Materials Science and Engineering C 59 (2016) 310–318

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Materials Science and Engineering C

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Synthesis of gelatin nano/submicron particles by binary nonsolvent aidedcoacervation (BNAC) method

Shamayita Patra ⁎, Piyali Basak, D.N. TibarewalaSchool of Bioscience and Engineering, Jadavpur University, Kolkata - 700032, India

⁎ Corresponding author.E-mail address: shamayitapatraster@gmail.com (S. Pa

http://dx.doi.org/10.1016/j.msec.2015.10.0110928-4931/© 2015 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 9 July 2015Received in revised form 18 September 2015Accepted 5 October 2015Available online 8 October 2015

Keywords:Gelatin nano/submicron particles (GN/SPs)Modified single step coacervation methodNitrofurazone

A newly developed modified coacervation method is utilized to synthesize gelatin nano/submicron particles(GN/SPs) as a drug carrier. Binary nonsolvent aided coacervation (BNAC) method is a modified single step coac-ervation method, which has yielded approximately a threefold lower particle size and higher average yield interms of weight percentage of around 94% in comparison to the conventional phase separation methods. Inthis study 0.5% (w/v) gelatin aqueous solution with a binary nonsolvent system of acetone and ethanol wasused. Nanoparticle synthesis was optimized with respect to nonsolvent system type and pH. pH 7 has resulteda minimum particle size of 55.67 (±43.74) nm in anhydrous medium along with a swollen particle size of776 nm (±38.57) in aqueousmediumwith a zeta potential of (−16.3± 3.51)mV in aqueousmedium. Swellingratio of 13.95 confirms the crosslinked hydrogel nature of the particles. Furthermore, drug loading efficiency ofthe gelatin particles prepared at 7 pHwas observedwith nitrofurazone as themodel drug. Results of drug releasestudy indicate the potential use of GN/SPs as drug loading matrix for wound management such as burn woundmanagement.

© 2015 Elsevier B.V. All rights reserved.

1. Introduction

Nano materials are powerful means to deal with different bottle-necks of existing scientific fields. High specific surface area, high surfaceenergy and low particle size have bestowed unique properties to thesenano particles and made them useful in smart material fabrication forelectronics, healthcare, defence and many other research areas [1,2].Nano particles are synthesized from a variety of materials such as,metals like gold (Au), silver (Ag), copper (Cu), salts like titanium diox-ide (TiO2), sodium alginate and soft polymeric materials [1,3–7]. Poly-meric nano particles are widely utilized as carrier matrix for drugmolecules, proteins, plasmid DNA and vaccines [8–11]. These carriermatrixes facilitate targeteddelivery, sustained release and lowermagni-tude of dosage [12]. Among natural polymers, proteins, polysaccharides,serum albumin, starch, chitosan, and liposomes are employed for nano-particle synthesis [13–18]. Also, several synthetic polymers such aspoly(lactic acid) (PLA), polycaprolactone (PCL), poly(cyanoacrylate)(PCA) and poly(lactic-co-glycolic) acid (PLGA) are utilized as carriermatrix [19–22].

Particle size, surface characteristics, and drug release profile arethe controlling factors for nano particle synthesis [23]. Conventionally,nanoparticles are prepared by dispersion of preformed polymers,polymerization of monomers, ionic gelation or coacervation method

tra).

along with their modified versions such as nanoprecipitation [23,24].Supercritical fluid technology and particle replication in non-wettingtemplates (PRINT) are also used for the same [25,26]. Synthesis of nano-particles of 100 to 500 nm size range, even below 50 nm is abundant[27–35]. However, majority of research papers have not mentionedabout the yield. Most of the high yield and lower polydispersity particleproducingmethods are based upon sophisticated and costly instrumentmediated processes such as template, lithography, 3D hydrodynamicflow focusing in single-layer microchannels based methods [26,36,37].Most of the processes related to nonsolvent based phase separationhave utilized supernatant or emulsifiers to produce lower particle size[34,35]. Microwave aided surfactant based method have resultedhighestweight percentage or yield of 40% for polystyrene nano particleswith particle size of 60 nm [30]. Thus, lower yield and/or additional stepof emulsifier separation are characteristics of conventional phase sepa-ration based synthesis methods. In this paper, a new approach isascertained to prepare GN/SPs with high yield. Low immunogenicity,biocompatibility, biodegradability, non-toxicity, simplicity in chemicalmodification, ease in cross-linking, rich amino acid content and pres-ence of multiple functional groups for different chemical anchoring(e.g. ionic, covalent) sites for drugs, proteins, growth factors andmacro-molecules have made gelatin, the low cost biopolymer, a lucrative can-didate for nano-carriers [27,28].

The new approach utilizes binary nonsolvent system to synthesizegelatin nanoparticles by traditional coacervation or phase separationmethod. Till now emulsion process yields the lowest particle size inthe range of 100–500 nm [27,28]. However, this slight modification

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has improved the efficiency of the phase separation method in terms oflower particle size and simplicity in the process as well as higher aver-age yield of about 94%. Modified phase separation method has yieldedan average minimum particle size of 55.67 (±43.74) nm in dry state.A probablemechanismalongwith optimization of themodifiedmethodhas been jotteddown in this researchwork. To show the effectiveness ofthe gelatin particles as drug reservoir, non strain resistant antimicrobialdrug nitrofurazone is utilized [38].

2. Materials and methods

2.1. Methods

2.1.1. Synthesis of GN/SPs by BNAC methodGN/SPs were synthesized by newmodified one step simple ionic ge-

lation or coacervationmethod. For this purpose gelatin, acetone, glutar-aldehyde (25% solution, Anhydrous) were procured from MERCK andethanol (EtOH) was obtained from Changshu City Yangyuan ChemicalCo., Ltd. In all experiments, double distilled water was utilized. Thenano/submicron particles were synthesized from 0.5% (w/v) gelatinaqueous solution at 40 °C and 750 rpm. Instead of single nonsolvent(acetone), binary nonsolvent system of acetone and ethanol was used.The volume ratio of aqueous medium, acetone and ethanol was kept1:1:1. Particles were cross linked by glutaraldehyde ethanol solutionwith a concentration of 1.6% (v/v). pH of the aqueous medium was setat different pH (3 to 11) to investigate its effect on particle size. Ineachwashing cycle, the systemwas ultrasonicated for 5min and follow-ed by centrifugation at 2000 rpm to separate out washed particles fromaqueous phase. The used ultrasonication instrument was RivotekTMmake Ultrasonic Processor (Sonicator) with ultrasonic power of120 W and 30 ± 3 kHz frequency.

2.1.2. Drug (nitrofurazone) loading in GN/SPsNitrofurazone (Sigma-Aldrich) has a minimum inhibitory concen-

tration (MIC50) of 19 mg/l along with maximum water solubility(MWS) of 240 mg/l. GN/SPs were submerged into nitrofurazone aque-ous solution for 48 h. To investigate the drug loading efficiency,nitrofurazone aqueous solution was prepared with 25, 50 and 100% ofMWS.

2.2. Material characterization

2.2.1. Identification of isoelectric point and type of the gelatinTo identify the type of uncategorized gelatin, isoelectric point of the

gelatin was determined by procedure adapted from WHO Pharmaco-poeia Library and amalgamated with ultraviolet–visible (UV–Vis)

Fig. 1. Calibration plot of optical density at 368 nm vs. nitrofurazone concentration.

spectroscopy [39,40]. In brief, 5 ml of 1% (w/v) gelatin aqueous solution(at 40 °C) was exposed with equivalent amount of buffer solutions ofdifferent pH (4.0, 5.0, 6.0, 7.0, 8.0, or 9.0) in glass bottles and kept at 4°C for 24 h after a gentle cooling. Type of gelatin was determined bythe yielded opalescence as identified through the measured maximum

Fig. 2. FESEM picture of gelatin particles synthesized at pH 7 from (a) acetone nonsolventsystem, (b) acetone ethanol nonsolvent system and (c) ethanol nonsolvent system. (Alldehydrated particles are represented here).

Fig. 3. FESEM picture of gelatin particles synthesized at pH 7 from (a) acetone nonsolventsystem, (b) acetone ethanol nonsolvent system and (c) ethanol nonsolvent system. (Allswelled particles are represented here).

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intensity at 360nm inUV–Vis spectrum [40]. pH7was obtained by pH 7buffer capsule and all other pHswere set by 1Mhydrochloric acid (HCL)solution and 1 N NaCl solution. Amaximum opalescence at pH 5.0 and amaximumopalescence between pH 7.0 and pH9.0were regarded as theindication of Type B and Type A gelatin respectively [39].

2.2.2. Quantification of swelling ratio of GN/SPsTo determine swelling ratio of the GN/SPs prepared from different

pHmediums, particles were submerged either in distilled water (to ob-tain the maximum swelled dimension) or in acetone (to acquire mini-mum dimension i.e. maximum collapsed dimension) for 72 h andlyophilized at (−40) °C. IIC Industrial Corporation manufactured labo-ratory freeze dryer was utilized for this purpose. Maximum swelledand maximum collapsed particle structures were then investigatedthrough particle size analyser (ZetaPlus Particle Sizer, ZetaPlus ParticleSizing Software Version 3.42, Brookhaven Instruments Corp.), scanningelectron microscope (SEM, JEOL–JSM–6360 scanning electron micro-scope with JEOL–JFC–1600 Autofine coater for Palladium coating fromInternational Equipment Trading Ltd. USA) and field emission scanningelectronmicroscope (FESEM, INSPECT F 50, FEI, USA) to determine theirindividual maximum and minimum particle size and swelling ratio.Also, same FESEM, SEM photographs and ADOBE Photoshop 7.0 wereused to determine particle size distribution for dried GN/SPs synthe-sized at pH 7. For this, dimension of at least 300 particles were calculat-ed through the software and their frequency in terms of percentagewere calculated by following simple formula.

Fn %ð Þ¼ InN�100

where, ‘n’ represents a particular particle size range, Fn represents fre-quency in percentage (%) of particles within a certain particle sizewith respect to total amount of particles, In indicates no. of particleswithin ‘n’ size range and N is the total number of particles. Correspond-ing particle size dispersion is represented in a frequency polygon in theresult and discussion segment.

2.2.3. Estimation of yield (weight %) of BNAC methodTo calculate yield of the BNAC method oven dry weight of the syn-

thesized GN/SPs and gelatin was employed. GN/SPs and gelatin wasdried at 105 °C for 2 h and weighed in predried and weighed weighingbottles with lids to measure oven dry weight. After 2 h drying, sampleswere left to cool in thedesiccator beforeweighing [41,42]. Yield of BNACmethod was calculated by the following equation.

Yield wt%ð Þ ¼ Oven dry weight of GN=SP gð ÞOven dry weight of Gelatin gð Þ � 100:

For this calculation GN/SPs synthesized at pH 7 was utilized.

2.2.4. Estimation of loaded drug (nitrofurazone) in GN/SPsOptical density (OD) of known aqueous solutions was measured by

UV–Vis spectrophotometer to obtain a standard calibration plot (Fig. 1)for nitrofurazone at 368 nm [43]. Intensity at this wavelength isemployed to prepare calibration plot as well as to measure the loadingefficiency of gelatin particles. To measure loading efficiency, opticaldensity of drug solutions with different concentration before and afterdrug incorporation in GN/SPs for 72 h was estimated by UV–Visspectrophotometer.

2.2.5. Release study of loaded nitrofurazone in GN/SPsSynthesized particles were envisioned to be utilized as drug carrier

into the burn wound site, thus release study was accomplished intothe simulated wound fluid (SWF), representing the burn wound fluid.For SWF foetal bovine serum (FBS) (Gibco), maximum recoverydiluent (MRD) (Fluka), peptone (Loba) and sodium chloride (NaCl)

(MERCK) were exploited. SWF composition comprises of 50% FBS, 50%MRD along with 0.1% (w/v) peptone and 0.9% (w/v) Sodium Chloride[45].

Optical density method is followed to measure the presence ofreleased macromolecules in SWF. For nitrofurazone, at 368 nm ODwas registered by UV–Vis spectrophotometer. Bacteria and fungi are

Fig. 4. a. Effect of synthesis medium compositions over GN/SP size. b. Frequency polygon for particle size distribution of dried GN/SP synthesized at pH 7 and acetone ethanol nonsolventmedium.

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themost common pathogens for burnwounds.Within initial 48–72 h ofburn injury various microbes form multi-species biofilms on burnwounds [46]. However, Peterson et al. [47] have identified initial 19 htime span as “golden period” for wound healing and burnwound dress-ings are usually removed after 24 h [48]. Thus, the time span of thisstudy set from 2min to 25 h from incorporation of nitrofurazone loadedGN/SPs into the SWF.

Fig. 5. Schematic representation of BNAC method

3. Results and discussion

3.1. Synthesis of GN/SPs by binary nonsolvent aided coacervation (BNAC)method

Binary nonsolvent aided coacervation (BNAC) method has beenemployed to prepare GN/SPs for drug delivery applications. Similar to

of GN/SP synthesis. (x = 3, 5, 7, 9 and 11).

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basic coacervation or phase separation methods, this method also uti-lizes fundamental principles of liquid–liquid phase separation. Unlikeconventional methods, BNAC utilizes binary nonsolvent system toinduce liquid–liquid phase separation in the homogenized solution ofionizedmacromolecule (gelatin). Termonia et al. [49] have described in-teraction between polymer, solvent and nonsolvent as the controllingfactor for phase separation behaviour of polymer. They have ascertainedabout dust like particle formation frommiscible solvent and nonsolventalong with poor miscibility (i.e. higher interaction energy) of polymerand nonsolvent. Acetone is strong nonsolvent for gelatin as well as itis miscible with water. Thus, it produces lower particle size of 71.79(±62.71) nm (dried) and 197.66 (±157.44) nm (swelled) in compari-son to the ethanol system. Ethanol provides comparatively higherinteraction opportunity with gelatin, in terms of hydrogen bond. Thus,ethanol system has produced bigger particle size of 237.21 (±197)nm (dried) and 449.04 (±128.63) nm (swelled) (Figs. 2,3).

Whereas, binary nonsolvent system of acetone and ethanol yieldedaverage particle size of 55.67 (±43.74) nm (dried) and 776 (±38.57)nm (swelled) (Figs. 2,3). These results definitely indicate generation ofsmaller polymer rich phases through BNAC method, which enables tosynthesize lower particle size (Fig. 4a) along with better homogeneousparticle size (Fig. 4b). Though there is a small second hump around 90–100 nm in Fig. 4b, still it is showingmaximumpopulation of particle sizearound 10–20 nm. Schematic presentation of the GN/SP synthesis byBNAC method is presented in the Fig. 5.

Reason behind the result can be elucidated by the light of solubilityparameter or Hildebrand solubility parameter (δ) of solvent (water)and nonsolvents (acetone and ethanol). Solubility parameter (δ) is nu-merical representation of the relative solvency profile of a particular sol-vent. Solubility parameter represents total van derWaals force betweentwomolecules. It comprises of threemajor components and can be rep-resented by the Eq. (1) as proposed by Hensen [50].

δ2T¼δ2Dþδ2Pþδ2H ð1Þ

where, δT is total solubility parameter and δD, δP and δH representsHensen solubility parameters i.e. dispersive force, polar cohesive energyand electron exchange or hydrogen bonding energy of any material[50].

R2a¼ 4 δD1‐δD2ð Þ2þ δP1‐δP2ð Þ2þ δH1‐δH2ð Þ2: ð2Þ

Further, solubility parameter distance (Ra) between two materialsbased on Hensen solubility parameters (Eq. (2)) gives a quantitative

Table 1Literature reported Hildebrand solubility parameter (δ), total solubility parameter (δT) andδH =electron exchange or hydrogen bonding energy) alongwith calculated values of averawater system. (All values are represented in MPa1/2).

Component Volume fraction (∅i) δ δT δD

Gelatin 24.5[50] – 17.1Water (W) ∅W 48.00[50] 50.80[50] 15.6Acetone (A) ∅A 19.70[50] 20.00[50] 15.5Ethanol (E) ∅E 26.20[50] 26.50[50] 15.8

W + A∅W=1/3∅A=2/3

– – –

W + E∅W=1/3∅E=2/3

– – –

A + E∅A=1/2∅E=1/2

– – –

W + A + E∅W=1/3∅A=1/3∅E=1/3

– – –

idea about the most favourable interaction between different compo-nents of a solution system [50]. In Eq. (2), subscript 1 and 2 representstwo differentmaterials. Solubility parameter of the blended solvents (δ)can be acquired by averaging their individual solubility parameter (δi)values by their volume fraction (∅i) contribution in the solvent system(Eq. (3)) [50].

δ ¼X

i

∅iδi: ð3Þ

Ra and other solubility parameters of different solvent, nonsolventand polymer combination are summarized in Table 1.

Presence of hydrophobic groups (amide groups) [51] in gelatinhinders its solubilization at room temperature in aqueous medium.The difference in the solubility parameter of gelatin and water clear-ly indicates the same. Main reason behind the solubility of gelatin inwater at higher temperature (say 40 °C) is the formation of hydrogenbonds and ionic interaction with medium. Gelatin transforms fromsol to gel state with very low viscosity above transition temperature(≈30 °C) at a polymer concentration, below the critical chain over-lapping concentration (≈2% (w/v)) [52]. With respect to the hydro-gen bond formation capacity (on the basis of δH value) one canarrange water, acetone and ethanol in the following ascendingorder acetone b ethanol b water. The ascending hydrogen bond for-mation capacity of these three liquid governs their interaction be-tween them and gelatin. Thus, addition of ethanol to the acetoneinduced destabilized aqueous gelatin solution dehydrates polymer(gelatin) rich phases further and forms more compact polymerchain arrangement (Fig. 6) to yield minimum particle size.

Introduction of glutaraldehyde, subsequently secures the three di-mensional arrangement to synthesize stable particles. However, maxi-mum compactness in binary nonsolvent system may have causedminimum accessible functional group for crosslinking. Hence, it gavemaximum particle size in swelled condition (Fig. 4a).

3.2. Optimization of particle size with respect to synthesizing medium pH

Gelatin is an amphoteric molecule [53]. It can sustain positiveor negative charges according to the pH of the medium. Therefore, theeffect of pH of the medium over the size of synthesized particles hasbeen studied (Fig. 7). Residual charge in polymeric chain will repulseother polymeric chains and will result bigger particle size and higheraccessibility to the crosslinker. Therefore, it will result in higher particlesize with lower magnitude of swelling. On the contrary, polymer

Hensen solubility parameters (δD =dispersive force, δP =polar cohesive energy andge solubility parameter (δ) and interaction radius (Ra) of gelatin, acetone, ethanol and

δP δH δ Ra

9 ± 1.34[50] 17.98 ± 4.66[50] – – –0[50] 16.00[50] 42.30[50] – –0[50] 10.40[50] 7.00[50] – –0[50] 8.80[50] 19.40[50] – –

– – 29.13 35.74

– – 33.47 24.01

– – 22.95 12.52

– – 31.30 –

Fig. 6. Schematic representation of different steps of BNAC aided GN/SP synthesis. St-1 gelatin at the gel state at 40 °C in aqueous medium at pH 7, St-2 Introduction of Acetone, St-3aIncorporation of ethanol and glutaraldehyde, St-3b & c crosslinking of gelatin and synthesis of GN/SPs.

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chains will assumemaximum compact structure in neutral charge con-dition i.e. at its isoelectric point along with lower accessibility to thecrosslinkermolecules. Due to the isoelectric point at pH7 of utilized gel-atin (Fig. 8), medium with pH 7 had yielded minimum particle size of55.67 (±43.74) nm (dehydrated) with maximum swelling ratio of13.95 (Fig. 9). Thus, it is showing a probable use of these particles asdrug carrier. These particles have shown a zeta potential value of−16.7 ± 3.51 mV at 25 °C (conductivity: 0.0139 mS/cm), which indi-cates eventual aggregation. However, 2 min ultrasonication (ultrasonic

power of 120 W and 30 ± 3 kHz frequency) has provided stable aque-ous dispersion for further application.

3.3. Loading of drug molecule

3.3.1. Optimization of drug (nitrofurazone) loading efficiency with respectto different drug concentration

Three different concentrations of nitrofurazone (in stock solutions)were applied to load drug in the particles and highest nitrofurazone

Fig. 7.Effect of pHover gelatin particle size in aqueousmediumat (○) swelled condition inaqueous medium and (■) dried condition in Acetone medium.

Fig. 8.UV–Vis Absorption (at 360 nm) of gelatin aqueous (1%w/v) solution at different pH.

Fig. 10. Effect of nitrofurazone concentration over (♦) magnitude and (□) efficiency ofnitrofurazone loading in GN/SPs.

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stock solution concentration (i.e. 24 mg/100 ml) have yielded maxi-mum loading efficiency (Fig. 10). Maximum magnitude of loaded drugwas 631.39 mg of Drug/mg of GN/SP.

3.3.2. Drug release study in simulated wound fluid (SWF)To observe actual drug release profile in burn wound, SWF was

opted as medium. Nitrofurazone loaded GN/SPs have shown controlledrelease profile, which even sustained an effective drug concentrationafter 24 h (Fig. 11). However, GN/SPs loaded from the stock solution

Fig. 9. Swelling ratio of GN/SPs synthesized in different pH medium.

prepared with 25% of the maximum nitrofurazone concentration (i.e.24 mg/100 ml) have lost their effectiveness against infection, whichhas been indicated by sudden increase in release profile (Fig. 11),changed transparency and lowered pH (Fig. 12). Lower pH indicatesbacterial activity.

4. Conclusions

Modified coacervationmethod or binary nonsolvent aided coacerva-tion (BNAC) method has demonstrated its capability to produce homo-geneous gelatin nanoparticles with an average size of 55.67 (±43.74)nm. Also, this method assures about high yield (average yield around94%) due to the utilization of entire gelatin aqueous system, instead ofsupernatant solution [35]. Hence, the proposed BNAC method can beutilized as one of the solution for poor nano particle synthesis yield,even without employing any costly and sophisticated instrumentor technology. On the other hand, result of drug release study ofnitrofurazone loaded gelatin particles have shown controlled releasebeyond24h. Thus, theymay be utilized as drug carriermatrixwith a po-tential for burn wound management. As, nitrofurazone is used to treatburn injuries.

Acknowledgement

This researchwork is underDr. D. S. Kothari Postdoctoral Fellowship,University Grants Commission, India.

Fig. 11. Drug Release profile of GN/SP loaded with nitrofurazone from (○) 25%, (□) 50%and (◊) 100% of maximum concentration of nitrofurazone aqueous solution. (Maximumconcentration: 24 mg/100 ml).

Fig. 12. Change in pHof SimulatedWound Fluid after incubation for 24 h at 37 °C (a)without any additive, with (b) virgin GN/SP, with GN/SP loaded from (c) 25%, (d) 50% and (e) 100% ofmaximum nitrofurazone concentration (24 mg/100 ml) in aqueous medium.

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