Multiple daily injections via subcutaneous route are the primary modes of insulin delivery for patients with Diabetes Mellitus. While this process is invasive, painful and may cause patients to develop lipohypertrophy at injection site, the perception of fear surrounding this process causes patients to delay in initiation and remain persistent with insulin therapy over time. Moreover, poor glycemic control may often lead to acute complications, such as severe hypoglycemia and nocturnal hypoglycemia, especially in older patients with diabetes. To address the imperative need for a patient-convenient non-invasive insulin therapy, an insulin-loaded arginine-coated self-emulsifying nanoglobule system (INS-LANano) was developed for nasal delivery of insulin with a biodegradable cationic surfactant—Lauroyl Ethyl Arginate (LAE). Incorporation of LAE resulted in formation of positively charged nanoglobules with L-arginine oriented on the surface. LANano enabled binding of insulin molecules on the surface of nanoglobules via an electrostatic interaction between negatively charged α-helix and LAE molecules at physiological pH. INS-LANano showed a hydrodynamic diameter of 23.38 nm with a surface charge of +0.118 mV. The binding efficiency of insulin on LANano globules was confirmed by zeta potential, circular dichroism (CD) spectroscopy and centrifugal ultrafiltration studies. The attachment of insulin with permeation-enhancing nanoglobules demonstrated significantly higher in vitro permeability of insulin of 15.2% compared to insulin solution across human airway epithelial cell (Calu-3) monolayer. Upon intranasal administration of INS-LANano to diabetic rats at 2 IU/kg insulin dose, a rapid absorption of insulin with significantly higher Cmax of 14.3 mU/L and relative bioavailability (BA) of 23.3% was observed. Therefore, the INS-LANano formulation significant translational potential for intranasal delivery of insulin

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.

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 (R) RH40 further confirmed that the growth inhibitory and cytotoxic effects were in the order of Capmul (R) MCM > Maisine (R) CC > Miglyol (R) 810 > Kolliphor (R) 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. (C) 2020 American Pharmacists Association (R). Published by Elsevier Inc. All rights reserved.

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.

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) approximate to 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.

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.

Literature review shows that various media have been used for in vitro transport study, but their impact on the monolayer integrity and permeability has been somehow neglected, and there is no systemic study on this subject. This study aims to investigate the impact of 6 commonly used media on Madin-Darby Canine Kidney cell viability and adherence, and the permeation of mannitol and propranolol. Dulbecco's modified Eagle medium (DMEM), cMedium (DMEM with serum), Hanks' balanced salt solution (HBSS), HBSS- (without Ca2+/Mg2+), Dulbecco's phosphate-buffered saline (DPBS), and DPBS- (without Ca2+/Mg2+) exhibited no cytotoxicity. Cell detachment was observed in 4 media in the order of DPBS- approximate to HBSS- > DPBS > HBSS. HBSS-/DPBS- caused 70% TEER reduction and 4-fold higher apparent permeability (P-app) of mannitol than the other media. The addition of D-glucose in DPBS- decreased the P-app of mannitol. Cations and glucose negligibly influenced the P-app of propranolol. The addition of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) in HBSS increased the P-app of both mannitol and propranolol for 2-3 folds without TEER reduction. In conclusion, the media compositions (Ca2+/Mg2+, glucose, vitamins, amino acids, and HEPES) can affect the monolayer integrity and permeability. DMEM and cMedium without HEPES are hence suggested as the media in transport study to avoid the potential impact on the monolayer integrity and permeability by the media. (c) 2020 American Pharmacists Association (R). Published by Elsevier Inc. All rights reserved.

This study aimed to investigate the following factors affecting the cytotoxicity of Capmul®MCM (C8/10MD) in self-emulsified nanoemulsions (SENs): concentration, triglycerides, and droplet size, and how these factors influence permeability of lipid droplets. Two triglycerides (C8T and C18T) and six formulations were used: SEN1(C18T:C8/10MD:Kolliphor®RH40 = 7:3:10, 257 nm), SEN2(C8T:C8/10MD:Kolliphor®RH40 = 1:1:2, 30 nm), SEN3(C18T:Kolliphor®RH40 = 1:4, 26 nm), SEN4(C8T:Kolliphor®RH40 = 1:4, 27 nm), SEN5(C8/10MD:Kolliphor®RH40 = 1:1, 120 nm) and SEN6(C8/10MD:Kolliphor®RH40 = 1:4, 15 nm). There was no cytotoxicity from SEN3-4 (5% preconcentrate), but there was concentration-dependent cytotoxicity from the SENs containing C8/10MD. The presence of triglycerides in SEN1-2 reduced the toxicity of C8/10MD as compared to SEN5-6. SEN2 and SEN6 showed higher toxicity than SEN1 and SEN5, respectively, due to the smaller size. 14C-Triolein-loaded droplets from SEN1 (0.45-0.6% C8/10MD) and SEN2 (0.3-0.6% C8/10MD) could permeate across the MDCK monolayer, resulted in intact droplets and radioactivity in the receiver chamber. The TEER was reduced as the C8/10MD concentration increased, and not recovered after 24 h from SEN1 (0.6% C8/10MD) and SEN2 (0.45-0.6% C8/10MD), resulted in significantly higher (p < 0.05) permeability of 14C-mannitol and 3H-propranolol compared to the treatment by the medium. In conclusion, Capmul®MCM caused concentration-dependent cytotoxicity and permeation enhancement, which were reduced with the presence of triglycerides and increase in droplet size.

Fluorescein isothiocyanate-labeled insulin (FITC-insulin) has been widely used for bioanalytical applications. Due to the high cost of commercial FITC-insulin and tedious labeling procedures described in the literature, there is still a need to develop a cost effective, reliable and quick labeling method for insulin. The purpose of the present work was to develop a quick and affordable method for FITC labeling of human insulin and to determine the effect of different conjugations of FITC to human insulin on its permeability through the MDCK cell monolayer. FITC labeling of insulin gives mono-, di- or tri-conjugates depending on the reaction time and the molar ratio of FITC:insulin. Mono-conjugate with unlabeled insulin, mixture of di- and tri-conjugate, and tri-conjugate with very little amount of di-conjugate were synthesized in less than 4 h. Degree of conjugation had an effect on the permeability of insulin through the MDCK cell monolayer. Mono-conjugate had higher permeability than the unlabeled insulin due to increase in partition coefficient. However, tri-conjugate showed lower permeability than the unlabeled insulin due to the increase in molecular weight.

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.

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.

This study aims to investigate the transport of lipid nano-droplets through MDCK epithelial cell monolayer. Nanoemulsions of self-nano-emulsifying drug delivery systems (SNEDDS) labeled with radioactive C18 triglyceride were developed. The effect of droplet size and lipid composition on the transport was investigated. The results showed that the lipid nano-droplet transport through MDCK cell monolayer was as high as 2.5%. The transport of lipid nano-droplets was higher for nanoemulsions of medium chain glycerides than the long chain glycerides. The transport was reduced by more than half when the average lipid nano-droplet size increased from 38nm to 261nm. The droplet size measurement verified the existence of lipid nano-droplets in the receiver chamber only when the nanoemulsions were added to the donor chamber but not when the surfactant or saline solution was added. Cryo-TEM images confirmed the presence of lipid nano-droplets in both donor and receiver chamber at the end of transport study. In conclusion, lipid nano-droplets can be transported through the cell monolayer. This finding may help to further explore the oral and other non-invasive delivery of macromolecules loaded inside SNEDDS.

The aim of this study is to develop a simple, reliable, timesaving and cost-effective method for separation of the external aqueous phase from o/w nanoemulsions. The method is based on centrifugation through a hydrophilic membrane. Five o/w nanoemulsions (droplet size of 20-200nm) with radio-labeled C18 triglyceride were prepared. These nanoemulsions were placed in tubes with cellulose membranes (10-50K molecular weight cut-off) and centrifuged at 250-3000g for 5-15min. The filtrates were analyzed for the presence of lipid droplets and radioactivity. The applicability of this method was tested on FITC-dextran-loaded nanoemulsion (FITC-dextran was expected to be in the lipid droplets) and a blank nanoemulsion mixed with FITC-dextran (FITC-dextran was expected to be in the external aqueous phase). The results show that no droplets were detected in the filtrate. The radioactivity in the filtrate was in the range of 0.3-2.9%. The volume of the filtrate increased with increase in the membrane pore size, centrifugal force and time, and decrease in droplet size. There was 41.6±4.2% and 98.4±0.8% FITC-dextran in the aqueous phase of the FITC-dextran-loaded nanoemulsion and the blank nanoemulsion mixed with FITC-dextran, respectively. In conclusion, centrifugation with hydrophilic membrane is an efficient and convenient method for the separation of the external aqueous phase from o/w nanoemulsions with droplet sizes in the range of 20-200nm.

To develop a live oral delivery system of Glucagon like peptide-1 (GLP-1), for the treatment of Type-2 Diabetes.

PURPOSE:To establish the biodistribution profile of the PLGA nanoparticles with dual surface modifications of PEG and folic acid (FA) in mice xenografted with MDA-MB-231 human breast cancer cells with high expression of folate receptor (FR); and to illustrate that the modified nanoparticles can target the loaded indocyanine green (ICG) to the tumor with high FR expression. METHODS:ICG-loaded nanoparticles were prepared with PLGA (non-modified nanoparticles, NM-NP) or mPEG-PLGA and FA-PLGA (dual modified nanoparticles, DM-NP). Biodistribution of the ICG-loaded nanoparticles (1.25 mg/kg) after i.v. injection was investigated on athymic mice transplanted with MDA-MB-231 tumor. RESULTS:ICG concentration in plasma from the DM-NP group was significantly (p<0.05) higher than the NM-NP group from 90 min to the end of the study (12 h). After 4 h, the drug concentration in the tumor tissue from the DM-NP started to be significantly (p<0.05) higher than the NM-NP until 12 h. Compared to the NM-NP, the DM-NP increased the AUC(0-12 h) in plasma by 245% and the AUC(0-12 h) in tumor by 194%, while decreased the AUC(0-12 h) in liver by 13%. CONCLUSION:The accumulation of DM-NP into the tumor was significantly higher than NM-NP due to the long circulation and FR-mediated uptake.

Hedyotis diffusa or Oldenlandia diffusa (spreading hedyotis) is one of the most commonly used anticancer herbs. Its clinical use has a history more than several thousand years. It contains flavones, anthraquinones, polysaccharides, and other compounds possessing anticancer activities. In most cases, it is used together with other herbs. About 15% of the anticancer herbal formulas used in China contain this herb. Both pre-clinical and clinical studies have established the efficacy and safety of spreading hedyotis in treating various cancers including stomach cancer, liver cancer, lung cancer, esophagus cancer, and leukemia. It can directly inhibit the growth of various cancer cells and induce apoptosis both in vitro and in vivo. It shows selective cytotoxicity against cancerous cells. It can suppress some oncogenes and up-regulate anti-oncogenes. It also has immune modulation functions against cancer. It enhances the activities of natural killer cells and macrophages, promotes the proliferation of spleen cells, and up-regulates interleukin-2 and tumor necrosis factor-alpha. Clinical outcomes have demonstrated that it can enhance the efficacies and reduce the adverse effects (i.e. white blood cell decrease, nausea/vomit) by the conventional chemotherapies. It is also effective in relieving cancerous pain and fever. The commonly used clinical doses of 30–60 g/day usually do not cause any considerable adverse effects.

For the determination of in vivo beta-lactamase activity, a high-performance liquid chromatographic (HPLC) method was established, and the pharmacokinetics of beta-lactamase after intravenous administration to the rats was analyzed using this standardized HPLC method. The plasma samples containing beta-lactamase were reacted with ampicillin (substrate) and further processed to make them fluorescent. The fluorescent compound of interest was separated using HPLC at room temperature using the excitation and the emission wavelengths of 410 nm and 475 nm, respectively. For the pharmacokinetic studies, 252 mU of beta-lactamase solution was administered to the rats through the tail vein injection (n = 6). The blood samples were withdrawn from the tail vein at different time points and analyzed by HPLC for beta-lactamase activity. For the HPLC method of beta-lactamase in plasma samples, the peak area showed a good correlation within the concentration ranges of 0.126-12.6 mU/mL (10-1000 ng/mL). The coefficients of variations were within 0.56-6.24, and the percentage recovery were within 102-107. After the intravenous injection, plasma concentration at the time zero (C(p0)) was 11.47 +/- 0.48 mU/mL, and no beta-lactamase was detected 24 h after the injection. The volume of distribution (V(d)) was 22 mL. An elimination half-life (t(1/2)) of 4.12 +/- 0.5 h and AUC of 79.4 +/- 12.9 mU.hr/mL were also calculated. The HPLC fluorimetric method was a very sensitive and reproducible method for the detection of beta-lactamase in plasma. The disposition of beta-lactamase after intravenous administration followed one-compartment and first-order kinetics.

A probiotic bacterium, Lactococcus lactis subsp. lactis (L. lactis) transformed with plasmid ss80, which made it capable of synthesizing and secreting β-lactamase, a 29 kDa protein, was used to deliver β-lactamase via vaginal route. The vaginal absorption of β-lactamase in rats was studied when delivered by this L. lactis system and compared to the β-lactamase solution with or without the untransformed L. lactis. The vaginal administration of 1.2 × 10(7), 3 × 10(7), and 8 × 10(7) colony forming units (cfu) of L. lactis resulted in the amount absorbed of 77, 194, and 216 mU, with the respective doses. C(max), mean retention time and mean absorption time of β-lactamase were also increased with the increase in the cfu of L. lactis administered. These results have demonstrated that L. lactis can significantly increase (p < 0.01) the β-lactamase vaginal absorption as compared to the β-lactamase solution, which is probably due to the adhesion of L. lactis to and continuous synthesis and delivery of β-lactamase directly to the vaginal mucosa. In conclusion, transformed normal flora may be an efficient method to deliver protein drugs through the vaginal route.

Genetically modified Lactococcus lactis (L. lactis), a probiotic bacterium, able to secrete beta-lactamase (29 kDa), was used as a vector for the oral delivery of beta-lactamase to the rats. Three different doses of L. lactis were administered to the rats, and the resulted beta-lactamase oral bioavailability was studied, and compared to the solution form. The oral administration of 1.2 x 10(7), 3 x 10(7), and 8 x 10(7) colony-forming units of L. lactis led to 145, 209, and 364 mU of beta-lactamase absorbed, and the corresponding bioavailability was 8.7%, 15.5%, and 20.8% based on the in vitro production of beta-lactamase by L. lactis. The oral administration of 504 mU and 1008 mU beta-lactamase free solution resulted in 30 and 47 mU absorbed, a bioavailability of 5.9% and 4.7%, respectively. L. lactis significantly (p < 0.01) increased the oral bioavailability compared to the free solution form. A significant (p < 0.01) increase in the MAT value as compared to the solution, demonstrated that L. lactis can be used as a sustained delivery system. In conclusion, there is a linear relationship between L. lactis dose and these absorption PK parameters within L. lactis dose range of the current study.

To develop a self-nanoemulsifying drug delivery system (SNEDDS) for protein drugs, and particularly, to test the in vitro transport of beta-lactamase (BLM) by SNEDDS across the cell monolayer. Fluorescently labeled BLM (FITC-BLM), a model protein, formulated into 16 SNEDDS preparations through a solid dispersion technique were studied for transport across MDCK monolayer. All the SNEDDS nanoemulsions resulted in higher transport rate than the free solution. The transport rate by SNEDDS depends on the SNEDDS composition. SNEDDS NE-12-7 (oil: Lauroglycol FCC, surfactant: Cremophor EL and a cosurfactant: Transcutol HP) at the ratio of 5:4:3, rendered the highest transportation rate, 33% as compared to negligible transport by the free solution. FITC-BLM solution mixed with the surfactant and the cosurfactant of SNEDDS NE-12-7 or with blank SNEDDS NE-12-7 increased the transport only by 3.3 and 1.5 folds, respectively, compared to free solution alone. It was found that the monolayer integrity was not compromised in the presence of SNEDDS NE-12-7 or its surfactant/cosurfactant. The SNEDDS significantly increased the transport of FITC-BLM across MDCK monolayer in vitro. SNEDDS may be a potential effective delivery system for non-invasive protein drug delivery.