of two formed in vivo experiments, imaging, and data analysis. A.M.G., L.M.A., distinct D and G bands at 1352 and 1594 cm−1 , respectively, and a less J.C.N., and N.L. developed the complexation protocol and performed the projecting 2D band at 2707 cm−1 . That spectrum was used as a finger- characterization of the complex. A.D. performed tissue processing and print to validate the presence of GO in the tumor area. Raman mapping assisted in image analysis. K.B. performed bioinformatics analysis for was performed using a HORIBA XploRA Raman microscope working with the quantification of the necrotic tissue and nanomaterial distribution. a 638 nm laser operating at 25% of power, passed through a 300 μm hole M.S., T.K., P.S., L.M.A., N.S., and K.K. contributed to the writing of the and a 100 μm slit. A 50x objective was used to collect the Raman spectrum manuscript. at each single pixel of the region of interest.[27] Immunofluorescence (IF) Staining: For immunofluorescence analysis, 20 μm cryo-sections samples were air-dried and fixed for 10 min in ice- cold acetone. After washing the samples with PBS, sections were incu- Data Availability Statement bated for 1 h with 1% bovine serum albumin and 5% donkey serum in The data that support the findings of this study are available in the sup- PBS-Triton X 0.2% to remove any non-specific binding. Rabbit anti-mouse plementary material of this article. IBA1 antibody (dilution 1:1000, Fujifilm, 019–19741 Wako) was incubated overnight at 4 °C, for staining activated macrophage and microglia cells. For secondary antibody staining, Alexa 488-conjugated donkey anti-rabbit IgG (dilution 1:1000, Abcam, ab150073) was used. Sections were washed Keywords and Prolong Gold medium with DAPI was added and covered with cover- slips. Images were taken with a Histec Pannoramic250 slide scanner. 2D materials, chemotherapy, glioblastoma, graphene, nanomedicine Statistical Analysis: Statistical analysis was performed using Graph- Pad Prism 8.1.2 (332). For the comparison of two groups, an unpaired Received: August 4, 2022 Student’s t-test was used and for comparison of three or more groups, Revised: October 3, 2022 an ordinary one-way ANOVA (Tukey’s multiple comparison test) or two- Published online: November 11, 2022 way ANOVA (Tukey’s and Sidak’s multiple comparisons test) were utilized. 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