Uricase alkaline enzymosomes with enhanced stabilities and anti-hyperuricemia effects induced by favorable microenvironmental changes

Abstract

Enzyme therapy is an effective strategy to treat diseases. Three strategies were pursued to provide the favorable microenvironments for uricase (UCU) to eventually improve its features: using the right type of buffer to constitute the liquid media where catalyze reactions take place; entrapping UCU inside the selectively permeable lipid vesicle membranes; and entrapping catalase together with UCU inside the membranes. The nanosized alkaline enzymosomes containing UCU/(UCU and catalase) (ESU/ESUC) in bicine buffer had better thermal, hypothermal, acid-base and proteolytic stabilities, in vitro and in vivo kinetic characteristics, and uric acid lowering effects. The favorable microenvironments were conducive to the establishment of the enzymosomes with superior properties. It was the first time that two therapeutic enzymes were simultaneously entrapped into one enzymosome having the right type of buffer to achieve added treatment efficacy. The development of ESU/ESUC in bicine buffer provides valuable tactics in hypouricemic therapy and enzymosomal application.

Figure 1. Schematic diagrams of how ESU/ESUC in different buffers changed the biochemical and biological features of UCU as the microenvironments changed.

Figure 2. Principal characteristics of ESU and ESUC.

(A) Optical photographs of UCU, ESU and ESUC; (B) Principal parameters of ESU and ESUC (mean, n = 3); (C) Transmission electron photomicrographs of ESU-D (bar: 200 nm) and ESUC-D (bar: 500 nm); (D) Graphs depicting size distribution and zeta potential of ESU-D and ESUC-D. The maximum activity of free UCU-A was taken as 100%.

Figure 3. The optimal temperature and pH of UCU, ESU and ESUC.

The effects of temperature (A) and pH (B) on the activity of UCU, ESU and ESUC. The maximum activity of free UCU-A was taken as 100%. The data were presented as mean value, n = 3. Standard deviation (SD) was lower than 5% (data not shown).

Figure 4. The stabilities of UCU, ESU and ESUC.

The effects of (A) high temperature (55 °C, thermal stabilities), (B) low temperature (4 °C, hypothermal stabilities), (C) trypsin (proteolytic stabilities), (D) acidity-alkalinity (pH stabilities) and (E) chemical agents (chemical stabilities) on the UCU activities. In Figure A–C and E, the original activity of UCU, ESU or ESUC was taken as 100%; in Figure D, the remaining activity of UCU-A at pH 8.5 was taken as 100%. The data were presented as mean value, n = 3. Standard deviation (SD) was lower than 5% (data not shown).

Figure 5. FITC fluorescence changes of UCU.

(A) Interaction of UCU (in the absence or presence of CAT) with enzymosomal (ESU or ESUC) membranes in buffer-B; (B) Interaction of UCU with enzymosome membranes in buffer-D. (C) Fluorescence change of UCU (in the absence or presence of CAT) in buffer-B induced by heat treatment; (D) Fluorescence change of ESU (or ESUC) in buffer-B induced by heat treatment. (E) Fluorescence change of UCU (in the absence or presence of CAT) in buffer-D induced by heat treatment; (F) Fluorescence change of ESU (or ESUC) in buffer-D induced by heat treatment. The UCU or CAT concentration was fixed at 100 μg/mL. The FITC concentration was fixed at 0.51 μg/mL. UCU + CAT: mixture of UCU and catalase; Blk-ESU (or ESUC): blank ESU or blank ESUC, the blank ESU was exactly the same as blank ESUC, they were both blank enzymosomes; Blk-ESU (or ESUC) + UCU: mixture of blank enzymosome with free UCU; Blk-ESU (or ESUC) + UCU + CAT: mixture of blank enzymosome with free UCU and CAT;

Figure 6. Fluorescence changes of free UCU, ESU, free UC and ESUC.

(A) Fluorescence, (B) Changes of the maximum fluorescence wavelengths. (C) Changes of the fluorescence intensities determined at the maximum wavelengths. Free UC: mixture of free UCU and CAT. The data were shown as mean ± SD. n = 3.

Figure 7. Circular dichroism and gel electrophoresis of free UCU, ESU, UC and ESUC.

(A) Circular dichroism curves. (B,C) Changes of the circular dichroism millidegree at the wavelength of 208 nm and 222 nm. (D) Polyacrylamide gel electrophoresis. (E) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Free UC: mixture of free UCU and CAT. BSA: bovine serum albumin; OVA: ovalbumin. The molecular weights of BSA and OVA were 66 kD and 45 kD, respectively.

Figure 8. Pharmadynamics of free UCU, ESU and ESUC.

(A) Plasma uric acid or (B) hydrogen peroxide concentration versus time profiles after intravenous injection of UCU, ESU and ESUC. The data were shown as mean ± SD. n = 6 mice per group.

Figure 9. The in vitro and in vivo kinetic characteristics of UCU, ESU and ESUC.

(A) Enzymatic kinetic characteristics of UCU, ESU and ESUC: Lineweaver-Burk profiles and Enzyme kinetic constants of UCU, ESU and ESUC (n = 3); (B) Plasma UCU activity versus time profiles after intravenous injection of UCU-D, ESU-D and ESUC-D at the same dose (2000 mU/kg of UCU). (C) Plasma CAT activity versus time profiles after intravenous injection of CAT-D, ESC-D and ESUC-D at the same dose (1000 U/kg of CAT). (D) Main pharmacokinetic parameters of UCU-D, ESU-D and ESUC-D. (E) Main pharmacokinetic parameters of CAT-D, ESC-D and ESUC-D. The data were shown as mean ± SD. n = 6 rats per group. *P<0.05 indicated significant differences between ESUC-D (or ESU-D, ESC-D), free UCU-D or free CAT-D.