Diosmin is known to possess poor water solubility and low bioavailability. But in this study, Chitosan and Poly(d,llactide-co-glycolide) (PLGA) nanoparticles were successfully used to improve diosmin’s solubility and bioavailability. Coating of PLGA nanoparticles with chitosan demonstrated that this biopolymer matrix can ameliorate their gastric retention and cellular uptake and have potential anti-ulcer activity.

Diosmin, a bioflavonoid complex, is a plant chemical found mainly in citrus fruits used for treating various disorders of blood vessels including hemorrhoids, varicose veins, poor circulation in the legs, blood clots, and bleeding (hemorrhage) in the eye or gums. It is also used to treat swelling of the arms (lymphedema) following breast cancer surgery, to protect against liver toxicity, and a type of pain called radicular pain. It is often taken in combination with hesperidin, another plant chemical. But under normal conditions it demonstrates poor water solubility and low bioavailability.

Walaa Ebrahim Abd El Hady et al. (Department of Pharmaceutics, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt) formulated a biopolymer matrix using Chitosan and PLGA nanoparticles with diosmin.  The matrix was successfully used to improve the solubility and bioavailability of diosmin in vitro and in vivo. The result demonstrated potentiation of gastric retention, cellular uptake, and anti-ulcer activity of the chitosan-PLGA nanoparticles-diosmin matrix.

Methodology:   PLGA nanoparticles of diosmin were prepared using different drug and polymer amounts. Nanoparticles were selected based on entrapment efficiency% (EE%) and particle size measurements to be coated with chitosan. The selected nanoparticles either uncoated or coated were evaluated regarding morphology, ζ-potential, solid-state characterization, in vitro release, storage stability, and mucoadhesion. The anti-ulcer activity (AA) against ethanolinduced ulcer in rats was assessed through macroscopical evaluation, histopathological examination, immunohistochemical localization of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and transmission electron microscopic examination of gastric tissues compared to free diosmin (100 mg/kg) and positive control. Results: Based on EE% and particle size measurements, the selected nanoparticles, either uncoated or coated with 0.1% w/v chitosan, were based on 1:15 drug-PLGA weight ratio and 20 mg diosmin employing methylene chloride as an organic phase. Examination by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) revealed nanoscopic spherical particles. Drug encapsulation within the selected nanoparticles was suggested by Fourier transforminfrared, differential scanning calorimetry (DSC) and X-ray diffractometry results. Chitosan-coated nanoparticles were more stable against size enlargement probably due to the higher ζ-potential. Only coated nanoparticles showed gastric retention as revealed by SEM examination of stomach and duodenum. The superior AA of coated nanoparticles was confirmed by significant reduction in average mucosal damage, the majority of histopathological changes and NF-κB expression in gastric tissue when compared to positive control, diosmin and uncoated nanoparticles as well as insignificant difference relative to normal control. Coated nanoparticles preserved the normal ultrastructure of the gastric mucosa as revealed by TEM examination.

Conclusion: The optimized chitosan-coated PLGA nanoparticles can be represented as a potential oral drug delivery system of diosmin.

There are some endogenous aggressive factors that can cause gastric ulcer such as overproduction of hydrochloric acid and pepsin, leukotrienes, refluxed bile, and stress oxygen species.¹ The defensive endogenous mechanisms against the damage of the gastric mucosa include the surface mucus, the regulation of gastric mucosal blood flow, bicarbonate, antioxidants, surface active phospholipids, the acceleration of epithelial regeneration, and the preservation of epithelial hemostasis. Excessive gastric acid secretion was considered to be the major reason of the gastric ulcer for decades; thus, anti-cholinergic drugs, antacids, histamine H2-receptor antagonists, and proton pump inhibitors were the main therapy regimens. Nevertheless, the limited efficacy and the adverse effects of most of the current therapies limited their application.² Therefore, there is a great necessity for safe and effective anti-ulcer agents. Diosmin (3,5,7-trihydroxy-4-methoxyflavone 7-rutinoside) is a natural flavonoid glycoside that can be obtained from different plant sources or derived from the flavonoid hesperidin.³ Diosmin has been widely used as a vascular protector for the treatment of hemorrhoids and venous leg ulcers.⁴ It also exhibited anti-inflammatory, free-radical scavenging,⁵ and anti-ulcer activities.⁶ This drug showed gastro-protection against ethanol-induced gastric ulcer in rats by inhibiting the mitochondrial damage and MMP-9 upregulation.⁷ However, diosmin is poorly soluble, thus low dissolution rate and impaired gastrointestinal absorption were observed.⁸ Following oral administration, diosmin is quickly hydrolyzed by enzymes produced by intestinal microflora into its aglycone diosmetin that is absorbed through the intestinal wall to be then enzymatically esterified to its metabolite of 3,7-O-diglucuronide.⁸ Consequently, a large oral dose (500 mg twice daily) is usually required.⁹ However, the amount of diosmetin detected in plasma after a single oral administration of diosmin is low and highly inconsistent. The variability of absorption could be reduced by adherence to the gastrointestinal wall to allow a rapid replenishment of the absorbed drug. Small particles tend to adhere well to the mucus layer and then penetrate this layer to bind to the underlying epithelium.¹⁰ It has been reported that oral administration of diosmin in micronized form can ameliorate its plasma concentrations due to the larger surface area and subsequent improved intestinal absorption.¹¹

Different strategies have been attempted to improve diosmin solubility, such as complexation with β-cyclodextrin,⁶ as well as particle size reduction by formulation into nanosuspension with hydroxypropyl methylcellulose⁹ and electrospinning to nanofibers.⁵  Poly(d,l-lactide-co-glycolide) (PLGA) is a synthetic copolymer that has been approved by FDA for various medical and pharmaceutical applications including drug delivery.¹² PLGA is biocompatible and biodegradable since it is hydrolyzed into non-toxic oligomer and monomer of lactic and glycolic acids that are hydrophilic and finally eliminated as carbon dioxide and water.^13,14 In addition, the degradation rate of this copolymer can be modified by controlling the molar ratios of lactic and glycolic acids in the polymer chain and the degree of crystallinity, as well as the molecular weight and stereochemistry of the polyester.¹⁵ PLGA nanoparticles can increase the drug penetration across the different biological barriers, such as the blood–brain barrier, gastrointestinal mucosa, nasal mucosa, and ocular tissue.¹⁶ Therefore, this copolymer has been extensively used as nanoparticulate drug delivery system to enhance the biological activity, water solubility, and bioavailability of drugs.¹³ PLGA produces negatively charged, smooth surfaced, and spherical particles that are relatively resistant to salt- and pHinduced instability, and can slowly release the entrapped drugs by polymer hydrolysis. Yet, unsuccessful results have been observed in some cases possibly due to the lack of mucoadhesiveness and immune-stimulating factors.¹⁷  Also, the negative surface charge of PLGA nanoparticles can hinder the interaction with the negatively charged plasmids and hence limit their intracellular uptake and the bioavailability of the loaded drugs.¹⁸ Chitosan is a naturally occurring linear amino polysaccharide (poly 1, 4-day-glucoamine) which can be obtained from crustacea shells, insects cuticles, and cell walls of some fungi.¹⁹ Chitosan is an interesting biomaterial for entrapment of bioactive materials in nanoparticulate delivery systems due to its water solubility under acidic conditions, biocompatibility, non-toxicity as well as its mucoadhesive and permeability-enhancing properties.^18–20 Modification of PLGA nanoparticles surface with a mucoadhesive polymer, such as chitosan, can offer several advantages. Among these, increased stability of macromolecules such as proteins, providing a positive surface charge that can promote cellular adhesion and delivery system retention at the target site, as well as conjugating targeting ligands to chitosan free amine groups.21 Mucoadhesive delivery systems increase the residence time of dosage forms at the delivery site which may lead to improved bioavailability, lower drug dose, less dosing frequency, and minimal side effects.²² The successful use of chitosan-based gastrointestinal mucoadhesive delivery systems has been reported in some studies.^18,23 Longer residence time in the stomach is advantageous in the treatment of gastric ulcer.²⁴

Therefore in this study, it was worth to prepare, characterize, and optimize polymeric nanoparticulate delivery systems of diosmin with the biodegradable and biocompatible PLGA. Coating of the selected PLGA nanoparticles with chitosan was attempted and its effects on the gastric retention of diosmin were assessed. Additionally, the effects of the selected PLGA nanoparticles either uncoated or coated on cytoprotective activity of diosmin against ethanol-induced ulcer in rats were investigated through macroscopical, histopathological, and transmission electron microscope examination of gastric tissues as well as immunohistochemical localization of gastric nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB).

Characterization of diosmin nanoparticles Entrapment efficiency% (EE%) The results indicated that drug:polymer weight ratio, loaded drug amount and the organic phase nature affected EE% of uncoated diosmin-PLGA nanoparticles. At the same drug-loaded amount (10 or 20 mg), the increase in the polymer content from 1:10 to 1:15 drug:polymer weight ratio resulted in a rise in EE%. This behavior was more pronounced on the use of methylene chloride rather than ethyl acetate as organic phase. The increase in the polymer concentration could cause an elevation of the organic phase viscosity providing more resistance against the drug diffusion from the organic phase to the aqueous phase as well as the expected faster polymer precipitation might allow less time for drug molecules to diffuse out of nanoparticles.³⁸ Similarly, as the initial drug concentration in the organic phase especially methylene chloride increases from 10 mg to 20 mg keeping the drug:polymer weight ratio constant either 1:10, 1:15 or 1:20, the drug entrapment increased. The increase in the concentration of either drug or polymer could result in more drug–polymer interaction promoting the drug encapsulation.³⁸ Surprisingly, a further increase in the concentration of either the polymer to 1:20 compared to 1:15 or diosmin (30 mg) relative to the corresponding based on 20 mg caused a decrease in EE%. These results may be attributed to reaching the limit of drug miscibility in the polymer beyond which no increase in the drug entrapment occurs possibly due to the attraction of free drug molecules within the polymer matrix toward those in the aqueous phase.³⁹ Thus, it can be said that the limit of drug miscibility in the polymer was attained at drug:polymer weight ratio of 1:15 and loaded drug amount of 20 mg.

Use of methylene chloride as organic solvent imparted higher EE% at the same drug:polymer weight ratio, particularly when the loaded drug amount was 20 mg, in comparison with ethyl acetate (F11–F13). The higher water miscibility of ethyl acetate (8.70%) than methylene chloride (1.32%) and the expected faster partitioning in the aqueous phase and polymer precipitation in ethyl acetate could diminish the drug incorporation into PLGA nanoparticles.40 At drug:polymer weight ratio of 20:300, the highest EE% was obtained, and hence diminished material loss, improved particle production, and lower manufacturing cost can be expected.⁴¹  Therefore, drug:polymer mg 20:300 was selected to be coated with chitosan at different concentrations (0.10%, 0.15%, and 0.30% w/v) and further investigated. Those coated with 0.10% w/v chitosan (F14) showed mean EE% of 67.40 ±2.90% that insignificantly changed on the increase in chitosan concentration to 0.15 w/v or 0.30% w/v since chitosan concentration was much smaller than PLGA, and hence it might have not affected the organic phase viscosity and drug diffusion to the aqueous phase. Thus, 0.10% w/v chitosan-coated nanoparticles containing 20:300 drug PLGA using methylene chloride was further examined compared to the corresponding uncoated PLGA nanoparticles and the free drug.

The selected uncoated nanoparticles consisted of 1:15 drug-PLGA weight ratio using 20 mg diosmin and methylene chloride as an organic phase because they showed the highest EE% (75.30±2.60%) and particle size with 0.10% w/v were further investigated in comparison with the selected uncoated PLGA nanoparticles. The nanoscopic size and spherical shape were confirmed using SEM and TEM. FT-IR, DSC, and XRD results clarified the absence of drug peaks in the recorded data of the medicated nanoparticles suggesting drug encapsulation within them. The selected uncoated nanoparticles possessed lower potential (−10.50±0.20 mV) than those coated with 0.10% w/v chitosan (+27.40±2.90 mV). This may explain the higher stability of coated nanoparticles against size enlargement on storage for 3 months. The optimized coated nanoparticles exhibited gastric retention as indicated by SEM examination. As well, these nanoparticles caused a significant decrease in mucosal damage, the majority of histopathological alterations and NF-κB expression in glandular and non-glandular portions of gastric tissue when compared to positive control, free diosmin and uncoated nanoparticles. The superiority of coated nanoparticles was revealed by the insignificant difference of the macroscopical damage, histopathological alterations, and NF-κB expression relative to normal control as well as the preservation of normal ultrastructure of the gastric mucosa as revealed by TEM. Therefore, the optimized chitosan-coated nanoparticles can be suggested as a promising oral drug delivery system of diosmin.

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Study Citation: Walaa Ebrahim Abd El Hady, Elham Abdelmonem Mohamed, Osama Abd El-Aazeem Soliman, Hassan Mohamed EL-Sabbagh., Department of Pharmaceutics, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt. “In vitro–in vivo evaluation of chitosan-PLGA nanoparticles for potentiated gastric retention and anti-ulcer activity of diosmin”., International Journal of Nanomedicine 2019:14 7191–7213

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