The aim of the present study was preparation of hyaluronan (HA) targeted solid lipid nanoparticles (SLNs) of etoposide. SLNs were prepared by an emulsification-solvent evaporation method and physically coated with HA. Four variables, including the ratio of cetyl alcohol to cationic lipid, cationic lipid type (stearylamine (SA) or dodecylamine (DDA)), lipid to HA ratio, and organic to aqueous phase ratio, were studied in an irregular fraction factorial design. Four responses, including particle size, zeta potential, drug loading, and 24-hour release efficiency percent, were measured for each formulation and then the optimization was carried out. The percent of HA coated on the SLNs was calculated by CHN elemental analysis which was shown to be about 55.89%. The cationic lipid type and the ratio of cetyl alcohol to cationic lipid had the highest influence on particle size and zeta potential, respectively. The highest effects of the ratio of lipid to HA and the organic to aqueous phase ratio were on the drug loading efficiency of SLNs. The optimized formulation of SLNs was obtained by SA, the equal proportion of cetyl alcohol and cationic lipid, the ratio of 1.5 for lipid to HA, and 10% of organic phase to aqueous phase.
Etoposide (VP-16) is a hydrophobic anticancer agent inhibiting topoisomerase II. Unfortunately, despite its appropriate solubilization in vehicle solvents, its poor bioavailability concurs to disappointing results requiring the development of new delivery system forms. Etoposide is used in the treatment of ovarian cancer [
Nanoparticles are one of the most promising ways in decreasing side effects of anticancer drugs. After injecting the nanoparticles, they tend to accumulate in tumor tissue. This phenomenon owes to more ratios of endocytosis in cancerous cells than healthy cells.
Solid lipid nanoparticles (SLNs) that are often considered for intravenous use are colloidal submicron carriers sized 50 to 1000 nm, composed of solid lipids dispersed in water or surfactant aqueous solutions. These nanoparticles have particular features like small size, high surface of contact, and high loading of drug that makes them as potential and beneficial carriers for improving drug efficacy [
SLNs are similar to o/w emulsions used for total parenteral nutrition, with the difference that emulsion liquid lipid has been replaced with a solid lipid. SLNs have advantages such as controlled drug release in considered site, excellent biocompatibility, increase in drugs’ stability, high drug content, easy industrialization and sterilization, better control on drug release kinetics, high bioavailability for bioactive drugs, chemical protection of sensitive drugs, easier producing process compared to bio-polymeric nanoparticles, being producible by common emulsification methods, long-time stability, and various applications [
There are some reports on the successful production of etoposide containing nanoparticles. In a study [
Etoposide formulated with poly(butyl cyanoacrylate) nanoparticles and polysorbate 80 exhibited the highest cytotoxicity toward adenocarcinoma cells [
Hyaluronan (HA) (Figure
Chemical structure of hyaluronan: polymeric repeat of
Because of high expression of CD44 by cancer cells, targeting of drugs to CD44 by HA is an effective strategy to treat cancers. HA as bound to the nanoparticles, in addition to its targeting roll, can protect from nanoparticles against body phagocytosis system [
Stearylamine (SA), dodecylamine (DDA), and cetyl alcohol were all from Sigma-Aldrich Company (US). Acetone, dichloromethane, dialysis bags with molecular weight cut-off of 12,400 Da, and Tween 80 were from Merck Chemical Company (Germany). Etoposide was a gift from Nippon Kayaku Co., Ltd. (Tokyo, Japan), and sodium hyaluronate (
Four variables, including the ratio of cetyl alcohol to cationic lipid, cationic lipid type, lipid to HA ratio, and organic to aqueous phase ratio, were studied, each with 2 levels in an irregular fraction factorial design (Table
Description and trial levels of studied factors in irregular fraction factorial design used in preparation of etoposide loaded in SLNs.
Studied variables | Level | |
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I | II | |
C: cetyl alcohol/amine ratio | 1 | 2 |
L: lipid/HA ratio | 1.5 | 3 |
A: amine type | Dodecylamine (DDA) | Stearylamine (SA) |
O: organic/aqueous phase (%) | 10 | 20 |
Twelve different formulations were proposed (Table
Different formulations of SLNs loaded with etoposide proposed by Design-Expert software according to an irregular fraction factorial design.
Formulation code | Cetyl alcohol/amine ratio | Lipid/HA ratio | Amine type | Organic/aqueous phase percent |
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C2L1.5DO20 | 2 | 1.5 | DDA | 20 |
C1L3SO20 | 1 | 3 | SA | 20 |
C1L3DO20 | 1 | 3 | DDA | 20 |
C2L3DO10 | 2 | 3 | DDA | 10 |
C1L3SO10 | 1 | 3 | SA | 10 |
C1L1.5SO10 | 1 | 1.5 | SA | 10 |
C1L1.5DO20 | 1 | 1.5 | DDA | 20 |
C2L1.5SO20 | 2 | 1.5 | SA | 20 |
C1L1.5DO10 | 1 | 1.5 | DDA | 10 |
C2L3DO20 | 2 | 3 | DDA | 20 |
C2L3SO10 | 2 | 3 | SA | 10 |
C2L1.5SO10 | 2 | 1.5 | SA | 10 |
SLNs were produced by emulsification-solvent evaporation method. The lipid phase, including 30 mg of etoposide and 60 mg of lipids consisting of cetyl alcohol and SA or DDA, was dissolved in 1.8 or 3.6 mL of the 1 : 1 mixture of acetone-dichloromethane. Then this solution was added during 3 minutes to 18 mL of deionized water containing 1 w/v% of Tween 80 while being stirred at 1200 RPM. Ultimately, produced nanoemulsion was stirred in 600 RPM at room temperature for 75 minutes in order to evaporate the organic solvents [
In order to produce targeted nanoparticles after 15 minutes of adding the organic phase to aqueous phase of the nanoemulsion dispersion, HA was dissolved in deionized water containing Tween 80 (1 w/v%) and added to the mixture of nanoparticles during 5 minutes while being stirred at 600 RPM [
Unbound HA was separated from nanoparticles mixture by dialyzing the SLNs suspension against 100 mL of deionized water containing 1 w/v% of Tween 80 using a dialysis bag with molecular weight cut-off of 12,400 Da for 40 minutes and the dialysis solution was replaced every 10 minutes.
The particle size, polydispersity index, and zeta potential of nanoparticles were measured by a Zetasizer (Zetasizer 3000; Malvern Instruments, Malvern, UK), after 1 : 10 dilution of the samples with deionized water.
The loading efficiency percent of HA targeted SLNs was determined by centrifugation (Eppendorf 5430 centrifuge, Germany) using centrifugal filter tubes (Amicon Ultra, Ireland) with a 10 kDa molecular weight cut-off to separate the aqueous medium [
Drug release profiles from the nanoparticles were determined in phosphate buffer saline (PBS, 0.01 M, pH 7.4 containing 1 w/v% of Tween 80) at 37°C. A total of 2 mL of HA coated nanoparticles suspension was placed in dialysis bags with molecular weight cut-off of 12,400 days and the bag was then completely submerged in a beaker containing 50 mL of PBS on a magnetic stirrer with a speed of 200 RPM. Samples were withdrawn periodically and replaced with the same volume of PBS at the same temperature. The content of etoposide in the samples was determined spectrophotometrically by measuring their absorbance in 268.7 nm from the equation of
The gathered data for responses were analyzed by Design-Expert software and the optimum formulation was suggested by the software. The constraints of particle size were
Morphology of the nanoparticles was characterized by scanning electron microscopy (SEM). The nanoparticles were mounted on aluminum stabs, sputter-coated with a thin layer of Au/Pd, and examined using an SEM (Philips XL30, Almelo, The Netherlands).
After separation of unbound HA, some part of the targeted nanoparticles mixture was dried under vacuum and subjected to elemental analysis (CHN) (CHNS-932, Leco, USA) in order to determine the percentage of HA coated on the sample. By subtracting the total amount of HA from gaining value, the amount of HA bound on the SLNs surface was calculated.
Table
Particle size and polydispersity index (PdI) of nontargeted and HA targeted SLNs (
Formulation code | Particle size (nm) ± SD | Polydispersity index (PdI) ± SD | ||
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Nontargeted | HA targeted | Nontargeted | HA targeted | |
C2L1.5DO20 |
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C1L3SO20 |
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C1L3DO20 |
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C2L3DO10 |
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C1L3SO10 |
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C1L1.5SO10 |
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C1L1.5DO20 |
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C2L1.5SO20 |
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C1L1.5DO10 |
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C2L3DO20 |
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C2L3SO10 |
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C2L1.5SO10 |
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As it could be observed in Table
Effect of studied variables on particle size and zeta potential of nontargeted SLNs.
Increase in cetyl alcohol/amine ratio in DDA containing SLNs caused decrease in particle size (Figure
It could be understood that SA had more effects upon particle size reduction, when used with the 1 : 1 ratio with the lipid (Figure
Contribution percent of studied variables on responses of nontargeted and HA targeted SLNs of etoposide.
As Table
Effect of studied variables on particle size and zeta potential of HA targeted SLNs.
The effect of increasing of cetyl alcohol/amine ratio in both types of targeted SLNs containing SA and DDA was similar to nontargeted SLNs (Figures
Table
Zeta potential of nontargeted and HA targeted SLNs of etoposide (
Formulation code | Nontargeted (mV) ± SD | HA targeted (mV) ± SD |
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C2L1.5DO20 |
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C1L3SO20 |
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C1L3DO20 |
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C2L3DO10 |
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C1L3SO10 |
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C1L1.5SO10 |
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C1L1.5DO20 |
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C2L1.5SO20 |
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C1L1.5DO10 |
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C2L3DO20 |
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C2L3SO10 |
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C2L1.5SO10 |
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The formulations of C1L1.5DO20 and C2L1.5SO20 had the largest and the smallest values of zeta potential, respectively (Table
The formulations of C2L3DO10 and C1L1.5DO20 had the largest and the smallest values of zeta potential among the targeted SLNs, respectively (Table
Table
Etoposide loading and release efficiency percent in 24 hours from HA targeted SLNs (
Formulation code | Loading efficiency (%) | RE24 (%) |
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C2L1.5DO20 | 54.76 ± 5.71 | 52.89 ± 7.06 |
C1L3SO20 | 59.76 ± 3.22 | 65.05 ± 4.11 |
C1L3DO20 | 42.56 ± 6.32 | 59.71 ± 8.17 |
C2L3DO10 | 58.31 ± 8.79 | 57.49 ± 6.92 |
C1L3SO10 | 65.70 ± 5.36 | 69.06 ± 5.62 |
C1L1.5SO10 | 66.58 ± 4.56 | 65.36 ± 4.53 |
C1L1.5DO20 | 39.53 ± 7.16 | 55.22 ± 7.86 |
C2L1.5SO20 | 37.84 ± 6.19 | 54.47 ± 7.26 |
C1L1.5DO10 | 40.66 ± 5.52 | 59.71 ± 6.44 |
C2L3DO20 | 53.55 ± 8.49 | 54.24 ± 7.39 |
C2L3SO10 | 43.72 ± 8.27 | 59.28 ± 6.83 |
C2L1.5SO10 | 41.93 ± 4.95 | 55.35 ± 5.92 |
The formulations of C2L1.5SO20 and C1L1.5SO10 had the least and the highest etoposide loading efficiency percent, respectively (Table
Effect of studied variables on etoposide loading efficiency percent in HA targeted SLNs.
In DDA SLNs, increase in the cetyl alcohol/amine ratio (or reduction of DDA which increases the ratio of cetyl alcohol/amine amount) increased drug loading efficiency percent (Figure
The formulations of C2L1.5DO20 and C1L3SO10 had the lowest and the highest release efficiencies, respectively (Table
Effect of studied variables on etoposide release efficiency percent in 24 h from HA targeted SLNs.
The physicochemical properties of the drug are of the most important factors in the drug release rate through nanoparticles [
The most effective variable on the
Etoposide release profiles from different HA targeted SLNs of dodecylamine and stearylamine are shown in Figures
Etoposide release profiles from different HA targeted SLNs of (a) dodecylamine and (b) stearylamine.
The optimization was conducted by the Design-Expert software according to the gained responses discussed earlier. The suggesting formulation by the software with desirability of 86.8% was a formulation prepared using SA as amine type, cetyl alcohol/amine ratio of 1 : 1, lipid/HA ratio equal to 1.5 : 1, and organic/aqueous phase percent equal to 10%. This situation proposed by the software is in accordance with the formulation of C1L1.5SO10.
The predicted and actual values of responses for this formulation are compared in Table
Comparison of predicted values of different responses by Design-Expert software and their actual values.
Responses | Particle size (nm) | Zeta potential (mV) | Drug loading efficiency (%) | RE24 (%) |
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Predicted | 403.96 | −12.99 | 65.76 | 64.94 |
Actual |
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Error % | 3.08 | 2.62 | −1.28 | 0.82 |
The optimized formulation containing HA was compared with those lacking coating. The particle size of these SLNs was 170.3 nm before coating and was enhanced to 416.42 nm after HA coating. Zeta potential of these SLNs was also 11.94 mV which changed to −12.65 mV after coating with HA. Drug release profile from the optimum formulation has been shown in Figure
The morphology of the optimized HA coated SLNs is seen in Figure
SEM image of HA coated C1L1.5SO10 nanoparticles as the optimized SLNs.
Results of the CHNS/O elemental analysis are seen in Table
The results of elemental analysis of nontargeted and HA targeted SLNs.
SLN type | Elements % | ||
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C | H | N | |
Nontargeted SLNs |
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HA targeted SLNs |
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Based on the amount of C, H, and N (carbon, hydrogen, and nitrogen) percentage in the conjugate, the conjugation efficiency percent of HA attached per SLNs weight was determined. C, N, and H% of each conjugate were calculated based on the data found for uncoated SLNs plus the amount calculated for the percent of the HA molecules attached to the SLNs.Considering the calculated ratio of C/N ratio in different theoretical percentages of HA attached to the SLNs a polynomial equation was obtained and then by using the experimental ratio of C/N of CHN analysis the percent of HA was calculated to be about 55.89 percent of HA bound to the nanoparticles surface physically, which is a considerable percentage.
In conclusion, preparation of the HA targeted SLNs of etoposide using solvent diffusion method was optimized statistically by irregular fraction factorial design. The most desirable SLN formulation contained stearylamine, cetyl alcohol/amine ratio of 1 : 1, lipid/HA ratio equal to 1.5 : 1, and organic/aqueous phase percent of 10%. HA was coated successfully on the SLNs physically and about 55.89% of HA was attached to the nanoparticles. These SLNs had the particle size of 416.42 nm after HA coating, zeta potential of 12.65 mV, with release efficiency of 65.47% after 24 hours and drug loading efficiency was about 64.92%. This formulation seems promising from the physicochemical properties point of view and may be considered for further studies in drug targeting to ovarian cancer cells overexpressing CD44 receptors.
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors would like to thank Vice Chancellor for Research of Isfahan University of Medical Sciences that accepted project finances.