Corrigendum: HSA-nanobinders crafted from bioresponsive prodrugs for combined cancer chemoimmunotherapy-an in vitro exploration.

[This corrects the article DOI: 10.3389/fchem.2024.1378233.].

HSA-nanobinders crafted from bioresponsive prodrugs for combined cancer chemoimmunotherapy-an in vitro exploration.

Introduction: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer still lacking effective treatment options. Chemotherapy in combination with immunotherapy can restrict tumor progression and repolarize the tumor microenvironment towards an anti-tumor milieu, improving clinical outcome in TNBC patients. The chemotherapeutic drug paclitaxel has been shown to induce immunogenic cell death (ICD), whereas inhibitors of the indoleamine 2,3- dioxygenase 1 (IDO1) enzyme, whose expression is shared in immune regulatory and tumor cells, have been revealed to enhance the anti-tumor immune response. However, poor bioavailability and pharmacokinetics, off-target effects and hurdles in achieving therapeutic drug concentrations at the target tissue often limit the effectiveness of combination therapies. Methods: This work describes the development of novel biomimetic and carrier-free nanobinders (NBs) loaded with both paclitaxel and the IDO1 inhibitor NLG919 in the form of bioresponsive and biomimetic prodrugs. A fine tuning of the preparation conditions allowed to identify NB@5 as the most suitable nanoformulation in terms of reproducibility, stability and in vitro effectiveness. Results and discussion: Our data show that NB@5 effectively binds to HSA in cell-free experiments, demonstrating its protective role in the controlled release of drugs and suggesting the potential to exploit the protein as the endogenous vehicle for targeted delivery to the tumor site. Our study successfully proves that the drugs encapsulated within the NBs are preferentially released under the altered redox conditions commonly found in the tumor microenvironment, thereby inducing cell death, promoting ICD, and inhibiting IDO1.

Two Beats One: Osteosarcoma Therapy with Light-Activated and Chemo-Releasing Keratin Nanoformulation in a Preclinical Mouse Model.

Osteosarcoma treatment is moving towards more effective combination therapies. Nevertheless, these approaches present distinctive challenges that can complicate the clinical translation, such as increased toxicity and multi-drug resistance. Drug co-encapsulation within a nanoparticle formulation can overcome these challenges and improve the therapeutic index. We previously synthetized keratin nanoparticles functionalized with Chlorin-e6 (Ce6) and paclitaxel (PTX) to combine photo (PDT) and chemotherapy (PTX) regimens, and the inhibition of osteosarcoma cells growth in vitro was demonstrated. In the current study, we generated an orthotopic osteosarcoma murine model for the preclinical evaluation of our combination therapy. To achieve maximum reproducibility, we systematically established key parameters, such as the number of cells to generate the tumor, the nanoparticles dose, the design of the light-delivery device, the treatment schedule, and the irradiation settings. A 60% engrafting rate was obtained using 10 million OS cells inoculated intratibial, with the tumor model recapitulating the histological hallmarks of the human counterpart. By scheduling the treatment as two cycles of injections, a 32% tumor reduction was obtained with PTX mono-therapy and a 78% reduction with the combined PTX-PDT therapy. Our findings provide the in vivo proof of concept for the subsequent clinical development of a combination therapy to fight osteosarcoma.

Free and Poly-Methyl-Methacrylate-Bounded BODIPYs: Photodynamic and Antimigratory Effects in 2D and 3D Cancer Models.

Several limitations, including dark toxicity, reduced tumor tissue selectivity, low photostability and poor biocompatibility hamper the clinical use of Photodynamic therapy (PDT) in cancer treatment. To overcome these limitations, new PSs have been synthetized, and often combined with drug delivery systems, to improve selectivity and reduce toxicity. In this context, BODIPYs (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) have recently emerged as promising and easy-to-handle scaffolds for the preparation of effective PDT antitumor agents. In this study, the anticancer photodynamic effect of newly prepared negatively charged polymethyl methacrylate (nPMMA)-bounded BODIPYs (3@nPMMA and 6@nPMMA) was evaluated on a panel of 2D- and 3D-cultured cancer cell lines and compared with free BODIPYs. In particular, the effect on cell viability was evaluated, along with their ability to accumulate into the cells, induce apoptotic and/or necrotic cell death, and inhibit cellular migration. Our results indicated that 3@nPMMA and 6@nPMMA reduce cancer cell viability in 3D models of HC116 and MCF7 cells more effectively than the corresponding free compounds. Importantly, we demonstrated that MDA-MB231 and SKOV3 cell migration ability was significantly impaired by the PDT treatment mediated by 3@nPMMA and 6@nPMMA nanoparticles, likely indicating the capability of this approach to reduce metastatic tumor potential.

Co-Density Distribution Maps for Advanced Molecule Colocalization and Co-Distribution Analysis.

Cellular and subcellular spatial colocalization of structures and molecules in biological specimens is an important indicator of their co-compartmentalization and interaction. Presently, colocalization in biomedical images is addressed with visual inspection and quantified by co-occurrence and correlation coefficients. However, such measures alone cannot capture the complexity of the interactions, which does not limit itself to signal intensity. On top of the previously developed density distribution maps (DDMs), here, we present a method for advancing current colocalization analysis by introducing co-density distribution maps (cDDMs), which, uniquely, provide information about molecules absolute and relative position and local abundance. We exemplify the benefits of our method by developing cDDMs-integrated pipelines for the analysis of molecules pairs co-distribution in three different real-case image datasets. First, cDDMs are shown to be indicators of colocalization and degree, able to increase the reliability of correlation coefficients currently used to detect the presence of colocalization. In addition, they provide a simultaneously visual and quantitative support, which opens for new investigation paths and biomedical considerations. Finally, thanks to the coDDMaker software we developed, cDDMs become an enabling tool for the quasi real time monitoring of experiments and a potential improvement for a large number of biomedical studies.

Mesenchymal stromal cells mediated delivery of photoactive nanoparticles inhibits osteosarcoma growth in vitro and in a murine in vivo ectopic model.

BACKGROUND: Osteosarcoma (OS) is an aggressive malignant neoplasm that still suffers from poor prognosis in the case of distal metastases or occurrence of multi-drug resistance. It is therefore crucial to find novel therapeutic options able to go beyond these limitations and improve patients' survival. The objective of this study is to exploit the intrinsic properties of mesenchymal stromal cells (MSCs) to migrate and infiltrate the tumor stroma to specifically deliver therapeutic agents directly to cancer cells. In particular, we aimed to test the efficacy of the photoactivation of MSCs loaded with nanoparticles in vitro and in a murine in vivo ectopic osteosarcoma model. METHODS: AlPcS4@FNPs were produced by adding tetra-sulfonated aluminum phthalocyanine (AlPcS4) to an aqueous solution of positively charged poly-methyl methacrylate core-shell fluorescent nanoparticles (FNPs). The photodynamic therapy (PDT) effect is achieved by activation of the photosensitizer AlPcS4 in the near-infrared light with an LED source. Human MSCs were isolated from the bone marrow of five donors to account for inter-patients variability and used in this study after being evaluated for their clonogenicity, multipotency and immunophenotypic profile. MSC lines were then tested for the ability to internalize and retain the nanoparticles, along with their migratory properties in vitro. Photoactivation effect was evaluated both in a monolayer (2D) co-culture of AlPcS4@FNPs loaded MSCs with human OS cells (SaOS-2) and in tridimensional (3D) multicellular spheroids (AlPcS4@FNPs loaded MSCs with human OS cells, MG-63). Cell death was assessed by AnnexinV/PI and Live&Dead CalceinAM/EthD staining in 2D, while in the 3D co-culture, the cell killing effect was measured through ATP content, CalceinAM/EthD staining and TEM imaging. We also evaluated the effectiveness of AlPcS4@FNPs loaded MSCs as delivery systems and the ability of the photodynamic treatment to kill cancer cells in a subcutaneous mouse model of OS by bioluminescence imaging (BLI) and histology. RESULTS: MSCs internalized AlPcS4@FNPs without losing or altering their motility and viability in vitro. Photoactivation of AlPcS4@FNPs loaded MSCs induced high level of OS cells death in the 2D co-culture. Similarly, in the 3D co-culture (MSCs:OS ratios 1:1 or 1:3), a substantial decrease of both MSCs and OS cells viability was observed. Notably, when increasing the MSCs:OS ratio to 1:7, photoactivation still caused more than 40% cells death. When tested in an in vivo ectopic OS model, AlPcS4@FNPs loaded MSCs were able to decrease OS growth by 68% after two cycles of photoactivation. CONCLUSIONS: Our findings demonstrate that MSCs can deliver functional photosensitizer-decorated nanoparticles in vitro and in vivo and inhibit OS tumor growth. MSCs may be an effective platform for the targeted delivery of therapeutic nanodrugs in a clinical scenario, alone or in combination with other osteosarcoma treatment modalities.

Keratin nanoparticles co-delivering Docetaxel and Chlorin e6 promote synergic interaction between chemo- and photo-dynamic therapies.

The combination of chemotherapy and photodynamic therapy (PDT) is considered a valuable strategy for increasing therapeutic response in cancer treatment, and the re-formulation of pharmaceuticals in biocompatible nanoparticles (NPs) is particularly appealing for the possibility of co-loading drugs exerting cytotoxicity by different mechanisms, with the aim to produce synergic effects. We report the in-water synthesis of a novel keratin-based nanoformulation for the co-delivery of the antimitotic Docetaxel (DTX) and the photosensitizer Chlorin e6 (Ce6). The drug-induced aggregation method allowed the formation of monodisperse NPs (DTX/Ce6-KNPs) with an average diameter of 133 nm and loaded with a drug ratio of 1:1.8 of Ce6 vs DTX. The efficacy of DTX/Ce6-KNPs was investigated in vitro in monolayers and spheroids of DTX-sensitive HeLa (HeLa-P) and DTX-resistant HeLa (HeLa-R) cells. In monolayers, the cytotoxic effects of DTX/Ce6-KNPs toward HeLa-P cells were comparable to those induced by free DTX + Ce6, while in HeLa-R cells the drug co-loading in KNPs produced synergic interaction between chemotherapy and PDT. Moreover, as respect to monotherapies, DTX/Ce6-KNPs induced stronger cytotoxicity to both HeLa-P and HeLa-R multicellular spheroids and reduced their volumes up to 50%. Overall, the results suggest that KNPs are very promising systems for the co-delivery of chemotherapeutics and PSs, favoring synergic interactions between PDT and chemotherapy.

Functionalized Keratin as Nanotechnology-Based Drug Delivery System for the Pharmacological Treatment of Osteosarcoma.

Osteosarcoma therapy might be moving toward nanotechnology-based drug delivery systems to reduce the cytotoxicity of antineoplastic drugs and improve their pharmacokinetics. In this paper, we present, for the first time, an extensive chemical and in vitro characterization of dual-loaded photo- and chemo-active keratin nanoparticles as a novel drug delivery system to treat osteosarcoma. The nanoparticles are prepared from high molecular weight and hydrosoluble keratin, suitably functionalized with the photosensitizer Chlorin-e6 (Ce6) and then loaded with the chemotherapeutic drug Paclitaxel (PTX). This multi-modal PTX-Ce6@Ker nanoformulation is prepared by both drug-induced aggregation and desolvation methods, and a comprehensive physicochemical characterization is performed. PTX-Ce6@Ker efficacy is tested on osteosarcoma tumor cell lines, including chemo-resistant cells, using 2D and 3D model systems. The single and combined contributions of PTX and Ce6 is evaluated, and results show that PTX retains its activity while being vehiculated through keratin. Moreover, PTX and Ce6 act in an additive manner, demonstrating that the combination of the cytostatic blockage of PTX and the oxidative damage of ROS upon light irradiation have a far superior effect compared to singularly administered PTX or Ce6. Our findings provide the proof of principle for the development of a novel, nanotechnology-based drug delivery system for the treatment of osteosarcoma.

Fabrication of Innovative Silk/Alginate Microcarriers for Mesenchymal Stem Cell Delivery and Tissue Regeneration.

The aim of this study was to exploit silk fibroin's properties to develop innovative composite microcarriers for mesenchymal stem cell (MSCs) adhesion and proliferation. Alginate microcarriers were prepared, added to silk fibroin solution, and then treated with ethanol to induce silk conformational transition. Microcarriers were characterized for size distribution, coating stability and homogeneity. Finally, in vitro cytocompatibility and suitability as delivery systems for MSCs were investigated. Results indicated that our manufacturing process is consistent and reproducible: silk/alginate microcarriers were stable, with spherical geometry, about 400 µm in average diameter, and fibroin homogeneously coated the surface. MSCs were able to adhere rapidly onto the microcarrier surface and to cover the surface of the microcarrier within three days of culture; moreover, on this innovative 3D culture system, stem cells preserved their metabolic activity and their multi-lineage differentiation potential. In conclusion, silk/alginate microcarriers represent a suitable support for MSCs culture and expansion. Since it is able to preserve MSCs multipotency, the developed 3D system can be intended for cell delivery, for advanced therapy and regenerative medicine applications.

Extracellular matrix and α5ß1 integrin signaling control the maintenance of bone formation capacity by human adipose-derived stromal cells.

Stromal vascular fraction (SVF) cells of human adipose tissue have the capacity to generate osteogenic grafts with intrinsic vasculogenic properties. However, adipose-derived stromal/stem cells (ASC), even after minimal monolayer expansion, display poor osteogenic capacity in vivo. We investigated whether ASC bone-forming capacity may be maintained by culture within a self-produced extracellular matrix (ECM) that recapitulates the native environment. SVF cells expanded without passaging up to 28 days (Unpass-ASC) deposited a fibronectin-rich extracellular matrix and displayed greater clonogenicity and differentiation potential in vitro compared to ASC expanded only for 6 days (P0-ASC) or for 28 days with regular passaging (Pass-ASC). When implanted subcutaneously, Unpass-ASC produced bone tissue similarly to SVF cells, in contrast to P0- and Pass-ASC, which mainly formed fibrous tissue. Interestingly, clonogenic progenitors from native SVF and Unpass-ASC expressed low levels of the fibronectin receptor α5 integrin (CD49e), which was instead upregulated in P0- and Pass-ASC. Mechanistically, induced activation of α5ß1 integrin in Unpass-ASC led to a significant loss of bone formation in vivo. This study shows that ECM and regulation of α5ß1-integrin signaling preserve ASC progenitor properties, including bone tissue-forming capacity, during in vitro expansion.

MicroRNA let-7d is a target of cannabinoid CB1 receptor and controls cannabinoid signaling.

Cannabinoid CB1 receptor, the molecular target of endocannabinoids and cannabis active components, is one of the most abundant metabotropic receptors in the brain. Cannabis is widely used for both recreational and medicinal purposes. Despite the ever-growing fundamental roles of microRNAs in the brain, the possible molecular connections between the CB1 receptor and microRNAs are surprisingly unknown. Here, by using reporter gene constructs that express interaction sequences for microRNAs in human SH-SY5Y neuroblastoma cells, we show that CB1 receptor activation enhances the expression of several microRNAs, including let-7d. This was confirmed by measuring hsa-let-7d expression levels. Accordingly, knocking-down CB1 receptor in zebrafish reduced dre-let-7d levels, and knocking-out CB1 receptor in mice decreased mmu-let-7d levels in the cortex, striatum and hippocampus. Conversely, knocking-down let-7d increased CB1 receptor mRNA expression in zebrafish, SH-SY5Y cells and primary striatal neurons. Likewise, in primary striatal neurons chronically exposed to a cannabinoid or opioid agonist, a let-7d-inhibiting sequence facilitated not only cannabinoid or opioid signaling but also cannabinoid/opioid cross-signaling. Taken together, these findings provide the first evidence for a bidirectional link between the CB1 receptor and a microRNA, namely let-7d, and thus unveil a new player in the complex process of cannabinoid action.

Rapid and efficient magnetization of mesenchymal stem cells by dendrimer-functionalized magnetic nanoparticles.

AIM: Rapid and efficient magnetization of human bone marrow stromal cells (BMSC) through functionalized magnetic nanoparticles (MNP). METHODS: MNP were functionalized with poly(epsilon-lysine) dendrons exposing carboxybetaine residue (CB-MNP) to enhance binding to the cellular glycocalix. BMSC were incubated with CB-MNP or non-functionalized PAA-MNP for 5-30 min in suspension. RESULTS: CB-MNP functionalization increased the magnetization efficiency by threefold. Remarkably, 66% of cells were magnetized after only 5 min and the maximum efficiency of >80% was reached by 15 min. BMSC viability, proliferation and differentiation were not impaired: actually, adipogenic and osteogenic differentiation were even improved. CONCLUSION: Carboxybetaine-dendron functionalization ensured rapid and efficient BMSC magnetization and allowed innovative suspension labeling, with a potential for bypassing adhesion culture of progenitors for regenerative medicine.

Secreted adiponectin as a marker to evaluate in vitro the adipogenic differentiation of human mesenchymal stromal cells.

BACKGROUND AIMS: Multipotency is one of the hallmarks of mesenchymal stromal cells (MSCs). Given the widespread adoption of MSC-based clinical applications, the need for rapid and reliable methods to estimate MSC multipotency is demanding. Adipogenic potential is commonly evaluated by staining cell lipid droplets with oil red O. This cytochemical assay is performed at the terminal stage of adipogenic induction (21-28 days) and necessitates the destruction of the specimen. In this study, we investigated whether it is possible to assess MSC adipogenic differentiation in a more efficient, timely and non-destructive manner, while monitoring in vitro secretion of adiponectin, a hormone specifically secreted by adipose tissue. METHODS: A commercially available enzyme-linked immunosorbent assay kit was used to quantify adiponectin secreted in the culture medium of adipo-induced human bone marrow-derived MSCs. Oil red O staining was used as a reference method. RESULTS: Adiponectin is detectable after 10 days of induction at a median concentration of 5.13 ng/mL. The secretion of adiponectin steadily increases as adipogenesis proceeds. Adiponectin is undetectable when adipogenic induction is pharmacologically blocked, inefficient or when human MSCs are induced to differentiate toward the osteogenic lineage, proving the specificity of the assay. Furthermore, the results of adiponectin secretion strongly correlate with oil red O quantification at the end of induction treatment. CONCLUSIONS: Our results demonstrate that quantification of secreted adiponectin can be used as a reliable and robust method to evaluate adipogenic potential without destroying samples. This method provides a useful tool for quality control in the laboratory and in clinical applications of human MSCs.

The posterior iliac crest outperforms the anterior iliac crest when obtaining mesenchymal stem cells from bone marrow.

BACKGROUND: The clinical application of freshly isolated connective-tissue progenitors, as well as the potential preparation of culture-expanded mesenchymal stem cell populations for therapeutic applications, will benefit from clinical methods that maximize the yield of the starting population. We compared the number of cells, concentration, and prevalence of colony-founding connective-tissue progenitors from the anterior and posterior iliac crest. In addition, we compared the expansion kinetics and multilineage differentiation potential of their culture-expanded progeny when processed to form mesenchymal stem cells. METHODS: Marrow aspirate was collected from both the anterior and posterior iliac crest of twenty-two patients. The concentration and prevalence of colony-founding connective-tissue progenitors were estimated with use of a colony formation assay. The expansion kinetics and multilineage differentiation potential of the culture-expanded mesenchymal stem cell populations derived from these starting samples were compared. RESULTS: The yield of colony-founding connective-tissue progenitors was 1.6 times greater in the posterior compared with the anterior iliac crest. No differences were found with respect to the viability, phenotype, expansion kinetics, or multilineage differentiation potential of mesenchymal stem cell populations derived from these two sites. CONCLUSIONS: The concentration and yield of colony-founding connective-tissue progenitors were greater when aspirate was obtained from the posterior compared with the anterior iliac crest, whereas the biological potential of the cells derived from these sites appeared comparable. CLINICAL RELEVANCE: The harvesting of bone marrow from the posterior iliac crest appears to be preferred, as it provided a modestly higher concentration of colony-founding connective-tissue progenitors than comparable aspirate from the anterior iliac crest.

Regulation of opioid and cannabinoid receptor genes in human neuroblastoma and T cells by the epigenetic modifiers trichostatin A and 5-aza-2'-deoxycytidine.

OBJECTIVE: The aim of this study was to investigate the effect of the epigenetic modifiers trichostatin A and 5-aza-2'-deoxycytidine on the expression of the cannabinoid receptors CB1 and CB2 and µ-opioid receptors in human SH SY5Y neuroblastoma cells and human Jurkat T lymphocytes. METHODS: Using quantitative real-time RT-PCR, mRNA specific for the aforementioned receptors was determined. The functionality of the induced receptors was determined by analyzing the effect of the ligands to regulate intracellular cAMP. RESULTS: We demonstrated that treatment of SH SY5Y cells, which endogenously express µ-opioid receptors and CB1, but not CB2, resulted in de novo induction of CB2, while mRNA levels of CB1 and µ-opioid receptors were not significantly altered. In contrast, treatment of Jurkat lymphocytes, which endogenously express CB2, but not CB1 and µ-opioid receptors, resulted in de novo induction of CB1 and µ-opioid receptors, while mRNA levels of CB2 were not significantly altered. Furthermore, the functionality of the induced µ-opioid receptors and CB1 in the Jurkat cells was demonstrated. CONCLUSIONS: Our data suggest an epigenetically regulated expression of cannabinoid receptors and µ-opioid receptors. Their induction by epigenetic modifiers in distinct cells of the nervous and immune system might result in increased effects of the cognate drugs on neuronal and immune functions. Such modifications might be useful for novel therapies for various disorders, e.g. multiple sclerosis, where the elevated transmission of cannabinoid or opioid signals is beneficial.