Enhancing Photothermal/Photodynamic Therapy for Glioblastoma by Tumor Hypoxia Alleviation and Heat Shock Protein Inhibition Using IR820-Conjugated Reduced Graphene Oxide Quantum Dots.

We use low-molecular-weight branched polyethylenimine (PEI) to produce cytocompatible reduced graphene oxide quantum dots (rGOQD) as a photothermal agent and covalently bind it with the photosensitizer IR-820. The rGOQD/IR820 shows high photothermal conversion efficiency and produces reactive oxygen species (ROS) after irradiation with near-infrared (NIR) light for photothermal/photodynamic therapy (PTT/PDT). To improve suspension stability, rGOQD/IR820 was PEGylated by anchoring with the DSPE hydrophobic tails in DSPE-PEG-Mal, leaving the maleimide (Mal) end group for covalent binding with manganese dioxide/bovine serum albumin (MnO2/BSA) and targeting ligand cell-penetrating peptide (CPP) to synthesize rGOQD/IR820/MnO2/CPP. As MnO2 can react with intracellular hydrogen peroxide to produce oxygen for alleviating the hypoxia condition in the acidic tumor microenvironment, the efficacy of PDT could be enhanced by generating more cytotoxic ROS with NIR light. Furthermore, quercetin (Q) was loaded to rGOQD through π-π interaction, which can be released in the endosomes and act as an inhibitor of heat shock protein 70 (HSP70). This sensitizes tumor cells to thermal stress and increases the efficacy of mild-temperature PTT with NIR irradiation. By simultaneously incorporating the HSP70 inhibitor (Q) and the in situ hypoxia alleviating agent (MnO2), the rGOQD/IR820/MnO2/Q/CPP can overcome the limitation of PTT/PDT and enhance the efficacy of targeted phototherapy in vitro. From in vivo study with an orthotopic brain tumor model, rGOQD/IR820/MnO2/Q/CPP administered through tail vein injection can cross the blood-brain barrier and accumulate in the intracranial tumor, after which NIR laser light irradiation can shrink the tumor and prolong the survival times of animals by simultaneously enhancing the efficacy of PTT/PDT to treat glioblastoma.

Chitosan-coated magnetic graphene oxide for targeted delivery of doxorubicin as a nanomedicine approach to treat glioblastoma.

In this study, magnetic graphene oxide (mGO) was first prepared and modified with chitosan to prepare chitosan-coated mGO (mGOC). Gastrin-releasing peptide (GRP)-conjugated mGOC (mGOCG) was then prepared from mGOC. The chemo drug doxorubicin (DOX) was adsorbed to mGOCG surface for dual active/magnetic targeted drug delivery. The DOX loading to mGOCG is 1.71 mg/mg, and drug release is pH-sensitive to facilitate drug delivery in endosomes. In vitro studies confirmed enhanced mGOCG endocytosis by U87 glioblastoma cells, with which enhanced cytotoxicity towards cancer cells could be achieved. This could be revealed from the drastically reduced half-maximal inhibitory concentration of mGOCG/DOX compared with DOX and mGOC/DOX. Furthermore, mGOCG/DOX can be localized under the influence of a magnetic field (MF) to exert this cytotoxic effect. An orthotopic brain tumor model by implanting U87 cells in the intracranial area of BALB/c nude mice was used to study the in vivo anti-tumor efficacy by intravenous injection of different samples and followed with bioluminescence imaging. The tumor size in the mGOCG/DOX + MF group demonstrated the best potency to suppress tumor growth and prolong animal survival time compared with mGOCG/DOX, mGOC/DOX, or DOX groups, indicating this new dual-targeting delivery system for DOX can effectively treat glioblastoma.

Functionalized Nanomaterials for Inhibiting ATP-Dependent Heat Shock Proteins in Cancer Photothermal/Photodynamic Therapy and Combination Therapy.

Phototherapies induced by photoactive nanomaterials have inspired and accentuated the importance of nanomedicine in cancer therapy in recent years. During these light-activated cancer therapies, a nanoagent can produce heat and cytotoxic reactive oxygen species by absorption of light energy for photothermal therapy (PTT) and photodynamic therapy (PDT). However, PTT is limited by the self-protective nature of cells, with upregulated production of heat shock proteins (HSP) under mild hyperthermia, which also influences PDT. To reduce HSP production in cancer cells and to enhance PTT/PDT, small HSP inhibitors that can competitively bind at the ATP-binding site of an HSP could be employed. Alternatively, reducing intracellular glucose concentration can also decrease ATP production from the metabolic pathways and downregulate HSP production from glucose deprivation. Other than reversing the thermal resistance of cancer cells for mild-temperature PTT, an HSP inhibitor can also be integrated into functionalized nanomaterials to alleviate tumor hypoxia and enhance the efficacy of PDT. Furthermore, the co-delivery of a small-molecule drug for direct HSP inhibition and a chemotherapeutic drug can integrate enhanced PTT/PDT with chemotherapy (CT). On the other hand, delivering a glucose-deprivation agent like glucose oxidase (GOx) can indirectly inhibit HSP and boost the efficacy of PTT/PDT while combining these therapies with cancer starvation therapy (ST). In this review, we intend to discuss different nanomaterial-based approaches that can inhibit HSP production via ATP regulation and their uses in PTT/PDT and cancer combination therapy such as CT and ST.

Cetuximab-Conjugated Magnetic Poly(Lactic-co-Glycolic Acid) Nanoparticles for Dual-Targeted Delivery of Irinotecan in Glioma Treatment.

A glioma is the most common malignant primary brain tumor in adults and is categorized according to its growth potential and aggressiveness. Within gliomas, grade 4 glioblastoma remains one of the most lethal malignant solid tumors, with a median survival time less than 18 months. By encapsulating CPT-11 and oleic acid-coated magnetic nanoparticles (OMNPs) in poly(lactic-co-glycolic acid) (PLGA) nanoparticles, we first prepared PLGA@OMNP@CPT-11 nanoparticles in this study. After conjugating cetuximab (CET) with PLGA@OMNP@CPT-11, spherical PLGA@OMNP@CPT-11-CET nanoparticles with 250 nm diameter, 33% drug encapsulation efficiency, and 22% drug loading efficiency were prepared in a single emulsion/evaporation step. The nanoparticles were used for dual-targeted delivery of CPT-11 to U87 primary glioblastoma cells by actively targeting the overexpressed epidermal growth factor receptor on the surface of U87 cells, as well as by magnetic targeting. The physicochemical properties of nanoparticles were characterized in detail. CET-mediated targeting promotes intracellular uptake of nanoparticles by U87 cells, which can release four times more drug at pH 5 than at pH 7.4 to facilitate drug release in endosomes after intracellular uptake. The nanovehicle PLGA@OMNP-CET is cytocompatible and hemocompatible. After loading CPT-11, PLGA@OMNP@CPT-11-CET shows the highest cytotoxicity toward U87 compared with free CPT-11 and PLGA@OMNP@CPT-11 by providing the lowest drug concentration for half-maximal cell death (IC50) and the highest rate of cell apoptosis. In orthotopic brain tumor-bearing nude mice with U87 xenografts, intravenous injection of PLGA@OMNP@ CPT-11-CET followed by guidance with a magnetic field provided the best treatment efficacy with the lowest tumor-associated signal intensity from bioluminescence imaging.

Immunotherapeutic Approaches for the Treatment of Glioblastoma Multiforme: Mechanism and Clinical Applications.

Glioma is one of the most aggressive types of primary brain tumor with a high-grade glioma known as glioblastoma multiforme (GBM). Patients diagnosed with GBM usually have an overall survival rate of less than 18 months after conventional therapy. This bleak prognosis underlines the need to consider new therapeutic interventions for GBM treatment to overcome current treatment limitations. By highlighting different immunotherapeutic approaches currently in preclinical and clinical trials, including immune checkpoint inhibitors, chimeric antigen receptors T cells, natural killer cells, vaccines, and combination therapy, this review aims to discuss the mechanisms, benefits, and limitations of immunotherapy in treating GBM patients.

Hyaluronic Acid-Modified Cisplatin-Encapsulated Poly(Lactic-co-Glycolic Acid) Magnetic Nanoparticles for Dual-Targeted NIR-Responsive Chemo-Photothermal Combination Cancer Therapy.

Combination chemo-photothermal therapy with nanomaterials can reduce the dose of chemotherapeutic drugs required for effective cancer treatment by minimizing toxic side effects while improving survival times. Toward this end, we prepare hyaluronic acid (HA)-modified poly(lactic-co-glycolic acid) (PLGA) magnetic nanoparticles (MNP) for the CD44 receptor-mediated and magnetic field-guided dual-targeted delivery of cisplatin (CDDP). By co-encapsulating the CDDP and oleic acid-coated iron oxide MNP (IOMNP) in PLGA, the PMNPc was first prepared in a single emulsification/solvent evaporation step and successively surface modified with chitosan and HA to prepare the HA/PMNPc. Spherical HA/PMNPc nanoparticles of ~300 nm diameter can be prepared with 18 and 10% (w/w) loading content of CDDP and IOMNP and a pH-sensitive drug release to facilitate the endosomal release of the CDDP after intracellular uptake. This leads to the higher cytotoxicity of the HA/PMNPc toward the U87 glioblastoma cells than free CDDP with reduced IC50, a higher cell apoptosis rate, and the enhanced expression of cell apoptosis marker proteins. Furthermore, the nanoparticles show the hyperthermia effect toward U87 after short-term near-infrared (NIR) light exposure, which can further elevate the cell apoptosis/necrosis rate and upregulate the HSP70 protein expression due to the photothermal effects. The combined cancer therapeutic efficacy was studied in vivo using subcutaneously implanted U87 cells in nude mice. By using dual-targeted chemo-photothermal combination cancer therapy, the intravenously injected HA/PMNPc under magnetic field guidance and followed by NIR laser irradiation was demonstrated to be the most effective treatment modality by inhibiting the tumor growth and prolonging the survival time of the tumor-bearing nude mice.

PLGA/Gelatin/Hyaluronic Acid Fibrous Membrane Scaffold for Therapeutic Delivery of Adipose-Derived Stem Cells to Promote Wound Healing.

Hyaluronic acid (HA) has been suggested to be a preferential material for the delivery of adipose-derived stem cells (ASCs) in wound healing. By incorporating HA in electrospun poly (lactide-co-glycolide) (PLGA)/gelatin (PG) fibrous membrane scaffolds (FMS), we aim to fabricate PLGA/gelatin/HA (PGH) FMS to provide a milieu for 3D culture and delivery of ASCs. The prepared FMS shows adequate cytocompatibility and is suitable for attachment and growth of ASCs. Compared with PG, the PGH offers an enhanced proliferation rate of ASCs, shows higher cell viability, and better maintains an ASC-like phenotype during in vitro cell culture. The ASCs in PGH also show upregulated expression of genes associated with angiogenesis and wound healing. From a rat full-thickness wound healing model, a wound treated with PGH/ASCs can accelerate the wound closure rate compared with wounds treated with PGH, alginate wound dressing, and gauze. From H&E and Masson's trichrome staining, the PGH/ASC treatment can promote wound healing by increasing the epithelialization rate and forming well-organized dermis. This is supported by immunohistochemical staining of macrophages and α-smooth muscle actin, where early recruitment of macrophages, macrophage polarization, and angiogenesis was found due to the delivered ASCs. The content of type III collagen is also higher than type I collagen within the newly formed skin tissue, implying scarless wound healing. Taken together, using PGH FMS as a topical wound dressing material for the therapeutic delivery of ASCs, a wound treated with PGH/ASCs was shown to accelerate wound healing significantly in rats, through modulating immunoreaction, promoting angiogenesis, and reducing scar formation at the wound sites.

Hyaluronic acid-modified, IR780-conjugated and doxorubicin-loaded reduced graphene oxide for targeted cancer chemo/photothermal/photodynamic therapy.

We used reduced graphene oxide (rGO), which has two times higher photothermal conversion efficiency than graphene oxide (GO), as a photothermal agent for cancer photothermal therapy (PTT). By conjugating a photosensitizer IR780 to rGO, the IR780-rGO could be endowed with reactive oxygen species (ROSs) generation ability for concurrent photodynamic therapy (PDT). The IR780-rGO was coated with hyaluronic acid (HA) by electrostatic interaction to facilitate its intracellular uptake by U87 glioblastoma cells. The IR780-rGO/HA was loaded with doxorubicin (DOX) for chemotherapy (CT), to develop a pH-responsive drug delivery nano-platform for targeted multimodal cancer CT/PTT/PDT. We fully characterized the properties of all nanocomposites during the synthesis steps. The high loading efficiency of DOX on IR780-rGO-HA provides 3 mg/mg drug loading, while IR780-rGO-HA/DOX shows 3 times higher drug release at endosomal pH value (pH 5) than at pH 7.4. The mechanism for PTT/PDT was confirmed from the ability of IR780-rGO-HA to induce time-dependent temperature rise, synthesis of heat shock protein 70 (HSP70) and generation of intracellular ROSs, after exposure to 808 nm near infrared (NIR) laser light. The nano-vehicle IR780-rGO-HA shows high biocompatibility toward 3T3 fibroblast and U87 cancer cell lines, as well as enhanced intracellular uptake by U87 through active targeting. This translates into increased cytotoxicity of IR780-rGO-HA/DOX, by lowering the drug half-maximal inhibitory concentration (IC50) from 0.7 to 0.46 µg/mL. This IC50 is further decreased to 0.1 µg/mL by irradiation with NIR laser for 3 min at 1.5 W/cm2. The elevated cancer cell killing mechanism was supported from flow cytometry analysis, where the highest cell apoptosis/necrosis rate was observed in combination CT/PTT/PDT. Using xenograft tumor model created by subcutaneous implantation of U87 cells in nude mice, IR780-rGO-HA/DOX delivered through intravenous (IV) injection and followed with 808 nm laser treatment for 5 min at 1.5 W/cm2 results in the lowest tumor growth rate, with negligible change of tumor volume from its original value at the end 20-day observation period. The therapeutic efficacy was supported from inhibited cell proliferation rate, increased cell apoptosis rate, and increased production of HSP70 from immunohistochemical staining of tumor tissue slices. The safety of the NIR-assisted multimodal cancer treatment could be confirmed from non-significant change of body weight and hematological parameters of blood sample. Taken together, we conclude that IV delivery of IR780-rGO-HA/DOX plus NIR laser treatment is an effective nanomedicine approach for combination cancer therapy.

Functionalized Magnetic Nanoparticles for Alternating Magnetic Field- or Near Infrared Light-Induced Cancer Therapies.

The multi-faceted nature of functionalized magnetic nanoparticles (fMNPs) is well-suited for cancer therapy. These nanocomposites can also provide a multimodal platform for targeted cancer therapy due to their unique magnetic guidance characteristics. When induced by an alternating magnetic field (AMF), fMNPs can convert the magnetostatic energy to heat for magnetic hyperthermia (MHT), as well as for controlled drug release. Furthermore, with the ability to convert near-infrared (NIR) light energy to heat energy, fMNPs have attracted interest for photothermal therapy (PTT). Other than MHT and PTT, fMNPs also have a place in combination cancer therapies, such as chemo-MHT, chemo-PTT, and chemo-PTT-photodynamic therapy, among others, due to their versatile properties. Thus, this review presents multifunctional nanocomposites based on fMNPs for cancer therapies, induced by an AMF or NIR light. We will first discuss the different fMNPs induced with an AMF for cancer MHT and chemo-MHT. Secondly, we will discuss fMNPs irradiated with NIR lasers for cancer PTT and chemo-PTT. Finally, fMNPs used for dual-mode AMF + NIR-laser-induced magneto-photo-hyperthermia (MPHT) will be discussed.

Tension Stimulation of Tenocytes in Aligned Hyaluronic Acid/Platelet-Rich Plasma-Polycaprolactone Core-Sheath Nanofiber Membrane Scaffold for Tendon Tissue Engineering.

To recreate the in vivo niche for tendon tissue engineering in vitro, the characteristics of tendon tissue underlines the use of biochemical and biophysical cues during tenocyte culture. Herein, we prepare core-sheath nanofibers with polycaprolactone (PCL) sheath for mechanical support and hyaluronic acid (HA)/platelet-rich plasma (PRP) core for growth factor delivery. Three types of core-sheath nanofiber membrane scaffolds (CSNMS), consisting of random HA-PCL nanofibers (Random), random HA/PRP-PCL nanofibers (Random+) or aligned HA/PRP-PCL (Align+) nanofibers, were used to study response of rabbit tenocytes to biochemical (PRP) and biophysical (fiber alignment) stimulation. The core-sheath structures as well as other pertinent properties of CSNMS have been characterized, with Align+ showing the best mechanical properties. The unidirectional growth of tenocytes, as induced by aligned fiber topography, was confirmed from cell morphology and cytoskeleton expression. The combined effects of PRP and fiber alignment in Align+ CSNMS lead to enhanced cell proliferation rates, as well as upregulated gene expression and marker protein synthesis. Another biophysical cue on tenocytes was introduced by dynamic culture of tenocyte-seeded Align+ in a bioreactor with cyclic tension stimulation. Augmented by this biophysical beacon from mechanical loading, dynamic cell culture could shorten the time for tendon maturation in vitro, with improved cell proliferation rates and tenogenic phenotype maintenance, compared to static culture. Therefore, we successfully demonstrate how combined use of biochemical/topographical cues as well as mechanical stimulation could ameliorate cellular response of tenocytes in CSNMS, which can provide a functional in vitro environmental niche for tendon tissue engineering.

Magnetic and GRPR-targeted reduced graphene oxide/doxorubicin nanocomposite for dual-targeted chemo-photothermal cancer therapy.

Herein, we design a rGO-based magnetic nanocomposite by decorating rGO with citrate-coated magnetic nanoparticles (CMNP). The magnetic rGO (mrGO) was modified by phospholipid-polyethylene glycol to prepare PEGylated mrGO, for conjugating with gastrin-releasing peptide receptor (GRPR)-binding peptide (mrGOG). The anticancer drug doxorubicin (DOX) was bound to mrGO (mrGOG) by π-π stacking for drug delivery triggered by the low pH value in the endosome. The mrGOG showed enhanced photothermal effect under NIR irradiation, endorsing its role for dual targeted DOX delivery. With efficient DOX release in the endosomal environment and heat generation from light absorption in the NIR range, mrGOG/DOX could be used for combination chemo-photothermal therapy after intracellular uptake by cancer cells. We characterized the physico-chemical as well as biological properties of the synthesized nanocomposites. The mrGOG is stable in biological buffer solution, showing high biocompatibility and minimum hemolytic properties. Using U87 glioblastoma cells, we confirmed the magnetic drug targeting effect in vitro for selective cancer cell killing. The peptide ligand-mediated targeted delivery increases the efficiency of intracellular uptake of both nanocomposite and DOX up to ~3 times due to the over-expressed GRPR on U87 surface, leading to higher cytotoxicity. The increased cytotoxicity using mrGOG over mrGO was shown from a decreased IC50 value (0.70 to 0.48 µg/mL) and an increased cell apoptosis rate (19.8% to 47.1%). The IC50 and apoptosis rate changed further to 0.19 µg/mL and 76.8% in combination with NIR laser irradiation, with the photothermal effect supported from upregulation of heat shock protein HSP70 expression. Using U87 tumor xenograft model created in nude mice, we demonstrated that magnetic guidance after intravenous delivery of mrGOG/DOX could significantly reduce tumor size and prolong animal survival over free DOX and non-magnetic guided groups. Augmented with NIR laser treatment for 5 min, the anti-cancer efficacy significantly improves with elevated cell apoptosis and reduced cell proliferation. Together with safety profiles from hematological as well as major organ histological analysis of treated animals, the mrGOG nanocomposite is an effective nanomaterial for combination chemo-photothermal cancer therapy.

Photosensitizer-Functionalized Nanocomposites for Light-Activated Cancer Theranostics.

Photosensitizers (PSs) have received significant attention recently in cancer treatment due to its theranostic capability for imaging and phototherapy. These PSs are highly responsive to light source of a suitable wavelength for image-guided cancer therapy from generated singlet oxygen and/or thermal heat. Various organic dye PSs show tremendous attenuation of tumor cells during cancer treatment. Among them, porphyrin and chlorophyll-based ultraviolet-visible (UV-Vis) dyes are employed for photodynamic therapy (PDT) by reactive oxygen species (ROS) and free radicals generated with 400-700 nm laser lights, which have poor tissue penetration depth. To enhance the efficacy of PDT, other light sources such as red light laser and X-ray have been suggested; nonetheless, it is still a challenging task to improve the light penetration depth for deep tumor treatment. To overcome this deficiency, near infrared (NIR) (700-900 nm) PSs, indocyanine green (ICG), and its derivatives like IR780, IR806 and IR820, have been introduced for imaging and phototherapy. These NIR PSs have been used in various cancer treatment modality by combining photothermal therapy (PTT) and/or PDT with chemotherapy or immunotherapy. In this review, we will focus on the use of different PSs showing photothermal/photodynamic response to UV-Vis or NIR-Vis light. The emphasis is a comprehensive review of recent smart design of PS-loaded nanocomposites for targeted delivery of PSs in light-activated combination cancer therapy.

γ-Ray-Assisted Synthesis of a Pt-Sn Bimetallic Composite Loaded on Graphene-Graphitic Carbon Nitride Hybrid: A Cocktail Electrocatalyst for the Methanol Oxidation Reaction.

The composite of Pt with transition metals is viewed as the most promising anode material for direct methanol fuel cell (DMFC) applications. Besides the decrease in the Pt loading, these multimetallic structures help in circumventing CO poisoning issues associated with a Pt catalyst. Herein, we prepared and loaded Pt-Sn bimetallic nanoparticles on an electron-rich and stable substrate consisting of graphitic nitride (GCN) and graphene oxide (GO)/reduced graphene oxide (r-GO) hybrid composites. The γ-radiolysis method was employed for coreduction of metal salts to deposit the binary composite of metal nanoparticles over the substrates. These structures were tested as the anode material for the methanol oxidation reaction (MOR). Among various possible combinations, Pt-Sn-loaded rGO-GCN (Pt-Sn/rGO-GCN) demonstrated the current density of ca. 2.4 A/mgPt. To the best of our knowledge, this value is among the highest ones, reported for similar systems in the acidic pH. Furthermore, these composites demonstrated excellent stability in the repeated cycle test. The improved performance is associated to the plenty of -OH groups provided by the Sn counterpart and a large number of adsorption sites from the electron-reached GCN counterpart.

Functionalized Reduced Graphene Oxide as a Versatile Tool for Cancer Therapy.

Cancer is one of the deadliest diseases in human history with extremely poor prognosis. Although many traditional therapeutic modalities-such as surgery, chemotherapy, and radiation therapy-have proved to be successful in inhibiting the growth of tumor cells, their side effects may vastly limited the actual benefits and patient acceptance. In this context, a nanomedicine approach for cancer therapy using functionalized nanomaterial has been gaining ground recently. Considering the ability to carry various anticancer drugs and to act as a photothermal agent, the use of carbon-based nanomaterials for cancer therapy has advanced rapidly. Within those nanomaterials, reduced graphene oxide (rGO), a graphene family 2D carbon nanomaterial, emerged as a good candidate for cancer photothermal therapy due to its excellent photothermal conversion in the near infrared range, large specific surface area for drug loading, as well as functional groups for functionalization with molecules such as photosensitizers, siRNA, ligands, etc. By unique design, multifunctional nanosystems could be designed based on rGO, which are endowed with promising temperature/pH-dependent drug/gene delivery abilities for multimodal cancer therapy. This could be further augmented by additional advantages offered by functionalized rGO, such as high biocompatibility, targeted delivery, and enhanced photothermal effects. Herewith, we first provide an overview of the most effective reducing agents for rGO synthesis via chemical reduction. This was followed by in-depth review of application of functionalized rGO in different cancer treatment modalities such as chemotherapy, photothermal therapy and/or photodynamic therapy, gene therapy, chemotherapy/phototherapy, and photothermal/immunotherapy.

Mechanism of Nanoformulated Graphene Oxide-Mediated Human Neutrophil Activation.

Understanding the molecular mechanisms of graphene oxide (GO)-based biomaterials is important for logical biomedical applications. Previous studies have revealed biointeractions between GO and immune effector cells, but the effects on neutrophils, crucial cells in the immune system, have not been thoroughly discussed. In this study, GO nanoformulations were synthesized with different functional groups, including GO, GO-carboxylated (GO-COOH), and PEGylated GO (GO-PEG), with different surface features, which were elucidated using imaging methods and surface-sensitive quantitative spectroscopic techniques, including atomic force microscopy (AFM), transmission electron microscopy (TEM), and X-ray photoemission spectroscopy (XPS). The GO-based nanoformulations elicited reactive oxygen species (ROS) generation and neutrophil extracellular trap (NET) formation in human neutrophils. Nanoformulated GO stimulates NET development via the formation of ROS. An endocytosis study revealed that nanoformulated GO facilitated internalization by neutrophils via macropinocytosis and actin-dependent phagocytosis. Importantly, calcium mobilization and phosphorylation proteins such as mitogen-activated protein kinases (extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38) and AKT were involved in the activation of neutrophils. These findings offer the first verification that nanoformulated GO exhibits direct effects on human neutrophils.