Giorgia Adamo

Minimalism in Radiation Synthesis of Biomedical Functional Nanogels

A scalable, single-step, synthetic approach for the manufacture of biocompatible, functionalized micro- and nanogels is presented. In particular, poly(N-vinyl pyrrolidone)-grafted-(aminopropyl)methacrylamide microgels and nanogels were generated through e-beam irradiation of PVP aqueous solutions in the presence of a primary amino-group-carrying monomer. Particles with different hydrodynamic diameters and surface charge densities were obtained at the variance of the irradiation conditions. Chemical structure was investigated by different spectroscopic techniques. Fluorescent variants were generated through fluorescein isothiocyanate attachment to the primary amino groups grafted to PVP, to both quantify the available functional groups for bioconjugation and follow nanogels localization in cell cultures. Finally, a model protein, bovine serum albumin, was conjugated to the nanogels to demonstrate the attachment of biologically relevant molecules for targeting purposes in drug delivery. The described approach provides a novel strategy to fabricate biohybrid nanogels with a very promising potential in nanomedicine.

Self-organized environment-sensitive inulin–doxorubicin conjugate with a selective cytotoxic effect towards cancer cells

An inulin-based random copolymer bearing high dose doxorubicin (18.45% on a weight basis), INU-EDA-P,C-DOXO, was prepared by coupling doxorubicin with inulin though a citraconylamide bridge used as a pH sensitive spacer. A further conjugation with pentynoic acid via an amidic bond led to the hydrophobization of the copolymer which allows the acquisition of a self-assembling ability at low concentration (0.33 mg mL−1) combining both Π–Π stacking and London interactions. Drug release studies were carried out at different pH demonstrating a remarkable pH dependency, where the maximum release rate was observed at pH mimicking cancer tissue and lysosomal environments. Besides, by measuring ζ-potential variations as a function of the pH, INU-EDA-P,C-DOXO proved capable of undergoing charge reversal at acidic pH, changing its physicochemical and biological behavior. In vitro tests with cancer (MDA-MB 231) and normal (HB-2) breast cells were carried out to verify the conjugate aptitude to follow different routes to enter cells depending on the microenvironment. This finding was supported by quantitative up-take studies, which revealed that INU-EDA-P,C-DOXO released doxorubicin before entering cancer cells, as the entire copolymer diffused across normal cell membranes without relevant modifications.

Multi-Functional Nanogels for Tumor Targeting and Redox-Sensitive Drug and siRNA Delivery

(1) Background: A new family of nanosystems able to discern between normal and tumor cells and to release a therapeutic agent in controlled way were synthetized by e-beam irradiation. This technique permits to obtain biocompatible, sterile, carboxyl-functionalized polyvinylpyrrolidone (PVP-co-acrylic acid) nanogels (NGs); (2) Methods: Here, we performed a targeting strategy based on the recognition of over-expressed proteins on tumor cells, like the folate receptor. The selective targeting was demonstrated by co-culture studies and flow cytometry analysis, using folate conjugated NGs. Moreover, nanoparticles were conjugated to a chemotherapeutic drug or to a pro-apoptotic siRNA through a glutathione sensitive spacer, in order to obtain a controlled release mechanism, specific for cancer cells. The drug efficiency was tested on tumor and healthy cells by flow cytometric analysis, confocal and epifluorescence microscopy and cytotoxicity assay; the siRNA effect was investigated by RNAi experiment; (3) Results: The data obtained showed that the use of NGs permits a faster cargo release in cancer cells, in response to high cytosolic glutathione level, also improving their efficacy; (4) Conclusion: The possibility of releasing biological molecules in a controlled way and to recognize a specific tumor target allows overcoming the typical limits of the classic cancer therapy.

Novel inulin-based mucoadhesive micelles loaded with corticosteroids as potential transcorneal permeation enhancers.

Graphical abstract Figure. No caption available. ABSTRACT In this work a new copolymer of inulin (INU) derivatized with ethylendiamine (EDA) and retinoic acid (RA), named INU‐EDA‐RA, was synthetized, characterized and employed to produce micelles as carriers for topical administration of corticosteroids for the potential treatment of diseases of posterior eye segment. Spectroscopic analysis confirmed a molar derivatization degree of 11.30 and 4.30% in EDA and RA, respectively. INU‐EDA‐RA micelles are capable of strong mucoadhesive interactions which result time‐independent and stable over time but concentration depending. Moreover micelles are able to encapsulate efficiently from 3 to 13% (w/w) of lipophilic drugs, as dexamethasone, triamcinolone and triamcinolone acetonide. Drug loaded micelles are stable for three months when stored as freeze‐dried powders and able to release high amount of drug when compared to drug dissolution profiles from suspensions. Moreover, drug loaded micelles are compatible with different ocular cell lines that are also able to internalize fluorescent micelles. Finally, drug loaded micelles enhance drug fluxes and permeability coefficients across corneal epithelial cells, thus reducing drug loss due to retention inside the cells.

E-beam crosslinked nanogels conjugated with monoclonal antibodies in targeting strategies.

Abstract Poly(N-vinyl pyrrolidone)-based-nanogels (NGs), produced by e-beam irradiation, are conjugated with monoclonal antibodies (mAb) for active targeting purposes. The uptake of immuno-functionalized nanogels is tested in an endothelial cell line, ECV304, using confocal and epifluorescence microscopy. Intracellular localization studies reveal a faster uptake of the immuno-nanogel conjugate with respect to the ‘bare’ nanogel. The specific internalization pathway of these immuno-nanogels is clarified by selective endocytosis inhibition experiments, flow cytometry and confocal microscopy. Active targeting ability is also verified by conjugating a monoclonal antibody which recognizes the αvβ3 integrin on activated endothelial cells. Epifluorescence images of the ‘wound healing assay’ on ECV304 cells provide evidence of nanogels localization only in the target cells. Therefore, the immuno-nanogels produced have the potential to recognize specific cell types in heterogeneous systems, which makes them promising candidates for targeted drug delivery applications.

Polyaminoacid–doxorubicin prodrug micelles as highly selective therapeutics for targeted cancer therapy

An amphiphilic copolymer carrying high-dose doxorubicin (21% on a weight basis), PHEA–EDA–P,C–Doxo, was prepared by coupling doxorubicin with a biocompatible polyaminoacid through a pH-sensitive spacer. Additional derivatization with 4-pentynoic acid endows it with self-assembling properties by means of π–π stacking. These micelles can be triggered to promptly release drug in lysosomes (∼40% in 12 h) through pH-dependent micelle hydrolysis after uptake. In vitro tests on co-cultures of cancer (MDA-MB 231) and normal (HB-2) breast cells proved that the conjugate was selectively internalized into the former rather than normal cells, exploiting the caveolae-dependent endocytosis pathway, explaining the selective cytotoxic effect toward cancer cells. Intracellular trafficking study of MDA-MB 231 showed that the delivery of the endocytosed drug occurs through the direct fusion of caveosomes with late lysosomes, triggering a massive release in the cytoplasm, bringing about cell death. Dose-effectiveness and mechanistic data indicate that PHEA–EDA–P,C–Doxo is endowed with a distinctive combination of selectivity and pharmacological potency (EC50 13 μM, Emax = 77% and EC50 > 25 μM, Emax = 21% for cancer and healthy cells respectively) that makes it an excellent candidate for future preclinical studies.

Functionalization of nanoparticles in specific targeting and mechanism release

The development of various nanotechnologies have provided a new field of research, which allows the manipulation of molecular components of matter and covers a vast array of nanodevices. The “smart” multifunctional nanostructures should work as customizable, targeted drug-delivery vehicles capable of carrying large doses of therapeutic agents into malignant cells. Some nanomedical approaches are based on the use of functionalized nanoparticles (NPs), not only to reduce toxicity and side effects of drugs but, also in potential the biological barriers crossing on, such as: the blood–brain barrier, different cellular compartments, including the nucleus. Currently, many materials are used for nanoparticle preparation, several of biological derivation, such as albumin, gelatin, phospholipids, etc.; others of chemical nature, such as various polymers, hydrogels, solid metals. Covalent and noncovalent chemical linking using different molecules have been reported for NPs surface functionalization, confer them specific properties, such as targeting ability. Based on the strategy chosen to control release (pH or redox or temperature sensitive NPs), different drugs are linked to NPs by adsorption, incorporation, or chemical binding. Use of smart nanocarriers can be a successful approach to overcome the limits of drug delivery.

A novel enzyme blend for efficient tissue dissociation and primary cells isolation

Tissue dissociation/primary cell isolation and cell harvesting are principal appli- cations for enzymes in tissue culture research and cell biology studies. The goal of a cell isolation procedure is to maximize the yield of functionally viable dissoci- ated cells. Among the parameters which affect the outcome of any particular dissociating procedure there are enzyme(s) used and related impurities presents in crude enzyme preparation. ABIEL srl recently produced the recombinant collagenase class I (Col G) and II (Col H) from Clostridium histolyticum (PCT WO 2011/073925 A9). The enzymes were produced in Escherichia coli and purified by affinity chromatography. The method of production adopted allows absolute control of the final composition of these enzymes, as well as their stability, purity, activity, absence of toxicity and higher reproducibility of batches. The two collagenases produced separately have been used in conjunction according to precise proportions to dissociate calvaria, liver, pancreas, retina of the BALB/c mouse; and bovine hoof. The analyses carried out on all isolated cell populations suggest that the cells maintain the structural and functional integrity of specific tissues/organs originating. Recombinant Col G and Col H enzymes produced by ABIEL are promising in the context of the tissue/cells dissociation, with the aim to make innovation in the fields of tissue engineering and transplantation medicine.