High-yield production of nano-lateral size graphene oxide by high-power ultrasonication

Nanographene oxide (GOn) constitutes a nanomaterial of high value in the biomedical field. However, large scale production of highly stable aqueous dispersions of GOn is yet to be achieved. In this work, we explored high-power ultrasonication as a method to reduce particle size of GO and characterized the impact of the process on the physicochemical properties of the material. GOn was obtained with lateral dimensions of 99 ± 43 nm and surface charge of -39.9 ± 2.2 mV. High-power ultrasonication enabled an improvement of stability features, particularly by resulting in a decrease of the average particle size, as well as zeta potential, in comparison to GO obtained by low-power exfoliation and centrifugation (287 ± 139 nm; -29.7 ± 1.2 mV). Remarkably, GOn aqueous dispersions were stable for up to 6 months of shelf-time, with a global process yield of 74%. This novel method enabled the production of large volumes of highly concentrated (7.5 mg mL-1) GOn aqueous dispersions. Chemical characterization of GOn allowed the identification of characteristic oxygen functional groups, supporting high-power ultrasonication as a fast, efficient, and productive process for reducing GO lateral size, while maintaining the material’s chemical features.This work was financed by FEDER funds through the COMPETE 2020—Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by national funds (PIDDAC) through FCT/MCTES in the framework of the project POCI-01-0145-FEDER-031143, and Base Funding—UIDB/00511/2020 of the Laboratory for Process Engineering, Environment, Biotechnology and Energy—LEPABE. Authors would also like to thank the support of i3S Scientific Platforms and respective funding: HEMS, member of the national infrastructure PPBI—Portuguese Platform of Bioimaging: POCI-01-0145-FEDER-022122; and Biointerfaces and Nanotechnology (BN) Laboratory, Portuguese Funds through FCT, UID/BIM/04293/2019. Artur Pinto thanks the Portuguese Foundation for Science and Technology (FCT) for the financial support of his work contract through the Scientific Employment Stimulus—Individual Call—[CEECIND/03908/2017]

Graphene oxide topical administration: Skin permeability studies

Nanostructured carriers have been widely used in pharmaceutical formulations for der-matological treatment. They offer targeted drug delivery, sustained release, improved biostability, and low toxicity, usually presenting advantages over conventional formulations. Due to its large surface area, small size and photothermal properties, graphene oxide (GO) has the potential to be used for such applications. Nanographene oxide (GOn) presented average sizes of 197.6 ± 11.8 nm, and a surface charge of -39.4 ± 1.8 mV, being stable in water for over 6 months. 55.5% of the mass of GOn dispersion (at a concentration of 1000 µg mL-1 ) permeated the skin after 6 h of exposure. GOn dispersions have been shown to absorb near-infrared radiation, reaching temperatures up to 45.7¿ C, within mild the photothermal therapy temperature range. Furthermore, GOn in amounts superior to those which could permeate the skin were shown not to affect human skin fibroblasts (HFF-1) morphology or viability, after 24 h of incubation. Due to its large size, no skin permeation was observed for graphite particles in aqueous dispersions stabilized with Pluronic P-123 (Gt–P-123). Altogether, for the first time, Gon’s potential as a topic administration agent and for delivery of photothermal therapy has been demonstrated.This work was financed by FEDER funds through the COMPETE 2020–Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by national funds (PIDDAC) through FCT/MCTES in the framework of the project POCI-01-0145-FEDER-031143, and Base Funding-UIDB/00511/2020 of the Laboratory for Process Engineering, Environment, Biotechnology and Energy–LEPABE. Additional funding included FCT/MCTES in the framework of the project “Institute for Research and Innovation in Health Sciences” (UID/BIM/04293/2019). Authors would also like to thank the support of i3S Scientific Platforms and respective funding: HEMS, member of the national infrastructure PPBI–Portuguese Platform of Bioimaging: POCI-01-0145-FEDER-022122; and Biointerfaces and Nanotechnology (BN) Laboratory, Portuguese Funds through FCT, UID/BIM/04293/2019. Artur Pinto thanks the Portuguese Foundation for Science and Technology (FCT) for the financial support of his work contract through the Scientific Employment Stimulus-Individual Call–[CEECIND/03908/2017]. Soraia Pinto (SFRH/BD/144719/2019) would like to thank FCT, Portugal for financial support

Near-infrared radiation-based mild photohyperthermia therapy of non-melanoma skin cancer with PEGylated reduced nanographene oxide

Using a one-step thermal reduction and non-covalent chemical functionalization process, PEGylated reduced nanographene oxide (rGOn-PEG) was produced from nanographene oxide (GOn) and characterized in terms of particle size, dispersion stability, chemistry, and photothermal properties, in view of its use for photothermal therapy (PTT) of non-melanoma skin cancer. GOn infrared spectrum presented more intense bands assigned to oxygen containing functional groups than observed for rGOn-PEG. GOn C/O ratio decreased more than 50% comparing with rGOn-PEG and nitrogen was present in the latter (N at % = 20.6) due to introduction of PEG-NH2. Thermogravimetric analysis allowed estimating the amount of PEG in rGOn-PEG to be of about 56.1%. Simultaneous reduction and PEGylation increased the lateral dimensions from 287 ± 139 nm to 521 ± 397 nm, as observed by transmission electron microscopy and dynamic light scattering. rGOn-PEG exhibited ˜ 13-fold higher absorbance in the near-infrared radiation (NIR) region, as compared to unmodified GOn. Low power (150 mW cm-2) NIR irradiation using LEDs resulted in rGOn-PEG heating up to 47 °C, which is within the mild PTT temperature range. PEGylation strongly enhanced the dispersibility of rGOn in physiological media (phosphate buffered saline, fetal bovine serum, and cell culture medium) and also improved the biocompatibility of rGOn-PEG, in comparison to GOn (25-250 µg mL-1). After a single NIR LED irradiation treatment of 30 min, a decrease of ˜38% in A-431 cells viability was observed for rGOn-PEG (250 µg mL-1). Together, our results demonstrate the potential of irradiating rGOn-PEG using lower energy, cheaper, smaller, and safer LEDs, as alternative to high power lasers, for NIR mild hyperthermia therapy of cancer, namely non-melanoma skin cancer.This work was financed by FEDER funds through the COMPETE 2020 – Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by national funds (PIDDAC) through FCT/MCTES in the framework of the project POCI-01-0145-FEDER-031143, and Base Funding - UIDB/00511/2020 of the Laboratory for Process Engineering, Environment, Biotechnology and Energy – LEPABE. Authors would also like to thank the support of i3S Scientific Platforms and respective funding: HEMS, member of the national infrastructure PPBI – Portuguese Platform of Bioimaging: POCI-01-0145-FEDER-022122; and Biointerfaces and Nanotechnology (BN) Laboratory, Portuguese Funds through FCT, UID/BIM/04293/2019

Near-Infrared Radiation-Based Mild Photohyperthermia Therapy of Non-Melanoma Skin Cancer with PEGylated Reduced Nanographene Oxide

Using a one-step thermal reduction and non-covalent chemical functionalization process, PEGylated reduced nanographene oxide (rGOn-PEG) was produced from nanographene oxide (GOn) and characterized in terms of particle size, dispersion stability, chemistry, and photothermal properties, in view of its use for photothermal therapy (PTT) of non-melanoma skin cancer. GOn infrared spectrum presented more intense bands assigned to oxygen containing functional groups than observed for rGOn-PEG. GOn C/O ratio decreased more than 50% comparing with rGOn-PEG and nitrogen was present in the latter (N at % = 20.6) due to introduction of PEG-NH2. Thermogravimetric analysis allowed estimating the amount of PEG in rGOn-PEG to be of about 56.1%. Simultaneous reduction and PEGylation increased the lateral dimensions from 287 ± 139 nm to 521 ± 397 nm, as observed by transmission electron microscopy and dynamic light scattering. rGOn-PEG exhibited ≈13-fold higher absorbance in the near-infrared radiation (NIR) region, as compared to unmodified GOn. Low power (150 mW cm−2) NIR irradiation using LEDs resulted in rGOn-PEG heating up to 47 °C, which is within the mild PTT temperature range. PEGylation strongly enhanced the dispersibility of rGOn in physiological media (phosphate buffered saline, fetal bovine serum, and cell culture medium) and also improved the biocompatibility of rGOn-PEG, in comparison to GOn (25–250 μg mL−1). After a single NIR LED irradiation treatment of 30 min, a decrease of ≈38% in A-431 cells viability was observed for rGOn-PEG (250 μg mL−1). Together, our results demonstrate the potential of irradiating rGOn-PEG using lower energy, cheaper, smaller, and safer LEDs, as alternative to high power lasers, for NIR mild hyperthermia therapy of cancer, namely non-melanoma skin cancer

Pharmaceutical Formulations Containing Graphene and 5‑Fluorouracil for Light-Emitting Diode-Based Photochemotherapy of Skin Cancer

Nonmelanoma skin cancer (NMSC) is the most common cancer worldwide, among which 80% is basal cell carcinoma (BCC). Current therapies’ low efficacy, side effects, and high recurrence highlight the need for alternative treatments. In this work, a partially reduced nanographene oxide (p-rGOn) developed in our laboratory was used. It has been achieved through a controlled reduction of nanographene oxide via UV–C irradiation that yields small nanometric particles (below 200 nm) that preserve the original water stability while acquiring high light-to-heat conversion efficiency. The latter is explained by a loss of carbon–oxygen single bonds (C–O) and the re-establishment of sp2 carbon bonds. p-rGOn was incorporated into a Carbopol hydrogel together with the anticancer drug 5-fluorouracil (5-FU) to evaluate a possible combined PTT and chemotherapeutic effect. Carbopol/p-rGOn/5-FU hydrogels were considered noncytotoxic toward normal skin cells (HFF-1). However, when A-431 skin cancer cells were exposed to NIR irradiation for 30 min in the presence of Carbopol/p-rGOn/5-FU hydrogels, almost complete eradication was achieved after 72 h, with a 90% reduction in cell number and 80% cell death of the remaining cells after a single treatment. NIR irradiation was performed with a light-emitting diode (LED) system, developed in our laboratory, which allows adjustment of applied light doses to achieve a safe and selective treatment, instead of the standard laser systems that are associated with damages in the healthy tissues in the tumor surroundings. Those are the first graphene-based materials containing pharmaceutical formulations developed for BCC phototherapy