Development and characterization of a temozolomide-loaded nanoemulsion and the effect of ferrocene pre and co-treatments in glioblastoma cell models.

BACKGROUND: Glioblastoma is a severe brain tumor that requires aggressive treatment involving surgery, radiotherapy, and chemotherapy, offering a survival rate of only 15 months. Fortunately, recent nanotechnology progress has enabled novel approaches and, alongside ferrocenes' unique properties of cytotoxicity, sensitization, and interaction with reactive oxygen species, have brought new possibilities to complement chemotherapy in nanocarrier systems, enhancing treatment results. METHODS: In this work, we developed and characterized a temozolomide-loaded nanoemulsion and evaluated its cytotoxic potential in combination with ferrocene in the temozolomide-resistant T98G and temozolomide-sensitive U87 cell lines. The effects of the treatments were assessed through acute assays of cell viability, cell death, mitochondrial alterations, and a treatment protocol simulation based on different two-cycle regimens. RESULTS: Temozolomide nanoemulsion showed a z-average diameter of 173.37 ± 0.86 nm and a zeta potential of - 6.53 ± 1.13 mV. Physicochemical characterization revealed that temozolomide is probably associated with nanoemulsion droplets instead of being entrapped within the nanostructure, allowing a rapid drug release. In combination with ferrocene, temozolomide nanoemulsion reduced glioblastoma cell viability in both acute and two-cycle regimen assays. The combined treatment approach also reversed T98G's temozolomide-resistant profile by altering the mitochondrial membrane potential of the cells, thus increasing reactive oxygen species generation, and ultimately inducing cell death. CONCLUSIONS: Altogether, our results indicate that using nanoemulsion containing temozolomide in combination with ferrocene is an effective approach to improve glioblastoma therapy outcomes.

Polymeric nanoparticles containing babassu oil: A proposed drug delivery system for controlled release of hydrophilic compounds.

Different drug delivery systems are prepared on the nanoscale to improve performance in drug formulations, such as nanoparticles or nanoemulsions. Polymeric nanoparticles have been used to encapsulate drugs for several applications because of some characteristics of these carriers to control drug delivery, transport molecules to a specific tissue, protect the drugs, and increase drug bioavailability. When using nanocapsules, an essential parameter for encapsulating different hydrophilic or lipophilic molecules is the characteristics of the core. Babassu oil (BBS) is a natural product from Brazil, composed majoritary of short-chain saturated fatty acids. BBS has an elevated hydrophilic-lipophilic balance (HLB), which may promote interaction of the oil with hydrophilic drugs. In this study, we developed and characterized particles containing babassu oil, solely or combined with sorbitan monostearate (Span® 60) or medium chain triglycerides (MCT) in the core to test different HLB and evaluated the encapsulation of a model hydrophilic molecule. Different techniques were used to characterize all formulations in terms of size and distribution, and in vitro drug release by dialysis technique was performed. The BBS was also characterized and presented 46,05 ± 1,11% and 15,38 ± 0,06% of lauric and myristic acid, respectively; saponification index of 248.87 ± 0.64 mg of KOH per gram of BBS, and no oxidation of the oil was indicated by means of peroxide index. Evaporation of solvent carried in the room or reduced pressure influenced the particles' size; nevertheless, all had a z-average smaller than 220 nm. Nanoparticles with a ratio among aqueous phase and organic phase of 2.8 were considered adequate to encapsulate diclofenac sodium. The particles size/zeta potential were 189.83 ± 7.86 nm / - 10.39 ± 2.52 mV, 156.80 ± 4.77 nm / - 9.27 ± 4.61 mV, and 168.87 ± 5.22 nm / - 12.98 ± 4.66 mV to nanoparticles prepared with BBS + MCT, BBS, and BBS + Span® 60, respectively. All formulations exhibited an amount of drug content close to the theoretical amount (1.0 mg mL-1), and no difference was observed in the release profile among the three nanoparticles. Formulation containing only babassu oil in the core displayed 66.78 ± 15.62% of encapsulation efficiency to diclofenac sodium, the highest value among all formulations tested. Results demonstrate that the innovative nanoparticles containing BBS promote the encapsulation of a model hydrophilic molecule, and other components can be evaluated to change the core's hydrophilicity and encapsulation of molecules.

Development and immunobiological evaluation of nanoparticles containing an immunodominant epitope of herpes simplex virus.

Herpes simplex virus (HSV) 1 and 2 are viruses that infect individuals worldwide and for which there is no cure or vaccine available. The protective response against herpes is mostly mediated by CD8 T lymphocytes that respond to the immunodominant SSIEFARL epitope. However, there are some obstacles concerning the use of free SSIEFARL for vaccine or immunotherapy. The aim of this study was to evaluate the feasibility of nanoencapsulation of SSIEFARL and its immunostimulatory properties. Nano/SSIEFARL was produced by interfacial polymerization in methylmetacrylate, and the physico-chemical properties, morphology and immunobiological parameters were evaluated. To evaluate the ex vivo capacity of Nano/SSIEFARL, we used splenocytes from HSV-1-infected mice to enhance the frequency of SSIEFARL-specific CD8 T lymphocytes. The results indicate that Nano/SSIEFARL has a spherical shape, an average diameter of 352 ± 22 nm, the PDI was 0.361 ± 0.009 and is negatively charged (-26.30 ± 35). The stability at 4°C was 28 days. Also, Nano/SSIEFARL is not toxic for cells at low concentrations in vitro and it is taken up by JAWS II dendritic cells. No histopathological changes were observed in kidneys, liver and lymph nodes of animals treated with Nano/SSIEFARL. Nan/SSIEFARL increased the production of IL-1ß, TNF-α and IL-12 by the dendritic cells. Finally, Nano/SSIEFARL expanded the frequency of SSIEFARL-specific CD8+T lymphocytes at the same rate as free SSIEFARL. In conclusion all data together indicate that SSIEFARL is suitable for nanoencapsulation, and the system produced presents some immunoadjuvant properties that can be used to improve the immune response against herpes.

Nasal Drug Delivery of Anticancer Drugs for the Treatment of Glioblastoma: Preclinical and Clinical Trials.

Glioblastoma (GBM) is the most lethal form of brain tumor, being characterized by the rapid growth and invasion of the surrounding tissue. The current standard treatment for glioblastoma is surgery, followed by radiotherapy and concurrent chemotherapy, typically with temozolomide. Although extensive research has been carried out over the past years to develop a more effective therapeutic strategy for the treatment of GBM, efforts have not provided major improvements in terms of the overall survival of patients. Consequently, new therapeutic approaches are urgently needed. Overcoming the blood-brain barrier (BBB) is a major challenge in the development of therapies for central nervous system (CNS) disorders. In this context, the intranasal route of drug administration has been proposed as a non-invasive alternative route for directly targeting the CNS. This route of drug administration bypasses the BBB and reduces the systemic side effects. Recently, several formulations have been developed for further enhancing nose-to-brain transport, mainly with the use of nano-sized and nanostructured drug delivery systems. The focus of this review is to provide an overview of the strategies that have been developed for delivering anticancer compounds for the treatment of GBM while using nasal administration. In particular, the specific properties of nanomedicines proposed for nose-to-brain delivery will be critically evaluated. The preclinical and clinical data considered supporting the idea that nasal delivery of anticancer drugs may represent a breakthrough advancement in the fight against GBM.

Chitosan-Coated Nanoparticles: Effect of Chitosan Molecular Weight on Nasal Transmucosal Delivery.

Drug delivery to the brain represents a challenge, especially in the therapy of central nervous system malignancies. Simvastatin (SVT), as with other statins, has shown potential anticancer properties that are difficult to exploit in the central nervous system (CNS). In the present work the physico⁻chemical, mucoadhesive, and permeability-enhancing properties of simvastatin-loaded poly-ε-caprolactone nanocapsules coated with chitosan for nose-to-brain administration were investigated. Lipid-core nanocapsules coated with chitosan (LNCchit) of different molecular weight (MW) were prepared by a novel one-pot technique, and characterized for particle size, surface charge, particle number density, morphology, drug encapsulation efficiency, interaction between surface nanocapsules with mucin, drug release, and permeability across two nasal mucosa models. Results show that all formulations presented adequate particle sizes (below 220 nm), positive surface charge, narrow droplet size distribution (PDI < 0.2), and high encapsulation efficiency. Nanocapsules presented controlled drug release and mucoadhesive properties that are dependent on the MW of the coating chitosan. The results of permeation across the RPMI 2650 human nasal cell line evidenced that LNCchit increased the permeation of SVT. In particular, the amount of SVT that permeated after 4 hr for nanocapsules coated with low-MW chitosan, high-MW chitosan, and control SVT was 13.9 ± 0.8 µg, 9.2 ± 1.2 µg, and 1.4 ± 0.2 µg, respectively. These results were confirmed by SVT ex vivo permeation across rabbit nasal mucosa. This study highlighted the suitability of LNCchit as a promising strategy for the administration of simvastatin for a nose-to-brain approach for the therapy of brain tumors.

Chitosan hydrogels containing nanoencapsulated phenytoin for cutaneous use: Skin permeation/penetration and efficacy in wound healing.

Although phenytoin is an antiepileptic drug used in the oral treatment of epilepsy, its off-label use as a cutaneous healing agent has been studied in recent years due to the frequent reports of gingival hyperplasia after oral administration. However, the cutaneous topical application of phenytoin should prevent percutaneous skin permeation. Therefore, the aim of this study was to evaluate the in vitro skin permeation/retention and in vivo effects of nanocapsules and nanoemulsions loaded with phenytoin and formulated as chitosan hydrogels on the healing process of cutaneous wounds in rats. The hydrogels had adequate pH values (4.9-5.6) for skin application, drug content of 0.025% (w/w), and non-Newtonian pseudoplastic rheological behaviour. Hydrogels containing nanocapsules and nanoemulsions enabled improved controlled release of phenytoin and adhesion to skin, compared with hydrogels containing non-encapsulated phenytoin. In vitro skin permeation studies showed that phenytoin permeation to the receptor compartment, and consequently the risk of systemic absorption, may be reduced by nanoencapsulation without any change in the in vivo performance of phenytoin in the wound healing process in rats.

Coated minispheres of salmon calcitonin target rat intestinal regions to achieve systemic bioavailability: Comparison between intestinal instillation and oral gavage.

Achieving oral peptide delivery is an elusive challenge. Emulsion-based minispheres of salmon calcitonin (sCT) were synthesized using single multiple pill (SmPill®) technology incorporating the permeation enhancers (PEs): sodium taurodeoxycholate (NaTDC), sodium caprate (C10), or coco-glucoside (CG), or the pH acidifier, citric acid (CA). Minispheres were coated with an outer layer of Eudragit® L30 D-55 (designed for jejunal release) or Surelease®/Pectin (designed for colonic release). The process was mild and in vitro biological activity of sCT was retained upon release from minispheres stored up to 4months. In vitro release profiles suggested that sCT was released from minispheres by diffusion through coatings due to swelling of gelatin and the polymeric matrix upon contact with PBS at pH6.8. X-ray analysis confirmed that coated minispheres dissolved at the intended intestinal region of rats following oral gavage. Uncoated minispheres at a dose of ~2000I.U.sCT/kg were administered to rats by intra-jejunal (i.j.) or intra-colonic (i.c.) instillation and caused hypocalcaemia. Notable sCT absolute bioavailability (F) values were: 5.5% from minispheres containing NaTDC (i.j), 17.3% with CG (i.c.) and 18.2% with C10 (i.c.). Coated minispheres administered by oral gavage at threefold higher doses also induced hypocalcaemia. A highly competitive F value of 2.7% was obtained for orally-administered sCT-minispheres containing CG (45µmol/kg) and coated with Eudragit®. In conclusion, the SmPill® technology is a potential dosage form for several peptides when formulated with PEs and coated for regional delivery. PK data from instillations over-estimates oral bioavailability and poorly predicts rank ordering of formulations.

In vitro and in vivo preclinical evaluation of a minisphere emulsion-based formulation (SmPill®) of salmon calcitonin.

Salmon calcitonin (sCT, MW 3432Da) is a benchmark molecule for an oral peptide delivery system because it is degraded and has low intestinal epithelial permeability. Four dry emulsion minisphere prototypes (SmPill®) containing sCT were co-formulated with permeation enhancers (PEs): sodium taurodeoxycholate (NaTDC), sodium caprate (C10) or coco-glucoside (CG), or with a pH acidifier, citric acid (CA). Minispheres protected sCT from thermal degradation and the released sCT retained high bioactivity, as determined by cyclic AMP generation in T47D cells. Pre-minisphere emulsions of PEs combined with sCT increased absolute bioavailability (F) compared to native sCT following rat intra-jejunal (i.j.) and intra-colonic (i.c.) loop instillations, an effect that was more pronounced in colon. Minispheres corresponding to ~2000I.U. (~390µg) sCT/kg were instilled by i.j. or i.c. instillations and hypocalcaemia resulted from all prototypes. The absolute F (i.j.) of sCT was 11.0, 4.8, and 1.4% for minispheres containing NaTDC (10µmol/kg), CG (12µmol/kg) or CA (32µmol/kg) respectively. For i.c. instillations, the largest absolute F (22% in each case) was achieved for minispheres containing either C10 (284µmol/kg) or CG (12µmol/kg), whilst the absolute F was 8.2% for minispheres loaded with CA (32µmol/kg). In terms of relative F, the best data were obtained for minispheres containing NaTDC (i.j.), a 4-fold increase over sCT solution, and also for either C10 or CG (i.c.), where there was a 3-fold increase over sCT solution. Histology of instilled intestinal loops indicated that neither the minispheres nor components thereof caused major perturbation. In conclusion, selected SmPill® minisphere formulations may have the potential to be used as oral peptide delivery systems when delivered to jejunum or colon.

Investigation of coco-glucoside as a novel intestinal permeation enhancer in rat models.

Due to instability in the GI tract and low intestinal permeability, peptides invariably have oral bioavailabilities below 1% and this has prevented the development of oral formulations. A mild plant-derived naturalalkyl polyglycoside (APG), coco-glucoside (CG), was studied for its capacity to enable rat intestinal permeation of the paracellular sugar marker, fluorescein isothiocyanate-dextran 4000 (FD4), across isolated rat jejunal and colonic mucosae mounted in Ussing chambers, as well as the polypeptide, salmon calcitonin (sCT) following intra-intestinal instillations in rats. 0.1% (w/v) CG enabled a 2.9-fold increase in the apparent permeability coefficient (Papp) of FD4 over the basal Papp across colonic mucosae, but it was without effect in jejunal mucosae. In situ intestinal instillations revealed that although sCT was absorbed across rat colonic loops to a greater extent than jejunal, CG still improved sCT absolute bioavailability(F) from both segments. Histopathology of rat intestinal mucosae following exposure to CG indicated only minor perturbation with adequate maintenance of secretory function. High content analysis(HCA) on Caco-2 showed that acute and chronic exposure to a range of concentrations of CG did not cause sub-lethal damage at concentrations at which it was effective as an enhancer. Overall, CG increased bioavailability of sCT across rat jejunal and colonic loops without indication of tissue damage. Thus, CG has potential as a safe and effective intestinal enhancer for oral delivery of proteins and peptides.

In vitro and in vivo characterisation of a novel peptide delivery system: amphiphilic polyelectrolyte-salmon calcitonin nanocomplexes.

The cationic peptide, salmon calcitonin (sCT) was complexed with the cationic amphiphilic polyelectrolyte, poly(allyl)amine, grafted with palmitoyl and quaternary ammonium moieties at pH 5.0 and 7.4 to yield particulates (sCT-QPa). The complexes were approximately 200 nm in diameter, had zeta potentials ranging from +20 to +50 mV, and had narrow polydispersity indices (PDIs). Differential scanning calorimetry revealed the presence of an interaction between sCT and QPa in the complexes. Electron microscopy confirmed the zeta-size data and revealed a vesicular bilayer structure with an aqueous core. Tyrosine- and Nile red fluorescence indicated that the complexes retained gross physical stability for up to 7 days, but that the pH 5.0 complexes were more stable. The complexes were more resistant to peptidases, serum and liver homogenates compared to free sCT. In vitro bioactivity was measured by cAMP production in T47D cells and the complexes had EC50 values in the nM range. While free sCT was unable to generate cAMP following storage for 7 days, the complexes retained approximately 33% activity. When the complexes were injected intravenously to rats, free and complexed sCT (pH 5.0 and 7.4) but not QPa reduced serum calcium over 120 min. Free and complexed sCT but not QPa also reduced serum calcium over 240 min following intra-jejunal administration. In conclusion, sCT-QPa nanocomplexes that have been synthesised are stable, bioactive and resistant to a range of peptidases. These enhanced features suggest that they may have the potential for improved efficacy when formulated for injected and oral delivery.