Process optimization for the preparation of oligomycin-loaded folate-conjugated chitosan nanoparticles as a tumor-targeted drug delivery system using a two-level factorial design method

Abstract

Oligomycin-A (Oli-A), an anticancer drug, was loaded to the folate (FA)-conjugated chitosan as a tumor-targeted drug delivery system for the purpose of overcoming the nonspecific targeting characteristics and the hydrophobicity of the compound. The two-level factorial design (2-LFD) was applied to modeling the preparation process, which was composed of five independent variables, namely FA-conjugated chitosan (FA-CS) concentration, Oli-A concentration, sodium tripolyphosphate (TPP) concentration, the mass ratio of FA-CS to TPP, and crosslinking time. The mean particle size (MPS) and the drug loading rate (DLR) of the resulting Oli-loaded FA-CS nanoparticles (FA-Oli-CSNPs) were used as response variables. The interactive effects of the five independent variables on the response variables were studied. The characteristics of the nanoparticles, such as amount of FA conjugation, drug entrapment rate (DER), DLR, surface morphology, and release kinetics properties in vitro were investigated. The FA-Oli-CSNPs with MPS of 182.6 nm, DER of 17.3%, DLR of 58.5%, and zeta potential (ZP) of 24.6 mV were obtained under optimum conditions. The amount of FA conjugation was 45.9 mg/g chitosan. The FA-Oli-CSNPs showed sustained-release characteristics for 576 hours in vitro. The results indicated that FA-Oli-CSNPs obtained as a targeted drug delivery system could be effective in the therapy of leukemia in the future.

Keywords: chitosan; folate; nanoparticles; oligomycin-A; targeted drug delivery system; two-level factorial design.

Figure 1

Schematic description of the preparation procedure of FA-Oli-CSNPs. Abbreviations: Oli-A, oligomycin-A; FA-CS, the folate-conjugated chitosan; TPP, sodium tripolyphosphate; FA-Oli-CSNPs, folate-conjugated chitosan nanoparticles loading oligomycin-A; NHS-folate, N-Hydroxysuccinimide ester of folate.

Figure 2

The FTIR spectroscopy figure of NHS-FA compared with FA. Notes: NHS-FA presents two remarkable absorption peaks at 1660 cm⁻¹ due to the N–O bond, and at 1700 cm⁻¹ because of the C=O bond. This phenomenon proves that FA has been successfully transferred into NHS-FA. Abbreviations: FA, Folate; NHS-FA, N-hydroxysuccinimide ester of folate; NHS, N-hydroxysuccinimide.

Figure 3

Determination of NHS-FA conjugation with CS. Notes: (a) CS has an extremely weak absorption peak at 300 nm. (b) FA-CS has three absorption peaks, at 281 nm, 300 nm, and 343 nm, respectively. (c) NHS-FA has a strong absorption peak at 281 nm and a weak absorption peak at 343 nm. Abbreviations: CS, chitosan; FA-CS, the folate-conjugated chitosan; NHS-FA, N-hydroxysuccinimide ester of folate.

Figure 4

Pareto chart of the standardized effects for (A) MPS and (B) DLR – full model. Abbreviations: MPS, mean particle size; DLR, drug loading rate.

Figure 5

Pareto chart of the standardized effects for (A) MPS and (B) DLR – reduced model. Abbreviations: MPS, mean particle size; DLR, drug loading rate.

Figure 6

Main effects plot for (A) MPS and (B) DLR. Abbreviations: MPS, mean particle size; DLR, drug loading rate.

Figure 7

Interaction plot for (A) MPS and (B) DLR. Abbreviations: MPS, mean particle size; DLR, drug loading rate.

Figure 8

Normal probability plot of residual values for (A) MPS and (B) DLR. Abbreviations: MPS, mean particle size; DLR, drug loading rate.

Figure 9

Comparison of SEM images of original Oli-A, FA-CS and FA-Oli-CSNPs. (A) Irregular lamelliform crystals of raw Oli-A particles. (B) FA-CS lyophilized powder. (C) FA-Oli-CSNPs dispersed with deionized water. (D) FA-Oli-CSNPs containing mannitol dispersed with deionized water. Abbreviations: Oli-A, oligomycin-A; FA-CS, folate-conjugated chitosan; FA-Oli-CSNPs, folate-conjugated chitosan nanoparticles which encapsulate oligomycin-A.

Figure 10

The particle size distributions of FA-Oli-CSNPs which were determined by DLS. (A) FA-Oli-CSNPs dispersed with deionized water. (B) FA-Oli-CSNPs containing mannitol dispersed with deionized water. Abbreviations: FA-Oli-CSNPs, folate-conjugated chitosan nanoparticles which encapsulate oligomycin-A; DLS, dynamic laser light scattering.

Figure 11

FA-Oli-CSNPs accumulative drug release profile. Abbreviation: FA-Oli-CSNPs, folate-conjugated chitosan nanoparticles which encapsulate oligomycin-A.