Edited by: Hsien-Yeh Chen, National Taiwan University, Taiwan
Reviewed by: Guoqing Pan, Jiangsu University, China; Gloria Huerta-Angeles, Contipro Inc., Czechia
This article was submitted to Polymer Chemistry, a section of the journal Frontiers in Chemistry
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The multi-functional micelles poly(
Inflammation is the common response to injured vascular living tissue. Chemical and physical agents, microbial infections, inappropriate immunological responses or other issues may cause inflammation. The purpose of inflammation is to eliminate the microorganisms, enclose the injury, inactivate toxin so that the tissue or organ can be repaired (Cline M. J.,
In this study, the multi-targeting drug carrier micelles were synthesized as shown in
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.
Firstly, UA was dissolved in an aqueous sodium hydroxide solution (NaOH) and added NIPAAm and DMAAm in solution and stirred uniformly. Hydrochloric acid (HCl) was used to titrate the solution to pH 6.7, and the initiator potassium peroxydisulfate and the chain transfer methyl-3-mercaptopropionate agent were added to conduct a reaction for 4 h. After dialysis and lyophilization, the PNDU-COOCH3 was obtained. Since the functional group COOCH3 of synthesized PNDU-COOCH3 was not easily grafted to magnetic particles, the functional group was modified with NH2 by hydrazine. The consecutive step was to modify poly (NIPAAm-
Dextran was a biocompatible, biodegradable and hydrophilic material (Draye et al.,
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.
The drug (Hesperetin) embed procedure was to initially add 30 mg of PNDU/CM-Dex-Fe3O4 and 15 mg of Hesperetin in
The multi-functional drug carrier micelle was characterized by Fourier transform infrared spectroscopy (FT-IR, IR-4200; Jasco, Easton, MD, USA) which was mixed with potassium bromide (KBr), ground into a powder, and made into the pellet. The pellet placed in the FT-IR instrument and scanned wavelength from 400 to 4,000 cm−1, the absorption wavelength of the functional group was analyzed. The graft ratio of the micelles would be the quantitative conversion of the integral area measured in Nuclear Magnetic Resonance Spectroscopy (NMR, Ascend 600, Bruker, USA) and the nuclear specie was 1H. The morphology of the PNDU/CM-Dex-Fe3O4 was examined by transmission electron microscope (FEG-TEM, Tecnai F30; Philips, USA). The particle size of the samples was measured by dynamic light scattering (DLS) (LS series, Beckman Coulter, USA).
The critical micelles concentration (CMC) and low critical solution temperature (LCST) were used to evaluate the functionality of micelles. To measure the CMC, the hydrophobic fluorescent agent 1,6-diphenyl-1,3,5-hexatriene (DPH) was embedded in the hydrophobic layer of the PNDU/CM-Dex-Fe3O4 and measured the fluorescence intensity. The LCST of the multi-functional drug carrier micelles was derived by measuring the permeability of micelles dissolved in Phosphate Buffered Saline (PBS). In order to verify the magnetic properties of the micelles, the micelles were analyzed by the superconducting quantum interference device (SQUID, MPMS 5, Quantum Design, San Diego, CA, USA) and the thermogravimetric analysis (TGA).
Dimethylsulfoxide (DMSO) was used to disrupt the micellar structure to be then analyzed spectrophotometrically (ultraviolet-visible [UV-Vis] spectrophotometer; JASCO, Tokyo, Japan) at 231 nm for drug content. The equations we used for the estimation of LE and EE% of Hesperetin in P5DF10, P10DF10, P20DF10 micelles were as follows (Zu et al.,
The Hesperetin release experiment was to dissolve the lyophilized Hesperetin-embedded PNDU/CM-Dex-Fe3O4 micelles in a PBS buffer solution of pH 6.6 and pH 7.4, respectively, at a concentration of 1 mg/ml. The two different pH solutions respectively heated to a fixed temperature which were the previously examined LCST. The extracted sample solution and the ethanol solution were configured as test solutions in ethanol/PBS (v:v = 20:80), uniformly stirred, and detected by UV-Vis, and further compared to calculate the release amount of the Hesperetin in micelles.
Micelles
This study used lipopolysaccharide (LPS) to induce the inflammatory response in RAW264.7 mouse macrophages/monocyte and co-culture with multi-functional drug carrier micelles for inflammatory evaluation. The amount of 5 × 104 cells/ml of RAW264.7 mouse macrophage/monocyte was cultured in 96 well-plate, and the positive control group, the negative control group and the experimental group were distinguished. The positive control group contained only macrophage/monocyte and the culture medium; the negative control group was the macrophage/monocyte, the LPS, and the culture medium. The Hesperetin-embed PNDU/CM-Dex-Fe3O4 micelles were added at 250, 500, and 1,000 μg/ml to the experimental group, respectively, and cultured for 24 h. After the reaction was completed, the scanning wavelength of 540 nm was set in the ELISA to measure the absorbance.
To visualize the expression of NF-κB in cultured RAW264.7, cells were cultured in 24-well plates (1 × 106 cells per well) and pretreated with PNDU/CM-Dex-Fe3O4 micelles P10DF10 (250, 500, and 1,000 μg/ml) for 1 h at 38°C and stimulated with LPS for 30 min. The cells were then washed and fixed with a 4% paraformaldehyde for 30 min at 37°C, permeabilized with 0.3% Triton X-100 for 15 min, blocked with PBS containing 5% bovine serum albumin (BSA) for 30 min. Next, the cells were processed for immunofluorescent staining with primary NF-κB p65 antibody for 1 h, and followed by incubation with a fluorescein (FITC)-labeled secondary antibody for 1 h before observation. Protein expressed of p65 in RAW264.7 exhibited green fluorescence and observed using a microscope. To create a phase-fluorescence mapping image, the phase contract, and green fluorescence images were overlaid, producing mapping fluorescence in areas of co-localization.
This study was based on the pH and temperature response polymer of poly(NIPAAm-
In this study, FT-IR was used to detect the vibrational absorption peak of the functional group of the material, thereby confirming that the synthesized micelles of PNDU-COOCH3. PNDU-COOCH3 micelles are synthesized from three monomers: NIPAAm, DMAAm, and UA.
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.
The PNDU/CM-Dex-Fe3O4 (PDF) functional group vibration absorption peak was also detected by FT-IR to confirm the synthesized micelles. The PDF micelles were synthesized by PNDU-NH2 and CM-Dex/Fe3O4, and the different ratios were adjusted: PNDU-NH2: CM-Dex/Fe3O4 = 5:10 (P5DF10), PNDU-NH2: CM-Dex/Fe3O4 = 10:10 (P10DF10), and PNDU-NH2: CM-Dex/Fe3O4 = 20:10 (P20DF10). The experimental results are shown in
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.
According to the FT-IR spectrum, 1,728, 1,664, and 1,551 cm−1 had the carbonyl group and nitrogen-hydrogen bond functional groups of PNDU-NH2, while 2,974, 2,930, and 1,470 cm−1 had the carbon-hydrogen bond functional group of PNDU-NH2. This indicates that P5DF10, P10DF10, and P20DF10 each have the characteristics of PNDU-NH2. The characteristic peaks of the carbon-oxyl and iron-oxyl of CM-Dextran/Fe3O4 at 1,111, 1,011, and 570 cm−1 meant that CM-Dextran/Fe3O4 had indeed been grafted to PNDU. The higher the iron content of the micelles, the larger the signal. This further indicated that CM-Dextran/Fe3O4 was indeed grafted to the PNDU.
The nuclear magnetic resonance spectrometer (1H-NMR) was used to confirm the composite of PNDU-COOCH3. The 1H-NMR spectrum is shown in
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.
The morphology of PNDU-COOCH3, P5DF10, P10DF10, and P20DF10 was examined by TEM and DLS, as shown in
TEM image of
The functional manifestation of the micelles includes the CMC, the LCST and the change in the particle size of the micelles under temperature changes in order to realize the feasibility of the micelles for drug release.
The LCST of the PNDU-COOCH3, P5DF10, P10DF10, and P20DF10.
PNDU-COOCH3 | 6.6 | 32.40 ± 1.00 | 4.10 |
7.4 | 36.50 ± 0.50 | ||
P5DF10 | 6.6 | 37.50 ± 1.00 | 2.83 |
7.4 | 40.33 ± 1.08 | ||
P10DF10 | 6.6 | 37.76 ± 0.59 | 3.94 |
7.4 | 41.70 ± 1.14 | ||
P20DF10 | 6.6 | 35.86 ± 0.51 | 3.14 |
7.4 | 39.00 ± 0.52 |
Drug loading and encapsulation efficiencies at different ratio of PNDU-Fe3O4 micelles.
P5DF10 | 2.83 ± 0.21 | 12.55 ± 3.77 |
P10DF10 | 3.44 ± 0.18 | 42.23 ± 2.11 |
P20DF10 | 3.51 ± 0.33 | 35.45 ± 5.33 |
This study verified that PDF micelles have magnetic properties for magnetic targeting. The saturation magnetization results detected by SQUID are shown in
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.
Thermo-gravimetric analysis was used to predict the structure and composition of the material. In this study, TGA was used to investigate the synthesis ratio of grafted CM-Dex/Fe3O4. The TGA results, shown in
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%.
The drug carrier Hesperetin-embed P10DF10 micelles controlled the release rate of the drug at different pH values through its LCST characteristics. The purpose was to release the drug (Hesperetin) at the inflammatory area wherein the temperature is slightly above that of the normal tissue; thus, if it was at the same temperature as the normal tissue, the drug was protected inside the micelles. The micelles drug release amounts at 1, 3, 6, 9, 12, 24, and 36 h of pH 6.6 at 39°C were 4.79816, 5.13189, 6.28425, 7.66239, 7.6419, 7.93533, and 8.59221 μg. The micelles drug release amounts at 1, 3, 6, 9, 12, 24, and 36 h of pH 7.4 at 39°C were 0.42561, 0.06561, 0.11299, 1.36382, 1.306, 1.29812, and 1.53725 μg, as shown in
Drug release experiment of hesperetin-embed P10DF10 micelles.
It can be seen from the results that when the ambient temperature was higher than the LCST, the drug (Hesperetin) was released due to the change of the micelles morphology. When the release time was ~9 h, the cumulative release amount reached a saturated state.
The micelles
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.
In addition to their drug release function, the multi-functional drug carrier micelles are expected to reduce inflammation in cell tissues.
The multi-functional drug carrier micelles were synthesized by PNDU-NH2 and CM-Dextran/Fe3O4, and the functional groups were confirmed by FT-IR and 1H-NMR. TGA and SQUID verified the CM-Dextran/Fe3O4 ratio and the magnetic properties of the micelles and we observed the morphology of micelles by using TEM. This study regulated the composition of CM-Dextran/Fe3O4 and PNDU-NH2 to optimize LCST finding that it was suitable for the lesion area at a slightly higher temperature than normal tissues; it also responded to the environments of normal tissue (pH7.4) and lesions (pH6.6), which confirmed that the micelles had pH and temperature response behaviors. The drug Hesperetin was selected in this study as the drug carrier to reduce the oxidation of LDL with the aim of reducing the occurrence of atherosclerosis; in addition, it was expected to increase the therapeutic effect as well. The Hesperetin released from the micelles reached saturation after 9 h, and the release amount of pH6.6 and pH7.4 after 36 h were 8.59221 μg and 1.53725 μg, respectively. This achieved the aim of drug release in the lesion area. The
This study successfully provided multi-functional drug carrier Hesperetin-embed micelles with good biocompatibility, while maintaining the characteristics of PH-dependent temperature response, and magnetic properties. The drug carrier magnetic micelles were synthesized by PNDU-NH2 and CM-Dextran/Fe3O4, and the functional groups were confirmed. This study regulated the composition of CM-Dextran/Fe3O4 and PNDU-NH2 to optimize LCST finding in P10DF10 that it was suitable for the lesion area at a slightly higher temperature than normal tissues. In the drug hesperetin release experiment, P10DF10 micelles approximately higher 5.7 times in pH6.6 than in pH7.4 showed that using these magnetic micelles, moderately hydrophobic drugs can selectively deliver to acidic inflammation foci. Its iron content was nearly about 45% and it was provided magnetic properties (6.67 emu/g), it promoted the potential of magnetic targeting. Data provided the evidence for suggesting that 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 μg/ml in Hesperetin-embed P10DF10 micelles, the inflammatory ratio can be effectively reduced to 36.9%, suggesting that these magnetic micelles can be a good candidate for anti-inflammatory therapy in the further clinical trial.
W-JW contributed substantially to the conception and selection of the drug in micelles. W-JW is the PI of the project of magnetic micelles application with hesperetin treatment from Taoyuan General Hospital, Ministry of Health and Welfare. W-JW also assisted in carrying out
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
This manuscript is dedicated to the memory of Prof. Shyh-Liang Lou, who sadly passed away on 12 September 2016, whose insightful thoughts greatly impacted on this paper. This work was partly supported by the Ministry of Science and Technology, Taiwan, under Grant no. MOST 104-2112-M-033-008-MY2 and MOST 105-2221-E-033-006-, a grant from Taoyuan General Hospital, Ministry of Health and Welfare, and Changhua Christian Hospital, Changhua, Taiwan.