The Transformative Potential of Lipid Nanoparticle-Protein Corona for Next-Generation Vaccines and Therapeutics.

The integration of the lipid nanoparticle (LNP)-protein corona as a pioneering approach for the development of vaccines against the present and future SARS-CoV-2 variants of concern marks a significant shift in the field. This concept holds great promise, offering a universal platform that can be adaptable to combat future pandemics caused by unknown viruses. Understanding the complex interactions among the protein corona, LNPs, and receptors is crucial for harnessing its potential. This knowledge will allow optimal vaccine formulations and improve their effectiveness. Safety assessments are essential to ensure suitability for human use, compliance with regulatory standards, and rigorous quality control in manufacturing. This transformative workflow requires collaborative efforts, expanding our foundational knowledge and translating advancements from the laboratory to clinical reality. The LNP-protein corona approach represents a paradigmatic shift with far-reaching implications. Its principles and insights can be leveraged beyond specific applications against SARS-CoV-2, enabling a universal platform for addressing viral threats, cancer, and genetic diseases.

A novel 3'-tRNAGlu-derived fragment acts as a tumor suppressor in breast cancer by targeting nucleolin.

tRNA-derived fragments (tRFs) have been defined as a novel class of small noncoding RNAs. tRFs have been reported to be deregulated in cancer, but their biologic function remains to be fully understood. We have identified a new tRF (named tRF3E), derived from mature tRNAGlu, that is specifically expressed in healthy mammary glands but not in breast cancer (BC). Consistently, tRF3E levels significantly decrease in the blood of patients with epidermal growth factor receptor 2 (HER2)-positive BC reflecting tumor status (control > early cancer > metastatic cancer). tRF3E down-regulation was recapitulated in Δ16HER2 transgenic mice, representing a BC preclinical model. Pulldown assays, used to search for proteins capable to selectively bind tRF3E, have shown that this tRF specifically interacts with nucleolin (NCL), an RNA-binding protein overexpressed in BC and able to repress the translation of p53 mRNA. The binding properties of NCL-tRF3E complex, predicted in silico and analyzed by EMSA assays, are congruent with a competitive displacement of p53 mRNA by tRF3E, leading to an increased p53 expression and consequently to a modulation of cancer cell growth. Here, we provide evidence that tRF3E plays an important role in the pathogenesis of BC displaying tumor-suppressor functions through a NCL-mediated mechanism.-Falconi, M., Giangrossi, M., Elexpuru Zabaleta, M., Wang, J., Gambini, V., Tilio, M., Bencardino, D., Occhipinti, S., Belletti, B., Laudadio, E., Galeazzi, R., Marchini, C., Amici, A. A novel 3'-tRNAGlu-derived fragment acts as a tumor suppressor in breast cancer by targeting nucleolin.

Acetylshikonin isolated from Lithospermum erythrorhizon roots inhibits dihydrofolate reductase and hampers autochthonous mammary carcinogenesis in Δ16HER2 transgenic mice.

Breast cancer (BC) is the most common cancer in women and, among different BC subtypes, triple negative (TN) and human epidermal growth factor receptor 2 (HER2)-positive BCs have the worst prognosis. In this study, we investigated the anticancer activity of the root ethanolic and hexane extracts from Lithospermum erythrorhizon, a traditional Chinese herbal medicine known also as tzu ts'ao or tzu-ken, against in vitro and in vivo models of TNBC and HER2-positive BC. Treatment with L. erythrorhizon root extracts resulted in a dose-dependent inhibition of BC cell viability and in a significant reduction of the growth of TNBC cells transplanted in syngeneic mice. Acetylshikonin, a naphthoquinone, was identified as the main bioactive component in extracts and was responsible for the observed antitumor activity, being able to decrease BC cell viability and to interfere with autochthonous mammary carcinogenesis in Δ16HER2 transgenic mice. Acetylshikonin anticancer effect depends on its ability to act as a potent inhibitor of dihydrofolate reductase (DHFR), to down-regulate key mediators governing cancer growth and progression, such as HER2, Src and STAT3, and to induce apoptosis by caspase-3 activation. The accumulation of acetylshikonin in blood samples as well as in brain, kidney, liver and tumor tissues was also investigated by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) highlighting that L. erythrorhizon treatment is effective in delivering the active compound into the target tissues. These results provide evidence that L. erythrorhizon extract and in particular its main component acetylshikonin are effective against aggressive BC subtypes and reveal new acetylshikonin mechanisms of action.

Cationic lipid/DNA complexes manufactured by microfluidics and bulk self-assembly exhibit different transfection behavior.

Recent advances in biochemical and biophysical research have been achieved through the employment of microfluidic devices. Microfluidic mixing of therapeutic agents with biomaterials yields systems with finely tuned physical-chemical properties for applications in drug and gene delivery. Here, we investigate the role of preparation technology (microfluidic mixing vs. bulk self-assembly) on the transfection efficiency (TE) and cytotoxicity of multicomponent cationic liposome/DNA complexes (lipoplexes) in live Chinese hamster ovarian (CHO) cells. Decoupling TE and cytotoxicity allowed us to combine them in a unique coherent vision. While bulk self-assembly produces highly efficient and highly toxic MC lipoplexes, microfluidics manufacture leads to less efficient, but less cytotoxic complexes. This discrepancy is ascribed to two main factors controlling lipid-mediated cell transfection, i.e. the lipoplex concentration at the cell surface and the lipoplex arrangement at the nanoscale. Further research is required to optimize microfluidic manufacturing of lipoplexes to obtain highly efficient and not cytotoxic gene delivery systems.

Lyotropic Liquid-Crystalline Nanosystems as Drug Delivery Agents for 5-Fluorouracil: Structure and Cytotoxicity.

Lyotropic cubic liquid-crystalline systems have received increasing attention due to their unique microstructural and physicochemical properties as efficient nanocarriers for drug delivery. We report the preparation and characterization of bulk phases and cubosome dispersions of phytantriol loaded with the anticancer drug 5-fluorouracil, in neutral and anionic forms. In both cases, a Pn3m cubic phase was observed. The phytantriol phase behavior can be influenced by the addition of ionic agents, and, to this purpose, a positively charged lipid, such as N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride salt (DOTAP), was included in the studied formulations. It was found to induce a variation of the spontaneous membrane curvature of the phytantriol lipid bilayer, generating a transition from the Pn3m to the Im3m cubic phase. When 5-fluorouracil, in its anionic form (5-FUs), was encapsulated in these latter systems, a further transition to the HII hexagonal phase was observed as a consequence of the formation of a complex phytantriol/DOTAP/5-FUs. The physicochemical characterization was performed with various complementary techniques including synchrotron small-angle X-ray scattering, dynamic light scattering, and attenuated total reflection Fourier transform infrared and UV resonance Raman spectroscopies. Encapsulation of 5-fluorouracil in the corresponding nanodispersions was evaluated, and their in vitro cytotoxicity was assessed in MDA-MB-231 cell line. Phytantriol cubosomes containing 5-fluorouracil showed a higher toxicity compared with the bare drug solution, and hence they represent potential nanocarriers in the delivery of 5-fluorouracil for cancer therapy.

Manipulation of lipoplex concentration at the cell surface boosts transfection efficiency in hard-to-transfect cells.

To date, efficiency upon non-viral DNA delivery remains low and this implies the existence of unidentified transfection barriers. Here we explore the mechanisms of action of multicomponent (MC) cationic liposome/DNA complexes (lipoplexes) by a combination of reporter technologies, dynamic light scattering (DLS), synchrotron small angle X-ray scattering (SAXS), fluorescence activated cell sorting (FACS) analysis and laser scanning confocal microscopy (LSCM) in live cells. Lipofectamine - the gold standard among transfection reagents - was used as a reference. On the basis of our results, we suggest that an additional transfection barrier impairs transfection efficiency, that is: low lipoplex concentration at the cell surface. Based on the acquired knowledge we propose an optimized transfection protocol that allowed us to efficiently transfect DND41, JURKAT, MOLT3, P12-ICHIKAWA, ALL-SILL, TALL-1 human T-cell acute lymphoblastic leukemia (T-ALL) cell lines known to be difficult-to-transfect by using non-viral vectors and where LFN-based technologies fail to give satisfactory results.

The water soluble ruthenium(II) organometallic compound [Ru(p-cymene)(bis(3,5 dimethylpyrazol-1-yl)methane)Cl]Cl suppresses triple negative breast cancer growth by inhibiting tumor infiltration of regulatory T cells.

Ruthenium compounds have become promising alternatives to platinum drugs by displaying specific activities against different cancers and favorable toxicity and clearance properties. Here, we show that the ruthenium(II) complex [Ru(p-cymene)(bis(3,5-dimethylpyrazol-1-yl)methane)Cl]Cl (UNICAM-1) exhibits potent in vivo antitumor effects. When administered as four-dose course, by repeating a single dose (52.4mgkg-1) every three days, UNICAM-1 significantly reduces the growth of A17 triple negative breast cancer cells transplanted into FVB syngeneic mice. Pharmacokinetic studies indicate that UNICAM-1 is rapidly eliminated from kidney, liver and bloodstream thanks to its high hydrosolubility, exerting excellent therapeutic activity with minimal side effects. Immunohistological analysis revealed that the efficacy of UNICAM-1, mainly relies on its capacity to reverse tumor-associated immune suppression by significantly reducing the number of tumor-infiltrating regulatory T cells. Therefore, UNICAM-1 appears very promising for the treatment of TNBC.

Overcoming Chemoresistance in Cancer: The Promise of Crizotinib.

Chemoresistance is a major obstacle in cancer treatment, often leading to disease progression and poor outcomes. It arises through various mechanisms such as genetic mutations, drug efflux pumps, enhanced DNA repair, and changes in the tumor microenvironment. These processes allow cancer cells to survive despite chemotherapy, underscoring the need for new strategies to overcome resistance and improve treatment efficacy. Crizotinib, a first-generation multi-target kinase inhibitor, is approved by the FDA for the treatment of ALK-positive or ROS1-positive non-small cell lung cancer (NSCLC), refractory inflammatory (ALK)-positive myofibroblastic tumors (IMTs) and relapsed/refractory ALK-positive anaplastic large cell lymphoma (ALCL). Crizotinib exists in two enantiomeric forms: (R)-crizotinib and its mirror image, (S)-crizotinib. It is assumed that the R-isomer is responsible for the carrying out various processes reviewed here The S-isomer, on the other hand, shows a strong inhibition of MTH1, an enzyme important for DNA repair mechanisms. Studies have shown that crizotinib is an effective multi-kinase inhibitor targeting various kinases such as c-Met, native/T315I Bcr/Abl, and JAK2. Its mechanism of action involves the competitive inhibition of ATP binding and allosteric inhibition, particularly at Bcr/Abl. Crizotinib showed synergistic effects when combined with the poly ADP ribose polymerase inhibitor (PARP), especially in ovarian cancer harboring BRCA gene mutations. In addition, crizotinib targets a critical vulnerability in many p53-mutated cancers. Unlike its wild-type counterpart, the p53 mutant promotes cancer cell survival. Crizotinib can cause the degradation of the p53 mutant, sensitizing these cancer cells to DNA-damaging substances and triggering apoptosis. Interestingly, other reports demonstrated that crizotinib exhibits anti-bacterial activity, targeting Gram-positive bacteria. Also, it is active against drug-resistant strains. In summary, crizotinib exerts anti-tumor effects through several mechanisms, including the inhibition of kinases and the restoration of drug sensitivity. The potential of crizotinib in combination therapies is emphasized, particularly in cancers with a high prevalence of the p53 mutant, such as triple-negative breast cancer (TNBC) and high-grade serous ovarian cancer (HGSOC).

Protein corona alleviates adverse biological effects of nanoplastics in breast cancer cells.

Pollution from micro- and nanoplastics (MNPs) has long been a topic of concern due to its potential impact on human health. MNPs can circulate through human blood and, thus far, have been found in the lungs, spleen, stomach, liver, kidneys and even in the brain, placenta, and breast milk. While data are already available on the adverse biological effects of pristine MNPs (e.g. oxidative stress, inflammation, cytotoxicity, and even cancer induction), no report thus far clarified whether the same effects are modulated by the formation of a protein corona around MNPs. To this end, here we use pristine and human-plasma pre-coated polystyrene (PS) nanoparticles (NPs) and investigate them in cultured breast cancer cells both in terms of internalization and cell biochemical response to the exposure. It is found that pristine NPs tend to stick to the cell membrane and inhibit HER-2-driven signaling pathways, including phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathways, which are associated with cancer cell survival and growth. By contrast, the formation of a protein corona around the same NPs can promote their uptake by endocytic vesicles and final sequestration within lysosomes. Of note is that such intracellular fate of PS-NPs is associated with mitigation of the biochemical alterations of the phosphorylated AKT (pAKT)/AKT and phosphorylated ERK (pERK)/ERK levels. These findings provide the distribution of NPs in human breast cancer cells, may broaden our understanding of the interactions between NPs and breast cancer cells and underscore the crucial role of the protein corona in modulating the impact of MNPs on human health.

Loss of the extracellular matrix glycoprotein EMILIN1 accelerates Δ16HER2-driven breast cancer initiation in mice.

The extracellular matrix (ECM) is an important component of the tumor microenvironment and undergoes extensive remodeling during both initiation and progression of breast cancer (BC). EMILIN1 is an ECM glycoprotein, whose function has been linked to cancer and metastasis. However, EMILIN1 role during mammary gland and BC development has never been investigated. In silico and molecular analyses of human samples from normal mammary gland and BC showed that EMILIN1 expression was lower in tumors than in healthy mammary tissue and it predicted poor prognosis, particularly in HER2-positive BC. HER2+ BC accounts for 15-20% of all invasive BC and is characterized by high aggressiveness and poor prognosis. The Δ16HER2 isoform, a splice variant with very high oncogenic potential, is frequently expressed in HER2+ BC and correlates with metastatic disease. To elucidate the role of EMILIN1 in BC, we analyzed the phenotype of MMTV-Δ16HER2 transgenic mice, developing spontaneous multifocal mammary adenocarcinomas, crossed with EMILIN1 knock-out (KO) animals. We observed that Δ16HER2/EMILIN1 KO female mice exhibited an accelerated normal mammary gland development and a significantly anticipated appearance of palpable tumors (13.32 vs 15.28 weeks). This accelerated tumor initiation was corroborated by an increased number of tumor foci observed in mammary glands from Δ16HER2/EMILIN1 KO mice compared to the wild-type counterpart. Altogether our results underscore the centrality of ECM in the process of BC initiation and point to a role for EMILIN1 during normal mammary gland development and in protecting from HER2-driven breast tumorigenesis.

Enhancing Immune Responses against SARS-CoV-2 Variants in Aged Mice with INDUK: A Chimeric DNA Vaccine Encoding the Spike S1-TM Subunits.

Currently available vaccines against COVID-19 showed high efficacy against the original strain of SARS-CoV-2 but progressively lower efficacy against new variants. In response to emerging SARS-CoV-2 strains, we propose chimeric DNA vaccines encoding the spike antigen, including a combination of selected key mutations from different variants of concern. We developed two DNA vaccines, pVAX-S1-TM-D614G and pVAX-S1-TM-INDUK (INDUK), encoding the SARS-CoV-2 S1 spike subunit in fusion with the transmembrane region that allows protein trimerization as predicted by in silico analysis. pVAX-S1-TM-D614G included the dominant D614G substitution, while the chimeric vaccine INDUK contained additional selected mutations from the Delta (E484Q and L452R) and Alpha (N501Y and A570D) variants. Considering that aging is a risk factor for severe disease and that suboptimal vaccine responses were observed in older individuals, the immunogenicity of pVAX-S1-TM-D614G and INDUK was tested in both young and aged C57BL/6 mice. Two vaccine doses were able to trigger significant anti-SARS-CoV-2 antibody production, showing neutralizing activity. ELISA tests confirmed that antibodies induced by pVAX-S1-TM-D614G and INDUK were able to recognize both Wuhan Spike and Delta variant Spike as trimers, while neutralizing antibodies were detected by an ACE2:SARS-CoV-2 Spike S1 inhibitor screening assay, designed to assess the capacity of antibodies to block the interaction between the viral spike S1 protein and the ACE2 receptor. Although antibody titer declined within six months, a third booster dose significantly increased the magnitude of humoral response, even in aged individuals, suggesting that immune recall can improve antibody response durability. The analysis of cellular responses demonstrated that vaccination with INDUK elicited an increase in the percentage of SARS-CoV-2-specific IFN-γ producing T lymphocytes in immunized young mice and TNF-α-producing T lymphocytes in both young and aged mice. These findings not only hold immediate promise for addressing evolving challenges in SARS-CoV-2 vaccination but also open avenues to refine strategies and elevate the effectiveness of next-generation vaccines.

Transfection efficiency boost of cholesterol-containing lipoplexes.

Most lipid formulations require cholesterol for successful transfection, but the precise reason remains to be more clearly understood. Here, we have studied the effect of cholesterol on the transfection efficiency (TE) of lipoplexes in vitro. Addition of cholesterol to highly effective DC-Chol-DOPE/DNA lipoplexes increases TE, with 40 mol% cholesterol yielding about 10-fold improvement. The transfection mechanisms of cholesterol-containing lipoplexes have been investigated by combining dynamic light scattering, synchrotron small angle X-ray scattering, laser scanning confocal microscopy and transfection efficiency measurements. Our results revealed that cholesterol-containing lipoplexes enter the cells partially by membrane fusion and this mechanism accounts for efficient endosomal escape. We also found evidence that formulations with high cholesterol content are not specifically targeted to metabolic degradation. These studies will contribute to rationally design novel delivery systems with superior transfection efficiency.

Insights into the effect of polyethylene terephthalate (PET) microplastics on HER2 signaling pathways.

Plastic pollution poses a significant threat to both ecosystems and human health, as fragments of microscale size are daily inhaled and ingested. Such tiny specks are defined as microplastics (MPs), and although their presence as environmental contaminants is ubiquitous in the world, their possible effects at biological and physiological levels are still not clear. To explore the potential impacts of MP exposure, we produced and characterized polyethylene terephthalate (PET) micro-fragments, then administered them to living cells. PET is widely employed in the production of plastic bottles, and thus represents a potential source of environmental MPs. However, its potential effects on public health are hardly investigated, as the current bio-medical research on MPs mainly utilizes different models, such as polystyrene particles. This study employed cell viability assays and Western blot analysis to demonstrate cell-dependent and dose-dependent cytotoxic effects of PET MPs, as well as a significant impact on HER-2-driven signaling pathways. Our findings provide insight into the biological effects of MP exposure, particularly for a widely used but poorly investigated material such as PET.

SIRT6 promotes metastasis and relapse in HER2-positive breast cancer.

The histone deacetylase sirtuin 6 (SIRT6) has been endowed with anti-cancer capabilities in many tumor types. Here, we investigate the impact of SIRT6-overexpression (SIRT6-OE) in Delta16HER2 mice, which are a bona fide model of HER2-positive breast cancer. After an initial delay in the tumor onset, SIRT6-OE induces a more aggressive phenotype of Delta16HER2 tumors promoting the formation of higher number of tumor foci and metastases than controls. This phenotype of SIRT6-OE tumors is associated with cancer stem cell (CSC)-like features and tumor dormancy, and low senescence and oxidative DNA damage. Accordingly, a sub-set of HER2-positive breast cancer patients with concurrent SIRT6-OE has a significant poorer relapse-free survival (RFS) probability than patients with low expression of SIRT6. ChIP-seq, RNA-seq and RT-PCR experiments indicate that SIRT6-OE represses the expression of the T-box transcription factor 3 (Tbx3) by deacetylation of H3K9ac. Accordingly, loss-of-function mutations of TBX3 or low TBX3 expression levels are predictive of poor prognosis in HER2-positive breast cancer patients. Our work indicates that high levels of SIRT6 are indicative of poor prognosis and high risk of metastasis in HER2-positive breast cancer and suggests further investigation of TBX3 as a downstream target of SIRT6 and co-marker of poor-prognosis. Our results point to a breast cancer subtype-specific effect of SIRT6 and warrant future studies dissecting the mechanisms of SIRT6 regulation in different breast cancer subtypes.

p130Cas/Cyclooxygenase-2 axis in the control of mesenchymal plasticity of breast cancer cells.

INTRODUCTION: Intrinsic plasticity of breast carcinoma cells allows them to undergo a transient and reversible conversion into mesenchymal cells to disseminate into distant organs, where they can re-differentiate to an epithelial-like status to form a cohesive secondary mass. The p130Cas scaffold protein is overexpressed in human ER+ and HER2+ breast cancer where it contributes to cancer progression, invasion and resistance to therapy. However, its role in regulating mesenchymal aggressive breast cancer cells remains to be determined. The aim of this study was to investigate the molecular and functional involvement of this adaptor protein in breast cancer cell plasticity. METHODS: We used silencing strategies and rescue experiments to evaluate phenotypic and biochemical changes from mesenchymal to epithelial traits in breast tumor cell lines. In the mouse A17 cell model previously related to mesenchymal cancer stem cells and basal-like breast cancer, we biochemically dissected the signaling pathways involved and performed functional in vivo tumor growth ability assays. The significance of the signaling platform was assessed in a human setting through the use of specific inhibitors in aggressive MDA-MB-231 subpopulation LM2-4175 cells. To evaluate the clinical relevance of the results, we analyzed publicly available microarray data from the Netherlands Cancer Institute and from the Koo Foundation Sun Yat-Sen Cancer Center. RESULTS: We show that p130Cas silencing induces loss of mesenchymal features, by downregulating Vimentin, Snail, Slug and Twist transcriptional factors, resulting in the acquirement of epithelial-like traits. Mechanistically, p130Cas controls Cyclooxygenase-2 transcriptional expression, which in turn contributes to p130Cas-dependent maintenance of mesenchymal phenotype. This cascade of events also compromises in vivo tumor growth through inhibition of cell signaling controlling cell cycle progression. c-Src and JNK kinases are sequential players in p130Cas/ Cyclooxygenase-2 axis and their pharmacological inhibition is sufficient to downregulate Cyclooxygenase-2 leading to an epithelial phenotype. Finally, in silico microarray data analysis indicates that p130Cas and Cyclooxygenase-2 concomitant overexpression predicts poor survival and high probability of breast tumor recurrence. CONCLUSIONS: Overall, these data identify a new p130Cas/Cyclooxygenase-2 axis as a crucial element in the control of breast tumor plasticity, opening new therapeutic strategies leading to inhibition of these pathways in aggressive breast carcinoma.

Cholesterol-dependent macropinocytosis and endosomal escape control the transfection efficiency of lipoplexes in CHO living cells.

Here we investigate the cellular uptake mechanism and final intracellular fate of two cationic liposome formulations characterized by similar physicochemical properties but very different lipid composition and efficiency for intracellular delivery of DNA. The first formulation is made of cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the zwitterionic helper dioleoylphosphocholine (DOPC), while the second one is made of the cationic 3ß-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic lipid dioleoylphosphatidylethanolamine (DOPE). Combining pharmacological and imaging approaches we show that both DOTAP-DOPC/DNA and DC-Chol-DOPE/DNA lipoplexes are taken up in Chinese hamster ovary (CHO) living cells mainly through fluid-phase macropinocytosis. Our results also indicate that lipoplex macropinocytosis is a cholesterol-sensitive uptake mechanism. On the other side, both clathrin-mediated and caveolae-mediated endocytosis play a minor role, if any, in the cell uptake. Colocalization of fluorescently tagged lipoplexes and Lysosensor, a primary lysosome marker, reveals that poorly efficient DOTAP-DOPC/DNA lipoplexes are largely degraded in the lysosomes, while efficient DC-Chol-DOPE/DNA systems can efficiently escape from endosomal compartments.

Breast Cancer Treatment: The Case of Gold(I)-Based Compounds as a Promising Class of Bioactive Molecules.

Breast cancers (BCs) may present dramatic diagnoses, both for ineffective therapies and for the limited outcomes in terms of lifespan. For these types of tumors, the search for new drugs is a primary necessity. It is widely recognized that gold compounds are highly active and extremely potent as anticancer agents against many cancer cell lines. The presence of the metal plays an essential role in the activation of the cytotoxicity of these coordination compounds, whose activity, if restricted to the ligands alone, would be non-existent. On the other hand, gold exhibits a complex biochemistry, substantially variable depending on the chemical environments around the central metal. In this review, the scientific findings of the last 6-7 years on two classes of gold(I) compounds, containing phosphane or carbene ligands, are reviewed. In addition to this class of Au(I) compounds, the recent developments in the application of Auranofin in regards to BCs are reported. Auranofin is a triethylphosphine-thiosugar compound that, being a drug approved by the FDA-therefore extensively studied-is an interesting lead gold compound and a good comparison to understand the activities of structurally related Au(I) compounds.

Coinage Metal Compounds With 4-Methoxy-Diphenylphosphane Benzoate Ligand Inhibit Female Cancer Cell Growth.

In the continuous effort to find new metal-based compounds as alternatives to platinum-related anticancer drugs, 11th group metal phosphane compounds have been thoroughly taken into consideration. Tris-arylphosphane metal derivatives have been extensively considered as heteroleptic metal compounds exhibiting remarkable cytotoxic activities. Functional groups in the aryl moieties modulate the activity reinforcing or eliminating it. Previous works have highlighted that the presence of hydrophilic groups in the phosphane ligands, such as COOH or OH, hampers the anticancer activity of gold azolate/PPh3 compounds. To increase the polarity of the triarylphosphane ligand without affecting the activity, we considered the preparation of esters starting from the 4-diphenylphosphane-benzoic acid. The resulting phosphanes are poorer donators than the PPh3, leading to poly-phosphane M(I) compounds, and they exhibit intense emissive properties. A homologous series of L3MX-type compounds (where M = Au and X = Cl, M = Cu and X = BF4, and M = Ag and X = PF6) were obtained with the 4-methoxy-diphenylphosphane benzoate. The homologous metal compounds have been characterized by analytical and spectroscopic methods and, remarkably, their formation was associated with high frequencies of 31P NMR chemical shift variations (5-35 ppm in CDCl3). The new complexes and the ligand were evaluated on sensitive and cisplatin-resistant human tumor cell lines. The ligand is ineffective on cells while the complexes exert a notable antiproliferative effect. The homologous series of the L3MX complexes were able to significantly reduce the cell viability of human triple-negative breast cancer cells (MDA-MB-231), representing the most aggressive subtype of breast cancer, and of ovarian carcinoma (A2780). Among these coinage metal compounds, L3AgPF6 results the most interesting, showing the lowest GI50 values in all cell lines. Interestingly, this silver complex is more cytotoxic than cisplatin, taken as reference drug. The investigation of the mechanism of action of L3AgPF6 in A2780 cells highlighted the induction of the apoptotic pathway, the depolarization of the mitochondrial inner membrane, and a significant accumulation in cells.

The protein corona reduces the anticancer effect of graphene oxide in HER-2-positive cancer cells.

In the last decade, graphene oxide (GO)-based nanomaterials have attracted much attention for their potential anti-cancer properties against various cancer cell types. However, while in vitro studies are promising, following in vivo investigations fail to show any relevant efficacy. Recent research has clarified that the wide gap between benchtop discoveries and clinical practice is due to our limited knowledge about the physical-chemical transformation of nanomaterials in vivo. In physiological environments, nanomaterials are quickly coated by a complex dress of biological molecules referred to as the protein corona. Mediating the interaction between the pristine material and the biological system the protein corona controls the mechanisms of action of nanomaterials up to the sub-cellular level. Here we investigate the anticancer ability of GO in SK-BR-3 human breast cancer cells over-expressing the human epidermal growth factor receptor 2 (HER-2), which is functionally implicated in the cell growth and proliferation through the activation of downstream pathways, including the PI3K/AKT/mTOR and MAPK/ERK signaling cascades. Western blot analysis demonstrated that GO treatment resulted in a marked decrease in total HER-2, associated with a down-regulation of the expression and activation of protein kinase B (AKT) and extracellular signal-regulated kinase (ERK) thus indicating that GO may act as a potent HER-2 inhibitor. On the other side, the protein corona reverted the effects of GO on HER-2 expression and molecular downstream events to the control level. Our findings may suggest a mechanistic explanation of the reduced anticancer properties of GO-based nanomaterials in vivo. These results may also represent a good prediction strategy for the anticancer activity of nanomaterials designed for biomedical purposes, reaffirming the necessity of exploring their effectiveness under physiologically relevant conditions before moving on to the next in vivo studies.

Efficient Delivery of DNA Using Lipid Nanoparticles.

DNA vaccination has been extensively studied as a promising strategy for tumor treatment. Despite the efforts, the therapeutic efficacy of DNA vaccines has been limited by their intrinsic poor cellular internalization. Electroporation, which is based on the application of a controlled electric field to enhance DNA penetration into cells, has been the method of choice to produce acceptable levels of gene transfer in vivo. However, this method may cause cell damage or rupture, non-specific targeting, and even degradation of pDNA. Skin irritation, muscle contractions, pain, alterations in skin structure, and irreversible cell damage have been frequently reported. To overcome these limitations, in this work, we use a microfluidic platform to generate DNA-loaded lipid nanoparticles (LNPs) which are then characterized by a combination of dynamic light scattering (DLS), synchrotron small-angle X-ray scattering (SAXS), and transmission electron microscopy (TEM). Despite the clinical successes obtained by LNPs for mRNA and siRNA delivery, little is known about LNPs encapsulating bulkier DNA molecules, the clinical application of which remains challenging. For in vitro screening, LNPs were administered to human embryonic kidney 293 (HEK-293) and Chinese hamster ovary (CHO) cell lines and ranked for their transfection efficiency (TE) and cytotoxicity. The LNP formulation exhibiting the highest TE and the lowest cytotoxicity was then tested for the delivery of the DNA vaccine pVAX-hECTM targeting the human neoantigen HER2, an oncoprotein overexpressed in several cancer types. Using fluorescence-activated cell sorting (FACS), immunofluorescence assays and fluorescence confocal microscopy (FCS), we proved that pVAX-hECTM-loaded LNPs produce massive expression of the HER2 antigen on the cell membrane of HEK-293 cells. Our results provide new insights into the structure-activity relationship of DNA-loaded LNPs and pave the way for the access of this gene delivery technology to preclinical studies.