Multi-Functional Drug Carrier Micelles With Anti-inflammatory Drug

Abstract

The multi-functional micelles poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide-co-10 undecanoic acid)/CM-Dextran Fe₃O₄ (PNDU/CM-Dex Fe₃O₄) were poly (NIPAAm-co-DMAAm-co-UA) (PNDU) grafting hydrophilic CM-Dextran Fe₃O₄ which possess pH-dependent temperature response and magnetic response. In this research, anti-inflammation drug Hesperetin was encapsulated by micelles using membrane dialysis method to obtain the different ratio of Hesperetin-embedded P₅DF₁₀, P₁₀DF₁₀, and P₂₀DF₁₀. These micelles were characterized by Fourier transform infrared spectroscopy, ¹H-NMR, thermogravimetric analyzer, and superconducting quantum interference device magnetometer. The morphology and particle size of micelles was observed by transmission electron microscopy and dynamic light scattering. The low critical solution temperature of the P₁₀DF₁₀ micelles is in pH 6.6 at about 37.76°C and in pH 7.4 at about 41.70°C. The biocompatibility of micelles was confirmed by cytotoxicity study. Inflammatory inhibition of hesperetin-embedded P₁₀DF₁₀ micelles also studied through RAW264.7. Hesperetin-embed P₁₀DF₁₀ micelles suppressed LPS-induced inflammatory response. Via immunofluorescence cell staining demonstrate that Hesperetin-embed P₁₀DF₁₀ micelles inhibited the activation of NF-κB p60 and markedly attenuated in a drug dose-dependent manner. At a concentration of 1,000 ug/ml, an inflammatory rate can be reduced to 36.9%. Based on these results, the hesperetin-embed P₁₀DF₁₀ micelles had successfully synthesized and enable to carry and release the anti-inflammatory drugs, which instrumental for biomedical therapy and applications.

Keywords: anti-inflammatory drug; drug carrier; magnetic micelles; pH response micelles; temperature response micelles.

Figure 1

Synthesis diagram of the multi-targeting drug delivery micelle with hesperetin embedded. The multi-targeting micelles are composed of NIPAAm, DMAAm, and UA. The micelles are grafted CM-Dextran/Fe₃O₄ for magnetic manipulation. The inflammatory drug hesperetin are embedded into the micelles. Local inflammation area with relatively lower pH value and higher temperature led to the release of drug from the micelles. Target release to the inflammation area could be achieved by external magnetic field.

Figure 2

PNDU/CM-Dex-Fe₃O₄ chemical equation. First, the carboxyl group on the CM-Dextran/Fe₃O₄ was activated, and PNDU-NHNH₂ was added to enable the carboxyl group, and the amine group to be grafted. EDC and N-Hydroxysuccinimide were used to activate the carboxyl group of CM-Dex/Fe₃O₄.

Figure 3

Fourier transform infrared spectroscopy analysis of functional groups of NIPAAm, DMAAm, UA, and PNDU-COOCH₃. The characteristic peaks of NIPAAm, DMAAm, and UA can be seen in the spectrum of PNDU-COOCH₃, which proved the successful synthesis.

Figure 4

Fourier transform infrared spectroscopy analysis of functional groups of PNDU-NH₂, CM-Dex/Fe₃O₄, P₅DF₁₀, P₁₀DF₁₀, and P₂₀DF₁₀. The CM-Dex/Fe₃O₄ were successfully grafted onto the PNDU-NH₂ by Fourier transform infrared spectroscopy.

Figure 5

¹H-NMR spectrum showed the proton signal δ = 1.15 ppm of PNDU-COOCH₃; δ = 2.85 ppm of NIPAAm, and δ = 1.29 of DMAAm which verified the PNDU-COOCH₃ composed of NIPAAm, DMAAm, and UA.

Figure 6

TEM image of (a,e) PNDU-COOCH₃, (b,f) P₅DF₁₀, (c,g) P₁₀DF₁₀, and (d,h) P₂₀DF₁₀. The particles size of PNDU, P₅DF₁₀, P₁₀DF₁₀, and P₂₀DF₁₀ were 223.68, 422.07, 401.33, and 417.73 nm.

Figure 7

SQUID verification of CM-Dex/Fe₃O₄ and P₁₀DF₁₀. The saturation magnetic moment of CM-Dex/Fe₃O₄ and P₁₀DF₁₀ were 15.39 emu/g and 6.67 emg/g which represented the P₁₀DF₁₀ contained 43.33% CM-Dex/Fe₃O₄.

Figure 8

P₁₀DF₁₀ TGA analysis. The residual amount of CM-Dextran/Fe₃O₄, P₁₀DF₁₀, and PNDU-NH₂ was 37.76, 21.34, and 7.86% that extrapolated the content of CM-Dextran/Fe₃O₄ in P₁₀DF₁₀ was 45.08%.

Figure 9

Drug release experiment of hesperetin-embed P₁₀DF₁₀ micelles. (a) was the TEM image of the hesperetin-embed P₁₀DF₁₀ micelles released drug at pH 6.6 at 39°C; (b) was TEM image of the hesperetin-embed P₁₀DF₁₀ micelles at pH 7.4 at 39°C; (c) was the initial TEM image of hesperetin-embed P₁₀DF₁₀ micelles.

Figure 10

The cell viability test of P₅DF₁₀, P₁₀DF₁₀, and P₂₀DF₁₀. The cell viability was around 90% in different intake concentration of P₅DF₁₀, P₁₀DF₁₀, and P₂₀DF₁₀ that showed the good biocompatibility of micelles.

Figure 11

(A) P₁₀DF₁₀ prevented LPS-induced translocation of NF-κB p65 subunit by immunofluorescence studies. Cells were treated without P₁₀DF₁₀ (control) or with P₁₀DF₁₀ of 250, 500, and 1,000 μg/ml for 1 h and stimulated with LPS for 30 min, fixed, and incubated with anti-p65 antibody followed by FITC conjugated second antibody (green fluorescence). (B) Anti-Inflammatory experiment of the RAW264.7. The inflammatory ratio in the Hesperetin-embed P₁₀DF₁₀ micelles concentration at levels of 1,000, 500, and 250 μg/mL were 36.90, 56.58, and 72.43%. Data indicated that higher concentrations of Hesperetin-embed P₁₀DF₁₀ micelles could get higher anti-inflammatory ability.