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

1. 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.
2. 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.
3. 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

1. Leave the emulsion in a cold room (4 °C) for 2 hr to solidify/crystallize.
2. 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

1. 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,
2. 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)

1. 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.
2. 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.
3. 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.

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

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 com...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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