Investigation of Eutectic Mixtures of Fatty Acids as a Novel Construct for Temperature-Responsive Drug Delivery

Abstract

Background: Most of the traditional nanocarriers of cancer therapeutic moieties present dose-related toxicities due to the uptake of chemotherapeutic agents in normal body cells. The severe life-threatening effects of systemic chemotherapy are well documented. Doxorubicin, DOX is the most effective antineoplastic agent but with the least specific action that is responsible for severe cardiotoxicity and myelosuppression that necessitates careful monitoring while administering. Stimuli-sensitive/intelligent drug delivery systems, specifically those utilizing temperature as an external stimulus to activate the release of encapsulated drugs, have become a subject of recent research. Thus, it would be ideal to have a nanocarrier comprising safe excipients and controllable drug release capacity to deliver the drug at a particular site to minimize unwanted and toxic effects of chemotherapeutics. We have developed a simple temperature-responsive nanocarrier based on eutectic mixture of fatty acids. This study aimed to develop, physicochemically characterize and investigate the biological safety of eutectic mixture of fatty acids as a novel construct for temperature-responsive drug release potential.

Methods: We have developed phase change material, PCM, based on a series of eutectic mixtures of fatty acids due to their unique and attractive physicochemical characteristics such as safety, stability, cost-effectiveness, and ease of availability. The reversible solid-liquid phase transition of PCM is responsible to hold firm or actively release the encapsulated drug. The eutectic mixtures of fatty acids (stearic acid and myristic acid) along with liquid lipid (oleic acid) were prepared to exhibit a tunable thermoresponsive platform. Doxorubicin-loaded lipid nanocarriers were successfully developed with combined hot melt encapsulation (HME) and sonication method and characterized to achieve enhanced permeability and retention (EPR) effect-based solid tumor targeting in response to exogenous temperature stimulus. The cytotoxicity against melanoma cell lines and in vivo safety studies in albino rats was also carried out.

Results: Doxorubicin-loaded lipid nanocarriers have a narrow size distribution (94.59-219.3 nm), and a PDI (0.160-0.479) as demonstrated by photon correlation microscopy and excellent colloidal stability (Z.P value: -22.7 to -32.0) was developed. Transmission electron microscopy revealed their spherical morphology and characteristics of a monodispersed system. A biphasic drug release pattern with a triggered drug release at 41°C and 43°C and a sustained drug release was observed at 37°C. The thermoresponsive cytotoxic potential was demonstrated in B16F10 cancer cell lines. Hemolysis assay and acute toxicity studies with drug-free and doxorubicin lipid nanocarrier formulations provided evidence for their non-toxic nature.

Conclusion: We have successfully developed a temperature-responsive tunable platform with excellent biocompatibility and intelligent drug release potential. The formulation components being from natural sources present superior characteristics in terms of cost, compatibility with normal body cells, and adaptability to preparation methods. The reported preparation method is adapted to avoid complex chemical processes and the use of organic solvents. The lipid nanocarriers with tunable thermoresponsive characteristics are promising biocompatible drug delivery systems for improved localized delivery of chemotherapeutic agents.

Keywords: acute toxicity studies; doxorubicin; eutectic mixtures; fatty acids; hemolysis assay; lipid nanocarriers; nanostructured lipid carriers; phase change materials; thermoresponsive.

Graphical abstract

Figure 1

(A) FTIR spectroscopic analysis of eutectic mixture of myristic and stearic acid. (B) FTIR spectroscopic analysis of stearic acid. (C) FTIR spectroscopic analysis of myristic acid. (D) P-XRD patterns of eutectic mixture of myristic acid and stearic acid. (E) P-XRD patterns of physical mixture of myristic acid and stearic acid. (F) P-XRD patterns of stearic acid. (G) P-XRD patterns of myristic acid. All data were presented as mean ± S.D (n=3).

Figure 2

The particle size distribution by the intensity of optimized formulation characterized by dynamic light scattering technique.

Figure 3

Zeta potential distribution of optimized formulation characterized using electrophoretic mobility technique.

Figure 4

Transmission electron microscopic image of (A) blank lipid nanocarriers and (B) doxorubicin lipid nanocarriers. The scale bar is 100 nm.

Figure 5

FTIR spectrum of (A) doxorubicin, (B) physical mixture of doxorubicin and excipients (C) doxorubicin lipid nanocarriers.

Figure 6

The release profile (12-hours) of doxorubicin from optimized formulation and aqueous solution at temperatures 37 °C, 41°C, and 43°C in release media having pH 6.8. Data are presented as mean± SD.

Figure 7

Dissolution data modeling of Weibull model at (A) 37°C, (B) 41°C (C) 43°C and pH 6.8.

Figure 8

The percent viability in a monolayer of B16F10 cells characterized using cell titer blue assay treated with (A) free DOX at varying concentrations and incubation periods (B) doxorubicin lipid nanocarriers at varying concentrations of doxorubicin incubated at 37°C for 24 hours and with 1 hour at 41°C followed by 24 hours at 37°C, and (C) blank lipid nanocarriers at varying concentrations and incubation periods. Data are presented as mean ± SD, n=3, *p < 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Figure 9

Right panel: qualitative presentation of in-vitro hemolysis potential of blank and doxorubicin-loaded lipid nanocarriers with saline as a negative control and 2% triton-x 100 in saline as a positive control. Left panel: hemolysis percentage of blank and doxorubicin lipid nanocarriers at 250–2000 µg/mL of total lipid concentration. Data are presented as mean± SD, n=3, *p < 0.05.

Figure 10

Analysis of biochemical and hematological parameters in group-1 as the control group, group-2 tested with blank lipid nanocarriers, and group-3 tested with doxorubicin-loaded lipid nanocarriers. (A) Renal function test, (B) lipid profile, (C) hematological parameters, (D) liver function test, (E) total body weight changes. Data are presented as mean ± SD, n=3, *p < 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Figure 11

Histopathological analysis of heart, liver, spleen, and kidney of group-I animals as control treated with saline, group-II treated with carrier or blank lipid nanocarriers, group-III treated with doxorubicin-loaded lipid nanocarriers.