Incorporation of liquid lipid in lipid nanoparticles for ocular drug delivery enhancement This article has been downloaded from IOPscience. Please scroll down to see the full text article. 2010 Nanotechnology 21 025101 (http://iopscience.iop.org/0957-4484/21/2/025101) Download details: IP Address: 130.220.71.26 The article was downloaded on 06/10/2012 at 05:32 Please note that terms and conditions apply. View the table of contents for this issue, or go to the journal homepage for more Home Search Collections Journals About Contact us My IOPscience
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Incorporation of liquid lipid in lipid nanoparticles for ocular drug delivery enhancement
This article has been downloaded from IOPscience. Please scroll down to see the full text article.
2010 Nanotechnology 21 025101
(http://iopscience.iop.org/0957-4484/21/2/025101)
Download details:
IP Address: 130.220.71.26
The article was downloaded on 06/10/2012 at 05:32
Please note that terms and conditions apply.
View the table of contents for this issue, or go to the journal homepage for more
Home Search Collections Journals About Contact us My IOPscience
Received 24 August 2009, in final form 3 November 2009Published 3 December 2009Online at stacks.iop.org/Nano/21/025101
AbstractThe present work investigates the effect of liquid lipid incorporation on the physicochemicalproperties and ocular drug delivery enhancement of nanostructured lipid carriers (NLCs) andattempts to elucidate in vitro and in vivo the potential of NLCs for ocular drug delivery. TheCyA-loaded or fluorescein-marked nanocarriers composed of Precifac ATO 5 and Miglyol 840(as liquid lipid) were prepared by melting-emulsion technology, and the physicochemicalproperties of nanocarriers were determined. The uptake of nanocarriers by human cornealepithelia cell lines (SDHCEC) and rabbit cornea was examined. Ex vivo fluorescence imagingwas used to investigate the ocular distribution of nanocarriers. The in vitro cytotoxicity andin vivo acute tolerance were evaluated. The higher drug loading capacity and improved in vitrosustained drug release behavior of lipid nanoparticles was found with the incorporation of liquidlipid in lipid nanoparticles. The uptake of nanocarriers by the SDHCEC was increased with theincrease in liquid lipid loading. The ex vivo fluorescence imaging of the ocular tissues indicatedthat the liquid lipid incorporation could improve the ocular retention and penetration of oculartherapeutics. No alternation was macroscopically observed in vivo after ocular surface exposureto nanocarriers. These results indicated that NLC was a biocompatible and potential nanocarrierfor ocular drug delivery enhancement.
(Some figures in this article are in colour only in the electronic version)
1. Introduction
The most commonly used methods of drug treatment inocular diseases and diagnostics are topical delivery systems,however, the physiological constraints of the eye lead to poorabsorption of bioactive compounds. Appropriate deliverysystems should prolong the contact time of the drug with theeye surface and promote or facilitate the penetration of drugmolecules from the tear phase into the eye tissue withoutcausing any inconvenience to the patient [1]. Controlled andsustained delivery systems, such as liposomes, emulsions, andbiodegradable nanoparticles have been proved to improve thecorneal penetration of drugs [2] and prolong the retentionof drugs on the ocular surface as well [3–5]. Comparedwith other colloidal carriers, lipid nanoparticles combine many
1 Author to whom any correspondence should be addressed.
advantages including good biocompatibility, feasibility of largescale production by high pressure homogenization, controlleddrug release and drug targeting, etc [6, 7]. Recent reports haveindicated that lipid nanoparticles, e.g. the so-called solid lipidnanoparticles (SLN) and nanostructured lipid carriers (NLCs),increased the ocular bioavailability of tobramycin, ibuprofen,and other lipophilic drugs [8, 9]. Our previous research showedthat NLC with thiomer modification was a promising systemfor ocular drug delivery with prolonged residence time [10].
Due to the large extent of crystallization of solid lipids,the encapsulated drug in SLN was often expulsed from thecarrier during the storage period, and the drug loading capacityof SLN was very low [11–13]. Instead of taking solid lipidsas the lipid matrix for SLN, the NLC was produced byincorporating liquid lipids (oils) in solid lipids, which led tomore imperfections in the crystal structures, and higher drug
incorporation and drug release in comparison to SLN [14, 15].It has been reported that the incorporation of liquid lipids (oils)into solid lipids leads to a different inner structure of NLC,which influences the penetration depth of NLC into skin [16].
The aim of this study was to investigate the effect ofMiglyol 840 (liquid lipid) on the physicochemical propertiesand ocular drug delivery enhancement of NLC composedof ATO 5 as a solid lipid. Also the in vitro cytotoxicityand in vivo acute tolerance were examined. Cyclosporine A(CyA) was chosen as a model drug, which is used as first-line therapy in the prevention of xenograft rejection followingorgan transplantation. Topical application of CyA could beused in the treatment of a variety of immune-mediated oculardiseases like vernal conjunctivitis [17], dry eye syndrome [18],and the prevention of corneal allograft rejection [19]. Recentresearch has shown that CyA-loaded SLN could be a promisingcarrier to target CyA at the cornea [20].
2. Materials and methods
2.1. Materials
Precifac ATO 5 (glyceryl palmitostearate) was obtainedfrom Gattefosse’ (France). Miglyol 840 was obtainedfrom Sasol (Germany). Polyethylene glycol stearate (PEG-SA, the polymerization degree of ethylene glycol is 40)was kindly donated by Nanjing WELL Chemical Co. Ltd(China). Tween 80 (polysorbate 80, analytical reagent) waspurchased from the Shanghai Chemical Reagent Co. Ltd(China). Cyclosporine A (CyA) was purchased from Galena(Czech Republic). Fluorescein was purchased from Sigma(St Louis, MO, USA). the MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cytotoxicity assaykit and Coomassie Blue staining protein assay kit weresupplied by Nanjing Jiancheng Biological Technology Ltd(China). Methanol, acetonitrile, and other chemicalswere analytical reagent grade. The spontaneously derivedhuman corneal epithelia cell lines (SDHCEC) was kindlydonated by Key Laboratory of Ophthalmology, ZhongshanOphthalmic Center (China). Dulbecco-modified Eagle’smedium (DMEM), human epidermal growth factor (EGF), andhydrocortisone were obtained from Invitrogen-Gibco (BRL,UK). Heat inactivated fetal bovine serum (FBS), L-glutamine,streptomycin, and penicillin were purchased from HyClone(USA). Human transferrin was provided by Sigma (USA).Human insulin was purchased from Wako (Japan).
2.2. Preparation of nanocarriers
Taking the advantages of non-residue of organic solvent and noburst release at initial time, the melt-emulsification techniquewas employed to prepare lipid nanocarriers [21, 22]. Basedon previous studies, 2% Tween 80 was used as an emulsifierand 2% PEG-SA as the surface modifier of lipid nanoparticles.The total amount of solid lipid phase (ATO 5) and liquidlipid phase (Miglyol 840) was kept constant at 5%. Thepercentages of Miglyol 840 varied from 20% to 45% in NLCs,0% in SLN and 100% in NE. SLN and NE were used as
control nanocarriers. To prepare CyA-loaded or fluorescein-marked nanocarriers, drug or fluorescent dye were mixed withthe lipid matrix and melted at 80 ◦C. The melted mixturewas then dispersed in 10 ml of aqueous solution (heated upto 80 ◦C) containing Tween 80 and PEG-SA under stirring(Changzhou Electrical Engineering Instruments, China) toform the primary emulsion. The warm primary emulsion wasfurther treated under sonication at 400 W 90 times (work 2 sand stand 3 s) using a Lab ultrasonic cell pulverizer (JY92-II,Ningbo Scientz Biotechnology Co., Ltd China). The obtainednanoemulsion was rapidly cooled down and solidified with ice(0 ◦C) under continued agitation to form a uniform dispersionof nanoparticles. The resultant SLN and NLC dispersions werefast frozen under −75 ◦C for 5 h and then transferred to thefreeze-dryer (FD2.5, Heto, Denmark). The drying period was72 h, and the NLC and SLN powders were collected for theDSC and x-ray investigations.
2.3. Characterization of nanocarriers
The mean particle size and zeta potential of CyA-loaded orfluorescein-marked nanocarrier dispersions were determinedby Zetasizer (3000HS, Malvern Instruments, UK) after beingdiluted 20 times with distilled water. The experiment wascarried out in triplicate.
To characterize the thermal behavior of the lipidnanoparticles, DSC analysis was performed using NETZSCHDSC 204 F1 Phoenix® (NETZSCH-Geratebau GmbH,Germany). The scanning rate was 5 K min−1 ranging from30 to 160 ◦C. An empty pan was used as reference. Thecrystallinity index (CI) was calculated using the followingequation (1).
CI (%) =(
� EnthalpyFreeze-dried NLC or SLN
� EnthalpyBulk × Concentrationlipid phase
)× 100.
(1)The crystalline structures of the nanoparticles were
investigated using wide-angle x-ray scattering (WAXS) ona XD-3A x-ray diffractometer (SHIMADZU, Japan) witha copper anode (Cu Kα radiation, 40 kV, 40 mA, λ =0.154 18 nm), and a Goniometer VG-108R as a detector. Thediffraction pattern was recorded within a 2θ range of 5◦–60◦.The obtained data were typically collected with a step width of0.01◦ and a count time of 3 s. Bragg’s equation (equation (2))was used to transform the data from scattering angle θ to theinterlayer spacing d of lipid chains.
nλ = 2d sin θ (2)
where λ is the wavelength of the incident x-ray beam and n isa positive integer which describes the order of the interference.
2.4. Determination of drug entrapment efficiency and drugloading
The encapsulation efficiency (Ee) of CyA in nanocarrierswas determined by mass balance. Un-encapsulated free drugwas assessed by analyzing the drug content in the dispersionmedium after ultracentrifugation at 12 000 rpm for 10 min
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(Nanosep centrifugal devices, Pall Life Science, USA, witha 100 kDa molecular weight cut-off (MWCO) membrane).The diluted nanocarriers were dissolved in methanol andthe total drug content concentration was measured. All themeasurements were performed by HPLC. The HPLC systemconsisted of an Agilent 1100 HPLC series system (AgilentTechnologies, Palo Alto, CA), with an Agilent G1310Apump and an Agilent G1314A variable wavelength UV–visphotodiode array detector (DAD). The Diamonsil-C18 ODScolumn (150 mm × 4.6 mm, 5 μm) was operated at 60 ◦C.The mobile phase consisted of acetonitrile–water (90/10, v/v)and the flow rate was 0.6 ml min−1. Detection wavelength was210 nm. The drug entrapment efficiency (Ee) and drug loading(DL) of nanocarriers were calculated by equations (3) and (4).
Ee (%) =(
Wa − Wf
Wa
)× 100 (3)
DL (%) =(
Wa − Wf
Wa − Wf + 0.5
)× 100 (4)
where Wa was the analyzed amount of drug encapsulated by0.5 g lipid and Wf was the analyzed amount of drug in thefiltrate, respectively.
2.5. In vitro release study
The in vitro drug release profiles of the nanocarriers (0.5%CyA) were investigated by dialysis. The release mediumwas freshly prepared simulated tear fluid (STF, composition:NaCl 0.67 g, NaHCO3 0.20 g, CaCl2·2H2O 0.008 g, anddistilled, deionized water to 100.0 g) [23]. The fluid contained0.1% sodium dodecyl sulfate to assess sink conditions duringin vitro release studies. To start the release, 0.5 ml ofnanocarrier dispersion was transferred into the dialysis bagwith a membrane MWCO of 100 kDa, which was then put into50 ml of release medium stirred at 100 rpm at 34 ± 0.5 ◦C.The release medium was replaced (50 ml) constantly in thescheduled time intervals, and the CyA content was determinedby HPLC after filtration through 150 nm filters. As control, thediffusion profile of CyA dispersion across the dialysis bag wasalso evaluated. The experiment was carried out in triplicate.
2.6. Evaluation of the uptake and cytotoxicity using theSDHCEC
2.6.1. Cell cultivation. SDHCEC from 40 to 60 passageswere grown as a monolayer at 37 ◦C in a 5% CO2-95% airatmosphere, and only the cells showing a viability >97% wereused for the experiments. The culture medium was Dulbecco-modified Eagle’s medium (DMEM), supplemented with 15%heat inactivated fetal bovine serum (FBS), 10 ng ml−1
human epidermal growth factor (EGF), 5 μg ml−1 insulin,5 μg ml−1 human transferrin, 0.4 μg ml−1 hydrocortisone,2 mM L-glutamine, and 100 U ml−1 penicillin–100 μg ml−1
streptomycin.
2.6.2. Cellular uptake of nanocarriers. To investigate theeffect of Miglyol 840 on the ability of nanocarriers to crossthe corneal epithelium and enter the cells, the SDHCECwere exposed to fluorescent nanocarriers. In a 24-well plate,the SDHCEC were seeded on coverslips at a density of4 × 104 cells/well and incubated for 24 h to attach. Cellswere then incubated with a fluorescein-loaded nanocarrierdispersion (the fluorescein concentration was 0.01 mg ml−1
and the lipid concentration was 2 mg ml−1) in a supplement-free culture medium at different times (1, 2, 4, 8, and12 h). Cells were washed three times with phosphatebuffered saline (PBS, pH 7.4) and directly observed under afluorescence microscope (OLYMPUS America, Melville, NY).Control was incubated with fluorescein in a supplement-freeculture medium (0.01 mg ml−1). Each treatment was done intriplicate, and three independent fields were photographed foreach slide.
To assay the corneal epithelia cellular uptake ofCyA-loaded nanocarriers quantitatively, the SDHCEC wasincubated with CyA-loaded nanocarriers in a supplement-freeculture medium for different times (1, 2, 4, 8, and 12 h)and lipid concentrations (0.25, 0.5, 1, 1.5, and 2 mg ml−1).After the cells were washed three times with PBS (pH 7.4),200 μl of PBS was added and freeze-thawed. 100 μl ofcell suspension after freeze-thawing three times were mixedwith 100 μl of methanol and vortexed for 5 min, and thencentrifuged at 12 000 rpm for 10 min. The drug concentrationin the supernatant after centrifugation was measured by theHPLC method. The protein content in the cell suspensions wasmeasured using a Coomassie Blue staining protein assay kit.The uptake of drug was calculated from equation (5).
Drug uptake (μg mg−1 protein) = C/M (5)
where C is the intracellular concentration of CyA, M is theunit weight (milligram) of cellular protein. All experimentswere repeated three times.
2.6.3. Cytotoxicity test. The viability of SDHCEC afterexposure to CyA-loaded nanocarriers or fluorescein-loadednanocarriers in a supplement-free culture medium for 12 h (thelipid concentration were 0.25, 1, and 2 μg ml−1, respectively)were measured both immediately and 24 h post-incubation inculture medium by MTT assay [24, 25]. The experimentswere repeated at least four times. The potential toxic effectof the different nanocarriers tested was expressed as a viabilitypercentage calculated using the following equation (6).
Viability (%) =(
ODtest
ODc
)× 100 (6)
where ODtest was the optical density of those wells exposedto the nanocarriers, and ODc was the optical density of thosewells treated with supplement-free DMEM medium.
2.7. In vivo tolerance assay
Albino New Zealand rabbits weighing 2.0–2.5 kg, obtainedfrom Animal Center of China Pharmaceutical University, were
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Table 1. Properties of nanocarriers containing 1% CyA with different amounts of Miglyol 840. (Note: Data are represented with mean ±S.D. (n = 3). The number after NLC represents the weight percentage of Miglyol 840 to total lipid. For example, NLC-20 contains 20%Miglyol 840 in the total lipid.)
a P < 0.05: between 0, 20, 30, and 100 wt% Miglyol 840 incorporated nanocarriers.b P > 0.05: between 30 and 45 wt% Miglyol 840 incorporated nanoparticles.
used to study the acute ocular tolerance to nanocarriers.All animal treatments followed the recommendation of theRegulations for the Administration of Affairs ConcerningExperimental Animals. Rabbits were randomly dividedinto six groups of three animals, and each received 50 μlof fluorescein solution or fluorescein-loaded nanocarrierdispersion (the fluorescein concentration was 0.2 mg ml−1) inthe right eye every 30 min for 6 h. The contralateral eye wasused as control and received no treatment. The nineteenthanimal was untreated and served as a sham control. Sixhours after the first instillation, the animals were euthanizedby air embolism after being deeply anesthetized with anintramuscular overdose of anesthetic and a paralyzing mixtureof xylazine (20 mg kg−1) and ketamine (200 mg kg−1).The eyeball was removed and fixed with 10% neutralformalin. Following fixation, they were embedded in paraffinwith hematoxylineosin staining for pathology. Eyeballsections (6 mm) were evaluated in a masked fashionaccording to the following criteria: alteration in any ofthe ocular surface epithelia (cornea, limbus, conjunctiva),the presence of inflammatory cells (eosinophils, neutrophils,mast cells, and lymphocytes), and any other abnormalitycontrol.
2.8. In vivo uptake test
Paraffin-embedded eyeball sections without staining fromrabbits used for the in vivo tolerance assay were examinedby fluorescence microscopy (OLYMPUS America, Melville,NY) [25]. Both right (treated) and left (control) eyes from eachanimal were evaluated in duplicate.
The in vivo ocular distributions of fluorescein-loadednanocarriers in rabbit eyes were evaluated by ex vivofluorescence imaging. The cornea, the bulbar conjunctiva,the iris-ciliary body, the lens and the sclera of eyeballsenucleated in an in vivo tolerance assay were dissected andrinsed with normal saline, and then directly placed on the Invivo Imaging System (FX Pro, Kodak, USA) for fluorescenceimaging. The fluorescent signal intensities in the differentocular tissues were analyzed by Image Station in vivo FXsoftware.
2.9. Statistical analysis
One-way analysis of variance (ANOVA) was performed onall experimental data and the means were compared using
student’s t-test at the 5% level with SPSS 11.0 software (SPSS,Inc).
3. Results and discussion
3.1. Preparation and characterization of nanocarriers
The effects of Miglyol 840 incorporation on the physicochemi-cal properties of CyA-loaded nanocarriers are shown in table 1.As shown in the table, no significant effect of the oil contenton the zeta potentials of nanosystems studied was observed(P > 0.05). However, the size of CyA-loaded nanoparticlesdecreased when the liquid oil proportion exceeded 20 wt%(P < 0.05). In the production of lipid nanoparticles, thelipid and aqueous phases were heated up to 80 ◦C and thenformed nanoemulsion droplets. Thus the subsequent crystal-lization might influence the particle size. The excess oil in-hibited the crystallization of solid lipid and resulted in smallerparticles [26].
Though the viscosity of melted ATO 5 at temperaturesabove 80 ◦C (17 cP at 90 ◦C) is greater than that of Miglyol840 (9–12 cP at 20 ◦C), the largest particle size was observedfor NE (169.8 ± 6.3 nm), which was significantly larger thanthat of SLN and NLCs (P < 0.05). This result might beattributed to the better surface active properties of ATO 5 (witha HLB value of 2) and its combination with Miglyol 840, whichmay facilitate the emulsification efficiency and the formation ofmore fine dispersions [27].
3.2. Physical state of the core lipid and drug incorporated
Figure 1 and table 2 shows the DSC curves and related dataof the thermal peaks of bulk lipid, CyA and lyophilizedCyA-loaded lipid nanoparticles (1% CyA), respectively. Themelting point of CyA at 126.1 ◦C disappeared for CyA-loadedlipid nanoparticles. The bulk ATO 5 alone exhibited a sharpendothermic event with a melting peak at 57.6 ◦C. Whenthe lipid was formulated as blank SLN, ATO 5 revealed twopeaks at 53.3 and 56.4 ◦C, reflecting two distinct polymorphicforms. Furthermore, it could be seen that increasing Miglyol840 content led to the decrease of the melting point of lipidnanoparticles. There was a sharp decline in melting enthalpyfrom the bulk material to the nanoparticles. Considering theenthalpy of bulk lipid (ATO 5) at 178.1 J g−1 as 100%, thecrystallinity index (CI) of lipid nanoparticles was calculated
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Nanotechnology 21 (2010) 025101 J Shen et al
Figure 1. DSC curves of bulk materials, CyA-loaded lipidnanoparticles (1% CyA) with increasing Miglyol 840 content (from0% to 45%).
Figure 2. X-ray diffraction patterns of bulk materials, physicalmixtures and CyA-loaded lipid nanoparticles (1% CyA) withincreasing Miglyol 840 content (from 0% to 45%).
Table 2. Melting parameters and crystallinity indexes of thefreeze-dried CyA-loaded lipid nanoparticles (1% CyA) with differentMiglyol 840 content.
using equation (1) and is shown in table 2. As thelower melting enthalpy values suggested the lower orderedlattice arrangements, the CI (%) decrease with the increaseof Miglyol® 840 content in NLCs may be due to crystaldisturbance within the lipid matrix.
X-ray diffraction results shown in figure 2 were in goodagreement with the results obtained by DSC measurements.It was found that no typical peak of CyA was detected
Figure 3. Drug entrapment efficiency (Ee) and drug loading (DL) ofCyA-loaded nanocarriers containing 0, 20, 30, 45, and 100% Miglyol840 content. Each value represents the mean ± SD (n = 3).
Figure 4. In vitro release profiles of CyA from nanocarriers (0.5%CyA) containing 0, 20, 30, 45, and 100% Miglyol 840 content. Eachvalue represents the mean ± SD (n = 3).
in the diffractograms of lipid nanoparticles. These resultsconfirmed again that CyA was not in crystalline form inthe lipid nanoparticles investigated here. The diffractionpattern of ATO 5 displayed the characteristic short spacingsat 0.456 nm and at 0.383 nm, showing the typical signalsof the triglycerols [14]. The physical mixtures of CyA andblank NLC-30 showed sharp reflections with position andintensity ratios that were consistent with the tetragonal crystalform of CyA [28, 29]. All nanoparticles showed the samereflections at 0.46 and 0.38 nm, similarly to those of the bulklipid, however, the peak intensities of SLN and NLC particleswere weaker. This result indicated that the degree of thecrystallinity was lower in nanoparticles than pure lipids andthe presence of Miglyol 840 within the system did not changethe polymorphism of the lipid matrix (ATO 5). Moreover, thepeak intensity of NLC was less pronounced in comparisonto that of SLN and decreased with increasing Miglyol 840content. As recorded by DSC, the decrease of crystallinitydegree in WAXS profiles indicated a less ordered structure andpronounced crystal defects.
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Nanotechnology 21 (2010) 025101 J Shen et al
Figure 5. Fluorescence image after the cells were incubated with fluorescein-loaded nanocarriers composed of 0, 20, 30, 45, and 100%Miglyol 840 content. Scale bar: 25 μm.
Figure 6. The drug cellular uptake against incubation time (a) and lipid concentration (b) after the cells were incubated with CyA-loadednanocarriers composed of 0, 20, 30, 45, and 100% Miglyol 840 content mean ± SD, n = 3.
3.3. Drug entrapment efficiency and loading capacity
The effects of liquid lipid (Miglyol 840) incorporation ondrug entrapment efficiency (Ee) and loading capacity (DL) ofnanocarriers are shown in figure 3. The Ee of nanocarrierswas above 92%, and no significant effect of Miglyol 840incorporation on the Ee was observed (P > 0.05). TheDL was found to increase from 16.2% to 23.1% with theincreasing content of Miglyol 840 among SLN and NLCs.
As a very hydrophobic chemical, CyA is soluble in both thesolid lipid (ATO 5) and the liquid oil (Miglyol 840). Thesolubility in liquid lipids (oils) is higher than that in solid lipids.Furthermore, it was reported that the incorporation of liquidlipids disturbed the massive crystal order of solid lipids, andincreased the degree of imperfections in the lipid matrix. Thusit provided more space to accommodate drug molecules and ledto improved drug loading capacity [14, 30]. It was noticed thatthe DL was only 10% for NE, which was significantly lower
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Nanotechnology 21 (2010) 025101 J Shen et al
Figure 7. Light microscopy of rabbit corneal and conjunctival tissue sections: (A) control (untreated), (B) SLN, (C) NLC-20, (D) NLC-30,(E) NLC-45, and (F) NE. Staining: hematoxylineosin. Scale bar: 50 μm.
Figure 8. Fluorescence microscopy of rabbit corneal sections from fluorescent nanocarrier-treated rabbit eyes: (A) fluorescein solution;(B) SLN; (C) NLC-20; (D) NLC-30; (E) NLC-45; (F) NE. Scale bar: 50 μm. Ep, corneal epithelium; SP, corneal stroma; En, cornealendothelium.
than that of SLN and NLCs (P < 0.05). As described above,higher emulsifying efficiency and smaller particle size werereached for SLN and NLCs. The larger particle size of NEmight result from the limited phase volume of oil and interfacelayer for the solubilization of CyA, which lead to the lowerdrug loading.
3.4. In vitro release studies
The CyA release profiles from the nanocarriers are shown infigure 4. A relatively fast release was observed in the initial12 h, followed by a sustained release profile. The releaserate became faster upon the addition of Miglyol 840 and keptincreasing with higher content of Miglyol 840, and the drugrelease rate of NE was fastest among all formulations. Asshown in table 1, NLC-30 and NLC-45 had almost similarmean particle sizes. However, the drug release rate of NLC-
45 was significantly faster than that of NLC-30 (P < 0.05).It was obvious that the drug diffusion through the liquid lipidphase was faster than that via the solid lipid phase, in whichboth the diffusion and erosion mechanism would be the releaselimited factors.
3.5. Cellular uptake of nanocarriers
Figure 5 shows fluorescence image when the cells wereincubated with the fluorescein-loaded nanocarriers for differenttimes. No morphologic alterations in the cells treatedwere observed (figure 5(A)). For the fluorescein-loadednanosystems, the cellular uptakes of nanosystems were timedependent. There was no significant fluorescence insideSDHCEC in an incubation time of 2 h. After 4 h, the obviousfluorescence images were observed, and the fluorescenceintensity was enhanced with the incubation time up to 12 h.
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Nanotechnology 21 (2010) 025101 J Shen et al
Figure 9. Ex vivo fluorescence imaging of rabbit eye tissues from rabbits treated with fluorescent nanocarriers: (a) fluorescein solution;(b) SLN; (c) NLC-20; (d) NLC-30; (e) NLC-45; (f) NE. The color bar on the left side indicates the signal efficiency of the fluorescenceemission coming from the rabbit tissues.
As a control, there was no obviously green fluorescenceinside SDHCEC after the cells were incubated with fluoresceinin culture medium for 12 h (figure 5, F-s-12 h). Theintense fluorescence observed in the cells treated with thefluorescein-loaded nanocarriers indicated that these colloidalsystems could be internalized into SDHCEC. The cells treatedwith fluorescein-loaded NE showed the highest intensity offluorescence, followed in the order of fluorescein-loading byNLC-45, NLC-30, NLC-20, and SLN.
Our previous studies had demonstrated that the particlesize of CyA-loaded nanocarriers had no significant differencefrom that of fluorescein-loaded nanocarriers and the leakageof fluorescein from nanocarriers is slow with no significantdifference within 12 h (data not shown). It was reported that theuptake of SLN composed of lipid matrix materials with lowermelting point was higher than that of SLN with higher meltingpoint [31]. Based on this study, the highest cellular uptake offluorescein-loaded NE with largest particles size also suggestedthat the liquid lipid with lower melting point had the strongeraffinity to cell membrane, and was more easily taken by cellsin comparison with the solid lipid. In the fluorescein-loadedSLN and NLCs, the higher Miglyol 840 content in NLC-45 ledto the lower melting point and favored the cellular uptake.
As shown in figure 6, the cellular uptake of CyA-loadedNLCs was higher than that of SLN, and the highest intracellularCyA content was found after cells were treated with CyA-loaded NE. Furthermore, the cellular uptake of CyA increasedwith the increase in Miglyol 840 loading. Incubating cells withCyA-loaded nanocarriers, the intracellular drug concentrationincreased, with the incubation time prolonged to 12 h (thelipid concentration was 2 mg ml−1) as shown in figure 6(a)and the lipid concentration of nanocarriers with the sameincubation time (4 h) as shown in figure 6(b). When CyAdispersion was used as a reference, the amount of drug uptakeby cells was close to the limit of detection. Consideringthe results of the in vitro release, SLN and NLC-20 hadalmost the same CyA release rate, but the cellular uptake ofNLC-20 was significantly higher than that of SLN after 12 hincubation (P < 0.05). These result indicated that the abilityof lipid nanoparticles to be internalized into SDHCEC mightbe increased with the increasing amounts of liquid lipid.
3.6. Cell survival after exposure to nanocarriers
The viability of the cells after exposure to CyA-loaded orfluorescein-loaded nanocarriers for different concentrationswas directly evaluated by MTT assay (see figure A.1 of the
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Nanotechnology 21 (2010) 025101 J Shen et al
appendix). The cell viability was higher than 90% for allconcentrations after 12 h incubation (the lipid concentration�2 mg ml−1). No significant differences in cell survivalmeasured immediately and 24 h post-incubation were observedamong the different concentrations of nanocarriers, or whencompared with culture medium control (P > 0.05).
3.7. In vivo tolerance assay
Figure 7 illustrates ocular surface structures of untreated(control) and treated with fluorescein-loaded nanocarriers. Thecontrol and all of the experimental rabbits showed no signsof discomfort during the 6 h assay. No differences of cornealand conjunctival structure were observed between nanocarrier-treated eyes compared to the control or among the assayednanocarriers. The Miglyol 840 incorporation into the lipidmatrix of NLCs has no influence on corneal and conjunctivalcell structure and tissue integrity. Therefore, Miglyol 840and its resultant NLCs are well tolerated by ocular surfacestructures.
3.8. In vivo nanocarrier uptake
Figure 8 illustrates the fluorescence microscopy of eye tissuesections. As expected, no fluorescence was observed in thecontrol cornea sections (not shown). Corneas of fluoresceinsolution-treated eyes emitted weak fluorescent signals thatwere much less intense than those of nanocarrier-treated eyes.All sections from nanocarrier-treated eyes showed fluorescentsignals in corneal epithelium (figures 8(B)–(F)), and thefluorescent intensity increased with the increasing amount ofMiglyol 840 content. The fluorescent signal in the cornealstroma and endothelium can also be found in NLC 45- and NE-treated eyes. The particle size of NE was significantly higherthan that of SLN and NLCs (P < 0.05) and the penetrationability of NE into the cornea was significantly higher thanthat via SLN and NLCs. This means that the particle sizeof the nanocarriers studied is not a main factor affecting thepenetration ability of nanocarriers and the increasing amount ofMiglyol 840 improves the corneal penetration of nanocarriers.
Figure 9 shows the ex vivo fluorescence imaging of rabbitocular tissues of eyeballs enucleated in vivo tolerance assay.Weak fluorescent intensity was observed in the tissues offluorescein solution-treated eyes (figure 9(a)). The fluorescentsignals of NLC-treated eye tissues were stronger than thosewith SLN and NE, and even the signals in the lens and iris-ciliary body could be detected. The fluorescent intensityincreased with the increasing amount of oil content in NLCs.Among all tested groups, the NLC-45 group had the highestfluorescent intensity in all examined eye tissues (figure 9(e)).The weak fluorescence observed in the sclera and conjunctivafrom NE-treated eyes indicated that NE had short retentiontime in the ocular surface (figure 9(f)). Although thepenetration ability of NE into cornea was higher than NLCs,the short retention time limited its permeation into postcornealtissues. Due to the smaller particle size and more flexible corestructure resulting from the incorporation of liquid lipid, NLCscould be entrapped and retained in the mucin layer covering
the ocular surface, thus promoting the corneal penetration anddelivery of bioactive compounds to intraocular tissues in orderto realize controlled ocular drug delivery.
4. Conclusions
In this work, the influence of Miglyol 840 incorporation inlipid nanoparticles on the physicochemical properties, cornealepithelia cell uptake, and ocular distribution of NLCs wereevaluated. The increase of Miglyol 840 content in NLCsdisturbed the inner structures of lipid nanoparticles, thus,leading to higher drug loading capacity, smaller mean particlesize, and faster in vitro drug release profiles when comparedto SLN. The cellular uptake of nanocarriers appeared to betime and lipid concentration dependent. Moreover, the cellularinternalization of nanocarriers increases with the Miglyol840 loading. In vivo uptake has shown that this flexibleNLC prolonged the ocular surface retention and improvedthe penetration into ocular tissues of bioactive compounds.Furthermore, NLCs consist of biocompatible lipids, and liquidlipid exhibited a good tolerance in vivo. The results ofthis work have shown that NLC is a promising system forcontrolled ocular drug delivery.
Appendix
Figure A.1. The cell viability measured immediately (a) and 24 hpost-incubation (b) after the cells were exposed to differentnanocarriers (the lipid concentration is 0.25, 1, and 2 mg ml−1) for12 h. There were no significant differences in cell survival among thedifferent concentrations of nanocarriers, or when compared withculture medium control (P > 0.05). Mean ± SD, n = 4.
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Nanotechnology 21 (2010) 025101 J Shen et al
References
[1] Davies N M 2000 Biopharmaceutical considerations in topicalocular drug delivery Clin. Exp. Pharmacol. Physiol.27 558–62
[2] De Campos A M, Sanchez A and Alonso M J 2001 Chitosannanoparticles: a new vehicle for the improvement of thedelivery of drugs to the ocular surface. Application tocyclosporin A Int. J. Pharm. 224 159–68
[3] Durrani A M, Davies N M, Thomas M and Kellaway I W 1992Pilocarpine bioavailability from a mucoadhesive liposomalophthalmic drug delivery system Int. J. Pharm. 88 409–15
[4] Kaur I P, Garg A, Singla A K and Aggarwal D 2004 Vesicularsystems in ocular drug delivery: an overview Int. J. Pharm.269 1–14
[5] Vandervoort J and Ludwig A 2004 Preparation and evaluationof drug-loaded gelatin nanoparticles for topical ophthalmicuse Eur. J. Pharm. Biopharm. 57 251–61
[6] Yang S C, Lu L F, Cai Y, Zhu J B, Liang B W andYang C Z 1999 Body distribution in mice of intravenouslyinjected camptothecin solid lipid nanoparticles and targetingeffect on brain J. Control. Release 59 299–307
[7] Muller R H, Radtke M and Wissing S A 2002 Solid lipidnanoparticles (SLN) and nanostructured lipid carriers (NLC)in cosmetic and dermatological preparations Adv. DrugDeliv. Rev. 54 131–55
[8] Cavalli R, Gasco M R, Chetoni P, Burgalassi S andSaettone M F 2002 Solid lipid nanoparticles (SLN) as oculardelivery system for tobramycin Int. J. Pharm. 238 241–5
[9] Li X, Nie S F, Kong J, Li N, Ju C Y and Pan W S 2008 Acontrolled-release ocular delivery system for ibuprofen basedon nanostructured lipid carriers Int. J. Pharm. 363 177–82
[10] Shen J, Wang Y, Ping Q, Xiao Y and Huang X 2009Mucoadhesive effect of thiolated PEG stearate and itsmodified NLC for ocular drug delivery J. Control. Release137 217–23
[11] Freitas C and Muller R H 1999 Correlation between long-termstability of solid lipid nanoparticles (SLN) and crystallinityof the lipid phase Eur. J. Pharm. Biopharm. 47 125–32
[12] Muller R H, Radtke M and Wissing S A 2002 Nanostructuredlipid matrices for improved microencapsulation of drugs Int.J. Pharm. 242 121–8
[13] Souto E B, Wissing S A, Barbosa C M and Muller R H 2004Evaluation of the physical stability of SLN and NLC beforeand after incorporation into hydrogel formulations Eur. J.Pharm. Biopharm. 58 83–90
[14] Jenning V, Thunemann A F and Gohla S H 2000Characterisation of a novel solid lipid nanoparticle carriersystem based on binary mixtures of liquid and solid lipidsInt. J. Pharm. 199 167–77
[15] Hu F Q, Jiang S P, Du Y Z, Yuan H, Ye Y Q and Zeng S 2005Preparation and characterization of stearic acidnanostructured lipid carriers by solvent diffusion method inan aqueous system Colloids Surf. B 45 167–73
[16] Teeranachaideekul V, Boonme P, Souto E B, Muller R H andJunyaprasert V B 2008 Influence of oil content onphysicochemical properties and skin distribution of Nilered-loaded NLC J. Control. Release 128 134–41
[17] Gupta V and Sahu P K 2001 Topical cyclosporin A in themanagement of vernal keratoconjunctivitis Eye 15 39–41
[18] Kunert K S, Tisdale A S and Gipson I K 2002 Goblet cellnumbers and epithelial proliferation in the conjunctiva ofpatients with dry eye syndrome treated with cyclosporineArch. Ophthalmol. 120 330–7
[19] Perry H D, Doshi S J, Donnenfeld E D and Bai G S 2002Topical cyclosporin A in the management of therapeutickeratoplasty for mycotic keratitis Cornea 21 161–3
[20] Gokce E H, Sandri G, Bonferoni M C, Rossi S, Ferrari F,Guneri T and Caramella C 2008 Cyclosporine A loadedSLNs: evaluation of cellular uptake and corneal cytotoxicityInt. J. Pharm. 364 76–86
[21] Cortesi R, Esposjto E, Luca G and Nastruzzi C 2002Production of lipospheres as carriers for bioactivecompounds Biomaterials 23 2283–94
[22] Yuan H, Wang L L, Du Y Z, You J, Hu F Q and Zeng S 2007Preparation and characteristics of nanostructured lipidcarriers for control-releasing progesterone bymelt-emulsification Colloids. Surf. B 60 174–9
[23] Lin H R and Sung K C 2000 Carbopol/pluronic phase changesolutions for ophthalmic drug delivery J. Control. Release69 379–88
[24] Diebold Y, Jarrın M, Saez V, Carvalho E L, Orea M,Calonge M, Seijo B and Alonso M J 2007 Ocular drugdelivery by liposome–chitosan nanoparticle complexes(LCS-NP) Biomaterials 28 1553–64
[25] Enrıquez de Salamanca A, Diebold Y, Calonge M,Garcıa-Vazquez C, Callejo S, Vila A and Alonso M J 2006Chitosan nanoparticles as a potential drug delivery systemfor the ocular surface: toxicity, uptake mechanism and invivo tolerance Invest. Ophthalmol. Vis. Sci. 47 1416–25
[26] Lin X, Li X, Zheng L, Yu L, Zhang Q and Liu W 2007Preparation and characterization of monocapratenanostructured lipid carriers Colloids Surf. A 311 106–11
[27] Westesen K, Siekmann B and Koch M H J 1993 Investigationson the physical state of lipid nanoparticles by synchrotonradiation x-ray diffraction Int. J. Pharm. 93 189–99
[28] Lechuga-Ballestros D, Abdul-Fattah A, Stevenson C L andBennett D B 2003 Properties and stability of a liquid crystalform of cyclosporine—the first reported naturally occurringpeptide that exists as a thermotropic liquid crystal J. Pharm.Sci. 92 1821–31
[29] Muller R H, Runge S A, Ravelli V, Thunemann A F,Mehnert W and Souto E B 2008 Cyclosporine-loaded solidlipid nanoparticles (SLN): drug–lipid physicochemicalinteractions and characterization of drug incorporation Eur.J. Pharm. Biopharm. 68 535–44
[30] Souto E B, Wissing S A, Barbosa C M and Muller R H 2004Development of a controlled release formulation based onSLN and NLC for topical clotrimazole delivery Int. J.Pharm. 278 71–7
[31] Yuan H, Miao J, Du Y Z, You J, Hu F Q and Zeng S 2008Cellular uptake of solid lipid nanoparticles and cytotoxicityof encapsulated paclitaxel in A549 cancer cells Int. J.Pharm. 348 137–45
