Fabrication of Biopolymer Based Nanoparticles for the Entrapment of Chromium and Iron Supplements

processes

Article Fabrication of Biopolymer Based Nanoparticles for the Entrapment of Chromium and Iron Supplements

Nishay Patel 1, Mohammed Gulrez Zariwala 2 and Hisham Al-Obaidi 1,*

1 The School of Pharmacy, University of Reading, Reading RG6 6AD, UK; [email protected] 2 Faculty of Science & Technology, University of Westminster, 115 New Cavendish Street, London W1W 6UW, UK; [email protected] * Correspondence: [email protected]; Tel.: +44-11-8378-6261

Received: 20 May 2020; Accepted: 15 June 2020; Published: 19 June 2020

Abstract: The objective of this study was to encapsulate iron and chromium into novel nanoparticles formulated using chitosan (CS), dextran sulfate (DS) and whey protein isolate (WPI) for oral drug delivery. Empty and loaded CS-DS nanoparticles were prepared via complex coacervation whilst whey protein nanocarriers were produced by a modified thermal processing method using chitosan. The physiochemical properties of the particles were characterized to determine the effects of formulation variables, including biopolymer ratio on particle size and zeta potential. Permeability studies were also undertaken on the most stable whey protein–iron nanoparticles by measuring Caco-2 ferritin formation. A particle size analysis revealed that the majority of samples were sub-micron sized, ranging from 420–2400 nm for CS-DS particles and 220–1000 nm for WPI-CS samples. As expected, a higher chitosan concentration conferred a 17% more positive zeta potential on chromium-entrapped WPI nanoparticles, whilst a higher dextran volume decreased the size of CS-DS nanoparticles by 32%. The addition of iron also caused a significant increase in size for all samples, as seen where the loaded WPI samples were 296 nm larger than the empty particles. Caco-2 iron absorption revealed that one formulation, which had the lowest particle size (226 10 nm), caused a 64% greater iron ± absorption compared to the ferrous sulfate standard. This study describes, for the first time, the novel design of chromium- and iron-entrapped nanoparticles, which could act as novel systems for oral drug delivery.

Keywords: iron; chromium; whey protein isolate; nanoparticles; oral drug delivery

1. Introduction Iron and chromium have major roles in the metabolism and oxygen exchange processes [1]. Chromium is a trace element that has received a significant interest in recent years. It aids in the metabolism of carbohydrates and is thought to impair glucose tolerance in those with chromium deficiencies. This can lead to the development of maturity-onset diabetes-a chronic condition costing the national health service (NHS) over nine billion Pounds sterling annually [2,3]. Chromium supplements can be used to prevent insulin resistance and may be seen in the future as a cost effective strategy to reduce the onset of type 2 diabetes [4]. Iron is an essential mineral that plays a vital role in oxygen transport and DNA replication [5]. Almost 20% of the world’s population is iron deficient—a condition that is prevalent in most countries [6]. Iron is lost from the body through the natural shedding of skin, during pregnancy and heavy menstruation but cannot be synthesized in vivo [7]. Iron is also poorly absorbed from dietary sources meaning that supplementation may be needed to maintain healthy levels. In previous studies, iron-entrapped nanoparticles were formulated using ascorbic acid which augments the absorption of dietary non-haem iron by reducing ferric iron to the bioavailable ferrous form [8–10].

Processes 2020, 8, 707; doi:10.3390/pr8060707 www.mdpi.com/journal/processes Processes 2020, 8, 707 2 of 16 Processes 2020, 8, x FOR PEER REVIEW 2 of 17

Nanoparticles areare defineddefined as as solid solid colloidal colloidal structures structures ranging ranging in sizein size from from 1 to 1 1000 to 1000 nm. nm. They They can becan formed be formed of natural of natural polymers polymers such as such chitosan as chitosan and have and a number have a ofnumber advantages of advantages including enhancingincluding theenhancing stability the of drugs,stability increasing of drugs, encapsulation increasing encaps efficiencyulation and efficiency having better and uptakehaving inbetter the small uptake intestine. in the Thissmall is intestine. because nanoparticlesThis is because can nanoparticles permeate enterocyte can permeate membranes enterocyte easier thanmembranes microparticles easier than and canmicroparticles facilitate the and controlled can facilitate release the of thecontrolled encapsulated release drug of [11the,12 encapsulat]. These qualitiesed drug are [11,12]. of particular These importancequalities are for of particular this study importance because chromium for this stud is poorlyy because absorbed chromium (<2%) is [13 poor]. Therefore,ly absorbed nanoparticles (<2%) [13]. wereTherefore, formulated nanoparticles because were they canformulated improve because drug bioavailability they can improve and reduce drug toxicitybioavailability whilst maintaining and reduce therapeutictoxicity whilst eff ectsmaintaining [14]. therapeutic effects [14]. Biopolymers are naturally abundant polymers and possess a broad range of attributes which can be used to optimize the properties of nanoparticles. In In this this study, study, chitosan (CS), dextran sulfate (DS) and wheywhey protein protein isolate isolate (WPI) (WPI) were were chosen chosen due todu theme to being them cost being effective cost andeffective generally and recognized generally asrecognized safe (GRAS) as safe [15]. (GRAS) CS is a polysaccharide[15]. CS is a polysaccharide produced from produced the exoskeleton from the of exoskeleton crustaceans of by crustaceans the partial deacetylationby the partial ofdeacetylation chitin [16]. It of is composedchitin [16]. of It repeatingis composed glucosamine of repeating units glucosamine and is a biodegradable units and andis a pharmaceuticallybiodegradable and acceptable pharmaceutically excipient acceptable (Figure1). CSexcipient has a pKa (Figure of 6.5 1). and CS possesses has a pKa a high of ratio6.5 and of aminopossesses groups a high that ratio undergo of amino protonation groups in that acidic undergo conditions, protonation making itin soluble acidic inconditions, water (Figure making1)[ 17 it]. Itsoluble has mucoadhesive in water (Figure properties 1) [17]. It and has can mucoadhesi complexve with properties chromium and to can produce complex nanoparticles. with chromium DS to is aproduce polyanionic nanoparticles. polymer which DS is possesses a polyanionic negatively poly chargedmer which sulfate possesses groups, negatively dictating its charged ability tosulfate form stablegroups, coacervates dictating its with ability CS [to18 form]. It is stable highly coacervates hydrophilic with and CS has [18]. been It is used highly to control hydrophilic the release and has of drugsbeen used such to as control doxorubicin the release [19]. Complexof drugs coacervationsuch as doxorubicin was chosen [19]. as Complex opposed coacervation to ionic gelation was because chosen theas opposed nanoparticles to ionic have gelation enhanced because mechanical the nanopa strengthrticles and stabilityhave enhanced than those mechanical traditionally strength produced and usingstability CS than and those sodium traditionally triphosphate produced [20]. using CS and sodium triphosphate [20].

Figure 1. 1. ChemicalChemical structures structures of (a of) chitosan (a) chitosan and (b and) dextran (b) dextran sulfate sulfatewith indication with indication of their ionizable of their ionizablegroups. groups.

Whey protein is a by-product of the cheesecheese manufacturingmanufacturing process and consists of globular proteins, mainly β-lactoglobulin. It It has has excellent gelation gelation pr propertiesoperties and is widely used by athletes to stimulate muscle muscle growth. growth. Hence, Hence, it it can can work work synergistically synergistically with with chromium, chromium, which which is issuggested suggested it itcan can increase increase muscle muscle mass, mass, to to improve improve nutritional nutritional health health and and athletic athletic performance performance [21]. [21]. Therefore, chromium-entrapped nanoparticles were produced using whey protein isolate through thermal processing. Y. Y. Chen was the firstfirst to fabricate and entrap an arginine-richarginine-rich hexapeptide into CS-DS nanoparticles [[22].22]. ThisThis increasedincreased thethe popularitypopularity of of biopolymer-based biopolymer-based nanoparticle nanoparticle research research and and led led to furtherto further developments developments in the in fieldthe field such such as the as encapsulation the encapsulation of insulin of insulin into CS-based into CS-based nanoparticles nanoparticles [23,24]. [23,24].The aim of this study was to build on previous research by fabricating four types of novel chromium-The aim and of iron-entrappedthis study was nanoparticlesto build on previous using di researchfferent ratios by fabricating of biopolymers four types for oral of novel drug delivery.chromium- The and properties iron-entrapped of the nanoparticlesnanoparticlessuch using as di size,fferent charge ratios and of stability biopolymers were thenfor oral assessed drug todelivery. find the The optimum properties formulations. of the nanoparticles Permeability such studies as size, werecharge also and performed stability were using then Caco-2 assessed cells to determinefind the optimum the differences formulations. in ironabsorption Permeability between studies the were formulations. also performed using Caco-2 cells to determine the differences in iron absorption between the formulations.

2. Materials and Methods

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2. Materials and Methods

2.1. Materials Low and medium molecular weight chitosan (75% deacetylated, 50/190 kDa), dextran sulfate (190 kDa), chromium (III) chloride hydrate (96%), acetic acid and ferrous sulfate heptahydrate were purchased from Sigma-Aldrich Co. (Dorset, UK). Unﬂavored whey protein isolate was obtained from Davisco Foods International Inc. (Eden Prairie, MN, USA). Sodium hydroxide pellets were purchased from VWR international (Philadelphia, PA, USA). All nanosuspensions were prepared using deionized water. Caco-2-cells (HTB-37) were obtained from ATCC (Manassas, VA, USA). Ferritin ELISA kit (Catalog Number: S22) was purchased from Ramco (Chichester, UK) and Pierce BCA protein assay kit was from Thermo Fisher Scientiﬁc (Cramlington, Northumberland, UK). Permeability studies were undertaken using Milli-Q water.

2.2. Preparation of the Nanoparticles

2.2.1. Preparation of CS–DS Nanoparticles Empty CS–DS nanoparticles were prepared by complex coacervation method with some modification [19]. To study the effects of CS:DS ratio on particle properties, 0.1% (w/v) chitosan and dextran solutions were prepared. First 200 mg low molecular weight chitosan was dissolved in 0.175% (v/v) acetic acid whilst 200 mg dextran was dissolved in deionized water. Nanoparticles formed spontaneously when 10 mL chitosan was added to various volumes of dextran (Table1) whilst magnetically stirring (300 rpm) for 15 min at room temperature. Chromium-entrapped nanoparticles were formulated by adding 0.1% (w/v) chromium hydrate to different volumes of 0.1% dextran solution. In total, 10 mL chitosan was then mixed with the chromium-dextran solution under magnetic stirring to form the nanoparticles. Iron-entrapped nanoparticles were produced in a similar fashion where ferrous sulfate was substituted for chromium. Additional formulations were also prepared using low molecular weight chitosan (LMW CS) and medium molecular weight chitosan (MMW CS) for all samples. Empty preparations were coded: LMW CS and MMW CS whilst chromium and iron-entrapped samples were called: LMW CS-Cr, MMW CS-Cr, LMW CS-Fe and MMW CS-Fe.

Table 1. Composition of chitosan–dextran sulfate (CS-DS) nanoparticle formulations (not determined (ND)).

Type of CS:DS Chitosan Dextran Chromium Ferrous Loading Nanoparticle Ratio Volume (mL) Volume (mL) Hydrate (mg) Sulfate (mg) Eﬃciency (%) empty 4:1 10 2.5 - - - empty 4:2 10 5.0 - - - empty 4:3 10 7.5 - - - Cr entrapped 4:1 10 2.5 12.5 - ND Cr entrapped 4:2 10 5.0 15.0 - ND Cr entrapped 4:3 10 7.5 17.5 - ND Fe entrapped 4:1 10 2.5 - 12.5 13 2.3 ± Fe entrapped 4:2 10 5.0 - 15.0 21 4.5 ± Fe entrapped 4:3 10 7.5 - 17.5 47 5.2 ±

2.2.2. Preparation of Whey Protein-Chitosan Nanoparticles Empty nanoparticles were produced using an adapted method developed by Giroux et al. [25]. First 2.5% (w/v) whey protein isolate solution was prepared by dispersing 2 g WPI in 80 mL deionized water. This was magnetically stirred at room temperature for 2 h to ensure complete dissolution of the WPI powder. Next, 0.1% (w/v) low molecular weight (LMW) chitosan solution was added dropwise to various volumes of the WPI solution whilst stirring at room temperature (Table2). The sample was then heat-treated at 85 ◦C for 20 min and left to cool at room temperature for 15 min whilst magnetically stirring (~300 rpm). The pH was then adjusted to 7.5 using 10 mM NaOH. The same method was undertaken using medium MW chitosan. Processes 2020, 8, 707 4 of 16

Table 2. Composition of empty and chromium-loaded whey protein isolate (WPI)-CS nanoparticle formulations.

Sample WPI:CS Ratio Whey Protein (mL) Chitosan (mL) Chromium Hydrate (mg) empty 50:1 10.0 5.0 - empty 125:1 12.5 2.5 - empty 350:1 14.0 1.0 - Cr loaded 50:1 10.0 5.0 5 Cr loaded 125:1 12.5 2.5 5 Cr loaded 350:1 14.0 1.0 5

Chromium-entrapped nanoparticles were produced using a similar procedure. First 5 mg chromium hydrate was fully dissolved into 2.5% WPI solution. Then 0.1% chitosan was added dropwise whilst stirring. The temperature was then increased to 85 ◦C and the pH adjusted. Iron-entrapped nanoparticles were produced in a similar way to that stated above for chromium. Previous studies were used to determine the masses of iron and ascorbic acid to use [26,27]. These studies stated that an elemental iron to ascorbic acid ratio of 1:5 was required to suﬃciently increase iron absorption. Therefore, since 10 mg ferrous sulfate contained 2 mg elemental iron, 10 mg ascorbic acid was used (Table3). These were both dissolved in varying volumes of WPI solution following the same method shown above with a ﬁnal iron concentration of 2.3 mM.

Table 3. Composition of iron loaded WPI-CS nanoparticle formulations.

WPI:CS Ratio Whey Protein (mL) Chitosan (mL) Ferrous Sulfate (mg) Ascorbic Acid (mg) Loading Eﬃciency (%) 50:1 10.0 5.0 10 10 29 4.3 ± 125:1 12.5 2.5 10 10 38 4.1 ± 350:1 14.0 1.0 10 10 54 3.5 ±

Additional formulations were produced for chromium and iron WPI-CS nanoparticles using 0.1% (w/v) medium MW chitosan (MMW CS-Cr, MMW CS-Fe) and 0.2% (w/v) low MW chitosan (0.2% LMW CS-Cr, 0.2% LMW CS-Fe) at each ratio. These were prepared using the same quantities and volumes as seen in Tables2 and3.

2.3. Characterization of the Nanoparticles

2.3.1. Particle Size Analysis Particle size was determined using dynamic light scattering (Zeta Plus analyzer, Brookhaven instruments, Holtsvilleg, Suﬀolk County, NY, USA). CS-DS nanoparticles were diluted appropriately with deionized water and sonicated (Fisher brand) at 80 kHz (pulse mode) for 1 min before measurement to prevent particle aggregation. WPI-CS nanoparticles were diluted in the same manner before analysis to avoid multiple scattering. All measurements were carried out at room temperature with each result being reported as the mean of 10 runs.

2.3.2. Zeta Potential Measurements Zeta potential analysis was undertaken using electrophoretic light scattering (Zetasizer nanoseries, Malvern Instruments Ltd., Malvern, UK). Every sample was diluted to the same extent as in size analysis using deionized water before measurement. All results were recorded as the mean of three repeats.

2.3.3. Transmission Electron Microscopy (TEM) The morphology of the nanoparticles was observed with a Philips Tecnai-12 FEI Transmission Electron Microscope (TEM) using an accelerating voltage of 120 kV. The TEM samples were prepared by depositing a drop of suspension on a carbon-coated copper grid and evaporating the liquid for 5–10 min prior to examination in the microscope. Processes 2020, 8, 707 5 of 16

2.4. Caco-2 Cell Iron Absorption Iron absorption studies were undertaking using a method published by Zariwala et al. [8]. Four WPI-CS iron samples (which were the most physically stable over time) were assessed along with a ferrous sulfate standard. The four iron samples were—350:1 LMW CS-Fe, 350:1 MMW CS-Fe, 175:1 LMW CS-Fe and 125:1 LMW CS-Fe. Caco-2 cells were seeded onto a 6-well plate with an initial seeding density of 5 104 cells/cm2. × Permeability studies commenced on day 13 post seeding so that the cells expressed characteristics of a fully matured enterocyte lining. First, the growth media was removed, and the cells were washed with wash solution (140 mM NaCl, 5 mM KCl, 10 mM piperazine-N,N0-bis (2-ethanesulfonic acid) buffer, pH 6.7, 37 ◦C). They were then incubated in serum-free Minimum Essential Media (MEM) for 24 h. On day 14, test media was prepared by adding 1 M NaOH to 250 mL serum free MEM until a pH 5.8 solution was obtained. This pH was chosen due to it mimicking the pH of the duodenum. The media was then filter sterilized (0.2 µm pore size) and aliquoted into centrifuge tubes. Next, 226 µL of each nanoparticle sample was added into the different tubes containing test media so that a final elemental iron concentration of 20 µM was achieved. This iron concentration was chosen due to it being the optimum conditions for the expression of divalent metal transporters in Caco-2 cells [26]. This meant that slight changes in iron absorption between the samples could be detected [8]. The cells were washed with wash solution before test media was added in duplicate to a 6-well plate. The plate was then incubated for 2 h at 37 ◦C in a rotary shaker (Thermo Scientific 4450, Waltham, Massachusetts, United States) at 6 rpm. Growth media (serum free MEM, pH 5.8) was then added and the cells were incubated for 24 h. After this period, removal solution (5 µM sodium hydrosulfite and 1 µM bathophenanthroline disulfonic acid) was added to remove surface bound iron. Cell lysis was then undertaken by adding 350 µL lysis buffer (50 mM NaOH, 10 µL protease inhibitor cocktail, 1 µL benzonase) to each well. The plate was then incubated at 4 ◦C on a plate rocker (8 rpm) for 25 min. A cell scraper was used to collect cell lysate which was pipetted into vials. Each lysate was then passed six times through a 1 mL syringe (25G needle) to reduce their viscosity.

2.5. Iron Absorption Analysis and Determination of Loading Efficiency The total ferritin concentration was determined using a ferritin enzyme-linked immunosorbent assay. First, 8 standards (0–200 ng/mL) were produced using the kit diluent. In total, 30 µL of each standard and sample was pipetted into duplicate wells of a 96-well plate and 200 µL conjugated antihuman ferritin was then added into each well. The plate was incubated in a rotary shaker at 37 ◦C for 2 h after which 200 µL substrate solution was added into each well. After a further incubation period of 30 min at room temperature, 100 µL potassium ferricyanide was added to every well. A microplate reader (VersaMax, San Jose, CA, USA) was then used to measure the absorbance at 490 and 630 nm. The total protein concentration was determined using a bicinchoninic acid assay. Pre-diluted bovine serum albumin was used as the standards. In total, 25 µL of the standards and samples were pipetted into duplicate wells. Working reagent was prepared and 200 µL added per well. After incubation (30 min, 37 ◦C) the absorbance was then read at 562 nm. These results were then used to standardize the ferritin concentration to calculate iron absorption. The loading efficiency of the nanoparticles (Tables1 and3) was indirectly determined by preparing freeze dried samples of nanoparticles which were dissolved in in aqueous acetic acid solution (1% v/v) and the solutions were assayed for drug content using same essay above. The entrapment efficiency (%) was determined using the following equation:

Entrapment efficiency (%) = (weight of drug in nanoparticles)/(weight of initially used drug) 100 (1) × 2.6. Statistical Analysis Particle size measurements were the mean of 10 replicates and zeta potential results were the mean of 3 replicates. Means and standard deviations were calculated using Microsoft Excel. Data were Processes 2020, 8, x FOR PEER REVIEW 6 of 17

Entrapment efficiency (%) = (weight of drug in nanoparticles)/(weight of initially (1) used drug) × 100

Processes2.5. Statistical2020, 8, 707 Analysis 6 of 16 Particle size measurements were the mean of 10 replicates and zeta potential results were the analyzedmean of by 3 one-wayreplicates. analysis Means ofand variance standard analysis deviations of variance were calculated (ANOVA), using followed Microsoft by post-hoc Excel. Tukey’sData HSDwere tests analyzed using by SPSS one-way (version analysis 22, IBM,of variance Armonk, analysis NY, of USA). variance Results (ANOVA), were consideredfollowed by statisticallypost-hoc signiﬁcantTukey’s HSD if p tests0.05. using SPSS (version 22, IBM, Armonk, NY, USA). Results were considered statistically significant≤ if p ≤ 0.05. 3. Results 3. Results 3.1. Physiochemical Characteristics of Chromium-Entrapped CS-DS Nanoparticles 3.1. Physiochemical Characteristics of Chromium-Entrapped CS-DS Nanoparticles The particle size of empty and chromium-entrapped CS-DS nanoparticles was the ﬁrst dependent variableThe assessed. particle Figure size 2of shows empty how and the chromium-e particle sizentrapped changed CS-DS as the nanoparticles ratio of CS:DS was altered. the Firstly,first alldependent samples werevariable of submicronassessed. Figure dimensions 2 shows indicating how the particle the successful size changed generation as the ofratio nanoparticles. of CS:DS Thealtered. largest Firstly, particles all samples (4:1 MMW were CS-Cr) of submicron were 120% dimensions bigger than indicating the smallest the successful particles generation (4:3 LMW-CS). of nanoparticles. The largest particles (4:1 MMW CS-Cr) were 120% bigger than the smallest particles Secondly, as the molecular weight of chitosan increased, the particle size increased. This was seen at (4:3 LMW-CS). Secondly, as the molecular weight of chitosan increased, the particle size increased. a 4:1 ratio where the MMW CS particles were 19% larger than the LMW CS particles. More so, as the This was seen at a 4:1 ratio where the MMW CS particles were 19% larger than the LMW CS particles. volume of dextran increased, the size decreased. This was seen by there being a 32% decrease in size More so, as the volume of dextran increased, the size decreased. This was seen by there being a 32% when comparing 4:1 MMW CS-Cr to 4:2 MMW CS-Cr. Generally, there was a narrow size distribution decrease in size when comparing 4:1 MMW CS-Cr to 4:2 MMW CS-Cr. Generally, there was a narrow betweensize distribution the samples between with minimalthe samples variation. with minimal variation.

FigureFigure 2. 2.Particle Particle size size of of empty empty andand chromium-entrappedchromium-entrapped CS-DS CS-DS nanoparticles nanoparticles when when using using different different MWsMWs of of chitosan chitosan at at various various CS:DS CS:DS ratios. ratios. Results Results are ar showne shown as as mean mean ±standard standard deviation deviation (SD) (SD) (n (n= =10). ± Data10). were Data considered were considered statistically statistically different different when p when< 0.05. p *< indicates 0.05. * indicates significant significant difference difference comparing 4:1comparing medium molecular4:1 medium weight molecular (MMW) weight CS-Cr (MMW) to MMW CS-Cr CS-Cr to MMW at other CS-Cr ratios, at otherindicates ratios, † indicates a significant a † diffsignificanterence between difference low between molecular low weightmolecular (LMW) weight CS (LMW) and MMW CS and CS MMW at the CS same at the ratio, same ratio,indicates ‡ ‡ a significantindicates a disignificantfference betweendifference LMW between CS andLMW LMW CS and CS-Cr LMW at theCS-Cr same at the ratio. same ratio.

TheThe zeta zeta potential potential of of chromium-entrapped chromium-entrapped partic particlesles was was the the second second dependent dependent variable variable studied. studied. FigureFigure3 illustrates 3 illustrates how how particle particle charge charge was was aaffectedﬀected by by different diﬀerent CS:DS CS:DS ratios ratios and and molecular molecular weights weights ofof CS. CS. Firstly, Firstly, all all particles particles had had positive positive zetazeta potentialspotentials with with the the charge charge of of the the most most positive positive sample sample (4:1 LMW CS-Cr) being 1.4 times higher than that of the least positive sample (4:3 MMW CS). (4:1 LMW CS-Cr) being 1.4 times higher than that of the least positive sample (4:3 MMW CS). Secondly, the zeta potentials of the MMW CS samples were slightly lower than those produced using LMW CS. Furthermore, the chromium-entrapped samples had larger charges compared to empty samples at the same ratio. This was observed at 4:2 where the LMW CS-Cr sample had an 8% larger charge than LMW CS. Generally, when comparing Figures2 and3 it was seen that the largest particles (produced at a CS:DS ratio of 4:1) also had the largest zeta potentials. Processes 2020, 8, x FOR PEER REVIEW 7 of 17

Secondly, the zeta potentials of the MMW CS samples were slightly lower than those produced using LMW CS. Furthermore, the chromium-entrapped samples had larger charges compared to empty samples at the same ratio. This was observed at 4:2 where the LMW CS-Cr sample had an 8% larger Processescharge2020 than, 8, 707LMW CS. Generally, when comparing Figures 2 and 3 it was seen that the largest particles7 of 16 (produced at a CS:DS ratio of 4:1) also had the largest zeta potentials.

FigureFigure 3. 3.Zeta Zeta potential potential of of chromium-entrapped chromium-entrapped CS-DS nanoparticles nanoparticles when when using using different different MWs MWs of of chitosanchitosan at at various various CS:DS CS:DS ratios. ratios. ResultsResults areare shownshown as mean ± SDSD (n (n == 3).3). Data Data were were considered considered ± statisticallystatistically di differentfferent when whenp p< <0.05. 0.05. * indicates significant significant diffe differencerence comparing comparing 4:1 4:1 LMW LMW CS CS to toLMW LMW CSCS samples samples at at other other ratios, ratios, indicates† indicates a significanta significant di differencefference between between LMW LMW CS CS and and LMW LMW CS-Cr CS-Cr at at the † samethe ratio,same ratio,indicates ‡ indicates a significant a significant difference differe betweennce between MMW MMW CS and CS and MMW MMW CS-Cr CS-Cr at the at samethe same ratio. ratio. ‡ 3.2. Physiochemical Characteristics of Iron-Entrapped CS-DS Nanoparticles and Submicron Particles 3.2. Physiochemical Characteristics of Iron-Entrapped CS-DS Nanoparticles and Submicron Particles Iron-entrapped CS–DS nanoparticles and submicron particles were formulated, and their sizes measuredIron-entrapped using photon CS–DS correlation nanoparticles spectroscopy and submicro (Figure4).n First,particles it was were observed formulated, that iron and nanoparticles their sizes weremeasured only formed using at photon a 4:3 ratio correlation as opposed spectroscopy to in Figure 2(Figure where chromium4). First, it nanoparticles was observed were that produced iron atnanoparticles all biopolymer were ratios. only Similarly, formed at whena 4:3 ratio comparing as opposed Figures to in2 Figure and4, 2 all where iron-entrapped chromium nanoparticles nanoparticles werewere larger produced than those at all produced biopolymer using ratios. chromium. Similarly, More when so, bothcomparing Figures Figures indicated 2 thatand as4, theall volumeiron- entrapped nanoparticles were larger than those produced using chromium. More so, both Figures of dextran increased, the size decreased. Figure4 also shows that as the molecular weight of chitosan indicated that as the volume of dextran increased, the size decreased. Figure 4 also shows that as the increased, the particle size increased. However, this was not the case at a ratio of 4:3 where the opposite molecular weight of chitosan increased, the particle size increased. However, this was not the case at was true. Additionally, iron-entrapped particles were significantly larger than those which were empty a ratio of 4:3 where the opposite was true. Additionally, iron-entrapped particles were significantly at all ratios. This was seen at 4:1 where there was a 375% increase in size when iron was added to the larger than those which were empty at all ratios. This was seen at 4:1 where there was a 375% increase LMW CS sample. Moreover, the iron-entrapped particles at a 4:1 ratio had a large size distribution as in size when iron was added to the LMW CS sample. Moreover, the iron-entrapped particles at a 4:1 seenratio by had large a large error size bars. distribution as seen by large error bars. TheThe zeta zeta potential potential of of iron iron encapsulated encapsulated CS-DS CS-DS nanoparticles nanoparticles andand submicronsubmicron particlesparticles is shown in Figurein Figure5. Several 5. Several important important observations observations can becan made be made from from the graph. the graph. First, First, the presencethe presence of iron of iron led to a significantled to a significant decrease decrease in charge in forcharge all samplesfor all samp whenles comparedwhen compared to the to empty the empty particles. particles. This This is unlike is Figureunlike3 where Figure the3 where opposite the opposi was truete was when true chromium when chromium was added. was added. Moreover, Moreover, when when comparing comparing both figuresboth itfigures was seen it was that seen the iron-entrappedthat the iron-entrapped nanoparticles nanoparticles had lower had zeta lower potentials zeta potentials at all biopolymer at all ratiosbiopolymer than those ratios made than using those chromium. made using A similarity chromium between. A similarity Figures between3 and5 isFigures that samples 3 and 5 produced is that usingsamples medium produced MW using chitosan medium had MW slightly chitosan lower had zeta slightly potentials lower thanzeta potentials those made than using those low made MW chitosan.using low This MW was chitosan. illustrated This atwas 4:2 illustrated where MMW at 4:2 where CS-Fe MMW had an CS-Fe 11% lowerhad an charge 11% lower than charge LMW than CS-Fe. It wasLMW also CS-Fe. the caseIt was that also as the the case proportion that as the of propor dextrantion increased, of dextran the increased, charge decreased. the charge Additionally,decreased. when comparing Figures4 and5, the largest iron-entrapped particles (i.e., CS:DS 4:3) had the largest zeta potentials. This is reflected in loading efficiency as the CS:DS 4:3 sample showed highest loading efficiency (Table1). Processes 2020, 8, x FOR PEER REVIEW 8 of 17 Processes 2020, 8, x FOR PEER REVIEW 8 of 17

Additionally,Additionally, when when comparing comparing Figures Figures 4 4 and and 5, 5, the the largest largest iron-entrapped iron-entrapped particles particles (i.e., (i.e., CS:DS CS:DS 4:3) 4:3) had the largest zeta potentials. This is reflected in loading efficiency as the CS:DS 4:3 sample showed Processeshad the2020 largest, 8, 707 zeta potentials. This is reflected in loading efficiency as the CS:DS 4:3 sample showed8 of 16 highesthighest loading loading efficiency efficiency (Table (Table 1). 1).

Figure 4. Effects of CS:DS ratio, iron entrapment and chitosan molecular weight on particle size. FigureFigure 4. E4.ﬀ Effectsects of CS:DSof CS:DS ratio, ratio, iron iron entrapment entrapment and chitosanand chitosan molecular molecular weight weight on particle on particle size. Resultssize. Results are shown as mean ± SD (n = 10). Data were considered statistically different when p < 0.05. * areResults shown are as meanshown asSD mean (n = ±10). SD Data(n = 10). were Data considered were considered statistically statistically diﬀerent different when p

Figure 5. Zeta potential of iron-entrapped CS-DS nanoparticles when using different MWs of chitosan FigureFigure 5. 5.Zeta Zeta potential potential of of iron-entrapped iron-entrapped CS-DS nanoparticles when when using using different different MWs MWs of ofchitosan chitosan at various CS:DS ratios. Results are shown as mean ± SD (n = 3). Data were considered statistically at variousat various CS:DS CS:DS ratios. ratios. Results Results are areshown shown asas meanmean ± SD (n = 3)3).. Data Data were were considered considered statistically statistically different when p < 0.05. * indicates a significant differe± nce between LMW CS and LMW CS-Fe at the different whenp p < 0.05. * indicates a significant difference between LMW CS and LMW CS-Fe at the differentsame ratio, when † indicates< 0.05. *a indicatessignificant a difference significant between difference LMW between and MMW LMW CS-Fe CS andat the LMW same CS-Feratio, ‡ at thesame same ratio, ratio, † indicatesindicates a significant a significant difference difference between between LMW LMW and and MMW MMW CS-Fe CS-Fe at the at same the same ratio, ratio, ‡ indicates a significant† difference between MMW CS and MMW CS-Fe at the same ratio. indicatesindicates aa significantsignificant difference difference between between MMW MMW CS CS and and MMW MMW CS-Fe CS-Fe at atthe the same same ratio. ratio. ‡ 3.3. Physiochemical Characteristics of Chromium-Entrapped WPI-CS Nanoparticles 3.3.3.3. Physiochemical Physiochemical Characteristics Characteristics of ofChromium-Entrapped Chromium-Entrapped WPI-CS WPI-CS Nanoparticles Nanoparticles The sizes of chromium-entrapped whey protein nanoparticles are depicted in Figure 6. The TheThe sizes sizes of chromium-entrappedof chromium-entrapped whey whey protein protein nanoparticles nanoparticles are are depicted depicted in Figure in Figure6. The 6. FigureThe Figure summarizes the variation in particle size as the volume of whey protein and CS changed. All summarizesFigure summarizes the variation the variation in particle in particle size as size the as volume the volume of whey of whey protein protein and and CS CS changed. changed. All All the thethe particles particles were were submicron submicron in in size, size, ranging ranging from from 220 220 nm nm to to 503 503 nm nm and and as as expected, expected, the the particle particle particles were submicron in size, ranging from 220 nm to 503 nm and as expected, the particle size increased with chromium entrapment. In addition, particle size increased as the volume of chitosan increased and when the molecular weight of CS was increased. Furthermore, it was observed that as the chitosan concentration increased from 0.1 to 0.2%, the particle size increased. This was seen at 350:1 where 0.2% LMW CS-Cr particles were 12% larger than those at LMW CS-Cr. Processes 2020, 8, x FOR PEER REVIEW 9 of 17 Processes 2020, 8, x FOR PEER REVIEW 9 of 17 size increased with chromium entrapment. In addition, particle size increased as the volume of sizechitosan increased increased with andchromium when theentrapment. molecular In weight addition, of particleCS was sizeincreased. increased Furthermore, as the volume it was of Processeschitosanobserved2020, 8, 707increasedthat as the and chitosan when concentration the molecular increased weight fromof CS 0.1 was to 0.2%,increased. the particle Furthermore, size increased. it was 9 of 16 observedThis was seenthat atas 350:1the chitosan where 0.2% concentration LMW CS-Cr increased particles from were 0.1 12% to larger0.2%, thethan particle those at size LMW increased. CS-Cr. This was seen at 350:1 where 0.2% LMW CS-Cr particles were 12% larger than those at LMW CS-Cr.

FigureFigure 6. Particle 6. Particle size size of of chromium-entrapped chromium-entrapped WPI nanoparticles nanoparticles when when using using different different MWs MWs and and concentrations of chitosan at various WPI:CS ratios. Results are shown as mean ± SD (n = 10). Data concentrationsFigure 6. Particle of chitosan size atof variouschromium-entrapped WPI:CS ratios. WPI Results nanoparticles are shown when as meanusing differentSD (n = MWs10). Dataand were concentrationswere considered of statistically chitosan at different various whenWPI:CS p < rati 0.05.os. * Resultsindicates are a significantshown as meandifference± ± SD between (n = 10). LMW Data considered statistically different when p < 0.05. * indicates a significant difference between LMW wereCS and considered LMW CS-Cr statistically at the same different ratio, when † indicate p < 0.05.s a *significant indicates a difference significant between difference LMW between CS-Cr LMW and CS and LMW CS-Cr at the same ratio, indicates a significant difference between LMW CS-Cr and CSMMW and CS-CrLMW atCS-Cr the sameat the ratio, same ‡ratio, indicates† † indicate a significants a significant difference difference between between LMW LMW CS-Cr CS-Cr and 0.2% and MMW CS-Cr at the same ratio, indicates a significant difference between LMW CS-Cr and 0.2% LMW MMWLMW CS-Cr CS-Cr at at the the same same ratio, ratio,‡ § indicates ‡ indicates a sign a significantificant difference difference comparing between 350:1 LMW MMW CS-Cr CS andto MMW 0.2% CS-CrLMWCS at at the otherCS-Cr same ratios. at ratio,the same § indicates ratio, § indicates a significant a significant difference difference comparing comparing 350:1 350:1 MMW MMW CS CS to to MMW MMW CS at otherCS ratios. at other ratios. The zeta potential of chromium encapsulated WPI-CS nanoparticles is presented in Figure 7. As Theshown, zetaThe it zeta potentialwas potential observed of of chromium thatchromium all particles encapsulated exhibited WPI- WPI-CSa CSnegative nanoparticles nanoparticles charge andis presented that is presentedas thein Figure volume in 7. Figure Asof 7. As shown,shown,chitosan it it wasincreased, wasobserved observed the charge that all all increased. particles particles Similarly,exhibited exhibited aparticles negative a negative produced charge charge and using andthat 0.2% thatas theCS as volumehad the volumemore of of chitosanchitosanpositive increased, zetaincreased, potentials the charge the than charge increased. those increased. produced Similarly, Similarly, using particles 0.1% particles CS. producedThis produced was seen using usingat 350:1 0.2% 0.2% where CS CS had 0.2% had more LMWmore positive zeta potentialspositiveCS-Cr was zeta than17% potentials more those positive than produced those than produced LMW using CS-Cr. 0.1% usin CS.Mog 0.1%re This so, CS. all was This particles seen was seen at produced 350:1 at 350:1 where using where 0.2%medium 0.2% LMW LMW MW CS-Cr CS had a slightly more negative charge than those made using LMW CS. Moreover, chromium- was 17%CS-Cr more was positive17% more than positive LMW than CS-Cr. LMW More CS-Cr. so, Mo allre particlesso, all particles produced produced using using medium medium MW MW CS had CSentrapped had a slightlysamples more were negativemore positively charge chargedthan those than made the emptyusing LMWformulations, CS. Moreover, similar tochromium- that seen a slightly more negative charge than those made using LMW CS. Moreover, chromium-entrapped entrappedin Figure 3. samples were more positively charged than the empty formulations, similar to that seen samplesin Figure were 3. more positively charged than the empty formulations, similar to that seen in Figure3.

Figure 7. Zeta potential of chromium-entrapped WPI nanoparticles when using different MWs and concentrations of chitosan at various WPI:CS ratios. Results are shown as mean SD (n = 3). Data were ± considered statistically different when p < 0.05. * indicates a significant difference between 350:1 0.2% chromium-entrapped particles and 0.2% particles at other ratios, indicates a significant difference † between LMW CS-Cr and MMW CS-Cr at the same ratio, indicates a significant difference between ‡ LMW CS and LMW CS-Cr at the same ratio, § indicates a significant difference between LMW CS-Cr and 0.2% LMW CS-Cr at the same ratio. Processes 2020, 8, x FOR PEER REVIEW 10 of 17

Figure 7. Zeta potential of chromium-entrapped WPI nanoparticles when using different MWs and concentrations of chitosan at various WPI:CS ratios. Results are shown as mean ± SD (n = 3). Data were considered statistically different when p < 0.05. * indicates a significant difference between 350:1 0.2% chromium-entrapped particles and 0.2% particles at other ratios, † indicates a significant Processes 2020, 8, 707 10 of 16 difference between LMW CS-Cr and MMW CS-Cr at the same ratio, ‡ indicates a significant difference between LMW CS and LMW CS-Cr at the same ratio, § indicates a significant difference between 3.4. PhysiochemicalLMW CS-Cr Characteristics and 0.2% LMW CS-Cr of Iron-Entrapped at the same ratio. WPI–CS Nanoparticles The3.4. Physiochemical particle size forCharacteristics iron-entrapped of Iron-Entrapped WPI-CS nanoparticles WPI–CS Nanoparticles was assessed, and the results depicted in Figure8The. The particle results size showed for iron-entrapped that all except WPI-CS one nano sampleparticles produced was assessed, particles and the smaller results than depicted 1000 nm. The largestin Figure iron 8. The encapsulated results showed particles that all (50:1 except MMW one sample CS-Fe) produced were 4 times particles bigger smaller than than the 1000 smallest nm. iron particlesThe largest (350:1 iron LMW encapsulated CS-Fe). Chitosanparticles (50:1 inclusion MMW CS at-Fe) 0.2% were (w/ v4 )times resulted bigger in than the the formation smallest iron of larger particles,particles as seen(350:1 at LMW a 50:1 CS-Fe). ratio whereChitosan the inclusion particles at were0.2% ( 1.3w/v times) resulted bigger in the than formation those atof 0.1%larger LMW CS-Fe.particles, Additionally, as seen at iron-entrapped a 50:1 ratio where samples the particles were were larger 1.3 than times those bigger that than were those empty, at 0.1% as LMW illustrated CS- at 50:1 whereFe. Additionally, the MMW iron-entrapped CS-Fe particles samples were were 2.5 timeslarger largerthan those than that the were MMW empty, empty as illustrated particles. at Lastly, 50:1 where the MMW CS-Fe particles were 2.5 times larger than the MMW empty particles. Lastly, as as the molecular weight of CS increased, the size increased. This was seen at 350:1 where the MMW the molecular weight of CS increased, the size increased. This was seen at 350:1 where the MMW CS- CS-Fe particles were 33% larger than the LMW CS-Fe particles. Fe particles were 33% larger than the LMW CS-Fe particles.

FigureFigure 8. 8.Particle Particle size size ofof iron-entrappediron-entrapped WPI WPI nano nanoparticlesparticles when when using using different different MWs MWs and and concentrations of chitosan at various WPI:CS ratios. Results are shown as mean ± SD (n = 10). Data concentrations of chitosan at various WPI:CS ratios. Results are shown as mean SD (n = 10). were considered statistically different when p < 0.05. * indicates a significant difference between± LMW Data were considered statistically different when p < 0.05. * indicates a significant difference between CS-Fe and 0.2% LMW CS-Fe at the same ratio, † indicates a significant difference LMW CS-Fe and LMW CS-Fe and 0.2% LMW CS-Fe at the same ratio, indicates a significant difference LMW CS-Fe MMW CS-Fe at the same ratio, ‡ indicates a significant† difference between MMW CS and MMW CS- and MMW CS-Fe at the same ratio, indicates a significant difference between MMW CS and MMW Fe at the same ratio. ‡ CS-Fe at the same ratio. The zeta potential of iron-entrapped WPI nanoparticles at pH 7.5 is displayed in Figure 9. Firstly, Theall the zeta samples potential had ofa negative iron-entrapped charge, with WPI the nanoparticles most negative at pHnanocarriers 7.5 is displayed being produced in Figure using9. Firstly, all theLMW samples CS-Fe. had Secondly, a negative as the charge,CS concentration with the incr mosteased, negative the zeta nanocarriers potential became being more produced positive. using LMWThis CS-Fe. was observed Secondly, at as350:1 the where CS concentration 0.2% LMW CS-Fe increased, was one the time zeta more potential positive became than LMW more CS-Fe. positive. ThisMore was observedso, the MMW at 350:1 CS-Fe where formulations 0.2% LMW had signific CS-Feantly was more one timepositive more charges positive than thanthe LMW LMW CS- CS-Fe. MoreFe. so, This the differed MMW CS-Fefrom data formulations shown in Figure had significantly 7, where molecular more positiveweight generally charges had than little the effect LMW on CS-Fe. This dicharge.ffered Moreover, from data iron shown incorporation in Figure resulted7, where in molecular more positive weight zeta potentials generally for had MMW little particles e ffect on but charge. caused the opposite effect for LMW particles compared to the empty samples. The impact on loading Moreover, iron incorporation resulted in more positive zeta potentials for MMW particles but caused efficiency can be clearly seen when varying the WPI:CS ratio (Table 3). the opposite effect for LMW particles compared to the empty samples. The impact on loading efficiency can be clearly seen when varying the WPI:CS ratio (Table3).

3.5. Caco-2 Cell Iron Permeability Studies Permeability studies of the WPI-CS-Fe nanoparticles were undertaken using Caco-2 cells and the results shown in Figure 10. There was a large variation in iron uptake between the samples. The 350:1 LMW CS-Fe sample produced the highest iron absorption with 64% more ferritin being absorbed than from the ferrous sulfate reference. Similarly, the 175:1 LMW CS-Fe sample had an 8.7% higher iron absorption compared to the reference. In contrast, the sample produced using medium Processes 2020, 8, x FOR PEER REVIEW 11 of 17 Processes 2020, 8, x FOR PEER REVIEW 11 of 17 3.5. Caco-2 Cell Iron Permeability Studies 3.5. Caco-2 Cell Iron Permeability Studies Permeability studies of the WPI-CS-Fe nanoparticles were undertaken using Caco-2 cells and the resultsPermeability shown instudies Figure of 10. the There WPI-CS-Fe was a largenanopart variationicles werein iron undertaken uptake between using theCaco-2 samples. cells Theand Processes 2020 8 350:1the results LMW, , 707 shown CS-Fe in sample Figure 10.produced There was the ahighest large va ironriation absorption in iron uptake with between64% more the ferritinsamples. being The11 of 16 absorbed350:1 LMW than CS-Fe from thesample ferrous produced sulfate reference.the highest Similarly, iron absorption the 175:1 LMW with CS-Fe64% moresample ferritin had an being 8.7% higherabsorbed iron than absorption from the comparedferrous sulfate to the reference. reference. Similarly, In contrast, the 175:1 the sample LMW CS-Fe produced sample using had medium an 8.7% MW CS had the lowest iron absorption. Unexpectedly, the sample that contained a higher volume of MWhigher CS iron had absorption the lowest ironcompared absorption. to the Unexpectedly reference. In ,contrast, the sample the that sample contained produced a higher using volume medium of chitosan (125:1 LMW CS-Fe) performed worse than the standard (a 7.7% decrease in absorption). chitosanMW CS had (125:1 the LMW lowest CS-Fe) iron absorption. performed Unexpectedlyworse than the, the standard sample (a that 7.7% contained decrease a in higher absorption). volume of chitosan (125:1 LMW CS-Fe) performed worse than the standard (a 7.7% decrease in absorption).

FigureFigure 9. Zeta9. Zeta potential potential of of iron-entrapped iron-entrapped WPI nanoparticles nanoparticles when when using using different different MWs MWs and and Figure 9. Zeta potential of iron-entrapped WPI nanoparticles when using different MWs and concentrationsconcentrations of chitosanof chitosan at at variousvarious WPI:CS WPI:CS rati ratios.os. Results Results are shown are shown as mean as ± meanSD (n = 3).SD Data (n = 3). wereconcentrations considered of statisticallychitosan at differentvarious WPI:CSwhen p rati< 0.05.os. Results* indicates are showna significant as mean difference ± SD (n comparing ±= 3). Data Data were considered statistically different when p < 0.05. * indicates a significant difference comparing 350:1were consideredLMW CS to statistically LMW CS at different other ratios, when † pindi < 0.05.cates * a indicates significant a significantdifference differencebetween LMW comparing CS-Fe 350:1 LMW CS to LMW CS at other ratios, indicates a significant difference between LMW CS-Fe and and350:1 MMW LMW CS-Fe CS to atLMW the same CS at ratio, other ‡ ratios,indicates† † indi a significantcates a significant difference difference between LMWbetween CS-Fe LMW and CS-Fe 0.2% MMW CS-Fe at the same ratio, indicates a significant difference between LMW CS-Fe and 0.2% LMW LMWand MMW CS-Fe CS-Fe at the at same the same ratio,‡ ratio, § indicates ‡ indicates a sign aificant significant difference difference between between LMW LMW CS and CS-Fe LMW and CS-Fe 0.2% CS-FeatLMW atthe the sameCS-Fe same ratio. at ratio,the same § indicates ratio, § indicates a significant a significant difference difference between between LMW LMW CS CS and and LMW LMW CS-Fe CS-Fe at the sameat ratio. the same ratio.

Figure 10. Iron absorption by Caco-2 cells incubated with WPI-CS-Fe nanoparticle preparations. FigureFigure 10. Iron10. Iron absorption absorption by by Caco-2 Caco-2 cells cells incubatedincubated with with WPI-CS-Fe WPI-CS-Fe nanoparticle nanoparticle preparations. preparations.

4. Discussion Particle size was measured, as it can aﬀect drug release and absorption. As seen in previous studies [7,8], smaller particles have larger surface area to volume ratios, leading to improvement in intestinal drug permeability and thus an increase in absorption. The intestinal clearance mechanisms are also minimized and this prolongs the residence time of the formulation [9]. The zeta potential is an important assessment of formulation stability and the extent to which particles permeate through Processes 2020, 8, 707 12 of 16 membranes. Collectively, these factors play an integral role in the absorption of iron and chromium and can be tailored to give patients the most beneﬁt from supplement therapies.

4.1. Properties of CS–DS Nanoparticles Chitosan and dextran nanoparticles were produced via complex coacervation where the negatively charged sulfate groups of dextran interacted with the positively charge amino groups of chitosan (Figure1). The properties of the particles were altered based on the biopolymer ratios used. Previous observations demonstrated that increasing dextran ratio in the system decreased particle size [18]. This was in accordance with Figures2 and4 where there were inverse relationships between dextran ratio and size. A reason for this is due to the diﬀering molecular weights of the two biopolymers [18]. In this study chitosan had a molecular weight of 50/190 kDa whilst that of dextran was 15.6 kDa. When chitosan ratio was high, there was repulsion between its cationic groups, leading to more cross linking sites becoming available to form hydrogen bonding with water [18]. This caused water to penetrate into the polymer matrices, causing the particles to swell and increase in size. However, when the ratio of dextran was lowered into the system, sites for electrostatic interactions were fully occupied with chitosan, leading to less hydration occurring [22]. These interactions also caused a decrease in the positive charge of chitosan, causing folding to occur and the formation of smaller particles with lower zeta potentials (Figures2 and3). The higher levels of dextran also acted as a colloidal protectant, causing the retardation of inter/intra molecular interactions as a result of steric hindrance (1). This prevented aggregation occurring and could be the reason that smaller particles were formed at higher dextran volumes. As seen in other studies [28], particles made with MMW CS were larger than those produced with low MW CS. An explanation for this is that MMW CS contained longer chain meaning that the tertiary amine groups were harder to protonate. This meant that there were less electrostatic interactions between CS and DS, leading to the formation of larger particles [28]. The iron nanoparticles were generally considerably larger than those made using chromium. A reason for this is because rapid aggregation of the iron-entrapped particles occurred, as observed by the large error bars in Figure4. Other studies have explored the use of polyethylene glycol-40 to improve colloidal stability [29]. However, this approach may aﬀect drug release kinetics from the colloidal system.

4.2. Properties of WPI-CS Nanoparticles Proteins contain both acidic and basic amino acids, giving them the unique ability to form nanoparticles with polysaccharides such as chitosan in certain environmental conditions. Whey protein was heat treated to 85 ◦C in order to denature the protein secondary structures and improve their solubility. When the pH was adjusted to 7.5 the secondary structures reorganized and unfolding occurred, leading to more cross linking sites becoming available [30]. There were also more thiol-disulﬁde exchange reactions occurring which caused further particle formation and the discouragement of aggregation. More so, chitosan and β-lactoglobulin had opposite charges at pH 7.5 causing electrostatic attraction and complexation to occur. This was clearly seen there was inverse correlation between the pH and size of formed nanoparticles (Figure 11). It is interesting to observe the impact of pH on size of the nanoparticles as chitosan is only soluble at lower pH values; however, the size of the particles has decreased at higher pH. This indicates favorable interactions with whey protein chains preventing precipitation of chitosan as separate colloidal particles. Visual inspection of iron containing nanoparticles revealed stability dependent on WPI/CS ratio. As can be seen, there was a color change for samples that contained lower ratio of WPI/CS. WPI/CS 350:1 had similar color while the WPI/CS 50:1 showed noticeable color change. This is highlighted in Figure 12 where 50:1 LMW CS-Fe and 125:1 LMW CS-Fe changed color from orange to dark brown. As shown above, all samples contained similar amount of ascorbic acid which acts as antioxidant. Hence, this color change cannot be attributed to variable oxidation activity. Denaturing of whey protein isolate can also happen due to oxidative stress [31], which may contribute to this color change. Processes 2020,, 8,, xx FORFOR PEERPEER REVIEWREVIEW 13 of 17

350:1 had similar color while the WPI/CS 50:1 showed noticeable color change. This is highlighted in Figure 12 where 50:1 LMW CS-Fe and 125:1 LMW CS-Fe changed color from orange to dark brown. ProcessesAs shown2020, 8 ,above, 707 all samples contained similar amount of ascorbic acid which acts as antioxidant.13 of 16 Hence, this color change cannot be attributed to variable oxidation activity. Denaturing of whey protein isolate can also happen due to oxidative stress [31], which may contribute to this color change. ThisThis also also highlights highlights that that using using WPIWPI/CS/CS 350:1350:1 hashas produced particles particles with with optimum optimum stability stability and and encapsulationencapsulation eﬃ efficiency.ciency.

FigureFigure 11. 11.The The eﬀ effectect of pHof pH on on the the particle particle size size of iron-entrapped of iron-entrappediron-entrapped WPI-CS WPI-CSWPI-CS nanoparticles nanoparticlesnanoparticles produced producedproduced using lowusing MW low chitosan. MW chitosan.

FigureFigure 12. 12.Physical Physical stability stability of of iron-entrappediron-entrapped WPI-CS nanosuspensions nanosuspensions (a ()a 10) 10 min min and and (b) ( b10) 10days days post-production.post-production. From From left left to to right—50:1 right—50:1 LMWLMW CS-Fe, 125:1 LMW LMW CS-Fe, CS-Fe, 350:1 350:1 LMW LMW CS-Fe. CS-Fe.

LargerLarger particles particles were were formed formed when when the the molecular molecular weight weight of of chitosan chitosan increasedincreased (Figures(Figures 66 and and8 ). This8). agreedThis agreed with previouswith previous work andwork occurred and occurred because because the MMW the MMW CS had CS a lowerhad a aqueous lower aqueous solubility, meaningsolubility, that meaning aggregation that wasaggregation more likely was to more occur likely [32]. Conversely,to occur [32]. LMW Conversely, CS solution LMW was CS less solution viscous, meaningwas less that viscous, kinetic meaning energy that barrier kinetic for energy nanoparticle barrier for aggregation nanoparticle is aggregation enhanced is which enhanced can promotewhich formationcan promote of smaller formation nanoparticles. of smaller nanoparticles. The same explanation The same explanation was used to was explain used whyto explain the particle why the size increasedparticle size as the increased concentration as the concentration of chitosan of increased; chitosan increased; 0.2% CS was0.2% moreCS was viscous more viscous and less and soluble, less leadingsoluble, to leading inefficient to inefficient particle formation. particle formation. Furthermore, Furthermore, the zeta potentialthe zeta potential increased increased with increasing with increasing chitosan concentration (Figures 7 and 9). This was because 0.2% chitosan imparted a more chitosanincreasing concentration chitosan concentration (Figures7 and (Figures9). This 7 and was 9) because. This was 0.2% because chitosan 0.2% impartedchitosan imparted a more positivea more positive charge on the particle surfaces due to its positively charged amino groups [8]. Similarly, the charge on the particle surfaces due to its positively charged amino groups [8]. Similarly, the particle size of iron and chromium-entrapped particles increased with an increased volume of CS (Figures6 and8). Presence of chitosan chains and especially at the surface conferred the nanoparticles su fficient stability and promoted efficient encapsulation. This led to more chromium and iron being entrapped into particles, causing their size to increase. Moreover, when chromium and iron were added into the formulations, the sizes increased compared to empty samples. This is because chromium can adsorb Processes 2020, 8, x FOR PEER REVIEW 14 of 17 particle size of iron and chromium-entrapped particles increased with an increased volume of CS (Figures 6 and 8). Presence of chitosan chains and especially at the surface conferred the nanoparticles sufficient stability and promoted efficient encapsulation. This led to more chromium and iron being entrapped into particles, causing their size to increase. Moreover, when chromium and iron were Processes 2020, 8, 707 14 of 16 added into the formulations, the sizes increased compared to empty samples. This is because chromium can adsorb to chitosan chains, leading to enhanced entrapment [33]. More so, the MMW CS-Feto chitosan particles chains, had significantly leading to enhanced higher zeta entrapment potentials [than33]. those More produced so, the MMW with LMW CS-Fe CS-Fe particles (Figure had 9).significantly This was higherbecause zeta MMW potentials CS had than a larger those producednumber of with positively LMW CS-Fecharged (Figure amine9). Thisgroups was due because to its longerMMW chain CS had lengths. a larger number of positively charged amine groups due to its longer chain lengths.

4.3. Iron Iron Absorption in Caco-2 Cells Caco-2 cells werewere usedused because because they they resemble resemble intestinal intestinal enterocytes enterocytes and and the the assay assay investigates investigates the theintestinal intestinal permeability permeability of orally of orally absorbed absorbed drugs drugs [34]. As[34]. seen As in seen Figure in Figure 10, both 10, the both 350:1 the LMW 350:1 CS LMW and 175:1CS and CS 175:1 samples CS samples caused highercaused iron higher absorptions iron absorptions compared compared to the ferrous to the sulfate ferrous standard. sulfate standard. This was Thisin agreement was in agreement with the previous with the work previous which work stated which that ascorbic stated acidthat augmentsascorbic acid iron augments absorption iron [8]. Anotherabsorption reason [8]. Another for the enhancementreason for the isenhancement because chitosan is because was used chitosan in the was formulations. used in the formulations. Chitosan has Chitosanmucoadhesive has mucoadhesive properties and properties as a result and is aas permeation a result is a enhancer permeation which enhancer can cause which the can restructuring cause the restructuringof tight junction-associated of tight junction-associated proteins [35]. proteins Moreover, [35]. the Moreover, low MW CS the samples low MW caused CS samples higher ferritincaused higherabsorptions ferritin than absorptions the MMW than CS sample the MMW because CS samp the particlesle because exhibited the particles smaller exhibited size. This smaller meant size. they Thiscould meant permeate they intocould the permeate cells and into the the iron cells could and interact the iron with could the divalentinteract with metal the transporters divalent metal [36]. transportersThe size of formed [36]. The particles size of was formed submicron particles was was in the submicron nanometer was range in the (Figure nanometer 13). This range agreed (Figure with 13).the sizeThis analysis agreed with results the and size conﬁrmed analysis results that the and formed confirmed nanoparticles that the formed exhibited nanoparticles a size range exhibited suitable afor size oral range drug suitable delivery. for oral Due drug to challenges delivery. withDue to the challenges analysis ofwith chromium, the analysis which of chromium, requires the which use requiresof atomic the absorption use of atomic spectroscopy, absorption our spectroscopy future work, our will future focus work on the will analysis focus andon the optimization analysis and of optimizationchromium-loaded of chro carriers.mium-loaded carriers.

FigureFigure 13. TransmissionTransmission electron microscopy image of WPI-CS nanoparticles.

5. Conclusions Conclusions This studystudy reports reports the the formation formation of novelof novel iron andiron chromium-loaded and chromium-loaded nanoparticles nanoparticles using di ﬀusingerent differentbiopolymers. biopolymers. The properties The ofproperti the sampleses of the were samples characterized, were characterized, culminating in culminating an iron absorption in an assay.iron absorptionIt was seen assay. that increasing It was seen the chitosanthat increasing volume the and chitosan concentration volume signiﬁcantly and concentration increased significantly the particle increasedsize and zeta the potential,particle size whilst and thezeta opposite potential, was whils truet the for opposite dextran. was Permeability true for dextran. studies Permeability showed that studiesiron uptake showed was stronglythat iron correlated uptake was to particle strongly size correlated and that two to samplesparticle producedsize and higherthat two absorptions samples than ferrous sulfate. The results demonstrate the ability of the nanoparticles to act as novel delivery vehicles for supplementary therapy in the future.

Author Contributions: Conceptualization, H.A.-O. and M.G.Z.; methodology, H.A.-O.; validation, H.A.-O., M.G.Z. and N.P.; formal analysis, H.A.-O., M.G.Z. and N.P.; investigation, H.A.-O., M.G.Z. and N.P.; resources, Processes 2020, 8, 707 15 of 16

H.A.-O. and M.G.Z.; data curation, H.A.-O. and N.P.; writing—original draft preparation, H.A.-O. and N.P.; writing—review and editing, H.A.-O.; visualization, H.A.-O. and N.P.; supervision, H.A.-O.; project administration, H.A.-O. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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