Red blood cell membrane-camouflaged gold-core silica shell nanorods for cancer drug delivery and photothermal therapy.

Gold core mesoporous silica shell (AuMSS) nanorods are multifunctional nanomedicines that can act simultaneously as photothermal, drug delivery, and bioimaging agents. Nevertheless, it is reported that once administrated, nanoparticles can be coated with blood proteins, forming a protein corona, that directly impacts on nanomedicines' circulation time, biodistribution, and therapeutic performance. Therefore, it become crucial to develop novel alternatives to improve nanoparticles' half-life in the bloodstream. In this work, Polyethylenimine (PEI) and Red blood cells (RBC)-derived membranes were combined for the first time to functionalize AuMSS nanorods and simultaneously load acridine orange (AO). The obtained results revealed that the RBC-derived membranes promoted the neutralization of the AuMSS' surface charge and consequently improved the colloidal stability and biocompatibility of the nanocarriers. Indeed, the in vitro data revealed that PEI/RBC-derived membranes' functionalization also improved the nanoparticles' cellular internalization and was capable of mitigating the hemolytic effects of AuMSS and AuMSS/PEI nanorods. In turn, the combinatorial chemo-photothermal therapy mediated by AuMSS/PEI/RBC_AO nanorods was able to completely eliminate HeLa cells, contrasting with the less efficient standalone therapies. Such data reinforce the potential of AuMSS nanomaterials to act simultaneously as photothermal and chemotherapeutic agents.

Reduced graphene oxide-reinforced tricalcium phosphate/gelatin/chitosan light-responsive scaffolds for application in bone regeneration.

Bone is a mineralized tissue with the intrinsic capacity for constant remodeling. Rapid prototyping techniques, using biomaterials that mimic the bone native matrix, have been used to develop osteoinductive and osteogenic personalized 3D structures, which can be further combined with drug delivery and phototherapy. Herein, a Fab@Home 3D Plotter printer was used to promote the layer-by-layer deposition of a composite mixture of gelatin, chitosan, tricalcium phosphate, and reduced graphene oxide (rGO). The phototherapeutic potential of the new NIR-responsive 3D_rGO scaffolds was assessed by comparing scaffolds with different rGO concentrations (1, 2, and 4 mg/mL). The data obtained show that the rGO incorporation confers to the scaffolds the capacity to interact with NIR light and induce a hyperthermy effect, with a maximum temperature increase of 16.7 °C after under NIR irradiation (10 min). Also, the increase in the rGO content improved the hydrophilicity and mechanical resistance of the scaffolds, particularly in the 3D_rGO4. Furthermore, the rGO could confer an NIR-triggered antibacterial effect to the 3D scaffolds, without compromising the osteoblasts' proliferation and viability. In general, the obtained data support the development of 3D_rGO for being applied as temporary scaffolds supporting the new bone tissue formation and avoiding the establishment of bacterial infections.

In situ formation of alginic acid-gold nanohybrids for application in cancer photothermal therapy.

Gold-based nanoparticles present excellent optical properties that propelled their widespread application in biomedicine, from bioimaging to photothermal applications. Nevertheless, commonly employed manufacturing methods for gold-based nanoparticles require long periods and laborious protocols that reduce cost-effectiveness and scalability. Herein, a novel methodology was used for producing gold-alginic acid nanohybrids (Au-Alg-NH) with photothermal capabilities. This was accomplished by promoting the in situ reduction and nucleation of gold ions throughout a matrix of alginic acid by using ascorbic acid. The results obtained reveal that the Au-Alg-NHs present a uniform size distribution and a spike-like shape. Moreover, the nanomaterials were capable to mediate a temperature increase of ≈11°C in response to the irradiation with a near-infrared region (NIR) laser (808 nm, 1.7 W cm-2 ). The in vitro assays showed that Au-Alg-NHs were able to perform a NIR light-triggered ablation of cancer cells (MCF-7), being observed a reduction in the cell viability to ≈27%. Therefore, the results demonstrate that this novel methodology holds the potential for producing Au-Alg-NH with photothermal capacity and higher translatability to the clinical practice, namely for cancer therapy.

Injectable hydrogels for the delivery of nanomaterials for cancer combinatorial photothermal therapy.

Progress in the nanotechnology field has led to the development of a new class of materials capable of producing a temperature increase triggered by near infrared light. These photothermal nanostructures have been extensively explored in the ablation of cancer cells. Nevertheless, the available data in the literature have exposed that systemically administered nanomaterials have a poor tumor-homing capacity, hindering their full therapeutic potential. This paradigm shift has propelled the development of new injectable hydrogels for the local delivery of nanomaterials aimed at cancer photothermal therapy. These hydrogels can be assembled at the tumor site after injection (in situ forming) or can undergo a gel-sol-gel transition during injection (shear-thinning/self-healing). Besides incorporating photothermal nanostructures, these injectable hydrogels can also incorporate or be combined with other agents, paving the way for an improved therapeutic outcome. This review analyses the application of injectable hydrogels for the local delivery of nanomaterials aimed at cancer photothermal therapy as well as their combination with photodynamic-, chemo-, immuno- and radio-therapies.

Reduced graphene oxide-enriched chitosan hydrogel/cellulose acetate-based nanofibers application in mild hyperthermia and skin regeneration.

Asymmetric wound dressings have captured researchers' attention due to their ability to reproduce the structural and functional properties of the skin layers. Furthermore, recent studies also report the benefits of using near-infrared (NIR) radiation-activated photothermal therapies in treating infections and chronic wounds. Herein, a chitosan (CS) and reduced graphene oxide (rGO) hydrogel (CS_rGO) was combined with a polycaprolactone (PCL) and cellulose acetate (CA) electrospun membrane (PCL_CA) to create a new NIR-responsive asymmetric wound dressing. The rGO incorporation in the hydrogel increased the NIR absorption capacity and allowed a mild hyperthermy effect, a temperature increase of 12.4 °C when irradiated with a NIR laser. Moreover, the PCL_CA membrane presented a low porosity and hydrophobic nature, whereas the CS_rGO hydrogel showed the ability to provide a moist environment, prevent exudate accumulation and allow gaseous exchanges. Furthermore, the in vitro data demonstrate the capacity of the asymmetric structure to act as a barrier against bacteria penetration as well as mediating a NIR-triggered antibacterial effect. Additionally, human fibroblasts were able to adhere and proliferate in the CS_rGO hydrogel, even under NIR laser irradiation, presenting cellular viabilities superior to 90 %. Altogether, our data support the application of the NIR-responsive asymmetric wound dressings for skin regeneration.

Heptamethine Cyanine-Loaded Nanomaterials for Cancer Immuno-Photothermal/Photodynamic Therapy: A Review.

The development of strategies capable of eliminating metastasized cancer cells and preventing tumor recurrence is an exciting and extremely important area of research. In this regard, therapeutic approaches that explore the synergies between nanomaterial-mediated phototherapies and immunostimulants/immune checkpoint inhibitors have been yielding remarkable results in pre-clinical cancer models. These nanomaterials can accumulate in tumors and trigger, after irradiation of the primary tumor with near infrared light, a localized temperature increase and/or reactive oxygen species. These effects caused damage in cancer cells at the primary site and can also (i) relieve tumor hypoxia, (ii) release tumor-associated antigens and danger-associated molecular patterns, and (iii) induced a pro-inflammatory response. Such events will then synergize with the activity of immunostimulants and immune checkpoint inhibitors, paving the way for strong T cell responses against metastasized cancer cells and the creation of immune memory. Among the different nanomaterials aimed for cancer immuno-phototherapy, those incorporating near infrared-absorbing heptamethine cyanines (Indocyanine Green, IR775, IR780, IR797, IR820) have been showing promising results due to their multifunctionality, safety, and straightforward formulation. In this review, combined approaches based on phototherapies mediated by heptamethine cyanine-loaded nanomaterials and immunostimulants/immune checkpoint inhibitor actions are analyzed, focusing on their ability to modulate the action of the different immune system cells, eliminate metastasized cancer cells, and prevent tumor recurrence.

Poly(2-ethyl-2-oxazoline)-IR780 conjugate nanoparticles for breast cancer phototherapy.

Aims: To address the limitations of IR780 by preparing hydrophilic polymer-IR780 conjugates and to employ these conjugates in the assembly of nanoparticles (NPs) intended for cancer photothermal therapy. Materials & methods: The cyclohexenyl ring of IR780 was conjugated for the first time with thiol-terminated poly(2-ethyl-2-oxazoline) (PEtOx). This novel poly(2-ethyl-2-oxazoline)-IR780 (PEtOx-IR) conjugate was combined with D-α-tocopheryl succinate (TOS), leading to the assembly of mixed NPs (PEtOx-IR/TOS NPs). Results: PEtOx-IR/TOS NPs displayed optimal colloidal stability as well as cytocompatibility in healthy cells at doses within the therapeutic range. In turn, the combination of PEtOx-IR/TOS NPs and near-infrared light reduced heterotypic breast cancer spheroid viability to just 15%. Conclusion: PEtOx-IR/TOS NPs are promising agents for breast cancer photothermal therapy.

Conventional anticancer approaches are often associated with severe side effects. Herein, the authors assembled a novel nanoparticle whose therapeutic effect is triggered by laser light. In in vitro assays, the produced nanomaterial was able to, after interacting with laser light, reduce the viability of classic and advanced cancer models. In these conditions, but in the absence of laser light, no cytotoxicity was observed. In this way, the on-demand effect (triggered by laser light) may contribute to reduced side effects. Moreover, the produced nanoparticle revealed good stability, which is important for its future translation.

Poly(2-ethyl-2-oxazoline) functionalized reduced graphene oxide: Optimization of the reduction process using dopamine and application in cancer photothermal therapy.

The high near infrared (NIR) absorption displayed by reduced graphene oxide (rGO) nanostructures renders them a great potential for application in cancer photothermal therapy. However, the production of this material often relies on the use of hydrazine as a reductant, leading to poor biocompatibility and environmental-related issues. In addition, to improve rGO colloidal stability, this material has been functionalized with poly(ethylene glycol). However, recent studies have reported the immunogenicity of poly(ethylene glycol)-based coatings. In this work, the production of rGO, by using dopamine as the reducing agent, was optimized considering the size distribution and NIR absorption of the attained materials. The obtained results unveiled that the rGO produced by using a 1:5 graphene oxide:dopamine weight ratio and a reaction time of 4 h (termed as DOPA-rGO) displayed the highest NIR absorption while retaining its nanometric size distribution. Subsequently, the DOPA-rGO was functionalized with thiol-terminated poly(2-ethyl-2-oxazoline) (P-DOPA-rGO), revealing suitable physicochemical features, colloidal stability and cytocompatibility. When irradiated with NIR light, the P-DOPA-rGO could produce a temperature increase (ΔT) of 36 °C (75 µg/mL; 808 nm, 1.7 W/cm2, 5 min). The photothermal therapy mediated by P-DOPA-rGO was capable of ablating breast cancer cells monolayers (viability < 3%) and could reduce heterotypic breast cancer spheroids' viability to just 30%. Overall, P-DOPA-rGO holds a great potential for application in breast cancer photothermal therapy.

Injectable in situ forming hydrogels incorporating dual-nanoparticles for chemo-photothermal therapy of breast cancer cells.

Chemo-photothermal therapy (chemo-PTT) mediated by nanomaterials holds a great potential for cancer treatment. However, the tumor uptake of the systemically administered nanomaterials was recently found to be below 1%. To address this limitation, the development of injectable tridimensional polymeric matrices capable of delivering nanomaterials directly into the tumor site appears to be a promising approach. In this work, an injectable in situ forming ionotropically crosslinked chitosan-based hydrogel co-incorporating IR780 loaded nanoparticles (IR/BPN) and Doxorubicin (DOX) loaded nanoparticles (DOX/TPN) was developed for application in breast cancer chemo-PTT. The produced hydrogels (IR/BPN@Gel and IR/BPN+DOX/TPN@Gel) displayed suitable physicochemical properties and produced a temperature increase of about 9.1 °C upon exposure to Near Infrared (NIR) light. As importantly, the NIR-light exposure also increased the release of DOX from the hydrogel by 1.7-times. In the in vitro studies, the combination of IR/BPN@Gel with NIR light (photothermal therapy) led to a reduction in the viability of breast cancer cells to 35%. On the other hand, the non-irradiated IR/BPN+DOX/TPN@Gel (chemotherapy) only diminished cancer cells' viability to 85%. In contrast, the combined action of IR/BPN+DOX/TPN@Gel and NIR light reduced cancer cells' viability to about 9%, demonstrating its potential for breast cancer chemo-PTT.

Sulfobetaine methacrylate-albumin-coated graphene oxide incorporating IR780 for enhanced breast cancer phototherapy.

Aim: Enhance the colloidal stability and photothermal capacity of graphene oxide (GO) by functionalizing it with sulfobetaine methacrylate (SBMA)-grafted bovine serum albumin (BSA; i.e., SBMA-g-BSA) and by loading IR780, respectively. Materials & methods: SBMA-g-BSA coating and IR780 loading into GO was achieved through a simple sonication process. Results: SBMA-g-BSA-functionalized GO (SBMA-BSA/GO) presented an adequate size distribution and cytocompatibility. When in contact with biologically relevant media, the size of the SBMA-BSA/GO only increased by 8%. By loading IR780 into SBMA-BSA/GO, its photothermal capacity increased by twofold. The combination of near infrared light with SBMA-BSA/GO did not induce photocytotoxicity on breast cancer cells. In contrast, the interaction of IR780-loaded SBMA-BSA/GO with near infrared light caused the ablation of cancer cells. Conclusion: IR780-loaded SBMA-BSA/GO displayed an improved colloidal stability and phototherapeutic capacity.

Strategies to improve the photothermal capacity of gold-based nanomedicines.

The plasmonic photothermal properties of gold nanoparticles have been widely explored in the biomedical field to mediate a photothermal effect in response to the irradiation with an external light source. Particularly, in cancer therapy, the physicochemical properties of gold-based nanomaterials allow them to efficiently accumulate in the tumor tissue and then mediate the light-triggered thermal destruction of cancer cells with high spatial-temporal control. Nevertheless, the gold nanomaterials can be produced with different shapes, sizes, and organizations such as nanospheres, nanorods, nanocages, nanoshells, and nanoclusters. These gold nanostructures will present different plasmonic photothermal properties that can impact cancer thermal ablation. This review analyses the application of gold-based nanomaterials in cancer photothermal therapy, emphasizing the main parameters that affect its light-to-heat conversion efficiency and consequently the photothermal potential. The different shapes/organizations (clusters, shells, rods, stars, cages) of gold nanomaterials and the parameters that can be fine-tuned to improve the photothermal capacity are presented. Moreover, the gold nanostructures combination with other materials (e.g. silica, graphene, and iron oxide) or small molecules (e.g. indocyanine green and IR780) to improve the nanomaterials photothermal capacity is also overviewed.

Hyaluronic acid and vitamin E polyethylene glycol succinate functionalized gold-core silica shell nanorods for cancer targeted photothermal therapy.

Gold-core mesoporous silica shell (AuMSS) nanorods unique physicochemical properties makes them versatile and promising nanomedicines for cancer photothermal therapy. Nevertheless, these nanomaterials present a reduced half-life in the blood and poor specificity towards the tumor tissue. Herein, d-α-Tocopherol polyethylene glycol 1000 succinate (TPGS) and Hyaluronic Acid (HA) were combined for the first time to improve the AuMSS nanorods biological performance. The obtained results revealed that AuMSS surface functionalization induced the surface charge neutralization, from -28 ±â€¯10 mV to -3 ±â€¯5 mV and -10 ±â€¯4 mV for AuMSS-TPGS-HA (1:1) and (4:1) formulations, without impacting on nanomaterials' photothermal capacity. Moreover, the AuMSS functionalization improved the nanomaterials hemocompatibility and selectivity towards the cancer cells, particularly in the AuMSS-TPGS-HA (4:1) formulation. Furthermore, both formulations were able to mediate an on-demand photothermal effect, that induced the HeLa cancer cells death, confirming its potential for being applied as targeted multifunctional theragnostic nanomedicines.

Poly (vinyl alcohol)/chitosan layer-by-layer microneedles for cancer chemo-photothermal therapy.

The combination of photothermal and chemo- therapies displays a high potential to increase the efficacy of the cancer treatments or even promote their eradication. In this study, the micromoulding and electrospraying techniques were combined to produce polyvinylpyrrolidone microneedles coated with chitosan and poly (vinyl alcohol) for mediating the delivery of doxorubicin and AuMSS nanorods (Dox@MicroN) to cancer cells. The microneedles' physicochemical characterization demonstrated that the electrospraying technique can be used to produce a layer-by-layer coating consisting of layers of doxorubicin-loaded chitosan and AuMSS enriched poly (vinyl alcohol). Further, the Dox@MicroN patches presented a good photothermal capacity leading to a temperature increase of 12 °C under near-infrared irradiation (808 nm, 1.7 W/cm-2 for 5 min), which in conjugation with the chitosan' pH sensitivity could be used to control the doxorubicin release. Moreover, the microneedles were able to penetrate the tumor-mimicking agarose gel and promote a layer dependent drug release. Additionally, the Dox@MicroN patches' capacity to simultaneously mediate the chemo- and photothermal-therapies rendered a superior cytotoxic effect against the cervical cancer cells. Overall, the Dox@MicroN patches demonstrated to be a simple macroscale delivery device that can be used to mediate the local administration of new drug-photothermal combinations, avoiding all the issues related to the systemic administration of anti-cancer therapeutics.

Sulfobetaine methacrylate-functionalized graphene oxide-IR780 nanohybrids aimed at improving breast cancer phototherapy.

The application of Graphene Oxide (GO) in cancer photothermal therapy is hindered by its lack of colloidal stability in biologically relevant media and modest Near Infrared (NIR) absorption. In this regard, the colloidal stability of GO has been improved by functionalizing its surface with poly(ethylene glycol) (PEG), which may not be optimal due to the recent reports on PEG immunogenicity. On the other hand, the chemical reduction of GO using hydrazine hydrate has been applied to enhance its photothermal capacity, despite decreasing its cytocompatibility. In this work GO was functionalized with an amphiphilic polymer containing [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (SBMA) brushes and was loaded with IR780, for the first time, aiming to improve its colloidal stability and phototherapeutic capacity. The attained results revealed that the SBMA-functionalized GO displays a suitable size distribution, neutral surface charge and adequate cytocompatibility. Furthermore, the SBMA-functionalized GO exhibited an improved colloidal stability in biologically relevant media, while its non-SBMA functionalized equivalent promptly precipitated under the same conditions. By loading IR780 into the SBMA-functionalized GO, its NIR absorption increased by 2.7-fold, leading to a 1.2 times higher photothermal heating. In in vitro cell studies, the combination of SBMA-functionalized GO with NIR light only reduced breast cancer cells' viability to 73%. In stark contrast, by combining IR780 loaded SBMA-functionalized GO and NIR radiation, the cancer cells' viability decreased to 20%, hence confirming the potential of this nanomaterial for cancer photothermal therapy.

Development of poly-2-ethyl-2-oxazoline coated gold-core silica shell nanorods for cancer chemo-photothermal therapy.

AIM: Develop a new poly-2-ethyl-2-oxazoline (PEOZ)-based coating for doxorubicin-loaded gold-core mesoporous silica shell (AuMSS) nanorods application in cancer chemo-photothermal therapy. METHODS: PEOZ functionalized AuMSS nanorods were obtained through the chemical grafting on AuMSS of a PEOZ silane derivative. RESULTS: The PEOZ chemical grafting on the surface of AuMSS nanorods allowed the neutralization of nanodevices' surface charge, from -30 to -15 mV, which improved nanoparticles' biocompatibility, namely by decreasing the blood hemolysis to negligible levels. In vitro antitumoral studies revealed that the combined treatment mediated by the PEOZ-coated AuMSS nanorods result in a synergistic effect, allowing the complete eradication of cervical cancer cells. CONCLUSION: The application of the PEOZ coating improves the AuMSS nanorods performance as a multifunctional combinatorial therapy for cervical cancer.

Spheroids Formation on Non-Adhesive Surfaces by Liquid Overlay Technique: Considerations and Practical Approaches.

Scalable and reproducible production of 3D cellular spheroids is highly demanded, by pharmaceutical companies, for drug screening purposes during the pre-clinical evaluation phase. These 3D cellular constructs, unlike the monolayer culture of cells, can mimic different features of human tissues, including cellular organization, cell-cell and cell-extracellular matrix (ECM) interactions. Up to now, different techniques (scaffold-based and -free) have been used for spheroids formation, being the Liquid Overlay Technique (LOT) one of the most explored methodologies, due to its low cost and easy handling. Additionally, during the last few decades, this technique has been widely investigated in order to enhance its potential for being applied in high-throughput analysis. Herein, an overview of the LOT advances, practical approaches, and troubleshooting is provided for those researchers that intend to produce spheroids using LOT, for drug screening purposes. Moreover, the advantages of the LOT over the other scaffold-free techniques used for the spheroids formation are also addressed. Highlights • 2D cell culture drawbacks are summarized; • spheroids mimic the features of human tissues; • scaffold-based and scaffold-free technologies for spheroids production are discussed; • advantages of LOT over other scaffold-free techniques are highlighted; • LOT advances, practical approaches and troubleshooting are underlined.

Strategies to Improve Cancer Photothermal Therapy Mediated by Nanomaterials.

The deployment of hyperthermia-based treatments for cancer therapy has captured the attention of different researchers worldwide. In particular, the application of light-responsive nanomaterials to mediate hyperthermia has revealed promising results in several pre-clinical assays. Unlike conventional therapies, these nanostructures can display a preferential tumor accumulation and thus mediate, upon irradiation with near-infrared light, a selective hyperthermic effect with temporal resolution. Different types of nanomaterials such as those based on gold, carbon, copper, molybdenum, tungsten, iron, palladium and conjugated polymers have been used for this photothermal modality. This progress report summarizes the different strategies that have been applied so far for increasing the efficacy of the photothermal therapeutic effect mediated by nanomaterials, namely those that improve the accumulation of nanomaterials in tumors (e.g. by changing the corona composition or through the functionalization with targeting ligands), increase nanomaterials' intrinsic capacity to generate photoinduced heat (e.g. by synthesizing new nanomaterials or assembling nanostructures) or by optimizing the parameters related to the laser light used in the irradiation process (e.g. by modulating the radiation wavelength). Overall, the development of new strategies or the optimization and combination of the existing ones will surely give a major contribution for the application of nanomaterials in cancer PTT.