Chitosan-coated Nanoemulsions Research Articles

Evaluation of the in vitro Antileishmanial Activity of Chitosan-Coated Nanoemulsions of Ozonated Olive Oil

ABSTRACT Leishmaniasis is a neglected parasitic disease. Numerous studies suggested that nanoemulsions could improve drug delivery in the treatment of zoonotic cutaneous leishmaniasis. This study aims to determine the effect of chitosan-coated nanoemulsions of ozonated olive oil on Leishmania major promastigote/amastigote in vitro. Nanoemulsions (NEs) were prepared using a sonicator bath at 37 °C for one hour at 1000 W, followed by 24 hours of stirring at pH 5.5. The NE was then supplemented with chitosan and stirred all night long at a low speed. Droplet size, zeta potential, in vitro drug release, and cytotoxicity were examined to optimize the chitosan nanoemulsions (CNE) and chitosan ozonated nanoemulsions (CONE). CONE and CNE were found to have droplet sizes and zeta potentials of 16.86 nm, 19.03 nm, 19.7 mV, and 19.3 mV, respectively. Additionally, the final CONE’s drug loading and encapsulation efficiency were determined to be 100% and 3.21%, respectively. The lower doses of CONE had no cytotoxic effects on macrophages. Additionally, it has the ability to inhibit L. major promastigote/amastigote growth. Therefore, the lower doses of the CONE formulation can inhibit the growth of promastigote and amastigote of L. major in vitro while not being toxic to macrophages.

Metastatic Melanoma: A Preclinical Model Standardization and Development of a Chitosan-Coated Nanoemulsion Containing Temozolomide to Treat Brain Metastasis.

Melanoma is the most aggressive type of skin cancer. Brain metastasis is the worst scenario in metastatic melanoma and the treatment options for these patients are limited. Temozolomide (TMZ) is a chemotherapy agent used to treat primary central nervous system tumors. Our objective was to develop chitosan-coated nanoemulsion containing temozolomide (CNE-TMZ) for nasal route administration to melanoma brain metastasis treatment. A preclinical model of metastatic brain melanoma was standardized, and the efficiency of the developed formulation was further determined in vitro and in vivo. The nanoemulsion was done by spontaneous emulsification method and the formulation was characterized by size, pH, polydispersity index, and zeta potential. Culture assessments to determine cell viability were done in the A375 human melanoma cell line. To determine the safety of formulation, healthy C57/BL6 mice were treated with a nanoemulsion without TMZ. The model in vivo used B16-F10 cells implanted by stereotaxic surgery in C57/BL6 mice brains. The results demonstrate that the preclinical model used showed to be useful to analyze the efficiency of new candidate drugs to treat melanoma brain metastasis. The chitosan-coated nanoemulsions with TMZ showed the expected physicochemical characteristics and demonstrated safety and efficacy, reducing around 70% the tumor size compared to control mice, and presenting a tendency in mitotic index reduction, becoming an interesting approach to treat melanoma brain metastasis.

Nano-adjuvanted dry powder vaccine for the mucosal immunization against airways pathogens.

Nasal vaccination has been shown to provide optimal protection against respiratory pathogens. However, mucosal vaccination requires the implementation of specific immunization strategies to improve its effectiveness. Nanotechnology appears a key approach to improve the effectiveness of mucosal vaccines, since several nanomaterials provide mucoadhesion, enhance mucosal permeability, control antigen release and possess adjuvant properties. Mycoplasma hyopneumoniae is the main causative agent of enzootic pneumonia in pigs, a respiratory disease responsible for considerable economic losses in the pig farming worldwide. The present work developed, characterized, and tested in vivo an innovative dry powder nasal vaccine, obtained from the deposition on a solid carrier of an inactivated antigen and a chitosan-coated nanoemulsion, as an adjuvant. The nanoemulsion was obtained through a low-energy emulsification technique, a method that allowed to achieve nano droplets in the order of 200 nm. The oil phase selected was alpha-tocopherol, sunflower oil, and poly(ethylene glycol) hydroxystearate used as non-ionic tensioactive. The aqueous phase contained chitosan, which provides a positive charge to the emulsion, conferring mucoadhesive properties and favoring interactions with inactivated M. hyopneumoniae. Finally, the nanoemulsion was layered with a mild and scalable process onto a suitable solid carrier (i.e., lactose, mannitol, or calcium carbonate) to be transformed into a solid dosage form for administration as dry powder. In the experimental study, the nasal vaccine formulation with calcium carbonate was administered to piglets and compared to intramuscular administration of a commercial vaccine and of the dry powder without antigen, aimed at evaluating the ability of IN vaccination to elicit an in vivo local immune response and a systemic immune response. Intranasal vaccination was characterized by a significantly higher immune response in the nasal mucosa at 7 days post-vaccination, elicited comparable levels of Mycoplasma-specific IFN-γ secreting cells and comparable, if not higher, responsiveness of B cells expressing IgA and IgG in peripheral blood mononuclear cells, with those detected upon a conventional intramuscular immunization. In conclusion, this study illustrates a simple and effective strategy for the development of a dry powder vaccine formulation for nasal administration which could be used as alternative to current parenteral commercial vaccines.

Mucoadhesive nanoemulsion enhances brain bioavailability of luteolin after intranasal administration and induces apoptosis to SH-SY5Y neuroblastoma cells

Neuroblastoma is the most frequently diagnosed extracranial solid tumor in children and accounts for 7 % of all childhood malignancies and 15 % cancer mortality in children. Luteolin (LUT) is recognized by its anticancer activity against several types of cancer. The aim of this study was to prepare chitosan-coated nanoemulsion containing luteolin (NECh-LUT), investigate its potential for brain delivery following intranasal administration, and to evaluate its cytotoxicity against neuroblastoma cells. NECh-LUT was developed by cavitation process and characterized for its size, surface charge, encapsulation efficiency, and mucoadhesion. The developed formulation presented size 68 ± 1 nm, zeta potential + 13 ± 1 mV, and encapsulation efficiency of 85.5 ± 0.3 %. The NECh-LUT presented nearly 6-fold higher permeation through the nasal mucosa ex vivo and prolonged LUT release up to 72 h in vitro, following Baker-Lonsdale kinetic model. The pharmacokinetic evaluation of NECh-LUT revealed a 10-fold increase in drug half-life and a 4.4 times enhancement in LUT biodistribution in brain tissue after intranasal administration of single-dose. In addition, NECh-LUT inhibited the growth of neuroblastoma cells after 24, 48 and 72 h in concentrations starting from 2 µM. The NECh-LUT developed for intranasal administration proved to be a promising alternative for brain delivery of LUT, and a viable option for the treatment of neuroblastoma.

Enhancement of Storage Stability and Masking Effect of Curcumin by Turmeric Extract-Loaded Nanoemulsion and Water-Soluble Chitosan Coating.

This study focused on improving curcumin stability in various pHs and NaCl concentrations and reducing the strong scent of turmeric by the nanoemulsions system and further coating with water-soluble chitosan (WSC). Turmeric extract-loaded nanoemulsions (TE-NEs) were firstly prepared by mixing an oil phase containing turmeric extract, MCT oil, and lecithin, and an aqueous phase containing tween 80 using an ultrasonication method. TE-NEs were further coated with WSC in the ratio of TE-NEs and WSC (1:1 to 1:10). The optimum WSC-TE-NEs exhibited an average particle size of 182 nm, a PDI of 0.317, and a zeta potential of +30.42 mV when WSC-TE-NEs were prepared in the ratio of 1:1. The stability of the WSC-TE-NEs was also assessed by determining the remained curcumin content. The remained curcumin contents of the TE-NEs and the WSC-TE-NEs were higher than that of the turmeric extract (TE) at pH 2~7 and NaCl concentrations of 100~400 mM. Fourier transform infrared (FT-IR) spectra, transmission electron microscope (TEM), and confocal laser scanning microscope (CLSM) images confirmed that the TE-NEs were successfully encapsulated with a WSC coating. As a result of GC analysis, the content of aromatic-turmerone was significantly decreased in the TE-NEs and the WSC-TE-NEs compared to the pristine TE, but there was no significant difference between the TE-NEs and the WSC-TE-NEs. These results suggest that water-soluble chitosan-coated nanoemulsions may be suitable for improving the chemical stability and masking effect of curcumin to facilitate its application in food.

Synthesis and Characterization of Chitosan-Decorated Nanoemulsion Gel of 5-Fluorouracil for Topical Delivery.

(1) Background: The present study aimed to prepare chitosan-coated nanoemulsion gel containing 5-fluorouracil for enhanced topical delivery. (2) Methods: To formulate the nanoemulsion gel, oleic acid was used as the oil phase and Carbopol 940 as a gelling agent. Chitosan was used as a coating agent to control the release of 5-FU. Drug–excipient compatibility was evaluated using ATR-FTIR. The prepared nanoemulsion formulations were characterized based on particle size distribution, zeta potential, % encapsulation efficiency and drug content. In vitro drug release, skin drug retention and ex vivo permeation profiles were performed across rat skin using a Franz diffusion cell. Skin irritation experiments were also conducted on rats to examine the irritation potential of the formulations. (3) Results: It was found that the drug and excipients were compatible and chitosan successfully coated 5-FU, as demonstrated by ATR-FTIR results. The introduction of chitosan increased the size and zeta potential of the nanoemulsion. The 5-FU release in vitro was significantly lowered in the case of chitosan-decorated nanoemulsion (5-FU-C-NE), whereas the permeation and skin drug retention were higher in the case of 5-FU-C-NE. The formulations were proven non-irritant to the skin of the rats. The optimized formulation of the nanoemulsion was introduced into 1% Carbopol 940 gel. Incorporating the nanoemulsion into the gel further reduced the drug release in vitro and ex vivo permeation, whereas the retention of the drug in the skin was significantly increased (ANOVA; p < 0.05). The increase in skin retention was due to the presence of chitosan and Carbopol 940. The in vitro and ex vivo results were also confirmed with in vivo studies. Incorporating nanoemulsion into gel has resulted in higher Tmax, longer half-life and greater skin drug retention. (4) Conclusion: The results suggest that chitosan-decorated nanoemulsion gel is safe and can potentially be used to promote 5-FU skin retention, which is ideal for skin diseases such as melanoma.

Development of chitosan-coated nanoemulsions of two sulfides present in onion (Allium cepa) essential oil and their nematicidal activities against the pine wood nematode, Bursaphelenchus xylophilus.

Pine wood nematode, Bursaphelenchus xylophilus, is a plant parasitic nematode which causes severe damage to several Pinus species. Two natural compounds, dipropyl trisulfide (DPTS) and methyl propyl trisulfide (MPTS), showed strong nematicidal activity against the pine wood nematode, presenting 4.24 and 17.81 μg/mL LC50 values, respectively. However, hydrophobicity and low stability have limited their practical use in the field as nematicides. To overcome these problems, chitosan-coated nanoemulsions of DPTS and MPTS were developed. The optimum chitosan concentration for the delivery system of the two sulfides was 0.5%. Optimized chitosan-coated nanoemulsions of sulfides have a uniform size distribution (mean diameter = 203.7 and 207.7 nm, mean polydispersity index = 0.176 and 0.178) with sufficient colloidal stability (mean zeta potential = +40 and +45 mV). The LC50 values of DPTS and MPTS nanoemulsions coated with 0.5% chitosan against the pine wood nematode were 5.01 and 16.60 μg/mL, respectively. In addition, chitosan coating improved the long-term storage stability and persistence of nematicidal activity of the nanoemulsions. This study indicates that the chitosan-coated nanoemulsion is a suitable formulation for sulfides as novel nematicides against the pine wood nematode for field application.

Development and Optimization of Naringenin-Loaded Chitosan-Coated Nanoemulsion for Topical Therapy in Wound Healing.

The potential role of naringenin (NAR), a natural flavonoid, in the treatment of chronic wound has prompted the present research to deliver the drug in nanoemulsion (NE) form, where synergistic role of chitosan was achieved through development of chitosan-coated NAR NE (CNNE). The NE consisted of Capryol 90, Tween 20 and Transcutol P, which was fabricated by low-energy emulsification method to encapsulate NAR within the oil core. The optimization of the formulated NEs was performed using Box–Behnken statistical design to obtain crucial variable parameters that influence globule size, size distribution and surface charge. Finally, the optimized formulation was coated with different concentrations of chitosan and subsequently characterized in vitro. The size of the CNNE was found to be increased when the drug-loaded formulation was coated with chitosan. Controlled release characteristics depicted 67–81% release of NAR from the CNNE, compared to 89% from the NE formulation. Cytotoxicity study of the formulation was performed in vitro using fibroblast cell line (NIH-3T3), where no inhibition in proliferation of the cells was observed with CNNE. Finally, the wound healing potential of the CNNE was evaluated in an abrasion-created wound model in experimental animals where the animals were treated and compared histologically at 0 and 14 days. Significant improvement in construction of the abrasion wound was observed when the animals were treated with formulated CNNE, whereas stimulation of skin regeneration was depicted in the histological examination. Therefore, it could be summarized that the chitosan coating of the developed NAR NE is a potential platform to accelerate healing of wounds.

Sensitive ultra-fast liquid chromatography method for rosmarinic acid determination in Wistar rat’s plasma and brain

Rosmarinic acid (RA) is a polyphenolic compound recently associated to a neuroprotective potential. Nevertheless, besides the RA poor bioavailability and availability in the central nervous system (CNS) some alternatives to RA delivery, protection, and release been investigated. Recently, our research group optimized chitosan-coated nanoemulsions for RA nasal administration and demonstrated the glioprotective effect against LPS-induced damage in astrocytes. In this context, this study aimed to validate a fast, simplified and stability-indicating UFLC method previously reported by our research group for RA determination in Wistar rat’s plasma and brain, to be employed in further in vivo studies, since the nasal route for RA are not completely understood. The method was validated in terms of specificity, linearity, matrix effect, stability, precision, accuracy and extraction recovery for rat plasma and brain, according to the official guidelines. The method was sensitive, linear (0.1 - 10 µg.mL−1), precise and accurate, and showed RA recovery higher than 85% in plasma and brain. Overall results demonstrated that method was successfully validated for determination of RA in rat plasma and brain matrices with high sensibility and with high recovery using simple extraction processes.

Quaternized chitosan-coated nanoemulsions: A novel platform for improving the stability, anti-inflammatory, anti-cancer and transdermal properties of Plai extract

A new positively charged nanoemulsion using quaternized chitosan (QCS) as a protective layer was developed to improve the stability and bioactivity of lipophilic active components. The anti-inflammatory Plai extract was chosen as both an active ingredient and an oil phase of the system. Compared with chitosan-coated nanoemulsion (NE2-CS) and uncoated nanoemulsion (NE1), the QCS coating could improve the stability of the Plai extract during 28 days. The particle size of NE1 increased from 141 nm to 202 nm after coating with QCS, whereas zeta potential changed from -22.03 mV for NE1 to 20.23 mV for NE2-QCS, confirming the presence of QCS. A clear improvement in anti-inflammatory, anti-cancer, and transdermal properties of Plai extract was verified for NE2-QCS, which could be due to the NEs’ fineness and the permanent positive charge of the protective layer. Therefore, we suggested that QCS-coated NEs can be used as an effective transdermal delivery system for lipophilic active components.

Improved oral bioavailability of repaglinide, a typical BCS Class II drug, with a chitosan-coated nanoemulsion.

The aim of the present study was to develop modified nanoemulsions to improve the oral bioavailability and pharmacokinetics of a poor water-soluble drug, repaglinide (RPG). The repaglinide-loaded nanoemulsions (RPG-NEs) were prepared from olive oil as internal phase, span 80, tween 80, and poloxamer 188 as emulsifiers, using homogenization technique. The mean droplet size, zeta potential, and entrapment efficiency of RPG-NEs were 86.5 ± 3.4 nm, -33.8 ± 2.1 mV, and 96.3 ± 2.3%, respectively. The chitosan-coated RPG-NEs (Cs-RPG-NEs) showed an average droplet size of 149.3 ± 3.9 nm and a positive zeta-potential of +31.5 ± 2.8 mV. Drug release profile of RPG-NEs was significantly higher than free drug in the simulated gastrointestinal fluids (p < .005). The in vivo study revealed 3.51- and 1.78-fold increase in the AUC0-12h and Cmax of the drug, respectively, in RPG-NEs-receiving animals in comparison to the free drug. The pharmacokinetic analysis confirmed that Cs-RPG-NEs were more efficient than uncoated ones for the oral delivery of RPG. Cs-RPG-NEs showed a longer t1/2 and higher AUC0-∞ compared to control group. The relative bioavailability of Cs-RPG-NEs was higher than that of uncoated RPG-NEs and free drug. Collectively, these findings suggest that chitosan-coated nanoemulsions are promising carrier for improving the oral bioavailability of RPG.

Box-Behnken design optimization of mucoadhesive chitosan-coated nanoemulsions for rosmarinic acid nasal delivery—In vitro studies

Mucoadhesive chitosan-coated nanoemulsions for rosmarinic acid (RA) nasal delivery were optimized. The optimum ratio between the formulation components that led to minimum droplet size and PDI, and maximal ζ-potential and RA content was obtained using Box-Behnken design (BBD). Optimized conditions were 8.5% oil phase (w/v), 3:10 lecithin to oil phase ratio (w/w), and 0.1% chitosan final concentration (w/v). Physicochemical characterization, mucoadhesion measurement, in vitro release and permeation/retention were performed. Optimized chitosan-coated RA nanoemulsions presented adequate physicochemical characteristics, high mucoadhesive potential, prolonged drug release, and long-lasting permeation time with a higher RA penetration/retention through porcine nasal mucosa. Cell viability and death by necrosis in fibroblasts cells were also evaluated to investigate the formulations safety. Formulations did not induce cytotoxicity following 24 h (3.125–50 μM) or 48 h (3.125–25 μM) of treatments. Overall results demonstrated that optimized chitosan-coated nanoemulsion showed to be a suitable carrier for RA nasal delivery aiming neuroprotective therapies.

Protection of astaxanthin from photodegradation by its inclusion in hierarchically assembled nano and microstructures with potential as food

Astaxanthin is a light sensitive molecule which is already approved as a dietary supplement, with a wide range of biological properties in humans and fishes. In this paper, we provide strategies to photostabilize the pigment through its inclusion in easy-to-prepare and scale up formulations, comprising non-toxic excipients. Astaxanthin has been formulated in biocompatible hierarchically assembled structures in order to produce model food to humans and fishes. The structures present increasing complexity in the following order: 1) oily nanodroplets stabilized by phospholipids; 2) chitosan-coated nanoemulsions produced after coating the nanodroplets with chitosan; 3) carrageenan-coated nanoemulsions produced after coating chitosan-coated nanoemulsions with carrageenan; 4) calcium alginate and chitosan tripolyphosphate beads including one of the nanosystems. The sensitivity to UV light of astaxanthin in both the nanosystems and the hydrogels has been evaluated. The nanoformulations produce protection of astaxanthin from photodegradation in the order nanoemulsions > carrageenan-coated nanoemulsions > chitosan-coated nanoemulsions. Chitosan beads furnish higher protection to astaxanthin than alginate beads. In a preliminary assay in fishes, alginate beads of 2–3 mm and 160 μm containing the nutrient were supplied to adult Danio rerio and juvenile Eleginops maclovinus, respectively. Both living species showed high rates of capturing the hydrogels in the water column, corroborating the attractiveness of the formulations as food for fishes. Due to the easy preparation of the formulations (that exclude thermal treatment or the use of toxic solvents), and the flexibility of these hierarchically assembled materials to incorporate other active molecules (proteins, polyunsaturated fatty acids, oligoelements, oral vaccines, antibiotics, antiparasitic, etc.), we postulate these formulations as ideal platforms to create foods for humans and animal species.

Nano-ropinirole for the management of Parkinsonism: blood–brain pharmacokinetics and carrier localization

Aim: The aim of this study was to investigate the brain targeting potential of chitosan-coated oil in water nanoemulsions (CSNEROP) delivered intranasally in haloperidol-induced Parkinson's disease rat models. Methods: Chitosan-coated nanoemulsion (CSNEROP) was developed through aqueous titration followed by a high pressure homogenization method. Results: Gamma-scintigraphy study showed a significantly high mucoadhesive potential of CSNEROP and least for conventional and homogenized formulations. Confocal study showed deep localization of formulations in the brain confirming the permeation potential of CSNEROP. Pharmacokinetic results of CSNEROP in Wistar rat brain and plasma showed a significantly high (p** < 0.005) AUC0→24 and amplified Cmax over i.v treatment group. Neurobehavioral activity (rotarod and swim tests) and biochemical parameters (glutathion, TBARS and SOD) corroborated well with the pharmacokinetic results. The order of dopamine recovery in haloperidol-induced Wistar rats was found to be i.nCSNEROP group>i.nSolnROP group>i.vSolnROP group>haloperidol group. Conclusions: Finally, the investigation demonstrated that intranasal delivery of mucoadhesive nanocarrier might play as a potential candidate in the management of Parkinson's disease and related brain disorders.

Cationic nanoemulsions bearing ciprofloxacin surf-plexes enhances its therapeutic efficacy in conditions of E. coli induced peritonitis and sepsis.

Chitosan (CH) coated ciprofloxacin-sodium deoxycholate surfplex (CFn-SDC) loaded nanoemulsion (LE-CH-CFn-SDC) developed in order to improve tissue penetration of the CFn as well as to mop up the endotoxin (Lipopolysaccharides or LPS) released from bacteria during antibiotic treatment. Size and zeta potential was evaluated for nanoemulsions prepared by high-speed homogenization and sonication. Drug analysis in samples was done by HPLC equipped with fluorescence detector. All formulations were evaluated for any change in LPS induced NO and TNF-α release and ROS generation in J774 macrophages. The formulations were also evaluated for in-vitro killing efficiency on E-Coli. The efficacy of formulations in terms of survival and pharmacokinetics and inhibition of induction of cytokines was carried out in E-coli induced peritonitis model in rats. LE-CH-CFn-SDC interacted with LPS both by electrostatic and hydrophobic interactions. LE-CH-CFn-SDC resulted in reduction of endotoxin release and MIC values for E. coli. LE-CH-CFn-SDC also reduced NO and TNF-α as well as ROS generation by reducing the uptake of LPS in J774 macrophages. LE-CH-CFn-SDC improved CFn pharmacokinetics and tissue distribution, by reducing the bacterial burden, LPS and cytokines (TNF-α and IL-6) thereby improving survival in a rat model of E. coli induced peritonitis. In conclusion, this work highlights the effectiveness of the chitosan-coated nanoemulsion as intracorporeal approach for therapeutic intervention of E. coli induced peritonitis as well as in sepsis.