Transdermal delivery of fluvastatin sodium via tailored spanlastic nanovesicles: mitigated Freund's adjuvant-induced rheumatoid arthritis in rats through suppressing p38 MAPK signaling pathway

Figures & data

Table 1. The 2⁴ full factorial design and the observed responses of FVS-loaded SNVs.

Formulation	Independent variables				Dependent variables				PDI
	A: Span type	B: EA type	C: EA concentration (% w/w)^a	D: Sonication time (min)	Y1: EE%	Y2: Vesicle size (nm)	Y3: Q8h (%)	Y4: Q24 (µg/cm^2)
S1	Span 60	Tween 80	10	0	65.70 ± 2.55	692.57 ± 17.03	67.23 ± 11.75	229.44 ± 20.45	0.45
S2	Span 60	Tween 80	20	0	80.07 ± 3.10	388.40 ± 18.86	73.36 ± 11.50	284.45 ± 15.32	0.34
S3	Span 60	Brij 35	10	0	61.13 ± 1.42	569.40 ± 17.62	70.88 ± 9.00	255.51 ± 19.50	0.48
S4	Span 60	Brij 35	20	0	78.70 ± 1.21	322.07 ± 19.33	77.23 ± 5.25	292.12 ± 21.03	0.50
S5	Span 80	Tween 80	10	0	57.65 ± 2.09	275.07 ± 18.06	79.23 ± 3.75	313.96 ± 22.50	0.37
S6	Span 80	Tween 80	20	0	77.13 ± 2.26	220.53 ± 21.34	87.25 ± 3.25	349.42 ± 21.10	0.27
S7	Span 80	Brij 35	10	0	54.40 ± 1.49	250.57 ± 16.87	83.24 ± 3.25	324.42 ± 25.50	0.34
S8	Span 80	Brij 35	20	0	73.13 ± 2.30	215.00 ± 20.00	90.92 ± 4.00	392.67 ± 23.02	0.34
S9	Span 60	Tween 80	10	5	43.17 ± 1.77	576.67 ± 21.73	69.52 ± 2.50	265.99 ± 24.32	0.49
S10	Span 60	Tween 80	20	5	58.40 ± 2.04	329.33 ± 24.01	73.80 ± 3.75	290.06 ± 20.45	0.38
S11	Span 60	Brij 35	10	5	42.53 ± 2.45	380.00 ± 21.79	71.24 ± 3.75	277.54 ± 22.52	0.37
S12	Span 60	Brij 35	20	5	50.17 ± 2.02	280.20 ± 17.79	80.45 ± 2.50	308.77 ± 21.21	0.32
S13	Span 80	Tween 80	10	5	40.50 ± 2.50	264.17 ± 11.77	81.47 ± 3.51	325.06 ± 25.02	0.46
S14	Span 80	Tween 80	20	5	46.77 ± 3.03	170.00 ± 10.00	88.89 ± 3.25	402.48 ± 25.50	0.33
S15	Span 80	Brij 35	10	5	34.27 ± 2.15	230.53 ± 10.45	84.43 ± 4.50	338.50 ± 21.50	0.45
S16	Span 80	Brij 35	20	5	43.33 ± 3.06	167.83 ± 11.73	92.08 ± 3.00	460.59 ± 20.01	0.35

EA: edge activator; EE%: entrapment efficiency percent; Q_8h: cumulative release after 8 h (%); Q₂₄: cumulative amount permeated/unit area in 24 h; PDI: polydispersity index.

Data are mean values (n = 3) ± SD.

^a (% w/w) of the EA with respect to Span/EA total weight.

Figure 1. Response surface plot for the effect of Span type (A), EA type (B) at the middle levels of the 3^rd and 4^th variables (EA concentration and sonication time) on (a) EE%, (b) vesicle size, (c) Q_8h, (d) Q₂₄, and (e) desirability of the developed SNVs dispersions.

Figure 2. Transmission electron micrograph of the optimal SNVs formulation.

Table 2. Mean pharmacokinetic parameters for FVS in rat plasma following administration of oral solution, SNVs gel and traditional gel.

Pharmacokinetic parameter	Mean ± SD
	Oral solution	SNVs gel	Traditional gel
Cmax (ng/ml)	416.61 ± 25.92	339.88 ± 27.76^a	120.50 ± 17.52^a^,^b
Tmax (h)	1.17 ± 0.26	7.33 ± 1.03^a	3.58 ± 1.02^a^,^b
Kelim (h^–1)	0.2219 ± 0.0437	0.10678 ± 0.0101^a	0.1904 ± 0.0145^a^,^b
t1/2 (h)	3.12 ± 0.42	6.49 ± 0.67^a	3.64 ± 0.21^a^,^b
AUC0–24 (ng h/ml)	1126.91 ± 74.60	3093.82 ± 217.50^a	683.69 ± 78.38^a^,^b
AUC0–∞ (ng h/ml)	1131.42 ± 40.96	3156.93 ± 180.06^a	687.74 ± 66.01^a^,^b
Frel (%)	–	279.02	60.79

Values are means ± SD, with the number of animals = 6 for each group. Using one-way ANOVA followed by Tukey’s post hoc test.

^a p < .05 versus oral FVS solution.

^b p < .05 versus SNVs gel.

Table 3. Biochemical analysis in different studied groups.

Parameter	Normal control	Arthritis control	Arthritis + MTX	Arthritis + FVS	Arthritis + FVS- loaded
% of change	(group 1)	(group 2)	(group 3)	(group 4)	SNVs gel (group 5)
RF (IU/ml)	1.74 ± 0.47	12.60 ± 1.56^a	4.34 ± 0.34^a^,^b	6.33 ± 0.21^a^,^b	4.53 ± 0.29^a^,^b
% of change	–	(624.14%)	(–65.56%)	(–49.76%)	(–64.05%)
COMP (ng/ml)	1.47 ± 0.29	15.87 ± 1.42^a	3.40 ± 0.35^a^,^b	5.23 ± 0.32^a^,^b	4.20 ± 0.40^a^,^b
% of change	–	(979.59%)	(–78.58%)	(–67.04%)	(–73.53%)
ANA (ng/ml)	1.82 ± 0.22	19.53 ± 2.90^a	5.16 ± 0.82^b	8.20 ± 0.70^a^,^b	6.06 ± 1.00^a^,^b
% of change	–	(973.08%)	(–73.58%)	(–58.01%)	(–68.97%)
TNF-α (pg/ml)	34.53 ± 1.76	102.13 ± 14.77^a	44.87 ± 3.01^b	65.83 ± 6.93^a^,^b	46.33 ± 2.21^b
% of change	–	(195.77%)	(–56.07%)	(–35.54%)	(–54.64%)
IL-10 (pg/ml)	124.33 ± 2.21	48.73 ± 12.56^a	108.73 ± 6.33^b	99.83 ± 6.93^a^,^b	109.50 ± 3.82^b
% of change	–	(–60.81%)	(123.13%)	(104.86%)	(124.71%)
MDA (nmol/mg)	9.33 ± 0.75	46.07 ± 2.86^a	13.43 ± 1.35^b	12.23 ± 1.55^b	10.07 ± 0.75^b
% of change	–	(393.78%)	(–70.85%)	(–73.45%)	(–78.14%)
GSH (mmol/mg)	61.43 ± 2.72	28.77 ± 2.29^a	50.77 ± 4.35^a^,^b	47.23 ± 2.91^a^,^b	53.87 ± 4.19^b
% of change	–	(–53.17%)	(76.47%)	(64.16%)	(87.24%)

MTX: methotrexate; FVS: fluvastatin; SNVs: spanlastic nanovesicles; RF: rheumatoid factor; COMP: cartilage oligomeric matrix protein; ANA: antinuclear antibody; TNF-a: tumor necrosis factor-alpha; IL-10: interleukin-10; MDA: malondialdehyde; GSH: glutathione reduced.

Each value represents the mean of 8 values ± SD. Statistical analysis was performed using one-way ANOVA test followed by Tukey’s post hoc test. Differences between group means were considered significant at p < .05. Percentage (%) changes were calculated by comparing arthritic control group with normal control and arthritic-treated groups with the arthritic control group.

^a Significantly different from normal control group value at p < .05.

^b Significantly different from arthritic control group value at p < .05.

Figure 3. Radiological changes of joints. (a) Normal control group. (b1 and b2) Arthritis control group showing foot deformity, marked knee joint space narrowing and osteopenia. (c) Methotrexate-treated group showing normal joint space, but mild foot deformity. (d) Oral FVS-treated group showing mild narrowing of joint space, mild foot deformity and osteophytes. (e) FVS-loaded SNVs gel-treated group showing relative narrowing of knee joint space with mild foot deformity without osteopenia or osteophytes.

Figure 4. Photomicrographs of articular cartilage and splenic parenchymae of different sections in different groups (H&E ×100). (a–e) Articular cartilage of different groups. (a) Normal control rat section showing a smooth articular surface (black arrow) and a regular tide mark (blue arrow) separating the articular cartilage from the underlying subchondral bone. (b) Arthritis control rat section showing disrupted articular surface (black arrow), cartilage layer was thinner with cell loss, cell cloning and multicellular chondrocyte clusters and overall the cells appeared in a less ordered structure (blue arrow). The subchondral bone invaded the calcified cartilage (stars), top right. (c) Methotrexate-treated arthritic rat section showing slight smooth articular surface (black arrow), thickened articular cartilage with proliferating chondrocytes (blue arrow), hypercellularity and cloning. Regular tide mark can be seen (arrowhead). (d) Oral FVS-treated arthritic rat section showing slightly smooth articular surface (blue arrow) with thinner articular cartilage layer. The subchondral bone (star) can be observed. The proliferating chondrocytes showing hypercellularity, cloning, and slightly ordered distribution (black arrow). Regular tide mark can be seen (arrowhead). (e) FVS-loaded SNVs gel-treated arthritic rat section showing smooth articular surface (black arrow). Thickened articular cartilage and subchondral bone (star) can be observed. The proliferating chondrocytes showing hypercellularity, cloning, and slightly ordered distribution (blue arrow). Regular tide mark can be seen (arrowhead). (f–j) Splenic parenchymae of different sections. (f) Normal control rat section showing thin capsule (arrowhead), trabeculae (black arrow) and white pulp formed of lymphatic follicles (blue arrow) surrounded with red pulp (stars). Inserted box showing lymphatic follicles with central arteriole and well-defined marginal zone. (g) Arthritis control rat section showing marked atrophy of white pulp (black arrow) and red pulp (stars). (h) Methotrexate-treated arthritic rat section showing proliferating lymphatic follicles (white pulp) (blue arrow) surrounded with hyperplastic red pulp (black arrow). Lymphatic follicles showing central arteriole (arrowhead) and well-defined marginal zone. (i) Oral FVS-treated arthritic rat section showing slightly proliferating lymphatic follicles (white pulp) (blue arrow) surrounded with hyperplastic red pulp (black arrow). Lymphatic follicles showing central arteriole and well-defined marginal zone. (j) FVS-loaded SNVs gel-treated arthritic rat section showing slightly proliferating lymphatic follicles (white pulp) (blue arrow) surrounded with hyperplastic red pulp (black arrow). Lymphatic follicles showing central arteriole and well-defined marginal zone.