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. 2018 Apr 4;13:2051-2064.
doi: 10.2147/IJN.S151233. eCollection 2018.

Fabrication of a Triptolide-Loaded and Poly-γ-Glutamic Acid-Based Amphiphilic Nanoparticle for the Treatment of Rheumatoid Arthritis

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Fabrication of a Triptolide-Loaded and Poly-γ-Glutamic Acid-Based Amphiphilic Nanoparticle for the Treatment of Rheumatoid Arthritis

Li Zhang et al. Int J Nanomedicine. .
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Abstract

Triptolide (TP) exhibits immunosuppressive, cartilage-protective and anti-inflammatory effects in rheumatoid arthritis. However, the toxicity of TP limits its widespread use. To decrease the toxic effects, we developed a novel nano-drug carrier system containing TP using poly-γ-glutamic acid-grafted di-tert-butyl L-aspartate hydrochloride (PAT). PAT had an average diameter of 79±18 nm, a narrow polydispersity index (0.18), a strong zeta potential (-32 mV) and a high drug encapsulation efficiency (EE1=48.6%) and loading capacity (EE2=19.2%), and exhibited controlled release (t1/2=29 h). The MTT assay and flow cytometry results indicated that PAT could decrease toxicity and apoptosis induced by free TP on RAW264.7 cells. PAT decreased lipopolysaccharides/interferon γ-induced cytokines expression of macrophage (P<0.05). In vivo, PAT accumulated at inflammatory joints, improved the survival rate and had fewer side effects on tumor necrosis factor α transgenic mice, compared to TP. The blood biochemical indexes revealed that PAT did not cause much damage to the kidney (urea nitrogen and creatinine) and liver (alanine aminotransferase and aspartate aminotransferase). In addition, PAT reduced inflammatory synovial tissue area (P<0.05), cartilage loss (P<0.05), tartrate-resistant acid phosphatase-positive osteoclast area (P<0.05) and bone erosion (P<0.05) in both knee and ankle joints, and showed similar beneficial effect as free TP. In summary, our newly formed nanoparticle, PAT, can reduce the toxicity and guarantee the efficacy of TP, which represents an effective drug candidate for RA with low adverse side effect.

Keywords: drug carrier system; rheumatoid arthritis; triptolide; tumor necrosis factor α transgenic mice; γ-PGA.

Conflict of interest statement

Disclosure The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
Characterization of PAT. (A) H NMR spectra of Asp, PGA and PA dissolved in deuterium generation reagent DMSO. The nano-size of PAT was detected by (B) DLS and (C) TEM. (D) The controlled release of TP from PAT. Free TP or PAT was dissolved in dialysis bag and incubated at 37°C under shaking at 60 rpm. Abbreviations: Asp, di-tert-butyl L-aspartate hydrochloride; PGA, poly-γ-glutamic acid; PA, PGA-grafted Asp; TP, triptolide; PBS, phosphate buffer saline; PAT, TP-loaded PGA-grafted Asp; NMR, nuclear magnetic resonance; DMSO, dimethyl sulfoxide; DLS, dynamic light scattering; TEM, transmission electron microscopy.
Figure 2
Figure 2
PAT reduces the cytotoxicity of TP on RAW264.7 cells. (A) Growth curves of RAW264.7 cells were determined by MTT assay after incubation with TP (200, 100, 50, 25, 12.5 or 6.25 nM) or PAT (equal to TP concentration) for 24, 48 and 72 h. The values are the mean ± SD of 8 wells. (B) The apoptosis was assessed by FACS. Annexin V+/PI− cells represent early apoptotic cells, while Annexin V+/PI+ cells represent late apoptotic cells. The percentage of both early and late apoptotic cells is shown in the lower right corner. (C) The mRNA expression of TNFα, IL-1β, IL-6 and iNOS was analyzed by real-time PCR. The values are the mean ± SD of 3 wells. *P<0.05, compared with TP; **P<0.05, compared with PBS treated groups. Abbreviations: TP, triptolide; PAT, TP-loaded poly-γ-glutamic acid-grafted di-tert-butyl L-aspartate hydrochloride; PBS, phosphate buffer saline; PA, poly-γ-glutamic acid-grafted di-tert-butyl L-aspartate hydrochloride; FITC, fluorescein isothiocyanate; TNFα, tumor necrosis factor α; IL-1β, interleukin 1β; IL-6, interleukin 6; iNOS, inducible nitric oxide synthase; LPS/IFNγ, lipopolysaccharides/interferon γ; FACS, fluorescence-activated cell sorting; PI, propidium iodide; PCR, polymerase chain reaction.
Figure 3
Figure 3
PA and PAT target the inflammatory joints of TNFα-Tg mice. (A) Bioluminescent images of PA-ICG-treated WT mice, and PA-ICG- or PAT-ICG-treated TNFα-Tg mice were taken intermittently at 0.5, 1, 4, 24, 48 and 80 h after injection. (B) Fluorescence intensity of the knee joint at 24 h post-administration. The values are the mean ± SD of 6 legs from 3 mice. *P<0.05, compared with the WT + PA group. Abbreviations: WT, wild type; PA, poly-γ-glutamic acid-grafted di-tert-butyl L-aspartate hydrochloride; TNFα-Tg, tumor necrosis factor α transgenic; PAT, TP-loaded poly-γ-glutamic acid-grafted di-tert-butyl L-aspartate hydrochloride; ICG, indocyanine green.
Figure 4
Figure 4
PAT protects mice from the toxicity of TP. (A) The survival rate of different groups show PAT increased the survival rate of TNFα-Tg mice, compared with TP treatment. (B) The body weight of different groups at the last day of treatment shows PAT significantly prevented the body weight loss of TNFα-Tg mice, compared with TP treatment. Values are the mean ± SD of 7–10 mice per group. (C) HE staining sections (magnification ×200) of the liver, spleen and kidney show PAT protected those organs from injury caused by TP. The yellow arrow indicates the hepatic nucleus, and the blue arrow indicates the renal glomerulus. *P<0.05, vs TP group. Abbreviations: WT, wild type; TNFα-Tg, tumor necrosis factor α transgenic; PBS, phosphate buffer saline; PA, poly-γ-glutamic acid-grafted di-tert-butyl L-aspartate hydrochloride; TP, triptolide; PAT, TP-loaded poly-γ-glutamic acid-grafted di-tert-butyl L-aspartate hydrochloride; HE, hematoxylin–eosin.
Figure 5
Figure 5
Both TP and PAT reduce bone erosion in TNFα-Tg mice. (A) Longitudinal sections of 3-dimensional reconstructed ankle and knee joints by micro-CT show decreased bone erosion in TP- and PAT-treated mice. (B) Quantitation of bone volume of astragalus and patella. Values are the mean ± SD of 7–10 legs per group. *P<0.05, compared with PBS-treated group. Abbreviations: micro-CT, microcomputed tomography; WT, wild type; TNFα-Tg, tumor necrosis factor α transgenic; PBS, phosphate buffer saline; PA, poly-γ-glutamic acid-grafted di-tert-butyl L-aspartate hydrochloride; TP, triptolide; PAT, TP-loaded poly-γ-glutamic acid-grafted di-tert-butyl L-aspartate hydrochloride.
Figure 6
Figure 6
Both TP and PAT reduce ankle and knee joint tissue inflammatory damage in TNFα-Tg mice. (A) Representative ABOG-stained sections (magnification ×40) show decreased joint tissue damage, including decreased synovial inflammation and cartilage erosion in TP- and PAT-treated mouse. (B) Quantitation of inflammatory synovial tissue area percentage of ankle and knee joints, and cartilage area percentage of ankle and knee joints. Values are the mean ± SD of 7–10 legs per group. *P<0.05, compared with PBS treated group. Abbreviations: WT, wild type; TNFα-Tg, tumor necrosis factor α transgenic; PBS, phosphate buffer saline; PA, poly-γ-glutamic acid-grafted di-tert-butyl L-aspartate hydrochloride; TP, triptolide; PAT, TP-loaded poly-γ-glutamic acid-grafted di-tert-butyl L-aspartate hydrochloride; ABOG, Alcian blue/orange G.
Figure 7
Figure 7
Both TP and PAT reduce TRAP+ osteoclast area at ankle and knee joints in TNFα-Tg mice. (A) Representative TRAP-stained sections (magnification ×40) show decreased TRAP+ osteoclasts in TP- and PAT-treated mouse. (B) Quantitation of TRAP+ osteoclast area percentage of ankle and knee joints. Values are the mean ± SD of 7–10 legs per group. *P<0.05, compared with PBS treated group. Abbreviations: WT, wild type; TNFα-Tg, tumor necrosis factor α transgenic; PBS, phosphate buffer saline; PA, poly-γ-glutamic acid-grafted di-tert-butyl L-aspartate hydrochloride; TP, triptolide; PAT, TP-loaded poly-γ-glutamic acid-grafted di-tert-butyl L-aspartate hydrochloride; TRAP, tartrate-resistant acid phosphatase.

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