Self-emulsifying Nanoemulsions Research Articles

Development of a novel squalene/α-tocopherol-based self-emulsified nanoemulsion incorporating Leishmania peptides for induction of antigen-specific immune responses

Vaccination has emerged as the most effective strategy to confront infectious diseases, among which is leishmaniasis, that threat public health. Despite laborious efforts there is still no vaccine for humans to confront leishmaniasis. Multi-epitope protein/peptide vaccines present a number of advantages, however their use along with appropriate adjuvants that may also act as antigen carriers is considered essential to overcome subunit vaccines’ low immunogenicity. In the present study, a stable self-emulsified nanoemulsion was developed and double-adjuvanted with squalene and α-tocopherol. The prepared nanoemulsion droplets exhibited low cytotoxicity in a certain range of concentrations, while they were efficiently taken up by macrophages and dendritic cells in vitro as well as in vivo in secondary lymphoid organs. To further characterize nanoformulation’s potent antigen delivery capability, three multi-epitope Leishmania peptides were incorporated into the nanoemulsion. Peptide encapsulation resulted in dendritic cells’ functional differentiation characterized by elevated levels of maturation markers and intracellular cytokine production. Intramuscular administration of the nanoemulsion incorporating Leishmania peptides induced antigen-specific spleen cell proliferation as well as elicitation of CD4+ central memory cells, supporting the potential of the developed nanoformulation to successfully act also as an antigen delivery vehicle and thus encouraging further preclinical studies on its vaccine candidate potency.

Intranasal delivery of insulin by self-emulsified nanoemulsion system: In vitro and in vivo studies

The main objective of this research was to develop a self-emulsified nanoemulsion (SEN) dosage form of insulin where insulin is loaded into the lipid phase of the nanoemulsion for enhanced absorption through intranasal delivery. When loaded into the lipid droplets (oil phase), insulin can be protected from enzymatic degradation, can penetrate through the mucus gel barrier in a comparatively effective manner and can be absorbed through transcellular permeation along with paracellular route. To incorporate lipophilicity to insulin molecule, Ins-SPC (Soy-L-α-phosphatidylcholine) complex was prepared by solid dispersion method to load insulin into the oil phase. The cytotoxicity of SPC and the developed nanoemulsions was tested on the human nasal epithelial cells in vitro. An optimized formulation with high loading of insulin and low in vitro cytotoxicity was developed and characterized. To predict the absorption of insulin through nasal mucosa in vivo by the nanoemulsion system, the insulin-loaded SEN along with controls was tested for the transport through human nasal epithelial cell monolayer in vitro. The insulin-loaded SEN significantly (p < 0.01) enhanced the permeation of insulin by three times as compared to the insulin solution. The in vivo absorption of insulin after intranasal delivery of the insulin-loaded SEN was evaluated in anesthetized rats. The results show that the Cmax (maximum plasma concentration) and the bioavailability (relative to the subcutaneous delivery) of the insulin-loaded SEN was 255.9 µU/ml and 68 %, respectively, while the intranasal delivery of the insulin solution resulted in only 5.8 µU/ml of Cmax and 5% of relative bioavailability. Intranasal delivery of 3.6 IU/kg insulin-loaded SEN decreased the plasma glucose level remarkably, achieving a maximum reduction of 70%, and the glucose reduction activity lasted for the whole experimental period of 4 h. Histological examination of the nasal mucosa showed no apparent signs of toxicity at the site of administration after single dose of the insulin-loaded SEN. These results demonstrate that the insulin-loaded SEN significantly enhanced insulin absorption through intranasal delivery, indicating that the developed nanoemulsion system offers a favorable approach for intranasal delivery of insulin.

Effect of double bond in unsaturated long-chain monoglyceride in self-emulsified nanoemulsion on tight junction opening

Unsaturated long chain monoglycerides (LCMs) used to formulate self-emulsified nanoemulsion (SEN) are varied in their degree of unsaturation which may influence their activities on membrane permeability. Therefore, this study highlights the impact of double bond in LCMs on the tight junctions of Madin-Darby Canine Kidney (MDCK) cell monolayer. Two LCMs, Glyceryl monooleate (one double bond) and Glyceryl monolinoleate (two double bonds) were formulated into SENs (SEN2 and SEN3, respectively) with Glyceryl tricaprylate and PEG-40 hydrogenated castor oil (1:1:2), and compared to the SEN (SEN1) consisted of only Glyceryl tricaprylate and PEG-40 hydrogenated castor oil (1:4). Lucifer Yellow (LY, MW 457) or Fluorescein isothiocyanate–Dextran4K (FD4, MW 3000-5000) was added to the aqueous phase of the SENs and tested for their transport across the cell monolayer. The cumulative transport of LY and FD4 achieved by SEN2 and SEN3 were significantly (p < 0.05) higher than those in phosphate buffer, 0.5% PEG-40 hydrogenated castor oil, and SEN1, indicating that the tight junctions were opened by the SENs containing LCMs. The cumulative transport of FD4 achieved by SEN3 was also 2-3 folds higher than SEN2 (p < 0.05). Therefore, LCM with two double bonds have greater potency of opening tight junction than LCM with a single double bond in SEN formulation.

Self-Emulsified Nanoemulsion for Vaginal Administration: In Vitro Study of Effect on Lactobacillus acidophilus

Self-emulsified nanoemulsions (SENs), one of the promising lipid-based drug delivery systems may be used to deliver drugs through vaginal route. Vaginal cavity remains healthy because of the defensive action by its microflora against the pathogenic infections, and any disturbance to this microflora by the delivery systems gives invitation to the infections. In the present study, the growth inhibition and cytotoxic effects of two SENs and their components on L. acidophilus were evaluated. The two SENs showed inhibitory effects on the growth of L. acidophilus in a concentration-dependent manner when tested at the concentration range of 0.1–5.0%. The SEN composed of medium chain mono/di-glyceride had greater inhibitory effect than the one composed of long chain monoglyceride. The study on the effect by the individual lipids with the surfactant Kolliphor® RH40 further confirmed that the growth inhibitory and cytotoxic effects were in the order of Capmul® MCM > Maisine® CC > Miglyol® 810 > Kolliphor® RH40. Both OD600 and CFU counting were used to measure the viability of the culture. The results from the two methods were in good correlation except when there was no growth, suggesting OD600 can be used when there is no complete growth inhibition.

Effect of fatty acid ester structure on cytotoxicity of self-emulsified nanoemulsion and transport of nanoemulsion droplets

Although the cytotoxic and permeation enhancing activities of some fatty acid esters (FAEs) have been studied individually, there lacks systemic studies on how their molecular structures can affect these activities and how the activities would change when FAEs form micelles or nanoemulsion droplets. Therefore, this study aims to address these issues by investigating the cytotoxic and permeation enhancing effects of twenty-six FAEs in lipid droplets on Madin-Darby Canine Kidney (MDCK) cell monolayer. The effect of FAEs on the cytotoxicity and the transport of nanoemulsion droplets depends on how easy the FAE monomers can diffuse out of the droplets and perturbate into the cellular membrane, which determined by the critical structures of FAEs including hydrophilic head size, chain length, number of chains, and number of double bond(s) in the chain. The less the intermolecular attraction, the easier the monomer to diffuse out of the lipid droplets and the more interaction with the cell monolayer. Fatty acid monoester (FAME) with less intermolecular attraction than di- and tri-esters had more cytotoxic and higher permeation enhancing effects. Among the FAMEs, the cytotoxicity and permeation enhancing effects were in the order: medium-chain with small hydrophilic head (propylene glycol and glycol) > long-chain with two double bonds > medium-chain with large head (sorbitan, polyethylene glycol, sucrose, and PEGylated sorbitan) ≈ long-chain with single double bond > saturated long-chain, which are consistent with the intermolecular attraction force (low-high). However, the more kinks in the monomer’s tail caused by the double bond, the less the monomer can perturbate into the cell membrane, resulting in less cytotoxicity and permeation enhancement. In conclusion, the FAEs with less intermolecular attraction and straight chain have more cytotoxic and permeation enhancing effects when formulated in nanoemulsion.

Protection of Protein Drugs by Self-Emulsified Nanoemulsion Against Proteolysis

The present study aimed to develop a self-emulsified nanoemulsion for salmon calcitonin (sCT) for non-invasive delivery. Hydrophobic ion pairing method was used to form hydrophobic complex of sCT with 4 counter ions (oleate, deoxycholate, docusate and tripolyphosphate). The partition coefficient and dissociation of the complexes in water of various pH were investigated. The complex-loaded nanoemulsions were characterized for droplet size, leakage of sCT from the droplets, and protection of sCT from enzymatic degradation. The results show that all the counter ions could form complexes with sCT with a complexation efficiency about 95% at pH 8.0. The complexes significantly increased the partition coefficient of sCT. The dissociation of the complexes in water was pH-dependent. At pH 6.8 and 7.4, the dissociation was negligible. At pH 1.0, the dissociation was 71%, 8%, 37% and 50% for sCT-polyphosphate, sCT-docusate, sCT-oleate and sCT-deoxycholate, respectively. The developed nanoemulsions had a size in the range of 27–62 nm. The leakage of sCT from the nanodroplets into the aqueous phase depended on the lipophilicity of the counter ions: 60%, 56%, and 24% leakage for sCT-docusate, sCT-oleate and sCT-deoxycholate, respectively. The nanoemulsion protected sCT from enzyme degradation when loaded inside the droplets, but not the leaked sCT.

Potentiation of raloxifene cytotoxicity against MCF-7 breast cancer cell lines via transdermal delivery and loading on self-emulsifying nanoemulsions

Purpose: To enhance raloxifene (RLX) delivery and cytotoxicity against breast cancer (MCF-7) cell lines.&#x0D; Methods: This was a solubility study of RLX in different oils, surfactants, and co-surfactants. Twelve formulae were tested to reach the smallest globular size, and hydroxypropyl methylcellulose, (HPMC), and Carbopol 947 polymers were tested for formation of transdermal films. The formula with the lowest size was compared with raw RLX in diffusion studies using a Franz diffusion cell. Finally, a cytotoxicity study against MCF-7 breast cancer cell lines was conducted.&#x0D; Results: The maximum solubility of RLX was in Tween 80, peppermint oil, and PEG 200; therefore, these were the main components of the 12 formulations. The release of RLX loaded on the selfnanoemulsion drug delivery system (SNEDDS) was increased 3-fold compared with raw RLX.Cytotoxicity results revealed that RLX SNEDDs decreased MCF-7 cell survival by approximately 40 %, compared with raw RLX (control), which augmented the RLX suppression of breast cancer cell lines.&#x0D; Conclusion: Improvement in RLX cytotoxicity is a novel strategy to suppress breast cancer.&#x0D; Keywords: Raloxifene, Osteoporosis, Bioavailability, Nanoemulsion, Nanoparticles

Novel cuminaldehyde self-emulsified nanoemulsion for enhanced antihepatotoxicity in carbon tetrachloride-treated mice

Cuminaldehyde self-emulsified nanoemulsion (CuA-SEN) was prepared and optimised to improve its oral bioavailability and antihepatotoxicity. Cuminaldehyde self-emulsified nanoemulsion was developed through the self-nanoemulsification method using Box-Behnken Design (BBD) tool while appropriate physicochemical indices were evaluated. The optimised CuA-SEN was characterised via droplet size (DS), morphology, polydispersity index (PDI), zeta potential (ZP), entrapment efficiency, in-vitro release, and pharmacokinetic studies while its antihepatotoxicity was evaluated. Cuminaldehyde self-emulsified nanoemulsion with acceptable characteristics (mean DS-48.83 ± 1.06 nm; PDI-0.232±0.140; ZP-29.92±1.66mV; EE-91.51±0.44%; and drug-loading capacity (DL)-9.77±0.75%) was formulated. In-vitro drug release of CuA-SEN significantly increased with an oral relative bioavailability of 171.02%. Oral administration of CuA-SEN to CCl4 -induced hepatotoxicity mice markedly increased the levels of superoxide dismutase, glutathione and catalase in serum. Also, CuA-SEN reduced the levels of tumour necrosis factor-alpha and interleukin-6 in both serum and liver tissues while aspartate aminotransferase, alanine aminotransferase and malonaldehyde levels were significantly decreased. These findings showed that the improved bioavailability of cuminaldehyde via SEN provided an effective approach for enhancing antioxidation, anti-inflammation and antihepatotoxicity of the drug.

Novel liquid-liquid extraction and self-emulsion methods for simplified isolation of extra-virgin olive oil phenolics with emphasis on (-)-oleocanthal and its oral anti-breast cancer activity.

Epidemiological and clinical studies compellingly documented the ability of Mediterranean diet rich in extra-virgin olive oil (EVOO) to reduce breast and colon cancers incidence, cardiovascular diseases, and aging cognitive functions decline. (-)-Oleocanthal (OC) and other EVOO phenolics gain progressive research attention due to their documented biological effects against cancer, inflammations, and Alzheimer’s disease. There is no simple, reliable, and cost-effective isolation protocol for EVOO phenolics, which hinder their therapeutic applications. This study develops novel methods to isolate OC and other EVOO phenolics. This includes the use of ultra-freezing to eliminate most EVOO fats and the successful water capacity to efficiently extract OC and EVOO phenolics as self-emulsified nano-emulsion. Subsequent resin entrapment and size exclusion chromatography afforded individual EVOO phenolics in high purity. OC in vitro and in vivo oral anti-breast cancer (BC) activities validated its lead candidacy. Effective isolation of EVOO phenolics provided in this study will facilitate future preclinical and clinical investigations and stimulate the therapeutic development of these important bioactive natural products.

Mechanism and Structural Factors of Lipid and Surfactant in the Formation of Self-Emulsified Nanoemulsion

The study aims to elucidate the mechanism and the role of the molecular structure of surfactants and lipids in the formation of oil-in-water (o/w) self-emulsified nanoemulsions (SENs). The hypothesis is that the overall change of Gibb's free energy (ΔGf) during the mixing of the lipid and surfactant, the formation of the interface between the lipid nanodroplets and water, and the dispersion of the lipid nanodroplets into the water are the determinants of the formation of SEN, which are the result of the intermolecular interactions between the excipients involved. Various lipids and surfactants of different structures were studied for the possible formation of SEN. The results demonstrate that the formation of SEN requires (1) enough hydrophobic attractions between the surfactant molecule and the lipid molecule, which can break up the lipid-lipid and surfactant-surfactant intermolecular binding forces and (2) the surfactant to be able to associate with enough water molecules which can effectively cover the lipid droplets' surface to prevent coalescence.

Impact of digestion on the transport of dextran-loaded self-emulsified nanoemulsion through MDCK epithelial cell monolayer and rat intestines

Many of the lipids and surfactants used to prepare the self-emulsified nanoemulsion (SEN) are subjected to the gastro-intestinal enzymatic digestion, which may affect the absorption of the loaded drug. The present study was to investigate the impact of such digestion on the transport of hydrophilic macromolecules (10-kDa dextran as the model compound) loaded in SEN through the MDCK cell monolayer and ex-vivo rat intestines. FITC-labeled dextran (FD) was loaded inside the inner oil phase of SEN by the formation of FD-phospholipid solid dispersion (FDPS). After digestion, the droplet size increased from 31.06 ± 2.10 nm to 494.6 ± 22.1 nm, and the FD content in the external aqueous phase increased from 41.6 ± 4.2% to 61.1 ± 4.4%. Compared to the FD solution, SEN without digestion enhanced the transport of FD through MDCK cell monolayer 4.1 times and through rat intestines 3.0–7.4 times. However, the digestion reduced the transport of FD 3.5 times through MDCK cell monolayer and 1.3–2.0 times through rat intestines, compared to that without digestion. This reduction was due to the destruction of lipid nano-droplets and release of FD to the external aqueous phase of SEN. This finding should be considered when SEN is used as a delivery system for hydrophilic macromolecules.

Enhancement of physical stability and bioaccessibility of tangeretin by soy protein isolate addition.

Soy protein isolate (SPI) was selected to fabricate supersaturated self-emulsifying nanoemulsions, aiming to enhance physical stability and bioaccessibility of hydrophobic tangeretin. Dissolution studies demonstrated that tangeretin had the highest solubility in Tween 80, followed by oil phase solutions, and polymer solutions. Supersaturated tangeretin in oil phases easily formed crystals. That metastable zone was found to vary with its initial concentrations. After encapsulation by nanoemulsions, the addition of glycerol compressed the retention amount of tangeretin from 76% to 53%, but benefited the transparency. Whereas, the combination of glycerol and SPI could not only maintain high-loading tangeretin (>85%), but also provide high transparency for nanoemulsions. When tangeretin concentration was 4.83mM, combination of 50% glycerol and 1% SPI could maintain around 88% tangeretin in the nanoemulsion within one month. Its bioaccessibility of different systems were at 60-65%. These findings can provide useful information for protein to be a potential precipitation inhibitor.

Abstract 5405: Self-emulsifying nanoemulsions target intracranial glioblastoma tumors: In vivo optical imaging and fluorescence microscopy evidences

Abstract Introduction: As the most aggressive form of brain tumor, glioblastoma has limited treatment options due to highly invasive nature, the largely impermeable blood-brain barrier (BBB) and presence of efflux transporters at BBB. We aimed to investigate the ability of self-emulsifying nanoemulsions to delivery BBB-impermeable molecules to the brain of orthotopic U87-luc human glioblastoma xenografts. Methods: Oil-in-water nanoemulsions containing medium-chain triglycerides oil and co-surfactants (polysorbate-80:transcutol 6:4 w/w) were prepared by a spontaneous self-emulsification method. Luciferase transfected U87-luc human glioblastoma cells were injected intracranially as well as subcutaneously to Nu/Nu mice and tumor growth was monitored by bioluminescence imaging. Seventeen days after tumor implantation, near infrared dye DiD labeled nanoemulsions and size matched pegylated stealth liposomes were injected intravenously in U87-luc xenografts and the real-time biodistributions of formulations were monitored by whole animal imaging. Results: Nanoemulsions had a mean particle size ∼120 nm with slightly negative charge (-10 mV). It is biocompatible with no hemolysis in vitro. In orthotopic U87-luc xenograft tumor model, intravenous administration of DiD labeled nanoemulsions revealed enhanced accumulation of the nanoemulsions in the brain and subcutaneous tumor at 1-9 hours post injection, comparing to that of the free dye and pegylated liposomes. The brain accumulations of nanoemulsions were persistent till 96 hours. The ex vivo fluorescence microscopy analysis showed that the DiD-nanoemulsions specifically accumuolated at the U87-luc tumor region, not at the surrounding normal brain tissue. In the normal CD-1 mice with intact BBB, the nanoemulsions accumulation in the brain was still significantly higher than the free dye, albeit lower than the brain level achieved in the U87-luc xenografts. The nanoemulsions were non-toxic to both CD-1 and Nu/Nu mice after daily repeated doses for three weeks. Conclusion: Our data indicates that nanoemulsion is a promising nanocarrier to deliver imaging or therapeutic agents for the diagnosis or systemic treatment of GBM. Citation Format: Xinli Liu, Hussaini Syed Sha Qhattal, Tanvirul Hye. Self-emulsifying nanoemulsions target intracranial glioblastoma tumors: In vivo optical imaging and fluorescence microscopy evidences. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5405. doi:10.1158/1538-7445.AM2014-5405