Carboxylated Graphene Quantum Dots-Mediated Photothermal Therapy Enhances Drug-Membrane Permeability, ROS Production, and the Immune System Recruitment on 3D Glioblastoma Models.

doi:10.21203/rs.3.rs-2312481/v1

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Research Article

Carboxylated Graphene Quantum Dots-Mediated Photothermal Therapy Enhances Drug-Membrane Permeability, ROS Production, and the Immune System Recruitment on 3D Glioblastoma Models.

https://doi.org/10.21203/rs.3.rs-2312481/v1

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published 24 Feb, 2023

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Graphene quantum dots (GQDs) are biocompatible nanoparticles employed in biomedical field, thanks to their size and photophysical properties. GQDs have shown the capability to cross biological barriers, including the blood-brain barrier, which makes them promising agents for brain diseases therapy. It has been shown that surface-functionalized GQDs enhance membrane fluidity and intracellular uptake, exerting a synergistic effect with antitumor drugs at subtherapeutic doses. Here we tested GQDs effects in combination with chemotherapeutic agents doxorubicin and temozolomide, on a complex 3D spheroid model of glioblastoma. We observed that the capability of GQDs to absorb and convert near-infrared light into heat is a key factor in membrane permeability enhancement on 3D model. This non-invasive therapeutic strategy named photothermal therapy (PTT), combined to chemotherapy at subtherapeutic doses, significantly increased the effect of antitumor drugs, by reducing tumor growth and viability. Furthermore, the increase in membrane permeability due to GQDs-mediated PTT enhanced the release of reactive oxygen species with strong migration of the immune system towards irradiated cancer spheroids. Our data indicate that the increase in membrane permeability can enhance the efficacy of antitumor drugs at subtherapeutic doses against glioblastoma, reducing side effects and directing immune response, ultimately improving quality of life for patients.

Graphene

Glioblastoma

Photothermal

Phtotodinamic

3D-bioprinting

Graphene quantum dots (GQDs) are small semiconducting nanoparticles with a size around 10 nm.¹ Thanks to their great photophysical properties, GQDs have widely been exploited in several fields.^1,2 GQDs have been employed as bioimaging molecules, carriers for drug delivery and as theranostic nanomolecules.^3,4 Thanks to their capability of easily crossing biological barriers, particularly the blood-brain barrier, GQDs are also being used in the field of neuroscience.^5,6 In our preivous works, we have tested the effect of surface-chemically functionalized biocompatible GQDs in combination with chemotherapy on a traditional 2D model of glioblastoma, the most lethal and aggressive brain cancer.^7,8 We demonstrated that GQDs functionalized with carboxylic acid could synergistically enhance chemotherapy against glioblastoma.⁸ However, an issue that still must be overcome is the lack of reliable experimental models, different from standard 2D cultures, to provide representation of tumor’ behavior.⁹ We observed that this synergystic tumor killing efficacy of GQDs and chemotherapy was reduced on 3D glioblastoma spheroids indeed.¹⁰ We therefore expolited another photophysical property of GQDs: the capability of absorbing near-infrared light and convert it into heat, namely phototherapy. Phototherapy, which comprises photothermal therapy (PTT) and photodynamic therapy (PDT), is a non-invasive treatment that can either sensitize cells to chemotherapy by inducing a local increase of temperature mediated by light and that can induce a local increase in the production of reactive oxygen species, respectively.^11–13 Here, we tested GQDs-mediated combined chemotherapy and phototherapy on a reliable 3D tumor spheroid model of glioblastoma. For this purpose, we focused on GQDs functionalized with carboxylic acid groups.¹⁴ We demonstrated that GQDs exert a strong killing effect as a chemo-photosensitizer agent on tumor spheroids when combining chemotherapy with an approach based on hyperthermia. We observed that the increase in efficacy of tumor killing was related to a strong increase in membrane permeability induced by GQDs and, in particular, by irradiated GQDs. We therefore proposed a mechanism of action in which the increase in membrane permeability induced by GQDs improves intracellular uptake of antitumor drugs inside glioblastoma cells.^7,8,15 Another relevant approach for the treatment of glioblastoma is immunotherapy^16–18 due to the paucity of infiltrated leukocytes and downregulation of immunological response, facilitated by intratumoral hetereogeneity.¹⁶ The purpose of immunotherapy is to reactivate the immune system, stimulating it to recognize tumor-associated antigens, in order to directly target tumor region, minimizing the non-specific inflammatory response. For this purpose, PTT and PDT can be auxiliary approaches for immunotherapy, due to the high release of tumor-associated antigens and the production of reactive oxygen species, which induce a strong activation of the immune system.^19,20 Here we observed a strong migration of immune cells towards cancer region after irradiation with a near-infrared laser in the presence of GQDs. Taken together, our findings on a reliable 3D tumor model demonstrate the crucial clinical relevance of GQDs, which could take part in the fight against brain diseases thanks to their multifunctional chemo-photosensitizing effect, and to their ability of stimulating the migration of immune cells towards cancer region, exerting immunotherapy enhancement.

Characterization of GQDs

GQDs were characterized by DLS, TEM, absorbance, fluorescence intensity ATR-FTIR. Results of spectroscopic and microscopic characterization are reported in Fig. 1. TEM images depicted an average diameter of GQDs around 10 nm (Fig. 1A), which is in accordance with DLS analysis, that showed a broad size distribution having a hydrodynamic radius peaked at 6 nm (Fig. 1B). Absorbance measurements were performed by recording optical density (OD) from 230 nm to 800 nm (Fig. 1C). We observed a peak at 350 nm, which is typically due to π − π^∗ transitions, and it is associated with the presence of lone pairs within oxygens. Fluorescence intensity was recorded by exciting from 260 to 480 nm with a step size of 20 nm and reading emission from 300 to 700 nm with a step size of 5 nm (Fig. 1D). GQDs showed an emission peak at 450 nm when excited at 320 nm. Surface chemical characterization was performed by ATR-FTIR (Fig. 1E). The infrared spectra of GQDs showed characteristic frequencies of the carboxylic acid group, such as a broad band around 3000 cm^− 1 for the O-H stretch, and a very strong band at 1705 cm^− 1 for the C = O stretch. Furthermore, we observed a peak at 1200 cm^− 1 due to epoxide C-O stretching vibrations. We then evaluated the biocompatibility of GQDs on the U87 human glioblastoma cell line, to confirm the high biocompatibility of these nanomaterials. We administered GQDs at different concentrations, ranging from 50 to 250 µg/mL. Results are reported in Fig. 1F. No evident cytotoxicity was depicted even at the highest tested concentration, highlighting the biocompatibility of bare GQDs.

Effect of Changes in Membrane Permeability Mediated by GQDs on 2D Model of Glioblastoma

We then moved to evaluate the effect of GQDs on changes in the permeability of cell membrane, to test whether these changes could improve the effect of two chemotherapeutic agents: temozolomide (TMZ) and doxorubicin (DOX). For this purpose, we administered GQDs to cells and, after incubation, we labeled cells with Laurdan.^21,22 This molecule is a fluorescent probe with a hydrophilic head and a hydrophobic tail that can be used to describe the lipid-phase state in membranes with high sensitivity. Membrane fluidity quantified by Laurdan is given by calculation of the generalized polarization (GP) index, which goes from − 1 (highest membrane fluidity) to + 1 (lowest membrane fluidity).²³ Representative Laurdan confocal microscopy images are reported in Fig. 2A, while quantification of the GP is shown in Fig. 2B. Data are reported as mean ± standard deviation. We observed a GP value of 0.48 ± 0.05 for control (untreated) U87 cells. Cells treated with GQDs had a GP value of 0.21 ± 0.02. Cells treated with GQDs showed a GP value significantly lower than control cells, highlighting a strong increase in membrane fluidity (p < 0.01, one-way ANOVA and Turkey post-hoc test). We then moved to test the combined effect of GQDs and the two chemotherapeutics, to test whether the increase of membrane permeability could augment the effectiveness of the antitumor drugs due to a higher intracellular uptake. First, we evaluated the concentratin of chemotherapeutic agent capable of inhibiting 50% of cell growth (namely IC50). Data for DOX are reported in Fig. 2C. We observed a reduction in cell viability corresponding to the IC50 at 2 µM. We then set the concentration of DOX in combination with GQDs at a subtherapeutic dose, which was half of the IC50 (1 µM). We incubated cells with GQDs at concentration ranging from 50 up to 250 µg/mL for 24 hours. After incubation, we removed GQDs from the supernatant, to avoid possible extracellular interactions between GQDs and antitumor drugs that coul affect the effectiveness of the latter. We then added DOX at half of its IC50. After further incubation, we measured cell viability. Results of the combined effect of GQDs and DOX are reported in Fig. 2D as % of death (1-viability). DOX and GQDs at 50 µg/mL depicted a cell mortality of 37 ± 7.1%. This reduction was similar to that of DOX alone. We observed a strong reduction in viability with the combined treatment of GQDs at concentrations higher than 50 µg/mL and DOX compared to the anticanced drug alone. Cells treated with DOX and GQDs at 100 µg/mL had a mortality of 48 ± 4.5%. Cells treated with DOX and GQDs at 200 µg/mL showed a mortality of 73 ± 5.1%. Finally, cells treated with DOX and GQDs at the highest tested concentration of 250 µg/mL had a mortality of 75 ± 3.2%. Mortality resulted significantly higher for cells treated with DOX and GQDs at the two highest tested concentrations (200 and 250 µg/mL, with a p < 0.01, one-way ANOVA and Turkey post-hoc test). To verify the hypothesis of a higher intracellular uptake of the antitumor drug, we evaluated the fluorescence intensity of DOX inside cells through confocal microscopy. Results of the fluorescence intensity are reported in Fig. 2E. We observed a significant increase in the fluorescence intensity of DOX inside cells at the two highest tested concentrations, in accordance with cell viability. Our data show a clear correlation between intracellular uptake of the anticancer drug and changes in membrane permeability due to the treatment of GQDs. Therefore, we moved to test the effectiveness of TMZ, since it is known to be the most widely used chemotherapy for patients with glioblastoma.^24–26 As for DOX, we first evaluated the IC50 of TMZ, which resulted to be 200 µM (Fig. 2F). We then proceeded with the same experimental protocol of DOX. We administered GQDs at the same previous concentrations, then after incubation we removed GQDs from supernatant and we dispensed TMZ at half of its IC50. Results are reported in Fig. 2G. As for DOX, no significant changes in mortality were pointed out after administration of GQDs at the lowest concentration of µg/mL when compared to TMZ alone (28 ± 3.5%). Cells treated with GQDs at 100 µg/mL and TMZ had an increase in mortality up to 47 ± 4.8%. GQDs at 200 µg/mL combined with TMZ increased mortality of cells to 71 ± 7.8%. Finally, cells treated with GQDs at 250 µg/mL and TMZ had a mortality of 73 ± 5.3%. As expected, the combined treatment of GQDs and TMZ resulted in a significant effect at the two highest tested concentrations of GQDs (p < 0.01, one-way ANOVA and Turkey post-hoc test). Our data strongly indicate that biocompatible GQDs can interact with cell membrane in a concentration-dependent manner. This interaction leads to an increase in membrane permeability, due to a higher membrane fluidity that we measured through GP. The changes in membrane permeability allow the entrance of the chemotherapeutic agent, significantly enhancing its efficacy (Fig. 2H).

Effect of the Combined Treatment of GQDs and Chemotherapy on a 3D Model of Glioblastoma

We have tested the effect of GQDs in combination with chemotherapy on a 2D model of glioblastoma. We demonstrated that GQDs could synergistically enhance chemotherapy against cancer. We explained the mechanism underlying the synergy between GQDs and antitumor drugs by measuring an increased membrane permeability of U87 cells. However, an issue that still must be overcome is the lack of reliable experimental models, different from standard two-dimensional cultures, to provide a faithful representation of tumor behavior.^27–29 For this purpose, we moved to test the enhancement of chemotherapy mediated by changes in membrane permeability induced by GQDs on a reliable 3D tumor spheroid model of glioblastoma. We monitored spheroid growth in terms of size and viability for a timespan of two weeks. Results are reported in Fig. 3. Spheroid growth was monitored in the timespan of two weeks for all treatments by acquiring bright field images over time (Fig. 3A). After formation of spheroids, we administered GQDs at 200 µg/mL. After incubation, we administered antitumor drugs. Results of the combined treatment of GQDs and DOX in terms of spheroid growth is reported in Fig. 3B. Data are normalized by the area at day 0 for each treatment. Control (untreated) spheroids increased their size up to 173 ± 10.4%. Spheroids treated with GQDs showed a similar increase in their size, reaching 152 ± 11.1%. The chemotherapeutic drug alone exerted a reduction in spheroid growth over time, decreasing to 78 ± 7.6% with respect to the initial size. Importantly, the most evident effect was exerted by coadministration of GQDs and DOX. Spheroids treated with the combination of DOX and GQDs had a reduction in their growth to 73 ± 2.8% indeed. Both treatments with DOX and GQDs combined with DOX resulted in a significant loss of tumor mass when compared to untreated spheroids (p < 0.01, one-way ANOVA and Turkey post-hoc test). However, despite experimental evidence on 2D model, no significant difference between the two groups was observed on spheroids. Results of cell viability at 7 and 14 days from administration of GQDs is reported in Fig. 3C as % of control spheroids. Spheroids treated with GQDs at 7 and 14 days did not show relevant loss in viability, remaining at 96 ± 7.4% and 93 ± 5.1% respectively. Spheroids treated with DOX alone at half of its IC50 had a reduction in cell viability up to 63 ± 10.3% and 43 ± 2.1% at 7 and 14 days respectively. Spheroids treated with the combination of GQDs and DOX depicted a loss of viability at 7 and 14 days up to 45 ± 7.6% and 28 ± 3.1% respectively. Both treatments with DOX alone or in combination with GQDs resulted in a significant loss in cell viability with respect to control (untreated) spheroids (p < 0.01, one-way ANOVA and Turkey post-hoc test). Although there was a clear decreasing trend, in accordance with data of tumor growth, indicating an enhanced efficacy of the antitumor drug in the presence of GQDs, we did not observe significant differences between the two treatment groups. As for the 2D model, we then moved to test the combined effect of GQDs and TMZ. Results of spheroid growth are reported in Fig. 3D. After two weeks, glioblastoma spheroids treated with TMZ had a reduction in their growth up to 87 ± 8.3%. When TMZ was combined with GQDs, spheroids reduced their growth to 83 ± 2.8%. Both treatments resulted to be significantly different with respect to control spheroids (p < 0.01, one-way ANOVA and Turkey post-hoc test). Even in this case, we did not observe significant differences between the two treatment groups. Results of cell viability after the treatment with TMZ and GQDs at 7 and 14 days is reported in Fig. 3E. Spheroids treated with the antitumor drug alone had a loss in viability to 75 ± 3.3% and 25 ± 1.2% at 7 and 14 days respectively. Spheroids treated with TMZ and GQDs showed a reduction in viability to 63 ± 6.1% and 17 ± 0.4% at 7 and 14 days respectively. Both treatments significantly reduced cell viability of spheroids (p < 0.01, one-way ANOVA and Turkey post-hoc test). As for DOX, despite the decreasing trend indicating an enhanced efficacy of TMZ in combination with GQDs, we did not observe significant differences between the two treatment groups.

Increase in Membrane Permeability of Glioblastoma Spheroids Through Photothermal Conversion Mediated by GQDs

We have tested coadministration of GQDs and antitumor drugs on a tumor spheroid, which allows reproducing a more reliable model of glioblastoma with respect to standard cultures. As a fact, several studies reported that most of the tests carried out on 2D models show a significant change in their outcomes when compared to to 3D cultures.^{10, 28–33} Therefore, a multidisciplinary approach for the treatment of glioblastoma, which guarantees effective tumor cell targeting and killing and is coupled with an efficient experimental model is mandatory. For this purpose, we exploited another feature of GQDs, which is their ability to absorb near-infrared (NIR) light and convert it into heat, exerting a photothermal effect.^{10, 34–36} This effect can be used in a therapeutic approach, namely PTT. PTT is a non-invasive technique that can be an intriguing auxiliary approach for chemotherapy. PTT is based on a local increase in temperature mediated by light-responsive molecules, which in turn exerts a cytotoxic effect on cancer cells. GQDs have widely demonstrated their high photothermal conversion.^34,37 Therefore, in this work, we used GQDs as chemotherapy enhancers and photothermal converting molecules (Fig. 4A). For this purpose, we used a near-infrared laser, with an emission wavelength of 808 nm. Characterization of the laser power is reported in Fig. 4B. Once characterized, we irradiated GQDs at all tested concentrations for 5 minutes at a power density of 6 W/cm², in accordance with previous data.^10,38 We found a concentration-dependent increase in temperature over time. GQDs at 50 µg/mL increased local temperaturefrom RT (25°C) up to 28°C after 5 minutes of irradiation, similarly to culture medium alone. GQDs at 100 µg/mL depicted a higher temperature raise, up to 38°C. GQDs at the two highest tested concentrations (200 and 250 µg/mL), showed a similar increase in temperature, up to 46 and 49°C respectively. Therefore, we performed further experiments by setting our working concentration of GQDs at 200 µg/mL. It has already been hypothesized that mild near-infrared irradiation can induce an augmented membrane permeability. Therefore, we tested the capability of GQDs to increase membrane permeability after photothermal conversion. For this purpose, we administered GQDs to glioblastoma spheroids and, after incubation, we irradiated them with the 808 nm laser. We then administered calcein to spheroids, in order to monitor its uptake (Fig. 4D). Results of calcein uptake are reported in Fig. 4E. Results are reported as fluorescence intensity of each group normalized by its intensity at the beginning of the experiment. After 30 minutes, fluorescence intensity of calcein in spheroids treated with GQDs and irradiated with near-infrared laser was 32 ± 1.6 times higher compared to t0. Fluorescence intensity of spheroids treated with only GQDs had an increase of 27 ± 1.3 with respect to t0. We observed a significant increase in fluorescence intensity of calcein in spheroids treated with GQDs and irradiated (p < 0.01, one-way ANOVA and Turkey post-hoc test). Taken together, our data suggest that GQDs can increase membrane permeability even in a reliable 3D tumor spheroid model through photothermal conversion.

Photothermal-Enhanced Chemotherapy Through GQDs on Glioblastoma Spheroids

We therefore repeated our experiment of chemotherapy enhancement on glioblastoma spheroids by implementing PTT. We administered GQDs and, after incubation, we added the antitumor drugs. We then irradiated spheroids with near-infrared light, and we evaluated the combined effect of PTT and chemotherapy in terms of spheroid growth and reduction of cell viability. Data are reported in Fig. 5 for both DOX and TMZ. We acquired bright field images of spheroids over time for all treatments (Fig. 5A), from which we evaluated spheroid growth in the same timespan of two weeks. Results of spheroid growth after the treatment with GQDs-mediated PTT and DOX is reported in Fig. 5B. Administration of GQDs and consequent PTT slackened spheroid growth to 115 ± 11.1% with respect to their initial size. Spheroids treated with photothermal-mediated enhancement of chemotherapy through GQDs had a critical reduction in spheroid growth to 40 ± 5.7%. Both GQDs-mediated PTT and photothermal-mediated enhancement of chemotherapy resulted significantly different from control untreated spheroids (p < 0.01, one-way ANOVA and Turkey post-hoc test). Importantly, the combination of PTT and chemotherapy resulted significantly more effective than the antitumor drug alone or coadministered with GQDs. Viability measurements furtherly confirmed the effectiveness of PTT and chemotherapy (Fig. 5C). We observed a reduction in viability of cells treated with GQDs and irradiated. This was particularly evident at 14 days from administration, which resulted in a loss of viability to 65 ± 7.1% with respect to control spheroids (p < 0.01, one-way ANOVA and Turkey post-hoc test). PTT through GQDs combined with DOX exerted a strong reduction at both 7 and 14 days from administration of GQDs, resulting in 33 ± 3.1% and 13 ± 3.2% respectively. Importantly, this combined treatment resulted significantly more effective than the chemotherapeutic drug alone (p < 0.01, one-way ANOVA and Turkey post-hoc test). We then moved to test TMZ combined with GQDs-mediated PTT. Results of spheroid growth are reported in Fig. 5D. Spheroids treated with the combination of TMZ and PTT had a significant reduction in spheroid growth to 69 ± 1.1% compared to the initial size and to the antitumor drug alone. Cell viability furtherly confirmed data of TMZ and PTT (Fig. 5E), highlighting a reduction up to 55 ± 4.6% and 7 ± 0.4% at 7 and 14 days respectively. As for DOX, this loss in viability was significant at both 7 and 14 days with respect not only to control spheroids, but also to TMZ alone (p < 0.05 and p < 0.01, one-way ANOVA and Turkey post-hoc test). Taken together, our data indicate that GQDs alone are not capable of significantly changing membrane permeability on a reliable model. However, by exploiting the photothermal conversion ability of these nanoparticles, it is possible to alter membrane permeability on a reliable spheroid model, increasing the efficacy of antitumor drugs.

Increase in Spheroid Membrane Permeability Through GQDs-Mediated PTT Induce an Improved Uptake of Chemotherapy

We hypothesized that the lack of effectiveness in absence of PTT could be due to a poor penetration capability of chemotherapeutic agents through the core of spheroids.^{9, 39–42} Therefore, we measured the penetration depth of DOX along with its uptake inside spheroids after PTT. For this purpose, we administered GQDs and, after incubation, we added DOX. We then irradiated spheroids, and we measured the penetration of DOX and its fluorescence intensity. Results are reported in Fig. 6. Figure 6A depicts representative confocal microscopy images of spheroids incubated with DOX. When treated with PTT-enhanced chemotherapy, spheroids displayed a significantly higher penetration depth of the antitumor drug with respect to DOX alone or without PTT (Fig. 6B, p < 0.01, one-way ANOVA and Turkey post-hoc test). In accordance with penetration depth, fluorescence intensity of DOX resulted significantly higher in spheroids treated with PTT and DOX (Fig. 6C). These data strongly confirm our proposed mechanism of action of irradiated GQDs, which can induce an increase in membrane permeability of spheroids, improving penetration depth of the antitumor drug, along with its uptake. Taken together, our findings suggest that biocompatible GQDs can increase membrane permeability through photothermal conversion in a reliable tumor model. The change in membrane permeability allows the use of subtherapeutic doses of antitumor drugs, enhancing its efficacy specifically in cancer region and, at the same time, strongly reducing side effects, potentially improving quality of life for patients.

PTT-Mediated Induction of Migration of Immune Cells Towards Cancer Region

Another relevant feature of glioblastoma multiforme is the downregulation of immune response.^20,43,44 Glioblastoma-mediated immune suppression is hypothesized to be related to a combination of multiple factors, such as the presence of suppressive CD163 + tumor-associated macrophages or direct induction of apoptosis of leukocytes.⁴⁵ Moreover, the high intratumoral heterogeneity of glioblastoma, along with its low number of somatic mutations, furtherly facilitates immune evasion. Immunotherapy aims at reactivating the immune system, stimulating it to recognize tumor-associated antigens, in order to directly target glioblastoma region, minimizing the non-specific inflammatory response that could seriously affect brain architecture and physiology. Recent immune-based therapies have been validated by FDA.^46–51 Nevertheless, first clinical trials of immunotherapeutic drugs displayed negative results, mainly due to defects in antigen presentation of tumor associated lymphocytes.¹⁷ Therefore, auxiliary approaches play a key role to produce specific and long-lasting antitumor immune response against glioblastoma-immune evasion.⁵¹ For this purpose, PTT can be an intriguing auxiliary cytotoxic approach for immunotherapy for two main reasons. First, it is known to cause a release of tumor-associated antigens by directly targeting cancer cells.^52,53 Second, depending on the presence of oxygen groups on the photosensitizing agent, it can induce production of ROS.^54–59 Furthermore, increase in membrane permeability due to GQDs-mediated PTT can augment release of both tumor-associated antigens and ROS produced within cells.^60–62 For these reasons, we tested the capability of GQDs-mediated PTT to induce migration of immune cells towards cancer region. We performed PTT on glioblastoma spheroids incubatedi with GQDs and, after the treatment, we co-cultured spheroids with THP-1 human monocytic cells stained in red. We then evaluated migration of immune cells by monitoring red fluorescence intensity in cancer region (Fig. 7A). Results are reported in Fig. 7B, in which fluorescence intensity is normalized by t0 for each treatment. Importantly, we observed that spheroid’s exposure to combination of GQDs-mediated PTT allowed a clearly faster recruitment of immune cells than untreated ones. As described above, release of tumor-associated antigens and enhanced ROS production are the two main explored mechanisms underlying reactivation of the immune system. Here we employed GQDs bearing carboxyl surface functional groups, which are responsible for the production of oxygen species after photothermal treatment.^63,64 We evaluated the production of ROS after exposure of spheroids to GQDs-mediated PTT. Results are displayed in Fig. 7C as % of control spheroids. Cells treated with PTT had a clear increase in the production of ROS (151 ± 14.7%), which was significantly higher than control (untreated) spheroids and than spheroids exposed only to GQDs (p < 0.01, one-way ANOVA and Turkey post-hoc test). From this evidence we can assume that the effect of rapid migration of immune cells is due to a greater production of ROS species.

In this work, we tested the effect of GQDs with surface carboxyl groups on gliobastoma multiforme. We demonstrated that GQDs can induce an increase in membrane permeability on a 2D model of brain cancer. We observed that the alteration of membrane permeability enhances the effect of two chemotherapeutic drugs, despite administered at subtherapeutic doses: doxorubicin and temozolomide. We therefore proposed a mechanism of action in which the increase in membrane permeability induced by GQDs improves intracellular uptake of antitumor drugs inside glioblastoma cells. We observed an increased uptake of doxorubicin after exposure of tumor cells to GQDs indeed. We then moved to test the efficacy of GQDs on a more complex and reliable glioblastoma model: 3D cancer spheroids. We monitored the evolution of spheroids exposed to GQDs and the antitumor drugs for two weeks. We observed a strong reduction both in growth and viability. However, this reduction resulted similar to that of chemotherapeutic drug alone. We hypothesized that GQDs are not capable of inducing a significant change in membrane permeability on a more complex spheroid model. We therefore exploited another feature of GQDs: their capability of absorbing near-infrared light and converting it into heat. This therapeutic approach is called photothermal therapy. We observed that spheroids exposed to GQDs and irradiated with a 6 W/cm² near-infrared laser display a significantly higher membrane permeability compared to the treatment with GQDs alone. We then combined GQDs-mediated PTT and chemotherapy at subtherapeutic doses on glioblastoma spheroids. We observed a significant reduction both in spheroid growth and viability in the timespan of two weeks, along with a considerably higher penetration depth and uptake of antitumor drug inside the more reliable glioblastoma model. Taken together, our findings suggest that biocompatible GQDs can increase membrane permeability through photothermal conversion in a reliable tumor model. The change in membrane permeability allows the use of subtherapeutic doses of antitumor drugs, enhancing its efficacy specifically in cancer region and, at the same time, strongly reducing side effects, potentially improving quality of life for patients.

Another crucial issue of glioblastoma is the downregulation of immune response. For this reason, reactivation of immune response through immunotherapy represents an intriguing approach. However, until now, immunotherapy displayed negative results on glioblastoma. Therefore, the use of auxiliary therapies plays a key role to produce specific and long-lasting antitumor immune response against glioblastoma-immune evasion. PTT can be an intriguing auxiliary cytotoxic approach due to the release of tumor-associated antigens and production of reactive oxygen species, which can strongly stimulate immune cells specifically against glioblastoma. We investigated the migration of human immune cells towards glioblastoma spheroids exposed to GQDs-mediated PTT. We found a rapid migration, which was associated with an increased production of reactive oxygen species from spheroids. Our evidence indicate that PTT can induce a strong and durable immune response specifically against glioblastoma, enhancing the efficacy of immunotherapy.

Characterization of GQDs

Graphene quantum dots in powder were purchased from ACS Material. GQDs were dissolved in double-distilled water (MilliQ) at a final concentration of 1 mg/mL. After dissolution, GQDs were microscopically and spectroscopically characterized. Transmission electron microscopy (TEM) was performed with a Zeiss Libra 120 electron microscope. For TEM, 50 µg/mL of GQDs were spotted on a TEM tab and let dry at room temperature. Dynamic light scattering measurements were performed with Zetasizer Nano ZS (Malvern), equipped with a 633 nm He − Ne laser and operating at an angle of 173°. UV-transparent cuvettes (Malvern) have been used for experiments with a sample volume of 500 µL and a concentration of 100 µg/mL. The measurements were performed at a fixed position (4.65 mm) with an automatic attenuator. For each sample, three measurements were averaged, the diffusion coefficient D has been retrieved through cumulants analysis from autocorrelation functions. The equivalent hydrodynamic radius (Z-Average size) was obtained by the Stokes − Einstein equation. Data analysis was performed by Malvern Zetasizer software. Optical density (OD) and fluorescence intensity were obtained by using a Cytation 3 Cell Imaging Multi-Mode Reader (Biotek). OD was recorded from 230 to 800 nm. Fluorescence intensity spectra were obtained by using excitation wavelengths from 260 to 480 nm with a step of 20 nm and reading the emission from 300 to 700 nm with a step of 5 nm. Fluorescence intensity spectra were normalized to their corresponding maximum emission. The chemical analysis of the GQDs was carried out using Attenuated Total Reflectance-Fourier Transform Infrared spectroscopy (ATR-FTIR) by Spectrum One spectrometer (Perkin Elmer). The material under investigation was directly laid upon the ATR crystal and the spectra were recorded in the wave number range of 4000 − 550 cm^− 1.

Near-Infrared Laser Characterization

A near-infrared laser (LaserEver) focused at 808 nm was used to perform photothermal treatments on cells. First, the laser was characterized by evaluating the laser power at every current intensity by using a power meter. The spot of the laser had a diameter of 0.8 cm. Power density was evaluated by normalizing the laser power to the area of the spot. To test the photothermal effect of GQDs, different concentrations of the nanomaterials were used, ranging from 50 to 250 µg/ mL. GQDs were irradiated at a power density of 6 W/cm² for 5 minutes. Thermal increase was monitored by using a thermal camera (Optris) focused on the well containing irradiated nanoparticles.

Cell Culture

U87 human glioblastoma cells were purchased from the American Type Culture Collection, (ATCC). Cells were maintained in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS, EuroClone), 2% penicillin-streptomycin (Sigma-Aldrich), and 2% L-glutamine (Sigma-Aldrich). Cells were kept in T75 flasks (Corning) at at 37°C, 5% CO₂ for further treatments.

Cell Viability Measurements on 2D Model

To measure cell viability on 2D cultures, U87 cells were seeded on 96-well plates (Corning) at a seeding density of 1 x 10⁵ cells/well. Plates were then incubated at 37°C, 5% CO₂. To test biocompatibility of GQDs, nanoparticles were administered at concentrations ranging from 50 to 250 µg/mL. After 24 hours of incubation, viability was measured by using CellTiter-Glo® (Promega). Briefly, an amount of CellTiter-Glo® reagent equal to the volume of culture medium was added to each well. Then, the plate was orbitally shaken for 2 minutes to ensure complete cell lysis and incubated in the dark at room temperature for 10 minutes. After incubation, luminescence was recorded by using Cytation 3. Results were reported as % of control (untreated) cells. To evaluate the concentration inhibiting 50% of cell growth (IC50), doxorubicin (DOX) or temozolomide (TMZ) were administered to cells at different concentrations. For DOX, concentration ranged from 0.5 to 10 µM. For TMZ, concentration ranged from 62.5 to 1000 µg/ mL. Untreated cells were used as a control group. Cells were then incubated at 37°C − 5% CO₂ for 48 hours, then viability was measured by using CellTiter-Glo® as described above. Results were reported as % of control (untreated) cells. The IC50 was determined by fitting with Logger Pro software the experimental results. To evaluate the combined effect of chemotherapeutic drugs and graphene quantum dots (GQDs), cells were incubated with GQDs at different concentrations ranging from 50 to 250 µg/ mL. After 24 hours of incubation GQDs were removed, and chemotherapeutic agents were added at half of their IC50. After further incubation, viability was measured by using CellTiter-Glo® as described above.

Confocal Microscopy on 2D Model

Confocal microscopy images of 2D cell cultures were acquired with an inverted microscope (Nikon A1 MP+, Nikon). Cells were seeded on sterile chamber slides (Ibidi) at a concentration of 1 × 10⁶ cell/ mL and then incubated at 37°C, 5% CO₂. After 24 hours of incubation, GQDs were administered to cells at the highest tested concentration. For both 6-dodecanoyl-2-dimethylamino-naphthalene (Laurdan) measurements and DOX uptake, GQDs were removed, and cells were resuspended in fresh medium containing Laurdan or DOX at a final concentration of 1 µM. Confocal images were acquired at 37°C for all measurements. For Laurdan excitation, cells were imaged with a wavelength of 400 nm. Laurdan is a fluorescent probe capable of detecting and distinguishing gel phase and liwuid phase of cell membrane. Laurdan intensity images were recorded simultaneously with emissions in the ranges of 425–475 nm (gel phase) and 500–550 nm (liquid phase). To quantify the images collected in this way, Fiji (ImageJ) software was used. The intensities of the two different channels were calculated, and the generalized polarization (GP) index was calculated as previously reported. For DOX uptake measurements, images were acquired after 1 hour of incubation with DOX. The excitation and emission wavelengths were, respectively, 488 nm and 590 nm. To quantify the uptake, Fiji (ImageJ) software was used by measuring the fluorescence intensity of DOX. Data were normalized by cells treated with chemotherapeutic agent alone.

Spheroid Preparation, Growth and Viability

For spheroid preparation, cells were seeded on 96-well, round bottom, ultra-low attachment plates (Corning) at a density of 0.25 × 10⁵ cells/mL. The plate was centrifuged at 300× g for 3 min to ensure the confluence of cells to the center of the wells. The so-formed single spheroids were incubated for three days at 37°C with 5% CO₂. Spheroids were treated for 2 weeks with GQDs at 200 µg/mL with or without chemotherapy administration. Culture medium was changed with fresh medium containing the same concentration of nanoparticles and chemotherapeuticals every three days. For photothermal treatments, spheroids were irradiated with an 808 nm laser for 5 minutes at 6 W/cm². Transmittance images of spheroids were collected using Cytation 3 and spheroid growth in terms of area were analyzed with INSIDIA 2.0 as described elsewhere. Spheroid viability was evaluated at 7 and 14 days from the beginning of the experiments by using CellTiter-Glo® luminescent assay as described above. For spheroid growth, data were normalized by the area at day 0 for each treatment. For viability, results were reported as % of control (untreated) spheroids.

Changes in Membrane Permeability on Spheroids

To test changes in membrane permeability on the 3D model, the uptake of calcein-AM (ThermoFisher) was measured. Briefly, spheroids were incubated with GQDs at a concentration of 200 µg/mL. After 24 hours of incubation, spheroids were irradiated with an 808 nm laser at 6 W/cm² for 5 minutes. Then, calcein was added at a final concentration of 10 µM and incubated in a Cytation 3 for 30 minutes at 37°C. Fluorescence intensity was recorded by exciting at 488 nm and recording emission at 525 nm. Data were then normalized by the fluorescence intensity at the beginning. To evaluate fluorescence intensity of calcein, spheroids at different timepoints were collected for each condition (untreated, incubated with GQDs and incubated with GQDs and irradiated) and imaged with confocal microscopy by using Nikon A1 MP+.

Production of Reactive Oxygen Species on Glioblastoma Spheroids

For the detection of reactive oxygen species (ROS), the fluorinated derivative of 2’,7’-di-chlorofluorescein (H₂DCFDA, Sigma-Aldrich) was employed. This probe is non-fluorescent until the acetate groups are removed by intracellular esterases and oxidation occurs within cells. Thus, oxidation can be detected by monitoring the increase in fluorescence intensity. spheroids were treated with GQDs at 200 µg/mL for 24 hours. After the treatment, cells were irradiated with an 808 nm laser with a power density of 6 W/cm² for 5 minutes. After a recovery time of 30 minutes, the medium containing GQDs was carefully washed and replaced with PBS containing 10 µM H₂DCFDA. Cells were incubated for an additional hour at 37°C, 5% CO₂. PBS containing H₂DCFDA was then removed, and spheroids were resuspended in complete medium. Fluorescence intensity of H₂DCFDA was recorded by using a Cytation 3 by exciting at 495 nm and recording emission at 528 nm. Results were expressed as % of control (untreated) spheroids.

Uptake of Chemotherapeutic Drug on Spheroids

The uptake of DOX through tumor spheroids was evaluated via quantification of fluorescence intensity in confocal microscopy images of spheroids. Briefly, 3D models were incubated with GQDs for 24 hours. After incubation, DOX was added and photothermal irradiation was performed with an 808 nm laser. Spheroids were then incubated with calcein for 20 minutes, then images were acquired with Nikon A1 MP + by exciting at 488 nm and reading at 525 nm (calcein) and by reading emission at 590 nm (DOX). Uptake of DOX was evaluated in terms of fluorescence intensity and penetration depth. Uptake was compared to cells incubated with only DOX or with DOX and GQDs.

Migration of THP-1 Towards Glioblastoma Spheroids

To evaluate migration of THP-1 human monocytic cell line towards cancer region, U87 spheroids were treated with GQDs at 200 µg/mL. After 24 hours of incubation, spheroids were irradiated with an 808 nm laser for 5 minutes at 6 W/cm². After a recovery time of 30 minutes, spheroids were co-cultured with THP-1 cells stained with Red Long-Term Cell Tracer Kit (Enzo), by following manufacturer’s instructions. Briefly, cells were centrifuged for 5 minutes at 400 g, then supernatant was discarded. Cells were then resuspended in 1 mL of Labeling Buffer. An equal volume of 2XCyto-ID™ RED Tracer Dye was added to cell suspension. The suspension was incubated for 5 minutes. Then, Stop Buffer was added to block the reaction. Finally, cells were centrifuged and resuspended in culture medium. Images were acquired in bright field (spheroids) and Texas red channel with a Cytation 3 for 48 hours at 37°C, 5% CO₂.

Statistical Analysis

Statistical analysis was performed using one-way ANOVA, followed by Turkey’s post-hoc test. Differences were considered significant when p < 0.01.

2D (Two dimensional)

3D (Three dimensional)

ANOVA (Analysis of variance)

ATR-FTIR (Attenuated Total Reflectance-Fourier Transform Infrared spectroscopy)

DLS (Dynamic light scattering)

DOX (Doxorubicin)

FDA (Food and Drug Administration)

GQDs (Graphene quantum dots)

IC50 (inhibiting 50% of cell growth)

NIR (near-infrared light)

OD (Optical density)

PBS (Phosphate buffered saline)

PDT (Photodynamic therapy)

PTT (Photothermal therapy)

ROS (Reactive oxygen species)

RT (Room temperature)

TEM (Transmission Electron Microscopy)

TMZ (Temozolomide)

GP (Generalized polarization)

UV (Ultraviolet)

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests

Funding

The research leading to these results has received funding from AIRC under IG 2019—ID. 23124 project—P.I. Massimiliano Papi and from AIRC Grant to Giordano Perini 26918-2021.

Authors' contributions

GP and GF performed the experiments. AA and GP realized the 3D bioprinting cellular models. GP VP MDS and MP analyzed and interpreted the data. MP designed the experiments. All authors read and approved the final manuscript

Acknowledgements

We would like to acknowledge the contribution of 3D Bioprinting Research Core Facility G-STeP of the Fondazione Policlinico Universitario “A. Gemelli” IRCCS for sample processing.

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No competing interests reported.

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Carboxylated Graphene Quantum Dots-Mediated Photothermal Therapy Enhances Drug-Membrane Permeability, ROS Production, and the Immune System Recruitment on 3D Glioblastoma Models.

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Journal Publication

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Abstract

Figures

Background

Results

Characterization of GQDs

Effect of Changes in Membrane Permeability Mediated by GQDs on 2D Model of Glioblastoma

Effect of the Combined Treatment of GQDs and Chemotherapy on a 3D Model of Glioblastoma

Increase in Membrane Permeability of Glioblastoma Spheroids Through Photothermal Conversion Mediated by GQDs

Photothermal-Enhanced Chemotherapy Through GQDs on Glioblastoma Spheroids

Increase in Spheroid Membrane Permeability Through GQDs-Mediated PTT Induce an Improved Uptake of Chemotherapy

PTT-Mediated Induction of Migration of Immune Cells Towards Cancer Region

Conclusions

Methods

Characterization of GQDs

Near-Infrared Laser Characterization

Cell Culture

Cell Viability Measurements on 2D Model

Confocal Microscopy on 2D Model

Spheroid Preparation, Growth and Viability

Changes in Membrane Permeability on Spheroids

Production of Reactive Oxygen Species on Glioblastoma Spheroids

Uptake of Chemotherapeutic Drug on Spheroids

Migration of THP-1 Towards Glioblastoma Spheroids

Statistical Analysis

List of abbreviations

Declarations

References

Additional Declarations

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Journal Publication

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