Rapidly separating dissolving microneedles with sustained-release colchicine and stabilized uricase for simplified long-term gout management

Abstract

Despite growing prevalence and incidence, the management of gout remains suboptimal. The intermittent nature of the gout makes the long-term urate-lowering therapy (ULT) particularly important for gout management. However, patients are reluctant to take medication day after day to manage incurable occasional gout flares, and suffer from possible long-term toxicity. Therefore, a safe and easy-to-operate drug delivery system with simple preparation for the long-term management of gout is very necessary. Here, a chitosan-containing sustained-release microneedle system co-loaded with colchicine and uricase liposomes were fabricated to achieve this goal. This microneedle system was confirmed to successfully deliver the drug to the skin and maintain a one-week drug retention. Furthermore, its powerful therapeutic potency to manage gout was investigated in both acute gouty and chronic gouty models. Besides, the drug co-delivery system could help avoid long-term daily oral colchicine, a drug with a narrow therapeutic index. This system also avoids mass injection of uricase by improving its stability, enhancing the clinical application value of uricase. In general, this two-drug system reduces the dosage of uricase and colchicine and improves the patient's compliance, which has a strong clinical translation.

Keywords: Colchicine; Gout management; Liposome; Long-term urate-lowering therapy; Microneedles; Sustained-release; Transdermal administration; Uricase.

Graphical abstract

Figure 1

The scheme of UAO-LPO/Col-MNs preparation and its gout management. The microneedle consisted of three layers: the needle tip layer, the separation layer, and the base layer. UAO-LPO was co-loaded with Col into chitosan-containing needle tip layer. When UAO-LPO/Col-MNs were administered topically in gouty joints, the separation layer would dissolve and the needle tip layer remained in the skin to continuously release Col and UAO-LPO. UAO-LPO entered the joint cavity to release UAO to catalyze the oxidation of MSU and reduce MSU crystals in the joint cavity. Col possessed potent anti-inflammatory effects by inhibiting neutrophil recruitment, reducing damage-associated molecular patterns (DAMPs) cytokines release, and blocking NLR family pyrin domain-containing 3 (NLRP3)/interleukin (IL)-1β inflammasome activation by MSU crystals, etc. Colchicine and uricase work together for gout management.

Figure 4

MSU-induced acute gouty arthritis model. (A) The experimental scheme in acute gouty arthritis model: groups of microneedles were administrated with corresponding microneedles at Day 0; the solution groups were given colchicine solution by intragastric administration every day for the first three days, and injected uricase solution intravenously on Day 0; then the rat right hind knee joints were injected with MSU crystals on Day 3; after MSU crystals injection, the right hind knee joint diameter of 9 groups at each time point (0, 6, 20, and 24 h) were measured; and the synovial fluid of 9 groups was extracted under anesthesia for analysis of inflammatory factor. (B) The right hind knee joint diameter of 9 groups at 0, 6, 20, and 24 h after MSU crystals injection, data are presented as mean ± SD, n = 5 (^∗P < 0.05, ^∗∗P < 0.01 and ^∗∗∗P < 0.001). The inflammatory factors of synovial fluid extracted from rat right hind knee joints of the 9 groups, including IL-1β (C₁), TNF-α (C₂), and IL-8 (C₃), data are presented as mean ± SD, n = 5 (The “∗” symbolizes significant variation between the untreated group and the other groups, respectively, ^∗P < 0.05, ^∗∗P < 0.01 and^∗∗∗P < 0.001; “#” symbolizes significant variation between the UAO-LPO/Col-MNs and the fourth group, ^#P < 0.05, ^##P < 0.01; and “&” symbolizes significant variation between the two groups of line connected, ^&&&P < 0.001).

Figure 5

MSU-induced chronic and recurrent attacks of gouty arthritis model. (A) The experimental scheme in repeated intra-articular MSU-induced gouty arthritis model. (B) Macroscopic images of the knee joints from the groups of control, untreated, UAO-LP/Col-MNs without CTS, UAO/Col-Sol, UAO-LP/Col-MNs. Time course of the effects of UAO-LP/Col-MNs without CTS, UAO/Col-Sol, UAO-LP/Col-MNs on rat knee swelling at 24 h (C) and 48 h. (D) after each MSU crystal injection, data are presented as mean ± SD, n = 5. (E) The swollen degree of knee joint of 5 groups of rats at the end of the experiment (72 h after the last intra-articular injection of MSU crystals), data are presented as mean ± SD, n = 5. (F) the representative H&E staining images for the skin at the right hind knee joint of two MNs groups (region administered with MNs) and control group. (G) The time course of body weight changes of the rats, data are presented as mean ± SD, n = 5. The inflammatory factors of blood collected by the heart punctures from rats of the 5 groups, including IL-1β (H₁), TNF-α (H₂), and IL-8 (H₃), data are presented as mean ± SD, n = 5. The “∗” symbolizes significant variation between the UAO-LPO/Col-MNs and the untreated group, respectively (^∗P < 0.05, ^∗∗P < 0.01 and ^∗∗∗P < 0.001); “#” symbolizes significant variation between the UAO-LPO/Col-MNs and the controlled group (^##P < 0.01); and “&” symbolizes significant variation between the two groups line connected.

Figure 2

Characterization of UAO-LPO and UAO-LPO/Col-MNs. Graphic illustration of the particle size distributions (A), appearance (B) and Tynadll effect (C) of Blank-LPO (A₁, B₁ and C₁) and UAO-LPO (A₂, B₂ and C₂). TEM images of Blank LPO (D₁) and UAO-LPO (D₂), scale bar = 100 nm. SFM images of UAO-LPO/Col-MNs at bright field (E₁ and E₂), at fluorescent light (E₃ and E₄), scale bar = 800 μm in E₁ and E_3, and scale bar = 500 μm in E₂ and E₄. (F) SEM images of UAO-LPO/Col-MNs, scale bars = 1 mm in F₁ and = 250 μm in F₂. (G) The mechanical strength of four MNs with different formulations, data are presented as mean ± SD, n = 3, ^∗P < 0.05. (H) Bright-field micrographs and fluorescence micrographs of UAO-LPO/Col-MNs separation layer dissolved over time in water, scale bar = 200 μm.

Figure 3

Characterization of UAO-LPO/Col-MNs. (A) Distribution of needle tip layer (RITC, red) and separation layer (FITC, green) in UAO-LPO/Col-MNs, scale bar = 500 μm. (B) Z-stack CLSM images of the fluorescent dyes-colored microneedles inserted in the rat dorsal skin, scale bar = 500 μm. The SFM images of rhodamine-6G-loaded UAO-LPO/Col-MNs inserted in the rat dorsal skin. (C₁) and its fluorescent images (C₂), scale bar = 2 mm. (D) In vivo fluorescence images at different time points after administration of the rhodamine-6G-loaded MNs. (E) In vitro drug permeation of UAO-LPO/Col-MNs and UAO-LPO/Col-MNs without CTS, data are presented as mean ± SD, n = 3. (F) In vivo drug retention of UAO-LPO/Col-MNs and UAO-LPO/Col-MNs without CTS, data are presented as mean ± SD, n = 3. The absorbance changes of uric acid over time (G₁), the absorbance changes of uric acid at end point, data are presented as mean ± SD, n = 3 (G₂), the fitted curves according to the enzyme kinetics model of E₁ profiles (G₃) when the fresh uricase in solutions (UAO-Sol, stored at 4 °C), liposomes (UAO-LPO, stored at 4 °C), and microneedles (UAO-LPO/Col-MNs stored at 4 or 25 °C) were added into the uric acid solution. (H) Uric acid degradation ability changes of uricase in UAO-Sol stored at 4 °C, UAO-LPO stored at 4 °C, and UAO-LPO/Col-MNs stored at 4 or 25 °C over two months, data are presented as mean ± SD, n = 3.

Figure 6

Micro-CT analysis on the knee joints of MSU-induced chronic and recurrent attacks of gouty arthritis model. The representative two-dimensional (A) and three-dimensional (B) CT reconstruction images of rat right hind knee joints in five different groups. The bone structure-related indicators of rat right hind knee joints in each group, including BMD (C), BV/TV (D), Tb.N, rd (E), and Tb. Sp, rd (F), data are presented as mean ± SD, n = 5. The “∗” symbolizes significant variation between the control group and the other four groups, respectively (^∗P < 0.05, ^∗∗P < 0.01 and ^∗∗∗P < 0.001); “#” symbolizes significant variation between the UAO-LPO/Col-MNs and untreated group (^##P < 0.01).

Figure 7

Histopathological evaluation of MSU-induced chronic and recurrent attacks of gouty arthritis model. Representative Safranin O/Fast green staining images and H&E staining images for knee joints, and TRAP staining images for osteoclasts of subchondral bone of the control, untreated, UAO-LPO/Col-MNs without CTS, UAO/Col-Sol and UAO-LPO/Col-MNs. Green arrows indicate TRAP-positive cells.

Figure 8

Double-immunofluorescent staining on the RANKL and osteocalcin of subchondral bone marrow. The results showed that RANKL (green) is colocalized with osteocalcin (red), scale bar = 50 μm.