International Journal of Pharmaceutics 493 (2015) 347–356

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International Journal of Pharmaceutics
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Pharmaceutical nanotechnology

Novel self-assembled nano-tubular mixed micelles of Pluronics P123,
Pluronic F127 and phosphatidylcholine for oral delivery of nimodipine:
In vitro characterization, ex vivo transport and in vivo pharmacokinetic
Emad B. Basalious* , Rehab N. Shamma
Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Kasr El-aini street, Cairo 1156, Egypt



Article history:
Received 16 May 2015
Received in revised form 25 July 2015
Accepted 28 July 2015
Available online 1 August 2015

Subarachnoid hemorrhage (SAH) is a major cause of death in patients suffering from stroke. Nimodipine
(NM) is the only FDA-approved drug for treating SAH-induced vasospasm. However, NM suffers from
poor oral bioavailability (5–13%) due to its low aqueous solubility, extensive first pass metabolism and
short elimination half-life (1–2 h). The objective of this study was to develop NM-loaded Pluronic/
phosphatidylcholine/polysorbate 80 mixed micelles (PPPMM) that can solubilize NM in aqueous media
even after dilution, prolong its circulation time, improve its bioavailability and eventually help in
targeting it to the brain tissue. PPPMM formulations were prepared using the thin film hydration
technique, and evaluated for drug payload, solubilization efficiency (SE), micellar size, zeta potential,
transmission electron microscopy (TEM) and ex vivo transport through rat intestine. The selected NMloaded PPPMM, containing PC to Pluronics1 molar ratio of 75:25, showed a drug payload, SE, micellar size
and zeta potential of 1.06  0.03 mg/mL, 99.2  2.01%, 571.5  11.87 nm and 31.2  0.06 mv, respectively.
The selected formulation had a much larger hydrophobic core volume for solubilization of NM and
exhibited the highest NM transport. TEM micrographs illustrated the formation of highly flexible nanotubular mixed micelles (NTMM). The in vivo pharmacokinetic study showed greater bioavailability of NM
in plasma (232%) and brain (208%) of rats from NM-loaded PPPMM compared to that of the drug solution
due to the efficiency of flexible NTMM to enhance absorption of NM from the intestinal mucosa. The
significant increase in drug solubility, enhanced drug absorption and the long circulation time of the
NTMM could be promising to improve oral and parenteral delivery of NM.
ã 2015 Elsevier B.V. All rights reserved.

Nano-tubular mixed micelles
Thermodynamic stability of micelles
Subarachnoid hemorrhage

1. Introduction
Subarachnoid hemorrhage (SAH) is a serious, life-threatening
type of stroke where cerebral vasospasm remains a serious
complication and a major cause of death and disability in these
patients. Nimodipine (NM) is a dihydropyridine calcium antagonist
with therapeutic indications for cerebrovascular spasm, stroke and
migraine (Gelmers, 1985; Langley and Sorkin, 1989). Recently, NM
has been shown to be effective in ameliorating memory
degeneration and preventing senile dementia in the old age
(Pantoni et al., 2000; Zhang, 1993). NM is also used for cerebral
malaria, cerebral vasospasm, acute ischemic stroke, and migraines
(Ahmed et al., 2000; Cabrales et al., 2010; Togha et al., 2012; Wolf

* Corresponding author.
E-mail addresses:, (E.B. Basalious).
0378-5173/ ã 2015 Elsevier B.V. All rights reserved.

et al., 2010). NM is the only FDA-approved drug for treating SAHinduced vasospasm. The oral administration of NM has several
limitations and disadvantages. NM belongs to Class II of the
Biopharmaceutical Classification System (BCS) with the typical
characteristics of high permeability and poor solubility (Fu et al.,
2013). The substantial factors that limit its oral bioavailability (5–
13%) and clinical efficacy are the very low aqueous solubility
(3.86 mg/mL) and the extensive first pass metabolism in the liver
(Soliman et al., 2010; Sun et al., 2008). NM was also found to have a
very short half-life (1–2 h) with subsequent need for frequent
dosing (every 4 h) (Langley and Sorkin, 1989).
Among several drug carriers currently under investigation for
improved drug absorption and efficacy, nanocarriers hold the
greatest promise (Soliman et al., 2010). Inclusion of poorly soluble
drugs into polymeric mixed micelles has been found to be very
attractive concept for solubilization and bioavailability enhancement (Wei et al., 2009; Zhao et al., 2012). Polymeric micelles are

Formula Total Pluronics mixture (mg) PC (mg) PC: Pluronics molar ratio Tween 80 (mg) Payload (mg/ mL) SE (%) PPPMM-1 PPPMM-2 PPPMM-3 PPPMM-4 PPPMM-5 PPPMM-6 Liposome PMM 100 100 100 200 200 200 – 200 33 66 100 33 66 100 100 – 75:25 85:15 90:10 60:40 75:25 80:20 – – 6.5  11. Pluronics1 P123/F127 mixed micelles (PMM) are considered to be kinetically stable due to the stabilization effect of long polyethylene oxide (PEO) chains of hydrophilic F127 blended with P123 in micelles which might prevent the stacking of cylindrical aggregates formed by the long polypropylene oxide (PPO) chains of P123 (Wei et al. 2009).97  0.B.10  0..N. (Marczylo et al.35  0.56 92. and 100 mg) and Pluronic1 (F127 and P123) mixture in the ratio 2:1 (100. .06 30. Ethanol and Polysorbate 80 were from El-Nasr Chemical Co. These systems exhibit many advantages such as targeting ability. 2011. Shamma / International Journal of Pharmaceutics 493 (2015) 347–356 nanocarriers based on amphiphilic block copolymers of hydrophilic and hydrophobic chains that self-assemble in water above the critical micelle concentration (CMC) (Abdelbary and Tadros.08 98. Polysorbate 80 (20% of the phosphatidylcholine content) was dissolved in the solution. 2012).10 0. However.05 93.000 molecular weight cut off) was purchased from Spectrum Laboratories Inc.09 761. Investigations have been carried out to elucidate the formation of spherical and tubular micelles and studying the effect of the different morphologies on drug permeation.. Sugawara et al.08 0.99 549. they can also inhibit P-gp enhancing drug absorption (Chiappetta and Sosnik. solubilization efficiency (SE).03 0. no attempt has been reported to increase the solubilizing efficiency of PMM through incorporation of PC in the micellar structure. 2005...1  1. However. PMM and liposome and their characterization values. Pluronic mixed micelles have a core–shell structure which enables the system to incorporate poorly soluble drugs. it was reported that polysorbate 80 acts also as pglycoprotein and/or CYP450 enzymes inhibitors decreasing the intestinal efflux and drug biotransformation (Basalious et al.05 25. Flawil. the low stability of these polymeric micelles upon dilution in the bloodstream or gastrointestinal fluids and the consequent drug precipitation circumvent their use in drug delivery.7  2. micellar size.3  0.09 22. Disodium hydrogen phosphate was procured from Merck (Darmstadt. SE: solubilization efficiency. 2007). Switzerland) revolving at 120 rpm for 1 h until a thin dry film was formed on the inner wall of the flask.05 0. NMloaded PPPMM were evaluated for drug payload.01 0. L-a-Phosphatidylcholine (PC) from soybean was purchased from MP Biomedicals (Santa Ana. Egypt).9  2.97  0. California. 2001).05 31.02 1. 66.87 97. Spectra/Pore1 dialysis membrane (12.02 0.5  1. The mixture was then sonicated for 1 min and the volume was adjusted into 10 mL at room temperature (25  C) to obtain nanocarrier dispersions.2  0.4  0.01 0. 2013).000– 14. The objective of this study was to develop NM-loaded PPPMM that can solubilize NM in aqueous media at clinically relevant concentrations even after dilution.55  0.01 571. It was assumed that the incorporation of PC would increase the thermodynamic stability of the micelles due to the tight hydrophobic interactions with hydrophobic PPO blocks and the consequent reduction of CMC. In addition.03 0.02 23. long circulation and easy production.09 0.97 458.56  4. All the materials were used as received without any further modifications. it seems necessary to develop new hydrophobic thermodynamically stabilized PMM that could resist precipitation upon dilution through incorporation of phosphatidylcholine (PC) in micelle structure. respectively in an attempt to increase the thermodynamic and kinetic stabilities of these micelles.4  11.4  0.12 94. reduce its frequency of administration and eventually target it to the brain tissue.79  1. 2. USA). Materials NM and amlodipine besylate (IS) were kindly donated by Marcyrl for pharmaceutical industries (Cairo. (Cairo.04 28. The ability of mixed micelle formulation to keep the drug in solubilized form after dilution in the GI tract is a key factor in bioavailability enhancement rather than solubility of the drug in the formulation itself.2  2.03 1.87 123.98  0.6 13.3  0.31 665.04 0. R.1. For comparative purpose.06 0. PDI: polydispersity index. 2007). Materials and methods 2. USA). PC: phosphatidylcholine. Moreover. and 200 mg) were accurately weighed and dissolved in ethanol (10 mL) in a round-bottom flask.2 20 6.56 PS (nm) PDI Zeta potential (mV) 0. The solvent was slowly evaporated at 60  C under reduced pressure using a rotary evaporator (Buchi R-110 Rotavapor. Difunctional block copolymers of ethylene oxide/propylene oxide [Pluronic1 F127 and Pluronic1 P123] were purchased from Sigma chemicals company (St.2.6 13.08  0.1  15. USA). 2007. Mourao et al. liposomes containing 100 mg of phosphatidylcholine and PMM containing 200 mg Table 1 Composition of nimodipine-loaded PPPMM. A small concentration of vegetable oil was introduced into Pluronic solutions to decrease micelle degradation upon dilution while not compromising the drug loading capacity of oil-stabilized micelles (Rapoport. 1999).55  0. It was assumed that more hydrophobic mixed micelles display lower CMC and concentrations remain above those values even after high dilution (Chiappetta and Sosnik. Herein.6  9. Preparation of nimodipine-loaded Pluronic/PC/polysorbate 80 mixed micelles (PPPMM) NM (10 mg).04 2. PMM: Pluronic p123/F127 mixed micelles.1  14. zeta potential and ex vivo transport through intestinal membranes of rats. Egypt).05  0.6  0.43  0..8  2.21  0.4  2.6  1. Basalious.95  0. 2010)..06  0.04 24. Louis.5  0.24 87. (CA.2  12.1  0. these micelles will finally separate since they are thermodynamically unstable.48 550.4  8.53  1.01 1.348 E.2 20 – – 1. Shanmugam et al. To the best of our knowledge. Increasing the hydrophobic character of these copolymers favors also the transition of morphology of micellar systems in aqueous solutions from spherical into worm-like (Khimani et al.47 99. 2. PS: particle size. Germany).24  0. PC has the ability to enhance oral absorption and bioavailability through improving portal blood absorption and lymphatic delivery.89 81. prolong its circulation time.. phosphatidylcholine (33.35 359. The in vivo pharmacokinetic behavior of the optimum PPPMM formulation in plasma and brain tissue was compared to NM solution following oral administration to rats.01 All formulations contained 10 mg NM. Therefore. The dried film was treated with distilled water (9 mL) and the flask was allowed to revolve at a fixed hydration temperature of 60  C for 30 min under normal pressure. NM-loaded Pluronic/PC/polysorbate 80 mixed micelles (PPPMM) were developed after incorporation of PC and Polysorbate 80 as examples of hydrophobic and hydrophilic molecules.

5% sodium lauryl sulfate solution (receptor compartment) at 37  C (FDA dissolution methods). Study design. The shafts rotated at a speed of 50 rpm in 250 mL of 0. The samples were analyzed for drug content using the same UPLC method previously mentioned. The SE was calculated as the ratio between the drug amount in the filtered sample and that added in the formulation process. 30.9 cm2) tightly covered from one side with rat intestine. Determination of drug payload and solubilization efficiency (SE) Exact quantity of NM-loaded PPPMM dispersion was dissolved in ethyl alcohol. Powder X-ray diffraction (XRD) Diffraction patterns of NM powder. 1.7 mm. Three samples for each formula were used for size determination and the average values  SD were calculated. The protocol of this study was reviewed and approved by the research ethics committee (REC) at Faculty of Pharmacy. Group 1 received oral NM solution prepared (1 mg/mL) by dissolving NM in a solvent mixture composed of alcohol/PEG400/water in a weight ratio of 7. Determinations were done in triplicates for three independent samples of each formula and the average values  SD were calculated. a voltage of 45 kV. 2. before the experiment. The cumulative amount of drug permeated through the membrane per unit area (mg/cm2) was plotted as a function of time (h) for each formulation. 2. 2006).B. Twenty four Wister male albino rats weighing 200–250 g were divided into two groups. samples were lyophilized at 45  C and pressure of 7  102 mbar for 24 h (Novalyphe-NL 500. each comprising 18 rats. NM was administered as a single oral dose of 4 mg/kg using oral gavage (Wang et al. Thirty six Wister male albino rats weighing 200–250 g were divided into two groups.25 mL/min. 1. Kyoto. Shamma / International Journal of Pharmaceutics 493 (2015) 347–356 Pluronic1 (F127 and P123) mixture in the ratio 2:1 were prepared according to the thin film hydration technique. Cairo University.1. and a current of 40 mA. This assembly represents the donor compartment (Fouad et al. Group 2 received oral NM-loaded selected PPPMM.7.3. Morphological examination of PPPMM by transmission electron microscopy (TEM) The morphologic examination of the selected formulations was performed by transmission electron microscopy (TEM) operating at 80 kV (model JEM-1230.. Wistar rats (200  20 g) were anesthetized and a midline abdominal incision was made and the entire length of the intestine was removed.3. The selected PPPMM formulation was frozen at 20  C in presence of 5% w/v mannitol as a cryoprotectant. A sample of NM loaded PPPMM dispersion was filtered through 0. The SE was determined after storing the prepared NM loaded PPPMM dispersion at 5  3  C for 24 h for possible drug precipitation. Characterization of NM-loaded PPPMM 2. The excised intestine was flushed with cold saline to remove any intestinal contents and cut into segments (4 cm long).3.3. zeta potential (ZP) of the diluted samples.5.3. 2.5 and 6. In vivo pharmacokinetics (PK) of NM loaded PPPMM in Wistar rats 2. C18. The study performed in this section was approved by Research Ethics Committee (REC). One drop of the diluted nanocarrier dispersion was deposited on the surface of a carbon coated copper grid. 70% acetonitrile and 20% buffer (10 mL glacial acetic acid in 1 L water). Aliquots were taken at different time intervals and replaced instantly by equal amount of fresh medium in order to 349 maintain the same volume. Experiments were repeated three times and the results were expressed as the mean values  SD. 2. The drug payload was calculated as the milligrams of NM per milliliter of PPPMM dispersion. the animals were decapitated.45 mm filter.5 cm in diameter and 5 cm in length. Plasma was harvested immediately by 10 min of centrifugation at 6000 rpm and stored at 80  C until analysis. Zetasizer Nano ZS. having pH ranged between 5. The study was done using Wister albino rats (200–250 g).7.E. Tokyo. Egypt). The loaded cylindrical tubes were attached from the second side to the shafts of the USP dissolution tester apparatus. 2. with area = 4. Faculty of Pharmacy. before the experiment.2. Group 1 received oral NM solution (1 mg/ mL) and group 2 received oral NM-loaded selected PPPMM. 180 and 240 min after administration.3. 2. the animals were fasted for 10 h with free access to water. Accurate volumes of drug-loaded PPPMM dispersion (containing the equivalent to 2 mg of NM) were placed in double open-sided glass cylindrical tubes (2..3. Jeol. At different time intervals (0.3. Plasma PK study. Table 1 depicts the composition of the prepared formulations. Blood samples (1 mL) were obtained from the retro-orbital plexus of rats by a well-trained practitioner and single sample was obtained from each individual eye. 50 mm). The column temperature was 25  C and the flow rate was 0.6. Savant Instruments Corp. 2.5. polydispersity index (PDI) and zeta potential Size and size distribution of NM-loaded PPPMM were determined by the dynamic light scattering method using a Malvern Mastersizer (DLS. Chromatography was performed using a Waters Alliance system with a UV detector at 238 nm.N.4. 120. were analyzed for evaluation of their physical stability. physical mixture and lyophilized selected formulation were determined in a Scintag X-ray diffractometer (USA) using Cu Ka radiation with a nickel filter. The mobile phase composed of 10% methanol. 3.3.45 mm membrane filter to separate the crystallized NM. Malvern instruments.5:8:8.3. Ex vivo transport study On the day of the experiment. The Ex vivo transport studies of NM from PPPMM were performed in a USP dissolution apparatus.3. A 2 mL aliquot of filtrate was injected in triplicate into UPLC column (BEH. The animals were sacrificed by cervical dislocation at the end of the experiment. The filtrate was assayed for NM content using the same previously mentioned UPLC method. Cairo University (Cairo. Filtration was performed using a 0. 2. 2. USA). 2013). The in vivo study was carried out to determine the pharmacokinetics of NM in the plasma and brain after oral administration of PPPMM dispersion compared to oral solution. R. physical mixture (PM) and lyophilized selected formulation was determined using Shimadzu differential scanning calorimeter (DSC-50.7.3. Malvern. their skulls were cut open and the . Basalious.7. Before initiation of the experiment.1. The samples were diluted with distilled filtered water before measurement until being translucent. 4 and 6 h after administration). Approximately 4 mg of each sample was heated in aluminum pans in a temperature range of 30–300  C at a heating rate of 10  C/min under inert nitrogen flow (25 mL/min). The samples were collected into heparinized tubes prior dosing and at 15.. Japan). negatively stained with 2% phosphotungstic acid then allowed to dry at room temperature for 10 min for investigation by TEM. polydispersity index (PDI) was measured to assess the particle size distribution. 60. Brain PK study. Japan). Determination of particle size (PS). UK). each comprising 12 rats. Finally. Additionally. 2. Then. Differential scanning calorimetry (DSC) The thermal analysis of NM powder.

The low stability of solubilized micellar system may be also due to the highly hydrophobic nature of NM molecule which makes its full accommodation in the hydrophobic core of the micelles practically difficult.10 ! 238. 1. The column was maintained at 35  C and the pressure of the system was 6500 psi. 2. Pluronic micellar systems were reported to have certain drawbacks such as the formation of aggregates with a large particle size. The pharmacokinetic data obtained from different treatments were analyzed for statistical significance by one-way analysis of variance (ANOVA) adopting Kinetica1 software version 5 (Thermo Fisher Scientific Inc. Pharmacokinetic and statistical analysis. 3.. Plasma and brain concentration–time data of NM was analyzed by noncompartmental pharmacokinetic models using Kinetica1 software version 5 (Thermo Fisher Scientific Inc.3. Homogenized brain samples were stored at 80  C until assayed. The total amount of Pluronics1 relative to NM reached up to 1:36 (w/w) and Pluronic P123 and F127 were used in ratios of 1:2. 1:1 and 2:1 (w/ w). selective and accurate LC-MS/MS method. A volume of 50 mL of IS solution in methanol (250 ng/mL) was added to 200 mL plasma samples or 500 mL homogenized brain samples then the samples were vortexed for 30 s. Chromatographic separation of analytes was carried out on ACQuity UPLC HSS C18 (50  2. using Eppendorf sample concentrator (Eppendorf. 50 L/ h. Samples preparation.). Amphiphilic block copolymers generally self-assemble in dilute aqueous solution into spherical micelles and/or tubular micelles based on the relative weight fraction of hydrophilic and hydrophobic blocks.7. followed by the addition of 200 mL 1 M NaOH. respectively.. Plasma and brain samples were analyzed for NM adopting a sensitive. 1. However. v/v) and was delivered at a flow rate of 0. Concentration–time profiles were plotted and the peak concentration Cmax (ng/mL) and the necessary time Tmax (h) to attain Cmax were obtained directly from it. Results and discussion 3.. The terminal elimination rate constant was calculated by linear regression of the terminal portion of the natural logarithm of the concentration and the elimination half-life was calculated. respectively. rinsed with saline twice and blotted with filter paper.1% formic acid (85: 15. The isocratic mobile phase composed of acetonitrile—0. desolvation gas flow. and a volume of 2 mL from the reconstituted sample was injected into the UPLCMS/MS system.350 E.1. 2013). 2.25 mL/min. 3. It was reported that the hydrophilic long PEOshell of the micelles formed by F127 and P123 had a protective effect on the micelle dispersion. Basalious. SPSS Inc. 2. Residue was reconstituted in 150 mL mobile phase. Shamma / International Journal of Pharmaceutics 493 (2015) 347–356 brain tissue was removed. Statistical analysis The data obtained from different formulations were analyzed for statistical significance by one-way analysis of variance (ANOVA) adopting SPSS statistics program (version 16.7. The source dependent parameters maintained for both the analytes and internal standards (ISs) were: cone gas flow. 2009). 800 L/h.6.1 mm. Extraction was applied by the addition of 3 mL of mixture of diethyl ether:n-hexane (1:1 v/ v) followed by vortex for one minute. Germany) till dryness. Mass Lynx software version 4. then samples were centrifuged at 3000 rpm for 10 min A volume of 2. The area under the drug concentration–time curve (AUC) was calculated by the trapezoidal method. R. 2015). the mixed micelles were still in a dynamic state and presented only temporary stability (Wei et al. The organic layer was evaporated at 45  C. All formulations showed precipitation of NM crystals after storage for one week at 5  3  C (data not shown). The samples were further vortexed for 30 s. The mass transition ion pair.3. and then transferred into tubes. Quantitative analysis was performed on a Waters Acquity UPLC H-Class-Xevo TQD system (MA.8 mm) column.1 was used to control all parameters of UPLC and MS. performed in the multiple-reaction monitoring (MRM) mode.5 to 100 ng/mL.14 for IS.4. Preliminary studies and formulation of mixed micelles of different morphologies Vain attempts to solubilize NM were performed by preparation of several Pluronic P123/F127 mixed micelles (PMM). developed and validated before the study using UPLC-MS/MS system. USA) interfaced with a Waters Quattro Premier XE triple quadrupole mass spectrometer and equipped with electrospray ionization operated in the positive ionization mode. desolvation temperature.). The non-parametric test (Kruskal Wallis test) was used to compare the tmax for test and reference. By decreasing the weight fraction of the PEO block to just less than about 50%.5.7. The ex vivo transport profiles of NM from PPPMM-5. 2. Chicago. PMM and liposomes through rat intestine compared to that from aqueous NM suspension.4.B. capillary voltage. It was reported that Pluronics1 can self-assemble into spherical and non-spherical micelles in dilute aqueous solutions (Park et al. The optimum values for compound dependent parameters like cone voltage and collision energy were set at 25 V and 10 eV for NM and 30 V and 12 eV for IS. Spherical micelles form spontaneously when the hydrophilic block such as PEO is the largest block by mass. The total brain tissue samples were taken.5 mL of the upper organic layer were transferred accurately into another dry clean tube. 409. 350  C. homogenized with 1 mL of saline. USA) followed by post hoc multiple comparisons using the least square difference (LSD). Thus.N. respectively. hydration and swelling of the corona imparts just enough curvature to the copolymer assembly that tubular micelles that may reach up to microns in length and similar in diameter to .09 was followed for NM and m/z Fig. our objective was to increase the thermodynamic and kinetic stabilities of PMM through incorporation of PC and polysorbate 80.5 kV.3. The lower and upper limits of quantification of NM in plasma and brain samples were 5 to 200 ng/mL and 0.05. 120  C. Differences were considered to be significant at p  0. the low stability and the possible reversion to the phase separated state (Abdelbary and Tadros. source temperature. of m/ z 419.24 ! 343. Chromatographic conditions.

The drug payload of all NM-loaded PPPMM ranged from 0.01%. Liposomes and PMM.48% to 99. The presence of different morphologies such as spherical and worm- . As shown in Table 1. Transmission electron microscopy (TEM) of PPPMM-5 (a) and PPPMM-1 (b) showing the tubular morphologies of the micelles.2  2.. Contrary to liposomes and PMM. Drug payload and solubilization efficiency (SE) Table 1 shows the drug payload and SE of PPPMM. thus indicating a wide size distribution of the measured dispersion (Essa et al. the PDI of all PPPMM formulations show a wide range of values starting from 0. Particle size distribution and zeta potential Fig.79.4  11.56 nm.35 to 0. for example through the incorporation of PC in the structure of PMM.95 to 1. The aggregation characteristics of Pluronics1 in water are modified quite significantly in the presence of additives that have a strong influence on their solubilization characteristics. The relatively low SE of systems containing 100 mg total Pluronics1 resulted from the release of NM from PPPMM and the formation of crystals of NM in the external aqueous phase. Their SE after storage at 5  3  C for 24 h ranged from 92. the more homogenous are the particle population.87. Basalious. 3. The PS of liposomes and PMM were 359. respectively.5  1.35% and 87.. The polydispersity index (PDI) is a dimensionless number expressing the particle size distribution of the investigated system.2. respectively) confirmed the inability of both systems to fully accommodate NM in their structure leading to drug precipitation after storage at 5  3  C for 24 h. the spheres are the predominant morphology (Cai et al. The accommodation of NM in the core of micelles was very efficient as demonstrated by the high values of SE especially for PPPMM containing 200 mg total Pluronics1. Incorporation of PC would increase the thermodynamic stability of the micelles due to the tight hydrophobic interactions with hydrophobic PPO blocks and the consequent reduction of CMC.08 to 761. 2.3.. 2007).05). The closer the polydispersity value to zero. Increasing the hydrophobic character of these copolymers.N. On the other hand. Incorporation of PC and polysorbate 80 in the structure of PPPMM significantly increased their particle size (p < 0. critical micellar temperature (CMT).89 nm. 2012). Shamma / International Journal of Pharmaceutics 493 (2015) 347–356 351 Fig. incorporation of polysorbate 80 would increase the kinetic stability of the micelles due to the steric hindrance that minimize micelle aggregation.8  2.6  9. 3.9  2. 2002). R. NM-loaded PPPMM have mean particle diameters ranging from 458.1 15. favors micelle formation by reducing the critical micellar concentration (CMC).1 mg/mL. and the sphere-to-rod transition temperature of their aqueous solutions (Khimani et al. The ex vivo transport profiles of NM from the different formulations of Pluronic/PC/polysorbate 80 mixed micelles (PPPMM) through rat intestine compared to liposomes.B. The very low SE of liposomes and PMM (81.56  4. 3.24 and 123.E.

Moreover. As shown in Fig. 2a.37 mcg/cm2) and finally the aqueous suspension (10. 2009. 1). PMM is more capable to keep NM in solubilized form during the transport study than liposomes as previously confirmed by the higher values of SE (Table 1).25  12. The poor water solubility of NM is the main reason for its low transport through intestinal membrane.3 mcg/ cm2) followed by PMM (102.9 cm2.97  6.0 suggested good stability of the prepared systems with low probability of aggregation and particle growth (Shanmugam et al. NM-loaded PPPMM showed the highest cumulative amount of NM permeated after 20 h (110. transferosomes containing bile salts as edge activators can readily transform into mixed micelles in the gastrointestinal environment.352 E. Incorporation of poorly water soluble drugs into liposomes caused substantial enhancement in absorption and bioavailability.B. The zeta potential was measured and found to be in values ranging from 22. R. 2009).2  0. the similarity between liposomal lipid bilayers and biological membranes and the relatively small size of liposomes significantly facilitates permeation (Chen et al. 2007).81 mcg/cm2). 2011). It was assumed that physiological bile salts can interact with phospholipids in the liposomal vesicles inducing a vesicle–micelle transition to form mixed micelles that play important roles in enhancing absorption of poorly water-soluble drugs. TEM micrograph of PPPMM-5 illustrates the worm-like or tubular morphology of the micelles..53 mcg/cm2).. Values more than 20. like (tubular) micelles could explain the wide distribution of particle size of Pluronic dispersion containing PC.N.35  9. 1.. Chen et al. This conclusion could explain why the spherical PMM showed about 2 folds increase of NM transport compared to NM-loaded liposomes (Fig.3  0. Liposomes may provide increased solubility of their load and protection from the hostile environment in the gastrointestinal tract.4. Schematic illustration for the different micellar morphologies formed by Pluronics P123/F127 and their mixture with phosphatidylcholine (PC). The length and diameter of the nanotubular micelles are in the nano range. ex vivo transport from PPPMM-5. the liposomes (56. PMM and liposomes through rat intestine compared to that from aqueous NM suspension (1 mg/mL in 1% HPMC solution) was performed and illustrated in Fig. Hildebrand et al. Ex vivo transport study To investigate the impact of different morphologies of the nanocarrier systems on the drug transport. thus enhancing membrane penetration.. Exact volume of each formulation equivalent to 2 mg NM was placed on rat intestine attached to a cylindrical tube having surface area 4. 2009. The resultant mixed micelles have been shown to function as excellent vehicles for poorly water-soluble drug molecules and one of the most important mesophases before absorption (Andrieux et al. 2003). Basalious. Therefore. Shamma / International Journal of Pharmaceutics 493 (2015) 347–356 Fig. The flexible morphology of the nano-tubular mixed micelles (NTMM) could explain the high enhancement of NM transport through rat intestine (Giddi et al. It was reported that the flexibility of .06 mV. Incorporation of PC in the structure of Pluronic micelles increases the thermodynamic stability of the micelles preventing NM crystallization during the transport study and favors the conversion of micelles from spherical into tubular morphologies. Several researchers have studied the underlying mechanism for the facilitated oral absorption by liposomes. The highest enhancement of NM transport through rat intestine was achieved by PPPMM-5.. Amongst the formulations tested.23  5. 3. 4.05 to 31..

The DSC pattern of the PM showed the presence of the characteristic peak of pure drug. As a result.23 and 53. The concept of critical packing parameter (CPP) for surfactant systems by Israelachvili could be applied to predict and explain the change of morphology as being driven by a change in the spontaneous curvature of the system (Arleth et al.. It was reported that higher paclitaxel loading was possible by worm-like micelles (Cai et al. Rupp et al. This would indeed result in wormlike or tubular morphology of the micelles as confirmed by TEM. PC has two alkyl chains and therefore a higher CPP (close to unity). respectively)... Due to the optimum SE and the higher permeation properties. Thus. respectively) followed by PPPMM-6 and PPPMM-2 (73. Amongst the formulations tested. the thermogram of NM-loaded PPPMM revealed broadening. 2012.61 and 79. the vesicular liposomal shape is the main morphology of this formulation. The results of transport study confirmed that the morphology of micelles affected drug transport through biological membranes where the efficiency for drug transport could be ranked as follows. and consequently have a CPP near 1/3. vesicles when CPP is between 1/2 and 1.N..51 12. 2012a) or completely transformed from crystalline form to molecular state in the surfactant mixture. the drug transport slightly increased which may be due to the formation of some spherical Pluronic mixed micelles in addition to the drug loaded vesicles.30  14. .. liposomes and PPPMM-3 showed the lowest cumulative amount of NM permeated after 24 h (61.5  C. the DSC pattern of the PM displayed a sharp melting endothermic peak at 56  C. The tubular morphologies of PPPMM-5 and PPPMM-1 were elucidated by the TEM micrographs shown in Fig. The PPO of Pluronics1 and the alkyl chains of PC are assembled in a hydrophobic core.52 mcg/ cm2. There is no significant difference between the permeation profiles of NM from liposomes and PPPMM-3 through rat intestine (P < 0. As shown in Table 1.. wormlike micelles when CPP is between 1/3 and 1/ 2. When surfactants aggregate with each other. Thus. nano-tubular mixed micelles (NTMM)  spherical PMM > vesicles (liposomes).. 2007).73  16.6 mole fraction of PC. As shown in Table 1.B. Mixtures of the two types of molecules in optimum PC: Pluronics1 ratio (75:25 molar ratio) should give rise to CPP ranging from 1/3 to 1/2. and lamellar or bilayer structure when CPP is 1 (Chu et al. 2005. although with a lower intensity (due to dilution) indicating that the drug retained its crystallinity (Elsayed et al. Abdelbary and Tadros.05). The thermogram of the micellar system revealed complete disappearance of the peaks of the drug and phospholipid. DSC thermograms of the lyophilized optimized NM-loaded PPPMM-5 as well as the corresponding physical mixture (PM) and NM plain. Fig. 353 3. 2013).42 and 118. corresponding to the melting point of mannitol (Chalikwar et al. Elsayed et al. surprisingly containing the same molar ratio of PC to Pluronics1 (75:25). corresponding to the melting point of Pluronic F127 (Abdelbary and Makhlouf. the tubular micelles of PPPMM-5 have a much larger core volume for encapsulation. and shifting in the Pluronic F127 peak from 56  C to 46. they tend to form monolayers that have curvature allowing the most efficient packing of the molecules. 2005).61  4.E. and PPPMM-4 (91.85 and 0.95 mcg/cm2. 2010). 3 illustrates the ex vivo transport of NM from the different formulations of Pluronic/PC/polysorbate 80 mixed micelles (PPPMM) through rat intestine. sharp melting endothermic peak at 127  C corresponding to the melting point of NM which confirms its crystallinity (Fu et al.5. In addition. 2013).89 mcg/cm2) and finally PPPMM-5 and PPPMM-1 (112. Basalious. and a smaller intensity endothermic peak at 33  C corresponding to the melting point of the phospholipid. PPPMM5 and PPPMM-1.47  22. 5. respectively).. Pluronics1 P123/F127 form spherical or nearly spherical mixed micelles in dilute aqueous solution. Spherical micelles are formed when CPP is less than 1/3. PPPMM-5 formulation was selected for further investigation and in vivo PK study.54  15. As PC content decreased from 0.. PPPMM-3 had the highest PC content relative to Pluronics1 (90:10 molar ratio). the free drug was characterized by a single. As illustrated in Fig. This hydrophobic core is surrounded by a hydrophilic shell consisting of PC polar head groups and hydrophilic PEO of Pluronics1 and polysorbate (Fig 4).9 into 0. Shamma / International Journal of Pharmaceutics 493 (2015) 347–356 tubular micelles allow them to penetrate nanoporous gels where 100 nm sized vesicles cannot enter (Kim et al. 2014).8 mole fraction (PPPMM-2 and PPPMM-6. Differential scanning calorimetrical studies (DSC) DSC was performed for the lyophilized NM-loaded PPPMM-5 as well as the corresponding physical mixture and NM plain powder in order to evaluate the phase transformation of NM during the formation of the tubular micelles. The higher content of Pluronics1 and PC in PPPMM-5 formulations could explain the high SE. 2a and b. 2013). Beside the disappearance of the drug and phospholipid endothermic peaks. decrease in the intensity. respectively). 2014. The thermal behavior of NMloaded PPPMM was distinct from that found for the PM. As illustrated in Fig. PPPMM-4. showed a slight increase of drug transport which indicated the formation of large proportion of spherical Pluronic micelles due to the large content of Pluronic.73  19. containing 0. it tends to form vesicle structures. A new sharp endothermic peak appeared at 167. PPPMM-5 showed significantly higher solubilization efficiency than PPPMM-1 although both formed nanotubular mixed micelles (NTMM). 4.49 mcg/cm2. Disappearance of the characteristic endothermic peak of NM probably signifies that NM was either entrapped inside PPPMM (Salama et al. R.6  C. The similarity of the morphology of the nanocarrier of both formulations could explain why they gave the same lowest drug transport rate through membranes. 5. gave the highest NM transport due to the formation of highly flexible nanotubular mixed micelles (NTMM).. Fig.

were investigated using powder X-ray diffraction. The lower limits of NM quantification in plasma. the diffractogram of the prepared PPPMM showed a typical diffuse pattern with complete absence of the numerous distinctive peaks of NM indicating that NM was molecularly dispersed in the core of tubular micelles of PPPMM-5.. which was used as a cryoprotectant during the lyophilization process. whereas they disappeared in the corresponding PPPMM. .B. 2014). was Fig. Powder X-ray diffraction In order to further examine the physical form of the drug in the selected formulation. As shown in Fig. for the PM.37 was used for calculating the RDC. PM. 3. Crystallinity was assessed by comparing some representative peak heights in the diffractogram of the NM-loaded PPPMM-5 with those of a NM powder. respectively). The characteristic diffraction peaks of NM were detected in the PM.37. The UPLC-MS/MS assay was validated and showed acceptable inter. Figs.94 h.54 and. and 0. brain tissues were assayed for NM concentration. respectively. The non-parametric test (Kruskal Wallis test) showed a significant difference between Tmax of both treatments. where Isample is the peak height of the sample under investigation and Idrug is the peak height at the same angle for the drug (Shoukri et al. 2014).25 h and 0. In vivo pharmacokinetic studies Fig.354 E. The mean AUC0t estimated from PPPMM (42.. X-ray diffraction of the lyophilized optimized NM-loaded PPPMM-5 as well as the corresponding physical mixture (PM) and NM plain.7. reached after times (Tmax) 0.044 and 0.5 ng/mL. The calculated RDC value was found to be 0. 6. respectively. and the lyophilized NM-loaded PPPMM-5.6. R. and 26. after administration of single oral dose of NM solution and NM-loaded PPPMM formulation and the corresponding pharmacokinetic parameters are shown in Table 2.N. Basalious.21 ng h/mL). and in brain were 5 and 0. and the PPPMM. 3.18 ng/mL.21.1. 7. Pharmacokinetics of NM in blood The mean Cmax estimated in plasma from the drug solution and the PPPMM were 20. To quantify the extent of nano-tubular micelles accumulation in the brain. The diffractogram of NM revealed its crystalline nature as indicated by three prominent diffraction peaks with the Fig. 8. The differences between the two treatments for Cmax and tmax were found to be statistically significantly different (p = 0. 7 and 8 denote the mean NM concentrations in plasma and in brain of rats.and intra-day reproducibility.016. The mean NM concentrations in brain of rats after administration of single oral dose (4 mg/kg) of NM solution and NM-loaded PPPMM formulation. respectively. 3.05. Shamma / International Journal of Pharmaceutics 493 (2015) 347–356 highest intensity at 2u of 12. Pharmacokinetic parameters for NM in rat plasma and brain were assessed using UPLC-MS/MS method in order to understand the in vivo behavior of the NM-loaded PPPMM compared to the drug solution. The mean NM concentrations in plasma of rats after administration of single oral dose (4 mg/kg) of NM solution and NM-loaded PPPMM formulation. 20. 6. On the other hand. 33. Pure drug peak at 2u = 20. which reflects the total amount of drug absorbed over the 4 h time period. 2009).7. pure NM.89 . The relationship used to calculate the relative degree of crystallinity (RDC) was RDC = Isample/Idrug. respectively.33 (Zu et al.

20 2.72  0. Singh et al.28 33.50  0.41  14.42  0. The significant increase in drug solubility. 2012. The value of Cmax of NM in brain after administration of NM-loaded PPPMM was 2.7 times that of the NM solution. PPPMMs were prepared by incorporation of PC and polysorbate 80 in the structures of Pluronic P123/F127 micelles. 4.7167e-005* 0. Following oral administration of NM-loaded PPPMMs. References Abdelbary. Plasma data Brain data Parameter NM solution NM-loaded PPPMM Significance (p-value) NM solution NM-loaded PPPMM Significance (p-value) Cmax (ng/mL) Tmax (h) AUC0t (ng h/mL) AUC01 (ng h/mL) T1/2 (h) 20. 2014. 2014)..84  13.74  15. This could be due to the encapsulation of NM in the long circulating NTMMs resulting in retarded clearance of NM from plasma.94  0. respectively. These results were in accordance with previous studies that reported the long circulation time of worm-like micelles in the blood circulation.95 h).88 ng h/mL. 2014) through Peyer’s patches of the intestine which results in bioavailability improvement (Basavaraj and Betageri.18 0.016* 0. the P-gp efflux inhibition character of Pluronic and polysorbate enhanced the drug permeability and absorption into the systemic circulation and brain tissues.140 Values are expressed as mean  SD. it can be concluded that the greater bioavailability obtained from NM-loaded PPPMM.11 0.044* 0. Giddi et al.. Moreover. orally administered PC vesicles accumulate at the brush–border membrane of enterocytes. G. 2001. respectively). free NM penetrated BBB and reached the brain tissues in a non-encapsulated form resulting in high levels of drug in the cerebral tissue followed by its rapid clearance from the brain tissues. According to Iwanaga et al.373 0.85 69. 2007).64 0. and enhances the drug bioavailability.21  12.0221* 31. it is obvious that the values of Cmax and AUC0t for the NM-loaded PPPMM in brain tissue were significantly higher than those of the oral NM solution.2. presence of Pluronics1 further contributes to bioavailability improvement.. The relative bioavailability (frel) of PPPMM compared to the drug solution was estimated to be approximately 232%. then to the systemic circulation.. 3.031.88  14.71 42. The mean NM t1/2 estimate from PPPMM (2.E. enhanced drug absorption and the long circulation time of the prepared tubular micelles could be promising to improve oral and parenteral delivery of NM. and 232% (plasma) larger than that measured after administration of the drug solution might be attributed to efficiency of flexible nanotubular micelles to solubilize and enhance absorption of NM from the intestinal mucosa and their long circulation time. 2007.68  7.14 85.53  4.009* 0. The lymphatic transport increases drug bioavailability as the intestinal lymph vessels travel directly into the thoracic duct.61  13. The high values of Cmax and AUC0t of the PPPMM compared to that of the solution might be attributed to the long circulation time of the nano-tubular micelles and the presence of Pluronics1. Moreover. Further studies for elucidating the key formulation factors required for the preparation of intravenous NTMM for sustained delivery of NM into the brain tissue are presently investigated.34 70.42 h) was determined to be higher and statistically significantly different (p = 0. which results in enhancement of drug permeability and absorption to the systemic circulation (Abdelbary and Makhlouf.95  0..14 ng h/mL).28 70.140). Ryan et al.00 33.78  12.B.034* 0. The aqueous mixtures of Pluronics1 with CPP < 1/ 3 and PC with CPP > 1/2 showed the formation of nano-tubular mixed micelles (NTMMs). The improved bioavailability of NM from PPPMMs confirmed the ability of highly flexible NTMM to flow through the nanopores of the intestinal membrane.57 0. including stealthy vesicles bearing the same length of PEO chains (Cai et al.14  7.031* 0.0004* 6. The encapsulation of NM into NTMMs significantly improved the pharmacokinetic profiles of the drug in plasma and brain compared to the drug solution. 2014. hence bypassing the portal circulation (Porter and Charman.39 0. R. This finding opened the door for future preparation of thermodynamically and kinetically stable NTMMs for enhanced solubilization and absorption of poorly water soluble drugs compared to liposomes and spherical micelles.18  15. Conclusions In this study. Several researchers studied the mechanism of liposome transport across the blood brain barrier and reported that it might be lipidmediated free diffusion or lipid-mediated endocytosis (Shah et al.84 0.25  0. resulting in increased drug concentration gradient across the intestinal epithelium. determined to be about 2. 2013). Kimura et al. 2014).. 1995). It was reported that the oral absorption of nano-lipid based particles occurs via lymphatic transport (Chalikwar et al. Adoption of polymeric micelles to enhance the oral bioavailability of dexibuprofen: formulation.N. Basalious.91  0. From Table 2.31 0. Florence et al. This enables absorption of significant amount of the drug into the systemic circulation.32 folds higher and statistically significantly different (p = 0. (p = 0. These results could indicate the ability of the optimized nanotubular dispersion to achieve sustained plasma profile of NM after oral administration when compared to the oral drug solution containing the drug in solubilized form. (1997). Shamma / International Journal of Pharmaceutics 493 (2015) 347–356 355 Table 2 The mean pharmacokinetic parameters of NM in plasma and brain of rats after oral administration of single dose of NM solution and NM-loaded PPPMM.7. the presence of PC facilitates the lymphatic delivery of NM bypassing the hepatic first-pass effect.29 0.. Pluronic micelles were reported to have a P-gp efflux inhibition character. polysorbate 80 and PC in the PPPMM which acts as a penetration enhancer and inhibitor of P-gp in the blood brain barrier (BBB) (Salama et al.67  0.42 34. Moreover.009. in-vitro evaluation and in- .24 21.55  8.00 18.. n = 6 for plasma and n = 3 for brain. The AUC0t for NM in brain after administration of NM-loaded PPPMM and NM-loaded solution were 70. 1988). On the other hand. Pharmacokinetics of NM in brain It was important to study the pharmacokinetic behavior of NM in brain tissue since it is the target organ of NM. Makhlouf. The elimination half-life (t1/2) of ND from the brain tissues showed no significant differences between the NM-loaded PPPMM and NM-loaded solution (p = 0. Based on these results. also reported the uptake and transport of intact PC liposomes across the small intestine following oral administration via gut associated lymphoid tissue (Kimura.61 and 33. and 0.59 0.. which was about 208% (brain tissue).0004) compared to the mean AUC0t estimated from the drug solution (18.. A. 2014. which appears longer than any other synthetic particle.. the pharmacokinetic behavior of NM in plasma showed a higher sustained presence compared to that in brain. 2012b). Patil-Gadhe et al.0221) compared to the mean t1/2 estimate from the drug solution (0.

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