Thiol-ene click injectable in situ forming hydrogels incorporating graphene: optimization of the polymer-related features for the immuno-photothermal therapy of metastatic breast cancer

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Development of multifunctional gold core silica shell nanomedicines for chemo/photothermal therapy of cancer

Publication . Rodrigues, Ana Carolina Félix; Correia, Ilídio Joaquim Sobreira; Moreira, André Ferreira

Cancer remains one of the most diagnosed and deadliest diseases worldwide. The currently available cancer treatments, such as surgery, chemotherapy, and radiotherapy present low therapeutic efficacies and selectivity toward cancer cells, inducing several side effects. Particularly, the chemotherapeutic agents are characterized by high non-specific toxicity, and low solubility, being rapidly degraded or eliminated from blood circulation. Therefore, there is an urgent and increased need to develop new anti-cancer approaches with greater therapeutic efficacy, selectivity, and safety. In past years, nanoparticles’ application in medicine triggered a new era of cancer research, from diagnostic to therapeutic. These nanosystems can encapsulate drugs, prevent their premature degradation, and promote the drug's specific delivery to the target site. In addition, researchers have also developed nanosystems capable of mediating a photothermal effect in response to near-infrared (NIR; 700-1100 nm) light, as well as, promoting a tumor-specific delivery of the chemotherapeutic agents. In fact, photothermal therapy (PTT) mediated by nanomaterials has been explored as a standalone or combinatorial therapy, focusing on the hyperthermia of the tumor tissue, and avoiding damage in healthy tissues. PTT take advantage of the nanomaterials’ physicochemical properties, which confer them an intrinsic capacity to accumulate at the tumor site, by taking advantage of the defective vasculature of the tumor (e.g., the enhanced permeability and retention (EPR) effect and/or vascular bursts). Thus, under NIR irradiation of the tumor tissue, the accumulated’ nanoparticles can induce a localized hyperthermia which in turn can sensitize cancer cells to other therapeutic approaches (e.g., chemotherapy) or induce several cellular damages that can ultimately lead to cancer cells’ apoptosis or necrosis. Among the different nanostructures developed so far, gold-core mesoporous silica shell (AuMSS) nanorods have been widely explored for cancer therapy application due to their unique physical and chemical properties, that support their simultaneous application as imaging (e.g., computed tomography) and therapeutic agents (e.g., drug delivery and PTT). The anisotropic morphology of the gold core confers to AuMSS nanorods an inherent capacity to present high absorption in the NIR region, and consequently mediate a photothermal effect. Additionally, the inclusion of a mesoporous silica shell provides a biocompatible, inert, and large surface area that can be easily functionalized; tubular pores that improve the particle's ability to encapsulate and deliver bioactive molecules; protection and stabilization of the gold core from photodegradation when exposed to energetic radiations (e.g., NIR light). Despite all the efforts and several studies showing the huge potential of nanomedicines to act as drug delivery platforms and induce cancer cells death both in vitro and in vivo, the treatment of human tumors with this technology has led to modest improvements. In fact, only 0.7% of the injected nanoparticles dose effectively reaches the tumor site, hindering their real therapeutic potential. Thus, this suboptimal pharmacokinetic profile is pointed out as one of the main factors for the reduced therapeutic effectiveness of nanomedicines and their poor translation to clinical practice. Systemic administration is the main route of nanomedicines’ administration in cancer therapy applications. However, upon intravenous administration nanoparticles are highly susceptible to the adsorption of plasma proteins on their surface. Such protein corona can induce several modifications in the nanomedicines' initial physicochemical properties (e.g., immunogenicity, aggregation state, hydrodynamic size, surface chemistry), that will impact their blood retention, bioavailability, tumor accumulation, and even their biosafety. Therefore, there is a consensus that the first step to increase the nanoparticles’ accumulation in the tumor tissue should encompass the optimization of the nanoparticles’ pharmacokinetics, starting with the blood circulation time, which is crucial to increase the chances of their accumulation in the tumor tissue and exert their therapeutic effect. In recent years, different strategies have been developed to improve nanoparticles’ circulation time, mainly by the optimization of their physicochemical properties and/or functionalization with hydrophilic polymers or biomimetic coatings. In fact, due to the non-immunogenicity, biocompatibility, and biodegradability of hydrophilic polymers, they can resist to non-specific protein adsorption and cell adhesion, which significantly attenuates immune system recognition, improving the pharmacokinetic profile. Particularly, biomimetic coatings based on cell-derived vesicles are an emerging concept to improve the nanomaterials’ biological performance. These cell-derived vesicles inherit the unique features of the source cells, such as biocompatibility, long circulation time, homologous targeting, and immune evasion, showing a high potential to create novel and more effective cancer therapies. In this way, the main goal of this Doctoral thesis work plan was to develop and validate the potential of AuMSS nanorods functionalized with polymeric and biomimetic coatings to improve their applicability in chemo-photothermal combinatorial therapy of cancer. This Doctoral thesis includes an introduction section (Chapter 2) where the AuMSS nanorods general properties and application in cancer therapy are discussed. Moreover, an overview of nanoparticles' role in cancer therapy, particularly the impact of physicochemical properties and surface functionalization in nanoparticles’ blood circulation and tumor accumulation. Furthermore, this thesis includes two chapters presenting the research work developed during this doctorate. In the first study (Chapter 3), branched polyethylenimine (PEI) and hyaluronic acid (HA) were combined, for the first time, to functionalize AuMSS nanorods and create a tumor-targeted chemo-photothermal nanomedicine. AuMSS nanorods were produced using the “seed-mediated growth” and Stöber’s modified methods to obtain the gold nanorod core and mesoporous silica shell, respectively. The functionalization of the AuMSS nanorods' surface was achieved through the chemical linkage of PEI (modified with 3-(triethoxysilyl)propyl isocyanate) followed by electrostatic adsorption of HA. The inclusion of PEI and HA on the AuMSS surface promoted a controlled and sustained release of the chemotherapeutic agent – acridine orange (AO). HA functionalization promoted a neutralization of AuMSS surface charge (from 44 to -10 mV) and consequently improved the AuMSS’ biocompatibility by decreasing the blood hemolysis to safe levels. The in vitro assays demonstrated that the HA functionalization increased the nanoparticles’ internalization by cervical cancer cells. Additionally, the combinatorial treatment (i.e., chemotherapy and PTT) mediated by AuMSS/PEI/HA_AO nanorods presented an enhanced effect when compared to single PTT or chemotherapy regiments, leading to the almost complete elimination of the cancer cells (95%). In the second study (Chapter 4), PEI and Red blood cells (RBC)-derived membranes were combined for the first time to functionalize AuMSS nanorods. The RBC-derived membranes were further loaded with AO to allow the combined chemo-photothermal therapy of cervical cancer cells. PEI was chemically modified with 3-(triethoxysilyl)propyl isocyanate, to enable its grafting on the AuMSS nanorods, followed by the entrapment on RBC-derived membranes. PEI/RBC-derived membranes promoted a neutralization of AuMSS surface charge (-26 to -16 mV) and consequently improved the AuMSS nanorods’ colloidal stability and biocompatibility. Also, the AuMSS/PEI/RBC nanorods induced a photo-induced heat variation (ΔT ≈30°C) under NIR irradiation (808 nm, 1.7 W/cm2, 10 min). The in vitro uptake studies revealed that PEI/RBC-derived membranes’ functionalization improved the nanoparticles’ cellular internalization. Furthermore, the combinatorial chemo-PTT mediated by AuMSS/PEI/RBC_AO nanorods eliminated cervical cancer cells, in contrast to less efficient standalone therapies. Overall, the results obtained herein demonstrated that AuMSS nanorods functionalization with hydrophilic polymers (PEI and HA) or biomimetic coating (RBC-derived membranes) improved nanoparticles’ surface charge and colloidal stability, yielding nanomedicines with enhanced biological properties and therapeutic performance. Furthermore, these results support the therapeutic potential of AuMSS nanorods as multifunctional nanomedicines in cancer therapy and highlight that their therapeutic potential can be improved by combining different therapeutic approaches (i.e., chemotherapy and PTT). Altogether, these results reinforce the great potential of AuMSS nanorods, encouraging their continuous study and optimization. This will allow the development of increasingly precise and personalized anticancer therapies, in order to accelerate the translation of AuMSS nanorods into clinical practice, and thus be able to effectively contribute to the treatment of cancer and the reduction of the mortality rate associated with this disease.

2024-05-16Doctoral thesis

Embargoed access

Development of dual-crosslinked Pluronic F127/Chitosan injectable hydrogels incorporating graphene nanosystems for breast cancer photothermal therapy and antibacterial applications

Publication . Pouso, Manuel António do Rosário; Melo, Bruna L.; Gonçalves, Joaquim; Mendonça, António; Correia, I.J.; Diogo, Duarte de Melo

Nanomaterials with responsiveness to near-infrared light can mediate the photoablation of cancer cells with an exceptional spatio-temporal resolution. However, the therapeutic outcome of this modality is limited by the nanostructures’ poor tumor uptake. To address this bottleneck, it is appealing to develop injectable in situ forming hydrogels due to their capacity to perform a tumor-confined delivery of the nanomaterials with minimal off-target leakage. In particular, injectable in situ forming hydrogels based on Pluronic F127 have been emerging due to their FDA-approval status, biocompatibility, and thermosensitive sol–gel transition. Nevertheless, the application of Pluronic F127 hydrogels has been limited due to their fast dissociation in aqueous media. Such limitation may be addressed by combining the thermoresponsive sol–gel transition of Pluronic F127 with other polymers with crosslinking capabilities. In this work, a novel dual-crosslinked injectable in situ forming hydrogel based on Pluronic F127 (thermosensitive gelation) and Chitosan (ionotropic gelation in the presence of NaHCO3), loaded with Dopamine-reduced graphene oxide (DOPA-rGO; photothermal nanoagent), was developed for application in breast cancer photothermal therapy. The dual-crosslinked hydrogel incorporating DOPA-rGO showed a good injectability (through 21 G needles), in situ gelation capacity and cytocompatibility (viability > 73 %). As importantly, the dual-crosslinking improved the hydrogel’s porosity and prevented its premature degradation. After irradiation with near-infrared light, the dual-crosslinked hydrogel incorporating DOPA-rGO produced a photothermal heating (ΔT ≈ 22 °C) that reduced the breast cancer cells’ viability to just 32 %. In addition, this formulation also demonstrated a good antibacterial activity by reducing the viability of S. aureus and E. coli to 24 and 33 %, respectively. Overall, the dual-crosslinked hydrogel incorporating DOPA-rGO is a promising macroscale technology for breast cancer photothermal therapy and antimicrobial applications.

2024-08-28Journal article

Open access