RNA interference-based nanosystems for inflammatory bowel disease therapy

Figures & data

Figure 1 RNAi nanoparticles target to epithelial cells or macrophages in intestinal lumen.

Abbreviation: RNAi, RNA interference.

Table 1 Summary of siRNA nanosystems in IBD treatment

Delivery system	siRNA target	Formulation	Size and zeta potential	Targeting			Route of administration	Characteristics	Ref
				Moiety	Cell and ligand	Mechanism
Lipid-based nanoparticles
Neutral liposome–hyaluronan–integrin mAb	Cyclin D1	siRNA-protamine encapsulated into β7 I-tsNPs by rehydrating and lyophilized	877±110 nm −4.1±2.1 mV	FIB504 mAb	β7 integrin on leukocyte	Antibody-targeting integrin	Intravenous injection via tail veins	Protamine is a positively charged protein, which was used to enhance delivery of nucleic acids. Hyaluronan maintains the structural integrity in the cycle of lyophilization and rehydration.	^Citation30
Polysaccharide-based nanoparticles
Modified chitosan–UAC–PEG–scCD98	CD98	Complex coacervation technique	147–261 nm 7.9–17.3 mV	Single-chain CD98 Ab	CD98 protein on colonic epithelial cells and macrophages	Antibody-targeting delivery	Oral gavage NPs encapsulated into hydrogel of alginate and chitosan	PEI in NPs functions as “proton sponge” to escape degradation by lysosome.	^Citation41
Galactosylated trimethyl chitosan–cysteine	Map4k4	Ionic gelation with TPP and siRNA entrapment method	140–160 nm 20–42 mV	Galactosyl	MGL on macrophage	MGL-mediated targeting	Oral gavage administration	Trimethyl chitosan improves solubility and gene transfection efficiency in physiological conditions. Cysteine–chitosan enhances bioadhesion capacity via covalently bonding with mucin glycoproteins. Cationic delivery systems spontaneously conjugate with anionic cross-linker TPP without sonication protecting siRNA.	^Citation46
Mannose trimethyl chitosan–cysteine	TNF-α	Ionic gelation with TPP and siRNA entrapment method	100–150 nm	Mannose	MR on enterocytes and M-cells	MR-mediated targeting	–	Mannose moieties improve intestinal permeation of Peyer’s patches.	^Citation52
SC12-cyclodextrin-click-propylamine	TNF-α	Complex coacervation technique	Without L-PEI: 240 nm +42 mV With L-PEI: 420 nm +24 mV	–	–	Mucoadhesion	Intrarectal administration	NPs keep stability in simulated colonic fluids and α-amylase. B-PEI has more appropriate electrostatic attraction with siRNA than L-PEI at low N/P ratio.	^Citation57
β-1,3-d-Glucan	Map4k4	SiRNA absorbs into glucan shell with electrostatic attraction and coated with PEI	2–4 µm	–	Phagocytosis via dectin-1 receptor on M-cells and macrophages	Mucoadhesion	Oral gavage administration	First report of oral siRNA delivery. The silencing efficiency of NPs up to 250 times compared to previous studies of systemic siRNA delivery in vivo.	^Citation59
PLA-based nanoparticles
PLA	TNF-α	Double emulsion/solvent evaporation	380 nm −8 mV	–	–	Mucoadhesion	Oral gavage administration	Encapsulate NPs into hydrogen of alginate and chitosan at a weight ratio of 7/3 and administered orally to mice.	^Citation63
PLA	Klf4	Double emulsion/solvent evaporation	–	–	–	Mucoadhesion	Oral gavage administration	DSS increases epithelial permeation efficiency of NPs. NPs uptake more by proliferating cells.	^Citation65
PLA–PEG–maleimide–Ab	TNF-α	Double emulsion/solvent evaporation	Without Fab’: 609±37 nm With Fab’: 379±19 nm	F4/80 Ab	F4/80 antigens on macrophages	Antibody-targeting delivery	Oral gavage administration	SiRNA-PEI complex decreases the therapeutic dose of siRNA.	66
CaP/PLGA-based nanoparticles
CaP/PLGA/PEI	TNF-α IP-10 KC	Rapid precipitation and double emulsion/solvent evaporation	151.52 nm 22.08 mV	–	–	Mucoadhesion	Intrarectal administration	B-PEI absorbed on the surface of CaP/PLGA nanoparticles to enhance cell endocytosis and endosomal escape. Intestinal epithelial cells as target of CaP/PLGA nanoparticles.	^Citation72
NiMOS-based microspheres
NiMOS	TNF-α	Double emulsion-like technique	NPs: 279±3.2 nm MPs: 2.4±0.94 µm	–	–	Mucoadhesion	Oral gavage administration	Blank NiMOS and scramble siRNA NiMOS show off-target effects.	^Citation80
NiMOS	TNF-α/cyclin D1	Double emulsion-like technique	–	–	–	Mucoadhesion	Oral gavage administration	Combined siRNA treatment caused stronger downregulated efficiency than single siRNA. Dilution effect in dual siRNA treatment compared to same amount of single siRNA.	^Citation77
Thioketal-based nanoparticles
PPADT	TNF-α	siRNA-DOTAP/PPADT mixture by emulsification method	600 nm 5.84+0.8 mV	–	–	Mucoadhesion	Oral gavage administration	DOTAP enhances siRNA transfection and endosome escapes. Oral TKNs target to disease tissue and perform silencing efficiency in tenfold lower dose than GeRPs.	^Citation83
Polyethylenimine-based nanoparticles
p(CBA–B-PEI)–PEG–Man	TNF-α	Complex coacervation technique	Without TPP: 302–363 nm With TPP: 211–275 nm	Mannose	MR on macrophages	MR-mediated targeting	Ex vivo cell culture	TPP is non-toxic, enhances siRNA consideration with polycation and decreases the size of NPs.	^Citation90

Note: “–” Indicates data not available.

Abbreviations: Ab, antibody; B-PEI, branched-polyethylenimine; CaP, calcium phosphate; CBA, N,N′-bioreducible cystamine bisacrylamide; DOTAP, 1,2-dioleoyl-3-trimethylammonium-propane; GeRPs, β-1,3-d-glucan-encapsulated siRNA particles; IBD, inflammatory bowel disease; Klf4, Krüppel-like factor 4; L-PEI, liner-polyethylenimine; mAb, monoclonal antibody; Man, mannose; MGL, macrophage galactose-type lectin; MPs, microspheres; MR, mannose receptor; NiMOS, nanoparticles-in-microsphere oral system; NPs, nanoparticles; PEG, polyethylene glycol; PEI, polyethylenimine; PLA, polylactide; PLGA, poly(d,l-lactide-co-glycolide acid); Ref, reference; ScCD98, single-chain CD98; siRNA, short interfering RNA; TKNs, thioketal nanoparticles; TNF-α, tumor necrosis factor-alpha; TPP, tripolyphosphate; UAC, urocanic acid; β₇ I-tsNPs, β₇ antibody-equipped liposome-siRNA complexes.

Table 2 Silencing experiments of different siRNA nanosystems in vitro and vivo

Delivery system	In vitro cell model	In vitro gene silencing result	Animal model	In vivo dose and administration interval	In vivo gene silencing result	Ref
Lipid-based nanoparticles
Neutral liposome–hyaluronan–integrin mAb	TK-1 cells	Suppression of CyD1, proinflammatory TH1 cytokine of IFN-γ, IL-2, IL-12, TNF-α.	C57BL/6 mice with DSS-induced	CyD1-siRNA 2.5 mg/kg entrapped in β7 I-tsNPs at days 0, 2, 4, and 6	Reduction in mRNA expression of CyD1 and IL-12, TNF-α. Drastic suppression of intestinal damage, leukocyte infiltration, reversal body weight loss.	^Citation30
Polysaccharide-based nanoparticles
Modified chitosan–UAC–PEG–scCD98	RAW 264.7 cells and colon-26 cells with LPS-induced	CD98 expression decreases to 27% of the control level.	Chronic colitis: transfer wild-type CD4^+CD45RB^high T-cells into RAG1^−/− mice. Acute colitis: male C57BL/6 mice with DSS-induced	Chronic colitis: 1 mg/kg of CD98 siRNA-loaded NPs double gavage twice a week for 5 consecutive weeks. Acute colitis: 2 mg/kg of CD98 siRNA-loaded NPs double gavage twice daily for 4 consecutive days	Chronic colitis: mRNA expression of CD98, TNF-α, IL-6, and IL-12 was decreased to 65.0%, 59.9%, 80.4%, and 31.8%, respectively, compared with control. Significant reduction of weight loss and MPO (~65.7%). Acute colitis: mRNA expression of CD98, TNF-α, IL-6, and IL-12 was decreased to 47.7%, 26.0%, 81.2%, and 71.2%, respectively. Body weight and MPO activity were correspondingly decreased.	^Citation41
Galactosylated trimethyl chitosan–cysteine	RAW 264.7 cells with LPS-induced	Expressions of Map4k4 and TNF-α mRNA were sharply decreased by 79.9% and 78.9%.	Male C57BL/6 mice with DSS-induced	SiRNA dose of 250 mg/kg per day for 6 consecutive days	Expressions of Map4k4 and TNF-α mRNA were sharply decreased by 92.1% and 69.0%. Inhibition of MPO activity, body weight loss, and colon shortening.	^Citation46
SC12-cyclodextrin-click-propylamine	RAW 264.7 cells with LPS-induced	TNF-α and IL-6 mRNA drop ~21- and sevenfold compared to control.	Female C57BL/6JOlaHsD mice	The volume of 100 µL solution administered to mice on day 2 and day 4 post-DSS treatment	Proximal colon tissue shows more significant TNF-α and IL-6 silencing than distal colon. Drastic reduction of TNF-α and IL-6 mRNA expression by 73%±13% and 58%±19%.	^Citation57
β-1,3-d-glucan	Peritoneal exudate cells with LPS-induced isolated from C57BL6/J male mice	TNF mRNA and TNF-α protein were inhibited by 50% and 30%.	C57BL6/J mice	20 mg siRNA/kg	NPs increase survival rates of model mice. Reduction of TNF-α and IL-1β accompanied with TNF-α silencing.	^Citation59
PLA-based nanoparticles
PLA	RAW 264.7 cells with LPS-induced	Significant reduction of TNF-α (175.9 vs 559.8 pg/mL).	C57BL/6 mice	The dose of 5 mg NPs/mL hydrogel pre-treated for 4 days before LPS treatment	Significantly TNF-α reduction in colonic tissue and blood. (Colon tissue 7.5 vs 136.2 pg/mL blood: 1,751.5 vs 2,084.5 pg/mL)	^Citation63
PLA	Embryonic fibroblasts in mice	Efficient suppression of Klf4 protein.	C57BL/6 mice	The dose of 0.5 ng siRNA in 1 mg NPs and 100 mL hydrogel per mice daily for 7 consecutive days	Reduction of clinical score and MPO activity in mice with colitis.	^Citation65
PLA–PEG–maleimide–Ab	RAW 264.7 cells with LPS-induced	Significant reduction of TNF-α and enhanced kinetics of uptake.	C57BL/6 mice	Daily gavages of hydrogel-encapsulated NPs (10 mg/mL) during the 7/8 days with DSS treatment	60 µg/kg TNF-α siRNA silence, 60% TNF-α expression, and 30% NPs were uptaken in intestinal macrophages in vivo. Higher IKβα accumulation in the treatment group than control.	^Citation66
CaP/PLGA-based nanoparticles
CaP/PLGA/PEI	MODE-K	The expression of IP-10 gene was reduced 30% as well as the expression of TNF-α or KC gene was reduced 50%.	BALB/c mice	CaP/PLGA nanoparticles (12 µg) were administered intrarectally from days 2 to 5 after DSS treatment	The expression of TNF-α mRNA decreased 40% and the expression of KC and IP-10 decreased 50% in the colon tissue.	^Citation72
NiMOS-based microspheres
NiMOS	–	–	Female Balb/c mice	The dose of 1.2 mg/kg body weight in 200 µL final volume	Reduction of TNF-α, IL-1β, IFN-γ, and MCP-1. Increasing body weight and falling levels of MPO.	^Citation80
NiMOS	–	–	Female Balb/c mice	The dose of 1.2 mg/kg body weight	Reduction of TNF-α, IL-1, IFN-γ. Increasing body weight and falling levels of MPO.	^Citation77
Thioketal-based nanoparticles
PPADT	RAW 264.7 cells with LPS-induced	Significant reduction of TNF-α production.	C57BL/6 mice	The dose of 2.3 or 0.23 mg siRNA/kg per day for 6 consecutive days	Reduction of mRNA expression of TNF-α, IL-6, IL-1, and IFN-γ.	^Citation83
PEI-based nanoparticles
p(CBA–B-PEI)–PEG–Man	RAW 264.7 cells with LPS-induced	Reduction of TNF-α production and the effects depend on the weight ratio of TPP-PPM/siRNA	Colitis tissues from FVB male mice with DSS induced	NPs added to the wells contain culture medium and colonic tissues, final TNF-α siRNA concentration in the medium is set as 100, 200, and 300 nM	TNF-α expression was decreased 61.0%. NPs were taken up by 29.5% colon macrophages.	^Citation90

Note: “–” Indicates data not available.

Abbreviations: Ab, antibody; B-PEI, branched-polyethylenimine; CaP, calcium phosphate; CyD1, cyclin D1; DSS, dextran sulfate sodium; IFN, interferon; IL, interleukin; Klf4, Krüppel-like factor 4; LPS, lipopolysaccharides; mAb, monoclonal antibody; Map4k4, mitogen-activated protein kinase kinase kinase kinase 4; Man, mannose; MCP, monocyte chemotactic protein; MPO, myeloperoxidase; NiMOS, nanoparticles-in-microsphere oral system; NPs, nanoparticles; PEG, polyethylene glycol; PLA, polylactide; PLGA, poly(d,l-lactide-co-glycolide acid); ScCD98, single-chain CD98; siRNA, short interfering RNA; T_H, T-helper cell; UAC, urocanic acid.

Figure 2 Chitosan-based nanoparticle delivery systems for siRNA delivery in IBD treatment.

Notes: Chemical structures of chitosan (A) and chitosan-based delivery systems (B–D).

Abbreviations: IBD, inflammatory bowel disease; PEG, polyethylene glycol; ScCD98, single-chain CD98; siRNA, short interfering RNA; UAC, urocanic acid.

Figure 3 Other polysaccharides for siRNA delivery in IBD treatment.

Notes: Chemical structures of modified amphiphilic cyclodextrin (A), β-1,3-d-glucan (B), and konjac glucomannan (C).

Abbreviations: IBD, inflammatory bowel disease; siRNA, short interfering RNA.

Figure 4 PLA-based nanoparticle delivery systems for siRNA delivery in IBD treatment.

Notes: Chemical structures of PLA, PVA, and PLA–PEG–maleimide–Ab.

Abbreviations: Ab, antibody; IBD, inflammatory bowel disease; PEG, polyethylene glycol; PLA, polylactide; PVA, polyvinyl alcohol; siRNA, short interfering RNA.

Figure 5 Schematic illustration of CaP/PLGA-based nanoparticles.

Abbreviations: CaP, calcium phosphate; PEI, polyethylenimine; PLGA, poly(d,l-lactide-co-glycolide acid); siRNA, short interfering RNA.

Figure 6 Schematic illustration of nanoparticles in the microsphere system.

Abbreviations: PCL, poly-ε-caprolactone; siRNA, short interfering RNA.

Figure 7 Thioketal and PEI-based nanoparticulate delivery system for siRNA delivery in IBD treatment.

Notes: Chemical structures of PPADT, B-PEI, CBA, and p(CBA–B-PEI)–PEG–Man.

Abbreviations: B-PEI, branched-polyethylenimine; CBA, cystamine bisacrylamide; IBD, inflammatory bowel disease; Man, mannose; PEG, polyethylene glycol; siRNA, short interfering RNA.

Figure 8 Colon histological sections stained with H&E from mice receiving different kinds of nanoparticles.

Notes: Colon histological sections stained with H&E from mice receiving normal water and gavage of phosphate buffer saline (A). Colon histological sections stained with H&E from DSS-induced mice receiving daily gavage of phosphate buffer saline (B), scramble-siRNA-loaded TKNs (C), TNF-α-siRNA-loaded TKNs (D), TNF-α-siRNA-loaded PLGA nanoparticles (E), or TNF-α-siRNA-loaded β-glucan particles (F). Adapted by permission from Macmillan Publishers Ltd: [Nat Mater],⁸⁴ copyright (2010). The β-glucan particles (F) have demonstrated siRNA carrying ability by oral administration in inflammatory treatment at previous studies.⁵⁹

Abbreviations: DSS, dextran sulfate sodium; H&E, hematoxylin/eosin; siRNA, short interfering RNA; TKNs, thioketal nanoparticles; TNF, tumor necrosis factor.

Table 3 Challenges of RNAi molecular delivery in IBD

RNAi molecule	Physical barriers	Pathological changes in gastrointestinal tract of IBD
High molecular mass: ~13 kDa of siRNA Strong net negative charge (~40 negative phosphate charges) of siRNA leads to electrostatic repulsion with anionic cell membrane surface Short half-life in vivo Off-target effects Immunostimulation	Low pH stomach environment Endogenous nuclease in gut lumen Reticuloendothelial system uptake and aggregation with serum proteins Gradual decline in pH and hydrolytic nuclease in endosome and lysosome via clathrin-mediated endocytosis	Abnormal colon luminal pH: 2.3–5.5 Diarrhea, abnormal motility, and surgical resection of gastrointestinal tract accelerated nanoparticulate transit time Dysbiosis of microbial composition influences polysaccharide-based nanoparticle degradation Enterocyte damage enhances nanoparticulate accumulation and uptake in enterocytes and macrophages

Abbreviations: IBD, inflammatory bowel disease; RNAi, RNA interference; siRNA, short interfering RNA.

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