Luca Sorrentino

Ferritin nanocages: A biological platform for drug delivery, imaging and theranostics in cancer

Nowadays cancer represents a prominent challenge in clinics. Main achievements in cancer management would be the development of highly accurate and specific diagnostic tools for early detection of cancer onset, and the generation of smart drug delivery systems for targeted chemotherapy release in cancer cells. In this context, protein-based nanocages hold a tremendous potential as devices for theranostics purposes. In particular, ferritin has emerged as an excellent and promising protein-based nanocage thanks to its unique architecture, surface properties and high biocompatibility. By exploiting natural recognition of the Transferrin Receptor 1, which is overexpressed on tumor cells, ferritin nanocages may ensure a proper drug delivery and release. Moreover, researchers have applied surface functionalities on ferritin cages for further providing active tumor targeting. Encapsulation strategies of non metal-containing drugs within ferritin cages have been explored and successfully performed with encouraging results. Various preclinical studies have demonstrated that nanoformulation within ferritin nanocages significantly improved targeted therapy and accurate imaging of cancer cells. Aims of this review are to describe structure and functions of ferritin nanocages, and to provide an overview about the nanotechnological approaches implemented for applying them to cancer diagnosis and treatment.

Preoperative Localization and Surgical Margins in Conservative Breast Surgery

Breast-conserving surgery (BCS) is the treatment of choice for early breast cancer. The adequacy of surgical margins (SM) is a crucial issue for adjusting the volume of excision and for avoiding local recurrences, although the precise definition of an adequate margins width remains controversial. Moreover, other factors such as the biological behaviour of the tumor and subsequent proper systemic therapies may influence the local recurrence rate (LRR). However, a successful BCS requires preoperative localization techniques or margin assessment techniques. Carbon marking, wire-guided, biopsy clips, radio-guided, ultrasound-guided, frozen section analysis, imprint cytology, and cavity shave margins are commonly used, but from the literature review, no single technique proved to be better among the various ones. Thus, an association of two or more methods could result in a decrease in rates of involved margins. Each institute should adopt its most congenial techniques, based on the senologic equipe experience, skills, and technologies.

Nanometronomic treatment of 4T1 breast cancer with nanocaged doxorubicin prevents drug resistance and circumvents cardiotoxicity

Chemotherapeutic treatment of breast cancer is based on maximum tolerated dose (MTD) approach. However, advanced stage tumors are not effectively eradicated by MTD owing to suboptimal drug targeting, onset of therapeutic resistance and neoangiogenesis. In contrast, “metronomic” chemotherapy is based on frequent drug administrations at lower doses, resulting in neovascularization inhibition and induction of tumor dormancy. Here we show the potential of H-ferritin (HFn)-mediated targeted nanodelivery of metronomic doxorubicin (DOX) in the setting of a highly aggressive and metastatic 4T1 breast cancer mouse model with DOX-inducible expression of chemoresistance. We find that HFn-DOX administered at repeated doses of 1.24 mg kg−1 strongly improves the antitumor potential of DOX chemotherapy arresting the tumor progression. We find that such a potent antitumor effect is attributable to multiple nanodrug actions beyond cell killing, including inhibition of tumor angiogenesis and avoidance of chemoresistance. Multiparametric assessment of heart tissues, including histology, ultrastructural analysis of tissue morphology, and measurement of markers of reactive oxygen species and hepatic/renal conditions, provided evidence that metronomic HFn-DOX allowed us to overcome cardiotoxicity. Our results suggest that HFn-DOX has tremendous potential for the development of “nanometronomic” chemotherapy toward safe and tailored oncological treatments.

Oral delivery of insulin via polyethylene imine-based nanoparticles for colonic release allows glycemic control in diabetic rats.

In this study, insulin-containing nanoparticles were loaded into pellet cores and orally administered to diabetic rats. Polyethylene imine-based nanoparticles, either placebo or loaded with insulin, were incorporated by extrusion and spheronization technology into cores that were subsequently coated with three overlapping layers and a gastroresistant film. The starting and coated systems were evaluated in vitro for their physico-technololgical characteristics, as well as disintegration and release performance. Nanoparticles-loaded cores showed homogeneous particle size distribution and shape. When a superdisintegrant and a soluble diluent were included in the composition enhanced disintegration and release performance were observed. The selected formulations, coated either with enteric or three-layer films, showed gastroresistant and release delayed behavior in vitro, respectively. The most promising formulations were finally tested for their hypoglycemic effect in diabetic rats. Only the nanoformulations loaded into the three-layer pellets were able to induce a significant hypoglycemic activity in diabetic rats. Our results suggest that this efficient activity could be attributed to a retarded release of insulin into the distal intestine, characterized by relatively low proteolytic activity and optimal absorption.

Localization of nonpalpable breast lesions with sonographically visible clip: optimizing tailored resection and clear margins

BACKGROUND Achieving clear margins with adequate resection volumes is one of the principal goals of breast-conserving surgery. The aim of our study was to compare preoperative localization using 2 different clips, radiopaque or sonographically visible, to reach this goal. METHODS We reviewed 209 consecutive nonpalpable breast cancers that were treated with lumpectomy: 59 with radiopaque and 150 with sonographically visible clip positioned during biopsy procedure. In the former case, preoperative localization was performed with mammography and in the latter by ultrasonography. RESULTS Clear margins were achieved in 80.4% of patients: 57.6% in the first and 89.3% in the second group (P < .0001; odds ratio, 7.6; 95% confidence interval, 3.4 to 17.2). By using sonographically visible clips, the re-excision rate has decreased from 42.4% to 10.7%, (P < .0001), and resections resulted smaller with average calculated resection ratio of 3.54 vs. 5.08 (P = .03). CONCLUSIONS Preoperative localization using a sonographically visible clip allows a more tailored breast-conserving surgery and reduces the re-excision rate.

Nanoformulated Antiretrovirals for Penetration of the Central Nervous System: State of the Art

The central nervous system is a very challenging HIV-1 sanctuary. But, despite complete suppression of plasmatic viral replication with current antiretroviral therapy, signs of HIV-1 replication can still be found in the cerebrospinal fluid in some patients. The main limitation to achieving HIV-1 eradication from the brain is related to the suboptimal concentrations of antiretrovirals within this site, due to their low permeation across the blood–brain barrier. In recent years, a number of reliable nanotechnological strategies have been developed with the aim of enhancing antiretroviral drug penetration across the blood–brain barrier. The aim of this review is to provide an overview of the different nanoformulated antiretrovirals, used in both clinical and preclinical studies, that are designed to improve their delivery into the brain by active or passive permeation mechanisms through the barrier. Different nanotechnological approaches have proven successful for optimizing antiretrovirals delivery to the central nervous system, with a likely benefit for HIV-associated neurocognitive disorders and a more debated contribution to the complete eradication of the HIV-1 infection.

Is there a role for homeopathy in breast cancer surgery? A first randomized clinical trial on treatment with Arnica montana to reduce postoperative seroma and bleeding in patients undergoing total mastectomy -

Aim: This study aimed to evaluate the benefits of Arnica montana on post-operative blood loss and seroma production in women undergoing unilateral total mastectomy by administering Arnica Montana 1000 Korsakovian dilution (1000 K). Materials and Methods: From 2012 to 2014, 53 women were randomly assigned to A. montana or placebo and were followed up for 5 days. The main end point was the reduction in blood and serum volumes collected in drainages. Secondary end points were duration of drainage, a self-evaluation of pain, and the presence of bruising or hematomas. Results: The per-protocol analysis revealed a lower mean volume of blood and serum collected in drainages with A. montana (−94.40 ml; 95% confidence interval [CI]: 22.48-211.28; P = 0.11). A regression model including treatment, volume collected in the drainage on the day of surgery, and patient weight showed a statistically significant difference in favor of A. montana (−106.28 ml; 95% CI: 9.45-203.11; P = 0.03). Volumes collected on the day of surgery and the following days were significantly lower with A. montana at days 2 (P = 0.033) and 3 (P = 0.0223). Secondary end points have not revealed significant differences. Conclusions: A. montana 1000 K could reduce post-operative blood and seroma collection in women undergoing unilateral total mastectomy. Larger studies are needed with different dilutions of A. montana to further validate these data.

Key Role of the Adenylate Moiety and Integrity of the Adenylate-Binding Site for the NAD(+)/H Binding to Mitochondrial Apoptosis-Inducing Factor.

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein with pro-life and pro-death activities, which plays critical roles in mitochondrial energy metabolism and caspase-independent apoptosis. Defects in AIF structure or expression can cause mitochondrial abnormalities leading to mitochondrial defects and neurodegeneration. The mechanism of AIF-induced apoptosis was extensively investigated, whereas the mitochondrial function of AIF is poorly understood. A unique feature of AIF is the ability to form a tight, air-stable charge-transfer (CT) complex upon reaction with NADH and to undergo a conformational switch leading to dimerization, proposed to be important for its vital and lethal functions. Although some aspects of interaction of AIF with NAD(+)/H have been analyzed, its precise mechanism is not fully understood. We investigated how the oxidized and photoreduced wild-type and G307A and -E variants of murine AIF associate with NAD(+)/H and nicotinamide mononucleotide (NMN(+)/H) to determine the role of the adenylate moiety in the binding process. Our results indicate that (i) the adenylate moiety of NAD(+)/H is crucial for the association with AIF and for the subsequent structural reorganization of the complex, but not for protein dimerization, (ii) FAD reduction rather than binding of NAD(+)/H to AIF initiates conformational rearrangement, and (iii) alteration of the adenylate-binding site by the G307E (equivalent to a pathological G308E mutation in human AIF) or G307A replacements decrease the affinity and association rate of NAD(+)/H, which, in turn, perturbs CT complex formation and protein dimerization but has no influence on the conformational switch in the regulatory peptide.

H-Ferritin-nanocaged olaparib: A promising choice for both BRCA-mutated and sporadic triple negative breast cancer

Poly(ADP-ribose) polymerase (PARP) inhibitors represent a promising strategy toward the treatment of triple-negative breast cancer (TNBC), which is often associated to genomic instability and/or BRCA mutations. However, clinical outcome is controversial and no benefits have been demonstrated in wild type BRCA cancers, possibly due to poor drug bioavailability and low nuclear delivery. In the attempt to overcome these limitations, we have developed H-Ferritin nanoformulated olaparib (HOla) and assessed its anticancer efficacy on both BRCA-mutated and non-mutated TNBC cells. We exploited the natural tumor targeting of H-Ferritin, which is mediated by the transferrin receptor-1 (TfR1), and its physiological tropism toward cell nucleus. TNBC cell lines over-expressing TfR-1 were successfully recognized by H-Ferritin, displaying a fast internalization into the cells. HOla induced remarkable cytotoxic effect in cancer cells, exhibiting 1000-fold higher anticancer activity compared to free olaparib (Ola). Accordingly, HOla treatment enhanced PARP-1 cleavage, DNA double strand breaks and Ola delivery into the nuclear compartment. Our findings suggest that H-Ferritin nanoformulation strongly enhances cytotoxic efficacy of Ola as a stand-alone therapy in both BRCA-mutated and wild type TNBC cells, by promoting targeted nuclear delivery.

An improved expression system for the VC1 ligand binding domain of the receptor for advanced glycation end products in Pichia pastoris.

The receptor for the advanced glycation end products (RAGE) is a type I transmembrane glycoprotein belonging to the immunoglobulin superfamily and binds a variety of unrelated ligands sharing a negative charge. Most ligands bind to the extracellular V or VC1 domains of the receptor. In this work, V and VC1 of human RAGE were produced in the methylotrophic yeast Pichia pastoris and directed to the secretory pathway. Fusions to a removable C-terminal His-tag evidenced proteolytic processing of the tag by extracellular proteases and also intracellular degradation of the N-terminal portion of V-His. Expression of untagged forms was attempted. While the V domain was retained intracellularly, VC1 was secreted into the medium and was functionally active in binding AGEs. The glycosylation state of VC1 was analyzed by mass spectrometry and peptide-N-glycosidase F digestion. Like RAGE isolated from mammalian sources, the degree of occupancy of the N-glycosylation sites was full at Asn25 and partial at Asn81 which was also subjected to non-enzymatic deamidation. A simple procedure for the purification to homogeneity of VC1 from the medium was developed. The folded state of the purified protein was assessed by thermal shift assays. Recombinant VC1 from P. pastoris showed a remarkably high thermal stability as compared to the protein expressed in bacteria. Our in vivo approach indicates that the V and C1 domains constitute a single folding unit. The stability and solubility of the yeast-secreted VC1 may be beneficial for future in vitro studies aimed to identify new ligands or inhibitors of RAGE.