Correction: Ibarra et al. Selective Photo-Assisted Eradication of Triple-Negative Breast Cancer Cells through Aptamer Decoration of Doped Conjugated Polymer Nanoparticles. Pharmaceutics 2022, 14, 626.

In the original publication [...].

Enhancing Therapeutic Approaches in Glioblastoma with Pro-Oxidant Treatments and Synergistic Combinations: In Vitro Experience of Doxorubicin and Photodynamic Therapy.

Glioblastoma (GBM) is an aggressive brain cancer characterized by significant molecular and cellular heterogeneity, which complicates treatment efforts. Current standard therapies, including surgical resection, radiation, and temozolomide (TMZ) chemotherapy, often fail to achieve long-term remission due to tumor recurrence and resistance. A pro-oxidant environment is involved in glioma progression, with oxidative stress contributing to the genetic instability that leads to gliomagenesis. Evaluating pro-oxidant therapies in brain tumors is crucial due to their potential to selectively target and eradicate cancer cells by exploiting the elevated oxidative stress levels inherent in these malignant cells, thereby offering a novel and effective strategy for overcoming resistance to conventional therapies. This study investigates the therapeutic potential of doxorubicin (DOX) and photodynamic therapy (PDT) with Me-ALA, focusing on their effects on redox homeostasis. Basal ROS levels and antioxidant gene expression (NFE2L2, CAT, GSR) were quantitatively assessed across GBM cell lines, revealing significant variability probably linked to genetic differences. DOX and PDT treatments, both individually and in combination, were analyzed for their efficacy in inducing oxidative stress and cytotoxicity. An in silico analysis further explored the relationship between gene mutations and oxidative stress in GBM patients, providing insights into the molecular mechanisms underlying treatment responses. Our findings suggest that pro-oxidant therapies, such as DOX and PDT in combination, could selectively target GBM cells, highlighting a promising avenue for improving therapeutic outcomes in GBM.

Advances in the Development of New Drugs and Treatment Targets for Brain Cancers.

Brain tumors are a significant concern for the global medical community, with over 300,000 cases reported annually worldwide [...].

Metronomic Photodynamic Therapy with Conjugated Polymer Nanoparticles in Glioblastoma Tumor Microenvironment.

Alternative therapies such as photodynamic therapy (PDT) that combine light, oxygen and photosensitizers (PSs) have been proposed for glioblastoma (GBM) management to overcome conventional treatment issues. An important disadvantage of PDT using a high light irradiance (fluence rate) (cPDT) is the abrupt oxygen consumption that leads to resistance to the treatment. PDT metronomic regimens (mPDT) involving administering light at a low irradiation intensity over a relatively long period of time could be an alternative to circumvent the limitations of conventional PDT protocols. The main objective of the present work was to compare the effectiveness of PDT with an advanced PS based on conjugated polymer nanoparticles (CPN) developed by our group in two irradiation modalities: cPDT and mPDT. The in vitro evaluation was carried out based on cell viability, the impact on the macrophage population of the tumor microenvironment in co-culture conditions and the modulation of HIF-1α as an indirect indicator of oxygen consumption. mPDT regimens with CPNs resulted in more effective cell death, a lower activation of molecular pathways of therapeutic resistance and macrophage polarization towards an antitumoral phenotype. Additionally, mPDT was tested in a GBM heterotopic mouse model, confirming its good performance with promising tumor growth inhibition and apoptotic cell death induction.

Exploring Monocytes-Macrophages in Immune Microenvironment of Glioblastoma for the Design of Novel Therapeutic Strategies.

Gliomas are primary malignant brain tumors. These tumors seem to be more and more frequent, not only because of a true increase in their incidence, but also due to the increase in life expectancy of the general population. Among gliomas, malignant gliomas and more specifically glioblastomas (GBM) are a challenge in their diagnosis and treatment. There are few effective therapies for these tumors, and patients with GBM fare poorly, even after aggressive surgery, chemotherapy, and radiation. Over the last decade, it is now appreciated that these tumors are composed of numerous distinct tumoral and non-tumoral cell populations, which could each influence the overall tumor biology and response to therapies. Monocytes have been proved to actively participate in tumor growth, giving rise to the support of tumor-associated macrophages (TAMs). In GBM, TAMs represent up to one half of the tumor mass cells, including both infiltrating macrophages and resident brain microglia. Infiltrating macrophages/monocytes constituted ~ 85% of the total TAM population, they have immune functions, and they can release a wide array of growth factors and cytokines in response to those factors produced by tumor and non-tumor cells from the tumor microenvironment (TME). A brief review of the literature shows that this cell population has been increasingly studied in GBM TME to understand its role in tumor progression and therapeutic resistance. Through the knowledge of its biology and protumoral function, the development of therapeutic strategies that employ their recruitment as well as the modulation of their immunological phenotype, and even the eradication of the cell population, can be harnessed for therapeutic benefit. This revision aims to summarize GBM TME and localization in tumor niches with special focus on TAM population, its origin and functions in tumor progression and resistance to conventional and experimental GBM treatments. Moreover, recent advances on the development of TAM cell targeting and new cellular therapeutic strategies based on monocyte/macrophages recruitment to eradicate GBM are discussed as complementary therapeutics.

Development and biological evaluation of pNIPAM-based nanogels as vaccine carriers.

"Smart" nanogels are an attractive tool for the development of new strategies of immunization in veterinary medicine. Here, we reported the synthesis and physicochemical characterization of thermoresponsive nanogels based on poly(N-isopropylacrylamide) (pNIPAM) and theirin vitro, ex vivoand in vivo (mice model) performance. Smart nanogels of ca. 250 nm, with a transition temperature of 32 °C were obtained by precipitation polymerization. Assays to evaluatepNIPAM nanogels cytotoxicity were performed in different cell lines showing high biocompatibility (>70 %). The efficient internalization of the system was studied by confocal microscopy as well as flow cytometry. The ability to protect and deliver antigens was analyzed using the outer membrane lipoprotein A (OmlA), an important virulence factor ofActinobacillus pleuropneumoniae(App)cause of porcine pleuropneumonia. This lipoprotein was synthesized by recombinant technology and its technique was also described. The biodistribution ofpNIPAM nanogels administered intranasally was performedinvivo and ex vivo through Pearl Imaging System, which showed that nanogels were kept mostly in the lungs during the evaluated time. Besides, the efficacy of the proposal nanogel-based vaccine was studiedin vivoby measuring the antibody titers of BALB/c mice inoculated with OmlA encapsulated intopNIPAM nanogels compared to OmlA plus aluminum hydroxide adjuvant. The results proved the ability of nanogels to stimulate a humoral immune response. Therefore, we have demonstrated thatpNIPAM nanogels can be used as an efficient platform for vaccine nanocarriers.

Optimization of Short RNA Aptamers for TNBC Cell Targeting.

Triple-negative breast cancer (TNBC) is an aggressive cancer with limited targeted therapies. RNA aptamers, suitably chemically modified, work for therapeutic purposes in the same way as antibodies. We recently generated 2'Fluoro-pyrimidines RNA-aptamers that act as effective recognition elements for functional surface signatures of TNBC cells. Here, we optimized three of them by shortening and proved the truncated aptamers as optimal candidates to enable active targeting to TNBC. By using prediction of secondary structure to guide truncation, we identified structural regions that account for the binding motifs of the full-length aptamers. Their chemical synthesis led to short aptamers with superb nuclease resistance, which specifically bind to TNBC target cells and rapidly internalize into acidic compartments. They interfere with the growth of TNBC cells as mammospheres, thus confirming their potential as anti-tumor agents. We propose sTN145, sTN58 and sTN29 aptamers as valuable tools for selective TNBC targeting and promising candidates for effective treatments, including therapeutic agents and targeted delivery nanovectors.

Selective Photo-Assisted Eradication of Triple-Negative Breast Cancer Cells through Aptamer Decoration of Doped Conjugated Polymer Nanoparticles.

Photodynamic therapy (PDT) may be an excellent alternative in the treatment of breast cancer, mainly for the most aggressive type with limited targeted therapies such as triple-negative breast cancer (TNBC). We recently generated conjugated polymer nanoparticles (CPNs) as efficient photosensitizers for the photo-eradication of different cancer cells. With the aim of improving the selectivity of PDT with CPNs, the nanoparticle surface conjugation with unique 2'-Fluoropyrimidines-RNA-aptamers that act as effective recognition elements for functional surface signatures of TNBC cells was proposed and designed. A coupling reaction with carbodiimide was used to covalently bind NH2-modified aptamers with CPNs synthetized with two polystyrene-based polymer donors of COOH groups for the amide reaction. The selectivity of recognition for TNBC membrane receptors and PDT efficacy were assayed in TNBC cells and compared with non-TNBC cells by flow cytometry and cell viability assays. Furthermore, in vitro PDT efficacy was assayed in different TNBC cells with significant improvement results using CL4, sTN29 and sTN58 aptamers compared to unconjugated CPNs and SCR non-specific aptamer. In a chemoresistance TNBC cell model, sTN58 was the candidate for improving labelling and PDT efficacy with CPNs. We proposed sTN58, sTN29 and CL4 aptamers as valuable tools for selective TNBC targeting, cell internalization and therapeutic improvements for CPNs in PDT protocols.

Iron Oxide Incorporated Conjugated Polymer Nanoparticles for Simultaneous Use in Magnetic Resonance and Fluorescent Imaging of Brain Tumors.

Conjugated polymer nanoparticles (CPNs) have emerged as advanced polymeric nanoplatforms in biomedical applications by virtue of extraordinary properties including high fluorescence brightness, large absorption coefficients of one and two-photons, and excellent photostability and colloidal stability in water and physiological medium. In addition, low cytotoxicity, easy functionalization, and the ability to modify CPN photochemical properties by the incorporation of dopants, convert them into excellent theranostic agents with multifunctionality for imaging and treatment. In this work, CPNs were designed and synthesized by incorporating a metal oxide magnetic core (Fe3O4 and NiFe2O4 nanoparticles, 5 nm) into their matrix during the nanoprecipitation method. This modification allowed the in vivo monitoring of nanoparticles in animal models using magnetic resonance imaging (MRI) and intravital fluorescence, techniques widely used for intracranial tumors evaluation. The modified CPNs were assessed in vivo in glioblastoma (GBM) bearing mice, both heterotopic and orthotopic developed models. Biodistribution studies were performed with MRI acquisitions and fluorescence images up to 24 h after the i.v. nanoparticles administration. The resulting IONP-doped CPNs were biocompatible in GBM tumor cells in vitro with an excellent cell incorporation depending on nanoparticle concentration exposure. IONP-doped CPNs were detected in tumor and excretory organs of the heterotopic GBM model after i.v. and i.t. injection. However, in the orthotopic GBM model, the size of the nanoparticles is probably hindering a higher effect on intratumorally T2-weighted images (T2WI) signals and T2 values. The photodynamic therapy (PDT)-cytotoxicity of CPNs was not either affected by the IONPs incorporation into the nanoparticles.

Understanding the glioblastoma tumor biology to optimize photodynamic therapy: From molecular to cellular events.

Photodynamic therapy (PDT) has recently gained attention as an alternative treatment of malignant gliomas. Glioblastoma (GBM) is the most prevalent within tumors of the central nervous system (CNS). Conventional treatments for this CNS tumor include surgery, radiation, and chemotherapy. Surgery is still being considered as the treatment of choice. Even so, the poor prognosis and/or recurrence of the disease after applying any of these treatments highlight the urgency of exploring new therapies and/or improving existing ones to achieve the definitive eradication of tumor masses and remaining cells. PDT is a therapeutic modality that involves the destruction of tumor cells by reactive oxygen species induced by light, which were previously treated with a photosensitizing agent. However, in recent years, its experimental application has expanded to other effects that could improve overall performance against GBM. In the current review, we revisit the main advances of PDT for GBM management and also, the recent mechanistic insights about cellular and molecular aspects related to tumoral resistance to PDT of GBM.

Photodynamic therapy of Glioblastoma cells using doped conjugated polymer nanoparticles: An in vitro comparative study based on redox status.

Due to their superb light absorption and photostability conjugated polymer nanoparticles are promising photosensitizers (PS) for their use in Photodynamic therapy (PDT). Recently, we developed metallated porphyrin-doped conjugated polymer nanoparticles (CPNs) for PDT that efficiently eliminate tumor cells through reactive oxygen species (ROS) mediated photoinduced damage of apoptotic nature. These nanoaggregates act as densely packed multi-chromophoric systems having exceptional light harvesting and (intra-particle) energy transfer capabilities which lead to efficient photosensitized formation of ROS. In general, three key components; light, PS, and oxygen; are considered in the prediction of the PDT outcome. However, recent studies led to the discovery of a profound genetic heterogeneity among glioblastoma (GBM) cells which include the adaptation to ROS. Thus, tumor heterogeneity and their associated difference in sensitivity to ROS-producing therapeutic agents must be considered in the design of PDT protocols for the prediction of its outcome. In this study, anticancer activity through ROS-mediated PDT using CPNs was compared in three GBM cell lines with different initial redox status. T98G cells were the most effective incorporating nanoparticles but also were the most resistant to CPN-PDT effect. In part, this feature could be attributed to the differential basal and PDT-induced antioxidant enzyme levels found in these cells measured by gene expression analysis. Furthermore, considering that cell-specific antioxidant enzyme status is a significant feature of GBM heterogeneity, establishing its correlation with CPN-PDT outcome might be important for designing novel and improved CPN-based treatments.

Metallated porphyrin-doped conjugated polymer nanoparticles for efficient photodynamic therapy of brain and colorectal tumor cells.

AIM: Assess biocompatibility, uptake and photodynamic therapy (PDT) mechanism of metallated porphyrin doped conjugated polymer nanoparticles (CPNs) in human brain and colorectal tumor cells and macrophages. MATERIALS & METHODS: CPNs were developed employing 9,9-dioctylfluorene-alt-benzothiadiazole, an amphiphilic polymer (PS-PEG-COOH),  and platinum octaethylporphyrin. T98G, SW480 and RAW 264.7 cell lines were exposed to CPNs to assess uptake and intracellular localization. Additionally, a PDT protocol using CPNs was employed for the in vitro killing of cancer and macrophage cell lines. RESULTS & CONCLUSION: CPNs were well incorporated into glioblastoma and macrophage cells with localization in lysosomes. SW480 cells were less efficient incorporating CPNs with localization in the plasma membrane. In all cell lines PDT treatment was efficient inducing oxidative stress that triggered apoptosis.