Natural Product-Based Nanomedicine in Treatment of Inflammatory Bowel Disease

Inflammatory bowel disease (IBD), which includes Crohn’s disease (CD) and ulcerative colitis (UC), is a highly debilitating chronic inflammatory disorder of the small intestine and colon with enhanced risk of colorectal cancer (CRC). IBD affects millions of people and mainly occurs in genetically predisposed individuals having dysregulated immune response to various environmental conditions [ 1 , 2 , 3 ]. The burden of IBD is quite high, both on patient’s quality of life and on the health care system, with estimated hospitalization rates of 8.2–17 per 100,000 annually and annual treatment costs of $6.8 billion [ 4 ]. Genetic, immunological, and environmental factors play important roles in its etiology [ 5 ]. The most common symptoms include regular abdominal pain, fever, vomiting, diarrhea, blood in the stool, and weight loss with enhanced risk of colorectal cancer [ 6 , 7 ]. The factors involved in intestinal inflammation include altered synthesis and release of pro-inflammatory cytokines (interleukin (IL)-1β, IL-6, and IL-12), tumor necrosis factor α (TNF-α), interferon γ (IFN-γ), transforming growth factor (TGF)-β, and increased reactive oxygen species (ROS) that results in excessive damage to the intestinal tissues [ 6 , 8 , 9 ]. In IBD, intestinal permeability is predominantly abnormal with increased appearance of immune cells and increased mucus production [ 10 , 11 , 12 ]. The drug therapy in IBD principally induces either remission of acute attacks or limits the acute attacks during remission. Aminosalicylates, steroids, and immunosuppressants are some of the conventional anti-inflammatory drugs used for treating IBD. Although these medications are efficient, the systemic absorption of these anti-inflammatory medications results in side effects (short- and long-term), such as allergic responses, diarrhea, vomiting, lymphopenia, raised liver enzymes, and inflammation of the pancreas [ 1 , 13 ]. In addition to these conventional therapeutics, monoclonal antibody-based biological therapies are also recommended in IBD treatment. Several such USFDA (U.S. Food and Drug Administration) approved TNF-α antibodies include infliximab, adalizumab, certolizumab, vedolizumab, and golimumab, whereby infliximab was the first product approved for the management of IBD. However, huge costs associated to biological therapies, parenteral administration, higher number of nonresponders, and immune complications are major challenges limiting their potential in IBD treatment [ 14 , 15 , 16 ]. Therefore, curative strategies that may be more safe and effective in the treatment and management of IBD are required [ 5 , 6 , 17 , 18 ] Several recent reports document use of natural phytochemicals in IBD that possess anti-inflammatory and antioxidant activity such as flavonoids and phenolic compounds. They modulate various inflammatory mediators, such as IL-1β, IL-6, IL-10, TNF-α, prostaglandin E2 (PGE-2), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) [ 13 , 19 ]. Additionally, natural macromolecules are also tested in suppression of IBD-associated biochemical and molecular inflammatory pathways. Recently, antioxidant and anti-inflammatory properties of biophenols derived from fruits and vegetables have been considered beneficial for IBD through various in vitro and in vivo studies [ 20 ]. Biophenols like oleuropein, hydroxytrosol, and ligstroside derived from olive trees have been reported to possess aforesaid activities [ 21 ]. In particular, oleuropein has been widely studied for its anti-inflammatory and immunomodulatory activities, whereby an oleuropein-treated colon biopsy sample of a colitis patient revealed restoration of the typical microscopic damage with strong recovery of mucin-forming goblet cells against untreated colon biopsy sample [ 22 ]. The same compound has also resulted in a reduction of pro-inflammatory cytokine level (IL-1β, IL-6, and IL-8) in ex-vivo organ cultures of mucosal explants from CD patients, thus demonstrating immunomodulatory activity in IBD [ 23 ]. Similarly, polyphenols derived from green tea have great therapeutic potential against IBD treatment due to their antioxidant properties, regulation of inflammatory mediator TNF-α, and COX-2 synthesis, thereby playing a vital role in downregulating the aberrant signaling pathways in IBD [ 24 ]. A possible barrier to this area is that the administration of natural drugs in conventional manner is restricted because of their little solubility, permeability, and bioavailability. However, micronization and nanonization of these natural drugs may be an acceptable strategy to improve their physical and chemical properties to overcome the challenges faced otherwise [ 25 ]. In the following pages, we will review encapsulated plant-based medicines and natural macromolecules in the treatment of colitis and colitis-associated colorectal cancer (CAC). As use of natural products is an immensely vast field in IBD, we have focused this review

3.1. Thymoquinone Thymoquinone (TQ) in the seed oil extract of Nigella sativa has several anti-inflammatory and immunomodulating activities targeting nuclear factor kappa B (NF-κB), IL-1β, and TNF-α signaling. Liposome or nanoparticle-based formulations of TQ are effective against various diseases in animal models [ 35 ]. Treatment of dextran sodium sulfate (DSS)-induced colitis in mice with TQ suppresses malondialdehyde (MDA) levels and myeloperoxidase (MPO) activity with concomitant increase in glutathione levels indicating improvement in colitis-associated tissue damage [ 36 ]. In addition, there is significant reduction in the expression of inflammatory markers Cox-2, iNOS, Nrf2, KEAP1, and pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) both at the mRNA and protein levels [ 37 ]. In vitro treatment of HT-29 colon cancer cells with a combination of TQ and lipopolysaccharide (LPS) also reduced inflammatory markers [ 38 ]. Further, oral administration of alginate microcapsule encapsulated N. sativa extract (NSE) is an efficient strategy for the delivery of TQ to the colon for the treatment of IBD [ 38 ].

Curcumin (Cur) is derived from the roots of a plant Curcuma longa, a member of the Zingiberaceae family. It is composed of 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione, a polyphenolic hydrophobic compound, and 2%–5% turmeric, a bioactive pigment giving it a yellow color. Its anti-inflammatory [ 41 , 42 ], immunomodulatory [ 43 , 44 , 45 ], and antioxidant [ 46 ] properties are documented in several human diseases including cancers [ 47 , 48 , 49 ]. Traditional use of curcumin in the treatments is limited because of its poor absorption in the gastrointestinal tract, poor stability, low bioavailability, and rapid systemic elimination [ 50 ]. However, use of curcumin in nano-formulations with albumin, histone, solid lipids, polylactide-coglycolide, liposomes, and polybutylcyanocrylate improves its bioavailability, solubility, and stability, making it therapeutically stronger with no adverse effects [ 50 , 51 ]. Curcumin-primed and curcumin-encapsulated exosomes have shown profound anti-inflammatory activities by decreasing expression of IL-6 and TNF-α in murine macrophage RAW 264.7 cells when induced by lipopolysaccharide (LPS) [ 51 ]. Moreover, curcumin-primed and curcumin encapsulated exosomes are promising agents in treating inflammation-related diseases by affecting NF-κB-, Nrf2-, and activator of transcription-3 (STAT3)-dependent signaling pathways [ 52 ]. Qiao et al. characterized amphiphilic curcumin polymer (PCur), which is made of hydrophilic polyethylene glycol (PEG) and hydrophobic curcumin joined together by a disulfide bond. Due to nano-scaled sizes with sufficient solubility and neutral surface potential, PCur efficiently accumulates at the inflamed sites of the gut. Further, low cytotoxicity and increased membrane permeability of the PCur improves its oral bioavailability. Oral administration of PCur in DSS-induced mice results in amelioration of progression of inflammation in the colon and possible prevention from IBD and colitis-associated cancer (CAC) [ 53 ]. In another study, water-insoluble curcumin is chemically engineered into hydrophilic mucoadhesive chitosan and used in a preclinical dextran sodium sulfate (DSS) colitis model and azoxymethane (AOM)-DSS-induced CAC mouse models. Orally delivered curcumin-chitosan NPs accumulate in inflamed intestinal regions and tumor tissues. Treatment significantly protects mice from ulcerative colitis (UC) and CAC [ 54 ].

Quercetin (3,3′,4′,5,7-pentahydroxyflavone), a polyphenol found in abundance in onions, has anti-inflammatory and antioxidant activity [ 56 ]. It occurs commonly in both glycoside and aglycone forms [ 57 ]. PEG-coated vesicles with chitosan and nutriose-loaded quercetin are most suitable for the drug delivery in colon and thus improve and ameliorate symptoms of trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats [ 58 ]. Glycoside-rutin is proven to be very effective in treating IBD in DSS-induced experimental animals by regulating body weight and oxidative stress in terms of MPO and by reducing GSH (glutathione), malondialdehyde, and serum nitrous oxide (NO) concentrations [ 59 , 60 ]. Lin et al. noticed that dietary quercetin alleviates the effects of Citrobacter redentium -induced colitis in mice by inhibiting the pro-inflammatory cytokines (IL-6, IL-17, and TNF-α) and by promoting anti-inflammatory cytokine IL-10 in colon tissues as well as by modifying gut microbiota [ 61 ]. Moreover, quercetin has bactericidal capacity and anti-inflammatory activity in macrophages via Heme Oxygenase-1 (HO-1)-mediated pathways and thus is useful in IBD therapy by restoring hemostasis and by balancing the enteric commensal microflora [ 62 ]. Another study by Dicarlo et al. revealed the benefit of quercetin in suppressing the inflammatory pathway in an in vitro intestinal organoid model. The authors stated that such a model is a novel tool to investigate epithelial response in models of chronic inflammation. Intestinal organoids of the Winnie model (ulcerative colitis mice model) treated with quercetin showed suppression of inflammation through downregulation of TNF-α and lipocalin-2 and upregulation of the heme oxygenase 1 and ferroportin 1 against the untreated model, thus mimicking the characteristics of the in vivo model in evaluating the gut epithelial inflammatory responses [ 63 ].

In multicellular organisms, in addition to secretion of proteins, release of exosomes is another method of intercellular communication. Exosomes can haul proteins, lipids, mRNAs, and miRNAs from one cell to another and thus are involved in cell–cell communication. Recent research has shown that nanosized particles of plant origin can act as exosomes [ 70 ] and are involved in cell–cell communication, thereby regulating innate immunity [ 71 ]. The gastrointestinal tract is continuously in contact with digested edible plant derived exosome-like nanosized particles. However, the role of these plant-derived exosome-like nanoparticles as an interspecies messenger has never been addressed. Ju et al. identified exosome-like nanoparticles from grapes known as grape exosome-like nanoparticles (GELNPs) and demonstrated that GELNPs are 380.5 ± 37.47 nm in size and contain many miRNAs and proteins [ 72 ]. However, mammalian exosomes contain even greater numbers of miRNAs and proteins. The lipid profile of mammalian exosomes is also much different than that of GELNPs. GELNPs are composed of 2% galactolipids (typical plant lipids) and 98% phospholipids (50% of which is phosphatidic acid (PA)). Interaction of PA with the mammalian target of rapamycin (mTOR) is reported to initiate cell growth and proliferation [ 73 ]. Ju et al. also demonstrated that GELNPs can penetrate the intestinal mucus barrier and are involved in protection of colon against DSS-induced colitis in mice. In addition, lipids from GELNPs and Liposome-like nanoparticles (LLNs) are required for targeting nanoparticles towards intestinal stem cells. The signaling pathway mediated by β-catenin is blocked in GELNP recipient cells, which in turn modulates renewal and remodeling of intestinal tissues in response to pathological triggers [ 72 ].

Honey bee propolis is found to contain a phenolic compound Caffeic acid phenethyl ester (CAPE) that is reported to possess anti-inflammatory activity mainly by inhibiting NF-κB [ 76 ]. In DSS-induced experimental mouse model of colitis, CAPE is effective in suppressing pro-inflammatory cytokines and MPO activity, which improves epithelial barrier function [ 77 ].

4.1. Natural Peptides Tuftsin, a natural tetrapeptide (Thr-Lys-Pro-Arg) with immunomodulating and anti-inflammatory activities, is a part of immunoglobulin G (IgG) heavy chain generated by enzymatic cleavage in the spleen. Several analogs of tuftsin are affective in colitis treatment in animal models [ 83 ]. Tuftsin-phosphocholine (TPC) is responsible for maintaining normal gut microbiota in collagen-induced arthritis model [ 84 ]. TPC immunomodulates by stimulating colon anti-inflammatory cytokines (IL-10) and by downregulating pro-inflammatory cytokines (IL-1β, IL-17, and TNF-α) in collagen-induced arthritis model and DSS-induced colitis model [ 85 , 86 ]. Lysine-proline-valine (KPV), a naturally occurring tripeptide, attenuates inflammatory responses of colonic cells. KPV entrapped into hyaluronic acid (HA)-functionalized polymeric nanoparticles and encapsulated in a hydrogel (chitosan/alginate) prevents mucosa damage and downregulates TNF-α [ 87 ].

Generally, natural polysaccharides such as cellulose, dextran, pectin, and chitosan have been used as drug delivery systems for colon because of their ease to work, nontoxic nature, and approval by the USFDA. The use of natural polysaccharides as delivery system also prevents premature release of drug in the small intestine and stomach and favors selective degradation in colon. Modified apple polysaccharide inhibits colitis by decreasing the level of IL-22 and by increasing the expression of IL-22BP [ 93 ]. Additionally, polysaccharide-rich extracts of Eucheuma cottonii and Acmella oleracea modulate inflammatory response and suppress colonic damage in DSS-induced colitis [ 94 , 95 ]. Nie et al. used non-starch polysaccharides to treat IBD in both in vivo and in vitro models [ 96 ]. The mechanisms involved in amelioration of the signs and symptoms and in suppression of reoccurrence rates are anti-inflammation, immune stimulation, and gut-microbiota modulation.

Insect-derived bioactive compounds such as Bombyx mori haemocyte, Gryllus bimaculatus extract, Tetragonula carbonaria extract, Nasonia vitripennis venom, glycosaminoglycan, cecropin A, silk fibroin, SibaCec, Cecropin-TY1, N -acetyldopamine dimers, papiliocin, and Melittin have been reported to be active against inflammatory diseases. These compounds inhibit the secretion of cytokines and downregulate expression of signaling molecules involved in the pathophysiology of inflammation [ 101 , 102 ]. They inhibit activation of MAPK pathways and NF-κB, thereby acting against inflammation under both in vitro and in vivo conditions [ 103 ]. However, the knowledge behind their regulatory activities is not sufficient. Further studies related to their ability to cross the blood–brain barrier along with toxicological and pharmacodynamical properties will direct them to be used as potential drugs for future clinical trials in colitis and CAC [ 103 ].

4.7.1. CD98 CD98 is chosen in the studies conducted by Xiao et al. as the therapeutic targeting molecule [ 105 ]. Earlier studies reported upregulated expression of CD98 in mice colonic tissues suffering from UC [ 106 ]; in intestinal B cells, CD4 + T cells, and CD8 + T cells from IBD patients [ 107 ]; and in colonic biopsies from CD patients [ 108 ]. CD98 is reported to be redistributed to the apical surface of intestinal epithelium during inflammation, resulting in loss of epithelial barrier [ 109 , 110 ]. Also reported are the findings that increased expression of CD98 has an important role to play in the progression and development of IBD [ 111 ]. Administration of chitosan/alginate hydrogel nanoparticles linked with CD98 antibody and loaded with siRNA CD98 significantly reduces expression of CD98 in epithelial cells and macrophages. These NPs decrease severity of colitis in mice, suggesting future use of chitosan/alginate nanoparticle for IBD therapy [ 105 ]. Zhang et al. used natural nanoparticles known as ginger-derived lipid vehicles (GDLVs) produced from lipids found in ginger for the delivery of siRNAs. Oral administration of GDLVs loaded with a very low dose of siRNA-CD98 targets them specifically and effectively to colon tissues, which results in decreasing expression of CD98. Moreover, GDLVs are biocompatible and production on a large scale can be achieved easily, thus making them a very safe and cost-effective delivery system for siRNA in UC treatment [ 7 ].

Aouadi et al. revealed that mitogen-activated protein kinase 4 (MAPK4) plays an important role in mediating the production of inflammatory cytokines in macrophages. Gene silencing by administration of MAPK4 siRNA encapsulated in β1,3- d -glucan shells in LPS-induced mouse model suppresses the production of TNF-α and IL-1β, thereby protecting the animals from systemic inflammation induced by LPS [ 120 ].

Increased expression of Cyclin D1 (CyD1) is reported in epithelial and immune cells during IBD [ 124 ]. siRNA CyD1 linked to targeted stabilized NPs (tsNPs) directed against leukocytes inhibits CyD1 mRNA and inflammatory responses in DSS-induced mouse model of colitis. CyD1 silencing affects induction of inflammatory cytokines TNF-α and IL-12 in TH1 cells; however, no effect on IL-10 cytokine in TH2 cells is observed [ 125 ].

Inhibition of Interferon Regulatory Factor-8 (IRF-8), an immunomodulatory protein, has therapeutic potential in IBD. siRNA-loaded lipid-based nanoparticles (siLNPs) block IRF-8 mRNA and significantly regulate differentiation, polarization, and activation of mononuclear phagocytic cells. To silence IRF8 in vivo, siLNPs are coated with anti-Ly6C antibodies to achieve selectivity for inflammatory leukocytes. The immunomodulatory effect is observed with a significant decrease in pro-inflammatory cytokines [ 130 ].

5.1. Novel Unproven Anti-Inflammatory Nano-Formulations and Patents in Colitis More recently, a new terminology “nutraceutical” has emerged, which is described as “A food or parts of food that provide medical or health benefits, including the prevention and/or treatment of disease”. This term originated from two words: “nutrition” and “pharmaceutical”. A recent publication summarized various novel nano-formulations and several patents, which include curcumin, resveratrol, quercetin, epigallocattchin-3-gallate, β-carotene, fish oil, and gallic acid. These novel formulations, which have anti-inflammatory and antioxidant activities, are fully characterized by efficacy and toxicity in laboratory models [ 134 ]. However, these formulations have never been investigated in colitis research. Future investigations using these novel nano-formulations and patents are warranted.

Phytochemicals can act on various targets of pathogenesis and inflammation. Owing to immense pharmacological activities, at present, the key limitations and the barriers observed with natural molecules in IBD therapy are that the therapeutic activity is compromised before reaching to the inflamed colon. To overcome these challenges, various synthetic polymer-based nanoparticles are being utilized that are toxic to biological systems. To minimize the risk of side effects associated with synthetic polymer-based nanocarriers, future studies should be aimed at delivering and/or targeting these molecules by encapsulating them into natural protein and/or polysaccharide-based delivery systems. Natural biopolymer-based nanoparticulate formulations can protect active phytochemical from gastrointestinal instability, can maximize colon targeting, and can get degraded by colonic microflora without altering their composition; hence, such a biodegradable carrier system can be an ideal formulation for colon targeting in the future. In this regard, a natural-lipid nanoparticle drug delivery system is created to encapsulate and release 6-shogaol (biologically active compound of ginger) to the colon [ 140 ]. Ulva lactuca polysaccharide-selenium nanoparticles offer therapeutic potential for reducing the symptoms of acute colitis through its anti-inflammatory actions [ 141 ]. Nanoparticles isolated from broccoli extracts provide protection against colitis by activation of adenosine monophosphate-activated protein kinase in dendritic cells [ 142 ]. Budesonide-entrapped krill oil-incorporated liposomes suppressed TNF-α in colitis models. This natural delivery platform has great potential as a nanovehicle for oral delivery of IBD drugs [ 143 ]. An exhaustive discussion on the different categories of nanocarriers is beyond the scope of this article.

IBD therapy with biologics is favored for mucosal healing and for maintaining clinical remission. Invariably, biologics lead to adverse side effects, a key limitation observed in treatment of IBD. This review demonstrates that, as an alternate, natural products when entrapped in nanoparticles hold a huge potential in the prophylaxis, management, and treatment of IBD. In future, new untested patents and novel nano-formulations should be investigated. Further, combinatorial mixtures of phytochemicals or phytochemicals with macromolecules may have additive effects as compared to single agent treatment strategy. More importantly, though limited plant-based nano-formulations have been tested in humans, new clinical trials are urgently needed to analyse nontoxic natural nanoparticles targeted to colon for delivery of natural products.