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Drying TechnologyAn International Journal

ISSN: 0737-3937 (Print) 1532-2300 (Online) Journal homepage: https://www.tandfonline.com/loi/ldrt20

Optimization of nano spray drying parametersfor production of α-amylase nanopowder forbiotheraputic applications using factorial design

Heidi M. Abdel-Mageed, Shahinaze A. Fouad, Mahmoud H. Teaima, Azza M.Abdel-Aty, Afaf S. Fahmy, Dalia S. Shaker & Saleh A. Mohamed

To cite this article: Heidi M. Abdel-Mageed, Shahinaze A. Fouad, Mahmoud H. Teaima, Azza M.Abdel-Aty, Afaf S. Fahmy, Dalia S. Shaker & Saleh A. Mohamed (2019) Optimization of nano spraydrying parameters for production of α-amylase nanopowder for biotheraputic applications usingfactorial design, Drying Technology, 37:16, 2152-2160, DOI: 10.1080/07373937.2019.1565576

To link to this article: https://doi.org/10.1080/07373937.2019.1565576

Published online: 15 Feb 2019.

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Optimization of nano spray drying parameters for production of a-amylasenanopowder for biotheraputic applications using factorial design

Heidi M. Abdel-Mageeda,d, Shahinaze A. Fouadb, Mahmoud H. Teaimac, Azza M. Abdel-Atya,Afaf S. Fahmya, Dalia S. Shakerd, and Saleh A. Mohameda

aMolecular Biology Department, Genetic Engineering and Biotechnology Division, National Research Centre, Cairo, Egypt; bDepartmentof Pharmaceutics and Industrial Pharmacy Faculty of Pharmacy, Ahram Canadian University, Cairo, Egypt; cDepartment ofPharmaceutics and Industrial Pharmacy Faculty of Pharmacy, Cairo University, Cairo, Egypt; dDepartment of Pharmaceutics andPharmaceutical Technology Faculty of Pharmaceutical Sciences and Pharmaceutical Industries, Future University in Egypt (FUE),Cairo, Egypt

ABSTRACTThis study was designed to optimize the effect of operating conditions and formulationparameters using various additives to develop a-amylase nanoparticles. a-Amylase waschosen due to its importance in the substantial number of industrial processing withemphasis on pharmaceutical industry. Factorial statistical design was adopted to effectivelyoptimize the size, yield value, residual enzyme activity, and morphology of a-amylase nano-particles using Nano Spray Dryer B€UCHI B-90. The physicochemical characterization of theprepared nanopowder was carried out using zetasizer and scanning electron microscopy(SEM) and enzyme activity assay. Results showed that the type of additive and mesh sizesignificantly influenced the particles size and yield value. SEM images showed three differentstructure patterns where particle morphology was influenced by TweenVR 80 or sucrose atlow concentration (0.05%). Optimized spherical nanoparticles (600 nm) was obtained using7 mm mesh cap size, sucrose (0.15%), 95% yield value, drying flow rate (100 L/min), and inlettemperature of 80 �C. Higher storage stability was detected for enzyme spray-dried usinglarger cap size. It was concluded that nano spray drying of aqueous enzyme solution underdetermined operating conditions produced stable a-amylase powders. This would extendthe application of the enzyme in a variety of pharmaceutical products.

ARTICLE HISTORYReceived 12 September 2018Revised 1 January 2019Accepted 1 January 2019

KEYWORDSNanoparticles; a-amylase;nano spray dryer; drugdelivery; protein delivery;biotheraputics

1. Introduction

As the fields of biotechnology and nanotechnology arecontinuously integrating, more attention has beengiven to the use of proteins and enzymes as biother-aputics. Effective production methods are being devel-oped to meet the requirements for this progressivelyrising field. The highly sensitive nature of enzymesand proteins to physical and chemical stresses duringvarious stages of processing and storage represents themajor limitation and challenge for their pharmaceut-ical applications. The most common methods used toincrease the stability of enzymes and proteins are dry-ing and immobilization.[1–3] It has been reported thatwhen proteins and enzymes are in a dry state, theirthermal stability is markedly enhanced as a result ofwater evaporation. Water facilitates or mediates a var-iety of physical and chemical degradations. Hence, dry

solid formulations of enzymes are often sought afterto augment practical enzyme stability for commercialapplication.[4–6] Hence, protein-based drug is persist-ently formulated as solid dosage forms where greaterstability and higher activity can be achieved.[7]

Spray drying is a well-established versatile methodused in the pharmaceutical industry to generate drypowder from a liquid state where drug solution issprayed into air by atomization to evaporate the solv-ent.[8–9] Through utilizing the nano spray drying pro-cess, it is possible to produce spray-dried particles inthe submicron scale down to nanoscale (350–500 nm).In addition, it can produce remarkable high yields ofup to 94% for powder amounts down to the milligramscale (e.g. 3.0–500mg).[10] It is possible through utiliz-ing nano spray dryer to dry drug sample and producenanoparticles in a single step, within a continuous

CONTACT Heidi M. Abdel-Mageed heidi.abdelmageed@gmail.com; heidi.abdelmageed@fue.edu.eg Molecular Biology Department, GeneticEngineering and Biotechnology Division, National Research Centre, El Behoth St, Dokki, Cairo, Egypt.Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/ldrt.� 2019 Taylor & Francis Group, LLC

DRYING TECHNOLOGY2019, VOL. 37, NO. 16, 2152–2160https://doi.org/10.1080/07373937.2019.1565576

and scalable process. Spray drying has been consid-ered as an alternative to freeze-drying and is nowcommonly used to produce dried biotheraputics.Besides being a one-step process, spray drying offersthe advantage that no freezing or high vacuum isinvolved contrary to freeze-drying process.[11] Hence,spray drying techniques using nano spray dryer havebeen investigated by several authors as an optimummethod for preparation of thermosensitive com-pounds such as proteins and enzymes in a stable dryform, in the presence of different types of additivessuch as surfactants and sugars.[3,12–14] Stabilizing exci-pients are usually required during spray drying ofprotein formulations to prevent protein degradationduring processing and storage, and sugars has beencommonly chosen.[15] Sugars such as trehalose areusually used in pharmaceutical and biomedical appli-cations to prepare glassy matrices for long-term stor-age of biological membranes.[13] For a-amylasestabilization, sucrose was used in this study as a dry-ing aid to increase the glass transition temperature ofthe material, to reduce stickiness and to aid in theproduct recovery.

B€UCHI B-90 Nano Spray Dryer employs advancedpatent technology which uses vibrating mesh sprayhead, gentle laminar drying flow, and highly efficientelectrostatic particle collector. It offers the advantageof using sample amount or volume as small as 200mgor 2mL, with increased final product yields up to90%. Moreover, smaller particle size (PS) of 300 nmwith narrow size distribution can be obtained, andeventually, fast drying process (up to 150 L/min) wasattained.[9,12–15] However, manual collection of theproduct is a drawback of B-90 Nano Spray Dryer as itmight cause a variation in the results obtained asidefrom the formulation variables. Nonetheless, nanopar-ticle properties such as particle size, bulk density, andmorphology can be easily controlled via simplemanipulation of the process parameters or formula-tion. Producing nanopowder, especially in the field ofproteomics and genomics, is highly recommended toenhance their stability and therapeutic efficiency. Thiswould result in an optimal product for industrialprocessing in addition to minimizing the productioncost.[3,12,13,16]

a-Amylases (endo-1,4-a-D-glucan glucohydrolase;EC 3.2.1.1) are hydrolytic enzymes that catalyze thehydrolysis of starch into low molecular weight sugarmolecules. Amylases are of special biotechnologicalsignificance, constituting a class of industrial enzymeshaving approximately 65% of the enzyme market inthe world, and they are continuously increasing.[17]

They are used in the pharmaceutical industry asdigestive aid, for the production of cyclodextrins andin cross-linked polymer tablets to modulate the releasekinetics of drugs.[18] With advances in biotechnology,their spectrum of potential applications extends toclinical, medical, and analytical chemistry. In addition,they are used in various industrial processes includingfood, fermentation, and detergent.[19–20]

Using statistical analytical models provide an esti-mate of the output variables based on the selected setof input variables for the various parameters testedduring spray drying process.[21] In this study, frac-tional factorial design was used to evaluate the effi-ciency of B€UCHI B-90 Nano Spray Dryer to producenanopowder of a-amylase enzyme. The main objectivewas to investigate and optimize the operating condi-tions and formulation parameters for the productionof a-amylase nanopowder using nano spray dryerwith full enzyme activity and maximum possible yield.In addition, the influence of formulation parameterson particle morphology and shelf life was examined.The preparation of active and stable a-amylase nano-particles would facilitate and extend the robust appli-cation of this enzyme in the pharmaceutical industry.

2. Materials and methods

2.1. Materials

a-Amylase (EC 3.2.1.1) from Bacillus subtilis (�1,500units/mg protein A6380), 3, 5-dinitrosalicylic acid(DNS), soluble starch potato, sucrose, TweenVR 80(Polyoxyethylene sorbitan monooleate) were pur-chased from Sigma-Aldrich Chemical Co., St. Louis,MO. Polyethylene glycol 1000 (PEG 1000) was sup-plied from Adwic, El-Nasr Pharmaceutical ChemicalsCo., Egypt. PluronicVR F-127 was purchased fromBASF Corporation, Chemical division, N.J., USA. Allother chemicals were of reagent grade and purchasedfrom Merck (GmbH, Darmstadt, Germany).

2.2. Methods

Experiments were performed in triplicate, and the val-ues are the mean of at least three independent experi-ments. Standard deviations were always under 10%.

2.2.1. Experimental design and statistical analysisA fractional factorial design (Design ExpertVR software,Version 11, State-Ease Design, USA) was employedwith a total number of 21 experimental runs, includ-ing three replicates at the center point. The independ-ent process parameters were type of additive, additive

DRYING TECHNOLOGY 2153

concentration (%), and mesh cap size (mm), while theexamined response variables were particle size (nm),yield value (%), and residual enzymatic activity (%).The additives used in this study are TweenVR 80,PluronicVR F-127, PEG 1000, and sucrose. The levelstested for each variable are shown in Table 1.

2.2.2. Nano spray dryingAll experimental runs in this study were prepared byspray drying using Nano Spray Dryer B-90 (B€UCHILabortechnik AG, Switzerland) employing long-dryingchamber, spray cap with vibrating membrane to atom-ize the feed and allow the production of small particlesize, and electrostatic particle collector. Aqueous solu-tion of a-amylase and different additives tested atvarious concentrations at a final solid content concen-tration of 5% were spray-dried at the specified experi-mental conditions under normal humidity conditions.All samples were filtered to avoid blockage at thespray mesh.

Some of the operating conditions were chosen afterseveral preliminary trials and published work of otherauthors.[4,9] During spray drying, the operating condi-tions were set at inlet air temperature (80 �C), flowrate (95–105 L/min), relative spray rate (100%) wereheld constant. The outlet temperature increased withincreasing inlet temperature and is decreased withincreasing liquid feed rate which would result inincreased enzymatic activity.

For comparative studies, additional experimentswere carried out to investigate the morphology andthe residual enzymatic activity upon spray drying offree a-amylase, without the use of additives. Hence,aqueous free a-amylase solution (at a final solid con-tent 0.5, 1, and 2% w/v) was spray-dried under thepreviously mentioned process parameters. Sucrose waschosen as enzyme stabilizer in this study to avoid pos-sible interference with the enzyme assay. The spray-dried nanopowder was collected from the electrostaticparticle collector using a scraper and stored in a desic-cator at room temperature until further examination.

2.2.3. Physicochemical characterization2.2.3.1. Enzyme activity assay. The activity of a-amyl-ase was assayed according to the Bernfeld method,[22]

and determination was based on absorbance values readin an UV-Visible spectrophotometer (U-1800,Shimadzu, Japan) at 540nm, at room temperature. Theassay of free and spray-dried a-amylase included in onemL: 0.1mL of 1% (w/v) soluble starch as the enzymesubstrate, 100mM sodium phosphate buffer (pH = 7),and 0.1mL of enzyme sample. The assay mixture was

incubated at 37 �C for 15min in water bath. Theenzymatic reaction was stopped by the addition of 1mLof DNS reagent, and the reaction mixture was kept inboiling water bath for 10min. After 10min, 10mL ofdistilled water was added and reaction mixture wasallowed to cool to room temperature. The absorbance ofthe digested products of enzymatic reaction (reducedsugars) was measured spectrophotometrically at wave-length 540nm (a blank was prepared in the same man-ner without free a-amylase). A calibration curveestablished with maltose, 0.3–4 (mmol/mL) in 1mL ofdeionized water. One a-amylase activity unit (U) isdefined as the amount of the enzyme that liberates1.0 mmol of reducing sugar/min with maltose as astandard, under standard assay conditions. Residualenzyme activity was calculated as the percentage ofactivity of nano-sprayed enzyme divided by the activityof free enzyme. All assay experiments were carried outin triplicate.

2.2.3.2. Yield recovery. The efficiency of the nano-spraying process in producing a-amylase nanopowderwas calculated as the percentage weight nanopowderspray-dried compared to the total solid content ini-tially used.[23]

Yield value ¼ Total weight nano

�powder recovered=Total solid content �100

2.2.3.3. Particle size. Particle size was determinedusing the Mastersizer (Malvern Instruments, Hydro2000S, Malvern, UK). The calculations were based onMie theory. The dimensional information gained rep-resents the mean of five successive measurements of120 s. Every measured value is an average of 12 runs.All measurements were carried out at 25 �C.

2.2.4. Morphology of spray-dried particlesThe surface morphology of the spray-dried a-amylasenanoparticles was observed using scanning electronmicroscopy (SEM) (ESEMTM, Quanta 250-EFG,Thermo-Fischer Scientific, USA). Prior to imaging,samples were coated with gold for 60 s in a Jeol JSM-6400 (Tokyo, Japan) at National Research Centre,Cairo, Egypt.

Table 1. Levels of experimental parameters.Parameters 1 2 3 4

(A) Additive conc. (%) 0.05 0.1 0.2 0(B) Mesh cap size (mm) 4 – 7 –(C) Additive type Tween 80 Pluronic F127 PEG 1000 Sucrose

2154 H. M. ABDEL-MAGEED ET AL.

2.2.5. Shelf lifeFor stability evaluation, the prepared optimized nanospray-dried a-amylase powder and free amylase solu-tions (in 50mM potassium phosphate buffer, pH 7.0)were stored at 5 �C and at room temperature (25 �C),in tightly closed Eppendorf tubes. Enzyme activitymeasurements were made periodically for two monthsunder standard assay conditions. The percentageresidual activity was calculated as the proportion ofthe activity remaining at the end of the storage periodas compared to the initial enzyme activity at zerotime, which was defined as 100%.

3. Results and discussion

Encapsulating a-amylase within nanoparticles can aug-ment its usage in different industrial applications andprocesses. The results of the experimental runs arepresented in Table 2.

3.1. Effect of spray drying conditions

Spray drying conditions were carefully chosen after sev-eral initial trials. The inlet temperature was kept at 80 �Cto provide the lowest possible temperature for spray dry-ing of aqueous-based feed solution.[13] Using lower pos-sible temperature is also advantageous when spray dryingenzymes of thermosensitive nature. Lower inlet tempera-ture would result in lower outlet temperature. The outlet

temperatures recorded during this study ranged from 34to 46 �C. The outlet temperature is a critical variable inspray drying that can compromise the enzyme activity aswell, where reduction in both inlet and outlet tempera-tures is needed to preserve maximum enzyme activity andprevent product degradation.[21] Studies have shown thatthe loss of activity depended primarily on the inlet tem-perature and the temperature distribution within thedryer.[24] It has also been reported by Arpagaus that a lowoutlet temperature is necessary to avoid loss of enzymaticactivity in the dried enzyme sample.[10]

A high flow rate (95–105L/min) was maintained toprevent the adverse effects of temperature on enzymeactivity where the effect of drying air temperature onthe enzyme activity depends on feed flow speed. In add-ition, based on spray drying theory, when the atomizingflow rate is high, the drop exiting the spraying nozzlewould be smaller.[21] This would result in smaller par-ticle size as discussed later in Section 3.2. Ståhl et al.reported that the degradation of insulin dried at lowerfeed flow was much higher compared to that driedusing higher feed flow.[25]

3.2. Effect of formulation parameters onthe following

3.2.1. Residual enzyme activityIn this study, it was aimed to optimize formulation vari-ables during spray drying that would result in maximum

Table 2. Formulation parameters (A; B; C) and response variables (R1; R2; R3) of spray-dried nanopowder. Shelf-life stabilityresults are also presented.

Process parameters Response variables

A B C R1 R2 R3

Run Additive conc. (%) Mesh size (mm) Additive type PS (nm) Yield value (%) Enzyme residual activity (%)Activity after60 days (%)�

1 0.2 7 Sucrose 698 94 99 722 0.2 7 PEG 1000 NA NA NA NA3 0.05 4 PEG 1000 454 75 94 714 0.05 4 Sucrose 367 94 99 725 0.2 4 Sucrose 400 94 99 726 0.2 4 Tween 80 590 90 98 657 0.2 4 Pluronic F127 NA NA NA NA8 0.05 7 Pluronic F127 678 87 96 689 0.05 7 Sucrose 650 94 99 7210 0.05 7 PEG 1000 879 75 94 7111 0.2 7 Tween 80 1057 90 97 6512 0.2 7 Sucrose 698 94 99 7213 0.2 7 Tween 80 1057 90 97 6514 0.2 7 Pluronic F127 NA NA NA NA15 0.2 4 PEG 1000 NA NA NA NA16 0.2 4 Pluronic F127 NA NA NA NA17 0.05 7 PEG 1000 879 75 98 7118 0.05 7 Tween 80 602 90 98 6519 0.05 4 Tween 80 337 91 97 6520 0.05 4 Pluronic F127 345 87 96 6821 0.05 4 PEG 1000 454 75 97 71

NA: not applicable.�Shelf-life stability calculated as mean % residual enzyme activity, n¼ 3.

DRYING TECHNOLOGY 2155

enzyme activity. Statistical analysis (p< 0.0001) showedthat the mesh size and additive concentration had nosignificant effect on residual enzyme activity (Figure 1).Results presented in Table 2 show that the residualenzyme activity for all experimental runs carried outwas greater than 94%. The enzyme activity was fullypreserved (99%) in runs (R1, R4, and R5) in the pres-ence of sucrose. The lowest residual activity wasrecorded for run (R10). Samples of free a-amylase(without additive) spray-dried at same operational con-ditions showed greater loss of enzyme activity. Theresidual enzyme activity measured for 0.5%, 1%, and 2%w/v free enzyme samples were 80%, 82%, and 83.4%,respectively. During spray drying of protein formula-tions, thermal denaturation of proteins is not generallyobserved, as the temperature of the droplet hardlyexceeds the wet bulb temperature of water (�40 �C).Nonetheless, the denaturation temperature of proteins isa function of water content, increasing sharply withdecreasing water content.[26] Suggested mechanismsreported in the literature that would prevent proteindegradation during spray drying included glass immobil-ization and/or water replacement (water replacementhypothesis).[ 6,27] Additives such as surfactants or sugarchosen in this study are commonly used excipient forprotein-based pharmaceutical products. Surfactants werereported in the literature by several authors to inhibitprotein aggregation.[28,29] This was explained by Adleret al. who reported that upon adding TweenVR 80 to thea-amylase solution the balance of surface-to-viscousforces was altered which resulted in smooth particle sur-face after spray drying.[ 30] The used operating condi-tions in this study for the nano spray dryer B-90 proved

to be temperate and suitable for drying of a-amylasewithout significant activity loss.

3.2.2. Yield valueFigure 2 shows the response surface plot of the effect ofthe formulation variables on yield value. ANOVA statis-tical analysis (p< 0.0001) revealed that the three inde-pendent variables (mesh size, additive type, and additiveconcentration) had a significant effect on the yield value.The yield value obtained with the different experimentalruns ranged from 75% (R4 and R10) to 94% (R1, R4, R5and R9) for powder amounts equivalent to 500mg.

Higher yield values were observed in the presenceof TweenVR 80 or sucrose. Formulations containingPluronic F127 exhibited lower product recovery. Thiscan be attributed to the thermosensitive gel forming

Figure 3. Response surface plot of the effect of additive con-centration (%) and mesh size (mm) on particle size (nm).

Figure 1. Response surface plot of the effect of additive con-centration (%) and mesh size (mm) on enzyme residual activ-ity (%).

Figure 2. Response surface plot of the effect of additive con-centration (%) and mesh size (mm) on yield value (%).

2156 H. M. ABDEL-MAGEED ET AL.

character of Pluronic F127 which caused a higher lossof the product during recovery. Lower product recov-ery was also evident in case of PEG 1000 due to itsmelting inside the collecting chamber at the operatingoutlet temperature and sticking to the collector.Thereafter, as shown in Table 2, spray drying ofexperimental runs with 0.2% Pluronic F127 or PEG1000 was not determined. In fact, spray drying ofthese samples was stopped after few minutes as spraydrying was not possible where the increased concen-tration of these additives caused a higher density andkinematic viscosity of the feed solution. During spraydrying, product loss principally occurred through walldeposition and loss during manual collection of theproduct from the collecting electrode.

3.2.3. Particle sizeAs this study aimed to produce spray-dried a-amylasenanopowder for biotherapeutic application, obtainingnanoparticle size was of primary interest. Results of particlesize measurements revealed that under the used processparameters, values measured for all experimental runswere below 1 mm. As shown in Table 2, runs (R4 and R19)had measured particle size below 400nm which is a prettyremarkable result for a spray drying technique. ANOVA

analysis revealed the significant effect of formulation varia-bles on the particle size. The response surface plot of theeffect of the formulation variables on particle size (Figure3) shows that upon increasing the additive concentration,the particle size significantly increases (p¼ 0.0002) andalso upon increasing the mesh size, the particle size signifi-cantly increases (p< 0.0001). The type of additive had anaverage effect most pronounced with sucrose. These resultsare in agreement with previous reports where Lee et al.reported the dominance of spray mesh size parameter overother tested variables for particle size using Taguchi experi-mental design.[9] Kaerger and Prince suggested a weakdependence of the particle size on the protein concentra-tion as explained by the particle diameter being propor-tional to the cube root of the concentration.[31]

3.3. Particle morphology

A representative range of four different additivesincluding a stabilizer was tested. The spray-dried sam-ples were generally powdery. Three types of nanopar-ticle morphologies were observed by SEM (Figure 4):(A, B) smooth spherical, (C) fiber-like, (D) coral-like.Smooth spherical a-amylase particles were obtainedfrom experimental runs in the presence of sucrose orTweenVR 80, while the fiber-like samples were obtained

Figure 4. Representative SEM images of samples product with different morphologies: (A) smooth spherical with sucrose, (B)smooth spherical with Tween 80, (C) fiber-like with Pluronic F127and free enzyme, (D) coral like for free enzyme.

DRYING TECHNOLOGY 2157

with added PluronicVR F127. The coral-like structurewas obtained upon spray drying of free enzymesolution at the tested concentrations (0.5, 1, 2 w/v%) without any additives. It was also notable thatonly a small amount (0.05%) of either TweenVR 80 orsucrose was sufficient to cause radical improvementin particle morphology from irregular to spherical.This observation was obvious from SEM imagesshown in Figure 4. The fiber-like structure is typicalfor Pluronic F127 due to its thermoreversible gel-ation in water at outlet temperature during spray

drying. It is worthy to note that the obtained fiber-like structure nanopowder should be useful for fur-ther studies employing nano spray-dried a-amylasefor fast-dissolving tablets. Our results are in agree-ment with previous reports which supported thatthe addition of surfactant or sugar to the spray-dried proteins changed the deep hole morphology toa much smoother particle exterior.[8,30,32,33] Thesestudies also described a shriveled morphology forspray-dried protein which was changed to sphericalupon addition of surfactant.

Figure 5. Optimization of nano-spray drying parameters to reach the desirability value (¼ 1).

2158 H. M. ABDEL-MAGEED ET AL.

3.4. Shelf-life stability

Results of shelf life stability are tabulated in Table 2.Free enzyme showed 41% and 25%residual activityafter 15 and 60 days, respectively. On the other hand,the optimized nano spray-dried powder retained 85%of its activity after 15 days and 72% after 60 days.Several authors reported the use of additives toimprove stability of proteins during spray drying.[13,34]

Burki et al. also reported a higher residual enzymeactivity for powders with larger particles after storagethan powders with small particles.[35] On industriallevel, using nanopowder in comparison with solubleenzyme could reduce the cost by reducing the quan-tity required of the enzyme for same process.[36,37]

3.5. Optimized conditions

In this work, it was aspired to achieve smooth spher-ical nanoparticles of a-amylase that would producestable unaggregated particles. Figure 5 shows the val-ues of each independent variable to reach the desir-ability value (1). The suggested parameter values ofthe optimized formulation are very close to R1obtained using the employed factorial design. Toreach the optimal formula, an improved optimizedrun was carried out. The obtained nanoparticles weresmooth and spherical with 94% yield value, particlesize of 600 nm and residual enzyme activity of 98%.

4. Conclusion

The designed series of experimental trials aimed todetermine the drying parameters and formulationparameters that would produce a-amylase nanopow-der with highest preserved enzyme activity at thehighest yield. Optimized production of smooth spher-ical nanoparticles (600 nm, yield 94%, and residualenzyme activity 99%) was achieved using 7-mm spraycap and sucrose concentration 0.15% (w/v), dryingflow rate 100 L/min, and inlet temperature of 80 �C.The results revealed the capacity of the Nano SprayDryer B€UCHI B-90 to preserve the activity of thedried enzyme, a fundamental prerequisite for nanobiotherapeutics in the pharmaceutical industry. Theresults obtained in this study present a solid startingpoint for further studies for the formulationof a-amylase.

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