Nanocarriers in Veterinary Medicine: A Challenge for Improving Osteosarcoma Conventional Treatments.

In recent years, several nanocarrier-based drug delivery systems, such as polymeric nanoparticles, solid lipid nanoparticles, metallic nanoparticles, liposomes, and others, have been explored to target and treat a wide variety of diseases. Their employment has brought many benefits, not only to human medicine but also to veterinary medicine, albeit at a slower rate. Soon, the use of nanocarriers could revolutionize the animal health sector, and many veterinary therapies will be more effective as a result. The purpose of this review is to offer an overview of the main applications of nanocarriers in the veterinary field, from supplements for animal health and reproduction to nanovaccines and nanotherapies. Among the major pathologies that can affect animals, special attention is given to canine osteosarcoma (OSA): a comparison with human OSA is provided and the main treatment options are reviewed emphasizing the benefits that nanocarriers could bring in the treatment of this widespread disease.

Naked and Decorated Nanoparticles Containing H2S-Releasing Doxorubicin: Preparation, Characterization and Assessment of Their Antitumoral Efficiency on Various Resistant Tumor Cells.

Several semisynthetic, low-cardiotoxicity doxorubicin (DOXO) conjugated have been extensively described, considering the risk of cytotoxicity loss against resistant tumor cells, which mainly present drug efflux capacity. Doxorubicin 14-[4-(4-phenyl-5-thioxo-5H-[1,2]dithiol-3-yl)]-benzoate (H2S-DOXO) was synthetized and tested for its ability to overcome drug resistance with good intracellular accumulation. In this paper, we present a formulation study aimed to develop naked and decorated H2S-DOXO-loaded lipid nanoparticles (NPs). NPs prepared by the "cold dilution of microemulsion" method were decorated with hyaluronic acid (HA) to obtain active targeting and characterized for their physicochemical properties, drug entrapment efficiency, long-term stability, and in vitro drug release. Best formulations were tested in vitro on human-sensitive (MCF7) and human/mouse DOXO-resistant (MDA-MDB -231 and JC) breast cancer cells, on human (U-2OS) osteosarcoma cells and DOXO-resistant human/mouse osteosarcoma cells (U-2OS/DX580/K7M2). HA-decoration by HA-cetyltrimethyl ammonium bromide electrostatic interaction on NPs surface was confirmed by Zeta potential and elemental analysis at TEM. NPs had mean diameters lower than 300 nm, 70% H2S-DOXO entrapment efficiency, and were stable for almost 28 days. HA-decorated NPs accumulated H2S-DOXO in Pgp-expressing cells reducing cell viability. HA-decorated NPs result in the best formulation to increase the inter-cellular H2S-DOXO delivery and kill resistant cells, and therefore, as a future perspective, they will be taken into account for further in vivo experiments on tumor animal model.

Doxorubicin-Loaded Lipid Nanoparticles Coated with Calcium Phosphate as a Potential Tool in Human and Canine Osteosarcoma Therapy.

Osteosarcoma (OSA) is the most frequently diagnosed primary malignant bone tumor in humans and dogs. In both species, standard chemotherapy can be limited by multidrug resistance of neoplastic cells, which prevents intracellular accumulation of cytotoxic drugs, resulting in chemotherapy failure. In this study, a lipophilic ester of doxorubicin (C12DOXO) was loaded into nanoparticles (NPs) using the "cold microemulsion dilution" method. The resulting NPs were then coated with calcium phosphate (CaP) in two different ways to have calcium or phosphate ions externally exposed on the surface. These systems were characterized by determining mean diameter, zeta potential, and drug entrapment efficiency; afterward, they were tested on human and canine OSA cells to study the role that the coating might play in increasing both drug uptake into tumor cells and cytotoxicity. Mean diameter of the developed NPs was in the 200-300 nm range, zeta potential depended on the coating type, and C12DOXO entrapment efficiency was in the 60-75% range. Results of studies on human and canine OSA cells were very similar and showed an increase in drug uptake and cytotoxicity for CaP-coated NPs, especially when calcium ions were externally exposed. Therefore, applications in both human and veterinary medicine can be planned in the near future.

Calcium Phosphate-Coated Lipid Nanoparticles as a Potential Tool in Bone Diseases Therapy.

The treatment of bone diseases (including osteoporosis, osteoarthritis, and bone cancer) often results in reduced efficiency and/or adverse reactions due to the fact that it is not specifically targeted to the site of action. The employment of a suitable carrier should increase drug location to the site of bone disease. The purpose of this study is to prepare and characterize lipid nanoparticles (NPs) coated with calcium phosphate (CaP-NPs). A coating method, to date used only to obtain liposomes covered with CaP, is herein partially-modified to prepare CaP-coated lipid NPs. An extensive physico-chemical characterization was achieved by employing several techniques (DLS, SEM and TEM, and both combined with EDS, XRD, and FTIR) that confirmed the feasibility of the developed coating method. Preliminary uptake studies on human osteosarcoma cells (U-2OS) were performed by entrapping, as a lipid probe, Sudan Red III in NPs. The obtained data provided evidence that CaP-NPs showed higher cell accumulation than uncoated NPs. This result may have important implications for the development of drug loaded CaP-NPs to be tested in vitro with a view of planning future treatment of bone diseases, and indicate that CaP-NPs are potential vehicles for selective drug delivery to bone tissue.

Developing Actively Targeted Nanoparticles to Fight Cancer: Focus on Italian Research.

Active targeting is a valuable and promising approach with which to enhance the therapeutic efficacy of nanodelivery systems, and the development of tumor-targeted nanoparticles has therefore attracted much research attention. In this field, the research carried out in Italian Pharmaceutical Technology academic groups has been focused on the development of actively targeted nanosystems using a multidisciplinary approach. To highlight these efforts, this review reports a thorough description of the last 10 years of Italian research results on the development of actively targeted nanoparticles to direct drugs towards different receptors that are overexpressed on cancer cells or in the tumor microenvironment. In particular, the review discusses polymeric nanocarriers, liposomes, lipoplexes, niosomes, solid lipid nanoparticles, squalene nanoassemblies and nanobubbles. For each nanocarrier, the main ligands, conjugation strategies and target receptors are described. The literature indicates that polymeric nanoparticles and liposomes stand out as key tools for improving specific drug delivery to the site of action. In addition, solid lipid nanoparticles, squalene nanoparticles and nanobubbles have also been successfully proposed. Taken together, these strategies all offer many platforms for the design of nanocarriers that are suitable for future clinical translation.

Osteomyelitis: Focus on Conventional Treatments and Innovative Drug Delivery Systems.

Osteomyelitis is a bone marrow infection which generally involves cortical plates and which may occur after bone trauma, orthopedic/maxillofacial surgery or after vascular insufficiency episodes. It mostly affects people from the Third World Countries, the elderly and patients affected by systemic diseases e.g. autoimmune disorders, AIDS, osteoporosis and microvascular disease. The highest percentage of osteomyelitis cases (almost 75%) is caused by Staphylococcus spp., and in particular by Staphylococcus aureus (more than 50%). The ideal classification and the diagnosis of osteomyelitis are two important tools which help the physicians to choose the best therapeutic strategies. Currently, common therapies provide an extensive debridement in association with intravenous administration of antibiotics (penicillin or clindamycin, vancomycin and fluoroquinolones among all for resistant microorganisms), to avoid the formation of sequestra. However, conventional therapeutic approach involves several drawbacks like low concentration of antibiotics in the infected site, leading to resistance and adverse effects due to the intravenous administration. For these reasons, in the last years several studies have been focused on the development of drug delivery systems such as cements, beads, scaffolds and ceramics made of hydroxyapatite (HA), calcium phosphate (CaP) and ß-tricalcium phosphate (ß-TCP) which demonstrated to be biocompatible, poorly toxic and capable to allow osteointegration and a prolonged drug release. The aim of this review is to provide a focus on current therapies and latest developed drug delivery systems with particular attention on those based on CaP and its derivatives, hoping that this work could allow further direction in the field of osteomyelitis.

Bone Diseases: Current Approach and Future Perspectives in Drug Delivery Systems for Bone Targeted Therapeutics.

Bone diseases include a wide group of skeletal-related disorders that cause mobility limitations and mortality. In some cases, e.g., in osteosarcoma (OS) and metastatic bone cancer, current treatments are not fully effective, mainly due to low patient compliance and to adverse side effects. To overcome these drawbacks, nanotechnology is currently under study as a potential strategy allowing specific drug release kinetics and enhancing bone regeneration. Polymers, ceramics, semiconductors, metals, and self-assembled molecular complexes are some of the most used nanoscale materials, although in most cases their surface properties need to be tuned by chemical or physical reactions. Among all, scaffolds, nanoparticles (NPs), cements, and hydrogels exhibit more advantages than drawbacks when compared to other nanosystems and are therefore the object of several studies. The aim of this review is to provide information about the current therapies of different bone diseases focusing the attention on new discoveries in the field of targeted delivery systems. The authors hope that this paper could help to pursue further directions about bone targeted nanosystems and their application for bone diseases and bone regeneration.

Curcumin-Loaded Solid Lipid Nanoparticles Bypass P-Glycoprotein Mediated Doxorubicin Resistance in Triple Negative Breast Cancer Cells.

Multidrug resistance (MDR) is a critical hindrance to the success of cancer chemotherapy. The main thing responsible for MDR phenotypes are plasma-membranes associated with adenosine triphosphate (ATP) Binding Cassette (ABC) drug efflux transporters, such as the P-glycoprotein (Pgp) transporter that has the broadest spectrum of substrates. Curcumin (CURC) is a Pgp inhibitor, but it is poorly soluble and bioavailable. To overcome these limitations, we validated the efficacy and safety of CURC, loaded in biocompatible solid lipid nanoparticles (SLNs), with or without chitosan coating, with the goal of increasing the stability, homogeneous water dispersibility, and cellular uptake. Both CURC-loaded SLNs were 5-10-fold more effective than free CURC in increasing the intracellular retention and toxicity of doxorubicin in Pgp-expressing triple negative breast cancer (TNBC). The effect was due to the decrease of intracellular reactive oxygen species, consequent inhibition of the Akt/IKKα-ß/NF-kB axis, and reduced transcriptional activation of the Pgp promoter by p65/p50 NF-kB. CURC-loaded SLNs also effectively rescued the sensitivity to doxorubicin against drug-resistant TNBC tumors, without signs of systemic toxicity. These results suggest that the combination therapy, based on CURC-loaded SLNs and doxorubicin, is an effective and safe approach to overcome the Pgp-mediated chemoresistance in TNBC.

Thermosensitive Nanocomposite Hydrogels for Intravitreal Delivery of Cefuroxime.

Endophthalmitis is a rare, but serious, intravitreal inflammatory disorder that can arise after cataract surgery. The intracameral injection of 1 mg cefuroxime (CEF) followed by three-times daily antibiotic topical administration for a week is generally recognized as the routine method of prophylaxis after cataract surgery. This procedure is controversial because of both the low efficacy and the low adherence to therapy by elderly patients. A unique slow release antibiotic intravitreal injection could solve these problems. The objective of the present study was to design ophthalmic nanocomposite delivery systems based on in situ gelling formulations that undergo sol-to-gel transition upon change in temperature to prolong the effect of CEF. Oil in water (O/W) microemulsion (µE) and solid lipid nanoparticles (SLN), obtained with an innovative formulation technology called cold microemulsion dilution, were evaluated as ocular drug delivery systems for CEF. Drug entrapment efficiency up to 80% was possible by esterifying CEF with 1-dodecanol to obtain dodecyl-CEF (dCEF). Both dCEF-loaded SLN and µE were then added with Pluronic®F127 (20% w/v) to obtain a nanocomposite hydrogel-based long acting system. The prepared thermosensitive formulations were evaluated for their physical appearance, drug content, gelation temperature, injectability and rheological properties, in vitro release studies and stability studies. Moreover, cell proliferation assays on human retinal pigment epithelial ARPE-19 cells were performed to evaluate the influence of this innovative system on the cellular viability. In addition, minimal inhibitory concentration (MIC) was assessed for both CEF and dCEF, revealing the need of dCEF hydrolysis for the antimicrobial activity. Although further experimental investigations are required, the physico-chemical characterization of the nanocomposite hydrogels and the preliminary in vitro release studies highlighted the potential of these systems for the sustained release of CEF.

Development of Solid Lipid Nanoparticles by Cold Dilution of Microemulsions: Curcumin Loading, Preliminary In Vitro Studies, and Biodistribution.

Background: Solid lipid nanoparticles (SLNs) are attractive drug delivery systems for lipophilic molecules like curcumin (CURC) with low chemical stability. Methods: A simple, innovative, and cold-operating method, named "cold dilution of microemulsion" is developed by the authors to produce SLNs. An oil-in-water microemulsion (µE), whose disperse phase consisted of a solution of trilaurin in a partially water-miscible solvent, was prepared after mutually saturating solvent and water. Trilaurin SLNs precipitated following solvent removal upon water dilution of the µE. After SLN characterization (mean size, Zeta potential, CURC entrapment efficiency, and over time stability), they were tested for in vitro cytotoxicity studies on pancreatic adenocarcinoma cell lines and for in vivo preliminary biodistribution studies in Wistar healthy rats. Results: CURC loaded SLNs (SLN-CURC) had mean diameters around 200 nm, were negatively charged, stable over time, and able to entrap CURC up to almost 90%, consequently improving its stability. SLN-CURC inhibited in vitro pancreatic carcinoma cell growth in concentration-dependent manner. Their in vivo intravenous administration suggested a possible long circulation. Conclusions: These results, according to a concomitant study on chitosan-coated SLNs, confirm the possibility to apply the developed SLN-based delivery systems as a means to entrap CURC, to improve both its water dispersibility and chemical stability, facilitating its application in therapy.

Stearoyl-Chitosan Coated Nanoparticles Obtained by Microemulsion Cold Dilution Technique.

Chitosan is an excipient which has been studied thoroughly in research works thanks to its positive characteristics such as muco-adhesiveness and ability to open epithelial-tight-junctions. In this article, lipophilic stearoyl chitosan (ST-CS) was synthetized in order to anchor this polymer to lipid nanoparticles and prepare ST-CS-coated nanoparticles (ST-CS-NP) using the microemulsion cold dilution technique. Curcumin (CURC) was used as model drug. CURC-ST-CS-NP were characterized by dimensional analysis, zeta potential, drug entrapment, drug release; tested in vitro on Human Umbilical Vein Endothelial Cell (HUVEC) cells to study its cytotoxicity and on human pancreatic cancer cells (PANC-1) to determine inhibition ability; tested in rats to determine CURC blood profiles and biodistribution. CURC-ST-CS-NP had mean diameters in the range 200⁻400 nm and CURC entrapment up to 73%. These systems did not show cytotoxicity on HUVEC cells at all tested dilutions and revealed to be more effective than free CURC solution on PANC-1 cells at 5 and 10 µM CURC. Blood profile studies evidenced as CURC entrapment in NP prolonged the permanence of drug in the systemic circulation compared to CURC solution due to a certain stealth property of NP, probably attributable to hydrophilic chitosan coating. Biodistribution studies showed a smaller CURC concentration in RES organs when CURC-ST-CS-NP were administered.

Lipophilic Prodrug of Floxuridine Loaded into Solid Lipid Nanoparticles: In Vitro Cytotoxicity Studies on Different Human Cancer Cell Lines.

Floxuridine is a very effective drug with high potency in the treatment of various tumors but its utility is limited by its low efficiency of cellular uptake. In order to improve the floxuridine efficiency of cellular uptake, lipophilic prodrug of floxuridine (3',5'-distearoyl-5-fluoro-2'-deoxyuridine) was synthetized and loaded into behenic acid nanoparticles produced by fatty acid coacervation technique. Generally, spherical shaped SLN with mean diameters below 300 nm were obtained. Distearoyl-floxuridine was loaded in SLN with high entrapment efficiency (from 70.8 to 82.8%). In Vitro cytotoxicity studies on different human cancer cell lines (M14, HT-29 and MDA-MB231) were performed in order to test the ability of distearoyl-floxuridine-SLN to inhibit the cancer cell growth. In MTT test distearoyl floxuridine SLN showed a greater efficacy than floxuridine on all cancer cell lines revealing an efficiency about 100 times higher. Also clonogenic assay showed a higher cytotoxicity of distearoyl-floxuridine-SLN compared to floxuridine but the difference between the formulations was only about 10 times. In conclusion, SLN proved to be a promising vehicle to increase the floxuridine efficacy in cancer therapy.

Development and Characterization of Solid Lipid Nanoparticles Loaded with a Highly Active Doxorubicin Derivative.

Solid lipid nanoparticles (SLNs) comprise a versatile drug delivery system that has been developed for the treatment of a variety of diseases. The present study will investigate the feasibility of entrapping an active doxorubicin prodrug (a squalenoyl-derivative) in SLNs. The doxorubicin derivative-loaded SLNs are spherically shaped, have a mean diameter of 300-400 nm and show 85% w/w drug entrapment efficiency. The effects on cell growth of loaded SLNs, free doxorubicin and the prodrug have been examined using cytotoxicity and colony-forming assays in both human ovarian cancer line A2780 wild-type and doxorubicin-resistant cells. Further assessments as to the treatment's ability to induce cell death by apoptosis have been carried out by analyzing annexin-V staining and the activation of caspase-3. The in vitro data demonstrate that the delivery of the squalenoyl-doxorubicin derivative by SLNs increases its cytotoxic activity, as well as its apoptosis effect. This effect was particularly evident in doxorubicin-resistant cells.

Comparison of Allogeneic and Syngeneic Rat Glioma Models by Using MRI and Histopathologic Evaluation.

Research in neurooncology traditionally requires appropriate in vivo animal models, on which therapeutic strategies are tested before human trials are designed and proceed. Several reproducible animal experimental models, in which human physiologic conditions can be mimicked, are available for studying glioblastoma multiforme. In an ideal rat model, the tumor is of glial origin, grows in predictable and reproducible patterns, closely resembles human gliomas histopathologically, and is weakly or nonimmunogenic. In the current study, we used MRI and histopathologic evaluation to compare the most widely used allogeneic rat glioma model, C6-Wistar, with the F98-Fischer syngeneic rat glioma model in terms of percentage tumor growth or regression and growth rate. In vivo MRI demonstrated considerable variation in tumor volume and frequency between the 2 rat models despite the same stereotactic implantation technique. Faster and more reproducible glioma growth occurred in the immunoresponsive environment of the F98-Fischer model, because the immune response is minimized toward syngeneic cells. The marked inability of the C6-Wistar allogeneic system to generate a reproducible model and the episodes of spontaneous tumor regression with this system may have been due to the increased humoral and cellular immune responses after tumor implantation.

Solid lipid nanoparticles by coacervation loaded with a methotrexate prodrug: preliminary study for glioma treatment.

AIM: Methotrexate-loaded biocompatible nanoparticles were tested for preliminary efficacy in glioma treatment. MATERIALS & METHODS: Behenic acid nanoparticles, prepared by the coacervation method, were loaded with the ester prodrug didodecylmethotrexate, which was previously tested in vitro against glioblastoma human primary cultures. Nanoparticle conjugation with an ApoE mimicking chimera peptide was performed to obtain active targeting to the brain. RESULTS & CONCLUSION: Biodistribution studies in healthy rats assessed the superiority of ApoE-conjugated formulation, which was tested on an F98/Fischer glioma model. Differences were observed in tumor growth rate (measured by MRI) between control and treated rats. In vitro tests on F98 cultured cells assessed their susceptibility to treatment, with consequent apoptosis, and allowed us to explain the apoptosis observed in glioma models.

Solid lipid nanoparticles carrying lipophilic derivatives of doxorubicin: preparation, characterization, and in vitro cytotoxicity studies.

Doxorubicin (DOXO) lauroyl ester and amide were proposed as lipophilic derivatives and entrapped in SLNs. DOXO derivatives-loaded SLNs were spherical shaped, had 200-300 nm mean diameters and showed 80-94% w/w drug entrapment efficiencies. The effect of DOXO derivatives-loaded SLNs and free DOXO on cell growth was examined by MTT and colony-forming assays on four different tumour cell lines: a pancreatic, CFPAC-1, a lung, A549, and two ovarian, A2780 and A2780res (DOXO-resistant). The results obtained with MTT and colony-forming assay show that although DOXO displayed an inhibition of cell proliferation greater or similar to DOXO lauroyl amide-loaded SLNs on all cell types, the effect induced by DOXO lauroyl ester-loaded SLNs was higher and concentration-dependent, and it was the only one maintained at 10(-5 )mM concentration. Only DOXO lauroyl ester-loaded SLNs were able to induce a 40% inhibitory effect on A2780 res cell line up to 10(-4 )mM concentration.

Bevacizumab loaded solid lipid nanoparticles prepared by the coacervation technique: preliminary in vitro studies.

Glioblastoma, the most common primary brain tumor in adults, has an inauspicious prognosis, given that overcoming the blood-brain barrier is the major obstacle to the pharmacological treatment of brain tumors. As neoangiogenesis plays a key role in glioblastoma growth, the US Food and Drug Administration approved bevacizumab (BVZ), an antivascular endothelial growth factor antibody for the treatment of recurrent glioblastoma in patients whose the initial therapy has failed. In this experimental work, BVZ was entrapped in solid lipid nanoparticles (SLNs) prepared by the fatty-acid coacervation technique, thanks to the formation of a hydrophobic ion pair. BVZ activity, which was evaluated by means of four different in vitro tests on HUVEC cells, increased by 100- to 200-fold when delivered in SLNs. Moreover, SLNs can enhance the permeation of fluorescently labelled BVZ through an hCMEC/D3 cell monolayer-an in vitro model of the blood brain barrier. These results are promising, even if further in vivo studies are required to evaluate the effective potential of BVZ-loaded SLNs in glioblastoma treatment.

Positive-charged solid lipid nanoparticles as paclitaxel drug delivery system in glioblastoma treatment.

Paclitaxel loaded solid lipid nanoparticles (SLN) of behenic acid were prepared with the coacervation technique. Generally, spherical shaped SLN with mean diameters in the range 300­600 nm were obtained. The introduction of charged molecules, such as stearylamine and glycol chitosan into the formulation allowed to obtain positive SLN with Zeta potential in the 8-20 mV range and encapsulation efficiency in the 25­90% range.Blood­brain barrier (BBB) permeability, tested in vitro through hCMEC/D3 cells monolayer, showed a significantly increase in the permeation of Coumarin-6, used as model drug, when vehicled in SLN. Positive-charged SLN do not seem to enhance permeation although stearylamine-positive SLN resulted the best permeable formulation after 24 h.Cytotoxicity studies on NO3 glioblastoma cell line demonstrated the maintenance of cytotoxic activity of all paclitaxel-loaded SLN that was always unmodified or greater compared with free drug. No difference in cytotoxicity was noted between neutral and charged SLN.Co-culture experiments with hCMEC/D3 and different glioblastoma cells evidenced that, when delivered in SLN, paclitaxel increased its cytotoxicity towards glioblastoma cells.

Solid lipid nanoparticles loaded with fluorescent-labelled Cyclosporine A: anti-inflammatory activity in vitro.

FMOC-isocyclosporine A, a fluorescent labeled cyclosporine A, was encapsulated in solid lipid nanoparticles (SLN) prepared by the coacervation technique, and its anti-inflammatory activity was evaluated. The anti-inflammatory activity of the fluorescent labelled molecule, measured as inhibition of TNF-α secretion, is similar to the native one. SLN were compared to commercial formulations, through measurement of cytokine release and drug uptake in rat peripheral blood mononuclear cells. Drug-loaded SLN inhibit TNF-α secretion in a lower extent than commercial formulations, probably due to a lower uptake by the cells, but the increase of IL-10 secretion caused by the lipid matrix itself makes this formulation interesting for its anti-inflammatory activity.

Solid lipid nanoparticles for potential doxorubicin delivery in glioblastoma treatment: preliminary in vitro studies.

The major obstacle to glioblastoma pharmacological therapy is the overcoming of the blood-brain barrier (BBB). In literature, several strategies have been proposed to overcome the BBB: in this experimental work, solid lipid nanoparticles (SLN), prepared according to fatty acid coacervation technique, are proposed as the vehicle for doxorubicin (Dox), to enhance its permeation through an artificial model of BBB. The in vitro cytotoxicity of Dox-loaded SLN has been measured on three different commercial and patient-derived glioma cell lines. Dox was entrapped within SLN thanks to hydrophobic ion pairing with negatively charged surfactants, used as counterions. Results indicate that Dox entrapped in SLN maintains its cytotoxic activity toward glioma cell lines; moreover, its permeation through hCMEC/D3 cell monolayer, assumed as a model of the BBB, was increased when the drug was entrapped in SLN. In conclusion, SLN proved to be a promising vehicle for the delivery of Dox to the brain in glioblastoma treatment.