Integration of PEG 400 into a self-nanoemulsifying drug delivery system improves drug loading capacity and nasal mucosa permeability and prolongs the survival of rats with malignant brain tumors

Abstract

Introduction:

Kolliphor^® EL (K-EL) is among the most useful surfactants in the preparation of emulsions. However, it is associated with low hydrophobic drug loading in the resulting emulsified formulation.

Methods:

In this study, a formulation for intranasal administration of butylidenephthalide (Bdph), a candidate drug against glioblastoma (GBM), was prepared. Physical characteristics of the formulation such as particle size, zeta potential, conductivity, and viscosity were assessed, as well as its cytotoxicity and permeability, in order to optimize the formulation and improve its drug loading capacity.

Results:

The optimized formulation involved the integration of polyethylene glycol 400 (PEG 400) in K-EL to encapsulate Bdph dissolved in dimethyl sulfoxide (DMSO), and it exhibited higher drug loading capacity and drug solubility in water than the old formulation, which did not contain PEG 400. Incorporation of PEG 400 as a co-surfactant increased Bdph loading capacity to up to 50% (v/v), even in formulations using Kolliphor^® HS 15 (K-HS15) as a surfactant, which is less compatible with Bdph than K-EL. The optimized Bdph formulation presented 5- and 2.5-fold higher permeability and cytotoxicity, respectively, in human GBM than stock Bdph. This could be attributed to the high drug loading capacity and the high polarity index due to DMSO, which increases the compatibility between the drug and the cell. Rats bearing a brain glioma treated with 160 mg/kg intranasal emulsified Bdph had a mean survival of 37 days, which is the same survival time achieved by treatment with 320 mg/kg stock Bdph. This implies that the optimized emulsified formulation required only half the Bdph dose to achieve an efficacy similar to that of stock Bdph in the treatment of animals with malignant brain tumor.

Keywords:

• glioblastoma
• polyethylene glycol 400
• butylidenephthalide
• loading capacity
• permeability
• intranasal administration

Acknowledgment

Financial support was received from the National Science Council of the Republic of China, Taiwan (NSC 102-2221-E-259-017) and Ministry of Science and Technology of the Republic of China, Taiwan (MOST 103-2221-E-259-035 and MOST 107-2218-E-303-001-MY3).

Abbreviation list

BBB, blood-brain barrier; Bdph, butylidenephthalide; CNS, central nervous system; DMSO, dimethyl sulfoxide; EtOH, ethanol; GBM, glioblastoma; HLB, hydrophilic-lipophilic balance; IC₅₀, 50% inhibitory concentration; K-EL: Kolliphor^® EL; K-HS15, Kolliphor^® HS 15; PBS, phosphate-buffered saline; P_eff: permeability coefficient; PEG 400, Polyethylene glycol 400; PG, propylene glycol; R², coefficient of determination; SD, standard deviation; TEM: transmission electron microscopy.

Disclosure

The formulation has been patented in the USA (Patent No. US9504751 (B2)). Miss Yu-Shuan Chen reports non-financial support from National Science Council of the Republic of China, Taiwan, during the conduct of the study. Dr Shinn-Zong Lin reports non-financial support from National Science Council of the Republic of China, Taiwan, during the conduct of the study. Dr Tzyy-Wen Chiou reports grants from National Science Council, Taiwan, Republic of China, during the conduct of the study. The authors report no further conflicts of interest in this work.

Supplementary material

Supplementary material and method

Permeation study in an artificial cellulose membrane

The permeation of Bdph emulsion was evaluated across a cellulose membrane in a Franz diffusion cell (customized from San Mei, Taiwan). The diffusion area of cellulose membrane and the receptor chamber of a Franz diffusion cell were approximately 4.5 cm² and 23 mL, respectively. An artificial cellulose membrane was cut and immersed into HEPES buffer (118mM NaCl, 1.2 mM MgSO₄, 4.8 mM KCl, 5.5 mM D-glucose, 2.5 mM CaCl₂, mM Hepes) before the permeation study. The 2 ml of 3 mg/mL Bdph emulsion was added into the donor chamber and HEPES buffer was placed in the receptor chamber before the membrane was immersed into the buffer. A multipoint stirrer was set to 300 rpm, and the whole experiment took place in an incubator at 37 °C. After 10 h, 1 mL of sample was collected from the receptor chamber each hour, after which 1 mL fresh warmed HEPES buffer was added to the receptor chamber. The sampled solution was detected using absorption at 310 nm by ELISA reader. A permeability coefficient (P_eff) and flux (J) were obtained using the following supplementary Formula (S1) and (S2).¹

(S1) $P_{eff}=\frac{\mathrm{V}}{AC\mathbf{0}}\frac{\mathit{\text{dc}}}{\mathit{\text{dt}}}$(S1)

(S2) $\mathbf{J}=\left(\frac{\mathit{\text{dc}}}{\mathit{\text{dt}}}\right)\frac{\mathit{\text{V}}}{\mathit{\text{A}}}$(S2)

V：Capacity of receptor chamber (mL)

C₀：Concentration of drug in the donor compartment (mg/mL)

P_eff：Permeability coefficient (cm/s)

J：Flux (μg/cm²s)

A：Superficial area of diffusion (cm²)

$\frac{dc}{dt}$: Concentration changed over time in a pseudo steady-state (μg/mLs)

Permeation study in nasal mucosa

Human nasal septum quasi-diploid tumor (RPMI 2650) cells were seeded on a cell insert (0.4 μm pores size membrane) pre-coated with collagen and cultivated for 2 days. The cell insert contained monolayer cells placed in a 12 well plate with 1.5 mL HEPES buffer. The emulsified Bdph solution (0.5 mL) dissolved in HEPES buffer were added to the monolayer cells and incubated at 37 °C in 5% CO₂. The samples were harvested from the well at 1 hr intervals over 6 hrs and analyzed by UV-VIS spectroscopy. The concentration of Bdph was calculated by the absorption at 310 nm using a standard reference curve. The permeability coefficient (P_eff) and Flux were calculated using supplementary Formula (S1) and (S2).

Cultivation of cell lines

GBM 8401, human nasal septum quasidiploid tumor (RPMI 2650), and rat glioma cell lines 9L were purchased from Bioresources Collection and Research Center (BCRC, Taiwan). The GBM8401, RPMI 2650, and 9L cells were cultivated in basal medium of RPMI-1640 (Gibco), Minimum essential medium (Corning), and Dulbecco’s Modified Eagle Medium (Corning), respectively. All the cultural media were provided 10% fetal bovine serum (Gibco) and 1% Penicillin/Streptomycin (100 units/L Penicillin G and 100 μg/L Streptomycin Sulfate, Gibco). The procedures of cell passage were performed following the addition of 0.05% trypsin/EDTA (Biowest, Nuaille, France) to trypsinize the cells, and centrifugation at 240 g for 5 min to harvest the cells. The cellular suspensions were reconstituted in their cultivation medium and incubated at 37 °C in 5% CO₂.

MTT assay

GBM8401 Cells (6×10³) were seeded in a 96 well for overnight cultivation. Stock Bdph, emulsified Bdph, and medium alone (negative control group) were then added and left to incubate for 24 hrs. Thereafter, the supernatant was removed and 500 μg/mL thiazolyl bluetetrazolium blue (MTT, Sigma, MO, USA) was added to detect mitochondrial activity in any surviving cells. After 1 hr incubation, DMSO was added to dissolve the formazan crystal products. Absorption of the DMSO/formazan solution was detected via enzyme-linked immunosorbent assay (ELISA) reader at 595 nm.

Transmission electron microscopy (TEM)

For TEM observation, the emulsified formulation was diluted 500 times with pure water. Then, 10 μL of the diluted formulation was applied to carbon film-coated 400 mesh copper grids (Electron Microscopy Sciences, Hatfield, PA, USA) for 60 s and extra loaded solution was adsorbed by Kimwipe paper. Grids were negatively stained by incubation with 10 μL of 1.5% phosphotungstic acid for another 60 s. Again, extra loaded solution was adsorbed by Kimwipe paper. The grids of the emulsified formulation were stored in a desiccator for further analysis. Samples were visualized at 120 kV by a transmission electron microscope (JEOL JEM-1200CX II).

Reference

• Karasulu HY, Sanal ZE, Sozer S, Guneri T, Ertan G. Permeation studies of indomethacin from different emulsions for nasal delivery and their possible anti-inflammatory effects. AAPS PharmSciTech. 2008;9(2):342–348. doi:10.1208/s12249-008-9053-918431669
   PubMedGoogle Scholar