INTRODUCTION:

Due to advances in biotechnology and genetic engineering there is mass production of therapeutic macromolecules such as proteins, nucleic acids and polysaccharides. The oral route is the most convenient and comfortable means of administration of these hydrophilic macromolecules for patients. But the oral bioavailability¹ of these therapeutic agents is severely limited by their low stability in the gastrointestinal (GI) tract and their poor permeability across intestinal biological membranes. Hence, vehicles that can protect the loaded macromolecules from destruction in the GI tract and increase their intestinal absorption capacity are highly desired. Now CS -based nanoparticles are a promising vehicle for oral delivery of therapeutic macromolecules. Chitosan is a biological and cationic polysaccharide can be considered as a promising nanomaterial² with extensive medical applications. Chitosan is one of the most abundant biopolymers derived from natural chitin that commonly exists in the exoskeletons of arthropods, crustacean shells, insects, and fungal cell walls.

Structurally, CS is a representative polysaccharide with native amine groups that are positively charged. It possesses unique properties such as non toxicity, biocompatibility, biodegradability, bioactivity, and mucoadhesion. Chitosan can be degraded by internal enzymes, such as lysozymes and CSases, to acquire oligosaccharides and monosaccharides, which are subsequently absorbed by the body. Despite its unique physicochemical³ and biological properties, CS has not been extensively utilized in the clinic due to its low solubility and poor mechanical properties. Free amino and hydroxyl groups have been utilized to generate a wide range of CS derivatives with improved solubility based on its high affinity with functional proteins and the capability to self-assemble. Thus, CS has been widely employed in various biomedical and pharmaceutical processes, such as drug/gene/vaccine delivery, tissue engineering, wound healing, and manufacture of cosmetic products.

Nanomaterials successfully developed from CS having potential applications to targeted drug delivery based on the following features: (1) biocompatibility and ability to serve as reaction sites with other bioactive compounds, (2) protecting unstable drug molecules from strong gastric acids and blood flow responses, (3) ability to adhere to mucosal tissues to improve the absorption of specific drugs, (4) ease in combining with anionic biomacromolecules such as DNA by electrostatic action, and (5) colon-targeted administration. Some reviews about CS-based nanoparticle systems for disease treatment have been published and they talk about biological applications of CS. The present review comprehensively discusses recent advances in the development of the versatile nanomaterials derived from CS and its derivatives for well-controlled drug delivery.

Transport mechanisms of macromolecules across intestinal epithelium:

Intestinal epithelium is the major barrier for the absorption of macromolecules from the intestinal lumen to the systemic circulation. This epithelial cell layer consists of enterocytes, goblet cells and M cells. As the most abundant epithelial⁴ cells in the intestine, enterocytes are specialized cells responsible for transporting nutrients by active transport or passive diffusion.

Intestinal absorption of macromolecules occurs either through the transcellular or paracellular route. As an efficient absorption enhancer and drug carrier, CS increases both transcellular and paracellular transports of macromolecules across the intestinal epithelium.

Transcellular route:

Transcellular uptake of CS-based NPs occurs by transcytosis, a process by which carriers are taken up by enterocytes or M cells, mimicking the entry of pathogens. Transcellular transport can be enhanced by adapting the physicochemical properties of NPs, such as their particle size and mucoadhesivity. NPs under 100 nm are likely to be absorbed by the enterocytes, while particles larger than 500nm are likely to be taken up by the M cells of the Peyer's patches. CS is a well-known mucoadhesive polymer, whose mucoadhesive property is due to an electrostatic interaction between the positively charged CS and the negatively charged sialic-acid residues on the mucosal surface.

Paracellular route

Absorption via the paracellular route (i.e., uptake through the interstitial space between epithelial cells) is normally restricted by the relatively narrow width of the paracellular channels and the presence of Tight Junction (TJ)s. The TJs form a barrier that allows the absorption from the lumen of needed water and electrolytes, but prevents the passage of inflammatory and infectious agents into the systemic circulation⁵. They are composed of a complex combination of transmembrane integral proteins, including claudins (CLDNs), occludin and junctional adhesion molecules along with several intracellular plaque proteins and several regulatory proteins, which anchor the transmembrane proteins to the actin cytoskeleton. Transmembrane proteins, especially CLDNs, play a major role in forming the seal between adjacent cells. Plaque proteins are necessary to form a structural support for TJs, and regulatory proteins regulate signal transductions involving TJ permeability and cell differentiation.

Approaches in Generating CS Derivatives^6-10

CS derivatives mainly originate from the modification of hydroxyl and especially the free amino of CS skeleton.

1. General Modifications of CS to Improve Solubility:

CS only dissolves under acidic conditions and shows poor solubility⁵ under natural humoral or intracellular environments at a pH of 6.8 to 7.4, thereby significantly limiting its applications to drug delivery. Because of its poor solubility, CS cannot be modified by other bioactive compounds that are generally difficult to directly absorb and undergo normal blood circulation. Therefore, the primary purpose for modification of natural CS is to improve its solubility.

2. CS-Based Modifications for Targeted Drug Delivery:

CS derivatives harboring functional molecules can enhance their ability to target specific organs due to their high affinities to the organ’s surface. The human body is a huge factory built with complicated materials and cross-linked signals between different types of organs and cells, and the considerable efforts have been devoted on studying the profound mystery in association with human signaling.

3. CS-Based Modifications to Improve Environmental Responsiveness:

The pH, cytokine, enzymes, ion concentration, and cell membrane pore size may vary in different tissues and organs to some extent. The pH of the pure gastric juice is 1–1.5, the pH of the physiological environment is about 7.4, and the tumor extracellular microenvironment is 6.5, pH of endosomes and lysosomes is about 5.0–6.2 and 4.0–5.0, respectively. Therefore, the pH distribution in the human body supplies an excellent triggering condition for drug delivery and release. Simultaneously, different types of enzymes and cytokine would increase or decrease in acute and chronic arthritis, and the temperature at the site of inflammation is slightly higher.

4. Other Modifications of CS:

Prodrug and fluorescent tracer are also applied to the design of CS-based delivery vehicle to generate improved drug delivery systems. Prodrugs are compounds obtained by chemical modification of biomolecules or drug delivery vehicles and may be inactive or less active in vitro and exhibit pharmacological effects by enzymes or acids in vivo to release the active drug.

Synthesis and characterization of CS derivatives:

Chemical modification of CS is feasible because it has reactive amino and hydroxyl groups that can be readily modified with a diverse array of ligands, functional groups and moieties. Various chemical modifications, including quaternization, thiolation, carboxylation, alkylation, acylation, PEGylation and graft copolymerization, have been performed to further improve the beneficial properties of CS.

1. Quaternized CS:

Quaternization of CS can conserve its positive charge at a neutral pH value, thus increasing its aqueous solubility significantly. Various quaternized CS derivatives, trimethyl CS (TMC), dimethylethyl CS (DMEC), diethylmethyl CS (DEMC) and triethyl CS (TEC) have been synthesized. TMC is a partially quaternized derivative of CS prepared by the reductive methylation of CS, in which methyl iodide and sodium iodide are inserted in an alkaline solution of N-methyl pyrrolidinone (NMP). TMC-based NPs have been extensively studied for its potential to increase the absorption for oral delivery of macromolecules, owing to the effectiveness of TMC in mucoadhesion as well as in mediating a TJ opening at distinct intestinal pH environments.

DMEC, DEMC and TEC with different substituted N-alkyl groups can be made based on TMC synthesis with some modifications. The TJ opening ability of quaternized CS derivatives depends on their degree of quaternization; a higher degree of quaternization (or higher charge density) implies a higher transport capability.

2. Thiolated CS:

Thiolated CSs are synthesized by covalently coupling with sulfhydryl bearing agents such as cysteine, thioglycolic acid and glutathione onto the backbone of CS; in addition, they can be prepared through either a ring opening reaction of 2-iminothiolane or a direct imidoester reaction of isopropyl-S-acetylthioacetimidate. These reactions lead to the formation of different thiolated CS derivatives, including CS-thioglycolic acid (CS-TGA), CS-cysteine (CS-Cys) , CS-glutathione, CS-4-thio butyl-amidine (CS-TBA) and CS-thioethylamidine (CS-TEA).

3. Carboxylated CS:

Carboxymethyl CS derivatives can be prepared by introducing the −CH2COOH groups onto 6-O and 2-N atoms on CS. Another carboxylated CS derivative, N-succinyl CS, can be obtained by introducing succinyl groups into the glucosamine units of CS via ring-opening reactions with succinic anhydride.

4. Amphiphilic CS:

Amphiphilic CS derivatives can be prepared by grafting hydrophobic compounds such as aliphatic acids (C6–C16) via N-acylation or bile acids/fatty acids through amidating reaction on CS. In an aqueous environment, polymeric amphiphiles can self-assemble into NPs due to the interaction between their hydrophobic and hydrophilic segments.

5. CS derivatives bearing chelating agents:

Chelating agents, including nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA), can inhibit these peptidases owing to their deprivation of essential divalent cations (e.g., Ca2+ and Zn2+) out of the enzyme structures.

6. PEGylated CS:

PEGylated CS (CS-PEG) can be synthesized by using PEG-succinimidyl succinate or activated esters of PEG carboxylic acids capable of reacting with the primary amine groups on CS to form stable amides. Nanocapsules coated by CS-PEG improve their stability in simulated GI fluids and reduce their cytotoxicity.

General physiological considerations for colonic drug delivery^11-17

Considerations should be made during formulation design to the residence time of the formulation in the GI tract, how the GI environment affects the delivery of the formulation and dissolution of the drug at the site of action, the intestinal fluid volume, and the propensity of the formulation or drug to be metabolized in the GI tract through enzymatic or microbial degradation. For instance, consideration of the formulation transit time through the GI tract is critical to ensuring delivery of the drug to the site of action. Small intestinal transit time is generally accepted as 4 hours, 16 with individual variability ranging from 2 to 6 hours. In contrast, colonic transit times can vary significantly, with ranges from 6 to 70 hours reported.

Additional confounders influencing GI transit time include gender, with females having significantly longer colonic transit times, and the time of dosing with respect to an individual's bowel movements.

Differences in pH along the GI tract have been exploited for the purposes of delayed release therapies. The highly acid stomach environment rises rapidly to pH 6 in the duodenum and increases along the small intestine to pH 7.4 at the terminal ileum. Cecal pH drops below pH 6 and again rises in the colon reaching pH 6.7 at the rectum. However, pH ranges can exhibit variability between individuals, with factors such as water and food intake as well as microbial metabolism being major determinants. In addition to influencing pH, fluid: matter ratios may also affect the colonic delivery of drugs. Many acute GI infections will cause dysbiosis and drive increased intestinal fluid secretion, and may increase or decrease bowel motility, while more chronic conditions, such as Inflammatory bowel disease (IBD) can drastically and permanently alter the physiology of the GI tract.

Changes in the physiology of the GI tract during active IBD:

Oral delivery strategies in IBD are the change in the physiological condition of the GI tract associated with chronic inflammation. Mucosal inflammation in IBD causes pathophysiological changes, such as (i) a disrupted intestinal barrier due to the presence of mucosal surface alterations, crypt distortions and ulcers, (ii) increased mucus production and (iii) the infiltration of immune cells (e.g. neutrophils, macrophages, lymphocytes and dendritic cells). During relapse of IBD, patients suffering from severe mucosal inflammation may exhibit altered GI motility and diarrhea, which in turn affects intestinal volume, pH and mucosal integrity. The inflammatory response at the mucosa, along with severe diarrhea, will also disrupt the resident microbiome, which can alter microbial metabolism in the GI tract. Thus active inflammation significantly alters the physiology of the GI tract, which can affect the efficacy of conventional approaches to colon targeted drug delivery.

Transit time and microbial considerations:

Alterations to GI physiology in states of disease are often dynamic and inter-related, and therefore difficult to examine in isolation. For instance, orocecal transit time (OCTT), the time taken for the meal to reach the cecum, has been shown to be delayed. However, significantly faster OCTTs have been observed in IBD patients with the dysbiotic condition—small intestinal bacterial overgrowth (SIBO). These observations have been confirmed experimentally in humanized mice, following dietary manipulation of the gut microflora. Changes in the composition of the microbiome (dysbiosis) are common in GI disease, with alterations in physiology, inflammatory state, or as a result of treatment regimens. While it is generally accepted that the bacterial load is relatively static in IBD, the diversity of the microbiome is reduced with increases in major species such as Bacteroides, Eubacteria and Peptostreptococcus acting to the detriment of other populations.

Changes in colonic pH:

There is little evidence to suggest major alterations to small intestinal pH in IBD patients, however colonic pH is significantly lower in both UC and CD patients. In the colon, intestinal pH is influenced by microbial fermentation processes, bile acid metabolism of fatty acids, bicarbonate and lactate secretions, and intestinal volume and transit times. As all of these factors may be disrupted during active IBD, changes in luminal pH in the colon are not surprising. While normal colonic pH ranges from 6.8 in the proximal colon and rises to 7.2 in the distal colon, this can significantly vary in active UC patients from pH 5.5 to as low as 2.3. Similarly, reported colonic Ph values for CD patients are approximately 5.3, irrespective of disease activity. These pH changes are likely to affect the composition of the colonic microbiome and thus colonic transit times, which can influence the release of drug from formulations requiring bacterial fermentation or enzymatic activity. Likewise, pH changes can affect the release of compounds from pH-dependant release coatings.

Intestinal volume:

The composition of the intestinal biomass is altered in disease and is directly related to changes in microbial metabolism, intestinal transit time and luminal pH. In particular, increased fluid secretion and decreased reabsorption can dilute the digestive enzymes that control intestinal transit to allow nutrient absorption. This in turn may influence the intestinal microbiome, which can alter carbohydrate and polysaccharide digestion as well as contribute to changes in intestinal transit times. These changes in intestinal fluid volumes may alter the way conventional formulations are processed in the GI tract and the subsequent local delivery of drugs to the colon.

Mucosal integrity:

The epithelial barrier selectively regulates transport from the lumen to the underlying tissue compartments, restricting transport of smaller molecules across the epithelium, while virtually abolishing macromolecule transport. This selectivity is determined by apical transmembrane protein complexes known as tight junctions (TJ). These multi-protein complexes interact directly with underlying epithelium actomycin rings, influencing physiological and pathophysiological stimuli, such as ion transport, luminal glucose transport, water secretion and the transport of cytokines and leukocytes. While these properties make TJ an attractive pharmacological target for enhancing drug absorption, dysfunctional regulation of TJ complex formation is associated with a loss of epithelial integrity in intestinal inflammatory diseases, such as IBD. Active inflammation not only alters intestinal mucosal integrity, but also significantly alters mucosal metabolism as the tissue attempts to limit further damage and repair.

Intestinal resection in IBD patients:

Resection of bowel tissue is common among IBD sufferers, with over 70% of IBD patients undergoing at least one surgery in their lifetime. Removal of bowel tissue results in a shortening of the intestine and reduced transit distance through the GI tract, which potentially affects the way conventional oral formulations are processed. Beyond this, resection profoundly changes the physiology of the intestinal tract by altering pH, nutrient absorption, digestion and transit. In particular, resection of the terminal ileum alters water absorption and dilutes residual bile acids in the colon, therefore reducing net colonic fatty acid concentrations. This may profoundly alter microbial metabolism of fatty acids by hydroxylation to produce ricinoleic acid analogues that can drive diarrhea.

Current oral nano-delivery system strategies for drug delivery to inflamed colon^18-25:

Improved oral drug delivery design has drastically improved the colonic bioavailability of drugs, that is, these formulations are effective at reaching and releasing drug specifically in the colon. However, in order for a drug to have therapeutic efficacy it must be localized to the site of action within the colon. Nano-delivery systems have been designed to passively or actively target the site of inflammation. Current strategies for inflamed colon are

Size-dependent nano-delivery systems:

Reducing the size of drug delivery carriers to the nanometer scale has been shown to improve colonic residence time in inflamed intestinal regions and provide additional benefits for IBD therapy. This reduction in size enables enhanced and selective delivery of active molecules into the colitis tissue by exerting an epithelial enhanced permeability and retention (eEPR) effect, and allows the preferential uptake of the nano-sized particles by immune cells that are highly increased in number at the inflamed regions. By reducing the diameter of the particles, it is also possible to avoid rapid carrier elimination by diarrhea, which is a common symptom in IBD. Nanodelivery systems avoid rapid carrier elimination by being readily taken up into inflamed tissue and cells. Nanoparticles in the GI tract generally undergo cellular internalization by paracellular transport or endocytosis into epithelial cells in the GI tract. In IBD, specialized differentiated epithelial cells called M cells are involved in the predominant uptake of nanoparticles through transcytosis. Translocation of nanoparticles can also occur by persorption through gaps or holes at the villous tips. This accumulation is particle size dependent with an increasing effect for smaller particle diameters.

Surface charge-dependent nano-delivery systems:

Relatively little is known about how physicochemical parameters of drug carriers, other than particle size, influences adhesion to inflamed intestinal tissue. In particular, conflicting results have been reported on the influence of surface charge to colonic targeting, with results predominantly based on ex vivo tissue binding studies or in vivo studies following rectal administration. Modifying the surface charge of nano-delivery systems can influence the electrostatic interaction the nanocarriers have with components in the GI tract and theoretically should confer selectivity to diseased tissue. It should be noted however, that there is a potential for electrostatic interactions and subsequent binding of these nanoparticles with other chargemodifying substances during GI transit (e.g. bile acids and soluble mucins).

Positively charged nano-delivery systems:

Cationic nano-delivery systems adhere to the mucosal surface within inflamed tissue due to the interaction between the positively charged nanocarrier and the negatively charged intestinal mucosa. Colonic mucins carry a negative charge since their carbohydrates are substituted with numerous sulfate and sialic acid residues. Adhesion to the mucosa can be an advantage for GI tract targeting as it promotes better contact with the mucosal surface for cellular uptake and drug release. It can also reduce the clearance of nanocarriers when intestinal motility is increased, which is common in IBD.

Negatively charged nano-delivery systems:

Anionic nano-delivery systems were designed to preferentially adhere to inflamed tissue via electrostatic interaction with the higher concentration of positively charged proteins in inflamed regions. In particular, high amounts of eosinophil cationic protein and transferrin have been observed in inflamed colon sections of IBD patients.

pH-dependent nano-delivery systems:

pH-dependent drug delivery is to coat them with pH-sensitive biocompatible polymers. In addition to triggering release at specific pH range, the enteric-coating protects the incorporated active agents against the harsh GI tract environment (e.g. gastric juice, bile acid and microbial degradation), and creates an extended and delayed drug release profile to specific GI tract regions to enhance therapeutic efficiency. The most commonly used pH-dependent coating polymers for oral delivery are methacrylic acid copolymers (Eudragit®). Eudragit L100 and Eudragit S100, which dissolve at pH 6 and 7 respectively, are commonly used in combination in various ratios to manipulate drug release within the pH 6 to 7 range. Eudragit FS 30D is one of the more recently developed polymers and dissolves at pH above 6.5.

Biodegradable nano-delivery systems in the colon:

Colon-targeted nano-delivery system is the nanoparticle-in-microparticle oral delivery system (NiMOS). NiMOS are designed for oral administration of plasmid and siRNA by encapsulating them in type B gelatin nanoparticles, which are further entrapped in poly (epsiloncaprolactone) (PCL) microspheres. PCL is a synthetic hydrophobic polyester that is resistant to degradation by acid, therefore protecting nanoparticles during transit through the stomach. In addition, the coated microparticles are able to inhibit protein/enzyme adsorption, thereby avoiding the harsh environment of the GI tract. Release of the payload carrying nanoparticles occurs over time at inflamed sites in the intestine, via controlled degradation of the outer PCL layer by action of lipases abundantly present at this location, after which they can be endocytosed by enterocytes or other cells at these sites.

Redox nano-delivery systems:

This pharmaceutical strategy for targeted drug delivery to diseased colonic tissue takes advantage of the abnormally high levels of reactive oxygen species (ROS) produced at the site of intestinal inflammation. For example, biopsies taken from patients suffering from ulcerative colitis have a 10- to 100-fold increase in mucosal ROS concentrations, which are confined to sites of disease and correlate with disease progression. The unusually high concentrations of ROS localized to sites of intestinal inflammation are generated by activated phagocytes.

Active targeting-dependent nano-delivery systems:

This approach has been used to exploit disease-induced changes in the expression of receptors, adhesion molecules and proteins on the cellular surface of tissues affected by disease. The vast majority of research in active targeting-based nano-delivery systems has been studied using the parenteral route of administration to target a multitude of conditions, such as cancers, infections and sites of inflammation. Monoclonal antibodies and peptides are commonly used as targeting moieties, as they have been shown to have high specificity in targeting and potential mucopenetrative properties.

CONCLUSION:

Although CS-based nanoparticles are highly promising for use as carriers for oral delivery of therapeutic proteins, nucleic acids and polysaccharides, no clinical trials have been conducted. Additionally, toxicological issues of these newly emerging nanoparticle systems remain a major concern. Although CS alone is considered safe for oral administration, its properties may change completely after chemical modification. The toxicity of each derivative should be evaluated individually, both in the free form and nanoparticle form. Further pre-clinical studies are required to demonstrate the acceptable efficacy and safety of CS-based nanoparticle systems.

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Received on 10.12.2019 Modified on 16.03.2020

Research J. Pharm. and Tech. 2021; 14(7):3769-3774.

DOI: 10.52711/0974-360X.2021.00652