Li Chenglong,Lu Guanting,Jiang Yue,Su Huaiyu,Li Chen
Background: Rheumatoid arthritis (RA) is a systemic autoimmune disease (AD), and the
global incidence rate is 0.5 ~ 1%. Existing medications might reduce symptoms, however, there is no
known cure for this illness. Etanercept (EN) can competitively inhibit TNF-α binding to the TNF receptor
on the cell surface to treat RA. However, subcutaneous injection of free EN predisposes to systemic
distribution and induces immune system hypofunction. Draining lymph nodes (LNs) play a significant
role in the onset, maintenance, and progression of RA as they are the primary sites of aberrant immune
response and inflammatory cytokine production.
Aim: The purpose of this study was to successfully treat RA with etanercept by encapsulating it in nanoemulsions
(NEs/EN) and then delivering it specifically to draining LNs. The EN-loaded NEs were prepared
by high-pressure homogenization method and modified with DSPE-mPEG2000 and Ca(OH)2.
Methods: A novel nano-emulsion (NE) was constructed to deliver EN (NE/EN) to RA-draining LNs. To
decrease aggregation and load EN, DSPE-mPEG2000 and Ca(OH)2 were successively decorated on the
surface of the lipid injectable emulsions. The hydrodynamic diameter and morphology of NEs/EN were
investigated by using a laser particle size analyzer and transmission electron microscopy, respectively.
The in vivo fluorescence imaging system was used to study the in vivo LN targeting ability of the formulation.
In the therapeutic experiment, NEs/EN was subcutaneously administrated to inhibit the development
of the mouse arthritis model.
Results: Circular dichroism spectrum and L929 cell experiment confirmed that NEs encapsulation had
no impact on the biological activity of EN. In vivo investigation on collagen-induced arthritis (CIA)
mouse model showed that NEs/EN have good inguinal lymph node targeting capabilities, as well as,
anti-inflammatory effect against RA. Compared with the free group, the paw thickness and arthritic
score in NEs/EN group were significantly alleviated. Moreover, the concentration of pro-inflammatory
cytokines TNF-α and IL-1β in NEs/EN-treated mice was lower than that in free EN.
Conclusion: NEs/EN effectively improve the effectiveness of EN in the treatment of RA. Our work
provides an experimental foundation for expanding the clinical application of EN.
Freeze drying is one of the common methods to extend the long-term stability of biologicals. Biological products in solid form have the advantages of convenient transportation and stable long-term storage. However, long reconstitution time and extensive visible bubbles are frequently generated during the reconstitution process for many freeze-dried protein formulations, which can potentially affect the management efficiency of staff, patient compliance, and product quality. The reconstitution time has been extensively studied, but the influence of the formulations on the formation of visible bubbles is often overlooked. This paper investigated the effect of freeze-drying formulation factors (i.e., protein concentrations, surfactant concentrations, and sucrose/mannitol compositions) on product stability and visible bubbles generated during reconstitution of freeze-dried etanercept formulations. The generating and breakup mechanisms of visible bubbles were detected via internal microstructure of cake, surface tension, and viscosity measurement. Under the same protein concentration, the formulation of mannitol mixed with sucrose in a weight ratio of 4:1 produces fewer visible bubbles during the reconstitution process compared to the formulation of sucrose with the same total mass. This has been proven to be due to the large number of smaller radius pores distributed in the pores of the freeze-dried cake of the former, while the average internal structure pores of the latter are much larger than those of the former. As an amorphous stabilizer, sucrose can ensure the long-term stability of protein and greatly reduce the generation and maintenance of foams in the reconstitution process, making it a more robust excipient for freeze-dried protein formulations. Graphical
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