Silk nanoparticles: from inert supports to bioactive natural carriers for drug delivery.

Silk proteins have been studied and employed for the production of drug delivery (nano)systems. They show excellent biocompatibility, controllable biodegradability and non-immunogenicity and, if needed, their properties can be modulated by blending with other polymers. Silk fibroin (SF), which forms the inner core of silk, is a (bio)material officially recognized by the Food and Drug Administration for human applications. Conversely, the potential of silk sericin (SS), which forms the external shell of silk, could still be considered under evaluation. At the best of our knowledge, nanoparticles based on silk sericin "alone" cannot be produced, due to its physicochemical instability influenced by extreme pH, high water solubility and temperature; for these reasons, it almost always needs to be combined with other polymers for the development of drug delivery systems. In this review, we focused on silk proteins as bioactive natural carriers, since they show not only optimal features as inert excipients, but also remarkable intrinsic biological activities. SF has anti-inflammatory properties, while SS presents antioxidant, anti-tyrosine, anti-aging, anti-elastase and anti-bacterial features. Here, we give an overview on SF or SS silk-based nanosystems, with particular attention on the production techniques.

Design, synthesis and evaluation of biotin decorated inulin-based polymeric micelles as long-circulating nanocarriers for targeted drug delivery.

Here, long-circulating behaviors of Inulin-based nanomicelles are demonstrated for the first time in vivo. We show the synthesis and evaluation of biotin (BIO)-decorated polymeric INVITE micelles constituted of substances of natural origin, Inulin (INU) and Vitamin E (VITE), as long-circulating carriers for receptor-mediated targeted drug delivery. The resulting INVITE or INVITE-BIO micelles, nanometrically sized, did not reveal any cytotoxicity after 24h of incubation with Caco-2 cells. Moreover, in vitro studies on Caco-2 cells monolayers indicated that the transport of INVITE-BIO micelles was faster than surface unmodified INVITE micelles. In vivo optical imaging studies evidenced that, upon intravenous administration, INVITE-BIO micelles were quantitatively present in the body up to 48h. Instead, after oral administration, the micelles were not found in the systemic circulation but eliminated with the normal intestinal content. In conclusion, INVITE-BIO micelles may enhance drug accumulation in tumor-cells over-expressing the receptor for biotin through receptor mediated endocytosis.

A dry powder formulation from silk fibroin microspheres as a topical auto-gelling device.

With the aim of establishing the formulation of a new hydrophilic auto-gelling medical device for biomedical applications, fibroin-based microspheres were prepared. The proposed microspheres were produced by a cost-effective and industrially scalable technique, such as the spray-drying. Spray-dried silk fibroin microspheres were obtained and the effects of different hydrophilic polymer on the process yield, microsphere morphology and conformation transition of fibroin were evaluated. The final auto-gelling formulations were obtained by adding calcium gluconate (as a calcium source for alginate crosslinking) to the prepared microspheres and tested by an in vitro gelling test. This study showed that the combination of fibroin with sodium alginate and poloxamer produced the most promising auto-gelling formulation for specific biomedical applications, such as the treatment of pressure ulcers.

Inulin-D-α-tocopherol succinate (INVITE) nanomicelles as a platform for effective intravenous administration of curcumin.

The aim of this work was to evaluate the potential of INVITE-based nanomicelles, an amphiphilic polymer constituted by inulin (INU) and vitamin E (VITE), as a platform for improving the biopharmaceutical properties of hydrophobic drugs. For this purpose, curcumin was selected as a model and curcumin-INVITE nanomicelles were prepared. This drug delivery system was characterized both in vitro for what concerns the physicochemical properties, blood compatibility, and cellular uptake, and in vivo for the evaluation of the pharmacokinetic profile. It was found that these nanomicelles released curcumin in a controlled manner, and they were able to penetrate cellular membrane. Moreover, they showed an improved pharmacokinetic profile after intravenous administration. In conclusion, INVITE micelles might constitute promising nanocarriers for improving the biopharmaceutical performance of hydrophobic drugs.

Preliminary investigation on the design of biodegradable microparticles for ivermectin delivery: set up of formulation parameters.

The aim was to design sterile biodegradable microparticulate drug delivery systems based on poly(dl-lactide) (PLA) and poly(ε-caprolactone) (PCL) and containing ivermectin (IVM), an antiparasitic drug, for subcutaneous administration in dogs. The drug delivery system should: (i) ensure a full 12-month protection upon single dose administration; (ii) be safe with particular attention regarding IVM dosage and its release, in order to prevent over dosage side effects. This preliminary work involves: polymer selection, evaluation of the effects of γ-irradiation on the polymers and IVM, investigation and set up of suitable microparticle preparation process and parameters, IVM-loaded microparticles in vitro release evaluation. Results of gel permeation chromatography analysis on the irradiated polymers and IVM mixtures showed that combination of IVM with the antioxidant α-tocopherol (TCP) reduces the damage extent induced by irradiation treatment, independently on the polymer type. Solvent evaporation process was successfully used for the preparation of PLA microparticles and appropriately modified; it was recognized as suitable for the preparation of PCL microparticles. Good process yields were achieved ranging from 76.08% to 94.72%; encapsulation efficiency was between 85.76% and 91.25%, independently from the polymer used. The type of polymer and the consequent preparation process parameters affected microparticle size that was bigger for PCL microparticles (480-800 µm) and solvent residual that was >500 ppm for PLA microparticles. In vitro release test showed significantly faster IVM release rates from PCL microparticles, with respect to PLA microparticles, suggesting that a combination of the polymers could be used to obtain the suitable drug release rate.

Stability Evaluation of Ivermectin-Loaded Biodegradable Microspheres.

A stability study was performed on ivermectin (IVM)-loaded biodegradable microparticles intended for injection in dogs. The rational was to evaluate the performances upon irradiation of a drug, such as IVM, with a few criticalities with respect to its stability, and toxicity. The goal was to provide valuable information for pharmaceutical scientists and manufacturers working in the veterinary area. The microspheres based on poly(D,L-lactide) and poly-(ε-caprolactone) and loaded with IVM and with the addition of alpha-tocopherol (TCP) as antioxidant were prepared by the emulsion solvent evaporation method and sterilized by gamma irradiation. Microsphere characterization in term of size, shape, polymer, and IVM stability upon irradiation was performed. The results show that the type of polymer significantly affects microsphere characteristics and performances. Moreover, suitably stable formulations can be achieved only by TCP addition.

Carbon nanotubes-modified poly-high internal phase emulsions for pharmaceuticals pre-concentration and determination.

This paper deals with the preparation of new composites between polymerized/crosslinked high internal phase emulsions (polyHIPEs) and carbon nanotubes (CNTs), specifically designed for pharmaceutical analytical applications. While the composition of the polyHIPEs was maintained constant, the amount of CNTs was varied from 0.5% to 1% w/v. As proof-of-concept, the materials were tested for solid-phase extraction. Three drugs with different physical-chemical properties, namely 17ß-estradiol (E2), Naproxen (NPX), and Oxprenolol (OXP) were selected as probes to investigate the adsorption/elution conditions on/from the CNT/polyHIPE composites for future analytical applications. The sorption and desorption behavior of the three analytes was studied at different pH values. The experimental results are coherent with chemistry of the support and the physical-chemical characteristics of the considered analytes. The incorporation of CNTs into the polyHIPEs network strongly influences the sorption properties of these materials.

Mast Cell Diseases in Practice and Research: Issues and Perspectives Raised by Patients and Their Recommendations to the Scientific Community and Beyond.

BACKGROUND: Since 2010, patients and physicians have collaborated to understand unmet needs of patients with mast cell diseases, incorporating mastocytosis and mast cell activation disorders, which include mast cell activation syndromes. OBJECTIVE: This Open Innovation in Science project aims to expand understanding of the needs of patients affected by mast cell diseases, and encourage global communication among patient advocacy groups, physicians, researchers, industry, and government. A major aim is to support the scientific community's efforts to improve diagnosis, management, therapy, and patients' quality of life by addressing unmet needs. METHODS: In collaboration with mast cell disease specialists, 13 patient advocacy groups from 12 countries and regions developed lists of top patient needs. A core team of leaders from patient advocacy groups collected and analyzed the data and proposed possible actions to address patient needs. RESULTS: Findings identified similarities and differences among participating countries in unmet needs between patients with mastocytosis and those with mast cell activation syndromes. Issues emphasized struggles relating to the nature and rarity of mast cell diseases, their impact on quality of life, the diagnostic process, access to appropriate care, more effective treatment, and the need for research. CONCLUSIONS: Solutions vary across countries because situations differ, in particular regarding the existence of and access to centers of excellence and reference centers. Multifaceted mast cell activation syndrome barriers necessitate innovative approaches to improve access to appropriate care. The outcomes of this project should greatly support scientists and clinicians in their efforts to improve diagnosis, management, and treatment of patients with mastocytosis and mast cell activation disorders.

Monolithic Papain-Immobilized Enzyme Reactors for Automated Structural Characterization of Monoclonal Antibodies.

The characterization of monoclonal antibodies (mAbs) requires laborious and time-consuming sample preparation steps before the liquid chromatography-mass spectrometry (LC-MS) analysis. Middle-up approaches entailing the use of specific proteases (papain, IdeS, etc.) emerged as practical and informative methods for mAb characterization. This work reports the development of immobilized enzyme reactors (IMERs) based on papain able to support mAb analytical characterization. Two monolithic IMERs were prepared by the covalent immobilization of papain on different supports, both functionalized via epoxy groups: a Chromolith® WP 300 Epoxy silica column from Merck KGaA and a polymerized high internal phase emulsion (polyHIPE) material synthesized by our research group. The two bioreactors were included in an in-flow system and characterized in terms of immobilization yield, kinetics, activity, and stability using Nα-benzoyl-L-arginine ethyl ester (BAEE) as a standard substrate. Moreover, the two bioreactors were tested toward a standard mAb, namely, rituximab (RTX). An on-line platform for mAb sample preparation and analysis with minimal operator manipulation was developed with both IMERs, allowing to reduce enzyme consumption and to improve repeatability compared to in-batch reactions. The site-specificity of papain was maintained after its immobilization on silica and polyHIPE monolithic supports, and the two IMERs were successfully applied to RTX digestion for its structural characterization by LC-MS. The main pros and cons of the two supports for the present application were described.

Biomaterials for Soft Tissue Repair and Regeneration: A Focus on Italian Research in the Field.

Tissue repair and regeneration is an interdisciplinary field focusing on developing bioactive substitutes aimed at restoring pristine functions of damaged, diseased tissues. Biomaterials, intended as those materials compatible with living tissues after in vivo administration, play a pivotal role in this area and they have been successfully studied and developed for several years. Namely, the researches focus on improving bio-inert biomaterials that well integrate in living tissues with no or minimal tissue response, or bioactive materials that influence biological response, stimulating new tissue re-growth. This review aims to gather and introduce, in the context of Italian scientific community, cutting-edge advancements in biomaterial science applied to tissue repair and regeneration. After introducing tissue repair and regeneration, the review focuses on biodegradable and biocompatible biomaterials such as collagen, polysaccharides, silk proteins, polyesters and their derivatives, characterized by the most promising outputs in biomedical science. Attention is pointed out also to those biomaterials exerting peculiar activities, e.g., antibacterial. The regulatory frame applied to pre-clinical and early clinical studies is also outlined by distinguishing between Advanced Therapy Medicinal Products and Medical Devices.

Supramolecular Gelatin Networks Based on Inclusion Complexes.

Hydrogel forming physical networks based on gelatin are an attractive approach toward multifunctional biomaterials with the option of reshaping, self-healing, and stimuli-sensitivity. However, it is challenging to design such gelatin-based hydrogels to be stable at body temperature. Here, gelatin functionalized with desaminotyrosine (DAT) or desaminotyrosyl tyrosine (DATT) side chains is crosslinked with cyclodextrin (CD) dimers under formation of inclusions complexes. The supramolecular networks displayed at room temperature decreased water uptake (200-600 wt% for DAT-based systems, 200 wt% for DATT based systems), and increased storage moduli up to 25.6 kPa determined by rheology compared to DAT(T) gelatin. The gel-sol transition temperature increased from 33 up to 42 °C. The presented system that is completely based on natural building blocks may form the basis for materials that may potentially respond by dissolution or changes of properties to changes in environmental conditions or to the presence of CD guest molecules.

Inverse Poly-High Internal Phase Emulsions Poly(HIPEs) Materials from Natural and Biocompatible Polysaccharides.

This paper shows one of the few examples in the literature on the feasibility of novel materials from natural and biocompatible polymers like inulin (INU) or glycol chitosan (GCS) templated by the formation of o/w (inverse) high internal phase emulsion (HIPE). To the best of our knowledge, this is the first example of inverse polyHIPEs obtained from glycol chitosan or inulin. The obtained polyHIPEs were specifically designed for possible wound dressing applications. The HIPE (pre-crosslinking emulsion) was obtained as inverse HIPE, i.e., by forming a cream-like 80:20 v/v o/w emulsion by using the isopropyl myristate in its oil phase, which is obtained from natural sources like palm oil or coconut oil. The surfactant amount was critical in obtaining the inverse HIPE and the pluronic F127 was effective in stabilizing the emulsion comprising up to 80% v/v as internal phase. The obtained inverse HIPEs were crosslinked by UV irradiation for methacrylated INU or by glutaraldehyde-crosslinking for GCS. In both cases, inverse poly-HIPEs were obtained, which were physicochemically characterized. This paper introduces a new concept in using hydrophilic, natural polymers for the formation of inverse poly-HIPEs.

Synthesis and characterization of novel chitosan-dopamine or chitosan-tyrosine conjugates for potential nose-to-brain delivery.

This work aims to the synthesis of novel carboxylated chitosan-dopamine (DA) and -tyrosine (Tyr) conjugates as systems for improving the brain delivery of the neurotransmitter DA following nasal administration. For this purpose, ester or amide conjugates were synthesized by N,N-dicyclohexylcarbodiimide (DCC) mediated coupling reactions between the appropriate N-tert-butyloxycarbonyl (Boc) protected starting polymers N,O-carboxymethyl chitosan and 6-carboxy chitosan and DA or O-tert-Butyl-L-tyrosine-tert-butyl ester hydrochloride. The resulting conjugates were characterized by FT-IR and 1H- and 13C NMR spectroscopies and their in vitro mucoadhesive properties in simulated nasal fluid (SNF), toxicity and uptake from Olfactory Ensheathing Cells (OECs) were assessed. Results demonstrated that N,O-carboxymethyl chitosan-DA conjugate was the most mucoadhesive polymer in the series examined and, together with the 6-carboxy chitosan-DA-conjugate were able to release the neurotransmitter in SNF. The MTT assay showed that the starting polymers as well as all the prepared conjugates in OECs resulted not toxic at any concentration tested. Likewise, the three synthesized conjugates were not cytotoxic as well. Cytofluorimetric analysis revealed that the N,O-carboxymethyl chitosan DA conjugate was internalized by OECs in a superior manner at 24 h as compared with the starting polymer. Overall, the N,O-CMCS-DA conjugate seems promising for improving the delivery of DA by nose-to-brain administration.

Combination of inulin and ß-cyclodextrin properties for colon delivery of hydrophobic drugs.

Colon drug delivery is aimed at the administration of selected drugs to act locally or even systematically. Corticosteroid drugs are often used exerting even pronounced side effects due to systemic absorption. Here a new drug delivery system (DDS) based on the chemical conjugation of ß-cyclodextrin to inulin to form the INUCD bioconjugate is described. It was designed with the aim to provide this DDS with colon degradable portions (inulin) which degradation products have direct beneficial effects on the well-being of the colon and with a carrier that can solubilize hydrophobic drugs (ß-cyclodextrin). This system was specifically designed to promote a local/topical activity with a significant reduction of the drug systemic absorption. The INUCD bioconjugate was obtained by a simple chemistry binding ß-cyclodextrin to an inulin succinate previously synthesized. The bioconjugate was then characterized in terms of physicochemical properties by ATR-FTIR, 1H NMR, DSC and TGA, DLS and SEM. Furthermore phase-solubility test by using curcumin as a model drug were performed as well as biologic evaluations for cytocompatibility and drug transport across in vitro simulated physiological barriers. Moreover enzymatic degradation studies by inulinase were performed. From the gained results a predictable local drug release of the payload could be attained so allowing a local delivery of e.g. corticosteroids thus avoiding a systemic absorption especially in prolonged therapies.

Inulin as a multifaceted (active) substance and its chemical functionalization: From plant extraction to applications in pharmacy, cosmetics and food.

This review is aimed at critically discussing a collection of research papers on Inulin (INU) in different scientific fields. The first part of this work gives an overview on the main characteristics of native INU, including production, applications in food or cosmetics industries, its benefits on human health as well as its main nutraceutical properties. A particular focus is dedicated to the extraction techniques and to the specific effects of INU on intestinal microbiota. Other than in food industry, the number of INU applications increases dramatically in the pharmaceutical field especially due to its simple chemical functionalization. Thus, aim of this review is also to give practical examples of chemical functionalization performed on INU also by including critical comments based on the direct experience of the Authors. With this aim, a full paragraph is dedicated to practical chemical experiences useful to reduce the efforts when establishing new experimental conditions. Moreover, the pharmaceutical technology is also taken in special consideration by underlining the aspects leading at the preparation of formulations based on INU. At the end of the review, a critical paragraph is intended to feed the scientists' curiosity on this versatile polysaccharide.

Drug delivery of rifampicin by natural micelles based on inulin: Physicochemical properties, antibacterial activity and human macrophages uptake.

This work aims at designing a drug delivery system for rifampicin (RIF) to be used for the therapy of infections from mycobacterium tuberculosis or other lung-colonizing bacteria. We are proposing, in particular, the delivery of RIF by micelles based on inulin functionalized with vitamin E (INVITE). We previously demonstrated that INVITE micelles are formed from the self-assembling sustained by the interaction, within the hydrophobic core, of aromatic groups belonging to vitamin E. It points on the effectiveness of these biocompatible systems in incorporating aromatic-group-bearing hydrophobic drug such as RIF. The succinilated derivative of INVITE, namely INVITESA, was further studied. Other than a full physicochemical characterization, the obtained micelles containing RIF were tested for their antibacterial activity against Gram- or Gram+bacteria including mycobacterium smegmatis. Furthermore, uptake studies on human alveolar macrophages and MTT studies were performed.

Polyacrylate/polyacrylate-PEG biomaterials obtained by high internal phase emulsions (HIPEs) with tailorable drug release and effective mechanical and biological properties.

The paper focuses on the preparation of polyacrylate based biomaterials designed as patches for dermal/transdermal drug delivery using materials obtained by the high internal phase emulsion (HIPE) technique. In particular, butyl acrylate and glycidyl methacrylate were selected, respectively, as backbone and functional monomer while two different crosslinkers, bifunctional or trifunctional, were used to form the covalent network. The influence of PEG on the main properties of the materials was also investigated. The obtained materials show a characteristic and interconnected internal structure as confirmed by SEM studies. By an industrial point of view, an interesting feature of this system is that it can be shaped as needed, in any form and thickness. The physiochemically characterized materials showed a tailorable curcumin (model of hydrophobic drugs) drug release, effective mechanical properties and cell viability and resulted neither pro nor anti-angiogenic as demonstrated in vivo by the chick embryo choriallantoic membrane (CAM) assay. Based on these results, the obtained polyHIPEs could be proposed as devices for dermal/transdermal drug delivery and/or for the direct application on wounded skin.

Silk fibroin nanoparticles for celecoxib and curcumin delivery: ROS-scavenging and anti-inflammatory activities in an in vitro model of osteoarthritis.

This paper aims at demonstrating silk fibroin nanoparticles (SFNs) promote anti-inflammatory properties of celecoxib (CXB) or curcumin (CUR), and could be exploited for osteoarthritis (OA) treatment. Nanoparticles were prepared by desolvation method and physico-chemically characterized (FT-IR, DSC, TGA, SEM, size distribution and drug release); empty and drug loaded nanoparticles were tested for their ROS-scavenging activity, hemolytic properties, cytotoxicity, and anti-inflammatory potency in an OA in vitro model. Results indicate that a controlled drug release has been achieved by varying the drug loading. Curcumin plus SFNs exhibited a synergistic antioxidant effect, while CXB was, in some manner, inhibitory. Both free drugs resulted highly cytotoxic while cell viability reached high values when encapsulated in SFNs. No appreciable differences in anti-inflammatory activity was evidenced between CUR loaded SFNs and CXB. In conclusion, SFNs is an optimal carrier to improve cyto- and hemo-compatibility of both CUR and CXB.

In vitro and ex vivo studies on diltiazem hydrochloride-loaded microsponges in rectal gels for chronic anal fissures treatment.

Diltiazem hydrochloride, topically applied at 2% concentration, is considered effective for the treatment of chronic anal fissures, although it involves several side effects among which anal pruritus and postural hypotension. To test the hypothesis that a sustained delivery system of diltiazem hydrochloride may be helpful for the treatment of chronic anal fissures, in the present study we evaluated the potential of gels containing diltiazem hydrochloride entrapped in microsponges. Such microsponges were based on Eudragit RS 100 and the effect of some formulation variables was assessed by a 23 full factorial screening design. An optimized formulation of diltiazem hydrochloride microsponges was dispersed in Methylcellulose 2% or Poloxamer 407 20% and the resulting gels (micro-l-diltiazem hydrochloride 2%) were subjected to in vitro drug release, ex vivo permeability and drug deposition after application on porcine rectal mucosa. The results showed a prolonged release up to 24 h from micro-l-diltiazem hydrochloride at 2% in the gels. The permeation tests revealed up to 18% higher drug retention on the mucosal tissue after 24 h by the micro-l-diltiazem hydrochloride 2% gels compared to conventional diltiazem hydrochloride gels at 2%. These results suggest that diltiazem hydrochloride-loaded microsponges dispersed in rectal gels may be useful to overcome some limitations of conventional local chronic anal fissure therapy.

Effect of Methyl-ß-Cyclodextrin on the antimicrobial activity of a new series of poorly water-soluble benzothiazoles.

The antibacterial activity of the S-unsubstituted- and S-benzyl-substituted-2-mercapto-benzothiazoles 1-4 has been evaluated after complexation with Methyl-ß-Cyclodextrin (Me-ß-CD) or incorporation in solid dispersions based on Pluronic® F-127 and compared with that of the pure compounds. This with the aim to gain further insights on the possible mechanism(s) involved in the CD-mediated enhancement of antimicrobial effectiveness, a promising methodology to overcome the microbial resistance issue. Together with Differential Scanning Calorimetry, FT-IR spectroscopy and X-ray Powder Diffraction investigations, a molecular modeling study focused on compounds 2 and 4 showed that the S-unsubstituted compound 2/Me-ß-CD complex should be more stable than S-benzyl-substituted 4/Me-ß-CD. Only for 1/Me-ß-CD or, particularly, 2/Me-ß-CD complexes, the antibacterial effectiveness was enhanced in the presence of selected bacterial strains. The results herein presented support the mechanisms focusing on the interactions of the bacterial membrane with CD complexes more than those focusing on the improvement of dissolution properties consequent to CD complexation.