Encapsulation of Cancer Therapeutic Agent Dacarbazine Using Nanostructured Lipid Carrier
Summary
The most commonly used method for nanostructured lipid carrier (NLC) synthesis involves oil-in-water emulsion, homogenization and solidification. This was modified here by applying high shear dispersion after solidification to achieve a NLC with desirable size, improved drug encapsulation and drug loading efficiency as a potential carrier for dacarbazine delivery.
Abstract
The only formula of dacarbazine (Dac) in clinical use is intravenous infusion, presenting a poor therapeutic profile due to the low dispersity of the drug in aqueous solution. To overcome this, a nanostructured lipid carrier (NLC) consisting of glyceryl palmitostearate and isopropyl myristate was developed to encapsulate Dac. NLCs with controlled size were achieved using high shear dispersion (HSD) following solidification of oil-in-water emulsion. The synthesis parameters, including surfactant concentration, the speed and time of HSD were optimized to achieve the smallest NLC with size, polydispersion index and zeta potential of 155 ± 10 nm, 0.2 ± 0.01, and -43.4 ± 2 mV, respectively. The optimal parameters were also employed for Dac-loaded NLC preparation. The resultant NLC loaded with Dac possessed size, polydispersion index and zeta potential of 190 ± 10 nm, 0.2 ± 0.01, and -43.5 ± 1.2 mV, respectively. The drug encapsulation efficiency and drug loading reached 98% and 14%, respectively. This is the first report on encapsulation of Dac using NLC, implying that NLC could be a new potential candidate as drug carrier to improve the therapeutic profile of Dac.
Introduction
Dacarbazine (Dac) is an alkylating agent that exerts anti-tumor activity through nucleic acids methylation or direct DNA damage, leading to cell cycle arrest and cell death 1.
As a first line chemotherapeutic agent, Dac has been used alone or in combination with other chemotherapy drugs for treating various cancers 2-6. It is the most active agent so far used in treating cutaneous and metastatic melanoma, which is the most aggressive form of skin cancer 3,7,8. The response rate, however, is only 20% at best, and the therapeutic effects are often accompanied with severe systemic side effects.
In its natural form, Dac is hydrophilic and is unstable due to its photosensitivity 9. The only available formula for clinical use currently is a sterile powder to be used in suspension for intravenous infusion 7,8. The low response rate and high systemic toxicity rate of the drug is largely attributable to its poor water solubility, therefore low availability at target site, and high distribution at non-target sites, which limits the maximum dose of the drug 10. The rapid degradation and metabolism after intravenous admission together with the development of drug resistance limit the clinical application and therapeutic effect of the drug 11. Therefore, there is an urgent need to develop alternative Dac formulations for treating malignant melanoma.
Colloidal systems containing liposomes, micelles or nanostructured particles have been intensively investigated for their use in drug delivery as reviewed by Marilene et al. 12 Nanostructured particles as potential drug carriers have been attracting increasing attention in the last decade due to their ability to increase drug loading efficiency, control drug release, improve drug pharmacokinetics and biodistribution, and therefore reduce drug systemic toxicity 13. Only a few nanoformulations, however, have been investigated so far for Dac delivery, showing protection of the drug from photo degeneration, increased drug solubility, and improved therapeutic effect 10,14,15. However these formulations suffered from low encapsulating efficiency while some also using synthetic polymer nanoparticles that are not cost effective.
Nanostructured lipid carriers (NLC), made of a mixture of solid and liquid lipids, have been developed for drug delivery 16,17. The drugs to be encapsulated are often soluble in both the liquid lipids and solid lipids phases 18, resulting in a high loading and controlled release 19. This study aims to develop a new Dac formulation based on NLC-encapsulation using glyceryl palmitostearate and isopropyl myristate as lipids. The preparation involved oil-in-water emulsion, evaporation, solidification, and homogenization. The preparations have been characterized for NLC size, shape, ultrastructure, and dispersity, drug encapsulation efficiency and drug loading 20.
Protocol
1. Preparation of Oil-in-water Emulsion
- Weigh glyceryl palmitostearate (120 mg), isopropyl myristate (60 mg), d-α-tocopheryl polyethylene glycol succinate (30 mg) and soybean lecithin (30 mg), and add them to 12.5 ml of organic solvents (6.25 ml acetone and 6.25 ml ethanol). Quickly dissolve the mixture at the temperature 70 °C (5 °C above the melting point of the solid lipid) in water bath.
- Add either 125, 250 or 375 mg of Poloxamer188 in 12.5 ml of ddH2O to achieve 1-3% (respectively) of Poloxamer 188 solution, which is subject to heating at the same temperature as above.
- Add the aqueous phase solution from step 1.2 to the oil phase solution from step 1.1 dropwise to form emulsions under magnetic stirring at 400 rpm . Stir the emulsion at 400 rpm for another 4 hr to allow evaporation of the organic solvents.
2. Solidification and Homogenization
- Leave the emulsion in a cold room (4 °C) for 2 hr to solidify/crystallize.
- To obtain NLC, subject the emulsion to high sheer dispersion (HSD) with a homogenizer at 10,000-15,000 rpm for 10-40 min.
3. Optimization of the NLC Preparation
- Take samples from step 2.2 with a surfactant concentration of 1, 2 and 3% undergoing HSD at speeds of 10,000, 15,000 and 20,000 rpm, respectively, and time intervals of 10, 20, 30 and 40 min, respectively,
- Examine the samples for particle size (PS), poly dispersion index (PDI), morphology and ultrastructure 20.
Note: The parameters that produce particles with the smallest size (155 nm) and PDI (0.2) value are determined as optimal.
4. Preparation of Dac-loaded NLC (NLC-Dac)
- Prepare oil phase solution as described in step 1.1 with the addition of Dac (70 mg) before dissolving the mixture at 70 °C in water bath.
- Prepare an aqueous phase solution as described in step 1.2 with 1% surfactant, and add this solution to that prepared in step 4.1 dropwise to form an emulsion under magnetic stirring at 400 rpm. Stir the emulsion for further 4 hr to evaporate the organic solvents.
- Leave the emulsion in a cold room (4 °C) for 2 hr to solidify/crystallize as described in step 2, and finally, subject the emulsion to high sheer dispersion (HSD) using the optimal parameters determined in step 3.
Representative Results
The preparations of the NLC and NLC-Dac using glyceryl palmitostearate and isopropyl myristate with different parameters were characterized for PS, PDI, morphology and ultrastructure 20. The PS and PDI of the NLCs were surfactant concentration, HSD speed and duration dependent. As judged by PS and PDI of the NLCs, the best results were achieved with 1% of surfactant and a sheer dispersion speed of 15,000 rpm for 30 min (Figure 1A, B and C), which therefore wwew selected as the optimal parameters for NLC preparation in this study.
Figure 1. Optimization of parameters used in NLC preparation. The optimal surfactant concentration and the speed and time of HSD were determined according to their effect on PS and PDI. (A) Effect of surfactant concentration on PS; (B) Effect of HSD speed and time on PS; (C) Effect of HSD speed and time on PDI. This Figure has been modified from 20. The data are presented as mean value of 3 replicates ± standard deviation (mean ± SD).
The optimal parameters were used for NLC-Dac preparation. The smallest size achieved was 150 ± 10 nm for NLC (Figure 2A) and 190 ± 10 nm for NLC-Dac (Figure 2B), both with PDI of 0.2 ± 0.001, indicating a good uniformity.
Figure 2. DLS measurement of NLC. (A) The optimal size distribution of plain NLC; (B) the optimal size distribution of NLC-Dac.
Both NLC and NLC-Dac showed a spherical shape as observed under TEM (Figure 3).
Figure 3. TEM imaging of NLC and NLC-Dac. Both NLC and NLC-Dac showed a spherical shape. (A) Basic NLC structure comprises a surfactant layer (solid black arrow), liquid lipid matrix (white solid arrow), and solid lipid crystals (dotted white arrows); (B) NLC-Dac also exhibited the basic structure as seen in NLC but the surfactant layer, liquid lipid matrix and solid lipid crystals appeared expanded; an extra substructure could be seen inside the solid lipid crystals (indicated by dotted black arrows), indicative of drug loading. Bar scale: 50 nm, Magnification: 55,000X. This Figure has been modified from 20.
The uploading and encapsulation of Dac in NLC is indicated by the size and structure changes as seen in Figures 2 and 3 where NLC-Dac shows a larger size and altered internal structures as compared with NLC. The basic structure of NLC comprises a surfactant layer, a liquid lipid matrix and solid lipid crystals (Figure 3A). NLC-Dac also exhibited the basic structure as a NLC but with expanded surfactant layer, liquid lipid matrix and solid lipid crystals, together with an extra substructure inside the solid lipid crystals (Figure 3B), indicative of drug loading and encapsulation. The solid lipid crystals seen in NLC appeared denser than that in NLC-Dac, indicating that the solid lipid is less crystallized in NLC-Dac.
The drug encapsulation efficiency (EF) and drug loading (DL) percentage were derived from the following equations:
EE% = W1 – W2/W1 x 100 = 98.5%
DL% = W1 – W2/W3 x 100 = 14%
where W1 amount of Dac added in the NLC, W2 amount of un-entrapped Dac, W3 amount of the lipids added 20.
Discussion
Lipid-based nanostructured particles have been utilized to provide a highly lipophilic carrier for delivery of hydrophobic drugs. A NLC is the second generation of solid lipid nanostructured carrier, which are solid at room and body temperature. The incorporation of a solid lipid into a liquid lipid in a NLC results in a less perfect crystallization, thus increasing the drug loading efficiency and also reducing the expulsion of encapsulated drugs during storage.
For NLC synthesis, the most commonly used method involves oil-in-water emulsion, homogenization and solidification/crystallization 21,22. The homogenization allows NLCs to disperse thoroughly in an aqueous phase, whilst the solidification at low temperature allows the inner oil phase to crystallize. Different homogenization methods have been reported including magnetic stirring, ultrasonication, and HSD that are used before and/or during solidification 23,24.
In this study, the commonly used method was initially followed for NLC preparation. As the result was unsatisfactory, the method was modified such that HSD was applied after solidification. This modification proved highly effective in particle generation, PS and PDI control, while also making the NLC synthesis simpler, compared with previous reports on NLC preparation using the same lipids 23,25. It was worth noting that the length of the evaporation (protocol 1.3) and solidification (protocol 2.1) is very critical as too long or too short a time would have negative effects on the generation of NLC.
This study suggests that NLC particles and their aggregation were formed during solidification. The HSD could disrupt the aggregation, which was possibly due to hydrophobic interaction between NLCs, and also stabilize the particles by thoroughly remixing them with surfactant. The synthesis procedure was optimized such that the NLC and NLC-Dac were produced with a size 155 ± 10 nm and 190 ± 10 nm, respectively, and a PDI of 0.2 ± 0.01. The high level of uniformity with particle sizes and the small PDI values indicates that a sufficient dispersion energy was achieved and is well distributed within the solution for disruption of particle aggregates. It has been suggested that particles of 100-200 nm are not prone to uptake by non-targeted cells, including mononuclear phagocytic system, thus having a long blood circulation time in vivo 26,27, whilst a PDI of more than 0.5 is an indication of particle aggregation 28; the lower the PDI value, the higher the size homogeneity between the particles 29. Further increase of the HSD speed and time above the optimal point could result in further PS reduction, and consequently an increase in interactions between small particles as well as re-aggregation. The difference in size and structure between NLC and NLC-Dac suggests that the Dac loading and encapsulation was successful. The drug binding to the outer layer of the NLC and encapsulation inside lipid matrices provide the potential for prolonged drug release, that could involve drug release firstly from the outer layer, followed by the release from the liquid lipid matrix and then from the solid lipid crystals of the NLC 30.
Currently four nanoformulations have been attempted for delivery of Dac as a single agent. The latest formulation reported was designed for duel encapsulation of Dac and vitamin A 32. However these formulations suffered from a low encapsulation efficiency and/or relatively complex synthesis procedures. This is the first report for encapsulation of Dac with a NLC, proving advantageous over other encapsulations reported previously. NLC-Dac is easy to make and presents higher drug encapsulation and drug loading efficiency 20. The NLC-Dac showed nearly 50% of drug released within the first 2 hr whilst the remaining released slowly for up to 30 hr 20. The early release could be due to the binding of the drug with surfactant layer on the surface of the NLC, indicating that this formulation may not be ideal to replace the formulation currently in clinical use. However the drug in NLC-Dac appeared more stable compared with the nanoemulsion reported previously 10. In addition, lipid based vehicles have been proposed for treating cutaneous melanoma and epidermoid carcinoma through topical drug delivery 32,10, indicating that the NLC-Dac developed in this study could also be potentially beneficial for topical application where early drug release would not potentially lead to severe systemic toxicity.
Due to the collective limitations with the available drug delivery carriers developed so far, further research is needed to develop more advanced nanomaterials for Dac delivery for targeted cancer treatment.
Divulgations
The authors have nothing to disclose.
Acknowledgements
The authors acknowledge the Saudi Arabia-funded scholarship (I821) for making the research possible. The authors are grateful to Dr Xianwei Liu for expert support in TEM analysis at Cranfield University.
Materials
| Dacarbazine (DAC) | Sigma Aldrich (Gillingham Dorset, UK) | D2390-100MG | drug used for uploading |
| glyceryl palmitostearate | Gattefossé (Saint_Priest_cédex, France) | 85251-77-0 | solid lipid |
| d-α- Tocopherol polyethylene glycol succinate (TPGS) | Sigma Aldrich (Gillingham Dorset, UK) | 57668 | lipid phase surfactant |
| Poloxamer 188 | Sigma Aldrich (Gillingham Dorset, UK) | 15759-1KG | liqiud phase surfactant |
| Acetone | Sigma Aldrich (Gillingham Dorset, UK) | 650501-1L | organic solvent |
| Ethanol | Sigma Aldrich (Gillingham Dorset, UK) | 459836-1L | organic solvent |
| Soybean lecithin (SL) | Cuisine Innovation (Dijon, France) | SLL1402 | lipid phase surfactant |
| Double-distilled water was collected in our laboratory from | Millipore-Q Gradient A10 ultra-pure water system (Millipore, France) | SAS – 67120 | aqueous phase |
| T 25 digital ULTRA-TURRAX | IKA | 3725000 | as high shear disperser |
| Hotplate Magnetic Stirrer | Scientific Support, Inc | 1454 | emulsion homogenization |
References
- Loo, T. L., Housholder, G. E., Gerulath, A. H., Saunders, P. H., Farquhar, D. Mechanism of action and pharmacology studies with DTIC (NSC 45388). Cancer Treat Rep. 60 (2), 149-152 (1976).
- Behringer, K., et al. Omission of dacarbazine or bleomycin, or both, from the ABVD regimen in treatment of early-stage favourable Hodgkin’s lymphoma (GHSG HD13): An open-label, randomised, non-inferiority trial. The Lancet. 385 (9976), 1418-1427 (2014).
- Carvajal, R. D., et al. A phase 2 randomised study of ramucirumab (IMC-1121B) with or without dacarbazine in patients with metastatic melanoma. Eur J Cancer. 50 (12), 2099-2107 (2014).
- Jiang, G., Li, R., Sun, C., Liu, Y., Zheng, J. Dacarbazine combined targeted therapy versus dacarbazine alone in patients with malignant melanoma: A meta-analysis. PLoS ONE. 9 (12), (2014).
- Lazar, V., et al. Sorafenib plus dacarbazine in solid tumors: A phase i study with dynamic contrast-enhanced ultrasonography and genomic analysis of sequential tumor biopsy samples. Invest New Drugs. 32 (2), 312-322 (2014).
- Niemeijer, N. D., Alblas, G., Van Hulsteijn, L. T., Dekkers, O. M., Corssmit, E. P. M. Chemotherapy with cyclophosphamide, vincristine and dacarbazine for malignant paraganglioma and pheochromocytoma: Systematic review and meta-analysis. Clin Endocrinol (Oxf). 81 (5), 642-651 (2014).
- Bedikian, A. Y., Garbe, C., Conry, R., Lebbe, C., Grob, J. J. Dacarbazine with or without oblimersen (a Bcl-2 antisense oligonucleotide) in chemotherapy-naive patients with advanced melanoma and low-normal serum lactate dehydrogenase: ‘The AGENDA trial’. Melanoma Res. 24 (3), 237-243 (2014).
- Daponte, A., et al. Phase III randomized study of fotemustine and dacarbazine versus dacarbazine with or without interferon-a in advanced malignant melanoma. J Trans Med. 11 (1), (2013).
- Jiao, J., Rhodes, D. G., Burgess, D. J. Multiple Emulsion Stability: Pressure Balance and Interfacial Film Strength. J Colloid Interface Sci. 250 (2), 444-450 (2002).
- Kakumanu, S., Tagne, J. B., Wilson, T. A., Nicolosi, R. J. A nanoemulsion formulation of dacarbazine reduces tumor size in a xenograft mouse epidermoid carcinoma model compared to dacarbazine suspension. Nanomedicine. 7 (3), 277-283 (2011).
- Xie, T., Nguyen, T., Hupe, M., Wei, M. L. Multidrug resistance decreases with mutations of melanosomal regulatory genes. Cancer Res. 69 (3), 992-999 (2009).
- Estanqueiro, M., Amaral, M. H., Conceição, J., Lobo, J. M. S. Nanotechnological carriers for cancer chemotherapy: the state of the art. Colloids Surf., B. 126, 631-648 (2015).
- Koziara, J. M., Whisman, T. R., Tseng, M. T., Mumper, R. J. In-vivo efficacy of novel paclitaxel nanoparticles in paclitaxel-resistant human colorectal tumors. J Controlled Release. 112 (3), 312-319 (2006).
- Ding, B., et al. Biodegradable methoxy poly (ethylene glycol)-poly (lactide) nanoparticles for controlled delivery of dacarbazine: Preparation, characterization and anticancer activity evaluation. Afr J Pharm Pharacol. 5 (11), 1369-1377 (2011).
- Ding, B., et al. Anti-DR5 monoclonal antibody-mediated DTIC-loaded nanoparticles combining chemotherapy and immunotherapy for malignant melanoma: target formulation development and in vitro anticancer activity. Int J Nanomedicine. 6, 1991-2005 (2011).
- Jenning, V., Thünemann, A. F., Gohla, S. H. Characterisation of a novel solid lipid nanoparticle carrier system based on binary mixtures of liquid and solid lipids. Int J Pharm. 199 (2), 167-177 (2000).
- Müller, R. H., Radtke, M., Wissing, S. A. Nanostructured lipid matrices for improved microencapsulation of drugs. Int J Pharm. 242 (1-2), 121-128 (2002).
- Pouton, C. W. Lipid formulations for oral administration of drugs: Non-emulsifying, self-emulsifying and ‘self-microemulsifying’ drug delivery systems. Eur. J. Pharm. Sci. 11 (Suppl. 2), S93-S98 (2000).
- Jores, K., Mehnert, W., Drechsler, M., Bunjes, H., Johann, C., Mäder, K. Investigations on the structure of solid lipid nanoparticles (SLN) and oil-loaded solid lipid nanoparticles by photon correlation spectroscopy, field-flow fractionation and transmission electron microscopy. J Controlled Release. 95 (2), 217-227 (2004).
- Almousallam, M., Zhu, H. Encapsulation of cancer therapeutic agent dacarbazine using nanostructured lipid carrier. Int nano lett. , (2015).
- Ng, W. K., et al. Thymoquinone-loaded nanostructured lipid carrier exhibited cytotoxicity towards breast cancer cell lines (MDA-MB-231 and MCF-7) and cervical cancer cell lines (HeLa and SiHa). BioMed Research International. , (2015).
- Sun, M., et al. Quercetin-nanostructured lipid carriers: Characteristics and anti-breast cancer activities in vitro. Colloids Surf., B. 113, 15-24 (2014).
- Savla, R., Garbuzenko, O. B., Chen, S., Rodriguez-Rodriguez, L., Minko, T. Tumor-Targeted Responsive Nanoparticle-Based Systems for Magnetic Resonance Imaging and Therapy. Pharm Res. 31 (12), 3487-3502 (2014).
- Chen, Y., et al. Formulation, characterization, and evaluation of in vitro skin permeation and in vivo pharmacodynamics of surface-charged tripterine-loaded nanostructured lipid carriers. Int J Nanomedicine. 7, 3023 (2012).
- Sanna, V., Caria, G., Mariani, A. Effect of lipid nanoparticles containing fatty alcohols having different chain length on the ex vivo skin permeability of Econazole nitrate. Powder Technol. 201 (1), 32-36 (2010).
- Brigger, I., Dubernet, C., Couvreur, P. Nanoparticles in cancer therapy and diagnosis. Adv Drug Deliv Rev. 54 (5), 631-651 (2002).
- Visaria, R. K., et al. Enhancement of tumor thermal therapy using gold nanoparticle-assisted tumor necrosis factor-a delivery. Mol Cancer Ther. 5 (4), 1014-1020 (2006).
- Tripathi, A., Gupta, R., Saraf, S. A. PLGA nanoparticles of anti tubercular drug: Drug loading and release studies of a water in-soluble drug. Int J PharmTech Res. 2 (3), 2116-2123 (2010).
- Joshi, M., Patravale, V. Nanostructured lipid carrier (NLC) based gel of celecoxib. Int J Pharm. 346 (1-2), 124-132 (2008).
- Lim, W. M., Rajinikanth, P. S., Mallikarjun, C., Kang, Y. B. Formulation and delivery of itraconazole to the brain using a nanolipid carrier system. Int J Nanomedicine. 9 (1), 2117-2126 (2014).
- Bei, D., Zhang, T., Murowchick, J. B., Youan, B. C. Formulation of dacarbazine-loaded cubosomes. Part III. physicochemical characterization. AAPS PharmSciTech. 11 (3), 1243-1249 (2010).
- Lei, M., et al. Dual drug encapsulation in a novel nano-vesicular carrier for the treatment of cutaneous melanoma: Characterization and in vitro/in vivo evaluation. RSC Advances. 5 (26), (2015).
