Development of Biotinylated Liposomes Encapsulating Metformin for Therapeutic Targeting of Inflammation-Based Diseases.

Inflammation is a physiological response to a damaging stimulus but sometimes can be the cause of the onset of neurodegenerative diseases, atherosclerosis, and cancer. These pathologies are characterized by the overexpression of inflammatory markers like endothelial adhesion molecules, such as Vascular Cell Adhesion Molecule-1 (VCAM-1). In the present work, the development of liposomes for therapeutic targeted delivery to inflamed endothelia is described. The idea is to exploit a three-step pretargeting system based on the biotin-avidin high-affinity interaction: the first step involves a previously described biotin derivative bearing a VCAM-1 binding peptide; in the second step, the avidin derivative NeutrAvidinTM, which strongly binds to the biotin moiety, is injected; the final step is the administration of biotinylated liposomes that would bind to NeutravidinTM immobilized onto VCAM-1 overexpressing endothelium. Stealth biotinylated liposomes, prepared via the thin film hydration method followed by extrusion and purification via size exclusion chromatography, have been thoroughly characterized for their chemico-physical and morphological features and loaded with metformin hydrochloride, a potential anti-inflammatory agent. The three-step system, tested in vitro on different cell lines via confocal microscopy, FACS analysis and metformin uptake, has proved its suitability for therapeutic applications.

Development and validation of a GC-MS method for determination of metformin in normal brain and in glioblastoma tissues.

Metformin hydrochloride (MH) has recently been repurposed as an anticancer agent, showing antiproliferative activity in vitro and in vivo. In particular, experimental evidence has suggested its potential clinical efficacy in glioblastoma (GBM), a very aggressive tumor frequently characterized by gloomy prognosis. Unfortunately, the published literature concerning experimental applications of MH in glioblastoma animal models report no data on metformin levels reached in the brain, which, considering the high hydrophilicity of the drug, are likely very low. Therefore, new sensitive analytical methods to be applied on biological tissues are necessary to improve our knowledge of MH in vivo biodistribution and biological effects on tumors. In this research work, a GC-MS method for MH quantification in brain tissues is proposed. MH has been derivatized using N-methyl-bis(trifluoroacetamide), as already described in the literature, but the derivatization conditions have been optimized; moreover, deuterated MH has been selected as the best internal standard, after a comparative evaluation including other internal standards employed in published methods. After ascertaining method linearity, its accuracy, precision, specificity, repeatability, LOD and LOQ (0.373 µM and 1.242 µM, respectively, corresponding to 0.887 and 2.958 pmol/mg of wet tissue) have been evaluated on mouse brain tissue samples, obtained through a straightforward preparation procedure involving methanolic extraction from lyophilized brain homogenates and solid phase purification. The method has been validated on brain samples obtained from mice, either healthy or xenografted with GBM cells, receiving metformin dissolved in the drinking water. This analytical method can be usefully applied in preclinical studies aiming at clarifying MH mechanism of action in brain tumors.

Phytochemicals and Cancer Treatment: Cell-Derived and Biomimetic Vesicles as Promising Carriers.

The majority of anticancer agents currently used derive from natural sources: plants, frequently the ones employed in traditional medicines, are an abundant source of mono- and diterpenes, polyphenols, and alkaloids that exert antitumor activity through diverse mechanisms. Unfortunately, many of these molecules are affected by poor pharmacokinetics and limited specificity, shortcomings that may be overcome by incorporating them into nanovehicles. Cell-derived nanovesicles have recently risen to prominence, due to their biocompatibility, low immunogenicity and, above all, targeting properties. However, due to difficult scalability, the industrial production of biologically-derived vesicles and consequent application in clinics is difficult. As an efficient alternative, bioinspired vesicles deriving from the hybridization of cell-derived and artificial membranes have been conceived, revealing high flexibility and appropriate drug delivery ability. In this review, the most recent advances in the application of these vesicles to the targeted delivery of anticancer actives obtained from plants are presented, with specific focus on vehicle manufacture and characterization, and effectiveness evaluation performed through in vitro and in vivo assays. The emerging overall outlook appears promising in terms of efficient drug loading and selective targeting of tumor cells, suggesting further engrossing developments in the future.

Boron Vehiculating Nanosystems for Neutron Capture Therapy in Cancer Treatment.

Boron neutron capture therapy is a low-invasive cancer therapy based on the neutron fission process that occurs upon thermal neutron irradiation of 10B-containing compounds; this process causes the release of alpha particles that selectively damage cancer cells. Although several clinical studies involving mercaptoundecahydro-closo-dodecaborate and the boronophenylalanine-fructose complex are currently ongoing, the success of this promising anticancer therapy is hampered by the lack of appropriate drug delivery systems to selectively carry therapeutic concentrations of boron atoms to cancer tissues, allowing prolonged boron retention therein and avoiding the damage of healthy tissues. To achieve these goals, numerous research groups have explored the possibility to formulate nanoparticulate systems for boron delivery. In this review. we report the newest developments on boron vehiculating drug delivery systems based on nanoparticles, distinguished on the basis of the type of carrier used, with a specific focus on the formulation aspects.

Towards a better understanding of thermally treated polycarbophil matrix tablets for controlled release.

Polycarbophil (POL), a polyacrylic acid cross-linked with divinyl glycol, is widely used in semisolid and solid dosage forms. When undergoing a thermal treatment in the range 120-160 °C, POL shows interesting morphological modifications, related to changes in physical properties, such as swelling of the powder granules, or hardening and matrix formation if included in the composition of a tablet. Thermal analysis conducted on POL highlighted a thermal event (Z) that can be correlated both to the shrinking of the powder granules and to the matrix formation in compacted POL powder. Modulated differential scanning calorimetry (MDSC) allowed to distinguish, inside event Z, an irreversible process overlapping with a reversible glass transition, attributable to the volatilization of residual solvents identified, through a complex TGA-FTIR-GC-MS interface, as acetate esters used for the polymer production as very fine powder. A specific interaction between acetates and POL, capable of stabilizing the polymer chains in a given conformation, was highlighted. The molecular rearrangement of the POL chains, following the volatilization of the solvent-stabilizers, is therefore ascribable to a loss of energetic stability of this material, which justifies the shrinking phenomena in the granules of the powder and the matrix formation when POL is compacted.

Increased Water-Solubility and Maintained Antioxidant Power of Resveratrol by Its Encapsulation in Vitamin E TPGS Micelles: A Potential Nutritional Supplement for Chronic Liver Disease.

Children affected by chronic liver disease exhibit impaired neurocognitive development and growth due to the low absorption and digestion of nutrients. Furthermore, malnutrition is an adverse prognostic factor in liver transplantation as it is associated with an increase in morbidity and mortality. D-α-tocopheryl-polyethylene-glycol-succinate (TPGS) is currently administered per os as a vitamin E source to improve children's survival and well-being; however, TPGS alone does not reverse spinocerebellar degeneration and lipid peroxidation. To potentiate the effects of TPGS, we loaded micelles with resveratrol (RES), a natural polyphenol, with antioxidant and antiinflammatory activities, which has demonstrated protective action in the liver. Firstly, we investigated the suitability of TPGS to encapsulate RES in micelles by means of a phase-solubility study, then RES-TPGS formulations were prepared via solvent casting and solvent diffusion evaporation methods. RES-TPGS colloidal dispersions showed small mean diameters (12 nm), low polydispersity, and quite neutral Zeta potentials. The formulations showed a sustained drug release and a good drug loading capacity, further confirmed by infrared spectroscopy and differential scanning calorimetry. RES-TPGSs exhibited unaltered antioxidant activity compared to pristine RES via the DPPH assay and a significant reduction in toxicity compared to empty TPGS on HaCaT cells. Thus, RES-TPGS micelles may overcome the challenges of current liver disease therapy by providing more protective effects thanks to the antioxidant activity of RES and by reducing the surfactant toxicity on normal cells.

The Pharmaceutical Technology Approach on Imaging Innovations from Italian Research.

Many modern therapeutic approaches are based on precise diagnostic evidence, where imaging procedures play an essential role. To date, in the diagnostic field, a plethora of agents have been investigated to increase the selectivity and sensitivity of diagnosis. However, the most common drawbacks of conventional imaging agents reside in their non-specificity, short imaging time, instability, and toxicity. Moreover, routinely used diagnostic agents have low molecular weights and consequently a rapid clearance and renal excretion, and this represents a limitation if long-lasting imaging analyses are to be conducted. Thus, the development of new agents for in vivo diagnostics requires not only a deep knowledge of the physical principles of the imaging techniques and of the physiopathological aspects of the disease but also of the relative pharmaceutical and biopharmaceutical requirements. In this scenario, skills in pharmaceutical technology have become highly indispensable in order to respond to these needs. This review specifically aims to collect examples of newly developed diagnostic agents connoting the importance of an appropriate formulation study for the realization of effective products. Within the context of pharmaceutical technology research in Italy, several groups have developed and patented promising agents for fluorescence and radioactive imaging, the most relevant of which are described hereafter.

Two Novel PET Radiopharmaceuticals for Endothelial Vascular Cell Adhesion Molecule-1 (VCAM-1) Targeting.

Atherosclerosis is a chronic progressive disease involving inflammatory events, such as the overexpression of adhesion molecules including the endothelial Vascular Cell Adhesion Molecule-1 (VCAM-1). VCAM-1 is rapidly overexpressed in the first stages of atherosclerosis, thus representing a promising target for early atheroma detection. Two novel Positron Emission Tomography (PET) radiopharmaceuticals (MacroP and NAMP), based on the VCAM-1-binding peptide having sequence VHPKQHRGGSKGC, were synthesized and characterized. MacroP is derived from the direct conjugation of a DOTA derivative with the peptide, while NAMP is a biotin derivative conceived to be employed in a three-step pretargeting system, involving the use of a double-chelating derivative of DOTA. The identity of the newly synthesized radiopharmaceuticals was confirmed by mass spectrometry and, after radiolabeling with 68Ga, both showed high radiochemical purity; in vitro tests on human umbilical vein endothelial cells evidenced their VCAM-1 binding ability, with higher radioactive uptake in the case of NAMP. Moreover, NAMP might also be employed in a theranostic approach in association with functionalized biotinylated nanoparticles.

D-α-Tocopherol-Based Micelles for Successful Encapsulation of Retinoic Acid.

All-trans-retinoic acid (ATRA) represents the first-choice treatment for several skin diseases, including epithelial skin cancer and acne. However, ATRA's cutaneous side effects, like redness and peeling, and its high instability limit its efficacy. To address these drawbacks and to improve ATRA solubilization, we prepared ATRA-loaded micelles (ATRA-TPGSs), by its encapsulation in D-α-tocopheryl-polyethylene-glycol-succinate (TPGS). First, to explore the feasibility of the project, a solubility study based on the equilibrium method was performed; then, six ATRA-TPGS formulations were prepared by the solvent-casting method using different TPGS amounts. ATRA-TPGSs showed small sizes (11-20 nm), low polydispersity, slightly negative zeta potential, and proved good encapsulation efficiency, confirmed by a chemometric-assisted Fourier transform infrared spectroscopy (FTIR) investigation. ATRA-TPGS stability was also investigated to choose the most stable formulation. Using Carbopol® 980 as gelling agent, ATRA-TPGS-loaded gels were obtained and analyzed for their rheological profiles. Ex vivo release studies from ATRA-TPGSs were performed by Franz cells, demonstrating a permeation after 24 h of 22 ± 4 µ cm-2. ATRA-TPGSs showed enhanced cytotoxic effects on melanoma cells, suggesting that these formulations may represent a valid alternative to improve patient compliance and to achieve more efficacious therapeutic outcomes.

Anti-Vascular Cell Adhesion Molecule-1 Nanosystems: A Promising Strategy Against Inflammatory Based Diseases.

Inflammation underlays the onset and supports the development of several worldwide diffused pathologies, therefore in the last decades inflammatory markers have attracted a great deal of interest as diagnostic and therapeutic targets. Adhesion molecules are membrane proteins expressed by endotheliocytes and leukocytes, acting as mediators in the process of tethering, rolling, firm adhesion and diapedesis that leads the immune cells to reach an inflamed tissue. Among them, the adhesion molecule VCAM-1 has been investigated as a potential target because of its low constitutive expression and easy accessibility on the endothelium. Moreover, VCAM-1 is involved in the early stages of development of several pathologies like, among others, atherosclerosis, cancer, Alzheimer's and Parkinson's diseases, so a diagnostic or therapeutic tool directed to this protein would allow specific detection and efficacious intervention. The availability of monoclonal antibodies against VCAM-1 has recently fostered the development of various targeting technologies potentially suitable for imaging and drug delivery in VCAM-1 overexpressing pathologies. In this review we initially focus on the structure and functions of VCAM-1, giving also a brief overview of antibodies origin, structure and function; then, we summarize some of the VCAM-1 targeting nanosystems based on antibodies, gathered according to the carrier used, for diagnosis or therapeutic treatment of different inflammatory based pathologies.

Exosomes and Extracellular Vesicles as Emerging Theranostic Platforms in Cancer Research.

Exosomes are endosome-derived nanovesicles produced by healthy as well as diseased cells. Their proteic, lipidic and nucleic acid composition is related to the cell of origin, and by vehiculating bioactive molecules they are involved in cell-to-cell signaling, both in healthy and pathologic conditions. Being nano-sized, non-toxic, biocompatible, scarcely immunogenic, and possessing targeting ability and organotropism, exosomes have been proposed as nanocarriers for their potential application in diagnosis and therapy. Among the different techniques exploited for exosome isolation, the sequential ultracentrifugation/ultrafiltration method seems to be the gold standard; alternatively, commercially available kits for exosome selective precipitation from cell culture media are frequently employed. To load a drug or a detectable agent into exosomes, endogenous or exogenous loading approaches have been developed, while surface engineering procedures, such as click chemistry, hydrophobic insertion and exosome display technology, allow for obtaining actively targeted exosomes. This review reports on diagnostic or theranostic platforms based on exosomes or exosome-mimetic vesicles, highlighting the diverse preparation, loading and surface modification methods applied, and the results achieved so far.

Neuroinflammation in Aged Brain: Impact of the Oral Administration of Ellagic Acid Microdispersion.

The immune system and the central nervous system message each other to preserving central homeostasis. Both systems undergo changes during aging that determine central age-related defects. Ellagic acid (EA) is a natural product which is beneficial in both peripheral and central diseases, including aging. We analyzed the impact of the oral administration of a new oral ellagic acid micro-dispersion (EAm), that largely increased the EA solubility, in young and old mice. Oral EAm did not modify animal weight and behavioral skills in young and old mice, but significantly recovered changes in "ex-vivo, in vitro" parameters in old animals. Cortical noradrenaline exocytosis decreased in aged mice. EAm administration did not modify noradrenaline overflow in young animals, but recovered it in old mice. Furthermore, GFAP staining was increased in the cortex of aged mice, while IBA-1 and CD45 immunopositivities were unchanged when compared to young ones. EAm treatment significantly reduced CD45 signal in both young and old cortical lysates; it diminished GFAP immunopositivity in young mice, but failed to affect IBA-1 expression in both young and old animals. Finally, EAm treatment significantly reduced IL1beta expression in old mice. These results suggest that EAm is beneficial to aging and represents a nutraceutical ingredient for elders.

Author Correction: A new microdispersed albumin derivative potentially useful for radio-guided surgery of occult breast cancer lesions.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A new microdispersed albumin derivative potentially useful for radio-guided surgery of occult breast cancer lesions.

This paper describes a new nuclear imaging agent, 2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid of human albumin (HAC), potentially suitable for application in the Radio-guided Occult Lesion Localization (ROLL) of non-palpable mammalian cancerous lesions, as a tool to overtake the short radio-signal half-life of the technetium-99m based radiopharmaceutical currently used. This conjugate is a microsized powder aggregate, water-insoluble between pH 3 and 8.5, obtained by conjugating the protein with the macrocyclic chelating agent DOTA through a one-pot reaction in aqueous medium. The product has been fully characterized and is stable to the thermal conditions adopted for labeling; after radiolabeling with longer half-life radionuclides such as 177Lu or 111In, it has shown radiochemical purity (RCP) >90% and resulted stable when stored in saline or plasma for 6 days at 37 °C. A µPET/CT study, performed in vivo on adult female rats, showed that the radioactivity of HAC labeled with 64Cu remained located in the mammary glands for at least 40 h, without diffusion or drainage in healthy tissues or in the lymphatic circulation. This new imaging agent might make the ROLL procedure more accessible, safe and flexible, promoting a significant time and cost reduction of this intervention. Moreover, HAC might also be used in other radio-guided surgical procedures in oncology.

Development of an Injectable Slow-Release Metformin Formulation and Evaluation of Its Potential Antitumor Effects.

Metformin is an antidiabetic drug which possesses antiproliferative activity in cancer cells when administered at high doses, due to its unfavorable pharmacokinetics. The aim of this work was to develop a pharmacological tool for the release of metformin in proximity of the tumor, allowing high local concentrations, and to demonstrate the in vivo antitumor efficacy after a prolonged metformin exposition. A 1.2% w/w metformin thermoresponsive parenteral formulation based on poloxamers P407 and P124, injectable at room temperature and undergoing a sol-gel transition at body temperature, has been developed and optimized for rheological, thermal and release control properties; the formulation is easily scalable, and proved to be stable during a 1-month storage at 5 °C. Using NOD/SCID mice pseudo-orthotopically grafted with MDA-MB-231/luc+ human breast cancer cells, we report that multiple administrations of 100 mg of the optimized metformin formulation close to the tumor site cause tissue accumulation of the drug at levels significantly higher than those observed in plasma, and enough to exert antiproliferative and pro-apoptotic activities. Our results demonstrate that this formulation is endowed with good stability, tolerability, thermal and rheological properties, representing a novel tool to be pursued in further investigations for adjuvant cancer treatment.

Development and characterization of a mucoadhesive sublingual formulation for pain control: extemporaneous oxycodone films in personalized therapy.

OBJECTIVE: The aim of this work was the development of mucoadhesive sublingual films, prepared using a casting method, for the administration of oxycodone. MATERIALS AND METHODS: A solvent casting method was employed to prepare the mucoadhesive films. A calibrated pipette was used to deposit single aliquots of different polymeric solutions on a polystyrene plate lid. Among the various tested polymers, hydroxypropylcellulose at low and medium molecular weight (HPC) and pectin at two different degrees of esterification (PC) were chosen for preparing solutions with good casting properties, capable of producing films suitable for mucosal application. RESULTS AND DISCUSSION: The obtained films showed excellent drug content uniformity and stability and rapid drug release, which, at 8 min, ranged from 60% to 80%. All films presented satisfactory mucoadhesive and mechanical properties, also confirmed by a test on healthy volunteers, who did not experience irritation or mucosa damages. Pectin films based on pectin at lower degrees of esterification have been further evaluated to study the influence of two different amounts of drug on the physicochemical properties of the formulation. A slight reduction in elasticity has been observed in films containing a higher drug dose; nevertheless, the formulation maintained satisfactory flexibility and resistance to elongation. CONCLUSIONS: HPC and PC sublingual films, obtained by a simple casting method, could be proposed to realize personalized hospital pharmacy preparations on a small scale.

An innovative matrix controlling drug delivery produced by thermal treatment of DC tablets containing polycarbophil and ethylcellulose.

An innovative matrix, produced by thermal treatment on direct compression (DC) tablets containing polycarbophil (POL) and ethylcellulose (EC), identified as matrix forming polymers, and able to control the release of diltiazem hydrochloride, was developed. At pH 7.2, 72 ± 1.2% (w/w) of drug loaded was released in 25 h, mostly at constant rate. This swellable and unerodible matrix controls drug release by an anomalous transport mechanism. The modifications induced by the thermal treatment are irreversible and can be used to control and characterize the matrix. A 3-component constrained mixture design allowed the investigation of the experimental domain in which the matrix forms and the computation of a mathematical model that can be used to optimize the formulation properties. The release rate can be modulated (0.032-0.064% drug released/min) through the choice of suitable treatment conditions and tablet composition. The maximum amount of diltiazem hydrochloride released by zero-order kinetics, at the lowest release rate, occurs for POL:EC ratio in the range of 1:1-2:3 with 20-30% of diluent. The tablets are able to load up to 50% (w/w) of diltiazem hydrochloride without losing their properties. A stability study performed on a selected formulation containing DTZ showed stability for at least 2.7 years at RT conditions.

A chitosan lactate/poloxamer 407-based matrix containing Eudragit RS microparticles for vaginal delivery of econazole: design and in vitro evaluation.

A matrix based on chitosan lactate and poloxamer 407 was evaluated as a delivery system for the vaginal administration of the antifungal drug econazole. The matrix was investigated both containing the pure drug and after introducing microparticles of Eudragit RS 100 containing econazole. Eudragit RS 100 microparticles were prepared using an emulsion-extraction method and dispersed in a solution containing chitosan lactate (2% w/w) and poloxamer 407 (1.7% w/w). The microparticles, obtained with a yield of 64% w/w and an encapsulation efficiency of 42% w/w, had a diameter of less than 2 µm and a drug loading of 13% w/w. The compressed matrices, characterized by DSC, swelling, erosion, release and mucoadhesion studies, had behaviours dependent on the relative amounts of the contained microparticles. The matrix without microparticles (MECN) showed zero-order release kinetics, with a maximum drug-release of 60% w/w, while those containing 50 or 75% w/w microparticles showed a diffusion controlled release up to 8 and 16 h, respectively, and a linear trend after those time intervals, caused by the erosion process, which allowed reaching a drug-release of approximately 100% w/w at 22 h. In in vitro experiments, the matrices were mucoadhesive and active in inhibiting the growth of Candida albicans 796.

Preparation, characterisation and preliminary antitumour activity evaluation of a novel nanoparticulate system based on a cisplatin-hyaluronate complex and N-trimethyl chitosan.

In this work, nanoparticles with a positive surface charge were prepared through the electrostatic interaction of a new cisplatin-hyaluronate complex with N-trimethyl chitosan (substitution degree of 85%). Mean particle diameter was approximately 195 nm. Drug loading of nanoparticles, which had a zeta potential of about 27 mV, was equal to 6% w/w. After 24 h, while the cisplatin-hyaluronate complex released approximately 60% w/w drug in phosphate buffered saline at pH 7.4, approximately 40% w/w of total cisplatin was released from nanoparticles. The same cumulative amounts of released drug were found after 48 h. These nanoparticles, as well as the starting cisplatin-hyaluronate complex, were active on all cell lines tested (P388, A2780, A549), with an antiproliferative activity similar to that of cisplatin. Apoptosis was markedly induced in A2780 cells by nanoparticles. In a preliminary in vivo experiment, the antitumour activity against a murine tumour (P388 cells) subcutaneously implanted in mice, resulted similar to that of cisplatin for nanoparticles whereas the starting complex showed a non-significant activity at the cisplatin dose tested. Body weight change of treated mice suggested a significantly better tolerance of the nanoparticles compared to cisplatin, after an initial brief period of acute toxicity higher than the parent drug. These results indicate that such a particulate system could be useful as a carrier for cisplatin delivery.

Study of retinoic acid polymorphism.

Some authors recently hypothesized the existence of a new retinoic acid (RA) phase in addition to the two already known polymorphs. We investigated RA polymorphism and our results exclude the presence of new modifications and refine the properties of the known forms. By comparison of simulated and acquired X-Ray Powder Diffraction (XRPD) it was possible to identify only the known monoclinic (I) and the triclinic (II) modifications; the same were also characterized by DSC, IR, and Raman spectroscopy. A solubility study associated to DSC allowed establishing an enantiotropic relationship between the two forms, with form II being less stable (DeltaGII/I=0.71 kJ/mol at 37 degrees C) below the transition temperature (136.6 degrees C; DeltaH=3.2 kJ/mol). The intrinsic dissolution rate (IDR) (I=61 microg/cm2xmin-1; II=125 microg/cm2xmin-1) confirmed this energetic relationship. The kinetics of solid transition I-->II was examined and its activation energy estimated (356 kJ/mol). The attempts to produce new phases allowed the development of methods to obtain the two polymorphs with high chemical and polymorphic purity. A validated DSC method is presented that enables detection of the presence of form I at a level of 1% (w/w) when in mixture with form II.