isolation-primary-cancer-associated-fibroblasts-from-syngeneic-murine

Isolation of Primary Cancer-Associated Fibroblasts from a Syngeneic Murine Model of Breast Cancer for the Study of Targeted Nanoparticles

Cancer-associated fibroblasts (CAFs) are key actors in the context of the tumor microenvironment. Despite being reduced in number as compared to tumor ...

Università di Milano-Bicocca

Triple negative breast cancer (TNBC) is a highly aggressive, invasive, and metastatic tumor. Although it is reported to be sensitive to cytotoxic chemotherapeutics, frequent relapse and chemoresistance often result in treatment failure. In this study, we developed a biomimetic nanodrug consisting of a self-assembling variant (HFn) of human apoferritin loaded with curcumin. HFn nanocage improved the solubility, chemical stability, and bioavailability of curcumin, allowing us to reliably carry out several experiments in the attempt to establish the potential of this molecule as a therapeutic agent and elucidate the mechanism of action in TNBC. HFn biopolymer was designed to bind selectively to the TfR1 receptor overexpressed in TNBC cells. HFn-curcumin (CFn) proved to be more effective in viability assays compared to the drug alone using MDA-MB-468 and MDA-MB-231 cell lines, representative of basal and claudin-low TNBC subtypes, respectively. Cellular uptake of CFn was demonstrated by flow cytometry and label-free confocal Raman imaging. CFn could act as a chemosensitizer enhancing the cytotoxic effect of doxorubicin by interfering with the activity of multidrug resistance transporters. In addition, CFn exhibited different cell cycle effects on these two TNBC cell lines, blocking MDA-MB-231 in G0/G1 phase, whereas MDA-MB-468 accumulated in G2/M phase. CFn was able to inhibit the Akt phosphorylation, suggesting that the effect on the proliferation and cell cycle involved the alteration of PI3K/Akt pathway.

H-Ferritin Enriches the Curcumin Uptake and Improves the Therapeutic Efficacy in Triple Negative Breast Cancer Cells.

Nanoparticles are normally classified as "hard", mainly consisting of metal or metal oxide cores, or "soft", including polymer-based, liposomes and biomimetic nanoparticles. Soft nanoparticles have been studied in depth for drug formulation and therapeutic delivery applications, albeit hard nanoparticles may offer easier synthesis, smaller size and more effective tumor penetration. Among them, silica nanoparticles maintain excellent biocompatibility and biodegradability and can be finely adjusted in size and shape, easily produced in a large scale and functionalized or loaded with active molecules. To help filling the gap of a poor clinical translation of hard nanoparticles, we have designed and developed three different nonporous silica nanocarriers loading the chemotherapeutic doxorubicin within the core matrix, on the surface or both inside and outside, respectively. A comparative study was performed on drug loading and drug release, silica matrix degradation and nanodrug cytotoxic activity, highlighting unexpected correlation between the strategy adopted for drug incorporation and nanoparticle behavior in a physiological environment. This study offers a new insight on the impact of the choice of the prodrug nanoparticles on the kinetics and efficacy of drug delivery, which may encourage the scientific community in developing a new generation of drug delivery systems based on hard nanocarriers.

Impact of the strategy adopted for drug loading in nonporous silica nanoparticles on the drug release and cytotoxic activity.

The use of therapeutic monoclonal antibodies (mAbs) has revolutionized cancer treatment. The conjugation of mAbs to nanoparticles has been broadly exploited to improve the targeting efficiency of drug nanocarriers taking advantage of high binding efficacy and target selectivity of antibodies for specific cell receptors. However, the therapeutic implications of nanoconjugation have been poorly considered. In this study, half-chain fragments of the anti-EGFR mAb cetuximab were conjugated to colloidal nanoparticles originating stable nanoconjugates that were investigated as surrogates of therapeutic mAbs in triple negative breast cancer (TNBC). Three TNBC cell lines were selected according to EGFR expression, which regulates activation of MAPK/ERK and PI3K/Akt pathways, and to distinctive molecular profiling including KRAS, PTEN, and BRCA1 mutations normally associated with diverse sensitivity to treatment with cetuximab. The molecular mechanisms of action of nanoconjugated half-chain mAb, including cell targeting, interference with downstream signaling pathways, proliferation, cell cycle, and apoptosis, along with triggering of ADCC response, were investigated in detail in sensitive and resistant TNBC cells. We found that half-chain mAb nanoconjugation was able to enhance the therapeutic efficacy and improve the target selectivity against sensitive, but unexpectedly also resistant, TNBC cells. Viability assays and signaling transduction modulation suggested a role of BRCA1 mutation in TNBC resistance to cetuximab alone, whereas its effect could be circumvented using half-chain cetuximab nanoconjugates, suggesting that nanoconjugation not only improved the antibody activity but also exerted different mechanisms of action. Our results provide robust evidence of the potential of half-chain antibody nanoconjugates in the treatment of TNBC, which could offer a new paradigm for therapeutic antibody administration, potentially allowing improved curative efficiency and reduced minimal effective dosages in both sensitive and resistant tumors.

Half-Chain Cetuximab Nanoconjugates Allow Multitarget Therapy of Triple Negative Breast Cancer.

Colloidally stable nanoparticles-based magnetic agents endowed with very high relaxivity and specific absorption rate are extremely desirable for efficient magnetic resonance imaging and magnetic hyperthermia, respectively. Here, we report a water dispersible magnetic agent consisting of zinc-doped superparamagnetic iron oxide nanoparticles (i.e., Zn-SPIONs) of 15 nm size with high saturation magnetization coated with an amphiphilic polymer for effective magnetic resonance imaging and magnetic hyperthermia of glioblastoma cells. These biocompatible polymer-coated Zn-SPIONs had 24 nm hydrodynamic diameter and exhibited high colloidal stability in various aqueous media, very high transverse relaxivity of 471 mM s, and specific absorption rate up to 743.8 W g, which perform better than most iron oxide nanoparticles reported in the literature, including commercially available agents. Therefore, using these polymer-coated Zn-SPIONs even at low concentrations, T-weighted magnetic resonance imaging and moderate magnetic hyperthermia of glioblastoma cells under clinically relevant magnetic field were successfully implemented. In addition, the results of this in vitro study suggest the superior potential of Zn-SPIONs as a theranostic nanosystem for brain cancer treatment, simultaneously acting as a contrast agent for magnetic resonance imaging and a heat mediator for localized magnetic hyperthermia.

Colloidal polymer-coated Zn-doped iron oxide nanoparticles with high relaxivity and specific absorption rate for efficient magnetic resonance imaging and magnetic hyperthermia.

Nanoparticle assisted drug delivery to the cytoplasm is limited by sequestration of nanoparticles in endosomes. Endosomal escape through polymer-induced membrane destabilization is one of a few well characterized mechanisms to overcome it. Aiming to utilize this method in vivo, it is necessary to understand how modulating the structural and chemical features of the polymer and the presence of proteins in biological fluids can affect this activity. Here, as a model for the endosomal membrane, we use the membrane of red blood cells to evaluate the membrane destabilization ability of a model amphiphilic polymer in the presence of blood plasma using a hemolysis assay. This allows determination of red blood cells membrane permeabilization through the quantification of hemoglobin leakage. Our results showed a strong inhibitory effect of plasma, and that hemolytic activity can be improved by chemical modification of the polymeric micelle, reducing its interaction with plasma proteins. Finally, a second mechanism of pH-induced direct diffusion is proposed and tested using an oil/water partitioning model. These results offer a body of knowledge to improve delivery of drugs across biological membranes using carefully designed polymeric nanocarriers.

Maria Antonietta Rizzuto

Dipartimento di Biotecnologie e Bioscienze

Università di Milano-Bicocca

Isolation of Primary Cancer-Associated Fibroblasts from a Syngeneic Murine Model of Breast Cancer for the Study of Targeted Nanoparticles

Maria Antonietta Rizzuto

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Università di Milano-Bicocca

Isolation of Primary Cancer-Associated Fibroblasts from a Syngeneic Murine Model of Breast Cancer for the Study of Targeted Nanoparticles

Dipartimento di Biotecnologie e Bioscienze