Food Protein Based CoreâShell Nanocarriers for Oral Drug Delivery

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Food Protein-based Core-Shell Nanocarriers for Oral Drug Delivery: Effect of Shell Composition on in-vitro and in-vivo Functional Performance of Zein Nanocarriers Mohammed S Alqahtani, MD SAIFUL ISLAM, Satheesh Podaralla, Radhey S Kaushik, Joshua J. Reineke, Tofuko Woyengo, and Omathanu Perumal Mol. Pharmaceutics, Just Accepted Manuscript • Publication Date (Web): 19 Jan 2017 Downloaded from http://pubs.acs.org on January 19, 2017

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Molecular Pharmaceutics

Food Protein-based Core-Shell Nanocarriers for Oral Drug Delivery: Effect of Shell Composition on in-vitro and in-vivo Functional Performance of Zein Nanocarriers Mohammed S. Alqahtania1#, Md. Saiful Islama1, Satheesh Podarallaa, Radhey S. Kaushikb, Joshua Reinekea, Tofuko Woyengoc, Omathanu Perumala* a

1

Department of Pharmaceutical Sciences, bDepartment of Biology and Microbiology/Veterinary and Biomedical Sciences, cDepartment of Animal Science, South Dakota State University, Brookings, SD 57007

Both authors contributed equally to this work.

#Current Address: Department of Pharmaceutics College of Pharmacy King Saud University P.O.Box 2457 Riyadh 11451, Saudi Arabia

*Corresponding Author: Phone: 605-688-4745, Fax: 605-688-5993 Email address: [email protected]

ACS Paragon Plus Environment


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ABSTRACT The study was aimed at systematically investigating the influence of shell composition on the particle size, stability, release, cell uptake, permeability and in-vivo gastrointestinal distribution of food protein-based nanocarriers for oral delivery applications. Three different core-shell nanocarriers were prepared using food-grade biopolymers including zein-casein (ZC) nanoparticles, zein-lactoferrin (ZLF) nanoparticles and zein-PEG (ZPEG) micelles. Nile red was used as a model hydrophobic dye for in-vitro studies. The nanocarriers had negative, positive and neutral charge respectively. All the three nanocarriers had a particle size of less than 200 nm and a low polydispersity index. The nanoparticles were stable at gastrointestinal pH (2-9) and ionic strength (10-200mM). The nanocarriers sustained the release of Nile red in simulated gastric and intestinal fluids. ZC nanoparticles showed the slowest release followed by ZLF nanoparticles and ZPEG micelles. The nanocarriers were taken up by endocytosis in Caco-2 cells. ZPEG micelles showed the highest cell uptake and transepithelial permeability followed by ZLF and ZC nanoparticles. ZPEG micelles also showed P-gp inhibitory activity. All the three nanocarriers showed bioadhesive properties. Cy 5.5, a near IR dye was used to study the in-vivo biodistribution of the nanocarriers. The nanocarriers showed longer retention in the rat gastrointestinal tract compared to the free dye. Among the three formulations, ZC nanoparticles was retained the longest in the rat gastrointestinal tract (≥24 hours). Overall, the outcomes from this study demonstrate the structure-function relationship of core-shell protein nanocarriers. The findings from this study can be used to develop food protein based oral drug delivery systems with specific functional attributes.

Keywords: Food proteins, Core-shell nanocarriers, Zein, Lactoferrin, Casein, oral delivery


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INTRODUCTION Oral delivery is the most common and convenient route of drug administration with a high patient compliance. Various physicochemical and physiological barriers can limit the oral bioavailability of drugs.1 These include poor water solubility, permeability and stability of the drug, among other physicochemical factors. The physiological variables include the gastrointestinal pH, digestive enzymes, mucus, transit time and permeability of different sections of the gastrointestinal tract.2 In addition, the efflux transporters and metabolic enzymes in the intestinal epithelial cells can limit the oral bioavailability of drugs.3,4 From a formulation perspective, it is important to mask the taste of the drugs, especially for pediatric patients.5 Various carriers including nanoparticles, micelles, emulsions and lipid based carriers have been explored to address the oral delivery challenges.6-9 Nanoparticles are versatile carriers due to the wide range of materials that can be used for drug encapsulation. These include different types of synthetic and natural polymers.6,7 The natural polymers are attractive carriers due to their biocompatibility and biodegradability. To this end, protein biopolymers have a wide-range of amino acid composition that can be utilized for chemical modification for various drug delivery application.10 Especially, the food proteins are potential oral delivery vehicles due to their good sensory attributes.11 Although water-soluble proteins such as gelatin and albumin have been widely explored as drug delivery carriers, they have limited ability to sustain the drug release.10

Zein, is one of the few water-insoluble natural proteins that has a high proportion (>50%) of hydrophobic amino acids (proline, alanine and leucine). It is derived from corn and is composed of α, β, γ and δ zein, classified based on the amino acid composition and solubility in hydroalcoholic solvents.12 The commercial zein is mainly composed of α-zein (22-27kDa).12 Zein is an US-FDA approved GRAS material widely used in the food and packaging industries



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to provide a moisture impervious barrier.13 Zein has been used to encapsulate hydrophobic compounds and sustain the release from microspheres, nanoparticles and nanofibers.14-18 Zein nanoparticles per se exhibit poor colloidal stability leading to particle aggregation.19,20 Therefore, hydrophilic or amphiphilic materials are required to stabilize and enhance the waterdispersibility of zein nanoparticles.20-23 To this end, the goal is to develop effective oral zein nanocarriers using food-grade biocompatible polymers as stabilizers.

Milk proteins consists of casein and whey proteins (lactoglobulin, lactoferrin and lactalbumin), which differ in their physicochemical properties.24 Casein is the major milk protein and is widely used as a stabilizer and emulsifier in dairy and food products.25 Beta casein can form micelles, and it has been explored as an oral drug delivery vehicle for hydrophobic drugs.26,27 Although sodium casein has been investigated as a stabilizer for zein nanoparticles, the resultant nanoparticles showed limited stability to particle aggregation.20-22 As opposed to sodium casein which consists of a mixture of three different types of casein, the goal of this study is to use beta casein to prepare a more stable zein nanoparticles.

Lactoferrin (LF) is an iron-binding glycoprotein that belongs to the transferrin family. It has several biological properties including anti-bacterial, anti-viral, anti-oxidant and anti-cancer activity.28 Lactoferrin has high nutritional value for infants and children as an iron transporter.29 It also has been used as targeting ligand for improving the delivery across blood-brain barrier and intestinal epithelial barrier through lactoferrin receptors.30,31


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Polyethylene glycol (PEG) is an FDA approved polymer that is used to modify the surface of nanoparticles and proteins.32 Further, PEG is a widely used water-soluble excipient in oral and other formulations.32 Earlier, we have reported the synthesis of PEGylated zein and demonstrated its ability to form self-assembled micelles with hydrophobic zein as the core and hydrophilic PEG chains as the shell.33 The PEG-zein (ZPEG) micelles enhanced the aqueous solubility and chemical stability of curcumin, a water-insoluble anti-cancer agent. Further, the curcuminloaded PEG-zein micelles showed higher cell uptake than free curcumin in drug resistant cancer cells and reduced the IC50 value of curcumin by 3-fold.33

The main goal of this study is to systematically investigate the influence of shell composition on the physicochemical and functional performance of zein nanocarriers for oral delivery applications. Three different core-shell nanocarriers were prepared including zein-casein (ZC) nanoparticles, zein-lactoferrin (ZLF) nanoparticles and zein-polyethylene glycol (ZPEG) micelles. The three nanocarriers differ in their surface charge and hydrophilicity. The influence of shell composition on the physical stability, enzymatic stability, release kinetics, cell/tissue uptake, intestinal permeability and in-vivo biodistribution of zein nanocarriers were evaluated. Nile red was used as a model hydrophobic compound for in-vitro studies, while Cy 5.5, a near infra-red dye was used for the in-vivo imaging studies.



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MATERIALS AND METHODS Materials White zein (Zein F-4000) was purchased from Freeman Industries Inc, (Tuckahole, NY, USA). Nil red, β-casein, and D-α-Tocopherol polyethylene glycol 1000 succinate (TPGS 1000) were purchased from Sigma-Aldrich Company (St. Louis, MO, USA). Trehalose was from Acros Organics (Bridgewater, NJ, USA). Pepsin, trypsin, sodium azide and Dulbecco’s Modified Eagle Medium were purchased from Fisher Scientific (Bridgewater, NJ, USA). Dialysis membrane (MWCO 10 kDa) was purchased from Spectrapor (Houston,TX, USA). ProLong Gold Antifade mounting medium with DAPI was purchased from Invitrogen-Molecular Probes (Eugene, OR, USA). Polyethylene glycol (PEG, 5kDa) was purchased from Jenkem Technology (Plano, TX, USA). Bovine lactoferrin was provided by Dr. Hasmukh Patel from the Department of Dairy Science. Cyanine 5.5 NHS ester was purchased from Lumiprobe (Hallandale Beach, FL, USA).

Preparation of Zein-casein (ZC) and Zein-lactoferrin (ZLF) nanocarriers The core-shell nanoparticles were prepared using the phase separation method based on the differential solubility of zein and the milk protein.34 Briefly, 15 mg of zein was dissolved in 90% ethanol containing 1mg of Nile red (NR) or Cy5.5 dye. The alcoholic phase was added dropwise under probe sonication (Sonic & Materials, Inc., Danbury, CT, USA) to an aqueous phase consisting of 0.1M citrate buffer (pH 7.4) and 0.2% w/v of milk protein (β-casein or lactoferrin). The resulting colloidal dispersion was placed on a magnetic stirrer (200 rpm) for 3 hours to evaporate the ethanol. The nanoparticles were separated using Millipore (EMD Millipore, Billerica, MA, USA) centrifugal filters (30 kDa MWCO) at 4000 rpm for 1 hour and washed with deionized water (20ml) 3-5 times to remove free Nile red. Trehalose (30 mg) was added as a cryoprotectant and the formulation was lyophilized. The lyophilized formulations were stored in the desiccator at 4°C until further use.


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Preparation of Zein-PEG (ZPEG) micelles PEGylated zein was synthesized using the method developed in our laboratory.33 Briefly, an equal weight ratio of zein and methoxy PEG succnimidyl succinate (5KDa) was dissolved in 90% ethanol and incubated overnight. The reaction was stopped by adding 1M glycine followed by dialysis (10kDa MWCO) for 24 hours against water to remove the free PEG. To prepare the micelles, NR or Cy5.5 was dissolved in 20 ml of 90% alcohol along with zein-PEG (5.5 x10-2 g/L) and the mixture was stirred overnight in a magnetic stirrer to allow for partitioning of the dye into the hydrophobic zein core. Ethanol was removed using a rotary evaporator and the resultant film was hydrated with citrate buffer (pH 7.4) to form zein-PEG micelles. The mixture was dialyzed against deionized water (100ml) to remove the free dye. The dye loaded ZPEG micelles was lyophilized and stored in a desiccator at 4°C until further use.

Characterization of core-shell nanoparticles The particle size, size distribution and zeta potential were determined using Malvern Zetasizer Nano-ZS (Malvern Instruments Inc., Southborough, MA) by dispersing the lyophilized nanoparticles in deionized water (0.1 mg/mL). For morphological analysis, the nanocarriers were analyzed by transmission electron microscopy (TEM). The specimens were prepared by placing a dilute dispersion of the nanoparticles on carbon-coated 200 mesh copper grid and allowed to evaporate overnight. TEM images were acquired using a Tecnai Spirit G2 TEM (FEI, Hillsboro, OR, USA), operated at an accelerating voltage of 120 kV. The electron micrographs were recorded using Orius SC200 CCD camera coupled to the TEM. Image analysis was performed using Digital Micrograph software.



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Determination of Encapsulation Efficiency Briefly 2 mg of NR loaded nanoparticles was dispersed in deionized water and centrifuged at 14000 rpm for 10 min. The supernatant was discarded and the pellet was dissolved in 90% alcohol. The concentration of NR was determined using a calibration curve (0.2 to 5µg/ml) of NR in 90% alcohol. The samples were analyzed by spectrofluorimetry (SpectromaxM2, Molecular Devices, Sunnyvale, CA) using 559 nm and 629 nm as the excitation and emission wavelength respectively. The encapsulation efficiency was calculated using the following equation NR Encapsulation Efficiency (%) =

x 100

In vitro release of Nile red from core-shell nanocarriers The release of NR from the nanocarriers was determined using the dialysis method. The in-vitro release was determined in simulated gastric (SGF) and intestinal fluid (SIF). SGF was prepared using 0.1 M HCl and 0.32% w/v pepsin (pH 1.2), while SIF was prepared using 0.05M KH2PO4 with 0.1 M NaOH (pH 7.5) and 1% w/v pancreatin. For release studies, 20 mg of NR loaded nanoparticles was suspended in 5 ml release medium and placed in a dialysis cassette (EMD Millipore, 10KDa MWCO). The cassette was placed in 200 ml release medium containing Tween 80 (0.1% v/v) to maintain sink conditions. At pre-determined time intervals, 1 ml of the release medium was removed and replaced with an equal volume of pre-warmed release medium. The sample was mixed with 1ml of ethanol to maintain same solvent matrix as 50% ethanol as standard curve and the concentration of NR was determined by spectrofluorimetry as described earlier. The NR concentration was corrected for the replaced dissolution medium while calculating the cumulative amount of NR released from the nanocarriers.


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Cell culture Caco-2 cells (ATCC, Rockville, MD, USA) cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) (4.5 g/L glucose) supplemented with 20% FBS (HyClone; Thermo Scientiﬁc, Waltham, MA, USA), 1% non-essential amino acids, 1% L-glutamine, 1% streptomycin (100 µg/ml) and penicillin (100 IU/ml). The growth medium was changed every day in the first two weeks followed by replacement of the medium three times a week.

In-vitro transepithelial transport of core-shell nanocarriers Caco-2 cells (1×106 cells/well) were seeded onto Transwell polyester filter membranes (pore size 0.4 µm, surface area 4.67 cm2) in a 6-well plate (Transwell®, Corning Costar Corp., Cambridge, MA, USA). The cells were grown in an atmosphere of 5% CO2 and 95% air with 95% relative humidity at 37°C. The cells were cultured for three weeks and the formation of monolayer was confirmed from the transepithelial electrical resistance (TEER) (EVOM meter, Sarasota, FL). Before starting the experiment, the medium in the apical side was replaced with 2mg/ml of the nanoparticles/micelles in Hank’s balanced salt solution (HBSS) and the permeability of NR loaded nanoparticles/micelles was determined for 6 hours. In preliminary studies with other similar zein nanoparticles, 2mg/ml showed maximal transport (data not shown) and hence this concentration was used for the transport studies. Samples (1ml) were withdrawn from the basolateral compartment at pre-determined time points and replaced with fresh buffer solution. The apparent permeability coefficient (Papp) was calculated using the following equation Papp (cm/s) = (dQ/dt)/(A × C0)



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Where dQ/dt is the flux of NR across Caco-2 monolayer, C0 is the initial concentration of NR in the apical chamber, and A is the surface area of the Transwell membrane (4.67 cm2). Cell uptake of NR loaded nanocarriers Caco-2 cells were seeded in a 6-well plate at a concentration of 2 x 105 cells/well (passage number 10-15). Next day, the culture medium was removed and the cells were treated with nanocarriers (equivalent to1 µg of Nile red) in HBSS for 30 minutes to 2 hours at 37°C. At the end of the treatment period, the cells were washed with HBSS three times followed by trypsinization. The cells were washed with ice cold HBSS and fixed using 2% paraformaldehyde. The mean fluorescence intensity was analyzed by flow cytometry (FACS, BD biosciences, San Jose, CA). To determine if the cell uptake of the nanocarriers was energy dependent, the cell uptake at 4°C was compared with the cell uptake at 37°C. In a separate set of experiments, Caco-2 cells were pre-incubated with endocytosis inhibitors including 1µM phenylarsine oxide (PAO) and 4µM Filipin for 30 minutes followed by cell uptake experiments for 2 hours. For ZLF nanocarriers, the cells were treated with (2 mg/ml) bovine lactoferrin for 1 hour before incubation with ZLF nanocarriers to determine the involvement of lactoferrin receptors in cellular uptake.

P-gp inhibition assay Caco-2 cells were seeded in 96-well plate overnight at a cell density of 1x104cells/ml. The following day, cells were treated with 50 µL of blank nanocarriers diluted in HBSS buffer for 30minutes. Cells were washed three times followed by addition of 200µl of 0.5 µM calcein AM, a P-gp substrate (Molecular Probes, Eugene, OR, USA). Then the cells were washed two times with ice-cold HBSS and lysed using 0.5% Triton-X100. A control experiment was performed


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following the same procedure, but without the addition of nanocarriers. The calcein uptake was measured by spectrofluorimetry using 485 nm and 530 nm as excitation and emission wavelength respectively. To determine P-gp inhibition, the relative calcein fluorescence intensity was calculated for the nanocarrier treated and non-treated groups using the following equation. % Relative Fluorescence =

[ ] x 100

Cell uptake studies using confocal laser scanning microscopy To visualize the cell uptake of core-shell nanocarriers, Caco-2 cells (5x104 cells/well) were seeded in a chamber slide (Nunc Lab-TEK®II Chamber SlideTM system, Thermo Scientific, Waltham, MA) and was incubated overnight. Next day, the growth medium was replaced with HBSS buffer (pH 7.4), and equilibrated for 30 min at 37°C. Then, the nanoformulation (equivalent to 1µg of Nile red) was added to the wells and incubated for 30-60 min. At the end of treatment period, the cells were washed three times with cold HBSS to remove the surface adsorbed nanoparticles. Cells were then fixed using 4% paraformaldehyde for 20 min at room temperature followed by staining of F-actin with Alexa Fluor 488-Phalloidin (Life Technologies, Carlsbad, CA).

DAPI was used to stain the nucleus. Images were taken using Olympus

FluoView 1200 (FV 1200) confocal laser scanning microscope (CLSM) at a 60x magnification.

Ex vivo bioadhesion assay The mucoadhesive properties of the core-shell nanocarriers were determined using everted intestinal sac.35 Pig jejunum was procured from the Department of Animal Science at South Dakota State University. A section of the freshly collected pig jejunum (6 cm length) was



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everted using a glass rod to expose the mucosal side and the open ends were tied to form a sac. The loose mucus was removed from the intestine. The sac was filled with phosphate buffered saline-glucose (PBSG). The everted sac was placed in a suspension of the nanocarrier (100 mg/ml in PBSG) and incubated at 37˚C in a shaker water-bath for 1 hour to allow time for the nanoparticles to adhere to the tissue. Fluorescent labeled polystyrene nanoparticles (200nm, Phosphorex, Hopkinton, MA, USA) were used as a positive control. Following incubation, the everted sac was removed and processed. The adherent mucus and the remaining suspension collected as bound and unbound fractions, respectively for determining NR concentration by spectrofluorimetry. The ratio of NR concentration in the bound and unbound nanoparticles was used to calculate the percent bioadhesion.

Immunogenicity studies Female Balb/c mice (4 weeks old) were used for the immunogenicity study. The animal experiments were conducted with approval from the Institutional Animal Care and Use Committee (IACUC). Mice were divided into five groups with four animals in each group. Nanocarrriers equivalent to 100µg of total protein was administered orally using an oral feeding tube at 0 and 3rd week. Blood samples were collected from the retro orbital plexus at 0, 3rd and 5th week. Intestinal contents were also collected at the end of the study and processed for further analysis.

Serum samples were analyzed for anti-zein, anti-casein, anti-lactoferrin, anti-PEG IgG antibodies by measuring the optical density (OD) value. Briefly, 96 well plates were coated with 200ul of 0.1% (w/v) total protein containing ZC, ZLF nanoparticles and ZPEG micelles for overnight at


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4°C. After overnight incubation, the plates were washed with buffer (PBS in 1% tween 20) and blocked with 3% normal goat serum for 1 hour at 37°C. The second washing step was done using PBS buffer (pH 7.4), followed by incubation with 1/16 diluted mouse serum for 2 hours at room temperature. The intestinal samples were used directly. The plate was washed with PBS buffer (4 times) and incubated with Horseradish Peroxidase (HRP) conjugated goat anti-mouse IgG for 1 hour. HRP-conjugated goat anti-mouse IgA was used to determine IgA level in the intestinal contents. Then the plate was washed four times using PBS buffer and incubated with 100µL TMB (3, 3’, 5, 5’-tetramethylbenzidine) substrate. The reaction was stopped after 10 minutes using 50µL 1M H2SO4 and the optical density was measured at 450 nm in a plate reader (SpectraMax2, microplate reader, Molecular devices, Sunnyvale, CA).

In vivo oral biodistribution To determine the in-vivo oral biodistribution of core-shell nanoparticles, Cy 5.5, a near infra-red dye was used. Male Sprague Dawley rats (6–8 weeks of age, 200-250 g) were used for the study. The animal studies were conducted after approval from the Institutional Animal Care and Use Committee. The animals were acclimatized one week prior to start of the study and had free access to water and food. Cy 5.5 loaded nanoparticles (50µg) were dispersed in water (2ml) and administered using a flexible oral gavage tube under mild isoflurane anesthesia. The timedependent biodistribution (2, 6, 12, and 24 hrs) of nanoparticles were determined using the invivo Imaging System (Xtreme, Bruker, Billerica, MA, USA). The free dye was used as a control. The animal was placed in the imaging chamber and a short exposure of x-ray was used to record the anatomy followed by determination of fluorescence. After 6 and 24 hrs, the animals were



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sacrificed and the gastrointestinal tract (GIT) was separated and the fluorescence was recorded using the in-vivo imaging system.

To determine the tissue uptake of Cy 5.5 loaded nanoparticles, the rat was sacrificed at 6 hrs and the jejunum was collected. The tissue fixed by incubating with 4% paraformaldehyde overnight at 4°C. The fixed tissue was embedded using optimal cutting temperature medium (OCT) and 10µm sections were prepared using a cryomicrotome (Leica, Buffalo Grove, IL). F-actin was stained with Alexa Fluor 488-Phalloidin and the nucleus was stained with DAPI. The images were taken at 20x magnification.

Statistical analysis All the experiments were conducted in triplicate. Data is represented as the mean ± standard deviation. Statistical evaluation was performed using one-way analysis of variance (ANOVA) in Minitab® (Minitab Inc., State College, PA) at a significance level of p < 0.05.


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RESULTS AND DISCUSSION Formulation and characterization of core-shell nanocarriers The phase separation method was used to prepare ZC and ZLF nanoparticles. Zein is soluble in the hydroalcoholic solvent (90% alcohol), but is insoluble in the aqueous phase.12 On the other hand, β-casein and lactoferrin are insoluble in the hydroalcoholic phase.24 The phase change leads to the colloidal aggregation of hydrophobic zein and self-assembly of milk proteins (βcasein and lactoferrin) around the zein core. Stirring allowed for the evaporation of alcohol and self-assembly of core-shell nanoparticles. The zein nanoparticles prepared in the absence of a stabilizer resulted in a larger particles and formed aggregates that were difficult to disperse in water.19,20,23, 34, 36, 37 Similarly, the core/shell mass ratio of 1:1 of zein/ β-casein or zein/lactoferrin resulted in larger particles (data not shown). An increase in core/shell ratio to 1:2 and 1:4 respectively for ZC and ZLF nanoparticles resulted in stable particles in the size range of 100 to 200nm with a uniform size distribution (Table 1). The results suggest that at these ratios there is an optimal coverage of the zein hydrophobic core. The results are consistent with other polymer core-shell nanoparticles reported in the literature.38, 39

Since the proteins are amphiphilic, they can interact with other proteins selectively through hydrophobic or hydrophilic amino acids to form self-assembled structures.40 Similarly, the proteins can also interact with other polymers to form core-shell nanoparticles.39 Hu et al have reported the preparation and characterization of zein-pectin core-shell nanoparticles for oral delivery of curcumin.39 The ZPEG film when hydrated with water form self-assembled micelles with an average size of 100nm (Table 1). The hydrophobic zein forms the core and the PEG chains form the outer shell. Earlier, we have demonstrated the core-shell architecture of ZPEG



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micelles using NMR spectroscopy. 33 To ensure there are no residual traces of ethanol in the final formulation, we determined the ethanol concentration in the three formulations by GC-MS. There was no detectable concentration of ethanol in the final formulation (data not shown).

All the three nanocarriers had a uniform size distribution, as seen from the low polydispersity index (Table 1). Polydispersity index in the range of 0.1-0.4 is indicative of a monodisperse particles.41As can be seen from the TEM images, the nanocarriers were spherical with a smooth morphology and a core-shell structure (Fig. 1). Although the exact thickness of the shell layer was not determined, lactoferrin appeared to form a thicker shell around the zein core (Fig. 1B). Unlike β-casein, lactoferrin is a glycoprotein with a larger molecular weight (80 kDa), which may explain the difference in the shell thickness. This can also be attributed to the relatively larger particle size of ZLF nanoparticles (175nm) compared to the other two core-shell nanocarriers (Table 1). TEM is a widely-used method to characterize core-shell nanoparticles.42 The formation of core-shell architecture was further confirmed from the zeta potential values (Table 1). The negative zeta potential of ZC nanoparticles is attributed to β- casein, while lactoferrin imparted positive charge to the ZLF nanoparticles. The ZPEG micelles had a very weak negative charge. Particles with zeta potential values around ±30mV are stable, which is consistent with the zeta potential values of ZC and ZLF nanoparticles (Table 1). 41 On the other hand, although ZPEG micelles have a low zeta potential, the PEG chains can provide a steric barrier to prevent particle aggregation. 43

The size of the nanoparticles determined by TEM was relatively smaller than the particle size determined by dynamic light scattering. This is due to the difference between these two


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techniques. The dynamic light scattering method measures the hydrodynamic radius of the particles, while the TEM measures the projected diameter of the particle in the dry state. The drying can shrink the particles leading to reduction in the particle size. 41 Nevertheless, all the three nanocarriers have the optimal size range (100-200nm) for enhancing the drug solubility and permeability through the intestinal membrane.44,45

The nanocarriers were found to be stable at various pH encountered in the gastrointestinal tract with no significant particle aggregation (Table S1-S3). Casein (pI= 4.6) is positively charged at the acidic pH in stomach and is negatively charged in the alkaline pH of the intestine.46 Earlier studies have reported the use of sodium casein to stabilize zein nanoparticles, but the particles were found to aggregate at the isoelectric point of casein (pH 3-5).19-21 Sodium casein contains a mixture of α, β and κ caseins, which differ in physicochemical properties.20 The caseins differ in the number of hydrophilic and hydrophobic domains.21 Patel et al prepared zein-sodium casein nanoparticles using electrostatic interaction between zein and sodium casein by controlling the pH of the solution.21 Unlike sodium casein, the β-casein used in the present study formed stable zein nanoparticles through hydrophobic interactions. Since lactoferrin has a high pI (pI=8.7) it remains positively charged throughout the entire pH range of the gastrointestinal tract.28 Zein, which is negatively charged at pH 7.4 (pI=6.8) 34, is stabilized through electrostatic interaction with the positively charged lactoferrin.

At lower pH (pH

Food Protein Based CoreâShell Nanocarriers for Oral Drug Delivery

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Food Protein Based CoreâShell Nanocarriers for Oral Drug Delivery