Sprayable inflammasome-inhibiting lipid nanorods in a polymeric scaffold for psoriasis therapy

Abstract

Localized delivery of inflammasome inhibitors in phagocytic macrophages could be promising for psoriasis treatment. The present work demonstrates the development of non-spherical lipid nanoparticles, mimicking pathogen-like shapes, consisting of an anti-inflammatory inflammasome inhibiting lipid (pyridoxine dipalmitate) as a trojan horse. The nanorods inhibit inflammasome by 3.8- and 4.5-fold compared with nanoellipses and nanospheres, respectively. Nanorods reduce apoptosis-associated speck-like protein and lysosomal rupture, restrain calcium influx, and mitochondrial reactive oxygen species. Dual inflammasome inhibitor (NLRP3/AIM-2-IN-3) loaded nanorods cause synergistic inhibition by 21.5- and 59-folds compared with nanorods and free drug, respectively alongside caspase-1 inhibition. The NLRP3/AIM-2-IN-3 nanorod when transformed into a polymeric scaffold, simultaneously and effectively inhibits RNA levels of NLRP3, AIM2, caspase-1, chemokine ligand-2, gasdermin-D, interleukin-1β, toll-like receptor 7/ 8, and IL-17A by 6.4-, 1.6-, 2.0-, 13.0-, 4.2-, 24.4-, 4.3-, and 1.82-fold, respectively in psoriatic skin in comparison to Imiquimod positive control group in an in-vivo psoriasis-like mice model.

Fig. 1. Design and engineering of sprayable polymeric scaffold loaded with non-spherical pathogen-like trojan-horse lipid nanoparticles.

a Schematic for the synthesis of non-spherical pathogen-like trojan-horse lipid nanorods. The nanorods, nanoellipses, and nanospheres were prepared using 10:1, 5:1, and 2:1 molar ratios of pyridoxine dipalmitate and DSPE PEG 2000 (carboxylic acid). Created in BioRender. Kulkarni, A. (2024) BioRender.com/e03e101 b Mechanism of non-spherical lipid nanoparticle cellular internalization and NLRP3 inflammasome inhibition. The non-spherical lipid nanoellipses/rods were internalized by macropinocytosis and clathrin-mediated endocytosis, while the nanospheres were internalized by phagocytosis. The nanorods delayed lysosomal rupture and significantly reduced the IL-1β level by attenuating ASC speck formation, restraining calcium influx, mitochondrial ROS and NLRP3 inflammasome, whereas the nanoellipses and nanospheres reduced the mitochondrial ROS formation. Created in BioRender. Kulkarni, A. (2024) BioRender.com/q44z068. c Scaffold design and synthesis. The NLRP3/AIM2-IN-3 (NA3) was loaded within the nanorods, and later, Poloxamer 407 (10% w/v; thermogelling agent) and mucin (2.5 mg/mL; thickening agent) were dissolved in the NA3 nanorods aqueous dispersion to form a solution for topical spraying. When sprayed, the poloxamer solution forms micelles and depicts temperature-dependent gelling properties. Created in BioRender. Kulkarni, A. (2024) BioRender.com/i72q105. d In vivo effect of sprayable polymeric scaffold loaded with nanorods or NA3 nanorods on psoriasis. The NA3 nanorod polymeric scaffold, when sprayed, causes a thin film due to gelation. The NA3 nanorods and mucin reduce inflammation, psoriatic chemokines, and keratinocyte proliferation due to their effect on NLRP3 and AIM2 inflammasome inhibition. Created in BioRender. Kulkarni, A. (2023) BioRender.com/f54e160.

Fig. 2. Evaluation of lipid nanoparticle shape on internalization and inflammasome inhibition in macrophages.

a Synthesis and physicochemical characterization of non-spherical pathogen-like Trojan-horse lipid nanoparticles including Transmission electron microscopy, Particle size, and zeta potential distribution, and stability studies at 4 °C (Data represented as mean ± SD; n = 3 individual trials for particle size and zeta potential determination and n = 2 for stability studies). b, c Mechanism of internalization of nano-spheres/ellipses/rods using flow cytometer (Mean ± SD; n = 6, 3 biological and 2-technical replicates; data analyzed by two-way ANOVA and Tukey’s multiple comparison test). d, e Internalization of nano-spheres/ellipses/rods through CD14 and TLR-4 determined by flow cytometry and RT-PCR, respectively (Mean ± SD and mean ± SEM, respectively n = 6 with 3 biological replicates and 2 technical replicates were used for both the studies, data analyzed by ordinary one-way ANOVA Tukey’s multiple comparisons and Brown Forsythe Welche Dunnet’s T3 multiple comparisons, respectively. The Fig. 2(e) represents the minima and maxima as the whisker bounds, the 25th and 75th percentile as the bounds of boxes, and the median as the center). f Effect of nano-spheres/ellipses/rods on NLRP3 inflammasomes in iBMDM determined by IL-1β ELISA. (Mean ± SD; n = 6 with 3 biological replicate and two technical replicate per biological sample data analyzed by ordinary one-way ANOVA and Tukey’s multiple comparison test).

Fig. 3. Mechanistic evaluation of lipid nanoparticles (LNPs) shape on inflammasome inhibition in macrophages.

a Schematic representation of the effect of LNP shapes on ASC speck formation. Created in BioRender. Kulkarni, A. (2023) BioRender.com/i04d511. b Confocal microscopic images of ASC speck (CFP) in iBMDM with nano-spheres/ellipses/rods at 20X (Scale bar: 100 µm). c ASC speck number per cell when treated with nano-spheres/ellipses/rods. (Mean ± SD, n = 3 biological replicates with 4 technical replicates per biological sample using Brown Forsythe Welche one-way ANOVA and Dunnett’s T3 multiple comparison. The graph represents the minima and maxima as the whisker bounds, the 25th and 75th percentile as the bounds of boxes, and the median as the center) (d) Schematic representation of the effect of LNP shapes on lysosomal rupture. Created in BioRender. Kulkarni, A. (2023) BioRender.com/l77q945. e Confocal microscopic images of intact lysosomes (TRITC) in iBMDM treated with nano-spheres/ellipses/rods at 20X (Scale bar: 100 µm). f, g Analysis of intact lysosomes normalized against LNP internalization and live cells as indicated by Cy5 and DAPI signal, respectively when treated with nano-spheres/ellipses/rods. Each data represented as mean ± SD; n = 7; with 3 biological replicate and 2,2,3 technical replicates data analyzed by ordinary one-way ANOVA and Tukey’s multiple comparisons. h, i Effect of nano-spheres/ellipses/rods on calcium influx and mitochondrial ROS formation determined by flow cytometer. (Each data represented as mean ± SD; n = 5 with 2 biological and 2, 3 technical replicates and n = 10; with 4 biological and 2,2,3,3 technical replicates, data analyzed by Brown Forsythe Welche one-way ANOVA with Dunnets T3 multiple comparison and ordinary one-way ANOVA, respectively).

Fig. 4. Effect of NLRP3-AIM2 inhibitor (NA3) loaded nanorods on inflammasome inhibition.

a Schematic of NA3 nanorods synthesis. Created in BioRender. Kulkarni, A. (2023) BioRender.com/o21z452. b, c Particle size (mean ± SD; n = 3 individual trials) and zeta potential distribution (mean ± SD; n = 2 individual trial) of NA3 loaded nanorods (d) Time-dependent stability studies of NA3 nanorods at 4 °C (Data represented as mean ± SD; n = 2 individual trials). e % drug loading and % encapsulation efficiency of NA3 in nanorods determined by RP-HPLC (n = 5 individual replicates). f Effect of NA3 loaded nanorods on inflammasome inhibition in iBMDM determined by IL-1β ELISA (Each data represented as mean ± SD; n = 5 with 2 biological replicate and 2,3 technical replicate per biological replicate; data analyzed by two-way ANOVA with Tukey’s multiple comparisons). g Effect of NA3 nanorods on ASC speck formation determined by confocal microscopy (Each data represented as mean ± SD; n = 5 with 2 biological replicate and 2 and 3 technical replicate per biological replicate data analyzed by ordinary one-way ANOVA and Tukey’s multiple comparison). The images were observed at 60X (scale bar 20 µm).

Fig. 5. Effect of NLRP3-AIM-2 inhibitor (NA3) loaded nanorods polymeric scaffold on inflammasome inhibition.

a Screening of thermogelling polymer concentration (Data represents mean ± SD, n = 3 individual experiments analyzed by one-way ANOVA and Tukey’s multiple comparison test) (b, c) Particle size and zeta potential distribution, respectively of NA3 loaded nanorods polymeric scaffold. d Time-dependent stability studies of NA3 nanorods polymeric scaffold at 4 °C (Mean ± S.D; n = 2 individual formulations). e In-vitro drug release of NA3 from NA3-loaded nanorods polymeric scaffold in PBS and cell lysate. (Each data represents mean ± SD; n = 6 for NA3 nanorods polymeric scaffold, n = 3 and 2 for NA3 nanorods scaffold drug release in cell lysate and free NA3, respectively. Data analyzed by two-way ANOVA, p = 0.0182,0.040, 0.0010, 0.0486,0.0107,0.0021 for NA3 nanorods polymeric scaffold in PBS compared with NA3 nanorods polymeric scaffold in cell lysate) (f) Effect of NA3 loaded nanorods polymeric scaffold on inflammasome inhibition in iBMDM determined by IL-1β ELISA. (Each data represents mean ± SD; n = 5 with 2 biological replicate and 2,3 technical replicate and analyzed by two-way ANOVA and Tukey’s multiple comparisons). g Effect of NA3 loaded nanorods and blank nanorods polymeric scaffold on caspase-1 determined by western blot in iBMDM (1-untreated, 2-LPS (Negative control), 3-LPS+Nigericin (positive control), 4-Blank nanorods polymeric scaffold, 5-NA3 nanorods polymeric scaffold treated iBMDM) (Each data represents mean ± SD; n = 3 technical replicate per biological sample and analyzed by one-way ANOVA and Tukey’s multiple comparisons). The samples were obtained from same experiment and the blots were run in parallel on the same gel. The uncropped gels are available in the supplementary and source data file. h Effect of NA3 nanorods polymeric scaffold on ASC speck formation determined by confocal microscopy. The images were observed at 60X magnification (scale bar-20 µm) (Each data represented as mean ± SD; n = 5 with 2 biological and 2,3 technical replicates and analyzed by Brown Forsythe Welche one-way ANOVA with Dunnett’s T3 multiple comparison). i Rheological studies of NA3 nanorods polymeric scaffold.

Fig. 6. In-vivo evaluation in Imiquimod (IMQ) induced Psoriasis Balb/C mice model.

a Study paradigm schematic representation. Created in BioRender. Kulkarni, A. (2023) BioRender.com/h57d051. b Cumulative daily PASI score of back scaling, erythema, and thickness (Data represented as mean ± SEM, n = 4 for all other groups and 3 for negative control and analyzed by ANOVA and Tukey’s multiple comparisons between NA3 nanorods and blank nanorods scaffold with ** indicates p = 0.0075). c Daily body weight measurement of IMQ-induced psoriasis mice in each group (Data represented as mean ± SD, n = 4 for all other groups and 3 for negative control). d, e Back thickness and spleen to body weight ratio of IMQ-induced mice from each group (Each data represented as mean ± SD; n = 4 for all other groups and 3 for negative control group; data analyzed by two-way ANOVA and Tukey’s multiple comparisons). f RNA expression of different cellular components involved in NLRP3 and AIM-2 inflammasome activation extracted from the back skin of Balb/c mice in each group. (Each data represented for n = 4 for all other groups and 3 for negative control with 3 technical replicate per animal, data analyzed by Brown Forsythe and Welche one-way ANOVA Test). g Western blot analysis of IL-17A expressed in the mice back skin from each group (Each data represented as mean ± SD; n = 4, samples of 2 animals/sample were pooled and two different samples were run for western blot analysis, data analyzed by one-way ANOVA and Tukey’s multiple comparison test). The uncropped gels are available in the supplementary and source data file.

Fig. 7. Microscopic evaluation of in-vivo skin samples from Imiquimod (IMQ) induced Psoriasis Balb/C mice model.

a Light microscopic images of H&E, Ki67 (DAB), and CD31 antibody (DAB) stained preserved back skin transverse sections from euthanized mice on day 6 (Images represent Munro microabscess (black arrow), hyperplasia/hypogranulosis (blue arrow), Capillary proliferation (green arrow), Rete Ridges (orange arrow), Inflammatory infiltration (red arrow)). The H&E and DAB Ki 67 were analzyed at 20X (Scale bar: 100 µm) and the represented DAB CD31 were observed at 40X (scale bar: 300 pixels). b The integrated density of Ki67 DAB stain indicating cell proliferation measured from IHC sections imaged at 20X (n = 4 for all the groups and 3 for negative control group with 2 technical replicate for positive and negative control group and 3 technical replicates for blank and NA3 nanorods scaffold; data represented as mean ± SD; and analyzed by ordinary one-way ANOVA). c Mean grey value of CD31 marker indicating vascular hyperproliferation measured for IHC microscopic images at 20X and 40X (Data represented as mean ± S.D; n = 4 with 2 technical replicate per sample and analyzed by ordinary one-way ANOVA Tukey’s multiple comparison) (d) The inflammatory cell infiltrate count indicating cell proliferation for H&E images at 40X (n = 4 with 2 technical replicate) (Data represented as mean ± SD; and analyzed by Brown Forsythe Welche Dunnet’s T3 multiple comparison).