Monitoring the Fate of Orally Administered PLGA Nanoformulation for Local Delivery of Therapeutic Drugs.

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.

Intra-Articular Formulation of GE11-PLGA Conjugate-Based NPs for Dexamethasone Selective Targeting-In Vitro Evaluation.

Selectively targeted nanoscale drug delivery systems have recently emerged as promising intravenously therapeutic option for most chronic joint diseases. Here, a newly synthetized dodecapeptide (GE11)-polylactide-co-glycolide (PLGA)-based conjugate was used to prepare smart nanoparticles (NPs) intended for intra-articular administration and for selectively targeting Epidermal Growth Factor Receptor (EGFR). GE11-PLGA conjugate-based NPs are specifically uptaken by EGFR-overexpressed fibroblast; such as synoviocytes; which are the primarily cellular component involved in the development of destructive joint inflammation. The selective uptake could help to tune drug effectiveness in joints and to decrease local and systemic side effects. Dexamethasone (DXM) is a glucorticoid drug commonly used in joint disease treatment for both systemic and local administration route. In the present research; DXM was efficiently loaded into GE11-PLGA conjugate-based NPs through an eco-friendly nanoprecipitation method set up for this purpose. DXM loaded GE11-PLGA conjugate-based NPs revealed satisfactory ex vivo cytocompatibility; with proper size (≤150 nm) and good dimensional stability in synovial fluid. Intra-articular formulation was developed embedding DXM loaded GE11-PLGA conjugate-based NPs into thermosetting chitosan-based hydrogel; forming a biocompatible composite hydrogel able to quickly turn from liquid state into gel state at physiological temperature; within 15 min. Moreover; the use of thermosetting chitosan-based hydrogel extends the local release of active agent; DXM.

Conformational properties of intrinsically disordered proteins bound to the surface of silica nanoparticles.

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.

Investigation of antitumor activities of trastuzumab delivered by PLGA nanoparticles.

BACKGROUND: We report the development of an efficient antibody delivery system for the incorporation of trastuzumab (TZ) into poly(lactic-co-glycolic) acid nanoparticles (PLGA NPs). The aim of the work was to overcome the current limitations in the clinical use of therapeutic antibodies, including immunogenicity, poor pharmacokinetics, low tumor penetration and safety issues. MATERIALS AND METHODS: Trastuzumab-loaded PLGA NPs (PLGA-TZ) were synthesized according to a double emulsion method. The same protocol was used to produce control batches of nonspecific IgG-loaded NPs and empty PLGA NPs. After release of TZ from PLGA NPs, the effects on the main biological activities of the antibody were evaluated on SKBR3 (human epidermal growth factor receptor 2 [HER2]-positive breast cancer cell line), including specific binding to HER2, phosphorylation of HER2 (Y1248), degradation of HER2 protein and antibody-dependent cell-mediated cytotoxicity (ADCC) mechanism. In addition, an MTT assay was performed for treating SKBR3 cells with PLGA NPs loaded with TZ and doxorubicin to evaluate the cytotoxic activity of the combined treatment. RESULTS AND DISCUSSION: TZ was gradually released in a prolonged way over 30 days. The physical characterization performed with circular dichroism, Fourier transform infrared and fluorescence spectroscopy of TZ after release demonstrated that no structural alterations occurred compared to the native antibody. In vitro experiments using SKBR3 cells showed that TZ released from PLGA NPs maintained the same biological activity of native TZ. PLGA NPs allowed a good co-encapsulation efficiency of TZ and doxorubicin resulting in improved therapy. CONCLUSION: With the TZ case study, we demonstrate that the distinctive features of therapeutic monoclonal antibodies, including molecular targeting efficiency, capability to inhibit or properly affect the regulatory signaling pathways of cancer cells and stimulation of the ADCC, are fully preserved after loading into and release from PLGA NPs. In addition, PLGA NPs are shown to allow for the simultaneous incorporation of TZ and conventional chemotherapeutics, resulting in a potent antitumor nanodrug well suited for in situ combination and neoadjuvant therapy.

Impact of the strategy adopted for drug loading in nonporous silica nanoparticles on the drug release and cytotoxic activity.

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.

GE11 Peptide as an Active Targeting Agent in Antitumor Therapy: A Minireview.

A lot of solid tumors are characterized by uncontrolled signal transduction triggered by receptors related to cellular growth. The targeting of these cell receptors with antitumor drugs is essential to improve chemotherapy efficacy. This can be achieved by conjugation of an active targeting agent to the polymer portion of a colloidal drug delivery system loaded with an antitumor drug. The goal of this minireview is to report and discuss some recent results in epidermal growth factor receptor targeting by the GE11 peptide combined with colloidal drug delivery systems as smart carriers for antitumor drugs. The minireview chapters will focus on explaining and discussing: (i) Epidermal growth factor receptor (EGFR) structures and functions; (ii) GE11 structure and biologic activity; (iii) examples of GE11 conjugation and GE11-conjugated drug delivery systems. The rationale is to contribute in gathering information on the topic of active targeting to tumors. A case study is introduced, involving research on tumor cell targeting by the GE11 peptide combined with polymer nanoparticles.

Development of 99mTc-radiolabeled nanosilica for targeted detection of HER2-positive breast cancer.

The human epidermal growth factor receptor 2 (HER2) is normally associated with a highly aggressive and infiltrating phenotype in breast cancer lesions with propensity to spread into metastases. In clinic, the detection of HER2 in primary tumors and in their metastases is currently based on invasive methods. Recently, nuclear molecular imaging techniques, including positron emission tomography and single photon emission computed tomography (SPECT), allowed the detection of HER2 lesions in vivo. We have developed a 99mTc-radiolabeled nanosilica system, functionalized with a trastuzumab half-chain, able to act as drug carrier and SPECT radiotracer for the identification of HER2-positive breast cancer cells. To this aim, nanoparticles functionalized or not with trastuzumab half-chain, were radiolabeled using the 99mTc-tricarbonyl approach and evaluated in HER2 positive and negative breast cancer models. Cell uptake experiments, combined with flow cytometry and fluorescence imaging, suggested that active targeting provides higher efficiency and selectivity in tumor detection compared to passive diffusion, indicating that our radiolabeling strategy did not affect the nanoconjugate binding efficiency. Ex vivo biodistribution of 99mTc-nanosilica in a SK-BR-3 (HER2+) tumor xenograft at 4 h postinjection was higher in targeted compared to nontargeted nanosilica, confirming the in vitro data. In addition, viability and toxicity tests provided evidence on nanoparticle safety in cell cultures. Our results encourage further assessment of silica 99mTc-nanoconjugates to validate a safe and versatile nanoreporter system for both diagnosis and treatment of aggressive breast cancer.

Design of smart GE11-PLGA/PEG-PLGA blend nanoparticulate platforms for parenteral administration of hydrophilic macromolecular drugs: synthesis, preparation and in vitro/ex vivo characterization.

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 1:1weight ratio between GE11-PLGA and PEG-PLGA, obtaining smart nanoplatforms with proper size for parenteral administration (143.9±5.0nm). 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 60days. GE11-PLGA/PEG-PLGA blend nanoparticles showed good in vitro stability for 30days in physiological saline solution at 4°C and for 24h 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 4h. 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 .

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.

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.

Adhesive microbeads for the targeting delivery of anticaries agents of vegetable origin.

The formulation of quinic acid, a food constituent demonstrating potential anticaries and antigingivitis properties, was investigated in an adhesive microparticulate delivery system with the goal of improving its effect by prolonging its residence time at the site of action. Alginate and chitosan were selected as mucoadhesive polymers. The microspheres were prepared by coacervation. Different types of alginates, polymers blends and crosslinking agent concentrations were considered and evaluated. The best results in terms of encapsulation efficiency, in vitro active agent release profile and in vitro adhesive properties, both to oral mucosa and to teeth surface, were obtained with a blend of Alginate Protanal LF200S: Alginate Protanal LF120LS 1:1.5 w/w, 0.1M CaCl(2), and chitosan coating, prepared by a one-step complex coacervation method. This microparticulate delivery system showed prolonged release of quinic acid, and could be used as an active component in chewing gums or mouthwashes for both caries and gingivitis prevention.