The surface chemical structure of poly(β-hydroxybutyrate) microparticles produced by solvent evaporation process

149

of Controlled Release, 9 (1989) 149-157 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

Journal

THE SURFACE CHEMICAL STRUCTURE OF POLY(g-HYDROXYBUTYRATE) MICROPARTICLES PRODUCED BY SOLVENT EVAPORATION PROCESS F. Koosha, R.H. Muller, S.S. Davis and M.C. Davies* Department

of Pharmaceutical

(Received November

Sciences,

Nottingham

University,

15, 1988; accepted December

13, 1988)

Nortingham

NG72RD (Great Britain)

The surface chemistry of poly(f?-hydroxybutyrate) particles produced by the solvent evaporation approach has been examined, before and after cleaning, by zeta potential measurements and static secondary ion mass spectrometry @SIMS). The SSIMS analysis was able to detect the presence of the residual stabiliser (surfactant sodium dodecyl sulphate (SDS)) on the particle surface. The surface analysis showed that DS still predominates at the particle surface even after cleaning by dialysis and ultrafiltration methods. A significant reduction in DS surface content was seen after cleaning bygelpermeation chromatography. The implications of these findings are discussed with reference to the particle production technique and the cleaning processes.

INTRODUCTION Colloidal carriers in the form of microspheres and nanoparticles have been investigated as potential advanced drug delivery systems [ 11. Considerable interest has been shown in the formation of nanoparticles from biodegradable polymer materials [ 2-41. One of the principal techniques employed for the production of these particles is the solvent evaporation process employed for the encapsulation of a wide variety of bioactive compounds such as pesticides [ 51, steroids, local anesthetics [ 61, fertility control hormones [7] and cytotoxic agents [ 81. Most workers have reported average particle sizes varying over a wide range of 0.52,000 ,um for microspheres prepared by this method. To achieve stable latex particles in the nanometer range by the solvent evaporation process, a number of emulsifying agents have been *To whom correspondence should be addressed.

0168-3659/89/$03.50

employed [ 91. The role of surface-active agents is primarily to lower the interfacial tension between the organic and aqueous phases and to prevent the agglomeration and coalescence of the dispersed emulsion particles after their formation. The principal problem in the use of such surface-active agents (to reduce the average particle size) lies in potential toxicological problems in vivo. Clearly, some indication of the solid-state concentration of the surfactant on the polymer particle and its modification by any cleaning techniques will assist in the realistic development and characterisation of a biodegradable latex produced in this manner. In this paper we examine the surface structure of a poly(/3-hydroxybutyrate) (PHB) latex stabilised using a model surfactant, sodium dodecyl sulphate (SDS), before and after various cleaning procedures including dialysis, gel permeation chromatography and ultrafiltration. The surface chemical structure of the particles has been determined using static secondary ion mass spectrometry (SSIMS). The

0 1989 Elsevier Science Publishers B.V.

150

SSIMS process yields a mass spectrum of the surface region to a limited sampling depth of l2 nm which may be analysed using conventional mass spectrometry rules [lo]. SSIMS analysis has been successfully applied for the study of a range of polymeric systems [ 111 including drug delivery systems [ 121.

EXPERIMENTAL Materials

PHB (MW 21,000 D) was supplied by Marlborough Biopolymers, U.K., and SDS (“especially pure” grade) was obtained from BDH Chemicals (Poole, Dorset, U.K. ). All solvents were of analytical grade and distilled twice prior to use. Sephadex G200 was supplied by Pharmacia Ltd (Milton Keynes, U.K. ) . Particle production

and characterisation

PHB particles were prepared using a solvent evaporation technique based on the method described by Tice and Gilley [ 131. First, an oilin-water emulsion was formed by the emulsification of a chloroform phase containing 1.6% w/v PHB in an aqueous solution of 0.2% w/v SDS. After initial pre-mixing of the two phases using a Silverson Laboratory Homogeniser (Silverson Machine Ltd, Cheshunt, Buckinghamshire, U.K.), the emulsion was sonicated for 30 minutes using a Dawe Soniprobe (Type B7532, Dawe Instruments, Shipley, W. Yorkshire, U.K. ). After emulsion formation, the organic phase was removed slowly using a rotary evaporator employing an interrupted evaporation process to obtain microparticles with smooth homogeneous surfaces [ 141. In order to complete the evaporation, the particle suspension was left to stand for 24 hours. Particles were sized by photon correlation spectroscopy (PCS) using a Model K7025 multibit correlator (Malvern Instruments Ltd, Malvern, U.K.) in combination with a Com-

modore PET 3008 computer and a Malvern spectrometer employing a helium neon laser (Siemens, West Germany). Particles were found to have a mean diameter of 170 + 3 nm with a polydispersity index of 0.12, indicative of a narrow particle size distribution. Zeta potential measurements were obtained from electrophoresis measurements at pH 7.4 employing Laser Doppler Anemometry (LDA) (15) using a Malvern Zetasizer Mk II. The optical unit was connected to a K7027 Log-lin Correlator. In this part of the work, the emphasis was placed on looking at the changes of the potential of the particles during the cleaning process and not at an absolute value of potential. Therefore, the zeta potential (ZP ) was calculated by the Helmholtz-Smoluchowski equation [ 16,171 [= 4nu/e where n is the viscosity dium, e is the dielectric particle velocity.

(1) of the dispersion meconstant and u is the

Particle cleaning

Aliquots of the PHB latex were cleaned using a number of techniques as follows: ( 1) Dialysis - 10 ml particle suspension was placed in a dialysis tube (Spectropor, 6,000-8,000 D cut off) in double distilled water (11) which was stirred continuously. The water was changed every hour and the dialysis was completed over 8 hours. (2) Ultrafiltration - a 200 ml ultrafiltration cell fitted with an ultrafiltration membrane (Amicon, 1000 D cut off). Double distilled water was passed through the cell at a constant flow rate of 3 ml/min in a thermostatically controlled water bath at 25°C for 8 hours and the cell contents stirred continuously. (3 ) Size exclusion or gel permeation chromatography (GE) -particle suspensions were passed through a gel filtration system to remove free and loosely bound sodium and DS ions from the latex system by virtue of size exclusion. 5 ml samples of the latex were passed

151

down a Sephadex G200 gel column (dimensions 40 x 1.5 cm) at 250°C using double distilled water as eluent at a flow rate of 0.3 ml/ min. The elution of the particle band from the column was detected by turbidity measurements. Spectroscopy

Particle suspensions were coated onto cleaned aluminium foil and the solvent allowed to evaporate at ambient temperatures within a laminar flow cabinet. As a control, a film of PHB was formed by spin casting the polymer solution (0.1% w/v in chloroform) onto aluminium foil. The coated foil surfaces were mounted onto 1 cm2 analysis stubs for the SSIMS experiments. Static SIMS spectra were acquired using a modified VG Scientific SIMSLAB which has been described in detail elsewhere [ 181. SSIMS analyses were undertaken with 2 keV Ar atoms of flux density of 3 x 10’ particles cmm2 s-l. An electron flood gun was used to neutralise any surface charging in a manner described previously by Brown and Vickerman [ 191. The total dose for the SSIMS analysis was of the order of 2~ 1012 particles cmm2 sample-‘, which is within the limits established by Briggs and Hearn [ 201 for static SIMS spectra of “undamaged’ surfaces.

RESULTS Zeta potential

TABLE I Zeta potential of PHB powder and particles before and after cleaning Particles

Zeta potential (mV)

PHB PHB PHB PHB

-

powder particles (uncleaned) particles (dialysed) particles (GPC cleaned)

30.0 50.6 40.3 36.2

tion of ionic surfactant and/or to its adsorption on the surface. The uncleaned PHB particles showed a distinct increase in potential of - 20 mV (to the negative) relative to the powder (see Table 1) . This rise in potential is a reflection of an increase in the number of surface charges attributable to the presence of the surfactant. Cleaning of the PHB particles by dialysis showed a significant reduction in the zeta potential (of 10 mV) compared to the uncleaned system of - 40.3 mV. This may be attributable to the removal of the free and some surface-adsorbed DS. In the case of no desorption, the potential would remain uncharged or even slightly increase. Dialysis lowers the ionic strength of the dispersion medium, leading to an extended electric double layer and subsequently to an increase in the measured zeta potential. As shown in Table 1, GPC-cleaned particles possessed the lowest zeta potential at -36.4 mV, indicative of an improved cleaning efficiency of the technique over dialysis.

measurements SSIMS

The original PHB powder used for the preparation of the microparticles possessed a potential of - 30 mV. The value of this potential depends on the charges present on the particle surface (Nernst potential). An increase or decrease of the surface charged groups will lead to a higher or lower zeta potential [ 211. In the case of the microparticles, the change in the number of surface charges will be due to the incorpora-

analysis

In order to identify the diagnostic features of the PHB polymer in the particle spectra, the control PHB homopolymer film will be considered initially. The ion assignments of the SSIMS analysis of PHB and related polyesters have been described in detail elsewhere [ 221 but are summarised here to assist in interpretation of particle spectra. Both the positive (Fig. 1)

152 241 ~8

r

Fig. 1.Positive ion SSIMS spectrum of PHB polymer.

j x3

[nM +

59

57 5

CH3

85

Jr,

ti

H-O]+

100

,;, ‘lx !

200

Fig. 2. Negative ion SSIMS spectrum of PHB polymer.

CH,-

CH*-C[M +

i

0

_

HI-

CH3 HO-CH-CH,-C-O

i

_

[M + OH]-

and the negative (Fig. 2) SSIMS spectra are dominated by ions which are direct fragments of the monomer repeat units of PHB. nM? H ions (where M is the monomer unit) are observed in Fig. 1 (n=l-4) at 85/87, 171/173, 2571259 and 343/345 m/z for n=l-4, respectively. In the positive ion SSIMS spectrum of PHB, the nM - 0 & H ions are prominent at 69, 155/157,241/243 and 3251327 m/z for n= l-4. In Fig. 2, nM + 0 + H ions are observed at lOl/ 103 and 187/189 m/z for n= l-2. General structures for these ions are shown below:

[nM+

H]’

These assignments provide a molecularly specific analysis of the PHB polymer surface composition and may be employed as a “fingerprint” for the polymer molecule in the analysis of potentially contaminated particle surfaces. The positive and negative ion spectra of the uncleaned PHB nanoparticles are shown in Figs. 3a and 4a, respectively. As anticipated, few of the diagnostic PHB peaks noted above are present in either spectrum. The positive spectrum has become dominated by non-specific hydrocarbon fragments in the O-100 m/z region. No significant ions are observed at 69 and 85/87 m/z, corresponding to M- 0 2 H and M 2 H. Similarly, in the 100-200 m/z range, a number of unassignable ions emerge at 143 and

153

r X14

.

60

100

120

140

ml2 Fig. 3. Positive ion SSIMS spectra of PHB microparticles: (a) uncleaned; (b) dialysis cleaned; (c) ultrafiltration cleaned; (d) GPC cleaned.

165 m/z which are not seen in the corresponding pure PHB spectrum. A dominant signal attributable to sodium cations is noted at 23 m/z, probably arising from the SDS molecule. Similar observations may be made for the negative ion spectrum of the uncleaned particles (Fig. 4a). No prominent peaks attributable to PHB are observed in the entire spectrum. However, a number of significant ions are detected which may be interpreted as arising from the SDS molecule. The characteristic sulphate ions occur at 32 (S- ), 64 (SO,-), 80 (SO,-) and 96/97 m/z (SO,-/HSO,-). In the higher mass range, a dominant anion is detected at 265 m/z, which corresponds to the molecular ions

of the DS anion, CH, ( CH2)i10S03-. Thus, the surfactant may be readily detected on the particle surface to a high degree of molecular specificity. The SSIMS spectra of the uncleaned nanoparticles are dominated by the DS anion with little information relating to the PHB surface. The relative intensities of the characteristic ions from SDS (sodium, sulphate and DS) and PHB may be employed to analyse the relative efficiency of the removal of the surfactant from PHB particle surface using the various cleaning procedures. The positive and negative ion SSIMS spectra of the PHB particles after dialysis, ultrafiltra-

154 Xl80

r

265

(a)

Cd)

m/z Fig. 4. Negative ion SSIMS spectra of PHB microparticles: (a) uncleaned; (b) dialysis cleaned; (c) ultrafiltration cleaned; (d) GPC cleaned.

tion and GPC cleaning are shown in Figs. 3b-d and 4b-d. From the preceeding discussion, it is evident that the nanoparticle surfaces are still dominated by the sodium and DS ions after dialysis and ultrafiltration. The negative ion SSIMS spectra (Figs. 3b and c) are again dominated by the sulphate species at 32,64,80,96 and 97 m/z and the DS anion at 265 m/z. The corresponding positive ion spectra (Figs. 4b and c) are dominated by the hydrocarbon species with none of the prominent ions observed featured in the SSIMS analysis of the PHB homopolymer. In contrast, ions specific to both the PHB and SDS species are detected in the positive and negative ion SSIMS spectra (Figs. 3d and 4d, respectively) of the GPC-cleaned nanoparticles. The cations attributed to PHB at 69 (M-OH+), 87 (M+H+), 155 (M-O+H+)

and 171/173 m/z (2M?H+) are dominant in the positive ion spectrum. Sodium from SDS is present at 23 m/z. Similarly, sulphate species from DS are still detected but overshadowed by the more intense PHB anions at 59 (M - OH- ), 85 (M-H-), 103 (M+O-H-), 171(2MH-) and 187 m/z (2M+O-H-). No molecular ion for the DS fragment was analysed in Fig. 4d.

DISCUSSION The SSIMS analysis has proven to be a sensitive tool for probing the surface structure of the PHB nanoparticles. The molecular specificity of the technique demonstrates that it is possible to distinguish signals specific to either the contaminating surfactant or the polymer backbone within a limited sampling depth of l-

155

2 nm. In this case, the molecular specificity of the SSIMS technique enables the clear elucidation of the chemical structure of the surfactant on the particle surface. With regard to polymeric nanoparticles, we have previously successfully applied SSIMS analysis to study the nature of the steric stabilisation layer of dextran on poly (butylcyanoacrylate) latices [ 231 together with the characterisation of surface charged end groups on poly (butylmethacrylate) colloids [ 241. Similarly, previous SSIMS analyses have also permitted the chemical identification of small organic molecules on biomedical polymeric devices, e.g. drugs [ 121 and lubricants [ 111. Despite the fact that Hearn et al. [25] have demonstrated that quantitative SSIMS information may be gained from the analysis of wellcharacterised copolymer structures, it must be remembered that SSIMS is generally considered to provide qualitative information. However, it is possible to draw some interesting conclusions from the SSIMS analysis of the uncleaned and cleaned PHB nanoparticles. In the latex system, the SDS may be present in a number of environments: surface entrapped and immobilised within the polymer structure. This form of SDS will prove extremely difficult to remove. Secondly, surfaceadsorbed Na, and DS ions which are bound to the hydrophobic PHB surface predominantly by hydrophobic interactions. Finally, “free” SDS within the aqueous phase unassociated with the particle systems. An equilibrium between the adsorbed and “free” Na+ and DS- will undoubtedly exist. In the SSIMS characterisation of the uncleaned particles, it is not surprising that the spectra are dominated by DS. It must be remembered that in this case, both surface-adsorbed and/or entrapped DS exists on the PHB particle together with free SDS in the surrounding aqueous medium. Thus, “free” Na+ and DS- ions from the surrounding medium may simply deposit directly onto the surface of the particle bed (on the evaporation of solvent)

during sample preparation. Therefore, it is not possible to distinguish between the different states of bound and free surfactant in this sample but simply infer from the SSIMS spectra that there is gross contamination with DS anions. Interestingly, the cleaning procedures show marked differences in the removal of sodium and DS ions from the latex surface. While the GPC technique demonstrates a significant depletion of the surfactant, SDS still has a dominant effect on the spectra of the PHB particles after dialysis and ultrafiltration. In the latter case, the efficiency of removal of the sodium and DS ions from the surrounding aqueous environment must be low and hence some “free” DS (as well as adsorbed) may persist in the latex sample. Even where the free DS ions are removed, the equilibrium between the surface and aqueous phase DS will be rapidly re-established. Only when the surface-adsorbed DS has been depleted will the aqueous phase remain free of the surfactant. Undoubtedly some surfactant has been removed from the dialysis cleaned system by virtue of a drop in the zeta potential measurements from those for the uncleaned particles. Unfortunately, the SSIMS data appears to indicate that the residual DS remains at sufficient levels to dominate the surface analysis of the PHB nanoparticles after the dialysis and ultrafiltration treatments. A possible explanation may be supplied for the relative success of the GPC method of particle cleaning. The technique will obviously separate the particles and DS in terms of hydrodynamic volume throughout the GPC column length. Initially, the free DS molecules in the injection volume enter the pores of the gel beads from which the particles are excluded on the basis of their size. On the passage down the column, surface-adsorbed sodium and DS ions may also be depleted into the surrounding medium. This DS depletion is likely to be more efficient in GPC than that invoked during simple dialysis or ultrafiltration due to the vast available surface area of the column and also,

156

the separation is undertaken in close proximity to the microenvironment of each particle surface and occurs through the entire length of column. In contrast, the efficiency of the dialysis treatment relies on the diffusion of DS down the concentration gradient through the latex bulk, across a membrane and into the surrounding aqueous phase. Therefore, it is likely that any signals observed from DS on the particle surface after GPC cleaning may arise from the surfactant which is either strongly bound to the surface or more probably, physically entrapped within the latex surface structure. The SDS surfactant has been employed in this study to illustrate conveniently how the technique of SSIMS may be employed to characterise the surface chemistry of nanoparticles. It may be inferred from these studies that surfactant is likely to be present on the surface of particles prepared by the solvent evaporation technique even after extensive cleaning. To date, little information is available on the fate of the surfactants employed with solvent evaporation techniques of particle production. Therefore, one must consider the contamination levels for particles produced in this manner are likely to be significant. Clearly, judicious choice of the surfactant is required to minimise any potential problems in this area. The importance of this residual surfactant in terms of the potential toxicology problems is currently under investigation. ACKNOWLEDGEMENTS

SERC is gratefully acknowledged for the grant to MCD for the surface analysis studies. We would like to thank Dr P. Humphrey, Surface Analysis Unit, UMIST for advice and cooperation in the SSIMS studies. REFERENCES 1

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