Roberto Molinaro

The impact of nanoparticle protein corona on cytotoxicity, immunotoxicity and target drug delivery

In a perfect sequence of events, nanoparticles (NPs) are injected into the bloodstream where they circulate until they reach the target tissue. The ligand on the NP surface recognizes its specific receptor expressed on the target tissue and the drug is released in a controlled manner. However, once injected in a physiological environment, NPs interact with biological components and are surrounded by a protein corona (PC). This can trigger an immune response and affect NP toxicity and targeting capabilities. In this review, we provide a survey of recent findings on the NP-PC interactions and discuss how the PC can be used to modulate both cytotoxicity and the immune response as well as to improve the efficacy of targeted delivery of nanocarriers.

Biomimetic proteolipid vesicles for targeting inflamed tissues.

A multitude of micro- and nanoparticles have been developed to improve the delivery of systemically administered pharmaceuticals, which are subject to a number of biological barriers that limit their optimal biodistribution. Bioinspired drug-delivery carriers formulated by bottom-up or top-down strategies have emerged as an alternative approach to evade the mononuclear phagocytic system and facilitate the transport across the endothelial vessel wall. Here, we describe a method that leverages the advantages of bottom-up and top-down strategies to incorporate proteins derived from the leukocyte plasma membrane into lipid nanoparticles. The resulting proteolipid vesicles - which we refer to as leukosomes - retained the versatility and physicochemical properties typical of liposomal formulations, preferentially targeted inflamed vasculature, enabled the selective and effective delivery of dexamethasone to inflamed tissues, and reduced phlogosis in a localized model of inflammation.

Supramolecular devices to improve the treatment of brain diseases.

The blood-brain barrier (BBB) hinders the accumulation of active compounds in the central nervous system, thus decreasing their therapeutic effectiveness. To overcome this obstacle, interesting supramolecular nanodevices are herein considered. These systems have many advantages over the conventional formulations, such as having structures made up of biocompatible and biodegradable materials, the possibility of bypassing the BBB in a non-invasive manner (without structural modifications) and the possibility of being structurally modified to modulate the biopharmaceutical properties of the encapsulated compounds. Polymolecular (liposomes, niosomes, nanogels) and oligomolecular (cyclodextrins) devices have potential clinical applications in brain drug delivery, being capable of active targeting that can concentrate bioactives in the brain.

Evaluation of anticancer activity of celastrol liposomes in prostate cancer cells

Abstract Context: Celastrol, a natural compound derived from the herb Tripterygium wilfordii, is known to have anticancer activity, but is not soluble in water. Objective: Formation of celastrol liposomes, to avoid the use of toxic solubilising agents. Materials and methods: Two different formulations of PEGylated celastrol liposomes were fabricated. Liposomal characteristics and serum stability were determined using dynamic light scattering. Drug entrapment efficacy and drug release were measured spectrophotometrically. Cellular internalisation and anticancer activity was measured in prostate cancer cells. Results: Liposomal celastrol displayed efficient serum stability, cellular internalisation and anticancer activity, comparable to that of the free drug reconstituted in dimethyl sulfoxide. Discussion and conclusion: Liposomal celastrol can decrease the viability of prostate cancer cells, while eliminating the need for toxic solubilising agents.

Polyethylenimine and chitosan carriers for the delivery of RNA interference effectors

Introduction: Manipulating gene activity represents a promising approach for the treatment of cancer and other diseases. The relatively recent discovery of RNA interference (RNAi) revolutionized therapeutic approaches in this field. RNA effectors can now be used to modify the activity of genes and theoretically control any biological process. Area covered: However, the clinical application of RNAi has been limited by the inefficient delivery of RNA. Challenges associated with the in vivo use of RNAi mediators, include rapid degradation, uptake by the reticular endothelial system and inefficient cellular internalization. To date, various strategies have been developed in order to overcome these pitfalls. Among these approaches, non-viral delivery systems have gained increasing popularity, as they are generally considered safer than their viral counterparts. Expert opinion: The use of cationic polymers, especially polyethylenimine and chitosan, for the in vivo delivery of doubled-stranded RNAs is discussed in this review.

Mild Hyperthermia Enhances Transport of Liposomal Gemcitabine and Improves In Vivo Therapeutic Response

Obstructive biological barriers limit the transport and efficacy of cancer nanotherapeutics. Creative manipulation of tumor microenvironment provides promising avenues towards improving chemotherapeutic response. Such strategies include the use of mechanical stimuli to overcome barriers, and increase drug delivery and therapeutic efficacy. The rational use of gold nanorod-mediated mild hyperthermia treatment (MHT) alters tumor transport properties, increases liposomal gemcitabine (Gem Lip) delivery, and antitumor efficacy in pancreatic cancer CAPAN-1 tumor model. MHT treatment leads to a threefold increase in accumulation of 80-nm liposomes and enhances spatial interstitial distribution. I.v. injection of Gem Lip and MHT treatment lead to a threefold increase in intratumor gemcitabine concentration compared to chemotherapeutic infusion alone. Furthermore, combination of MHT treatment with infusion of 12 mg kg(-1) Gem Lip leads to a twofold increase in therapeutic efficacy and inhibition of CAPAN-1 tumor growth when compared to equimolar chemotherapeutic treatment alone. Enhanced therapeutic effect is confirmed by reduction in tumor size and increase in apoptotic index where MHT treatment combined with 12 mg kg(-1) Gem Lip achieves similar therapeutic efficacy as the use of 60 mg kg(-1) free gemcitabine. In conclusion, improvements in vivo efficacy are demonstrated resulting from MHT treatment that overcome transport barriers, promote delivery, improve efficacy of nanomedicines.

Physicochemical features and transfection properties of chitosan/poloxamer 188/poly(D,L-lactide-co-glycolide) nanoplexes.

The aim of this study was the evaluation of the effects of two emulsifiers on the physicochemical and technological properties of low molecular weight chitosan/poly (D,L-lactide-co-glycolide) (PLGA) nanoplexes and their transfection efficiency. Nanospheres were prepared using the nanoprecipitation method of the preformed polymer. The mean diameter and surface charge of the nanospheres were investigated by photocorrelation spectroscopy. The degree of binding of the plasmid with the nanoplexes was qualitatively and quantitatively determined. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) testing was performed using HeLa, RPMI8226, and SKMM1 cell lines. Flow cytometry and confocal laser scanning microscopy were used to determine the degree of cellular transfection and internalization of the nanoplexes into cells, respectively. The nanoplexes had a positive zeta potential, and low amounts of PLGA and poloxamer 188 showed a mean colloidal size of ~200 nm with a polydispersity index of ~0.14. The nanoplexes had suitable entrapment efficiency (80%). In vitro experiments showed that the colloidal nanodevices did not induce significant cytotoxicity. The nanoplexes investigated in this study could represent efficient and useful nonviral devices for gene delivery. Use of low amounts of PLGA and poloxamer 188 enabled development of a nanosphere able to transfect cells efficiently. These nanosystems are a helpful platform for delivery of genetic material while preserving therapeutic efficacy.

Biomimetic carriers mimicking leukocyte plasma membrane to increase tumor vasculature permeability

Recent advances in the field of nanomedicine have demonstrated that biomimicry can further improve targeting properties of current nanotechnologies while simultaneously enable carriers with a biological identity to better interact with the biological environment. Immune cells for example employ membrane proteins to target inflamed vasculature, locally increase vascular permeability, and extravasate across inflamed endothelium. Inspired by the physiology of immune cells, we recently developed a procedure to transfer leukocyte membranes onto nanoporous silicon particles (NPS), yielding Leukolike Vectors (LLV). LLV are composed of a surface coating containing multiple receptors that are critical in the cross-talk with the endothelium, mediating cellular accumulation in the tumor microenvironment while decreasing vascular barrier function. We previously demonstrated that lymphocyte function-associated antigen (LFA-1) transferred onto LLV was able to trigger the clustering of intercellular adhesion molecule 1 (ICAM-1) on endothelial cells. Herein, we provide a more comprehensive analysis of the working mechanism of LLV in vitro in activating this pathway and in vivo in enhancing vascular permeability. Our results suggest the biological activity of the leukocyte membrane can be retained upon transplant onto NPS and is critical in providing the particles with complex biological functions towards tumor vasculature.

Proteomic Profiling of a Biomimetic Drug Delivery Platform

Current delivery platforms are typically designed for prolonged circulation that favors superior accumulation of the payload in the targeted tissue. The design of efficient surface modifications determines both a longer circulation time and targeting abilities of particles. The optimization of synthesis protocols to efficiently combine targeting molecules and elements that allow for an increased circulation time can be challenging and almost impossible when several functional elements are needed. On the other hand, in the last decade, the development of bioinspired technologies was proposed as a new approach with which to increase particle safety, biocompatibility and targeting, while maintaining the synthesis protocols simple and reproducible. Recently, we developed a new drug delivery system inspired by the biology of immune cells called leukolike vector (LLV) and formed by a nanoporous silicon core and a shell derived from the leucocyte cell membrane. The goal of this study is to investigate the protein content of the LLV. Here we report the proteomic profiling of the LLV and demonstrate that our approach can be used to modify the surface of synthetic particles with more than 150 leukocyte membrane associated proteins that determine particle safety, circulation time and targeting abilities towards inflamed endothelium.

Effects of the protein corona on liposome–liposome and liposome–cell interactions

A thorough understanding of interactions occurring at the interface between nanocarriers and biological systems is crucial to predict and interpret their biodistribution, targeting, and efficacy, and thus design more effective drug delivery systems. Upon intravenous injection, nanoparticles are coated by a protein corona (PC). This confers a new biological identity on the particles that largely determines their biological fate. Liposomes have great pharmaceutical versatility, so, as proof of concept, their PC has recently been implicated in the mechanism and efficiency of their internalization into the cell. In an attempt to better understand the interactions between nanocarriers and biological systems, we analyzed the plasma proteins adsorbed on the surface of multicomponent liposomes. Specifically, we analyzed the physical properties and ultrastructure of liposome/PC complexes and the aggregation process that occurs when liposomes are dispersed in plasma. The results of combined confocal microscopy and flow cytometry experiments demonstrated that the PC favors liposome internalization by both macrophages and tumor cells. This work provides insights into the effects of the PC on liposomes’ physical properties and, consequently, liposome–liposome and liposome–cell interactions.