Bioadhesive Food Protein Nanoparticles as Pediatric Oral Drug

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Biological and Medical Applications of Materials and Interfaces

Bioadhesive Food-Protein Nanoparticles as Pediatric Oral Drug Delivery system Md Saiful Islam, Joshua Reineke, Radhey S Kaushik, Tofuko Woyengo, Aravind Baride, Mohammed S Alqahtani, and Omathanu Perumal ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 29 Apr 2019 Downloaded from http://pubs.acs.org on April 29, 2019

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ACS Applied Materials & Interfaces

Bioadhesive Food-Protein Nanoparticles as Pediatric Oral Drug Delivery system

Md Saiful Islam1, Joshua Reineke1, Radhey Kaushik2, Tofuko Woyengo3, Aravind Baride4, Mohammed S. Alqahtani1#, and Omathanu Perumal1* 1

Department of Pharmaceutical Sciences, 2Department of Biology and Microbiology, 3

4

Department of Animal Science, South Dakota State University, SD-57007, USA

Department of Chemistry, University of South Dakota, Vermillion, SD-57069, USA

*Corresponding Author: Phone: 605-688-4745, Fax: 605-688-5993 Email address: [email protected] #Current Address: Department of Pharmaceutics College of Pharmacy King Saud University P.O.Box 2457 Riyadh 11451, Saudi Arabia

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Abstract The goal of this study was to develop bioadhesive food protein nanoparticles using zein (Z), a hydrophobic corn protein as the core and whey protein (WP) as the shell for oral pediatric drug delivery applications. Lopinavir (LPV), an anti-retroviral drug and fenertinide, an investigational anti-cancer agent used were used as model drugs in the study. The particle size of ZWP nanoparticles was in the range of 200-250 nm and the drug encapsulation efficiency was > 70%. The nanoparticles showed sustained drug release in simulated gastrointestinal fluids. ZWP nanoparticles enhanced the permeability of LPV and fenretinide across Caco-2 cell monolayers. In both ex-vivo and in-vivo studies, ZWP nanoparticles were found to be strongly bioadhesive. ZWP nanoparticles enhanced the oral bioavailability of LPV and fenretinide by 4 and 7-fold respectively. ZWP nanoparticles also significantly increased the half-life of both drugs. The nanoparticles did not show any immunogenicity in mice. Overall, the study for the first-time demonstrates the feasibility of developing a safe and effective food protein-based nanoparticles for pediatric oral drug delivery. Keywords: Pediatric delivery system, Food protein nanoparticles, Bioadhesive, Zein, Whey protein, Biopolymers.


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1. INTRODUCTION

The lack of age appropriate formulations represents a major challenge in the delivery of many therapeutic agents in children.1 In the absence of age-appropriate formulations, often ad-hoc pediatric formulations are prepared by manipulating the adult drug products (e.g. crushing a tablet). These ad-hoc formulations carry the risk of under or over-dosing in children.2, 3. More importantly, the excipients regarded as safe in adults may represent a safety risk in children.4 In addition to the drug physicochemical properties, the pediatric formulation development should take into consideration age-related physiological changes, palatability, ease of swallowing and age appropriate formulations to meet the needs of children in various age groups.1,2,5,6. Several regulatory initiatives have been introduced to promote the development of new pediatric drug delivery systems and formulations.7-9 Although several delivery carriers have been explored for addressing the oral drug delivery challenges in adults, there has been limited focus on pediatric delivery systems.8 To this end, the food protein polymers are promising materials for developing oral pediatric drug delivery systems. The advantages of using protein biopolymers as carriers include the long-history of use as food, palatability, biodegradability and compatibility with a wide-range of food matrices.9 In addition, the chemical and structural diversity of protein polymers can be utilized to develop delivery systems with unique functional attributes.10 The study focuses on developing hybrid nanoparticles using food protein biopolymers, zein and whey protein. Zein (Z) is a water-insoluble corn protein with a high proportion of hydrophobic amino acids.11 Since zein is an edible protein, it has been widely used in food and packaging industry.12 It is an FDA approved GRAS excipient and has been used for film coating tablets to improve drug stability and sustained drug release.12,13 In addition, zein has also been used to develop nanoparticles and microspheres for encapsulation of drugs and nutraceuticals.14 However,



given the hydrophobic nature of zein, the nanoparticles tend to aggregate.15 Polymers and surfactants have been used to stabilize zein nanoparticles to prevent aggregation.15-18 Earlier, we reported the preparation of core-shell nanoparticles using zein as the hydrophobic core and milk proteins/polymers (casein, lactoferrin and PEG) as the shell.19,20 The zein core can be used to encapsulate water-insoluble drugs, while the hydrophilic/amphiphilic shell can be used to enhance the water solubility and also impart functional attributes to the nanoparticles. The focus of this study is to develop bioadhesive nanoparticles by using whey protein (WP) as the shell. Whey protein is isolated from bovine milk and is mainly composed of β-lactoglobulin (70%), along with other proteins (α-lactalbumin, lactoferrin, bovine serum albumin). It is widely used for its nutritional value in infants and children.21 WP have also been explored as a delivery carrier for water-insoluble drugs and nutraceuticals.22,23 More importantly, WP has been shown to have mucoadhesive properties.24 Mucoadhesive polymers help to increase the bioavailability of poorly absorbed drugs by prolonging the residence time in the gastrointestinal tract. 25 The improved oral bioavailability leads to reduced dose and dosing frequency, which in turn results in increase in therapy adherence, especially in children.6 Taken together, the present study aims to develop hybrid nanoparticles by combining the functional properties of zein and whey protein for pediatric oral drug delivery applications. Lopinavir (LPV) and fenretinide (FEN) were as model drugs in the study. LPV is a first-line protease inhibitor used in the treatment of HIV infection in children. This drug suffers from poor oral bioavailability due to its poor water solubility, poor membrane permeability and first-pass metabolism.26 Ritonavir (RTV), which is a CYP3A4 inhibitor is coadministered to prevent the first-pass metabolism of LPV. 26 Due to the poor solubility of LPV and ritonavir, a high proportion of propylene glycol and alcohol is used to solubilize these two drugs


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in the pediatric liquid formulation. However, these solvents have the potential to cause toxicity in children.27,28 In addition, the bitter taste of LPV and ritonavir is associated with poor adherence to therapy in children.29 The use of RTV with LPV also leads to multiple drug interactions and adverse effects.30 Taken together, there is a strong need to develop a safe and effective pediatric formulation for LPV. Fenretinide is a retinoid derivative and is currently under investigation for prevention and treatment of various cancers including pediatric neuroblastoma.31,32 Although fenretinide is a highly effective anti-cancer agent, the poor solubility and poor permeability limits its clinical application.31 Due to its poor oral bioavailability, large doses of fenretinide are required to achieve therapeutic concentrations.32 As a result, there is a strong unmet need for an effective oral formulation of fenretinide in general, and for children in particular. The main objective of this study is to evaluate the feasibility of enhancing the oral bioavailability of LPV and fenretinide using ZWP nanoparticles. In addition, the goal is to investigate the mechanism of oral absorption of ZWP nanoparticles using fluorescent dyes (Nile red and Cy 5.5). As per the authors knowledge this is the first report on the use of zein-whey protein based oral nanoparticles for oral drug delivery.



2. MATERIALS AND METHODS 2.1. Materials

Zein F-4400 was purchased from Flow Chemical (Tuckahole, NY). Whey protein isolate was provided by Dr. Husmukh Patel from the Dairy Science Department at South Dakota State University. Fenretinide was purchased from LC Laboratories (Woburn, MA, USA). Lopinavir was purchased from AvaChem Scientific (San Antonio, TX, USA). Saline and syringes were purchased from SAI Technologies (Lake Villa, Illinois, USA). Acetonitrile and glacial acetic acid were purchased from Fisher Scientific (Hampton, NH, USA). Nile red (NR) was purchased from SigmaAldrich Company (St. Louis MO). Trehalose was purchased from Acros Organics (NJ, US). Precast-Gels 12% SDS-PAGE gels were purchased from Bio-Rad (Hercules, CA). Pepsin, Pancreatin, Sodium azide and Eagle’s Minimum Essential Medium (EMEM) were purchased from American Type Culture Collection (ATCC) (Manassas, VA). Amicon Ultra-15 centrifugal filters (10 kDa molecular weight cutoff) was purchased from EMD Millipore (Billerica, MA). Antifade mounting medium with DAPI was purchased from VectaShield Laboratories (Burlingame, CA). 2.2. Preparation of Zein-whey protein nanoparticles Nanoparticles were prepared using the phase separation method by utilizing the differential solubility of zein and whey protein (WP) in hydro-alcoholic solvent and aqueous solution. Briefly, 30 mg of WP was dissolved in 0.1 M citrate buffer (pH 6.8) and heated at 60°C for 30 minutes to unfold the protein. Zein (15 mg) was dissolved in 2 mL of 90% ethanol containing 1 mg of the drug (LPV, or fenretinide) or fluorescent dyes (Nile red or Cy 5.5), and the solution was added dropwise to an aqueous phase containing WP under constant stirring (Figure 1). The resulting colloidal dispersion was stirred using a magnetic stirrer at 200 rpm for four hours to evaporate the ethanol. The nanoparticles were separated by centrifugation using Millipore centrifugal filters


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(10KD MWCO) at 4000 rpm for 1 hour and the nanoparticles were washed with deionized water three to five times to remove the free drug or the dye. Trehalose (30 mg) was added as a cryoprotectant and the nanoparticles were lyophilized. The nanoparticle formulation was stored in a desiccator at 4°C till further use.

Figure 1. Schematic representation for the preparation of Zein-Whey protein nanoparticles. 2.3. Characterization of Nanoparticles Nanoparticles were characterized for particle size, size distribution and zeta potential using Malvern Zetasizer-S 3600 (Malvern Instruments Inc., Southborough, MA). The morphology and core-shell structure of nanoparticles was characterized using transmission electron microscope (TEM). For TEM studies, the sample was prepared by placing a dilute dispersion of the nanoparticles on carbon-coated 200 mesh copper grids (Electron Microscopy Sciences, Fort Washington, PA) and dried overnight. Negative staining was performed using Uranyl acetate. TEM images were obtained using a Tecnai Spirit G2 TEM (FEI, Hillsbora, OR, USA) at an accelerating voltage of 120 kV. The electron micrograph images were captured using Orius SC200 CCD camera coupled to the TEM.



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2.3. Determination of encapsulation and loading efficiency Briefly, 5mg drug loaded nanoparticles was dispersed in deionized water followed by centrifugation for 10 minutes at 14,000 rpm. After decanting the supernatant, the pellet was dissolved in 90% ethanol for determining the drug concentration. The samples were analyzed by HPLC after filtration through a 0.2 µm filter. Encapsulation efficiency (EE%) and loading efficiency (LE%) were calculated using the following equations: EE% = LE% =

𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑑𝑟𝑢𝑔 𝑖𝑛 𝑛𝑎𝑛𝑜𝑝𝑎𝑟𝑡𝑖𝑐𝑙𝑒𝑠 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑑𝑟𝑢𝑔 𝑖𝑛𝑖𝑡𝑖𝑎𝑙𝑙𝑦 𝑎𝑑𝑑𝑒𝑑

x 100

𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑑𝑟𝑢𝑔 𝑜𝑟 𝑑𝑦𝑒 𝑖𝑛 𝑛𝑎𝑛𝑜𝑝𝑎𝑟𝑡𝑖𝑐𝑙𝑒𝑠 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑛𝑎𝑛𝑜𝑝𝑎𝑟𝑡𝑖𝑐𝑙𝑒𝑠

x 100

2.4. In vitro drug release from ZWP nanoparticles The release of LPV and FEN from ZWP nanoparticles was performed using a dialysis bag with simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) in the presence of pepsin and pancreatin enzymes respectively. Briefly, 50 mg of drug loaded nanoparticles was dispersed in 5mL of SGF or SIF and placed inside the dialysis tube (Snakeskin dialysis membrane, 10 kDa molecular weight cutoff). The dialysis sac was placed in a beaker containing 25 mL of SGF or SIF and 0.1% (w/v) of Tween 80 was added to the release medium to maintain sink conditions. The beaker was placed in a shaker at 37°C and agitated at 100 rpm. Around 400 µL of the sample was withdrawn at predetermined time intervals up to 24 hours, and an equal volume of pre-warmed SGF or SIF was replaced. The sample was diluted with an equal volume of ethanol and filtered through a 0.2 µm filter followed by HPLC analysis. 2.5. HPLC analysis of LPV and FEN HPLC analysis of LPV and fenretinide was performed on a Waters system (Milford, MA) equipped with an isocratic pump, a degasser, an autosampler and data processing software (Breeze version 3.30 SPA). The drug (50 µL injection volume) was separated on a symmetry® C18


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Column (Waters Corporation, Milford, USA) (5 µm, 4.6 mm X 150 mm). The mobile phase for LPV was a mixture of 10 mM ammonium acetate (pH 6.5) and acetonitrile (35:65 v/v).33 The mobile phase for fenretinide was a mixture of acetonitrile: water: glacial acetic acid (80:18:2, v/v). The mobile phase was pumped at a flow-rate of 1.0 and 0.8 mL/min for LPV and fenretinide respectively. LPV and fenretinide were monitored at 210 and 310 nm wavelength respectively. The calibration curve (peak area versus drug concentration) was linear (R2=0.999) in the concentration range of 0.4 to 3 μg/mL for both the drugs. 2.6. Cell culture The human intestinal Caco-2 cells (colon carcinoma cell line) was used to study the drug permeability and cellular uptake of ZWP nanoparticles. Caco-2 cells (ATCC, Rockville, MD, USA) were maintained in Eagle’s minimum essential medium (EMEM) supplemented with 10% FBS (HyClone; Thermo Scientiﬁc, Waltham, MA, USA), 1% non-essential amino acids, 1% Lglutamine, streptomycin (100 μg/ml) and penicillin (100 IU/ml). The growth medium was changed every other day in the first two weeks, and twice a week thereafter. 2.7. Transepithelial permeability of drug loaded ZWP nanoparticles Transepithelial permeability of free drug or drug loaded ZWP nanoparticles (10 µg/mL equivalent weight of drug) was determined using Caco-2 cell monolayers. Briefly, 5x104 cells (Passage number # 20-25) were cultured in a 12-well plate using transwell inserts (1.12 sq.cm; Transwell®, Corning Costar Corp. Cambridge, MA, USA). The growth medium was changed every other day for 15 days. The formation of a polarized Caco-2 cell monolayer was confirmed from transepithelial electrical resistance (TEER) values measured using EVOM instrument (World Precision Instrument, Sarasota, FL, USA). The formation of monolayer and barrier integrity was also further confirmed by immunostaining for tight junction protein, Zona Occludens-1 (Figure



S1). After the Caco-2 monolayers reached a TEER value >800 Ω/sq.cm, the donor solution was replaced with 500 μL of free drug suspension (10 μg/mL) or drug loaded ZWP nanoparticles dispersed in HBSS buffer. Around 100 μL of the sample was collected at each time point from the basolateral chamber for up to 4 hours. An equal volume of pre-warmed HBSS buffer was added to the basolateral chamber to maintain the volume. The samples were mixed with an equal volume of ethanol and processed for HPLC analysis. The apparent permeability coefficient (Papp) was calculated using the following equation

Papp (cm/s) =

[

(dQ/dt)

𝐴𝐶𝑜

] x 100

Where dQ/dt represents the flux across Caco-2 monolayer, C0 is the initial drug concentration in the apical chamber, and A is the surface area of the Transwell membrane (1.12 cm2). 2.8. In vitro cell uptake study Caco-2 cells were seeded (passage number# 20 to 30) in a 12-well plate (1x 105 cells/well). The cells after achieving 75-80% confluency (5 days) were treated with 0.5 to 2 µg/ml of free NR, or an equivalent NR loaded ZWP nanoparticles dispersed in 1 mL of HBSS buffer and was incubated for 30 minutes to 2 hours at 37°C with 5% CO2. The cells were washed three times with ice-cold HBSS, detached from the wells by trypsinization, and fixed using 2% formaldehyde. The cell uptake was quantified by flow cytometry (FACS, BD Biosciences, San Jose, CA) by analyzing mean fluorescence intensity obtained by compiling fluorescence of 50,000 cells. To determine if the nanoparticle uptake was energy-dependent, the cell uptake studies were conducted at low temperature (4°C) and the results were compared with the cell-uptake 37°C.


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To determine the mechanism of cell uptake, the Caco-2 ells were pre-incubated (30 minutes) with each endocytosis inhibitors (1 μM phenylarsine oxide, 4 μM filipin or 10 μM cytochalasin D) to for clathrin, caveolea, and macropinocytosis mediated cell uptake.19 2.9. Confocal laser scanning microscopy (CLSM) Caco-2 cells (5x104) were seeded in a chamber slide (Nunc Lab-TEK®II Chamber SlideTM system, Thermo Scientific, Waltham, MA). After 5 days, the growth medium was replaced with 1µg of free NR or equivalent NR loaded ZWP nanoparticles dispersed in HBSS buffer and incubated at 37˚C for 0.5 to 2 hours. At the end of the treatment, the cells were washed three times with ice-cold HBSS to remove nanoparticles adsorbed to the cell surface. Cells were then fixed in 4% paraformaldehyde for 20 minutes at room temperature, followed by staining of F-actin with Alexa Fluor 488 labeled Phalloidin (Life Technologies, Carlsbad, CA).

DAPI containing

mounting media (Vector Labs, Burlingame, CA) was added and sealed with a cover slip. The images were captured using Olympus FluoView 1200 (FV 1200) confocal laser scanning microscope (CLSM) at 60x magnification. 2.10. In-vitro metabolism studies To determine the metabolic stability of LPV, human intestinal microsomes was purchased from Sekisui Xenotech, LLC (Kansas City, KS, USA) and incubated with LPV loaded ZWP nanoparticles. The microsomal protein was diluted with rapid start solution (5mM magnesium chloride, 5mM glucose-6-phosphate, 1mM b-NADPI, and 1U/mL glucose-6-phosphate dehydrogenase) supplied by Sekisui Xenotech, LLC (Kansas City, KS, USA). Briefly, 0.3 mg/mL of microsomes was incubated with 10 μg/mL of free LPV, LPV/ritonavir, and ZWP-LPV nanoparticles. The study was performed for 30 minutes at 37°C under 100 rpm. The sample was



mixed with an equal volume of cold acetonitrile to stop the reaction. The LPV content was determined by HPLC after extraction with 90% ethanol. 2.11. Ex-vivo intestinal bioadhesion studies 2.11.1. Dye retention assay To determine the bioadhesive properties of NR loaded ZWP nanoparticles, everted sac method was used.33 The jejunum from 28 days old pig was procured from the Department of Animal Science at South Dakota State University. A section of the pig jejunum (6 cm length) was everted using a glass rod to expose the mucosal side, and the open ends were tied to form the sac. The sac was then filled with phosphate buffered saline-glucose (PBSG). The everted sac was immersed in 10.4% (w/v) of NR loaded ZWP nanoparticle suspension and incubated at 37˚C in a shaker for 1 hour to allow for spontaneous adhesion of nanoparticles. Fluorescent polystyrene (PS) nanoparticles (200nm; Phosphorex, Hopkinton, MA) was used as a positive control. Samples were processed as adhered particles to the tissue and non-adhered particles in the suspension. The samples were lyophilized followed by extraction of NR using 90% ethanol for analysis by fluorescence spectroscopy (Excitation wavelength 559nm and Emission wavelength 629nm). The percent bioadhesiveness was determined using the following equation: % 𝐵𝑖𝑜𝑎𝑑ℎ𝑒𝑠𝑖𝑣𝑒𝑛𝑒𝑠𝑠 =

𝐹𝑙𝑢𝑜𝑟𝑒𝑠𝑐𝑒𝑛𝑐𝑒 𝑖𝑛𝑡𝑒𝑛𝑠𝑖𝑡𝑦 𝑜𝑓 𝑡𝑖𝑠𝑠𝑢𝑒 𝑎𝑑ℎ𝑒𝑟𝑒𝑑 𝑛𝑎𝑛𝑜𝑝𝑎𝑟𝑡𝑖𝑐𝑙𝑒𝑠 𝑋100 𝑇𝑜𝑡𝑎𝑙 𝑓𝑙𝑢𝑜𝑟𝑒𝑠𝑐𝑒𝑛𝑐𝑒 𝑖𝑛𝑡𝑒𝑛𝑠𝑖𝑡𝑦 𝑜𝑓 𝑎𝑑ℎ𝑒𝑟𝑒𝑑 𝑎𝑛𝑑 𝑛𝑜𝑛 − 𝑎𝑑ℎ𝑒𝑟𝑒𝑑 𝑛𝑎𝑛𝑜𝑝𝑎𝑟𝑡𝑖𝑐𝑙𝑒𝑠

2.11.2. Bioadhesion assay To confirm the bioadhesive property of ZWP nanoparticles, additional studies were conducted using a texture analyzer (TA. HD Plus, Texture Technologies Corp. Scarsdale, NY). The bioadhesive property of nanoparticles was determined by measuring the maximum force required to separate the nanoparticles from mucosal surface of intestinal tissue.33 Polystyrene


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nanoparticle (PS) (200nm; Phosphorex Inc., Hopkinton, MA, USA) was used as a positive control. Briefly, the metal-head probe was coated by dip and dry method using 5% (w/v) aqueous nanoparticle dispersion.33 A thin uniform layer of the coat was obtained by repeated cycles of dipping and drying for eight times. The coated probe was then fitted in the loading arm of the texture analyzer. Freshly collected piglet jejunum was washed with pre-warmed oxygenated PBS (pH 7.4) and placed below the probe. The coated metal probe was programmed to descend at 0.5 mm/s, until a final force of 5 g was achieved between the coated probe and the intestinal tissue. The probe was incubated for 7 minutes to allow for interaction with the intestinal tissue. The probe was ascended and the peak loads were recorded during the start of the fracture between the tissue and the probe. The fracture strength was determined using the tensile load at the beginning of the fracture and the value was normalized for the projected surface area (PSA). The following equation was used for calculating the PSA [52]: 1

PSA = 2 × 6 × 𝑎 × ℎ Where ‘a’ is the length of each side of the hexagonal metal head surface and ‘h’ is the radius of the probe surface. 2.12. In vivo gastrointestinal distribution Male Sprague-Dawley rats (6–8 weeks of age) weighing 230-250 g were used for the study. The animal experiments were carried out with approval from the Institutional Animal Care and Use Committee (IACUC) at SDSU. The animals were acclimatized for one week before starting the study. The animals had free access to food and water. Cy 5.5 near infra-red dye (Lumiprobe) was used for studying the intestinal distribution of ZWP nanoparticcles after oral administration. Free Cy 5.5 or Cy 5.5 dye loaded ZWP nanoparticles (100 µg) was dispersed in 2ml of water and administered using a flexible oral gavage needle under mild anesthesia (isoflurane). The



gastrointestinal distribution of Cy 5.5 loaded nanoparticles was determined at different time points from 0 to 24 hrs using in-vivo Xtreme Bruker Preclinical Imaging System (Bruker, Billerica, MA). To determine the distribution of the nanoparticles in the gastrointestinal tract, the animals were sacrificed at 6 or 24 hours, and the whole GIT was imaged using the in-vivo imager. At the end of the experiment, the animals were euthanized by CO2 asphyxiation. In a separate study rats were sacrificed at 6 hours after treatment and the jejunum was sectioned for visualization by CLSM. 2.13. Immunogenicity studies The immunogenicity of ZWP nanoparticles was tested in Balb/c mice. The experiment was conducted with approval from the University IACUC. Mice were divided into three groups with four animals in each group. Around 100µg of blank ZWP nanoparticles as administered by oral gavage using a feeding tube at 1st and 3rd week. Blood was collected from the retro-orbital plexus at 1st, 3rd and 5th week to measure serum IgG levels. Intestinal contents were collected at the end of 5th week and processed to determine the mucosal IgA levels. Serum samples were analyzed using ELISA. Briefly, 96-well plate was coated with 200ul of 0.1% (w/v) of ZWP nanoparticles and kept overnight at 4°C. Next day, the plate was washed with PBS buffer with 1% Tween 20 and blocked with 3% normal goat serum for 1 hour at 37°C. The plate was then washed with PBS buffer (pH 7.4), followed by incubation with 1/16 diluted mouse serum for 2 hours at room temperature. Normal goat serum (3%) was used as a negative control. Serum was then removed followed by washing (4 times) with PBS buffer and incubation with horseradish peroxidase (HRP) conjugated goat anti-mouse IgG for 1 hour. Similarly, HRPconjugated goat anti-mouse IgA antibody was used to determine the mucosal IgA levels. The plate was then washed for four times using PBS buffer and incubated with 100µL TMB (3, 3’, 5, 5’tetramethylbenzidine). The reaction was stopped after 10 minutes using 50µL 1M sulfuric acid


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and the optical density was recorded at 450 nm in a plate reader (SpectraMax2, Microplate reader, Molecular Devices, Sunnyvale, CA). 2.13. Pharmacokinetics study in rats The pharmacokinetic studies of drug loaded ZWP nanoparticles were conducted in rats. The animal experiments were carried out with approval from the University IACUC. Male Sprague Dawley rats (3–4 weeks of age) were used for the study. Rats with surgically implanted jugular vein catheter were purchased from Charles River (Wilmington, MA) and acclimatized for one week. After a 12 hour fasting, free drug suspension with 2% Tween 20, or drug loaded ZWP nanoparticles in water (LPV: 52 mg/kg and fenretinide: 20 mg/kg body weight) was administered by oral gavage to rats. Blood samples (200 µL) were collected up to 24 hrs in heparinized tubes. After centrifugation (15 minutes at 4000 rpm), the plasma was collected and stored at -80 °C until further analysis. The drug concentration in the plasma samples was determined by HPLC using a calibration plot generated using LPV and fenretinide in rat plasma. The drugs were extracted from the plasma by liquid-liquid extraction method using a mixture of ethyl acetate and n-hexane (50:50, v/v). An equal volume of the organic solvent mixture was added to the plasma and centrifuged to separate the supernatant drug solution. The extraction was repeated for three times to ensure complete drug extraction. The organic solvent was evaporated using dry nitrogen. The residue was then reconstituted in 90% ethanol and used for HPLC analysis. The lower limit for the detection of LPV and fenretinide was 20 and 50 ng/mL respectively, and the extraction efficiency was > 90% for both drugs. The pharmacokinetic parameters including peak concentration (Cmax), time to reach peak concentration (Tmax), area under the plasma concentration-time (AUC), and half-life (t1/2) were calculated by non-compartmental analysis using PK-solution software. The percent relative bioavailability (Frel%) was calculated using the following formula,



Frel (%) =

𝐴𝑈𝐶 (𝑑𝑟𝑢𝑔 𝑙𝑜𝑎𝑑𝑒𝑑 𝑍𝑊𝑃 𝑛𝑎𝑛𝑜𝑝𝑎𝑟𝑡𝑖𝑐𝑙𝑒𝑠) 𝐴𝑈𝐶 (𝑓𝑟𝑒𝑒 𝑑𝑟𝑢𝑔 𝑠𝑢𝑠𝑝𝑒𝑛𝑠𝑖𝑜𝑛)

Page 16 of 46

𝑋100

2.14. Statistical analysis All the experiments were conducted in triplicates using three independent batches of nanoparticles. The data is represented as a mean ± standard deviation. Student's t-test and analysis of variance (ANOVA) was performed using Minitab® statistical software (Minitab Inc., State College, PA) at a significance level of p

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