A Better Anti-Diabetic Recombinant Human Fibroblast Growth Factor 21 (rhFGF21) Modified with Polyethylene Glycol

As one of fibroblast growth factor (FGF) family members, FGF21 has been extensively investigated for its potential as a drug candidate to combat metabolic diseases. In the present study, recombinant human FGF21 (rhFGF21) was modified with polyethylene glycol (PEGylation) in order to increase its in vivo biostabilities and therapeutic potency. At N-terminal residue rhFGF21 was site-selectively PEGylated with mPEG20 kDa-butyraldehyde. The PEGylated rhFGF21 was purified to near homogeneity by Q Sepharose anion-exchange chromatography. The general structural and biochemical features as well as anti-diabetic effects of PEGylated rhFGF21 in a type 2 diabetic rat model were evaluated. By N-terminal sequencing and MALDI-TOF mass spectrometry, we confirmed that PEG molecule was conjugated only to the N-terminus of rhFGF21. The mono-PEGylated rhFGF21 retained the secondary structure, consistent with the native rhFGF21, but its biostabilities, including the resistance to physiological temperature and trypsinization, were significantly enhanced. The in vivo immunogenicity of PEGylated rhFGF21 was significantly decreased, and in vivo half-life time was significantly elongated. Compared to the native form, the PEGylated rhFGF21 had a similar capacity of stimulating glucose uptake in 3T3-L1 cells in vitro, but afforded a significantly long effect on reducing blood glucose and triglyceride levels in the type 2 diabetic animals. These results suggest that the PEGylated rhFGF21 is a better and more effective anti-diabetic drug candidate than the native rhFGF21 currently available. Therefore, the PEGylated rhFGF21 may be potentially applied in clinics to improve the metabolic syndrome for type 2 diabetic patients

Asphaltene Inhibition and Flow Improvement of Crude Oil with a High Content of Asphaltene and Wax by Polymers Bearing Ultra-Long Side Chain

A high content of asphaltene and wax in crude oil leads to difficulties in the recovery and transportation of crude oil due to the precipitation of asphaltenes and the deposition of waxes. Comb-like polymers were found to be capable of inhibiting the aggregation of asphaltenes and crystallization of waxes. In this work, comb-like bipolymers of α-olefins/ultra-long chain (C18, C22 and C28) alkyl acrylate were synthesized and characterized by FT-IR and 1H NMR spectra. The results show that, for a model oil containing asphaltene, the initial precipitation point (IPP) of asphaltene was prolonged by UV, and the asphaltene particle size was reduced after adding the biopolymers, as revealed by dynamitic light scattering (DLS). The bipolymer containing the longer alkyl chain had a better asphaltene inhibition effect. However, DSC and rheological results show that the wax appearance temperature (WAT) of the typical high asphaltene and high wax content of crude oil was obviously reduced by adding bipolymers with shorter alkyl chains. The bipolymer (TDA2024-22) with a mediate alkyl chain (C22) reduced the viscosity and thixotropy of the crude oil by a much larger margin than others. Compared with the previously synthesized bipolymer with phenyl pendant (PDV-A-18), TDA2024-22 exhibited a better performance. Therefore, bipolymers with appropriate alkyl side chains can act as not only the asphaltene inhibitors but also wax inhibitors for high asphaltene and wax content of crude oil, which has great potential applications in the oil fields

Asphaltene Inhibition and Flow Improvement of Crude Oil with a High Content of Asphaltene and Wax by Polymers Bearing Ultra-Long Side Chain

A high content of asphaltene and wax in crude oil leads to difficulties in the recovery and transportation of crude oil due to the precipitation of asphaltenes and the deposition of waxes. Comb-like polymers were found to be capable of inhibiting the aggregation of asphaltenes and crystallization of waxes. In this work, comb-like bipolymers of α-olefins/ultra-long chain (C18, C22 and C28) alkyl acrylate were synthesized and characterized by FT-IR and 1H NMR spectra. The results show that, for a model oil containing asphaltene, the initial precipitation point (IPP) of asphaltene was prolonged by UV, and the asphaltene particle size was reduced after adding the biopolymers, as revealed by dynamitic light scattering (DLS). The bipolymer containing the longer alkyl chain had a better asphaltene inhibition effect. However, DSC and rheological results show that the wax appearance temperature (WAT) of the typical high asphaltene and high wax content of crude oil was obviously reduced by adding bipolymers with shorter alkyl chains. The bipolymer (TDA2024-22) with a mediate alkyl chain (C22) reduced the viscosity and thixotropy of the crude oil by a much larger margin than others. Compared with the previously synthesized bipolymer with phenyl pendant (PDV-A-18), TDA2024-22 exhibited a better performance. Therefore, bipolymers with appropriate alkyl side chains can act as not only the asphaltene inhibitors but also wax inhibitors for high asphaltene and wax content of crude oil, which has great potential applications in the oil fields

Structural and biochemical evaluation of PEGylated rhFGF21.

<p>Panel A: Far-UV CD spectra of native (dash line) and PEGylated rhFGF21 (black solid line). Panel B: Near-UV CD spectra of native (dash line) and PEGylated rhFGF21 (black solid line). The ellipticities are reported as mean residue ellipticitie (MRE) (deg cm² dmol⁻¹). Panel C: Cellular glucose uptake stimulated by native and PEGylated rhFGF21 on 3T3-L1, measured using Wallac 1450 MicroBeta counter (Perkin Elmer). Values (±SE) shown are the average of at least 3 independent measurements. *, P<0.001 vs vehicle control. Panel D: Thermal stability of native and PEGylated rhFGF21 incubated in mouse serum at 37 °C for indicated times, and then the serum-incubated rhFGF21 were added to 3T3-LI cells for which glucose uptake was measured to determine the functional integrity of each rhFGF21. Panel E: The relative resistance to trypsinization was examined, for which PEGylated and native rhFGF21 were incubated with trypsin at 2 mM for indicated times. Trypsin-treated rhFGF21s were examined for the protein integrity by SDS-PAGE. The bands representing PEGylated and native rhFGF21 were quantified by densitometry scanning. The band densities of non-trypsin-treated native and PEGylated rhFGF21 are considered as 100% (indicated by control), while the band densities of trypsin-treated PEGylated or native rhFGF21 are presented as relative percentage to the control. *, p<0.01 vs non-trypsin-treated native or PEGylated rhFGF21. Panel F: Pharmacokinetics study of PEGylated rhFGF21 in rat. Male Wistar adult rats were injected intravenously with 100 µg/kg native rhFGF21 (open circle), PEGylated rhFGF21 (close circle). Blood samples were collected at various time points. The amount of rhFGF21 was measured by ELISA. A standard curve was made for each rhFGF21. <i>n</i> = 5. Values are mean±SD. Panel G: Half-life time of rhFGF21 and PEGylated rhFGF21. Panel H. Immunogenicity of native and PEGylated rhFGF21 was examined by immunizing BALB/c mice with either PEGylated rhFGF21 or native rhFGF21 in 20 mM sodium acetate buffer by i.p. injection (2 µmol/mouse) twice (the 1^st day and the 14^th day) and then serum samples were collected on the 21^st day after the first immunization. Anti-rhFGF21 IgG levels were detected by indirect ELISA method using native FGF21 as the coating antigen. In addition, a group of mice given same volume of sodium acetate only for a same time schedule as rhFGF21 was included for sodium acetate control. *, p<0.01 and **, p<0.001vs native rhFGF21 group.</p

Long lasting anti-diabetic effects of PEGylated rhFGF21 in type 2 diabetic rats.

<p>Diabetic rat model and rhFGF21 treatment are same as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020669#pone-0020669-g005" target="_blank">Figure 5</a>. However, hypoglycemic (A) and hypolipidemic effects (B) as well as effect on plasma insulin levels (C) of native and PEGylated rhFGF21 were examined at different times [0 (1 h), 2 days, 4 days and 6 days] after the last treatment of the 7-day treatment with native or PEGylated rhFGF21 at the 4×10⁻⁸ mol/kg. Blank control: normal control rats; #, p<0.01 vs corresponding native rhFGF21 group; *, p<0.05 between indicated groups.</p

Examination for insulin resistance by IPGTT and IST.

<p>Type 2 diabetic rats were induced by 2-month HFD-feeding followed twice injection of low-dose of STZ at 35 mg/kg. At 8 weeks after STZ-induced hyperglycemia, IPGTT (A) and insulin sensitivity (B) were examined. For IPGTT, the rats with an overnight fast (12–16 h) were IP injected with 40% glucose (2 g/kg body weight) and blood samples were collected from the tail at 0, 30, 60, and 120 min for glucose measurement. For IST, insulin (0.75 IU/kg) was administered by IP injection and blood samples were collected at 0, 30, 60, and 120 minutes for the measurement of plasma glucose. The value is presented as a percentage of initial plasma glucose level.</p

Body weight and food intake of control group, DM group and TM group.

<p>CON: control group; DM: high-fat diet with STZ 35 mg/kg twice injection group; TM₁: Native rhFGF21-treated diabetic rats; TM₂: PEGylated rhFGF21-treated diabetic rats. The values shown are the average of the measurements of at least 5 animals in each group. *,p <0.05 vs corresponding TM₁ (native rhFGF21) groups.</p

Anti-diabetic effects of PEGylated rhFGF21 in type 2 diabetic rats.

<p>After defined the induction of insulin resistance in these type 2 diabetic rats with IPGTT and IST assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020669#pone-0020669-g005" target="_blank">Fig 5</a>, diabetic rats were treated with either native or PEGylated rhFGF21 at 1×10⁻⁸, 2×10⁻⁸, and 4×10⁻⁸ mol/kg daily for 2 weeks. On day 3 (A) and day 7 (B) after the first treatment of rhFGF21, plasma glucose levels were examined. Blank control: normal control rats; Negative control: 0.9% NaCl-treated diabetic group; a: native rhFGF21-treated diabetic rats; b: PEGylated FGF21-treated diabetic rats. *, P<0.01 vs corresponding pre-treatment groups.</p

Factors and levels of orthogonal tests.

<p>Factors and levels of orthogonal tests.</p