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.

SMYD3 Modulates AMPK-mTOR Signaling Balance in Cancer Cell Response to DNA Damage.

Cells respond to DNA damage by activating a complex array of signaling networks, which include the AMPK and mTOR pathways. After DNA double-strand breakage, ATM, a core component of the DNA repair system, activates the AMPK-TSC2 pathway, leading to the inhibition of the mTOR cascade. Recently, we showed that both AMPK and mTOR interact with SMYD3, a methyltransferase involved in DNA damage response. In this study, through extensive molecular characterization of gastrointestinal and breast cancer cells, we found that SMYD3 is part of a multiprotein complex that is involved in DNA damage response and also comprises AMPK and mTOR. In particular, upon exposure to the double-strand break-inducing agent neocarzinostatin, SMYD3 pharmacological inhibition suppressed AMPK cascade activation and thereby promoted the mTOR pathway, which reveals the central role played by SMYD3 in the modulation of AMPK-mTOR signaling balance during cancer cell response to DNA double-strand breaks. Moreover, we found that SMYD3 can methylate AMPK at the evolutionarily conserved residues Lys411 and Lys424. Overall, our data revealed that SMYD3 can act as a bridge between the AMPK and mTOR pathways upon neocarzinostatin-induced DNA damage in gastrointestinal and breast cancer cells.

SMYD3 Modulates the HGF/MET Signaling Pathway in Gastric Cancer.

Gastric cancer (GC) is the third most deadly cancer worldwide. Considerable efforts have been made to find targetable drivers in order to improve patient outcomes. MET is one of the most important factors involved in GC initiation and progression as it plays a major role in GC invasiveness and is related to cancer stemness. Unfortunately, treatment strategies targeting MET are still limited, with a proportion of patients responding to therapy but later developing resistance. Here, we showed that MET is a molecular partner of the SMYD3 methyltransferase in GC cells. Moreover, we found that SMYD3 pharmacological inhibition affects the HGF/MET downstream signaling pathway. Extensive cellular analyses in GC models indicated that EM127, a novel active site-selective covalent SMYD3 inhibitor, can be used as part of a synergistic approach with MET inhibitors in order to enhance the targeting of the HGF/MET pathway. Importantly, our data were confirmed in a 3D GC cell culture system, which was used as a surrogate to evaluate stemness characteristics. Our findings identify SMYD3 as a promising therapeutic target to impair the HGF/MET pathway for the treatment of GC.

Drug Delivery in Photodynamic Therapy.

Photodynamic therapy (PDT) has gained prominence as a non-invasive and selective treatment option for solid tumors and non-oncological diseases [...].

A non-clinical and clinical IUCLID database for 530 pharmaceuticals (part I): Methodological aspects of its development.

A new IUCLID database is provided containing results from non-clinical animal studies and human information for 530 approved drugs. The database was developed by extracting data from pharmacological reviews of repeat-dose, carcinogenicity, developmental, and reproductive toxicity studies. In the database, observed and no-observed effects are linked to the respective effect levels, including information on severity/incidence and transiency/reversibility. It also includes some information on effects in humans, that were extracted from relevant sections of standard product labels of the approved drugs. The database is complemented with a specific ontology for reporting effects that was developed as an improved version of the Ontology Lookup Service's mammalian and human phenotype ontologies and includes different hierarchical levels. The developed ontology contains novel and unique standardized terms, including ontological terms for reproductive and endocrine effects. The database aims to facilitate correlation and concordance analyses based on the link between observed and no-observed effects and their respective effect levels. In addition, it offers a robust dataset on drug information for the pharmaceutical industry and research. The reported ontology supports the analyses of toxicological information, especially for reproductive and endocrine endpoints and can be used to encode legacy data or develop additional ontologies. The new database and ontology can be used to support the development of alternative non-animal approaches, to elucidate mechanisms of toxicity, and to analyse human relevance. The new IUCLID database is provided free of charge at https://iuclid6.echa.europa.eu/us-fda-toxicity-data.

Discovery of the 4-aminopiperidine-based compound EM127 for the site-specific covalent inhibition of SMYD3.

Recent findings support the hypothesis that inhibition of SMYD3 methyltransferase may be a therapeutic avenue for some of the deadliest cancer types. Herein, active site-selective covalent SMYD3 inhibitors were designed by introducing an appropriate reactive cysteine trap into reversible first-generation SMYD3 inhibitors. The 4-aminopiperidine derivative EM127 (11C) bearing a 2-chloroethanoyl group as reactive warhead showed selectivity for Cys186, located in the substrate/histone binding pocket. Selectivity towards Cys186 was retained even at high inhibitor/enzyme ratio, as shown by mass spectrometry. The mode of interaction with the SMYD3 substrate/histone binding pocket was revealed by crystallographic studies. In enzymatic assays, 11C showed a stronger SMYD3 inhibitory effect compared to the reference inhibitor EPZ031686. Remarkably, 11C attenuated the proliferation of MDA-MB-231 breast cancer cell line at the same low micromolar range of concentrations that reduced SMYD3 mediated ERK signaling in HCT116 colorectal cancer and MDA-MB-231 breast cancer cells. Furthermore, 11C (5 µM) strongly decreased the steady-state mRNA levels of genes important for tumor biology such as cyclin dependent kinase 2, c-MET, N-cadherin and fibronectin 1, all known to be regulated, at least in part, by SMYD3. Thus, 11C is as a first example of second generation SMYD3 inhibitors; this agent represents a covalent and a site specific SMYD3 binder capable of potent and prolonged attenuation of methyltransferase activity.

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.

HSA-Binding Prodrugs-Based Nanoparticles Endowed with Chemo and Photo-Toxicity against Breast Cancer.

Exploiting the tumor environment features (EPR effect, elevated glutathione, reactive oxygen species levels) might allow attaining a selective and responsive carrier capable of improving the therapeutic outcome. To this purpose, the in situ covalent binding of drugs and nanoparticles to circulating human serum albumin (HSA) might represent a pioneering approach to achieve an effective strategy. This study describes the synthesis, in vitro and in vivo evaluation of bioresponsive HSA-binding nanoparticles (MAL-PTX2S@Pba), co-delivering two different paclitaxel (PTX) prodrugs and the photosensitizer pheophorbide a (Pba), for the combined photo- and chemo-treatment of breast cancer. Stable and reproducible MAL-PTX2S@Pba nanoparticles with an average diameter of 82 nm and a PTX/Pba molar ratio of 2.5 were obtained by nanoprecipitation. The in vitro 2D combination experiments revealed that MAL-PTX2S@Pba treatment induces a strong inhibition of cell viability of MDA-MB-231, MCF7 and 4T1 cell lines, whereas 3D experiments displayed different trends: while MAL-PTX2S@Pba effectiveness was confirmed against MDA-MB-231 spheroids, the 4T1 model exhibited marked resistance. Lastly, despite using a low PTX-PDT regimen (e.g., 8.16 mg/Kg PTX and 2.34 mg/Kg Pba), our formulation showed to foster primary tumor reduction and curb lung metastases growth in 4T1 tumor-bearing mice, thus setting the basis for further preclinical validations.

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.

Pheophorbide A and Paclitaxel Bioresponsive Nanoparticles as Double-Punch Platform for Cancer Therapy.

Cancer therapy is still a challenging issue. To address this, the combination of anticancer drugs with other therapeutic modalities, such as light-triggered therapies, has emerged as a promising approach, primarily when both active ingredients are provided within a single nanosystem. Herein, we describe the unprecedented preparation of tumor microenvironment (TME) responsive nanoparticles exclusively composed of a paclitaxel (PTX) prodrug and the photosensitizer pheophorbide A (PheoA), e.g., PheoA≅PTX2S. This system aimed to achieve both the TME-triggered and controlled release of PTX and the synergistic/additive effect by PheoA-mediated photodynamic therapy. PheoA≅PTX2S were produced in a simple one-pot process, exhibiting excellent reproducibility, stability, and the ability to load up to 100% PTX and 40% of PheoA. Exposure of PheoA≅PTX2S nanoparticles to TME-mimicked environment provided fast disassembly compared to normal conditions, leading to PTX and PheoA release and consequently elevated cytotoxicity. Our data indicate that PheoA incorporation into nanoparticles prevents its aggregation, thus providing a greater extent of ROS and singlet oxygen production. Importantly, in SK-OV-3 cells, PheoA≅PTX2S allowed a 30-fold PTX dose reduction and a 3-fold dose reduction of PheoA. Our data confirm that prodrug-based nanocarriers represent valuable and sustainable drug delivery systems, possibly reducing toxicity and expediting preclinical and clinical translation.

Nitric Oxide Photo-Donor Hybrids of Ciprofloxacin and Norfloxacin: A Shift in Activity from Antimicrobial to Anticancer Agents.

The potential anticancer effect of fluoroquinolone antibiotics has been recently unveiled and related to their ability to interfere with DNA topoisomerase II. We herein envisioned the design and synthesis of novel Ciprofloxacin and Norfloxacin nitric oxide (NO) photo-donor hybrids to explore the potential synergistic antitumor effect exerted by the fluoroquinolone scaffold and NO eventually produced upon light irradiation. Anticancer activity, evaluated on a panel of tumor cell lines, showed encouraging results with IC50 values in the low micromolar range. Some compounds displayed intense antiproliferative activity on triple-negative and doxorubicin-resistant breast cancer cell lines, paving the way for their potential use to treat aggressive, refractory and multidrug-resistant breast cancer. No significant additive effect was observed on PC3 and DU145 cells following NO release. Conversely, antimicrobial photodynamic experiments on both Gram-negative and Gram-positive microorganisms displayed a significant killing rate in Staphylococcus aureus, accounting for their potential effectiveness as selective antimicrobial photosensitizers.

Keratin nanoparticles and photodynamic therapy enhance the anticancer stem cells activity of salinomycin.

The high rates of aggressiveness, drug resistance and relapse of breast cancer (BC) are mainly attributed to the inability of conventional therapies to equally eradicate bulk differentiated cells and cancer stem cells (CSCs). To improve the effectiveness of BC treatments, we report the in-water synthesis of novel keratin-based nanoformulations, loaded with the CSC-specific drug salinomycin (SAL), the photosensitizer chlorin e6 (Ce6) and vitamin E acetate (SAL/Ce6@kVEs), which combine the capability of releasing SAL with the production of singlet oxygen upon light irradiation. In vitro experiments on BC cell lines and CSC-enriched mammospheres exposed to single or combined therapies showed that SAL/Ce6@kVEs determine synergistic cell killing, limit their self-renewal capacity and decrease the stemness potential by eradication of CSCs. In vivo experiments on zebrafish embryos confirmed the capacity of SAL nanoformulations to interfere with the Wnt/ß-catenin signaling pathway, which is dysregulated in BC, thus identifying a target for further translation into pre-clinical models.

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.

Light-Induced Therapies for Prostate Cancer Treatment.

Prostate cancer (PC) is one of the most widespread tumors affecting the urinary system and the fifth-leading cause from cancer death in men worldwide. Despite PC mortality rates have been decreasing during the last years, most likely due to an intensification of early diagnosis, still more than 300,000 men die each year because of this disease. In this view, researchers in all countries are engaged in finding new ways to tackle PC, including the design and synthesis of novel molecular and macromolecular entities able to challenge different PC biological targets, while limiting the extent of unwanted side effects that significantly limit men's life quality. Among this field of research, photo-induced therapies, such as photodynamic and photothermal therapies (PDT and PTT), might represent an important advancement in PC treatment due to their extremely localized and controlled cytotoxic effect, as well as their low incidence of side effects and tumor resistance occurrence. Based on these considerations, this review aims to gather and discuss the last 5-years literature reports dealing with the synthesis and biological activity of molecular conjugates and nano-platforms for photo-induced therapies as co-adjuvant or combined therapeutic modalities for the treatment of localized PC.

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.

2-Hydroxypropyl-ß-cyclodextrin-enhanced pharmacokinetics of cabotegravir from a nanofluidic implant for HIV pre-exposure prophylaxis.

Preexposure prophylaxis (PrEP) with antiretrovirals (ARV) can prevent human immunodeficiency virus (HIV) transmission, but its efficacy is highly dependent on strict patient adherence to daily dosing regimen. Long-acting (LA) ARV formulations or delivery systems that reduce dosing frequency may increase adherence and thus PrEP efficacy. While cabotegravir (CAB) long-acting injectable (CAB LA), an integrase strand transfer inhibitor (INSTI), reduces dosing frequency to bimonthly injections, variable pharmacokinetics (PK) between patients and various adverse reactions necessitate improvement in delivery methods. Here we developed a subcutaneously implantable nanofluidic device for the sustained delivery of CAB formulated with 2-hydroxypropyl-ß-cyclodextrin (ßCAB) and examined the pharmacokinetics (PK) in Sprague-Dawley rats for 3 months in comparison to CAB. Our study demonstrated ßCAB treatment group maintained clinically-relevant plasma CAB concentrations 2 times above the protein-adjusted concentration that inhibits viral replication by 90% (2 × PA-IC90) and drug penetration into tissues relevant to HIV-1 transmission. Further, we successfully fitted plasma CAB concentrations into a PK model (R2 = 0.9999) and determined CAB apparent elimination half-life of 47 days. Overall, our data shows the potential of sustained release of ßCAB via a nanofluidic implant for long-term PrEP delivery, warranting further investigation for efficacy against HIV infections.

Unprecedented Behavior of (9 R)-9-Hydroxystearic Acid-Loaded Keratin Nanoparticles on Cancer Cell Cycle.

Histone deacetylases, HDACs, have been demonstrated to play a critical role in epigenetic signaling and were found to be overexpressed in several type of cancers; therefore, they represent valuable targets for anticancer therapy. 9-Hydroxystearic acid has been shown to bind the catalytic site of HDAC1, inducing G0/G1 phase cell cycle arrest and activation of the p21WAF1 gene, thus promoting cell growth inhibition and differentiation in many cancer cells. Despite the ( R) enantiomer of 9-hydroxystearic acid (9R) displaying a promising in vitro growth-inhibitory effect on the HT29 cell line, its scarce water solubility and micromolar activity require novel solutions for improving its efficacy and bioavailability. In this work, we describe the synthesis and in vitro biological profiling of 9R keratin nanoparticles (9R@ker) obtained through an in-water drug-induced aggregation process. The anticancer activity of 9R@ker was investigated in the HT29 cell line; the results indicate an increased fluidity of cell membrane and a higher intracellular ROS formation, resulting in an unexpected S phase cell cycle arrest (25% increase as compared to the control) induced by 9R@ker with respect to free 9R and an induction of cell death.

Non-Steroidal Androgen Receptor Antagonists and Prostate Cancer: A Survey on Chemical Structures Binding this Fast-Mutating Target.

The Androgen Receptor (AR) pathway plays a major role in both the pathogenesis and progression of prostate cancer. In particular, AR is chiefly involved in the development of Castration-Resistant Prostate Cancer (CRPC) as well as in the resistance to the secondgeneration AR antagonist enzalutamide, and to the selective inhibitor of cytochrome P450 17A1 (CYP17A1) abiraterone. Several small molecules acting as AR antagonists have been designed and developed so far, also as a result of the ability of cells expressing this molecular target to rapidly develop resistance and turn pure receptor antagonists into ineffective or event detrimental molecules. This review covers a survey of most promising classes of non-steroidal androgen receptor antagonists, also providing insights into their mechanism of action and efficacy in treating prostate cancer.

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.