Iraqi J Pharm Sci, Vol.32( 1 ) 2023 Nanoparticle formulation design of piperine DOI: https://doi.org/10.31351/vol32iss1pp14-30 14 An update on Nanoparticle Formulation Design of Piperine to Improve its Oral bioavailability: A Review Nindya Kusumorini*, Akhmad Kharis Nugroho**,1, Suwijiyo Pramono*** and Ronny Martien** *Doctoral Program in Pharmaceutical Science, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta 55281 Indonesia **Department of Pharmaceutics, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta 55281 Indonesia ***Department of Pharmaceutical Biology, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta 55281Indonesia Abstract Piperine, a crystalline alkaloid compound isolated from Piper nigrum, Piper longum, and other types of pipers, has many excellent pharmacological advantages for preventing and treating some specific diseases, such as analgesic, anti-inflammatory, hepatoprotective, antimetastatic, antithyroid, immunomodulatory, antitumor, rheumatoid arthritis, osteoarthritis, Alzheimer's, and anticancer. However, its potential for clinical use through oral administration is hindered by water solubility and poor bioavailability. The low level of oral bioavailability is caused by low solubility in water and is photosensitive, susceptible to isomerization by UV light, which causes piperine concentration to decrease. A lot of nanoparticle formulation approaches have been applied to improve the poor oral bioavailability of piperine. Oral nanoparticle formulation strategies have been successfully implemented in increasing the solubility and bioavailability of piperine within the body, such as the formulation of nanoparticles, nanosuspensions, liposomes, complexation using polymers, and micro/nano-emulsions. This nanoparticle formulation approach has been successful in increasing the solubility, permeability, and bioavailability of piperine effectively. In addition, this nanoparticle formulated piperine can deliver piperine in a targeted manner and increase the efficacy of piperine treatments, such as for Alzheimer’s disease, epilepsy, rheumatoid arthritis, diabetes, breast cancer, colon cancer, and human brain cancer. Keywords: Piperine, Bioavailability, Solubility, Nano-formulation, Nanoparticle, Nanoemulsion Introduction Piperine is a crystalline, yellow, odorless, and pungent alkaloid compound isolated from Piper nigrum and Piper longum. Piperine is chemically known as (EE)-1-[5-(1,3-benzodioxol-5-yl)-1-oxo- 2,4-pentadienly]-piperidine, with the molecular formula of C17H19NO3, a molecular weight of 285.34 g/mol, and melting point at 128-130oC (1–3). The piperine structure consists of three subunits, namely methylenedioxyphenyl ring, side chain with conjugated double bonds, and basic piperidine moiety attached through carbonyl amide linkage to side chain (4). Piperine is unsTable in acidic, alkaline, oxidizing , and sunlight environments. Piperine can be degraded to trichostatin and cis- piperyline under acidic conditions (HCl 2 M) and to piperanine, piperettine, and piperyline under sunlight (5). Piperine contains four isomeric forms, namely trans-trans isomer (piperine), cis-trans isomer (isopiperine), cis-cis isomer (chavicine), and trans-cis isomer (isochavicine) (Fig. 1). Piperine contains four isomeric forms, namely trans-trans isomer (piperine), cis-trans isomer (isopiperine), cis-cis isomer (chavicine), and trans-cis isomer (isochavicine) (Fig. 1). The isomerization of piperine compounds increases along with the increasing light intensity and exposure. Transformation of piperine into chavicine isomers can be seen in long-term storage and spontaneously which causes a loss of spicy taste (6). The piperine ring influences the chemical stability and reactivity of piperine. This ring greatly influences steric-electronic properties, especially among the aromatic ring, carbonyl group, and alkene system (7,8). 1Corresponding author E-mail: a.k.nugroho@ugm.ac.id Received: 9/2 /2022 Accepted: 10/5 /2022 Iraqi Journal of Pharmaceutical Science https://doi.org/10.31351/vol32iss1pp14-30 Iraqi J Pharm Sci, Vol.32 (1) 2023 Nanoparticle formulation design of piperine 15 Figure 1. Structure of piperine and its isomers Piperine can also be isolated from white and black pepper (Piper nigrum) (9,10); root and fruit of Piper longum (11,12); Piper chaba (12,13); Piper guineense fruit (14); and roots, stems, leaves, and fruit of Piper sarmentosum (15). The alkaloid compound of piperine extracted from black and long pepper is commonly processed into traditional medicine in some Asian countries. Some medical products such as Ayurveda, Sidda, Unani, and Tibetan have used black pepper for treating cough, fever, sore throats, indigestion, insomnia, and heart disease (16–19). Through the development of science and technology, many pharmacological benefits of piperine compounds have been successfully revealed. Several studies show that piperine has therapeutic effects like antiallergic (20,21), anti- inflammatory (22–25), rheumatoid arthritis (26), osteoarthritis (27), anticonvulsants, anti-epileptic, antidepressants, neurodegenerative disorders, and Alzheimer's disease (24,28–32). Piperine can also inhibit cell proliferation by reducing cell variability, increasing reactive oxygen species (ROS), inducing cell shrinkage, fragmenting the DNA by blocking cell cycle and Caspase-3 activity (33), and effectively inhibiting cell proliferation and cell migration, inducing apoptotic processes in the HER-2 gene that expresses breast cancer cells, inducing MMP-9, and reducing Epithelial Growth Factor (EGF) (34). Piperine is safe to take orally at a 20 mg/kg BW (35). However, piperine is still not used clinically for disease prevention and treatment despite its broad pharmacological potential. Due to its low solubility and bioavailability, piperine is poorly absorbed in the body. However, many scientists have designed the right formulation to solve this issue. Recent strategies in oral nanoparticle formulated of piperine to increase their solubility and bioavailability are reviewed in detail. This review was conducted using several databases or computer-based electronic searches including Pubmed, Science Direct, and Google Scholar to access all journals related to the nano-formulation of piperine. All major research papers must be published between 2017 – March 2022 with the keywords “piperine”, “formulation”, “nanoparticle”, “polymeric”, “nanocarrier”, “phospholipid”, and “nanoemulsion”. Piperine physicochemistry Piperine is the main alkaloid found in white pepper, black pepper, and long pepper, having a weak base (pH 8.0 – 8.5) with a pKa of 12.22, and is poorly soluble in water with a solubility of 22.34 mg/L at 25oC (36). Piperine has a melting point at 128-130oC, is soluble in ethanol (1g/15mL), methanol, petroleum ether (1g/1.7mL), and chloroform (1g/1.7mL) (3). Piperine compounds are lipophilic with a log P value = 2.25 (36), which shows that they can be higher in octanol solvents than in water with a concentration ratio of 177.82:1. Piperine is slightly soluble in water and has a low dissolution level, thus it is less effective for oral use. Based on the results of piperine structure analysis, according to Lipinski’s and Veber’s rules, piperine compounds have good permeability in the membrane. The results of piperine permeability analysis are presented in the following section (37) (Table 1). Table 1. Piperine structural permeability analysis (37). Piperine structure analysis Parameter Results High permeability requirements log P 2.3 5 H-bonding donor 0 5 H-bonding acceptor 3 10 PSA (Polar Surface Area) 38.77 Ao 140 Ao Number of H- bonding donors & acceptors 3 12 Number of rotating bonds 2 10 Molecular weight 285.34 g/mol 500 Biopharmaceutics properties of piperine Based on the structural permeability analysis, piperine has good permeability across intestinal membranes. This is supported by some studies Khajuria et al. (38) and Suresh & Srinivasan (39) which showed that piperine can be easily absorbed across the intestinal membrane barrier through passive diffusion and no metabolic changes observed during absorption. Piperine is absorbed into the serosal fluid and intestinal tissue by 47-64% Iraqi J Pharm Sci, Vol.32 (1) 2023 Nanoparticle formulation design of piperine 16 and found to be transported predominantly to the duodenum (39–41). Piperine can be rapidly absorbed into the intestine probably because the molecule is non-polar and lipophilic so that it can easily cross and get through the intestinal barriers. Piperine does not experience biotransformation during intestinal absorption, 7- 12% of absorbed piperine is found in the serosal fluid (39,40). The results of the intestinal examination showed that the highest concentrations in the stomach and small intestine could be reached within 6 hours after application, where less than 0.15% was detected in serum, kidney, and spleen from 30 minutes to 24 hours. The piperine’s membrane permeability analysis conducted in a previous study used caco-2-monolayer model, where the results obtained a permeability coefficient of 5.41 x 10-5 cm/s and 4.78x10-5 cm/s for basolateral-to-apical and apical-to-basolateral (42). Piperine distribution begins after being absorbed into the intestine and then transported by serum albumin. Piperine is bound to subdomain-1B of human serum albumin which is an important factor in the transport of piperine in the blood (43). Based on a previous experiment conducted by Suresh et al. (43) in rats that had been given a dose of 170 mg/kg orally, the maximum concentration of piperine was reached after 6 hours of application with 10.8% found in the tissues, and most of the piperine was found in the duodenum (8.8%). However, the result is in contrast to a study conducted by Liu et al. (44), where the maximum concentration was obtained two hours after oral administration, with the highest concentration was in the liver. This difference may be due to the different forms of applied piperine, and other components may influence the distribution of piperine in the extraction process. Formulation design developed to increase piperine’s oral bioavailability The oral method is ideally applied for drug administration because it makes the patients easily consume the medicine. Oral administration can improve patients’ compliance, especially for long- term use compared to other consumption techniques (45). However, some latest chemical compounds being developed have low water solubility and low bioavailability, but high intra- and inter-subject variability (46), and one of which is piperine. Piperine has poor solubility, dissolution, and oral bioavailability (24% in rats) which are the main problems limiting its absorption (47). Piperine is classified into the Class II Biopharmaceutics Classification System (BCS) based on its poor solubility in aqueous media and good permeability across the intestinal membrane barrier. Many attempts and nanoparticle formulation designs have been developed to solve its low solubility and bioavailability. Details of recent developments in the oral nano-formulation of piperine are discussed in the following sections: Liposomes Liposomes are spherical vesicles with an aqueous core and vesicle sizes from 30 nm to several micrometers, consisting of one or more phospholipid bilayer membranes or lamellae in which the polar head leads to the inner and outer aqueous phase of the vesicle (48). Liposomes can deliver both hydrophilic and hydrophobic drugs. Liposomes contain a lipid bilayer similar to the cell membrane. Liposomes are made from a mixture of phospholipids, surfactants, and cholesterol. They function to increase the solubility of lipophilic and amphiphilic drugs, increase stability through encapsulation, reduce the toxicity of encapsulated drugs, are biodegradable, biocompatible, non-toxic, and non-immunogenic for systemic and non- systemic administration (49). Liposomes can increase the bioavailability of lipophilic drugs through oral administration by increasing cellular contacts and diffusion across epithelial and mucosal layers (50). The encapsulation of piperine into liposomes can increase the piperine’s dissolution and bioavailability levels. Dutta & Bhattacharjee (51) formulated piperine nanoliposomes for oral usage taken from soy phosphatidylcholine and Tween 80 in a ratio of 1:1.2 and generated 78.6% entrapment efficiency, 29.75 ± 0.84 nm vesicle size, and resulted in 70% of piperine release for 8 hours in pH 6.4 buffer. Imam et al. (52) formulated chitosan-coated piperine liposomes to improve the mucoadhesive properties of piperine liposomes. The liposomes were prepared through the thin-film dispersion method using a rotary evaporator and chitosan coating by electrostatic deposition. The chitosan functioned to increase the stability of piperine liposomes and protect them from chemical and enzymatic degradation in the digestive tract. The presence of chitosan on the liposomes’ surface showed an increase in stability and mucoadhesive properties by 2.8 times compared to those not coated with the chitosan. Microemulsion, self-emulsifying drug delivery system (SEDDS), and self-nano emulsifying drug delivery system (SNEDDS) Microemulsions are single-phase isotropic mixtures consisting of thermodynamically sTable and transparent oil, water, surfactant, and cosurfactant with an average droplet diameter of 10 to 140 nm (53,54). Microemulsions are good candidates for oral drug consumption which is poorly soluble in water. They function to increase drug solubility, absorption rate, and bioavailability and reduce inter- and intra-individual variability in drug pharmacokinetics (55,56). Microemulsions can improve the bioavailability of lipophilic drugs in several ways, such as protecting drugs from oxidation and enzymatic degradation and increasing Iraqi J Pharm Sci, Vol.32 (1) 2023 Nanoparticle formulation design of piperine 17 membrane permeability through lymphatic transport (57). Lymphatic transport is the most contributing element to drug absorption in the intestine because it can protect against first-pass metabolism (58). The findings of a study by Etman et al.(59) showed that piperine microemulsion for Alzheimer’s therapy via oral route has succeeded in supporting and accelerating its delivery to the brain and producing better therapeutic results than pure piperine. Piperine microemulsion has been successfully formulated with Capryol 90 oil, surfactants tween 80 and cremophor RH40, and cosurfactant transcutol, which finally generates a particle size of lower than 150 nm with a negative zeta potential -30.36 mV. The size of the SEDDS particles lower than 500 nm enables an increasing absorption level of piperine through the lymphatic route (60). Negatively charged nanoparticles can increase lymphatic uptake higher than positively and neutrally charged surfaces (61–63). A self-emulsifying drug delivery system (SEDDS) is an isotropic mixture of oils, surfactants, and cosurfactants that spontaneously forms an oil- in-water emulsion during contact with gastrointestinal tract (GIT) fluids with mild agitation. The SEDDS system was then modified into self-micro emulsifying drug delivery systems (SMEDDS) and self-nano emulsifying drug delivery systems (SNEDDS) (64). SEDDS, SMEDDS, and SNEDDS formulations have some specific benefits, such as increasing the drug solubility and bioavailability, increasing drug absorption rate through the lymphatic pathway, decreasing the effects of first-pass metabolism, and increasing physical and chemical stability in long-term storage (65). The SEDDS, SMEDDS, and SNEDDS formulations can improve the bioavailability of lipophilic drugs through some mechanisms. They include lipid droplets built within self-emulsifying dispersions that can directly facilitate drug absorption and protect drugs from chemical and enzymatic degradation. They are localized in aqueous environments, influence the changes in gastrointestinal membrane permeability, and increase the drug absorption level via lymphatic pathways (66,67). The lipids in the GIT (gastrointestinal tract) stimulate the excretion process of bile salts and endogenous bile lipids which also supports lipid emulsification to form micelles and improves drug solubility in the GIT (65).Surfactants can cause fluidization of intestinal membranes and opening of tight junctions which result in increased membrane permeability (68,69). The difference between SEDDS, SMEDDS, and SNEDDS systems can be viewed from oil and surfactant concentration. In this case, the SEDDS uses a surfactant with an HLB value lower than 12, while SMEDDS and SNEDDS use a surfactant with an HLB value higher than 12. Kusumorini et al. (70) have formulated SNEDDS piperine for oral administration using Miglyol 812N oil, surfactant Cremophor RH40, and cosurfactant PEG400 with a ratio of 1:5, 6:2.9 w/w and piperine content of 2% w/w, with nanoemulsion size of 33.35 ± 1.97 nm and zeta potential of -22.87 ± 3.31 mV. The size of the particles is less than 200 nm. Therefore, they can be easily absorbed by the intestinal epithelium and can prolong their living period which contributes to increased oral bioavailability. Then, negatively charged drug carriers can also easily penetrate and cross the mucus barrier in the digestive tract, so that the drug can be smoothly consumed and enters the blood circulatory system (71). Solid self-nano emulsifying drug delivery systems (S-SNEDDS) The solid self-nano emulsifying drug delivery systems (S-SNEDDS) are the changes of the SNEDDS from liquid to dry powder supported by an inert adsorbent through a solidification process which can then be used to produce solid preparations, such as Tablets, capsules, and pellets (72). S-SNEDDS has several advantages, like increasing solubility and bioavailability, increasing stability, being easier to use for the scale-up process, to obtaining content uniformity, increasing patients’ compliance, and providing more accurate dosage (24,73). S-SNEDDS, the solid form of liquid SNEDDS, can form nanoemulsions within the droplets sized lower than 300 nm in aqueous media. It has the same solubility and bioavailability enhancement mechanism as the liquid SNEDDS (74). Piperine has been successfully formulated in the form of S-SNEDDS using 9.39% Glyceryl Monolinoleic oil (GML), 17.38% Poloxamer 188 surfactants, 9.39% Transcutol cosurfactant HP, 56.33% adsorbent Avicel PH-101, and 7.51% piperine using adsorption technique (75). S-SNEDDS formulation results in a globule size of 73.56 ± 3.54 nm, a zeta potential of -28.12±2.54 mV, and bioavailability of 4.92 times higher than that of pure piperine dispersion. Meanwhile, S-SNEDDS piperine results in increased dissolution, bioavailability, hypertension effect, and better antioxidant and antibacterial activities than pure piperine. Polymeric nanocarriers Some polymeric nanocarriers such as dendrimers, polymer nanoparticles, micelles, nano gels, nanocapsules, and vesicles have recently been intensively used for drug delivery for their potential to modify the surface through chemical transformations to enhance drug loading and controlled release of specific ligands to reach specific sites where the drugs are designed (76,77). Polymeric nanocarriers can increase the solubility of hydrophobic drug compounds, increase drugs bioavailability, pharmacokinetics and Iraqi J Pharm Sci, Vol.32 (1) 2023 Nanoparticle formulation design of piperine 18 biodistribution, minimize drug toxicity at the targeted sites, increase the stability of various therapeutic agents, and deliver drugs to the central nervous system by penetrating the blood-brain barrier (BBB) (76,78,79). Ideally, polymeric nanocarriers should be biodegradable and non-toxic to minimize hypersensitivity reactions and provide good tissue compatibility (79). The polymeric nanocarrier systems are classified into nanocapsules and nanospheres. Nanocapsules are vesicular systems in which drug molecules are surrounded by a membrane (encapsulated and trapped in lipids), while nanospheres are matrix systems in which drug molecules are dispersed across the particle surface (79). Polymeric nanocarriers can increase the solubility and bioavailability of hydrophobic drugs in several specific ways, such as through diffusion- controlled drug release where the polymer matrix does not have a membrane that acts as a barrier to diffusion. The drug release is controlled by the solvent, in which polymeric materials having a three-dimensional connective tissue structure can control the drug release by biodegradable polymer degradation through enzymatic decomposition. Finally, the targeted drugs will be released at specific sites (80). Therefore, polymeric nanocarriers play a crucial function in the piperine formulation for oral drug and piperine delivery in treating cancer. Several formulations of piperine polymeric nanocarriers to increase piperine solubility are listed in Table 2. The piperine nanocarrier formulation enhances the anti-cancer effect compared to pure ones, such as MCF-7 breast cancer, triple-negative breast cancer (TNBC) and MDA-MB-468 cells, A549 cell lung cancer, HepG2 cell liver cancer, HeLa cervical cancer, and Hs683cell brain cancer (81–86). The piperine nanocarrier formulation can increase solubility and bioavailability, extend drug circulation time, decrease toxic effects on normal cells, deliver the drugs to specific sites, penetrate the blood-brain barrier (BBB), and increase the piperine storage time. Inorganic nanoparticle Inorganic nanoparticles have more benefits than polymer and lipid ones. They have higher stability levels and are non-toxic and non- immunogenic. There are inorganic carriers that have been used in drug delivery systems for drug therapy such as mesoporous silica, alumina, and zinc. Mesoporous silica enforces higher encapsulation efficiency of the active ingredients, controlled structural properties, and biocompatibility (79). Another example of an inorganic carrier for nanocarriers is hydroxyapatite. Hydroxyapatite is a component of teeth and bone that exhibits good biocompatibility for delivering of prolonged-release drugs (87). Piperine has been formulated with nanohydroxyapatite for anticancer therapy in vitro and performs a better anticancer effect on HCT116 monolayer colon cancer cells (88). Piperine nanohydroxyapatite produces spherical and amorphous nanoparticles with an average pore size of 9.7 ± 0.1 nm, drug loading of 16-22%, entrapment efficiency of 75-85%, and longer release at pH 7.4 (88). Inorganic nanoparticles have increased the solubility and bioavailability of piperine through particle size reduction. They target the piperine at specific sites and release it controllably (89). Solid lipid nanoparticle (SLN) and Nanostructured lipid carriers (NLCs) SLNs and NLCs are the two main types of lipid nanoparticles formulated by combining non- toxic lipid nanoparticles and excipients properties. SLNs and NLCs are promising drug carriers for delivering poorly water-soluble drugs (90,91). Muchow et al. (92) stated, that SLN is the first generation of lipid nanoparticles that structurally resembles an emulsion. SLNs are made of solid lipids at room and body temperatures stabilized by surface surfactants (93,94). Meanwhile, NLCs are the second generation of lipid nanoparticles where generated lipid particles are a mixture of solid and liquid lipids and will be solid at a temperature of around 40oC (92). Generally, the lipid excipients used to formulate SLNs and NLCs are biocompatible, biodegradable, and safe to use. SLNs and NLCs have some clinical advantages like increasing drug solubility and bioavailability, protecting drugs from chemical degradation, smoothly delivering drugs to specific sites, being easier to produce, and being sTable during the storage period (95). Bhalekar et al. (96) have developed an SLN formulation of piperine for treating rheumatoid arthritis which could be consumed orally. The SLN piperine formulation was prepared using the melt emulsification method with Glyceryl Monostearate and Span 80 as the main ingredients. The SLN piperine dispersion was successfully prepared with a drug and lipid ratio of 1:3 and a span concentration of 1% w/v. The results of the pharmacodynamic test showed that the piperine oral usage could provide a significant response compared to chloroquine suspension. Chaudhari et al. (97) found a piperine formulation in the form of NLCs. NLCs were chosen because they could load the drugs maximally and had higher stability during the storage period than SLN. Piperine NLCs were made by solvent evaporation through high shear homogenization using solid lipid Compritol 888 ATO (50% w/w), liquid lipid Squalene (25% w/w), piperine (20% w/w), Span 80 (2 .5% w/w), and Tween 80 (2.5% w/w). The piperine NLCs generated spherical particles, which were negatively charged, amorphous, resulting in higher drug release than pure piperine within 12 hours. Zafar et al. (98) formulated the surface modification of NLCs with chitosan to improve the Iraqi J Pharm Sci, Vol.32 (1) 2023 Nanoparticle formulation design of piperine 19 therapeutic effect of piperine for diabetes therapy. The characterization of piperine NLCs coated with 0.2% w/v chitosan resulted in piperine encapsulated in lipid and amorphous form, within the size of 149.34 ± 4.54 nm. This could increase the piperine bioavailability ten times higher than pure piperine, and decrease the blood glucose three and half times better than that of pure piperine. Quantum dots nanoparticle Quantum dots (QDs) are colloidal semiconductor nanocrystals that consist of atoms of groups II-VI or III-V of the periodic Table and possess unique optical and fluorescent properties. The nanocrystal size of QDs usually ranges from 2 to 10 nm and can be used for marking biological macromolecules, such as nucleosides and proteins (79). Some of the commonly used QDs include cadmium selenide (CdSe), cadmium telluride (CdTe), and indium arsenide (InAs). There have been the latest developments for targeted piperine delivery in the formulation of QDs nanoparticles. Piperine is synthesized with copper oxide (CuO) QDs and coated with hyaluronic acid (HA)/Poly (lactic-co-glycolic acid) (PLGA) (99). Piperine microspheres of CuOQDs aim to deliver the piperine to the brain for treating epilepsy. Piperine loaded in CuQDs HA/PLGA shows enhanced neuroprotection and promoted astrocyte activation in epilepsy- induced mice in vivo. The particle size of CuOQDS HA/PLGA is vital in delivering piperine through the BBB. Smaller particle sizes can easily facilitate the drug delivery process into the systemic circulation. Drug-phospholipids complexation Phospholipids are the main components of mammalian cell membranes, are amphiphilic, and perform good solubility in water and oil. Phospholipids are compatible with biological membranes and are hepatoprotective (100). They can increase the drug's bioavailability level with low water solubility and low membrane penetration, enhance drug absorption and release, protect against degradation in the gastrointestinal tract, reduce gastrointestinal side effects, and reduce the bitter taste of orally-consumed drugs (101). One of the benefits of phospholipids in drug delivery is their complexation with piperine compounds. Piperine is complexed with hydrogenated soy phosphatidylcholine (HSPC) with a molar ratio of 1:1.22 and exhibits complexation of the phospholipid group with the –C=O group of piperine through hydrogen bond interactions. The piperine/HSPC complexation generates amorphous particles, increases solubility and sustained release of piperine, and increases piperine bioavailability 10.4 times compared to pure piperine (102). Inclusion complexes Inclusion complexes mean the formation of some complexes between the ligand as a complexing agent with a lipophilic cavity and a hydrophilic outer surface, which can interact with hydrophobic drug molecules (103). Steric and thermodynamic factors function to form inclusion complexes of ligands and drug molecules. Inclusion complexes of ligands and drug molecules may occur through non-covalent interactions such as hydrogen bonds, van der Waals interactions, ion pairs, solvophobic effects, and/or hydrophobic interactions (104). The more hydrophobic the drug molecule, the more sTable the inclusion complexes will be. Applications of inclusion complexes in oral drug delivery include increasing oral solubility and bioavailability, increasing dissolution rate and rate, increasing drug stability at absorption sites, reducing drug-induced irritation, and masking unpleasant tastes (105). There are two types of formulations for forming inclusion complexes of ligands and drug compounds, namely binary and ternary inclusion complexes. Binary inclusion complexes consist of complexing agents and drug compounds, while the ternary ones include complexing agents, drug compounds, and hydrophilic polymers increase drugs solubility (106,107). The binary inclusion complexes of piperine compound with β- cyclodextrin with a molar ratio of 1:1 successfully increase solubility level, dissolution rate, and absorption rate of piperine in the intestine. The aromatic ring of piperine compounds interacts with the β -cyclodextrin cavity to form amorphous solids and work to increase the solubility level, dissolution rate, and bioavailability of piperine compounds (108,109). Other forms of cyclodextrin, i.e α - cyclodextrin and γ-cyclodextrin have also been successful in increasing the solubility and dissolution of piperine through hydrophobic interactions (110,111). The piperine inclusion complexes with ethylenediamine-β-cyclodextrin through hydrogen bonding at a molar ratio of 1:1 can completely increase the solubility and dissolution rate of the piperine. Piperine compounds are present in the ethylenediamine-β-cyclodextrin cavity and build a relatively sTable composite structure (112). The formation of ternary inclusion complexes assisted with hydrophobic polymers can increase the mixture of ligands and drug compounds. This can reduce ligands and the dose of the drug compounds (106,107). One of the polymers used for such complexes is hydroxypropyl methylcellulose (HPMC). The piperine ternary inclusion complexes with β-cyclodextrin and HPMC synergistically increase solubility and dissolution of piperine compared to the piperine binary inclusion complexes with β-cyclodextrin (106). Other ligands that have been commonly used for inclusion complexes with piperine are cucurbiturils (113). They are macrocyclic oligomers that have the potential to increase drug solubility and bioavailability and are non-cytotoxic (114). The interaction between piperine and cucurbiturils occurs due to the interaction of dipole charges and Iraqi J Pharm Sci, Vol.32 (1) 2023 Nanoparticle formulation design of piperine 20 hydrogen interactions between the hydrogen atoms of methylene cucurbiturils and the aromatic part of the piperine molecules (113). The formation of these inclusion complexes can prevent the piperine’s isomerization reactions. Table 2. Summaries some types of nanoparticle formulation design of piperine. Type Form Size (nm) Composition Method Results Reference Liposom Globular 243.4 ± 7.5 Cholesterol, Phospholipon® 90H, Sodium cholate, Chitosan Thin film evaporation method Increased solubility and permeability. Showed anti- breast cancer effect. (52) Globular 29.75 ± 0.84 Soya phosphatidylcholin e, Tween 80 Thin film evaporation method Increased solubility, stability, and sustained release. (51) Microemulsion Spherical 161.50 ± 4.06 Caproyl 90®, Tween 80, Cremophor RH 40, Transcutol HP - Increased solubility and bioavailability. Increased cognitive function in Alzheimer’s disease. (59) SEDDS (self- emulsifying drug delivery system) SNEDDS (self- nanoemulsifying drug delivery system) Spherical 89.82 ± 2.16 Ethyl oleate, tween 80, transcutol P - Increased solubility (5.90- fold), bioavailability (5.2-fold), and permeability. (41) Spherical 33.35 ± 1.97 Miglyol 812N, Cremophor RH40, PEG400 - Increased solubility. (70) S-SNEDDS Spherical 73.56 ± 3.54 Glyceryl monolinoleate (GML), poloxamer 188, transcutol HP, avicel PH-101 - Increased solubility (3.51- fold), bioavailability (4.92-fold), and sustained release. (75) Polymeric nanocarriers: - Albumin nanoparticle Globular polymer 187.3 5.7 Human serum albumin (HSA), ethanol, aqueous glutaraldehyde 8% (v/v) Desolvation method, self- assembly method Increased solubility and anticancer on MCF-7 cells. (81) - Micelle Spherical 61.9 Soluplus®, D-α- tocopherol polyethylene glycol succinate (TPGS) Thin-film hydration Increased sustained release, solubility piperine, bioavailability (2.56-fold), physicochemical stability, cellular uptake, anticancer efficacy in A549 and HegG2 cells. (82) Iraqi J Pharm Sci, Vol.32 (1) 2023 Nanoparticle formulation design of piperine 21 Continued table 2 . Type Form Size (nm) Composition Method Results Reference - Polymer nanoparticle Globular polymer 95 ± 10 Poly (D, L, - lactide-co-glycolic acid) (PLGA) Ultrasonic atomization Increased solubility, efficacy, and in vitro release (83) - Polymeric nanoparticle Globular polymer 53 ± 1 Methoxy poly (ethylene glycol)- poly(lactic-co- glycolic) (mPEGPLGA), dichloromethane, Lutrol® F68, sucrose Single emulsion solvent extraction and thin-film hydration Increased solubility and efficacy piperine inhibit breast cancer (TNBC) cells and MDA- MB-468 in breast cancer. (85) - Core-shell nanocarrier Spherical - Dimethyl sulfate (DMS), chitosan (C), Pluronic F- 127, sodium dodecyl sulfate (SDS), polyvinyl alcohol (PVA), dichloromethane (DCM), aceton, brain cancer cell line Hs683 Nanoprecipit ation technique, ionic gelation Increased sustained release and solubility. Induction of apoptosis and cell cycle arrect in brain cancer cells. (86) - Nanoparticle Globular polymer 130,20 ± 1,57 Piperine, Eudragit S100 (polymer), poloxamer 188 (surfactant) at 1:1:5 (w/w/w) Solvent : Ethanol Nanoprecipit ation Increased solubility, in vitro release, and bioavailability (2.7-fold). (115) - Starch nanoparticle Globular polymer 88 ± 20 Sago starch powder (natural biopolymer) In situ nanoprecipita tion Increased solubility and stability. (116) - Nanosuspensio n Spherical 172.5 HPMC (stabilizer), ethanol (solvent) (0.25% HPMC, 0.13% extract, rasio antisolvent- to-solvent 1:10) Bottom-up approach Increased solubility (5.13- fold) and bioavailability (3.65-fold). (117) - Nanosponge nanoencapsulat ion system Spherical - β-cyclodextrin, diphenyl carbonate, ethanol, acetone, diclorometane Microwave- assisted synthesis, solvent evaporation The ratio of -CD: DPC (1:10) resulted in the highest loading efficiency of piperine. (118) - Nanosponge nanoencapsulat ion system Spherical - β-cyclodextrin, diphenyl carbonate, ethanol Solvent method Increased loading efficiency and solubility. (119) - Core-shell nanoparticle Spherical - ĸ-Carrageenan, Zein, potassium chloride, Coenzyme Q10 Antisolvent precipitation method, electrostatic deposition, ionic gelation Increased photodegradation half-life (3.0- fold), control release, chemical and physichochemica l stability. (120) Iraqi J Pharm Sci, Vol.32 (1) 2023 Nanoparticle formulation design of piperine 22 Continued table 2 . Type Form Size (nm) Composition Method Results Reference - Co- encapculation microparticle Spherical 922 Zein, chitosan, pectin, Coenzyme Q10 Ultrasound- assisted method, electrostatic deposition, solvent evaporation Decreased chemical degradation. Increased shelflive and physicochemical stability. (121) - Starch nanoparticle Globular polymer 110 Sago starch powder, anhydrous sodium sulfate (Na2SO4), propylene oxide, ethanol In situ nanoprecipita tion Increased solubility and sustained release. (122) - Gelatin nanofiber Globular polymer - Gelatin, glutaraldehyde, acetic acid Electrospinni ng method Improved controlled release profile. Increased diffusion barrier and solubility. - Nanoencapsula tion Spherical 68.2 Gum rosin (polymer), acetone, oleic acid, pluronic-f Emulsion- diffusion method Increased sustained release and solubility. (123) Inorganic nanoparticle Spherical - Hydroxyapatite nanoparticle, gum arabic, folic acid Hydrotherma l method, precipitation method Increased solubility and inhibited monolayer HCT116 colon cancer cell. (88) SLN, NLC Spherical 115,7 ± 6.26 Compritol® 888 ATO, squalene (liquid lipid), Span 80, Tween 80 Solvent evaporation through high shear homogenizati on method Increased solubility (2.0- fold). (97) Spherical 128,80 Glyceryl monostearate, Compritol 888 ATO, Precirol ATO 5, Tween 80, Span 80 Melt emulsificatio n Increased solubility. (96) Spherical 149.34 ± 4.54 Chitosan 0.2% w/v - Increased piperine release (4.67-fold), bioadhesive, permeability (10.15-fold), bioavailability (3.76-fold), and antidiabetic. (98) QDs Spherical - Copper acetate dihydrate, 0.1 M NaOH, ethanol, PLGA, ethyl acetate, poly (vinyl alcohol) (PVA), hyaluronic acid Precipitation method, emulsificatio n solvent evaporation method Increased solubility and anticonvulsive efficiency. (99) Iraqi J Pharm Sci, Vol.32 (1) 2023 Nanoparticle formulation design of piperine 23 Continued table 2 . Type Form Size (nm) Composition Method Results Reference Drug- phospholipids complexation Globular - Hydrogenated soy phosphatidyl choline (HSPC), dichloromethane, n-hexane Precipitation method Increased bioavailability (10.40-fold) and half-life (20.55- fold). (102) Inclusion complexes Cyclic - Hydroxypropyl beta cyclodextrin (HP β CD), D-α- tocopherol polyethylene glycol 1000 succinate (TPGS) Solvent evaporation method Increased solubility (52.67- fold) and dissolution (5.45- fold). (124) Cyclic - Hydroxy propyl methyl cellulose (HPMC), β- cyclodextrin Solvent evaporation and microwave irradiation methods Increased stability, solubility and dissolution (4.43- fold), (106) Cyclic - β-cyclodextrin Freeze drying technique Increased stability, bioaccessability, and antioxidant activity. (109) Cyclic - α-cyclodextrin, β- cyclodextrin, γ- cyclodextrin Physical mixture (PM) & ground mixture (GM) Increased solubility. (110) Cyclic - Mono(6- ethylenediamine)- β-cyclodextrin Solvent evaporation method Increased solubility (2- fold). (112) Cyclic - Cucurbit[n]uril - Increased solubility and stability. (113) The nanoparticle systems, especially in lipid carriers and biodegradable polymers, have been succesful in increasing the solubility, permeability, and bioavailability of piperine. The polymer nanoparticle formulation is very sTable and can easily modify the surface; thus, drug release at specific sites can be managed well. Biodegradable polymer nanoparticles can act as a drug depot that provides a sustainable supply of therapeutic compounds at the targeted sites (125). The biodegradable polymers (PLGA and PLA) for nanoparticle production have been widely used for sustainable drug administration because they effectively deliver the drug to intracellular targeted sites (126). Polymeric nanoparticle piperine using PLGA can also smoothly deliver the piperine to the targeted sites and increase its efficacy on HEK-293 (human embryonic kidney cells) and MCF-7 (Michigan Cancer Foundation-T and breast cancer cells) (83,84). Lipid formulations can be easily absorbed by the body tissue after experiencing dissociation and release of the lipid system (127). The lipids in the gastrointestinal tract can also slow gastric emptying. Therefore, drug lipids are stored longer in the small intestine, allowing better drug dissolution at the absorption sites to enhance absorption level. A high lipophilic drug with high solubility in triglycerides can be transported by the lymphatic system. In this way, the metabolism process in the liver can be bypassed, which can increase the drug bioavailability (127,128). Lipid formulations can also increase drug bioavailability by inhibiting P- glycoprotein efflux at the luminal membrane of epithelial cells in the colon, jejunum, and liver (129). Although drug delivery on the nanoscale is very promising, nano-based formulations are still difficult to develop. For the pharmaceutical industry, drug formulation in nano form triggers complicated challenges because nanoformulations are easy to self-aggregate on long-term storage, affecting drug stability, variability, and efficacy (130– 132). Other factors are related to the mechanism of solubility increase by nonionic surfactants, co- solvents of propylene glycol and polyethylene glycol, and the use of cyclodextrins. Even though Iraqi J Pharm Sci, Vol.32 (1) 2023 Nanoparticle formulation design of piperine 24 nonionic surfactants, propylene glycol, polyethylene glycol, and cyclodextrin have shown to increase solubility, they can decrease permeability and lead to lower bioavailability levels despite high solubility (133–137). Ingels et al. (138) found that the increase in solubility was disproportionate to drug absorption, and drug preparations cannot go through the epithelial barrier. Although the solubility of the hydrobic drug was increased, it did not mean that there was an increase in its bioavailability. Besides, increased solubility due to micellization did not necessarily increase the amount of drug available for passive diffusion across membranes and epithelial barriers in the gastrointestinal tract (GIT) (139). Another challenge is related to scale-up and manufacturing processes. The nanoparticles are complex three-dimensional multicomponent products with a specific arrangement of components at the nanometer scale. Therefore, they present typical challenges during the scale-up and manufacturing processes. Nanoparticle formulation processes involving high-speed homogenization, sonication, organic solvents, cross-linking, milling, organic solvent evaporation, centrifugation, filtration, and lyophilization pose complex challenges during scale-up and manufacturing processes. Different processing conditions during initial development on a small scale can affect chemical structure and conformation changes, denaturation, cross-linking, coagulation, and degradation of the active substances and excipients (140). For the scale-up process from small to industrial level, it is necessary to understand the characterization, testing, and release of nanoparticle products to identify critical parameters and analytical criteria; thus, the scale-up and manufacturing processes can result in high reproducibility and consistent products. Identification of processing conditions involving polymer ratio, drug, target site, type of organic solvent, oil and water phase ratio, mixing temperature, pressure, pH of media, emulsifier, stabilizer, and cross-linker is also necessary before coming to the scale-up and manufacturing processes (141). Conclusions The brief review shows that various formulation approaches have successfully increased the solubility and absolute bioavailability of piperine within the body. Some approaches include solid dispersion, liposome, microemulsion, SEDDS, SNEDDS, S-SNEDDS, polymeric nanocarriers, inorganic nanoparticles, SLN, NLC, QDs, complexation with phospholipids, and inclusion complexes. However, most of the formulations do not consider the stability of piperine in dosage forms, especially for those in liquid preparations, because piperine compounds are very sensitive to light and can be easily degraded in liquid form. Therefore, it is essential to further study the stability of piperine in the dosage form. Some toxicity testing and demonstration of safety and efficacy should also be carried out to support the success of the piperine nano compounds formulation which later aims to increase its solubility and bioavailability. Conflict of Interest The authors have no conflict of interest. References 1. Ezawa T, Inoue Y, Tunvichien S, Suzuki R, Kanamoto I. Changes in the Physicochemical Properties of Piperine/β-Cyclodextrin due to the Formation of Inclusion Complexes. Int J Med Chem. 2016;1-9. 2. Gorgani L, Mohammadi M, Najafpour GD, Nikzad M. Piperine—The Bioactive Compound of Black Pepper: From Isolation to Medicinal Formulations. Comprehensive Reviews in Food Science and Food Safety. 2017;16(1):124–40. 3. Vasavirama K, Upender M. Piperine: a valuable alkaloid from piper species. Int J Pharm Pharm Sci. 2014;6(4):34–8. 4. Chopra B, Dhingra AK, Kapoor RP, Prasad DN. Piperine and Its Various Physicochemical and Biological Aspects: A Review. Open Chemistry Journal. 2016;3(1). 5. Kotte SCB, Dubey PK, Murali PM. Identification and characterization of stress degradation products of piperine and profiling of a black pepper (Piper nigrum L.) extract using LC/Q-TOF-dual ESI-MS. Anal Methods. 2014;6(19):8022–9. 6. Kozukue N, Park M-S, Choi S-H, Lee S-U, Ohnishi-Kameyama M, Levin CE, et al. Kinetics of light-induced cis-trans isomerization of four piperines and their levels in ground black peppers as determined by HPLC and LC/MS. J Agric Food Chem. 2007;55(17):7131–9. 7. Carosso S, Miller MJ. Nitroso Diels–Alder (NDA) reaction as an efficient tool for the functionalization of diene-containing natural products. Org Biomol Chem. 2014;12(38):7445– 68. 8. Wei K, Li W, Koike K, Nikaido T. Cobalt(II)- catalyzed intermolecular Diels-Alder reaction of piperine. Org Lett. 2005;7(14):2833–5. 9. Kanaki N, Dave M, Padh H, Rajani M. A rapid method for isolation of piperine from the fruits of Piper nigrum Linn. J Nat Med. 2008;62(3):281–3. 10. Kusumorini N, Nugroho AK, Pramono S, Martien R. Development of New Isolation and Quantification Method of Piperine from White Pepper Seeds (Piper Nigrum L) Using A Validated HPLC. Indonesian J Pharm. 2021;32(2):158–65. 11. Mohapatra M, Basak U. Evaluation of Piperine Content from Roots of Piper Longum Linn., Originated from Different Sources with Iraqi J Pharm Sci, Vol.32 (1) 2023 Nanoparticle formulation design of piperine 25 Comparison of Zonal Variation in Odisha, India. International Journal of Pharma Research & Review. 2015;4:1–8. 12. Rameshkumar KB, Aravind APA, Mathew PJ. Comparative Phytochemical Evaluation and Antioxidant Assay of Piper longum L. and Piper chaba Hunter Used in Indian Traditional Systems of Medicine. Journal of herbs, spices & medicinal plants. 2011; 17(4):351-60. 13. Khan M. Comparative Physicochemical Evaluation of Fruits and Anti depressant Potential of volatile oils of fruits of Local Piper Species. Oriental Journal of Chemistry. 2015;31:541–5. 14. Juliani HR, Koroch AR, Giordano L, Amekuse L, Koffa S, Asante-Dartey J, et al. Piper guineense (Piperaceae): Chemistry, Traditional Uses, and Functional Properties of West African Black Pepper. In: African Natural Plant Products Volume II: Discoveries and Challenges in Chemistry, Health, and Nutrition. American Chemical Society. 2013; 1127:33-48 15. Hussain K, Ismail Z, Sadikun A, Ibrahim P. Antioxidant, anti-TB activities, phenolic and amide contents of standardised extracts of Piper sarmentosum Roxb. Nat Prod Res. 2009;23(3):238–49. 16. 1Ahmad B, Akhtar J, Akhtar J, Akhtar J. Unani System of Medicine. Pharmacognosy Reviews. 2007;1(2):210–4. 17. Ansari KA, Akram M. Filfil Siyah (Piper nigrum Linn) an important Drug of Unani System of Medicine: A Review. J Pharmacogn Phytochem. 2014;2(6):219–21. 18. Johri RK, Zutshi U. An Ayurvedic formulation ‘Trikatu’ and its constituents. Journal of Ethnopharmacology. 1992;37(2):85–91. 19. Nadkarni KM, Nadkarni AK. (Indian materia medica ) ; Dr. K. M. Nadkarni’s Indian materia medica : with Ayurvedic, Unani-Tibbi, Siddha, allopathic, homeopathic, naturopathic & home remedies, appendices & indexes. 2. Popular Prakashan; 1994. 20. Aswar U, Shintre S, Chepurwar S, Aswar M. Antiallergic effect of piperine on ovalbumin- induced allergic rhinitis in mice. Pharm Biol. 2015;53(9):1358–66. 21. Kim J, Lee K-P, Lee D-W, Lim K. Piperine enhances carbohydrate/fat metabolism in skeletal muscle during acute exercise in mice. Nutrition & Metabolism. 2017;14(1):43-51. 22. Bang JS, Oh DH, Choi HM, Sur B-J, Lim S-J, Kim JY, et al. Anti-inflammatory and antiarthritic effects of piperine in human interleukin 1beta-stimulated fibroblast-like synoviocytes and in rat arthritis models. Arthritis Res Ther. 2009;11(2):1-9. 23. Dong Y, Huihui Z, Li C. Piperine inhibit inflammation, alveolar bone loss and collagen fibers breakdown in a rat periodontitis model. J Periodontal Res. 2015;50(6):758–65. 24. 24. Hu D, Wang Y, Chen Z, Ma Z, You Q, Zhang X, et al. The protective effect of piperine on dextran sulfate sodium induced inflammatory bowel disease and its relation with pregnane X receptor activation. J Ethnopharmacol. 2015;169:109–23. 25. Liu Y, Yadev VR, Aggarwal BB, Nair MG. Inhibitory effects of black pepper (Piper nigrum) extracts and compounds on human tumor cell proliferation, cyclooxygenase enzymes, lipid peroxidation and nuclear transcription factor- kappa-B. Nat Prod Commun. 2010;5(8):1253–7. 26. Umar S, Golam Sarwar AHM, Umar K, Ahmad N, Sajad M, Ahmad S, et al. Piperine ameliorates oxidative stress, inflammation and histological outcome in collagen induced arthritis. Cellular Immunology. 2013;284(1–2):51–9. 27. Li L, Liu H, Shi W, Liu H, Yang J, Xu D, et al. Insights into the Action Mechanisms of Traditional Chinese Medicine in Osteoarthritis. Evidence-based Complementary and Alternative Medicine. 2017;1–13. 28. Al-Baghdadi OB, Prater NI, Van der Schyf CJ, Geldenhuys WJ. Inhibition of monoamine oxidase by derivatives of piperine, an alkaloid from the pepper plant Piper nigrum, for possible use in Parkinson’s disease. Bioorg Med Chem Lett. 2012;22(23):7183–8. 29. Chonpathompikunlert P, Wattanathorn J, Muchimapura S. Piperine, the main alkaloid of Thai black pepper, protects against neurodegeneration and cognitive impairment in animal model of cognitive deficit like condition of Alzheimer’s disease. Food Chem Toxicol. 2010;48(3):798–802. 30. Essa MM, Vijayan RK, Castellano-Gonzalez G, Memon MA, Braidy N, Guillemin GJ. Neuroprotective effect of natural products against Alzheimer’s disease. Neurochem Res. 2012;37(9):1829–42. 31. Mishra A, Punia JK, Bladen C, Zamponi GW, Goel RK. Anticonvulsant mechanisms of piperine, a piperidine alkaloid. Channels. 2015;9(5):317–23. 32. Song Y, Cao C, Xu Q, Gu S, Wang F, Huang X, et al. Piperine Attenuates TBI-Induced Seizures via Inhibiting Cytokine-Activated Reactive Astrogliosis. Frontiers in Neurology. 2020;11:431-40. 33. Jafri A, Siddiqui S, Rais J, Ahmad MS, Kumar S, Jafar T, et al. Induction of apoptosis by piperine in human cervical adenocarcinoma via ROS mediated mitochondrial pathway and caspase-3 activation. EXCLI J. 2019;18:154–64. 34. Yoo ES, Choo GS, Kim SH, Woo JS, Kim HJ, Park YS, et al. Antitumor and Apoptosis- inducing Effects of Piperine on Human Iraqi J Pharm Sci, Vol.32 (1) 2023 Nanoparticle formulation design of piperine 26 Melanoma Cells. Anticancer Research. 2019;39(4):1883–92. 35. Singh P, Shrman K, Sharma R, Meena N, Kumar N, Kishor K. Safety assessment of piperine after oral administration in sirohi goats. Journal of Entomology and Zoology Studies. 2021;9(1):753–5. 36. Wu Z, Xia X, Huang X. Determination of equilibrium solubility and apparent oil/water partition coefficient of piperine. J Jinan Univ. 2012;33:473–6. 37. Kerns EH, Di L. Drug-like properties: concepts, structure design and methods ; from ADME to toxicity optimization ; (metabolism, solubility, pharmacokinetics, permeability, CYP inhibition, toxicity, prodrugs). Amsterdam: Elsevier, Acad. Press; 2008. 38. Khajuria A, Zutshi U, Bedi KL. Permeability characteristics of piperine on oral absorption--an active alkaloid from peppers and a bioavailability enhancer. Indian J Exp Biol. 1998;36(1):46–50. 39. Suresh D, Srinivasan K. Studies on the in vitro absorption of spice principles – Curcumin, capsaicin and piperine in rat intestines. Food and Chemical Toxicology. 2007;45(8):1437–42. 40. Ganesh Bhat B, Chandrasekhara N. Studies on the metabolism of piperine: Absorption, tissue distribution and excretion of urinary conjugates in rats. Toxicology. 1986;40(1):83–92. 41. 41. Shao B, Cui C, Ji H, Tang J, Wang Z, Liu H, et al. Enhanced oral bioavailability of piperine by self-emulsifying drug delivery systems: in vitro, in vivo and in situ intestinal permeability studies. Drug Delivery. 2015;22(6):740–7. 42. Ren T, Wang Q, Li C, Yang M, Zuo Z. Efficient brain uptake of piperine and its pharmacokinetics characterization after oral administration. Xenobiotica. 2018;48(12):1249–57. 43. Suresh DV, Mahesha HG, Rao AGA, Srinivasan K. Binding of bioactive phytochemical piperine with human serum albumin: a spectrofluorometric study. Biopolymers. 2007;86(4):265–75. 44. Liu H, Luo R, Chen X, Liu J, Bi Y, Zheng L, et al. Tissue distribution profiles of three antiparkinsonian alkaloids from Piper longum L. in rats determined by liquid chromatography- tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci. 2013;928:78– 82. 45. Baweja R. The theory and practlce of industrial pharmacy, 3rd ed. Edited by Leon Lachman, Herbert A. Lieberman and Joseph L. Kanig. Lea and Febiger, Philadelphia, PA. Journal of Pharmaceutical Sciences. 1987;76(1):90–1. 46. Tang J, Sun J, He Z-G. Self-Emulsifying Drug Delivery Systems: Strategy for Improving Oral Delivery of Poorly Soluble Drugs. Current Drug Therapy. 2007;2:85–93. 47. Sahu PK, Sharma A, Rayees S, Kour G, Singh A, Khullar M, et al. Pharmacokinetic study of Piperine in Wistar rats after oral and intravenous administration. International Journal of Drug Delivery. 2014;6(1):82–7. 48. Akbarzadeh A, Rezaei-Sadabady R, Davaran S, Joo SW, Zarghami N, Hanifehpour Y, et al. Liposome: classification, preparation, and applications. Nanoscale Research Letters. 2013;8(1):102-11. 49. Anwekar H, Patel S, Singhai A. Liposome-as Drug Carriers. International Journal of Pharmacy and Life Sciences. 2011;2:945–51. 50. Kalepu S, Nekkanti V. Insoluble drug delivery strategies: review of recent advances and business prospects. Acta Pharm Sin B. 2015;5(5):442–53. 51. Dutta S, Bhattacharjee P. Nanoliposomal encapsulates of piperine-rich black pepper extract obtained by enzyme-assisted supercritical carbon dioxide extraction. Journal of Food Engineering. 2017;201:49–56. 52. Imam SS, Alshehri S, Altamimi MA, Hussain A, Qamar W, Gilani SJ, et al. Formulation of Piperine–Chitosan-Coated Liposomes: Characterization and In Vitro Cytotoxic Evaluation. Molecules. 2021;26(11):1-13. 53. Danielsson I, Lindman B. The definition of microemulsion. Colloids and Surfaces. 1987;3:135–49. 54. Derle DV, Sagar BSH, Pimpale S. Microemulsion as a vehicle for transdermal permeation of nimesulide. Indian Journal of Pharmaceutical Sciences. 2006;68(5):1-4. 55. Kawakami K, Yoshikawa T, Hayashi T, Nishihara Y, Masuda K. Microemulsion formulation for enhanced absorption of poorly soluble drugs. II. In vivo study. Journal of controlled release : official journal of the Controlled Release Society. 2002;81:75–82. 56. Tartaro G, Mateos Cuadrado H, Schirone D, Angelico R, Palazzo G. Microemulsion Microstructure(s): A Tutorial Review. Nanomaterials. 2020;10:1-40. 57. Tang T-T, Hu X-B, Liao D-H, Liu X-Y, Xiang D-X. Mechanisms of microemulsion enhancing the oral bioavailability of puerarin: comparison between oil-in-water and water-in-oil microemulsions using the single-pass intestinal perfusion method and a chylomicron flow blocking approach. Int J Nanomedicine. 2013;8:4415–26. 58. Sanjula B, Shah FM, Javed A, Alka A. Effect of poloxamer 188 on lymphatic uptake of carvedilol-loaded solid lipid nanoparticles for bioavailability enhancement. J Drug Target. 2009;17(3):249–56. 59. Etman SM, Elnaggar YSR, Abdelmonsif DA, Abdallah OY. Oral Brain-Targeted Microemulsion for Enhanced Piperine Delivery Iraqi J Pharm Sci, Vol.32 (1) 2023 Nanoparticle formulation design of piperine 27 in Alzheimer’s Disease Therapy: In Vitro Appraisal, In Vivo Activity, and Nanotoxicity. AAPS PharmSciTech. 2018;19(8):3698–711. 60. Yuan H, Chen J, Du Y-Z, Hu F-Q, Zeng S, Zhao H-L. Studies on oral absorption of stearic acid SLN by a novel fluorometric method. Colloids Surf B Biointerfaces. 2007;58(2):157–64. 61. Kaminskas LM, Porter CJH. Targeting the lymphatics using dendritic polymers (dendrimers). Adv Drug Deliv Rev. 2011;63(10– 11):890–900. 62. Khan AA, Mudassir J, Mohtar N, Darwis Y. Advanced drug delivery to the lymphatic system: lipid-based nanoformulations. Int J Nanomedicine. 2013;8:2733–44. 63. Rao DA, Forrest ML, Alani AWG, Kwon GS, Robinson JR. Biodegradable PLGA based nanoparticles for sustained regional lymphatic drug delivery. J Pharm Sci. 2010;99(4):2018–31. 64. Chatterjee B, Hamed Almurisi S, Ahmed Mahdi Dukhan A, Mandal UK, Sengupta P. Controversies with self-emulsifying drug delivery system from pharmacokinetic point of view. Drug Deliv. 2016;23(9):3639–52. 65. Cerpnjak K, Zvonar A, Gašperlin M, Vrečer F. Lipid-based systems as a promising approach for enhancing the bioavailability of poorly water- soluble drugs. Acta Pharm. 2013;63(4):427–45. 66. Nanjwade BK, Patel DJ, Udhani RA, Manvi FV. Functions of Lipids for Enhancement of Oral Bioavailability of Poorly Water-Soluble Drugs. Scientia Pharmaceutica. 2011;79(4):705–28. 67. Pouton CW, Porter CJH. Formulation of lipid- based delivery systems for oral administration: materials, methods and strategies. Adv Drug Deliv Rev. 2008;60(6):625–37. 68. Cai S, Shi C-H, Zhang X, Tang X, Suo H, Yang L, et al. Self-microemulsifying drug-delivery system for improved oral bioavailability of 20(S)-25-methoxyl-dammarane-3β, 12β, 20- triol: preparation and evaluation. Int J Nanomedicine. 2014;9:913–20. 69. Singh S, Bajpai M, Mishra P. Self-Emulsifying Drug Delivery System (SEDDS): An Emerging Dosage Form to Improve the Bioavailability of Poorly Absorbed Drugs. Crit Rev Ther Drug Carrier Syst. 2020;37(4):305–29. 70. Kusumorini N, Nugroho AK, Pramono S, Martien R. Application of D-Optimal design for the optimization of isolated piperine from piper nigrum L-loaded self-nanoemulsifying drug delivery systems (SNEDDS). Acta Poloniae Pharmaceutica - Drug Research. 2021;78(3):415–23. 71. Wang Y, Pi C, Feng X, Hou Y, Zhao L, Wei Y. The Influence of Nanoparticle Properties on Oral Bioavailability of Drugs. Int J Nanomedicine. 2020;15:6295–310. 72. Taha EI, Al-Saidan S, Samy AM, Khan MA. Preparation and in vitro characterization of self- nanoemulsified drug delivery system (SNEDDS) of all-trans-retinol acetate. Int J Pharm. 2004;285(1–2):109–19. 73. Bari A, Chella N, Sanka K, Shastri NR, Diwan PV. Improved anti-diabetic activity of glibenclamide using oral self nano emulsifying powder. Journal of Microencapsulation. 2015;32(1):54–60. 74. Kim KS, Yang ES, Kim DS, Kim DW, Yoo HH, Yong CS, et al. A novel solid self- nanoemulsifying drug delivery system (S- SNEDDS) for improved stability and oral bioavailability of an oily drug, 1-palmitoyl-2- linoleoyl-3-acetyl-rac-glycerol. Drug Deliv. 2017;24(1):1018–25. 75. Zafar A, Imam SS, Alruwaili NK, Alsaidan OA, Elkomy MH, Ghoneim MM, et al. Development of Piperine-Loaded Solid Self-Nanoemulsifying Drug Delivery System: Optimization, In-Vitro, Ex-Vivo, and In-Vivo Evaluation. Nanomaterials. 2021;11(11):2920. 76. Karabasz A, Bzowska M, Szczepanowicz K. Biomedical Applications of Multifunctional Polymeric Nanocarriers: A Review of Current Literature. Int J Nanomedicine. 2020;15:8673– 96. 77. Venditti I. Morphologies and functionalities of polymeric nanocarriers as chemical tools for drug delivery: A review. Journal of King Saud University - Science. 2019;31(3):398–411. 78. Alexis F, Rhee J-W, Richie JP, Radovic-Moreno AF, Langer R, Farokhzad OC. New frontiers in nanotechnology for cancer treatment. Urol Oncol. 2008;26(1):74–85. 79. Mishra B, Patel BB, Tiwari S. Colloidal nanocarriers: a review on formulation technology, types and applications toward targeted drug delivery. Nanomedicine. 2010;6(1):9–24. 80. Son G-H, Lee B-J, Cho C-W. Mechanisms of drug release from advanced drug formulations such as polymeric-based drug-delivery systems and lipid nanoparticles. Journal of Pharmaceutical Investigation. 2017;47(4):287– 96. 81. Abolhassani H, Shojaosadati SA. A comparative and systematic approach to desolvation and self- assembly methods for synthesis of piperine- loaded human serum albumin nanoparticles. Colloids Surf B Biointerfaces. 2019;184:110534. 82. Ding Y, Wang C, Wang Y, Xu Y, Zhao J, Gao M, et al. Development and evaluation of a novel drug delivery: Soluplus®/TPGS mixed micelles loaded with piperine in vitro and in vivo. Drug Dev Ind Pharm. 2018;44(9):1409–16. 83. Kaur J, Singh RR, Khan E, Kumar A, Joshi A. Piperine-Loaded PLGA Nanoparticles as Cancer Drug Carriers. ACS Appl Nano Mater. 2021;4(12):14197-207. Iraqi J Pharm Sci, Vol.32 (1) 2023 Nanoparticle formulation design of piperine 28 84. Pachauri M, Gupta ED, Ghosh PC. Piperine loaded PEG - PLGA nanoparticles: Preparation, characterization and targeted delivery for adjuvant breast cancer chemotherapy. Journal of Drug Delivery Science and Technology. 2015; 29:269–82. 85. Rad JG, Hoskin DW. Delivery of Apoptosis- inducing Piperine to Triple-negative Breast Cancer Cells via Co-polymeric Nanoparticles. Anticancer Res. 2020;40(2):689–94. 86. Sedeky AS, Khalil IA, Hefnawy A, El-Sherbiny IM. Development of core-shell nanocarrier system for augmenting piperine cytotoxic activity against human brain cancer cell line. Eur J Pharm Sci. 2018;118:103–12. 87. Musumeci T, Ventura CA, Giannone I, Ruozi B, Montenegro L, Pignatello R, et al. PLA/PLGA nanoparticles for sustained release of docetaxel. Int J Pharm. 2006;325(1–2):172–9. 88. AbouAitah K, Stefanek A, Higazy IM, Janczewska M, Swiderska-Sroda A, Chodara A, et al. Effective Targeting of Colon Cancer Cells with Piperine Natural Anticancer Prodrug Using Functionalized Clusters of Hydroxyapatite Nanoparticles. Pharmaceutics. 2020;12(1):1-28. 89. Paul W, Sharma CP. 8 - Inorganic nanoparticles for targeted drug delivery (Internet). In: Sharma CP, editor. Biointegration of Medical Implant Materials. Woodhead Publishing; 2010. 90. Gasco MR. Method for producing solid lipid microspheres having a narrow size distribution. Patent. 1993. 91. Müller RH, Radtke M, Wissing SA. Nanostructured lipid matrices for improved microencapsulation of drugs. Int J Pharm. 2002;242(1–2):121–8. 92. Muchow M, Maincent P, Muller RH. Lipid nanoparticles with a solid matrix (SLN, NLC, LDC) for oral drug delivery. Drug Dev Ind Pharm. 2008;34(12):1394–405. 93. Ghasemiyeh P, Mohammadi-Samani S. Solid lipid nanoparticles and nanostructured lipid carriers as novel drug delivery systems: applications, advantages and disadvantages. Res Pharm Sci. 2018;13(4):288–303. 94. Yadav N, Khatak D, Sara UV. Solid lipid nanoparticles- A review. International Journal of Applied Pharmaceutics. 2013;5:8–18. 95. Talegaonkar S, Bhattacharyya A. Potential of Lipid Nanoparticles (SLNs and NLCs) in Enhancing Oral Bioavailability of Drugs with Poor Intestinal Permeability. AAPS PharmSciTech. 2019;20(3):121. 96. Bhalekar MR, Madgulkar AR, Desale PS, Marium G. Formulation of piperine solid lipid nanoparticles (SLN) for treatment of rheumatoid arthritis. Drug Development and Industrial Pharmacy. 2017; 43(6) :1003–10. 97. Chaudhari VS, Murty US, Banerjee S. Nanostructured lipid carriers as a strategy for encapsulation of active plant constituents: Formulation and in vitro physicochemical characterizations. Chem Phys Lipids. 2021;235:1-8. 98. Zafar A, Alruwaili NK, Imam SS, Alsaidan OA, Alharbi KS, Yasir M, et al. Formulation of Chitosan-Coated Piperine NLCs: Optimization, In Vitro Characterization, and In Vivo Preclinical Assessment. AAPS PharmSciTech. 2021;22(7):231. 99. Zhu D, Zhang W, Nie X, Ding S, Zhang D, Yang L. Rational design of ultra-small photoluminescent copper nano-dots loaded PLGA micro-vessels for targeted co-delivery of natural piperine molecules for the treatment for epilepsy. Journal of Photochemistry and Photobiology B: Biology. 2020;205:1-8. 100. Khan J, Alexander A, Ajazuddin, Saraf S, Saraf S. Recent advances and future prospects of phyto-phospholipid complexation technique for improving pharmacokinetic profile of plant actives. Journal of Controlled Release. 2013;168(1):50–60. 101. Fricker G, Kromp T, Wendel A, Blume A, Zirkel J, Rebmann H, et al. Phospholipids and lipid-based formulations in oral drug delivery. Pharm Res. 2010;27(8):1469–86. 102. Biswas S, Mukherjee PK, Kar A, Bannerjee S, Charoensub R, Duangyod T. Optimized piperine–phospholipid complex with enhanced bioavailability and hepatoprotective activity. Pharmaceutical Development and Technology. 2021;26(1):69–80. 103. Laza-Knoerr AL, Gref R, Couvreur P. Cyclodextrins for drug delivery. Journal of Drug Targeting. 2010;18(9):645–56. 104. Parmar V, Patel G, Abu-Thabit NY. 20 - Responsive cyclodextrins as polymeric carriers for drug delivery applications. In: Makhlouf ASH, Abu-Thabit NY, editors. Stimuli Responsive Polymeric Nanocarriers for Drug Delivery Applications, Volume 1. Woodhead Publishing; 2018, 555-80. 105. Challa R, Ahuja A, Ali J, Khar RK. Cyclodextrins in drug delivery: An updated review. AAPS PharmSciTech. 2005;6(2):E329– 57. 106. Alshehri S, Imam SS, Hussain A, Altamimi MA. Formulation of Piperine Ternary Inclusion Complex Using β CD and HPMC: Physicochemical Characterization, Molecular Docking, and Antimicrobial Testing. Processes. 2020;8(11):1450-65. 107. Kulkarni AS, Dias RJ, Ghorpade VS, Mali KK. Freeze dried multicomponent inclusion complexes of piperine with cyclodextrin and hydrophilic polymers: Physicochemical characterization and In vivo anti-inflammatory activity. Rese Jour of Pharm and Technol. 2020;13(10):1-8. Iraqi J Pharm Sci, Vol.32 (1) 2023 Nanoparticle formulation design of piperine 29 108. Ezawa T, Inoue Y, Tunvichien S, Suzuki R, Kanamoto I. Changes in the Physicochemical Properties of Piperine/β-Cyclodextrin due to the Formation of Inclusion Complexes. Int J Med Chem. 2016. 109. Quilaqueo M, Millao S, Luzardo-Ocampo I, Campos-Vega R, Acevedo F, Shene C, et al. Inclusion of piperine in β-cyclodextrin complexes improves their bioaccessibility and in vitro antioxidant capacity. Food Hydrocolloids. 2019;91:143–52. 110. Ezawa T, Inoue Y, Murata I, Takao K, Sugita Y, Kanamoto I. Characterization of the Dissolution Behavior of Piperine/Cyclodextrins Inclusion Complexes. AAPS PharmSciTech. 2018;19(2):923–33. 111. Ezawa T, Inoue Y, Murata I, Takao K, Sugita Y, Kanamoto I. Evaluation of the Molecular State of Piperine in Cyclodextrin Complexes by Near-Infrared Spectroscopy and Solid-State Fluorescence Measurements. International Journal of Medicinal Chemistry. 2019;2019:1-15. 112. Liu K, Liu H, Li Z, Li W, Li L. In vitro dissolution study on inclusion complex of piperine with ethylenediamine-β-cyclodextrin. J Incl Phenom Macrocycl Chem. 2020;96(3):233– 43. 113. Mohanty B, Suvitha A, Venkataramanan NS. Piperine Encapsulation within Cucurbit(n)uril (n=6,7): A Combined Experimental and Density Functional Study. ChemistrySelect. 2018;3(6):1933–41. 114. Wheate NJ, Limantoro C. Cucurbit(n)urils as excipients in pharmaceutical dosage forms. Supramolecular Chemistry. 2016;28(9–10):849– 56. 115. Ren T, Hu M, Cheng Y, Shek TL, Xiao M, Ho NJ, et al. Piperine-loaded nanoparticles with enhanced dissolution and oral bioavailability for epilepsy control. European Journal of Pharmaceutical Sciences. 2019;137:1-8. 116. Wan-Hong C, Suk-Fun C, Pang S-C, Kok K- Y. Synthesis and Characterisation of Piperine- loaded Starch Nanoparticles. JPS. 2020;31(1):57–68. 117. 1Zafar F, Jahan N, Khalil-Ur-Rahman, Bhatti HN. Increased Oral Bioavailability of Piperine from an Optimized Piper nigrum Nanosuspension. Planta Med. 2019;85(3):249– 57. 118. Guineo-Alvarado J, Quilaqueo M, Hermosilla J, González S, Medina C, Rolleri A, et al. Degree of crosslinking in β-cyclodextrin- based nanosponges and their effect on piperine encapsulation. Food Chem. 2021;340:1-7. 119. Garrido B, González S, Hermosilla J, Millao S, Quilaqueo M, Guineo J, et al. Carbonate-β- Cyclodextrin-Based Nanosponge as a Nanoencapsulation System for Piperine: Physicochemical Characterization. J Soil Sci Plant Nutr. 2019;19(3):620–30. 120. Chen S, Zhang Y, Qing J, Han Y, McClements DJ, Gao Y. Core-shell nanoparticles for co-encapsulation of coenzyme Q10 and piperine: Surface engineering of hydrogel shell around protein core. Food Hydrocolloids. 2020;103:1-11. 121. Chen S, Zhang Y, Han Y, McClements DJ, Liao W, Mao L, et al. Fabrication of multilayer structural microparticles for co-encapsulating coenzyme Q10 and piperine: Effect of the encapsulation location and interface thickness. Food Hydrocolloids (Internet) 2020 (cited 2021 Dec 2);109:106090. Available from: https://www.sciencedirect.com/science/article/p ii/S0268005X20311243 122. Chin SF, Salim A, Pang SC. Hydroxypropyl Starch Nanoparticles as Controlled Release. Journal of Nanostructures. 2020;10(2):327–36. 123. Rani R, Kumar S, Dilbaghi N, Kumar R. Nanotechnology enabled the enhancement of antitrypanosomal activity of piperine against Trypanosoma evansi. Exp Parasitol. 2020;219:108018. 124. Imam SS, Alshehri S, Alzahrani TA, Hussain A, Altamimi MA. Formulation and Evaluation of Supramolecular Food-Grade Piperine HP β CD and TPGS Complex: Dissolution, Physicochemical Characterization, Molecular Docking, In Vitro Antioxidant Activity, and Antimicrobial Assessment. Molecules. 2020;25(20):4716. 125. Singh R, Lillard JW. Nanoparticle-based targeted drug delivery. Exp Mol Pathol. 2009;86(3):215–23. 126. 1Panyam J, Labhasetwar V. Biodegradable nanoparticles for drug and gene delivery to cells and tissue. Adv Drug Deliv Rev. 2003;55(3):329–47. 127. Hauss DJ. Oral lipid-based formulations. Advanced Drug Delivery Reviews. 2007;59(7):667–76. 128. Date AA, Desai N, Dixit R, Nagarsenker M. Self-nanoemulsifying drug delivery systems: formulation insights, applications and advances. Nanomedicine. 2010; 5(10) :1595 – 616. 129. Karthika C, Sureshkumar R. P-Glycoprotein Efflux Transporters and Its Resistance Its Inhibitors and Therapeutic Aspects). IntechOpen. 2020. 130. Kharia AA, Singhai AK, Verma R. Formulation and Evaluation of Polymeric Nanoparticles of an Antiviral Drug for Gastroretention. IJPSN. 2011;4(4):1557–62. 131. Nagpal K, Singh SK, Mishra DN. Chitosan nanoparticles: a promising system in novel drug delivery. Chem Pharm Bull. 2010;58(11):1423– 30. Iraqi J Pharm Sci, Vol.32 (1) 2023 Nanoparticle formulation design of piperine 30 132. Niamprem P, Rujivipat S, Tiyaboonchai W. Development and characterization of lutein- loaded SNEDDS for enhanced absorption in Caco-2 cells. Pharm Dev Technol. 2014;19(6):735–42. 133. Fischer SM, Brandl M, Fricker G. Effect of the non-ionic surfactant Poloxamer 188 on passive permeability of poorly soluble drugs across Caco-2 cell monolayers. Eur J Pharm Biopharm. 2011;79(2):416–22. 134. Fischer SM, Flaten GE, Hagesæther E, Fricker G, Brandl M. In-vitro permeability of poorly water soluble drugs in the phospholipid vesicle-based permeation assay: the influence of nonionic surfactants. J Pharm Pharmacol. 2011;63(8):1022–30. 135. Miller JM, Beig A, Carr RA, Spence JK, Dahan A. A win-win solution in oral delivery of lipophilic drugs: supersaturation via amorphous solid dispersions increases apparent solubility without sacrifice of intestinal membrane permeability. Mol Pharm. 2012;9(7):2009–16. 136. Miller JM, Beig A, Carr RA, Webster GK, Dahan A. The solubility-permeability interplay when using cosolvents for solubilization: revising the way we use solubility-enabling formulations. Mol Pharm. 2012;9(3):581–90. 137. Miller JM, Dahan A. Predicting the solubility-permeability interplay when using cyclodextrins in solubility-enabling formulations: model validation. Int J Pharm. 2012;430(1–2):388–91. 138. Ingels F, Deferme S, Destexhe E, Oth M, Van den Mooter G, Augustijns P. Simulated intestinal fluid as transport medium in the Caco- 2 cell culture model. Int J Pharm. 2002;232(1– 2):183–92. 139. Buckley ST, Frank KJ, Fricker G, Brandl M. Biopharmaceutical classification of poorly soluble drugs with respect to “enabling formulations.” Eur J Pharm Sci 2013;50(1):8– 16. 140. Desai N. Challenges in development of nanoparticle-based therapeutics. AAPS J. 2012;14(2):282–95. 141. Feng S-S, Mu L, Win KY, Huang G. Nanoparticles of biodegradable polymers for clinical administration of paclitaxel. Curr Med Chem. 2004;11(4):413–24. This work is licensed under a Creative Commons Attribution 4.0 International License. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/