Modeling and optimization of nanoemulsion containing Sorafenib for cancer treatment by response surface methodology
© The Author(s) 2017
Received: 5 May 2016
Accepted: 20 February 2017
Published: 2 March 2017
KeywordsNanoemulsion Sorafenib Anti-cancer Parenteral delivery Response surface methodology
Cancer is well known as a fatal disease. It has been found that the rate of survival of cancer-stricken patients has not increased prominently over the last 30 years . Among the key challenges in the successful treatment of cancer patients is the issue of drug resistance over a long period of time. The advantage of nanotechnology has increased the number of research in this area and carriers of nanoemulsion have been found to be an effective method of resolving the issue of drug resistance to chemotherapy drugs for cancer . There are many benefits attached to the drug delivery systems, which include the increase of drug stability in vivo, improved effects of retention and permeability, as well as the ease of surface modification [3, 4]. Nanotechnology has been utilized in various ways over the past decade, including food technology, pesticide use in agriculture, cosmetics, as well as pharmaceuticals [5, 6]. Most pharmacy-related nanocarriers, such as nanoparticles, nanoemulsions, and nanocapsules have been developed to control active biological drugs. These drugs have been encapsulated with nanocarriers for treatments to deal with controlled release and different parenteral, intranasal, oral, as well as transdermal routes . Nanoemulsions are heterogeneous in the 20–200 nm range, whereas immiscible solutions consisting of oil and aqueous ingredients can lead to the dispersal stage [8, 9]. The above-mentioned system has the capability to dissolve large amounts of drugs with the lipophilic features. Moreover, it has the ability to reduce the degradation of drugs by enzymes . Nanoemulsion was utilized in this research as a Sorafenib nanocarrier, with the anticipated capability of reducing the clearance rate in future biological studies. Nanoemulsion similarly needs high-energy input, which is dissipated across massive areas during the process of emulsification .
Some main drawbacks of the specific drugs for anti-cancer use include poor solubility, intense cytotoxicity in healthy tissue , as well as an inability to accurately select tumor tissues; this results in harsh side effects, leading to poor cure rates. Therefore, it is difficult to use the conventional drug delivery approach for targeted abnormal cells . Most drugs are still being investigated for the development of a maximized therapeutic value as well as a minimized or negligible amount of side effects, which includes gastrointestinal (nausea, diarrhea, constipation, vomiting), dermatological, constitutional (loss of weight, exhaustion), cardiovascular (hypertension), as well as painful pulmonary occurrences. Research shows that the nanoparticle carriers for chemotherapy drugs are effective methods of overcoming the resistance to cancer drugs . Among the most widely used and effective path to the administration of drugs is the parenteral drug delivery system that is normally utilized for low bioavailability actives as well as for slim therapeutic indexes [10, 17]. Even though many nanoemulsion systems have been documented, only a few of these can be utilized for the parenteral delivery system due to the surfactant’s toxicity .
A common multivariate statistical technique used to determine optimal conditions is response surface methodology (RSM) . This is the statistical, mathematical, and technical model that is capable of assessing the interactions and relationship between independent variables (factors) and dependent variables (response) . RSM was employed to study the optimal conditions at the low composition of surfactant in nanoemulsion containing Sorafenib. The central composite rotatable design (CCRD) was applied to study the effects of four independent variables, time of stirring overhead, rate and time of high shear, and the cycle of the homogenizer of high pressure, on the one dependant variable (result), namely particle size. RSM allows nanoemulsion development to be completed in a decreasing number of tests with a desirable result in the optimal condition. The objective of this study was the optimization of nanoemulsion condition containing Sorafenib as a parenteral drug delivery system using RSM for the treatment of tumor-cell proliferation.
Results and discussion
Solubility of Sorafenib in selected oils
Screening the independent variables
A study was carried out to evaluate the levels of independent variables (not mentioned). Based on these results, the lower, central and higher levels of four independent variables, the range of overhead stirring time of 80–240 min, high shear stirring time of 10–30 min, high shear rate of 800–5600 rpm, and the cycle of high-pressure homogenizer of 8 cycles to 20 cycles, were selected. Within this range, nanoemulsion formulation containing Sorafenib produced the particle size below 121 nm, a polydispersity index of 0.270, and the zeta potential of more or less ± 25 mV.
Statistical analysis and model fitting
The matrix of actual and predicted values of particle size from CCRD experimental design
Overhead stirring time (min)
Shear rate (rpm)
Shear time (min)
Cycle of homogenizer (cycle)
Particle size (nm)
Analysis of variance of the fitted modify quadratic equation for particle size and regression coefficients of the final reduced models
X 1 2
X 2 2
X 3 2
X 4 2
X2 X3 X4
X 1 2 X2
Lack of fit
Adjusted R 2
Predicted R 2
Response surface analysis
The findings of this research revealed that the tiniest size of the particle could be retrieved during the overhead stirring duration of 4000 rpm and 10 min of rate and high shearing time for high shearing, respectively. A particular additional increase in the size of the particle was noted as being carried out by some people. Rate and time with higher destabilization mechanisms, including coalescence and sedimentation, result in a final larger size or particle. The formation of the bigger particles across a longer duration could be related to the over processing of the emulsification, which could lead to coalescence. As earlier mentioned, some findings recorded for the nanoemulsions emulsification used an ultrasonic approach. Some of the findings were recorded for nanoemulsions emulsification by utilizing the ultrasonic approach as mentioned above [22, 23].
Optimization of the preparation of nanoemulsion formulation containing Sorafenib
The actual and predicted response values for the optimized nanoemulsion
Particle size (nm)
High shear rate
High shear stirring time
High pressure homogenizers
Transmission electron microscopy (TEM) analysis
Stability of nanoemulsion formulation containing Sorafenib
Homogenizers of high shear and pressure were utilized in the nanoemulsion formulation because they performed better than homogenizers that had either high shear or high pressure. The response surface approach was used to optimize particle size. The variables consisted of the overhead stirring time, shear rate, shear time, and the cycle of high-pressure homogenizers. The high-pressure homogenizer’s cycle had the most significant (P < 0.0001) effect on the particle size. Based on the TEM image, the particles are spherical with the average optimum formulation of 82.14 nm, which is a crucial factor in the stability and penetration of the nanoemulsion system. The MTT result showed that the optimum formulation did not significantly affect a normal cell at low drug concentrations, but could eliminate cancer cells. The nanoemulsion showed potential as a safe and effective parenteral delivery system for anticancer drugs. The optimum formulation can deliver Sorafenib into the body with less drug and higher efficacy than when Sorafenib is delivered in tablet form. The formulation exhibited very good stability in 3 months of storage. Therefore, the Sorafenib nanoemulsion could be used as a parenteral formulation and provide parenteral nutrition.
MCT (Pharmaceutical Grade), Glycerol and Lecithin (Lipoid S75) were purchased from Numedica, JT Baker (USA) and GmbH (Germany) respectively. Polysorbate 80 (Tween80) was obtained from Fluka (Germany).The Sorafenib free base was obtained from Xi’an Yiyang Bio-Tech Co., Ltd (China).
Selection of oils
The solubility of Sorafenib with four oil bases such as olive, castor, and soybean oils was investigated to find best solubilizing capacity in present and absent of lecithin. First, Sorafenib was added into the oil phase, then the solution was kept under moderate magnetic stirring at 400 rpm for 24 h. Finally, the sample was centrifuged at 4000 rpm for 30 min.
Preparation of nanoemulsions
The nanoemulsion containing Sorafenib was formulated with MCT and lecithin as the disperse phase and deionized water, while Polysorbate 80 and glycerol was treated as the continuous phase. 0.5% (w/w) Sorafenib was first dissolved into 5% (w/w) MCT followed by 2% (w/w) lecithin with magnetic stirring, at 50 °C. This result was added into the aqueous phase containing 1% Polysorbate 80 and 2.5% glycerol and subsequently blended using overhead stirring time (IKA®RW 20 Digital, Nara, Japan). The samples were subjected to further processing using high shear and high-pressure homogenizer. The samples were subjected to further processing using high shear and high-pressure homogenizer.
Particle size measurement
A Nano ZS90 (Malvern, UK) was utilized for the particle size measurement. The sensitivity range was 1–6000 nm. The particle size distribution was characterized in terms of their mean particle size (Z-average diameter) at room temperature. The samples were diluted with distilled water (1:100 ratio), then they were placed in the capillary cell for measuring of particle size.
Level of independent variables for using RSM
Level of variables
Overhead stirring time
Cycle of homogenizer
Transmission electronic microscopy
Nanoemulsion formulation containing Sorafenib was characterized by high-resolution transmission electron microscope (TEM) with the operating system to capture the morphology of the colloidal system. The diluted nanoemulsion formulation was placed on a carbon-coated copper grid supported with formvar films and allowed to stand for 2 min. Filled carbon-coated copper grid was negatively stained with 1% (w/v) uranyl acetate allowed to stand for 2 min. The carbon-coated copper grids examined with transmission electron microscopy (Hitachi H-7100, Japan).
Stability of nanoemulsion containing Sorafenib was determined by observing the changes of particle size, drug precipitation, and color changing during storage. Studying The effect of the temperature on the long term stability of formulation were stored at 4 ± 2, 25 ± 2 and 45 ± 2 °C for 90 days. The particle size of the nanoemulsions was also was monitored for 1, 30, 60 and 90 days to identify the variation the size of the particle over time.
After the cell viability is determined, the graphs were plotted with the cell viability’s percentage compared to their respective concentration.
water in oil
oil in water
response surface methodology
central composite rotatable designs
medium chain triglycerides
transmission electron microscope
analysis of variance
ZI had a prominent role in the implementation of the experimental section and writing manuscript. MB supervised all the project. HFM had cooperation in teaching and leading response surface methodology part. RA, KS and NS had actively contribution in solving problems, scientific editing of manuscript and involvement in discussion about during whole project. All authors read and approved the final manuscript.
The authors are thankful to the Department of Chemistry and Institute of Bioscience of University Putra Malaysia for the facilities provided throughout this research.
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Yardley DA (2013) Drug resistance and the role of combination chemotherapy in improving patient outcomes. Int J Breast Cancer 2013:15View ArticleGoogle Scholar
- Gowda R, Jones NR, Banerjee S, Robertson GP (2013) Use of nanotechnology to develop multi-drug inhibitors for cancer therapy. J Nanomed Nanotechnol 4:1000184View ArticleGoogle Scholar
- Blanco E, Kessinger CW, Sumer BD, Gao J (2009) Multifunctional micellar nanomedicine for cancer therapy. Exp Biol Med 234:123–131View ArticleGoogle Scholar
- Guo H, Liu Y, Wang Y, Wu J, Yang X, Li R, Wang Y, Zhang N (2014) pH-sensitive pullulan-based nanoparticle carrier for adriamycin to overcome drug-resistance of cancer cells. Carbohydr Polym 111:908–917View ArticleGoogle Scholar
- Anton N, Benoit J-P, Saulnier P (2008) Design and production of nanoparticles formulated from nano-emulsion templates—a review. J Controll Release 128:185–199View ArticleGoogle Scholar
- Al-Edresi S, Baie S (2009) Formulation and stability of whitening VCO-in-water nano-cream. Int J Pharm 373:174–178View ArticleGoogle Scholar
- Tan SF, Masoumi HRF, Karjiban RA, Stanslas J, Kirby BP, Basri M, Basri HB (2016) Ultrasonic emulsification of parenteral valproic acid-loaded nanoemulsion with response surface methodology and evaluation of its stability. Ultrason Sonochem 29:299–308View ArticleGoogle Scholar
- Devarajan V, Ravichandran V (2011) Nanoemulsions: as modified drug delivery tool. Int J Compr Pharm 2:1–6Google Scholar
- Bhatt P, Madhav S (2011) A detailed review on nanoemulsion drug delivery system. Int J Pharm Sci Res. 2:2482–2489Google Scholar
- Lovelyn C, Attama AA (2011) Current state of nanoemulsions in drug delivery. J Biomater Nanobiotechnol 2:626View ArticleGoogle Scholar
- Kobus Z, Kusinska E (2008) Influence of physical properties of liquid on acoustic power of ultrasonic processor. TEKA Kom Mot Energy Roln 8:71–78Google Scholar
- Wilhelm SM, Carter C, Tang L, Wilkie D, McNabola A, Rong H, Chen C, Zhang X, Vincent P, McHugh M (2004) BAY 43-9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis. Cancer Res 64:7099–7109View ArticleGoogle Scholar
- Bracarda S, Ruggeri EM, Monti M, Merlano M, D’Angelo A, Ferraù F, Cortesi E, Santoro A (2012) Early detection, prevention and management of cutaneous adverse events due to Sorafenib: recommendations from the Sorafenib Working Group. Crit Rev Oncol Hematol 82:378–386View ArticleGoogle Scholar
- Wang X-Q, Fan J-M, Liu Y-O, Zhao B, Jia Z-R, Zhang Q (2011) Bioavailability and pharmacokinetics of Sorafenib suspension, nanoparticles and nanomatrix for oral administration to rat. Int J Pharm 419:339–346View ArticleGoogle Scholar
- Pulkkinen M, Pikkarainen J, Wirth T, Tarvainen T, Haapa-aho V, Korhonen H, Seppälä J, Järvinen K (2008) Three-step tumor targeting of paclitaxel using biotinylated PLA-PEG nanoparticles and avidin–biotin technology: formulation development and in vitro anticancer activity. Eur J Pharm Biopharm 70:66–74View ArticleGoogle Scholar
- Mathur V, Satrawala Y, Rajput MS, Kumar P, Shrivastava P, Vishvkarma A (2011) Solid lipid nanoparticles in cancer therapy. Int J Drug Deliv 2:192–199View ArticleGoogle Scholar
- Thiagarajan P (2011) Nanoemulsions for drug delivery through different routes. Res Biotechnol 2:01–13Google Scholar
- Bezerra MA, Santelli RE, Oliveira EP, Villar LS, Escaleira LA (2008) Response surface methodology (RSM) as a tool for optimization in analytical chemistry. Talanta 76:965–977View ArticleGoogle Scholar
- Song M-M, Branford-White C, Nie H-L, Zhu L-M (2011) Optimization of adsorption conditions of BSA on thermosensitive magnetic composite particles using response surface methodology. Coll Surf Biointerfaces 84:477–483View ArticleGoogle Scholar
- Liu JZ, Weng LP, Zhang QL (2003) Optimization of glucose oxidase production by Aspergillus Niger in a benchtop bioreactor using response surface methodology. World J Microbiol Biotechnol 19:317–323View ArticleGoogle Scholar
- Rezaee M, Basri M, Rahman RNZRA, Salleh AB, Chaibakhsh N, Karjiban RA (2014) Formulation development and optimization of palm kernel oil esters-based nanoemulsions containing sodium diclofenac. Int J Nanomed 9:539View ArticleGoogle Scholar
- Lim CJ, Basri M, Omar D, Rahman MB, Salleh AB, Rahman RN (2012) Physicochemical characterization and formation of glyphosate-laden nano-emulsion for herbicide formulation. Ind Crops Prod 36(1):607–613View ArticleGoogle Scholar
- Rezaee M, Basri M, Rahman RNRA, Salleh AB, Chaibakhsh N, Masoumi HR (2014) A multivariate modeling for analysis of factors controlling the particle size and viscosity in palm kernel oil esters-based nanoemulsions. Ind Crops Prod 52:506–511View ArticleGoogle Scholar
- Araújo F, Kelmann R, Araújo B, Finatto R, Teixeira H, Koester L (2011) Development and characterization of parenteral nanoemulsions containing thalidomide. Eur J Pharm Sci 42:238–245View ArticleGoogle Scholar
- Latreille B, Paquin P (1990) Evaluation of emulsion stability by centrifugation with conductivity measurements. J Food Sci 55:1666–1668View ArticleGoogle Scholar
- Tadros T, Izquierdo P, Esquena J, Solans C (2004) Formation and stability of nano-emulsions. Adv Coll Interface Sci 108:303–318View ArticleGoogle Scholar
- Lee M-K, Chun S-K, Choi W-J, Kim J-K, Choi S-H, Kim A, Oungbho K, Park J-S, Ahn WS, Kim C-K (2005) The use of chitosan as a condensing agent to enhance emulsion-mediated gene transfer. Biomaterials 26:2147–2156View ArticleGoogle Scholar
- Rao AS, Reddy SG, Babu PP, Reddy AR (2010) The antioxidant and antiproliferative activities of methanolic extracts from Njavara rice bran. BMC Complement Altern Med. 10:4View ArticleGoogle Scholar