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Background Protein-nanoparticle (NP) interactions dictate properties of nanoconjugates relevant to bionanotechnology. Non-covalent adsorption generates a protein corona (PC) formed by an inner and an outer layer, the hard and soft corona (HC, SC). Intrinsically disordered proteins (IDPs) exist in solution as conformational ensembles, whose response to the presence of NPs is not known. Methods Three IDPs (α-casein, Sic1 and α-synuclein) and lysozyme are compared, describing conformational properties inside HC on silica NPs by circular dichroism (CD) and Fourier-transform infrared (FTIR) spectroscopy. Results IDPs inside HC are largely unstructured, but display small, protein-specific conformational changes. A minor increase in helical content is observed for α-casein and α-synuclein, reminiscent of membrane effects on α-synuclein. Frozen in their largely disordered conformation, bound proteins do not undergo folding induced by dehydration, as they do in their free forms. While HC thickness approaches the hydrodynamic diameter of the protein in solution for lysozyme, it is much below the respective values for IDPs. NPs boost α-synuclein aggregation kinetics in a dose-dependent manner. Conclusions IDPs maintain structural disorder inside HC, experiencing minor, protein-specific, induced folding and stabilization against further conformational transitions, such as formation of intermolecular beta-sheets upon dehydration. The HC is formed by a single layer of protein molecules. SC likely plays a key role stabilizing amyloidogenic α-synuclein conformers. General significance Protein-NP interactions can mimic those with macromolecular partners, allowing dissection of contributing factors by rational design of NP surfaces. Application of NPs in vivo should be carefully tested for amyloidogenic potential.

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.

One of the goals of the pharmaceutical sciences is the amelioration of targeted drug delivery. In this context, nanocarrier-dependent transportation represents an ideal method for confronting a broad range of human disorders. In this study, we investigated the possibility of improving the selective release of the anti-cancer drug paclitaxel (PTX) in the gastro-intestinal tract by encapsulating it into the biodegradable nanoparticles made by FDA-approved poly(lactic-co-glycolic acid) (PLGA) and coated with polyethylene glycol to improve their stability (PLGA-PEG-NPs). Our study was performed by combining the synthesis and characterization of the nanodrug with in vivo studies of pharmacokinetics after oral administration in mice. Moreover, fluorescent PLGA-nanoparticles (NPs), were tested both in vitro and in vivo to observe their fate and biodistribution. Our study demonstrated that PLGA-NPs: (1) are stable in the gastric tract, (2) can easily penetrate inside carcinoma colon 2 (CaCo2) cells, (3) reduce the PTX absorption from the gastrointestinal tract, further limiting systemic exposure, (4) enable PTX local targeting. At present, the oral administration of biodegradable nanocarriers is limited because of stomach degradation and the sink effect played by the duodenum. Our findings, however, exhibit promising evidence towards our overcoming these limitations for a more specific and safer strategy against gastrointestinal disorders.

Active drug targeting and controlled release of hydrophilic macromolecular drugs represent crucial points in designing efficient polymeric drug delivery nanoplatforms. In the present work EGFR-targeted polylactide-co-glycolide (PLGA) nanoparticles were made by a blend of two different PLGA-based polymers. The first, GE11-PLGA, in which PLGA was functionalized with GE11, a small peptide and EGFR allosteric ligand, able to give nanoparticles selective targeting features. The second polymer was a PEGylated PLGA (PEG-PLGA) aimed at improving nanoparticles hydrophilicity and stealth features. GE11 and GE11-PLGA were custom synthetized through a simple and inexpensive method. The nanoprecipitation technique was exploited for the preparation of polymeric nanoparticles composed by a 1:1 weight ratio between GE11-PLGA and PEG-PLGA, obtaining smart nanoplatforms with proper size for parenteral administration (143.9 ± 5.0 nm). In vitro cellular uptake in EGFR-overexpressing cell line (A549) demonstrated an active internalization of GE11-functionalized nanoparticles. GE11-PLGA/PEG-PLGA blend nanoparticles were loaded with Myoglobin, a model hydrophilic macromolecule, reaching a good loading (2.42% respect to the theoretical 4.00% w/w) and a prolonged release over 60 days. GE11-PLGA/PEG-PLGA blend nanoparticles showed good in vitro stability for 30 days in physiological saline solution at 4 °C and for 24 h in pH 7.4 or pH 5.0 buffer at 37 °C respectively, giving indications about potential storage and administration conditions. Furthermore ex vivo stability study in human plasma using fluorescence Single Particle Tracking (fSPT) assessed good GE11-PLGA/PEG-PLGA nanoparticles dimensional stability after 1 and 4 h. Thanks to the versatility in polymeric composition and relative tunable nanoparticles features in terms of drug incorporation and release, GE11-PLGA/PEG-PLGA blend NPs can be considered highly promising as smart nanoparticulate platforms for the treatment of diseases characterized by EGFR overexpression by parenteral administration.

Background: Smart nanoparticulate materials, namely tailored nanoparticles (NPs) with specific surface functionality, have recently attracted attention as useful tool for time- and site-specific drug delivery. Specifically, polymeric nanoparticles (NPs) can be chemically functionalized with different chemical entities, i.e. peptides, that selectively recognize biological substrates in vivo and target drug release. Divergent and very complex strategies can be pursued in order to obtain peptidedecorated NPs. Methods: A simple method was suggested for direct functionalization of poly-lactide-co-glicolide (PLGA) with small peptides. A solid-phase peptide synthesis was used to obtain a dodecapeptide (GE11) and a smaller tetrapeptide (FQPV). FQPV- and GE11-PLGA conjugates were obtained by optimized carbodiimide chemistry. Nanoprecipitation and solvent extraction/evaporation methods were purpose-built in order to prepare FQPV-PLGA NPs. NPs were characterized in terms of size, surface charge and adsorbed peptide amount; ex-vivo cytotoxicity studies were performed on FQPV-PLA NPs using adult fibroblasts. Results: Custom GE11, recently known as efficient Epidermal Growth Factor Receptor targeting agent, and FQPV, used as model peptide, were synthesized by solid-phase peptide synthesis achieving good purity (95%) and satisfactory process yields (70-85%). Then, FQPV- and GE11-PLGA conjugates were obtained by optimized carbodiimide chemistry achieving an high degree of functionalization (> 85%). Aware of different physico-chemical properties of peptide-PLGA conjugates with respect to plain PLGA, two different NPs preparation techniques, nanoprecipitation and solvent extraction/ evaporation methods, were purpose-built in order to prepare FQPV-PLGA NPs. Both methods revealed suitable to obtain NPs with proper dimensions for the parenteral administration (< 250nm), narrow size distribution (P.I. about 0.1), good morphological features and negative charge (about –20mV). A peptide adsorption protocol onto NPs was considered as additional strategy to increase peptide expression on NPs surface aimed at improving the targeting effectiveness. A Design of Experiment approach (DoE) has been successfully applied to the more versatile solvent extraction/ evaporation method in order to systematically highlight the influence of process parameters (organic solvent mixture, PVA concentration and polymeric solution volume) on NPs sizes. Conclusion: PLGA was successfully functionalized with two different peptides, FQPV and GE11, following the a versatile and simple carbodiimmide chemistry without further modifying polymer and/or peptide structure. Both nanoprecipitation and solvent extraction/evaporation NPs preparation methods were properly optimized in order to obtain peptide-PLGA based NPs and they can be alternatively selected according to solubility properties of both peptide-polymer conjugate and drug intended for encapsulation. Peptide adsorption on preformed PLGA NPs could be efficiently used to increase peptide expression on NPs surface thus improving cellular recognition in case of active targeting. Preliminary cytocompatibility evaluation of the selected peptide-PLGA based nanoparticulate materials shows a potential feasibility of the set-up synthetic procedures and NPs preparation methods for pharmaceutical purposes.