In Situ Coating—an Approach for Particle Modification and Encapsulation of Proteins During...

International Journal of Pharmaceutics 323 (2006) 5263

In situ coatingAn approach for parrinlqvSE-1580,May

006

Abstract

In this pa les inthe particle/p onalmethod used formand either o ) andpolymer (Po oscopconcentratio ted fras measured by the pendant drop method. Further, particle properties such as: size, dissolution time, powder flowability, and apparent particledensity, as measured by gas pycnometry, were affected by the type and concentration of the polymer. In addition, the particle surface morphologycould possibly be correlated to the surface elasticity of the droplet surface during drying. Moreover, an extensive investigation (Fourier transforminfrared spectroscopy, circular dichroism and size exclusion chromatography) of the structural effects of protein encapsulated in a polymeric coatingsuggested th 2006 Else

Keywords: Po

1. Introdu

Particleality or fordients, is iis an indusparticles (96MO

, Swe, WPEOonce

0.01uffe

oreCheEu

in ist. Loglassoluwitz

pray

roxufferhematic illustration of the formulation concept in situ coating.

mpetition during spray-drying implies adsorption ofive components to the air/liquid interface of drying

contrast to steady-state adsorption the time-scalee in spray-drying is restricted. The average life-plet surfaces, in a laboratory dryer was estimatedms (Elversson and Millqvist-Fureby, 2005b). Con-ransport and attachment to the interface is importantpetitiveness during spray-drying. At the surface, a

polymer that can rapidly rearrange and expose non-ns towards the air phase would be expected to be

studies establish the fact that the composition ofsurface is preserved during spray-drying (Faldt andl, 1994; Millqvist-Fureby et al., 1999; Landstrom etdler et al., 2000; Elversson and Millqvist-Fureby,

r example, in mixtures of BSA or sodium caseinate, protein is accumulated at the air/water interfaceying and thus appears on the powder surface (Faldtstahl, 1994). In contrast, neither of the components

tially accumulated in a mixture of glycine and lac-us, the surface composition of the powder reflects

sition of the spray solution (Faldt and Bergenstahl,hence possible to utilize surface competition dur-

drying in order to create desired powder proper-ttability, dissolution (Elofsson and Millqvist-Fureby,urface morphology (Elversson and Millqvist-Fureby,

tend to denature when exposed to an air/water inter-the protein is frequently unfolded. Considering the

ce area in a spray, the potential for surface-inducedn is substantial (Mumenthaler et al., 1994; Maa et

illqvist-Fureby et al., 1999), in particular at a low. Possibly, addition of a polymeric coating to proteins prepared by spray-drying can enhance protein sta-

eventing/reducing proteinsurface interactions. This

than stMillqvsitu cosimult

Conbilizatmethofied reformatconcer

homogimizedparticlformatcoating

2. Ma

2.1. M

Solmer w

tion VLouis,BuchswaukePPO27final cand 0,phate bMillip(Fluka(Merckorosce

Co., SAll

washwil, Swater.

2.2. S

Hydcold bserved for protein formulations with addition of low-eight surfactants such as polyoxy ethylene sorbitansorbate 20 (Maa et al., 1998) and Polysorbate 80ler et al., 1994; Broadhead et al., 1994; Millqvist-l., 1999), or sodium dodecyl sulphate (Adler et al.,ided the coating material, added to the protein for-

s the most efficiently adsorbing component it willtective layer at the droplet surface minimizing theic interaction between protein and air/liquid inter-

drying (Fig. 1). It has previously been observed

and once d24 h beforethe polymespray-drye(Elverssonand the ou5 ml/min, aflow was 0drying airat room temureby, 2005a). This formulation concept we call ing since both coating and particle formation occurusly, during spray-drying.ently, in situ coating is a formulation concept for sta-f protein formulations during spray-drying but thisalso be used for pure coating reasons, e.g. modi-

or oxidative protection. Since spray-drying enablesand coating of individual particles in a single step,n adhesion or wetting capability of coatings, coatingty and agglomeration of primary particles are min-e objective of this study was to influence specificperties and to reduce or prevent surface-induced con-l changes of protein during spray-drying by in situ

ls & methods

ials

s with different concentrations of non-ionic poly-prepared from stock solution of BSA (Cohn frac-% purity, Mw 67 kDa, Sigma Chemical Co., St.), d(+)-trehalose dihydrate (Fluka Chemie GmbH,itzerland) and HPMC (Mw 10 kDa, Aldrich, Mil-I) or Poloxamer 188 (Mw 76809510 Da, PEO80-80, Uniqema, Gouda, The Netherlands) to obtain antration of 0.5% (w/w) BSA, 9.5% (w/w) trehalose, 0.1 and 1% (w/w) polymer. A 10 mM sodium phos-r (pH 7.0) prepared from ultra-purified water (MilliQ,Systems, 18.2 MS resistivity), HNa2PO412H2Omie GmbH, Buchs, Switzerland) and NaH2PO4H2Orolab, Stockholm, Sweden) was used as solvent. Flu-othiocyanate (FITC) labeled BSA (Sigma Chemicaluis, MO) was used for CLSM imaging.sware was cleaned thoroughly by a surfactant-freetion (Deconex 20NS, Borer Chemie AG, Zuch-erland) and rinsed thoroughly with hot and cold

-drying

ypropyl methylcellulose (HPMC) was dispersed in(10 mM phosphate, pH 7.0) under vigorous stirringissolved the stock solution was kept for more thanuse to allow complete hydration and swelling of

r. Particles were prepared in a co-current lab-scaler (construction of the Institute for Surface Chemistry)et al., 2003). The inlet air temperature was 180 C

tlet temperature was kept at 70 C. Liquid feed wastomization airflow was 28 l/min and the drying air-.8 m3/min. The particles were separated from the

by a cyclone. Samples were stored in a desiccatorsperature and

54 J. Elversson, A. Millqvist-Fureby / International Journal of Pharmaceutics 323 (2006) 5263

Table 1Temperature cycle for DSC analysis of spray-dried powders

Segment Rate (C/min) Residence time (min)1 10 23456789

The nitrogen

2.3. Dynam

Surfacemer solutio(First TenVA). In thissize and shblunt-end mSyringes, Hwas calibra(72.3 mN/mtions werepoint was cdrop.

2.4. Scann

The parined with sCompany,ation voltaSEM-stub(SCD 050,of 640 A th

2.5. Confo

Coatedlaser-scannwith a 63was used tosion filter.the laser refirmed thatreflected li

2.6. Electr

The elewas assess

Kratos AnaAl K X-r

andprox

00 Aithisur

ativeientset alccor

sson

as p

apped wmerilledeforand

as 34

isso

n efpart

er (1otat

ific 1s detCycle Temperature cycle ( C)Heating 25140Cooling 14090Isothermal 90Heating 90160Isothermal 160Cooling 160140Heating 140240Isothermal 240Cooling 24025

flow was 40 ml/min.

ic surface tensiometry (pendant drop)

tensions at the air/water interface of protein and poly-ns were measured by the pendant drop technique

Angstroms AccuSoft, Version 1.961B, Portsmouth,technique, the surface tension is calculated from the

ape of a droplet hanging from a tip of a syringe with aetal or Teflon-coated needle (Hamilton MicrolitreTMamilton Bonaduz AG, Switzerland). The tensiometerted with 95% ethanol (23.1 mN/m) and MilliQ-water), at room temperature (23 C). The sample solu-monitored for approximately 14 s. The first dataollected approximately 0.07 s after formation of the

ing electron microscopy (SEM)

ticle shape and the surface morphology were exam-canning electron microscopy (SEM) (XL30TMP, FeiHillsboro, ON) in high vacuum mode. The acceler-ge was typically 25 kV. Powder was sprinkled on acovered by adhesive carbon tape and sputter coatedBalzers Union AG, Balzers, Lichtenstein) with Auickness (180 s).

cal laser-scanning microscopy (CLSM)

and uncoated particles were imaged in a confocaling microscope (Zeiss LSM 510 Meta, Germany)/1.3 oil objective. The 488 nm line of an Ar laser

cibles,was apthan 1spots w

Thethe relingred(Faldtlated a(Elver

2.7. G

TheanalyzMicrocell, fitimes bpurgesrate w

2.8. D

In acoatedof watously rScienttion, aimage FITC-labeled BSA, using a LP 505 nm emis-The pinhole was set to 0.7m. A higher effect ofsulted in photo bleaching of the particles, which con-the detected signal was from fluorescence and not

ght.

on spectroscopy for chemical analysis (ESCA)

mental surface composition of spray-dried particlesed by ESCA (AXIS HS photoelectron spectrometer,lytical, UK). The instrument used a monochromaticay light source. Powder was filled into DSC cru-

2.9. Differ

The thepowders w(DSC) (8225 mg of sampans. The ptransition tdeterminedbetween saindium (mp20 1

10 1

20 10

150

placed under vacuum overnight. The analysis areaimately 1 mm2 and the depth of analysis was less. Analysis was performed in triplicates at differentn a total area of 20 mm2.face composition of the powder was estimated fromamounts of carbon, oxygen and nitrogen in the pure(BSA and excipients) and in the spray-dried samples

., 1993). The percentage surface coverage was calcu-ding to a matrix model described in detail elsewhereand Millqvist-Fureby, 2005a).

ycnometry

arent particle density of spray-dried powders wasith nitrogen gas pycnometry (AccuPyc 1330,

tics, USA), with a 1-cm3 sample cell. The sampleto 2/3 with powder, was purged with nitrogen tene performing ten analysis runs. The pressure duringruns were 134.4 kPa (19.5 psig) and the equilibriumPa/min (0.005 psig/min).

lution properties

fort to illustrate the dissolution behavior of in situicles 50 mg of spray-dried sample was added to 1 ml8 C) in a 1.5 ml vial. The sealed vials were continu-ed on a Heidolph Duomax 1030 rocking table (Rose030, Edmonton, Canada) and the time for dissolu-ermined by visual inspection, was recorded.ential scanning calorimetry (DSC)

rmal properties of raw materials and spray-driedere examined with differential scanning calorimetrye, STARe System, Mettler Toledo, USA). Typicallyple was carefully weighted into 40l pinholed Al

owders were scanned twice around the expected glasso eliminate the effect of enthalpic relaxation. The Tgin the second heating scan was used for comparison

mples (Table 1). The instrument was calibrated using156.7 C, Hmelt = 28 J/g).

J. Elversson, A. Millqvist-Fureby / International Journal of Pharmaceutics 323 (2006) 5263 55

2.10. Fourier transform infrared spectrometry (FTIR)

Fourierspectrometdetector inpowder watein:KBr raat a load owas recons

BSA and apwas 4 cm

The abstion of abs(Dong et alfrom the prderivative sfunction (ODerivative(Kaleida Gamide I reparisons of

2.11. Circu

Far UVlarimeter (Jand 275 nmobtain a prto the concples were dof 0.12 mgspectrum w

2.12. Gel

Gel filtrumn with AUppsala, Swfiltered sama flow ratephosphate buble aggregafter appropeak perceinsoluble aUV absorbPerkin-Elm

3. Results

3.1. Dynam

Hydroxactive agen2003; Arboair/water in

Dynamic surface tension of BSA/trehalose solution after addition ofng amounts (indicated by darker spots) of: (a) 0.01%, 0.1% and 1%f HPMC, and (b) 0.01%, 0.1%, 1% (w/w) and 3.6% (w/w) of Poloxamer.mer added (squares). No BSA added (unfilled circles).

imately 44 mN/m (Machiste and Buckton, 1996). How-e time to reach equilibrium can be long, 25 h, dependingpolymer bulk concentration and polydispersity (Avranasou, 2003). Dynamic surface tension recorded by the pen-rop method demonstrated that complete adsorption oflymer to the air/water interface appeared already after547 mN/m) even at the lowest concentrations tested herea). Arboleya and Wilde (2005) obtained similar resultsring the surface tension after 1020 min, in solution of0.75% (w/w) of HPMC. However, for coating purposes,trations less than 0.001% (w/w) appeared insufficienthe surface tension remains unchanged for times as longin (induction phase) (Avranas and Iliou, 2003). Our mea-nts suggested that approximately 0.1% (w/w) (1% (w/w)weight) of HPMC was needed for efficient coating of

ince then the initial surface tension (


alone was considerably lower (>15 mN/m) than that of BSA andthe dynamic surface tension in the mixture of BSA and HPMCwas similar to that of HPMC alone (Fig. 2a). However, at aconcentration of 0.01% (w/w) of HPMC the initial surface ten-sion of the HPMCBSA mixture was only 5 mN/m lower thanthat of BSA alone and it was unclear whether this would besufficient for coating of BSA (Fig. 2a). Further, a polymer con-centration higher than 1% (w/w) does not necessarily lead toa more efficient coating. An unusual behavior with increasingdynamic surface tension with increasing concentration (15%,w/w) of HPMC was first reported by Machiste and Buckton(1996), which attracted attention to the influence of solution vis-cosity on the polymer diffusion rate. Later Arboleya and Wilde(2005) found a similar behavior even at concentrations as lowas 0.02 wt.%, possibly connected to the polydispersity or degree

Fig. 3. SurfaPercentage coof the polyme01% (w/w) d

of substitution. However, this was not confirmed in our experi-ments.

3.2. Dynamic surface tension of poloxamer

The equilibrium surface tension of poloxamer is compa-rable to that of HPMC (40 and 44 mN/m, respectively)(Alexandridis et al., 1994; Machiste and Buckton, 1996). How-ever, the dynamics of the polymers in relation to surface tensionappeared substantially different, at the times studied here. WhileHPMC gradually lowered the surface tension of the fresh sur-face, the initial adsorption of the poloxamer appeared muchfaster, observed as an instant reduction of the surface tension(Fig. 2b). The surface tension of HPMC then converged to near-equilibrium values while the poloxamer solutions appeared com-paratively slow in reaching the equilibrium surface tension. This

Surfage co

olyme/w) dce composition of in situ coated particles estimated by ESCA.verage by BSA (), trehalose (), and HPMC () as a functionr concentration: (a) 09% (w/w) dry weight of polymer, and (b)ry weight of polymer.

Fig. 4.Percentaof the p01% (wce composition of in situ coated particles estimated by ESCA.verage by BSA (), trehalose (), and poloxamer () as a functionr concentration: (a) 09% (w/w) dry weight of polymer, and (b)ry weight of polymer.


Table 2Atomic concentration determined by ESCA for spray-dried particles coated with HPMC and Poloxamer, respectively

Sample Atomic concentration (%)C (1s) O (1s) N (1s)

BSA 67.1 0.3 17.3 0.3 15.7 0.6Trehalose, spray-dried 57.1 0.0 42.9 0.0 HPMC 64.5 0.3 35.5 0.3 Poloxamer 188 71.7 0.4 28.3 0.4 BSA/trehalos, spray-dried (5:95) 61.6 02 28.5 0.3 10.0 0.2BSA/trehalos/HPMC, spray-dried (9.1% polymer) 63.6 0.1 36.5 0.1 0.00BSA/trehalos/poloxamer, spray-dried (9.1% polymer) 67.0 0.5 33.0 0.5 0.00Mean value S.D. (n = 3).

is in accordance with literature (Blomqvist et al., 2005), and thetime to reach equilibrium surface tension can be extremely long(days), due to polydispersity and slow exchange rates betweenthe adsorbed layer and the bulk. All formulations containingboth BSA and poloxamer followed the dynamics of the poly-mer which was expected from the reported solution diffusioncoefficients of Poloxamer 188 (9.2 109 m2/s) (Munoz et al.,2000b) and BSA (6.7 1011 m2/s) (Shen and Probstein, 1977).

3.3. Chemical surface composition of in situ coatedparticles

The atomic surface composition of pure materials andselected samples are shown in Table 2, and these data were used

to calculate the surface coverage of the different materials onsample powders as presented in Figs. 3 and 4. The percentagesurface coverage of polymer and protein correlated well withthe findings of dynamic adsorption as measured by the pendantdrop technique. In particles spray-dried from solutions of 1%(w/w) of dry weight of polymer no protein or only low levels(1.7% surface coverage) of protein were detected on the surfaceof the particles coated by HPMC and poloxamer, respectively(Figs. 3 and 4). At concentrations lower than 1% (w/w) of dryweight increasing levels of BSA were detected at the powder sur-face, and at the lowest amount of coating polymer used (0.1% insolids), it was not possible to calculate the surface coverage ofthe coating polymer. The reason for this is presumably that theC/O ratios in trehalose and polymer are not sufficiently differ-

Fig. 5. CLSMto the intensitcross-section illustrating the distribution of FITC-BSA in a particle before (a), and afy profile showed underneath.ter (b) in situ coating. The distribution of FITCBSA is correlated


ent to a give a low detection limit of either of these compoundswhen using the patch model. However, the surface coverage ofBSA could be estimated with good accuracy since N is exclu-sive to BSA. The decreasing level of BSA at the surface at eventhe lowest coating polymer concentration, and the appearanceof the corresponding placebo particles, suggest that the coat-ing polymer indeed was present at the powder surface, albeitat a low level. Uncoated particles displayed a mixed surfaceof BSA and trehalose, containing approximately 57% of BSA(Figs. 3 and 4). This level is lower compared to what has beenobserved for BSA spray-dried from other carbohydrate solutions(Faldt and Bergenstahl, 1994; Landstrom et al., 2000). Increas-ing the polymer content above 1% (w/w) resulted in higher levelsof polymer at the surface. However, at a polymer concentrationof 9.1% (w/w) of dry weight the surface coverage of polymer was30% higher with HPMC compared to poloxamer (Figs. 3 and 4).Even at a concentration as high as 26% (w/w) of dry weightpoloxamer the surface level was comparable to that of HPMCat 9.1% (w/w) of dry weight (data not shown). This can be pre-sumably explained by the poloxamer forming a thinner film thanHPMC, thus the ESCA signal is a combination of a (reasonably)complete surface film of poloxamer and the underlying mate-rial containing carbohydrate as well as protein. Interestingly, nosignal is detected from the protein. This might indicate a stericeffect from the adsorbed poloxamer layer, with the PEO-tails

pointing towards the solution at higher concentrations (Munozet al., 2000a; Blomqvist et al., 2005) Thereby, globular BSAbut not trehalose might be excluded from the (sub)surface layer.The CLSM pictures in Fig. 5 illustrate suppression of BSA fromthe particle surface by addition of HPMC. The concentrationof BSA was much higher near the particle surface in uncoatedparticles whereas the coated particles have a particularly highconcentration of BSA towards the centre of the particle.

3.4. Surface composition and correlations to particleproperties

As part of this investigation we wanted to investigate whetherspecific properties of the particles were affected at the polymerconcentrations appropriate for in situ coating. Indeed, variationin the surface composition induced changes in a number ofparticle properties such as the particle surface morphology,particle shape, particle size, dissolution time and powderflowability.

As expected, spray-dried particles containing proteins dis-played a characteristic raisin-like morphology (corrugated par-ticles), due to the adsorption of protein at the air/liquid interfaceof the droplets in the spray (Fig. 6a). During drying, proteinsform a visco-elastic adsorbed layer covering the surface of thedroplets. Addition of a low-molecular weight surfactant, such as

Fig. 6. Micro PMCweight.graphic pictures of BSA-containing in situ coated particles: (a) uncoated, (b) H , and (c) Poloxamer. The polymer content was 1% (w/w) of dry


Table 3Literature data on surface rheology of polymer and protein films

Polymer Surface concentration(mg/m2)

ElasticmodulusG(mN/m)

Dilatational modulusE (mN/m)

Reference

HPMC 120 Arboleya and Wilde (2005)

BSA1.37 59 Benjamins and Lucassen-Reynders (1998)1.95 69

60 Pereira et al. (2003)

PVA2.2 11 Benjamins and Lucassen-Reynders (1998)3.1 11

Poloxamer P85 0.1 2 Blomqvist et al. (2005)

polysorbateface; it alsosmooth sphIn terms ofsurface elasadsorbed lapolymers, sand the chato smooth,(Elverssonformer anddroplet as pa similar wsibly thereof the dropcles. To vein literaturlected andtheir flexibthe elasticWilde, 200and poloxaand Lucass2005), respof approxim1998; PerePVA. In adbates are vewhich mayafter additi2000). Conmers are likthe surfacehalose) wa

on owrinBSA

th anm c

es (ncoaties (Feolo

loxamd likat tsizeaffetennd Med aere

larlyand

gher), whsurfring

dieslymeay-dly m

polong tandpar

ionsion

Table 4Apparent part

Polymer (% o00.119

26

Mean value , not only expels protein molecules from the inter-produces a less cohesive film, which may result inerical particles (Maa et al., 1998; Adler et al., 2000).surface rheology, this means a film with negligibleticity due to the high mobility of the surfactant in theyer (Arboleya and Wilde, 2005). Linear film forminguch as polyvinyl alcohol (PVA), have a similar effectnge in particle surface morphology, from corrugatedis correlated to the PVA content of the particle surfaceand Millqvist-Fureby, 2005a). HPMC is a strong filmappears to form a similar type of film at the sprayroteins, since the particle morphology is affected inay as for protein containing particles (Fig. 6b). Pos-is a correlation between the visco-elastic propertieslet surface and the morphology of spray-dried parti-rify this hypothesis, data on surface viscosity founde for HPMC, BSA, PVA and poloxamer were col-it was possible to rank these polymers according toility and surface rheology (Table 3). For example,modulus of HPMC was 130 mN/m (Arboleya and5) whereas the dilatational modulus (E) of both PVAmer was comparatively lower, 11 mN/m (Benjaminsen-Reynders, 1998) and 2 mN/m (Blomqvist et al.,ectively. Consequently, with a dilatational modulusately 60 mN/m (Benjamins and Lucassen-Reynders,

ira et al., 2003) this places BSA between HPMC anddition, low-molecular surfactants, such as polysor-ry mobile and hence, without surface visco-elasticity,explain the frequent observations of smooth spheres

on of, e.g. polysorbates (Maa et al., 1998; Adler et al.,sequently, it might be possible to predict which poly-ely to change the surface morphology. For example,of particles without any BSA or HPMC (pure tre-

s smooth and particles were spherical (not shown).

Additihighlyulus ofbut wiogy froparticlin situparticlface rhthe poface anmobile

Thecan besurface(Kim auncoatticles wparticuweightand hishowninitialsize du

Stuthe pothe sprsiderabto theassessiextentcoatedreductcentraticle density by gas pycnometry of in situ coated particles

f dry weight) BSA/trehalose/HPMC (g/cm3) Trehalose/1.51 1.541.50 1.531.46 1.471.23 1.24

S.D.


Fig. 7. Dissoland the bulk c

spray-driedwith an incsity decreasThe increa(Elversson

A possimodified relow compaincrease intration spalated withUncoatedthe lowestdepended oin the bulkate dissoludesired dis

Interestipoloxamerdrying andtions wereshape andwater sorp(Kibbe, 20was not poand close iparticles in

erated into small aggregates, which may in part account for theimproved flow properties.

Inclusion of polymeric material can influence the re-crystallization behavior of, e.g. spray-dried lactose (Stubberudand Forbes, 1998; Corrigan et al., 2002; Berggren andAlderborn, 2004). However, it was unclear whether very lowconcentrations of polymer as in in situ coating would affect thesolid-state properties of the particles. The spray-dried particlesconsisted mainly of trehalose. Consequently, it was the transi-tions of trehalose that appeared from DSC analysis. Addition 5%of BSA induced a moderate (3 C) increase of the glass transi-tion temperature of the dried powder from 118.5 to 121.5 C(Table 5). However, addition of HPMC, which has a Tg around

, did not, at the studied concentrations, affect the Tg of thelationed oan ber t

merg trathe axam

ray-dst, adamo

each

tructes

inenati

as obromlix c, 19ble acom

etecPM

Table 5DSC results o

Polymer (% o

00.119

26

HPMC: Tg,ution time as a function of the level of HPMC at the particle surfaceoncentration of HPMC.

aqueous two-phase systems (ATPS). In formulationsreasing content of PVA the apparent particle den-ed linearly (Elversson and Millqvist-Fureby, 2005a).

sing surface load of PVA was confirmed by ESCAand Millqvist-Fureby, 2005a).ble application for in situ coated particles is forlease purposes. Although the amount of polymer isred to conventional coating solutions, a three-foldthe dissolution time was observed in the concen-

n investigated (Fig. 7). The dissolution time corre-the surface load of HPMC, as determined by ESCA.particles dissolved similar to particles coated withconcentration of HPMC. Hence, the dissolution timen the surface coverage rather than the concentration. Consequently, choosing a polymer with appropri-tion properties in situ coating can be used to obtain asolution profile of the powder.ngly, better flow properties of particles coated withcompared to HPMC were noticed during spray-powder handling. Possibly, interparticulate interac-affected by either surface composition or particle

morphology (Hickey et al., 1994). For example, the

175 Cformuobservamer c

i.e. lowpoloxameltinently,or poloing spcontraformstent of

3.5. Sparticl

Bovhad aBSA,plied fa -he(Petersof soluple. Inwere dwith Htion isotherm of poloxamer is below that of HPMC00), resulting in a less sticky powder. However, itssible to confirm these results by flowability tests

nspections of SEM images implied that the primarysamples with a high poloxamer content were agglom-

tion of natiFTIR was c(Table 6).FTIR but bthat a nativ

n thermal transitions in spray-dried powders coated with HPMC and poloxamer, resp

f dry weight) BSA/trehalose/HPMC Trehalose/HPMCTg (C) Tg (C)121.6 0.1 118.5 0.5121.1 0.3 122.1121.4 0.5 122.2 0.1121.6 0.2 123.0 1.5

170180 C (Kibbe, 2000); Poloxamer: Tm, 55 C, Tm, 115J/g, Tc, 30 C. Mean vas that remained amorphous. Neither was any effectn Tg from addition of poloxamer. The Tg of polox-e assumed to be similar to that of PEG (Kibbe, 2000),han all other constituents, and particles coated withshowed the presence of crystalline polymer, with ansition at approximately 5053 C (Table 5). Appar-ddition of an amphiphilic polymer, such as HPMCer result in phase separation close to the surface dur-rying, due to the surface activity of the polymer. Indition of, e.g. dextran, which has no surface activity,

rphous particles with a Tg dependant of the mass con-excipient (Elversson and Millqvist-Fureby, 2005a).

ural integrity of protein of in situ coatedFTIR, CD and gel ltration

serum albumin (BSA) in all in situ coated particlesve-like structure comparable to that of rehydratedserved from CD, FTIR and gel filtration. BSA sup-the commercial source and dissolved in buffer, hadontent of approximately 55%, which was expected95). Further, the content of 89% monomer and 11%ggregates were values typical of a commercial sam-parison, only slightly lower levels of -helix (47%)ted by FTIR in the liquid-state, for samples coatedC (Table 6). In the dried state, the high preserva-ve structure in in situ coated samples as displayed byonfirmed by oforandrade levels of soluble aggregatesPoloxamer-coated samples were not analyzed withoth CD (Fig. 8) and gel filtration (Fig. 9) confirmede structure was most likely. The structural integrity

ectively

BSA/trehalose/poloxamer

Tg (C) Tm (C) Hm (J/g)121.6 0.1 121.6 120.5 0 50.5 0.1 0.2 0.1121.4 51.1 0 4.1 0.1121.4 0.2 52.6 0.1 24.8 0.5

lue S.D. (n = 3).


Table 6Structural integrity of protein in in situ coated particles as analyzed with CD, FTIR and gel filtration

Excipients Polymer (% of dry weight) Protein:trehalose mss a b c

BSA, native

Trehalose, dextran 1:3.8Trehalose 0 1:19

In situ HPMC, trehalose0.1 1:191 1:199 1:19

In situ poloxamer, trehalose

0.1 1:191 1:199 1:1926 1:19

na, not analyzed; N, native BSA; -helix content (%); Aggr, soluble aggregates (insoluble); Frag,

of BSA in uncoated particles was higher than anticipated, andcomparable to that of BSA in coated particles. BSA spray-driedwith trehalose alone had a slightly lower -helix content (43%),and approximately 12% of soluble aggregates, compared to 11%in native or in situ coated samples (Table 6). In comparison, both-helix and34%, respetrehalose athe stabilizeffect, sincder only apexpected atprotein conin a higheras concentever, due toneeded forsolutions w

Fig. 8. CD spline), uncoateof dry weightmonomer content was substantially lower (33% andctively) as BSA was spray-dried in a formulation withnd dextran in ratio 1:5 (Table 6 and Fig. 9). Possibly,ing effect of trehalose dominated over the coatinge at a protein concentration of 5% (w/w) in the pow-proximately 4% of the total content of protein can bethe particle surface (Landstrom et al., 1999). A lowercentration in the spray solution would have resultedadsorbed fraction, and possibly a significant as well

ration dependent effect of the in situ coating. How-the limitations regarding the protein concentration

liquid FTIR, the protein concentration of the sprayas set to 5 mg/ml.ectra of in situ coated particles after rehydration. Native BSA (boldd (), 0.1% (w/w) ( ), 1% (w/w) ( ), 26% (w/w) (- - -)poloxamer.

Fig. 9. C

4. Conclu

Dynamisured by thdiction of tsurface. Duspray-dryinbecome mand hence,shell formathe correlatvery satisfyage of BSAof dry weigratio CD FTIR SEC

N Aggr Frag

N 55 89 11

33 34 2 (13) 51N 43 88 12

N 49 89 11 N 45 89 11 N 47 89 11

na na 86 14 N na 89 11 N na na na naN na na na na

fragments (%).ontent of monomeric and aggregated BSA by gel filtration.

ding remarks

c surface tension at the air/liquid interface, as mea-e pendant drop technique was very useful in pre-

he chemical composition of the spray-dried particlee to the very short lifetime of droplet surfaces duringg adsorption kinetics of surface-active components

ore important than the equilibrium surface tensionmolecules adsorbed at the surface in the moment oftion will remain there during drying. Consequently,ion between dynamic surface tension and ESCA wasing (Figs. 24). Efficient coating, with full cover-appeared at a polymer concentration 1% (w/w)

ht and the repression of BSA from the surface was


illustrated by CLSM of FITC-labeled BSA (Fig. 5). Further,changes in the surface morphology of particles (Fig. 6) mightbe correlatpolymers.several partime to dispycnometrcentrationpowder (Tameability oHPMC coamight indiAlthough imational stnot excludelow-dosageprotein is e

Acknowled

Financiagratefully aAndres Venology, Rouse of the A(AstraZenelarimeter,Chemistry,Institute ofmicrograph

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In situ coating-An approach for particle modification and encapsulation of proteins during spray-dryingIntroductionMaterials & methodsMaterialsSpray-dryingDynamic surface tensiometry (pendant drop)Scanning electron microscopy (SEM)Confocal laser-scanning microscopy (CLSM)Electron spectroscopy for chemical analysis (ESCA)Gas pycnometryDissolution propertiesDifferential scanning calorimetry (DSC)Fourier transform infrared spectrometry (FTIR)Circular dichroism (CD)Gel filtration

Results and discussionDynamic surface tension of HPMCDynamic surface tension of poloxamerChemical surface composition of in situ coated particlesSurface composition and correlations to particle propertiesStructural integrity of protein of in situ coated particles-FTIR, CD and gel filtration

Concluding remarksAcknowledgementsReferences

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