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Hydrophilic coating of mitotane-loaded lipid nanoparticles: Preliminary studies for mucosal adhesion
Patrícia Severino, Eliana B. Souto, Samantha C. Pinho & Maria H. A. Santana
To cite this article: Patrícia Severino, Eliana B. Souto, Samantha C. Pinho & Maria H. A. Santana (2013) Hydrophilic coating of mitotane-loaded lipid nanoparticles: Preliminary studies for mucosal adhesion, Pharmaceutical Development and Technology, 18:3, 577-581, DOI: 10.3109/10837450.2011.614250
To link to this article: https://doi.org/10.3109/10837450.2011.614250
Published online: 29 Sep 2011.
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Hydrophilic coating of mitotane-loaded lipid nanoparticles: Preliminary studies for mucosal adhesion
Patrícia Severino1,2, Eliana B. Souto2,3, Samantha C. Pinho4, and Maria H. A. Santana1
1School of Chemical Engineering, University of Campinas (UNICAMP), Campinas, São Paulo, Brazil, 2Faculty of Health Sciences, Fernando Pessoa University, Rua Carlos da Maia, Porto, Portugal, 3Institute for Biotechnology and Bioengineering, Centre of Genomics and Biotechnology, University of Trás-os-Montes e Alto Douro (IBB-CGB/UTAD), Portugal, and 4Department of Food Engineering, School of Animal Science and Food Engineering (FZEA), University of São Paulo (USP), Pirassununga, SP, Brazil
Abstract
The aim of the present work was to load mitotane, an effective drug for adrenocortical carcinoma treatment, in solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC). The SLN and NLC were successfully prepared by high shear homogenization followed by hot high pressure homogenization. Formulations were composed of cetyl palmitate as the solid lipid for SLN, whereas for NLC PEGylated stearic acid was selected as solid lipid and medium chain triacylglycerols as the liquid lipid. Tween® 80 and Span® 85 were used as surfactants for all formulations. The particle size, zeta potential, polydispersity index (PI), encapsulation efficiency (EE), and loading capacity (LC) were evaluated. The SLN showed a mean particle size of 150 nm, PI of 0.20, and surface charge -10 mV, and the EE and LC could reach up to 92.26% and 0.92%, respectively. The NLC were obtained with a mean particle size of 250 nm, PI of 0.30, zeta potential -15 mV and 84.50% EE, and 0.84% LC, respectively. Hydrophilic coating of SLN with chitosan or benzalkonium chloride was effective in changing zeta potential from negative to positive values. The results suggest that mitotane was efficiently loaded in SLN and in NLC, being potential delivery systems for improving mitotane LC and controlled drug release.
Keywords: Mitotane, solid lipid nanoparticles, nanostructured lipid carriers, PEGylated stearic acid, chitosan
Introduction
Adrenocortical carcinoma is a rare heterogeneous neo- plasm with an incompletely understood pathogenesis and a poor prognosis. Mitotane is the only drug approved by the Food and Drug Administration (FDA) for treat- ment of adrenocortical carcinoma.[1] It is marketed by Bristol Myers Squibb in the form of tablets containing 500 mg of the drug. The usual dose is 2 to 6g/day, which has been reported to cause gastrointestinal adverse effects (anorexia, nausea, vomiting, and diarrhea) in 80% of patients, and neurological toxicity (central ner- vous system depression, dizziness, vertigo, headache, confusion, weakness, and emotional ability) in 40% of patients.[2,3] Due to its lipophilic character, initially mito- tane suffers accumulation in adipose tissue and appears in low concentrations in the bloodstream.[4] The effect
of reducing the adrenocortical carcinoma has been reported for plasma concentrations greater than 14mg/ mL.[4] Mitotane has a reduced therapeutic range, and more severe reactions occur when plasma concentra- tions exceed 20 mg/mL.[5]
Mitotane (1-chloro-2-[2,2-dichloro-1-(4- chlorophenyl)-ethyl]-benzene) that has water solubility of 0.1 mg/L (Figure 1), shows low permeability in the cell, limiting its absorption.[6] Only 60% of the dose adminis- tered orally is absorbed, thus the development of new formulations to circumvent the problem of absorption is being envisaged.[6] For this purpose, colloidal systems have been used for the encapsulation of drugs to improve bioavailability when used in low doses[7,8] and reducing toxicity.[7,9] Solid lipid nanoparticles (SLN) have been
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proposed for several administration routes including for oral drug delivery. Drug encapsulation in SLN reduces the risk of toxicity, and produces a controlled release pro- file.[10-12] Thus, the objective was to encapsulate mitotane in SLN and nanostructured lipid carriers (NLC) coated with hydrophilic polymers to promote mucoadhesion, increase drug bioavailability, and reduce toxicity.
Material and methods
Materials
Cetyl palmitate (Crodamol CP®, Croda, Brazil), PEGylated stearic acid (Sigma, EUA), medium chain triacylglycerols (CB-C10) (GTCC Crodamol®, Croda, Brazil), polysorbate 80 (Tween® 80, Synth, Brazil), sorbitan trioleate (Span® 85, Croda, Brazil), chitosan (82% deacetylation degree, 296.6 kDa; Polymar, Brazil), benzalkonium chloride (Vetec, Brazil), and mitotane (commercialized as race- mic mixture) (Yick-Vic Chemicals & Pharmaceuticals, China) were used.
Methods
Hydrophilic coating of mitotane-loaded SLN and NLC
The SLN- and NLC- loading mitotane were prepared by hot high shear homogenization following high pressure homogenization (HPH) technique. The experimental pro- tocol consisted of melting the lipid phase at 80℃ following the addition of mitotane. An aqueous solution containing the surfactant combination Tween® 80/Span® 85 in the ratio 7:93 (wt%) was heated at the same temperature. The melting lipid phase was dispersed in the aqueous surfactant
under 10.000 rpm for 1 min using the Ultra-Turrax® (IKA, model T25, impeller 10G).[13] Then, the formed microemul- sion was transferred to the HPH (GEA Niro Soavi, model NS1001L2K, Panda 2K), coupled with a hot water system to the HPH input (80℃), where the formulation passed three times at 500 bar. After cycling, the formulation was cooled down to room temperature. The composition of developed SLN and NLC is shown in Table 1. All formulations were produced in triplicate.
Since NLC had already a hydrophilic coating provided by PEGylated stearic acid, for SLN, composed of cetyl pal- mitate as solid lipid, the coating was done using chitosan or benzalkonium chloride. Chitosan aqueous solution, 0.001% (w/v) containing glacial acetic acid 0.75% (v/v) was stirred for 24h at room temperature (25℃). A similar process was used for production of 0.001% benzalkonium chloride solution. The coating of particles was carried out using known quantities of chitosan or benzalkonium chloride and stirred for 2 min. For each added amount, the surface electrical charge was determined.
Particle size, polydispersity, and zeta potential analysis
The SLN and NLC were evaluated with respect to the hydrodynamic mean size, polydispersity index (PI), and zeta potential. The mean size was determined by Dynamic Light Scattering (DLS; Zetasizer Nano NS, Malvern, UK).[14] The samples were diluted with ultra-purified water to weaken the opalescence before particle size measure- ments. Zeta potential was analyzed in purified water adjusting conductivity to 50 µS/cm. The zeta potential was calculated from the electrophoretic mobility using the Helmholtz-Smoluchowski equation.[15] The analysis was performed using the software included in the system.
Encapsulation efficiency and loading capacity
To determine the encapsulation efficiency (EE) and load- ing capacity (LC), SLN and NLC (Tables 1 and 2) were frozen with liquid nitrogen (N2) and lyophilized (Liobras, L, Brazil) for 48 h.[16] Samples were dissolved in methanol
| Formulation | Oil phase | Aqueous phase | |||||
|---|---|---|---|---|---|---|---|
| *Cetyl palmitate | *Stearic acid | *Triacylglycerol | *Mitotane | *Span® 85 | *Tween® 80 | Water (mL) | |
| SLN 1 | 3 | – | – | – | 0.21 | 2.79 | 200 |
| SLN 2 | 3 | – | – | 0.05 | 0.21 | 2.79 | 200 |
| SLN 3 | 3 | – | – | 0.2 | 0.21 | 2.79 | 200 |
| SLN 4 | 3 | – | – | 0.3 | 0.21 | 2.79 | 200 |
| SLN 5 | 3 | – | – | 0.6 | 0.21 | 2.79 | 200 |
| SLN 6 | 3 | – | – | 1.2 | 0.21 | 2.79 | 200 |
| SLN 7 | 3 | – | – | 3.0 | 0.21 | 2.79 | 200 |
| NLC 1 | – | 2.1 | 0.9 | – | 0.42 | 5.58 | 200 |
| NLC 2 | – | 2.1 | 0.9 | 0.05 | 0.42 | 5.58 | 200 |
| NLC 3 | – | 2.1 | 0.9 | 0.2 | 0.42 | 5.58 | 200 |
| NLC 4 | – | 2.1 | 0.9 | 0.3 | 0.42 | 5.58 | 200 |
| NLC 5 | – | 2.1 | 0.9 | 0.6 | 0.42 | 5.58 | 200 |
| NLC 6 | – | 2.1 | 0.9 | 1.2 | 0.42 | 5.58 | 200 |
| NLC 7 | – | 2.1 | 0.9 | 3.0 | 0.42 | 5.58 | 200 |
*Values in percentage (wt%).
| Formulation | SLN | NLC | ||
|---|---|---|---|---|
| EE (%) | LC* | EE (%) | LC* | |
| 1 | 0.000 | 0.000 | 0.000 | 0.000 |
| 2 | 63.633 | 0.042 | 59.605 | 0.039 |
| 3 | 53.437 | 0.053 | 96.717 | 0.096 |
| 4 | 56.924 | 0.114 | 84.941 | 0.169 |
| 5 | 88.492 | 0.354 | 77.259 | 0.309 |
| 6 | 92.262 | 0.923 | 84.506 | 0.845 |
*(g mitotane/g lipid).
and kept in water bath at 65℃ for 30 min, then were main- tained at room temperature until complete cooling and solidification of the lipid phase. Subsequently, samples were centrifuged for 15 min at 20.000 rpm (Eppendorf, 5417R, USA), supernatant was collected and analyzed in the UV spectrophotometer (Malvern, S-MAM 5005, UK) at 230 nm for its free mitotane content. The EE was calcu- lated applying the following equation:
EE(%) 5
M2M, M.
where M, is initial mass of mitotane and M, is mass of mitotane determined in the supernatant by spectropho- tometry UV. The LC was calculated by
LC(%) 5
M incorporated mitotane
M lipid
where M incorporated mitotane stands for the mass of mitotane incorporated in SLN or NLC and M lipid stands for the mass of lipid used to produce the nanoparticles.
Results and discussion
Mitotane-loaded SLN and NLC were successfully pre- pared by high shear homogenization followed by HPH. This production process developed physically stable nanoparticle dispersions, being a reliable, simple, and reproducible lab process. The optimization of emulsifiers concentration was previously studied by Severino et al.[17] The DLS characterized SLN with hydrodynamic mean size of 150±3.5 nm and PI of 0.20±0.03. For NLC, the size was 250±3.3nm with a PI of 0.30±0.03. Smaller mean particle size was obtained for mitotane-loaded SLN than for NLC, attributed to the similar lipophilic characteris- tics of drug and the solid lipid (i.e. cetyl palmitate).
The surface electrical charge obtained for mitotane- loaded in SLN was -10±1.2 mV, whereas for NLC was -15±2.03mV. The SLN and NLC were, therefore, considered with acceptable properties for oral administration of mito- tane. The broad spectrum of mitotane depicted one maxi- mum, at the wavelength of 2=230nm, which was used to determine the EE and LC, applying the following equation:
y50.0759x20.011, R250.09944
(a) 1.00
0.80
D/L (Final)
0.60
0.40
0.20
0.00
0.00
0.20
0.40
0.60
0.80
1.00
1.20
D/L (Initial)
(b) 1.00
0.80
D/L (Final)
0.60
0.40
0.20
0.00
0.00
0.20
0.40
0.60
0.80
1.00
1.20
D/L (Initial)
(a) 12
Zeta potential (mV)
8
4
0
5
10
15
20
25
30
-4
Chitosan (mg)
-8
(b) 15
Zeta potential (mV)
10
5
0
0.03
0.05
0.07
0.09
0.11
-5
Benzallkonium chloride (mg)
-10
Figure 2A and B shows the drug-to-lipid (D/L) ratio of before (initial) and after (final) production of SLN and NLC. The effect of D/L in the final characteristics of the
nanoparticles was evaluated on the basis of the EE values. Results showed increase of final D/L ratio rising to initial D/L, following the increase of EE for both SLN and NLC.
Table 2 depicts the results of EE and LC obtained for SLN and NLC, showing improved EE and LC with the increase of D/L ratio. These results were expected due to the lipophilic character of mitotane.
From our results, SLN provided encapsulation param- eters (i.e. EE and LC) in comparison to NLC. This result was attributed to the high hydrophobic character of PEG-stearic acid used in NLC, in comparison to cetyl palmitate of the SLN matrix.[18] From the supplier data- base, PEG-stearic acid has a water solubility of 0.003 mg/ mL,[19] whereas the wax cetyl palmitate is reported to be insoluble in water.[20] Mitotane is a poor water soluble compound of low bioavailability. [21] Lipid screening of the drug in different solid lipids showed improved solubility in cetyl palmitate (selected as solid lipid for SLN) and in stearic acid (selected as solid lipid for NLC).
Figure 3 shows the variation of zeta potential versus the amount of benzalkonium chloride coated on the par- ticles. The coating was carried out to promote mucoad- hesive of lipid nanoparticles to the gut wall. It is known that positive surface charge promotes electrostatic inter- action with negative charges of sialic acid present in the mucin.[22] The results show that benzalkonium chloride is a suitable compound for coating lipid particles. The surface charge varied from negative to positive, and additional experiments were carried out adding exces- sive amounts of chitosan or benzalkonium chloride and no changes occurred on the maximum surface load onto particles. Benzalkonium chloride is a cationic surfactant widely used in ophthalmic[23] and oral disinfectant[24] formulations. The reported toxic doses are 15 and 250 mg/kg. [25,26] In the present work, much lower amount has been used for safety reasons. Under hot HPH, mito- tane is reported to be a stable drug. [27] In addition, the time that the drug is exposed at high temperature is short.[28-30] In our previous work, homogenous and stable emulsion was obtained with a combination of surfactants (Tween® 80 and Span® 85) creating HLB of 9.72.[31] Cetyl palmitate thermogram depicted a melting peak at 56.2℃, and mito- tane was shown stable during the production process.
Conclusion
This study shows that it is possible to prepare physico- chemically stable mitotane loaded in SLN and NLC for at least 30 days under storage at room temperature. The SLN provided improved encapsulation parameters and were coated with hydrophilic chitosan or benzalkonium chloride. The SLN coating improved surface charge toward the positive values suitable for gut intake.
Declaration of interest
The authors acknowledge the financial support obtained from the Fundação de Amparo a Pesquisa do Estado de
São Paulo (FAPESP/Brazil) and the Conselho Nacional de Pesquisa (CNPq, Brazil). The authors wish to acknowl- edge Fundação para a Ciência e Tecnologia do Ministério da Ciência e Tecnologia, under the reference PTDC/SAU- FAR/113100/2009.
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