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.

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.

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.

Optimization of the Preparation of Magnetic Liposomes for the Combined Use of Magnetic Hyperthermia and Photothermia in Dual Magneto-Photothermal Cancer Therapy.

In this work, we aimed to develop liposomal nanocomposites containing citric-acid-coated iron oxide magnetic nanoparticles (CMNPs) for dual magneto-photothermal cancer therapy induced by alternating magnetic field (AMF) and near-infrared (NIR) lasers. Toward this end, CMNPs were encapsulated in cationic liposomes to form nano-sized magnetic liposomes (MLs) for simultaneous magnetic hyperthermia (MH) in the presence of AMF and photothermia (PT) induced by NIR laser exposure, which amplified the heating efficiency for dual-mode cancer cell killing and tumor therapy. Since the heating capability is directly related to the amount of entrapped CMNPs in MLs, while the liposome size is important to allow internalization by cancer cells, response surface methodology was utilized to optimize the preparation of MLs by simultaneously maximizing the encapsulation efficiency (EE) of CMNPs in MLs and minimizing the size of MLs. The experimental design was performed based on the central composite rotatable design. The accuracy of the model was verified from the validation experiments, providing a simple and effective method for fabricating the best MLs, with an EE of 87% and liposome size of 121 nm. The CMNPs and the optimized MLs were fully characterized from chemical and physical perspectives. In the presence of dual AMF and NIR laser treatment, a suspension of MLs demonstrated amplified heat generation from dual hyperthermia (MH)-photothermia (PT) in comparison with single MH or PT. In vitro cell culture experiments confirmed the efficient cellular uptake of the MLs from confocal laser scanning microscopy due to passive accumulation in human glioblastoma U87 cells originated from the cationic nature of MLs. The inducible thermal effects mediated by MLs after endocytosis also led to enhanced cytotoxicity and cumulative cell death of cancer cells in the presence of AMF-NIR lasers. This functional nanocomposite will be a potential candidate for bimodal MH-PT dual magneto-photothermal cancer therapy.

Magnetic Graphene Oxide for Dual Targeted Delivery of Doxorubicin and Photothermal Therapy.

To develop a pH-sensitive dual targeting magnetic nanocarrier for chemo-phototherapy in cancer treatment, we prepared magnetic graphene oxide (MGO) by depositing Fe3O4 magnetic nanoparticles on graphene oxide (GO) through chemical co-precipitation. MGO was modified with polyethylene glycol (PEG) and cetuximab (CET, an epidermal growth factor receptor (EGFR) monoclonal antibody) to obtain MGO-PEG-CET. Since EGFR was highly expressed on the tumor cell surface, MGO-PEG-CET was used for dual targeted delivery an anticancer drug doxorubicin (DOX). The physico-chemical properties of MGO-PEG-CET were fully characterized by dynamic light scattering, transmission electron microscopy, X-ray diffraction, Fourier transform Infrared spectroscopy, thermogravimetric analysis, and superconducting quantum interference device. Drug loading experiments revealed that DOX adsorption followed the Langmuir isotherm with a maximal drug loading capacity of 6.35 mg/mg, while DOX release was pH-dependent with more DOX released at pH 5.5 than pH 7.4. Using quantum-dots labeled nanocarriers and confocal microscopy, intracellular uptakes of MGO-PEG-CET by high EGFR-expressing CT-26 murine colorectal cells was confirmed to be more efficient than MGO. This cellular uptake could be inhibited by pre-incubation with CET, which confirmed the receptor-mediated endocytosis of MGO-PEG-CET. Magnetic targeted killing of CT-26 was demonstrated in vitro through magnetic guidance of MGO-PEG-CET/DOX, while the photothermal effect could be confirmed in vivo and in vitro after exposure of MGO-PEG-CET to near-infrared (NIR) laser light. In addition, the biocompatibility tests indicated MGO-PEG-CET showed no cytotoxicity toward fibroblasts and elicited minimum hemolysis. In vitro cytotoxicity tests showed the half maximal inhibitory concentration (IC50) value of MGO-PEG-CET/DOX toward CT-26 cells was 1.48 µg/mL, which was lower than that of MGO-PEG/DOX (2.64 µg/mL). The IC50 value could be further reduced to 1.17 µg/mL after combining with photothermal therapy by NIR laser light exposure. Using subcutaneously implanted CT-26 cells in BALB/c mice, in vivo anti-tumor studies indicated the relative tumor volumes at day 14 were 12.1 for control (normal saline), 10.1 for DOX, 9.5 for MGO-PEG-CET/DOX, 5.8 for MGO-PEG-CET/DOX + magnet, and 0.42 for MGO-PEG-CET/DOX + magnet + laser. Therefore, the dual targeting MGO-PEG-CET/DOX could be suggested as an effective drug delivery system for anticancer therapy, which showed a 29-fold increase in therapeutic efficacy compared with control by combining chemotherapy with photothermal therapy.

Self-Targeting, Immune Transparent Plasma Protein Coated Nanocomplex for Noninvasive Photothermal Anticancer Therapy.

Cancer cells exhibit specific physiological differences compared to normal cells. Most surface membranes of cancer cells are characterized by high expression of given protein receptors, such as albumin, transferrin, and growth factors that are also present in the plasma of patients themselves, but are lacking on the surface of normal cells. These distinct features between cancer and normal cells can serve as a niche for developing specific treatment strategies. Near-infrared (NIR)-light-triggered therapy platforms are an interesting novel avenue for use in clinical nanomedicine. As a photothermal agent, conducting polymer nanoparticles, such as polypyrrole (PPy), of great NIR light photothermal effects and good biocompatibility, show promising applications in cancer treatments through the hyperthermia mechanism. Autologous plasma proteins coated PPy nanoparticles for hyperthermia therapy as a novel core technology platform to treat cancers through secreted protein acid and rich in cysteine targeting are developed here. This approach can provide unique features of specific targeting toward cancer cell surface markers and immune transparency to avoid recognition and attack by defense cells and achieve prolonged circulation half-life. This technology platform unveils new clinical options for treatment of cancer patients, supporting the emergence of innovative clinical products.

Alpha-Mangostin Reverses Multidrug Resistance by Attenuating the Function of the Multidrug Resistance-Linked ABCG2 Transporter.

The ATP-binding cassette (ABC) drug transporter ABCG2 can actively efflux a wide variety of chemotherapeutic agents out of cancer cells and subsequently reduce the intracellular accumulation of these drugs. Therefore, the overexpression of ABCG2 often contributes to the development of multidrug resistance (MDR) in cancer cells, which is one of the major obstacles to successful cancer chemotherapy. Moreover, ABCG2 is highly expressed in various tissues including the intestine and blood-brain barrier (BBB), limiting the absorption and bioavailability of many therapeutic agents. For decades, the task of developing a highly effective synthetic inhibitor of ABCG2 has been hindered mostly by the intrinsic toxicity, the lack of specificity, and complex pharmacokinetics. Alternatively, considering the wide range of diversity and relatively nontoxic nature of natural products, developing potential modulators of ABCG2 from natural sources is particularly valuable. α-Mangostin is a natural xanthone derived from the pericarps of mangosteen (Garcinia mangostana L.) with various pharmacological purposes, including suppressing angiogenesis and inducing cancer cell growth arrest. In this study, we demonstrated that at nontoxic concentrations, α-mangostin effectively and selectively inhibits ABCG2-mediated drug transport and reverses MDR in ABCG2-overexpressing MDR cancer cells. Direct interactions between α-mangostin and the ABCG2 drug-binding site(s) were confirmed by stimulation of ATPase activity and by inhibition of photolabeling of the substrate-binding site(s) of ABCG2 with [125I]iodoarylazidoprazosin. In summary, our findings show that α-mangostin has great potential to be further developed into a promising modulator of ABCG2 for reversing MDR and for its use in combination therapy for patients with MDR tumors.

EGRF conjugated PEGylated nanographene oxide for targeted chemotherapy and photothermal therapy.

Low accumulation of chemotherapeutic agent in tumor tissue and multidrug resistance (MDR) present a major obstacle to curing cancer treatment. Therefore, how to combine several therapeutics in one system is a key issue to overcome the problem. Here, we demonstrate epidermal growth factor receptor (EGFR) antibody-conjugated PEGylated nanographene oxide (PEG-NGO) to carry epirubicin (EPI) for tumor targeting and triple-therapeutics (growth signal blocking, chemotherapy, photothermal therapy) in tumor treatment. This synergistic targeted treatment simultaneously enhances the local drug concentration (6.3-fold) and performs the ultra-efficient tumor suppression to significantly prolong the mice survival (over the course of 50 days).

Magnetic gold-nanorod/ PNIPAAmMA nanoparticles for dual magnetic resonance and photoacoustic imaging and targeted photothermal therapy.

Nanomedicine can provide a multi-functional platform for image-guided diagnosis and treatment of cancer. Although gold nanorods (GNRs) have been developed for photoacoustic (PA) imaging and near infra-red (NIR) photothermal applications, their efficiency has remained limited by low thermal stability. Here we present the synthesis, characterization, and functional evaluation of non-cytotoxic magnetic polymer-modified gold nanorods (MPGNRs), designed to act as dual magnetic resonance imaging (MRI) and PA imaging contrast agents. In addition, their high magnetization allowed MPGNRs to be actively localized and concentrated by targeting with an external magnet. Finally, MPGNRs significantly enhanced the NIR-laser-induced photothermal effect due to their increased thermal stability. MPGNRs thus provide a promising new theranostic platform for cancer diagnosis and treatment by combining dual MR/PA imaging with highly effective targeted photothermal therapy.

Focused ultrasound-induced blood-brain barrier opening to enhance temozolomide delivery for glioblastoma treatment: a preclinical study.

The purpose of this study is to assess the preclinical therapeutic efficacy of magnetic resonance imaging (MRI)-monitored focused ultrasound (FUS)-induced blood-brain barrier (BBB) disruption to enhance Temozolomide (TMZ) delivery for improving Glioblastoma Multiforme (GBM) treatment. MRI-monitored FUS with microbubbles was used to transcranially disrupt the BBB in brains of Fisher rats implanted with 9L glioma cells. FUS-BBB opening was spectrophotometrically determined by leakage of dyes into the brain, and TMZ was quantitated in cerebrospinal fluid (CSF) and plasma by LC-MS\MS. The effects of treatment on tumor progression (by MRI), animal survival and brain tissue histology were investigated. Results demonstrated that FUS-BBB opening increased the local accumulation of dyes in brain parenchyma by 3.8-/2.1-fold in normal/tumor tissues. Compared to TMZ alone, combined FUS treatment increased the TMZ CSF/plasma ratio from 22.7% to 38.6%, reduced the 7-day tumor progression ratio from 24.03 to 5.06, and extended the median survival from 20 to 23 days. In conclusion, this study provided preclinical evidence that FUS BBB-opening increased the local concentration of TMZ to improve the control of tumor progression and animal survival, suggesting its clinical potential for improving current brain tumor treatment.

Self-protecting core-shell magnetic nanoparticles for targeted, traceable, long half-life delivery of BCNU to gliomas.

The successful delivery of anti-cancer drugs relies on the simultaneous capability to actively target a specific location, a sufficient lifetime in the active form in the circulation, and traceability and quantification of drug distribution via in vivo medical imaging. Herein, a highly magnetic nanocarrier (HMNC) composed of an Fe(3)O(4) core and an aqueous-stable, self-doped poly[N-(1-one-butyric acid)]aniline (SPAnH) shell was chemically synthesized. This nanocarrier exhibited a high capacity for 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) drug loading. BCNU and o-(2-aminoethyl)polyethylene glycol (EPEG) were covalently immobilized on the surface of the HMNC to form a self-protecting magnetic nanomedicine (i.e., SPMNM) that could simultaneously provide low reticuloendothelial system uptake, high active-targeting, and in vivo magnetic resonance imaging (MRI) traceability. Meanwhile, the SPMNM was found to reduce the phagocytosis by macrophages and reduce the hydrolysis rate of BCNU. The high magnetization (approximately 1.2-fold higher than Resovist) of the HMNC allowed efficient magnetic targeting to the tumor. The synergetic drug delivery approach provided approximately a 3.4-fold improvement of the drug's half-life (from 18 h to 62 h) and significantly prolonged the median survival rate in animals that received a low dose of BCNU, compared with those that received a high dose of free BCNU (63 days for those that received 4.5 mg BCNU/kg carried by the nanocarrier versus 50 days for those that received 13.5 mg of free-BCNU). This improvement could enhance the potential of magnetic targeting therapy in clinical applications of cancer treatments.