Controlled release of 4-nitroanisole from poly(lactic acid) nanoparticles

Journal of Controlled Release 82 (2002) 127–135 www.elsevier.com / locate / jconrel

Controlled release of 4-nitroanisole from poly(lactic acid) nanoparticles M.S. Romero-Cano a , *, B. Vincent b a

´ , 04120 Almerıa ´ , Spain Group of Complex Fluids Physics, Department of Applied Physics, University of Almerıa b School of Chemistry, University of Bristol, Cantock’ s Close, Bristol BS8 1 TS, UK Received 12 March 2002; accepted 14 May 2002

Abstract The controlled release of 4-nitroanisole from polylactide nanoparticles with different morphologies is reported. Two theoretical equations have been used in an attempt to fit the experimental results. Good agreement between theory and experiment was found for short release time. The estimated values of the diffusion coefficient of 4-nitroanisole in these nanoparticles, at short times (up to 50% release), were all |10 219 m 2 s 21 . At long time some differences in release behaviour were observed for different morphologies.  2002 Elsevier Science B.V. All rights reserved. Keywords: Nanoparticles; Nanocapsules; Core / shell morphology; Controlled release; Release kinetics

1. Introduction Controlled release technologies are used to deliver compounds like drugs, pesticides or fragrances at prescribed rates, together with improved efficacy, safety and convenience. Controlled drug release may be achieved using various devices, for example: mechanical pumps; osmotic pumps; diffusion-controlled systems containing reservoirs or matrix systems; chemically-controlled systems composed of biodegradable or non-biodegradable polymers; swelling-controlled systems and magnetically controlled systems [1].

*Corresponding author. Tel.: 134-950-015-912; fax: 134-950015-434. E-mail address: [email protected] (M.S. Romero-Cano).

The principal requirement of any controlled release system is that the release profile and rate are controlled. In this work diffusion-controlled systems, based on various kinds of polymeric nanoparticles, are described. The release of drug molecules from such nanoparticles depends on the value of the diffusion coefficient of the drug in the polymeric matrix. This, in turn, depends on the size and shape of the drug molecule, as well as the polarity of the matrix. In this paper three different nanoparticles morphologies were prepared and compared, as illustrated in Fig. 1. Structure (I) has a liquid core, which contains the active ingredient (AI) surrounded by a continuous polymer sheath. This is the so-called ‘‘core / shell structure’’. In structure (II) the AI is contained in the continuous polymeric matrix, but mainly confined to the central region. Structure (III)

0168-3659 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S0168-3659( 02 )00130-X

M.S. Romero-Cano, B. Vincent / Journal of Controlled Release 82 (2002) 127 – 135

128

1064367 M 21 cm 21 for e0 in water was obtained at 37 8C.

2.2. Preparation of PLA nanoparticles

Fig. 1. Different particles morphologies.

is similar to (II), but the AI is more evenly distributed. The controlled release of a model, small organic molecule representing the AI, has been studied in simulated physiological conditions from these different structures. Two, established, diffusion-based theoretical models have been used in attempts to fit the experimental results.

2. Experimental

2.1. Materials Poly ( L-lactide-co-D,Llactide) 70:30 was purchased from Boehringer Ingelheim as Resomer  LR708. According to the manufacturer’s specification the molecular weight of the polymer was |1.5310 3 kDa, as determined by gel permeation chromatography. Hexane (Aldrich, 99%) was employed as the core oil (structure I). Dichloromethane (DCM, Fischer Scientific, 99%) was used as polymer solvent. 4-nitroanisole (Aldrich, 97%, Mw 5153.14 g mol 21 ) was used as the active ingredient. The emulsifier used was poly(vinylalcohol) (PVA, 95 kDa, 95% hydrolysed, Acros Organic). Ultrapure water (resistivity 18.2 MV cm) was provided by a Millipore ‘Milli-Q plus’ unit. 4-nitroanisole was chosen as a representative AI because this compound is soluble in hexane but also slightly soluble in water (590 mg l 21 ) [2] and has a high molar light extinction coefficient (e0 ) at 318 nm, which allows the accurate determination of low concentrations using a spectrophotometer. A value of

Poly(lactide) (PLA) particles have been extensively used as a system for the controlled release of drugs. The reasons for their popularity are several. Firstly, PLA is biocompatible, and degrades to form non-toxic monomers. Secondly, the polymer matrix protects proteins and peptides against destructive environmental conditions, especially when administered orally. Thirdly, the release kinetics of entrapped drugs can be controlled by varying the molecular weight and the monomer ratio of PLA [3–6].

2.2.1. Nanocapsules with liquid core The method used to prepare nanocapsules with a liquid core (corresponding to structure I, Fig. 1) was proposed by Loxley and Vincent [7]. An O / W emulsion is first prepared, in which the oil phase consists of a mixture of the polymer, a volatile good solvent for the polymer and a non-volatile poor solvent for the polymer. Enough good solvent is present to ensure that all the polymer is dissolved initially. Then, as the good solvent for the polymer is removed, so a polymer-rich phase separates, initially as small droplets within the emulsion droplets. Provided the necessary wetting conditions are met, polymer-rich phase migrates to the oil / water interface and engulfs the original droplet. Further, solvent removal causes a polymer shell to form at the interface. This process leads to spherical core / shell micro or nanocapsules (structure I). The detailed procedure [7] that was used to prepare such core / shell nanocapsules, with a liquid core that contains the AI, is described below. The required amount of the PLA polymer (1.5 or 2.5 g) and the AI (0.055 g) were dissolved in a mixture of DCM (70.54 g) and hexane (3.88 g). Acetone (4 g) was added to this solution to aid the emulsification process. 80 ml of 2% PVA aqueous solution was placed into 200 ml jacketed glass vessel, maintained at 20.060.5 8C by circulated water. This aqueous phase was stirred at 12 000 rpm (Silverson stirrer), and the oil phase was added over 60 s to form a concentrated oil-in-water emulsion.

M.S. Romero-Cano, B. Vincent / Journal of Controlled Release 82 (2002) 127 – 135

Stirring was maintained for 1 h. Then the solution was poured into 120 ml of 2% PVA aqueous solution. This diluted emulsion was stirred, using a magnetic stirrer, at room temperature, overnight. The residual DCM and the water-soluble co-solvent (acetone) were removed by rotary evaporation, under vacuum, for 20 min at room temperature. The final volume was adjusted to 200 ml.

2.2.2. Nanoparticles without liquid core Two methods were used to prepare nanoparticles without the liquid core. (a) The nanocapsules prepared with hexane as liquid core (boiling point568 8C) as described in Section 2.2.1, were rotary evaporated further, under vacuum for 20 min at 55 8C. During this second step the volatile hexane was removed and thus the AI will be present mainly in the centre of the particle (structure II). (b) The organic non-solvent (hexane) is eliminated from the original recipe. With this procedure the AI is expected be distributed more homogeneously in the polymer matrix (structure III). It should be noted that in all three samples used for the PLA nanoparticles, at the end of the preparation process no 4-nitroanisole was detected in the aqueous outer phase, implying total drug loading efficiency.

2.3. Particle size measurements The size distributions of the nanoparticles were determined from photon correlation spectroscopy

129

(PCS) experiments, using a Zeta Plus (Brookhaven Instruments Corp.), equipped with a 30 mW laser source. Each sample was diluted with ultra-pure water until the appropriate particle concentration was achieved. Each measurement was performed at least in triplicate.

2.4. Release of 4 -nitroanisole from PLA nanoparticles in vitro 15 ml of the nanoparticles dispersion was put in a dialysis bag (Mw CO512 000–14 000 Da), which was then placed into 485 ml of phosphate buffer saline (pH 7 and 2 mM of NaCl) at 37.060.1 8C and stirred using a magnetic stirrer. The release of 4nitroanisole was followed as a function of time by measuring the light-absorbance of the outer aqueous phase at 318 nm using an UV/ VIS spectrometer (Perkin-Elmer).

3. Results and discussion The particle sizes of the nanoparticles, prepared by the various methods, are listed in Table 1. As expected [7], those prepared using 2.5 g of PLA are consistently larger than those prepared using 1.5 g. In each series, the size decreases: I.II.III. This again is what one would have predicted [7]. Figs. 2 and 3 show the time course of the release fraction of 4-nitroanisole in simulated physiological condition (pH 7 and 2 mM of NaCl at 37 8C) from the various types of PLA nanoparticles, containing 1.5 and 2.5 g of polymer, respectively. In all cases the final amount of 4-nitroanisole released at long

Table 1 Estimated diffusion coefficient of 4-nitroanisole Structure / code a

I (1.5) I (2.5) II (1.5) II (2.5) III (1.5) III (2.5) a b

Mean diameter (nm)b

549 601 530 585 511 558

The numbers in brackets refer to the weight of PLA (g) used in the preparation. In all cases the relative error was lower than 10%.

Diffusion coefficient, D 310 19 (m 2 s 21 ) From Eq. (1)

From Eq. (2)

9.560.4 4.5360.18 4.1060.16 4.3560.17 4.8860.20 3.8460.15

7.160.3 4.7360.13 3.8560.13 4.5460.15 5.0360.12 3.7760.12

130

M.S. Romero-Cano, B. Vincent / Journal of Controlled Release 82 (2002) 127 – 135

Fig. 2. Effect of particle morphology on 4-nitroanisole release from PLA nanoparticles with 1.5 g of PLA.

Fig. 3. Effect of particle morphology on 4-nitroanisole release from PLA nanoparticles with 2.5 g of PLA.

M.S. Romero-Cano, B. Vincent / Journal of Controlled Release 82 (2002) 127 – 135

times was less than 100%. This simply reflected the equilibrium partitioning of the AI between the aqueous phase and the polymer or polymer / hexane phase. It would seem that the final amount of 4nitroanisole released decreased when the nanocapsules were subjected to a second rotary evaporated step (it is forming structure II from structure I). This decrease was from |80% to |70% and |74% to |67%, for nanoparticles prepared with 1.5 g and 2.5 g of PLA, respectively. One possible explanation is that during the hexane evaporation process, a small amount of 4-nitroanisole was removed within the hexane. As expected the final amount of 4-nitroanisole released decreased, as increasing the PLA content from 1.5 to 2.5 g. It is well-known that the drug release rate from PLA polymer matrices is mainly controlled by the diffusion of drug in the matrices in the first time, crossing over to a regime controlled by the degradation of matrices. The latter mechanism depends on the molecular weight of the polymer. The hydrolytic degradation of low molecular weight PLA polymers starts in a few days, whereas for high molecular weight PLA polymers it takes much longer [8]. The

131

high molecular weight of the PLA used in this work permits us to assume that 4-nitroanisole principally releases by Fickian diffusion. In the case where the drug is molecularly dissolved in a spherical structure, the equation expressing the drug release by diffusion was derived from Fick’s second law by Crank [9].

O

S

M 6 ` 1 Dn 2 p 2 t ]t 5 1 2 ]2 ]2 exp 2 ]] M` p n51 n R s2

D

(1)

In Eq. (1), Mt /M` is the fraction of drug released at time t, D is the diffusion coefficient of the drug in the matrix and R s is the radius of the sphere. Figs. 4 and 5 show a plot of the quotient Mt /M` versus time for the various types of nanoparticles prepared with 1.5 g and 2.5 g of PLA, respectively. Eq. (1) was fitted to the experimental data. Good agreement between theory and experiment was found, for Mt /M` values of |0.8 (1.5 g) and |0.7 (2.5 g). There are some small, but systematic, differences. [d(Mt /M` ) / dt] t →0 is in the order I. III.II in both sets of data. Maybe this is expected. In type I the thin PLA shell, which contain some hexane, aiding release. In type III, the AI is more

Fig. 4. Release profile of 4-nitroanisole from PLA nanoparticles with 1.5 g of PLA. Lines are the fitting curves based on Eq. (1).

M.S. Romero-Cano, B. Vincent / Journal of Controlled Release 82 (2002) 127 – 135

132

Fig. 5. Release profile of 4-nitroanisole from PLA nanoparticles with 2.5 g of PLA. Lines are the fitting curves based on Eq. (1).

uniformly distributed than in II, again giving a higher [d(Mt /M` ) / dt] t →0 initially. The estimated values of the diffusion coefficients of 4-nitroanisole (Mw 5153.14 g mol 21 ), based on Eq. (1), for the various nanoparticles types are shown in Table 1. All the values are very low and of order of 10 219 m 2 s 21 . Unfortunately, there are few experimental data available in the literature for comparison. Polakovic et al. [10] obtained a diffusion coefficient of lidocaine (Mw 5234.3 g mol 21 ) in PLA of the order of 10 220 m 2 s 21 . Pitt et al. [11,12] have previously observed, that because of the enormous ‘hindrance’ effect of the PLA matrix, the release rate was so slow that a diffusion coefficient could not even be accurately determined [12]. One may also analyse the release kinetics, at short times, in terms of the linear relation between the fraction of released drug (Mt /M` ) and the square root of time [13–15], according to Eq. (2) [8].

S D

M D1 t ]t 5 6 ]] M` pR 2s

1/2

(2)

In Eq. (2), Mt /M` is the fraction of drug released at

time t, D1 is the diffusion coefficient of the drug in the matrix at short times and R s is the radius of the sphere. Higuchi [16] has shown that matrix-controlled release from a sphere is approximately linear for the first 50% of drug release. Fig. 6 shows the 4nitroanisole release data plotted against the square root of time. As with Higuchi’s data, a linear trend between Mt /M` and t 1 / 2 for the first 50% of drug release, is obtained. Eq. (2) permits us to determine the diffusion coefficient of 4-nitroanisole in the first stage of release. Fig. 7 shows the linear fit of Mt /M` versus t 1 / 2 for the first 50% of drug release. In all cases the linear regression coefficient fluctuates between 0.997 and 0.999. Diffusion coefficients for 4-nitroanisole from PLA nanoparticles, estimated from the linear region of these plots are shown in Table 1. With the possible exception of the type I (1.5 g) nanocapsules, the values obtained using Eqs. (1) and (2) are rather close. The small differences in release rates, referred to earlier in the text, are only seemingly apparent at longer times.

M.S. Romero-Cano, B. Vincent / Journal of Controlled Release 82 (2002) 127 – 135

133

Fig. 6. Release profile of 4-nitroanisole from PLA nanoparticles as a function of the square root of time: (a) with 1.5 g of PLA; (b) with 2.5 g of PLA.

134

M.S. Romero-Cano, B. Vincent / Journal of Controlled Release 82 (2002) 127 – 135

Fig. 7. Linear fit of Mt /M` versus t 1 / 2 for the first 50% of 4-nitroanisole release: (a) with 1.5 g of PLA; (b) with 2.5 g of PLA.

M.S. Romero-Cano, B. Vincent / Journal of Controlled Release 82 (2002) 127 – 135

The values of the diffusion coefficients shown in Table 1 indicate that in type I (1.5 g) nanocapsules the release rate is the fastest. Increasing the amount of polymer used in this core-shell structure, type I (2.5 g), reduces significantly the release rate. This difference should be related with a change in the polymer matrix properties. Higher polymer concentration in the oil phase leads to an increase in the shell thickness of type I nanocapsules [7]. In this way, the higher diffusion coefficients values obtained for type I (1.5 g) nanocapsules seem to indicate that the thinner polymer shell is also less dense. On the other hand, the similar diffusion coefficients values obtained for the rest of nanoparticles indicate that although the particle structure changes (type II and III) the polymer matrix properties are practically the same, thus yielding similar diffusion coefficients.

[2]

[3]

[4]

[5]

[6]

[7]

[8]

4. Conclusions The controlled release of 4-nitroanisole from various types of PLA nanoparticles may be explained by a diffusion mechanism, with a good agreement between theory and experiment in the limit of small release times. The very small diffusion coefficient values that have been obtained (|10 219 m 2 s 21 ) may be interpreted in terms of the very dense polymer matrix, which leads to a significant ‘hindrance’ effect. At long release times, some small, but significant differences in release rate kinetic are observed depending on the exact particle morphology. In no case is 100% release obtained; some of the ‘active ingredient’ is always left, partitioned inside the nanoparticles.

[9] [10]

[11]

[12]

[13]

[14]

Acknowledgements The financial support provided by CICYT under project No. MAT2001-2767 is greatly acknowledged. M.S. R-C expresses his gratitude to the Junta ´ and Plan Propio de Investigacion ´ de la de Andalucıa ´ for financing a seven-month Universidad de Almerıa stay at the University of Bristol.

References [1] F. Silver, C. Doillon, in: Polymers, Biocompatibility: Inter-

[15]

[16]

135

actions of Biological and Implantable Materials, Vol. 1, VCH Publishers, New York, 1989. S.H. Yalkowsky, R.M. Dannenfelser, Arizona Database of Aqueous Solubilities, University of Arizona, College of Pharmacy, 1992. ˆ F. Chabot, M. Veillard, G. Spenlehauer, M. Vert, J.-P. Benoıt, Biodegradable cisplatin microspheres prepared by the solved evaporation method: morphology and release characteristics, J. Control. Rel. 7 (1988) 217–229. D.L. Wise, D.J. Trantolo, R.T. Marino, J.P. Kitchell, Opportunities and challenges in design of implantable biodegradable polymeric systems for drug delivery of antimicrobial agents and vaccines, Adv. Drug Del. Rev. 1 (1987) 19–39. F.G. Hutchinson, B.J.A. Furr, Biodegradable polymer systems for the sustained release of polypeptides, J. Control. Rel. 13 (1990) 274–294. Y. Cha, C.G. Pitt, A one-week subdermal delivery system for L-methadone based on biodegradable microcapsules, J. Control. Rel. 7 (1988) 69–78. A. Loxley, B. Vincent, Preparation of poly(methylmethacrylate) microcapsules with liquid cores, J. Colloid Interface Sci. 208 (1998) 49–62. A.G. Andreopolus, E. Hatzi, M. Doxastakis, Synthesis and properties of poly(lactic acid), J. Mater. Sci. Mater. Med. 10 (1999) 29–33. J. Crank, The Mathematics of Diffusion, Clarendon Press, Oxford, 1975. ¨ M. Polakovic, T. Gorner, R. Gref, E. Dellacherie, Lidocaine loaded biodegradable nanospheres. II. Modelling of drug release, J. Control. Rel. 60 (1999) 169–177. C.G. Pitt, A.R. Jeffcoat, R.A. Zweindinger, A. Schindler, Sustained drug delivery systems. I. The permeability of poly(e-caprolactone), poly( DL-lactide acid) and their copolymers, J. Biomed. Mat. Res. 13 (12) (1979) 497–507. C.G. Pitt, M.M. Gratzi, A.R. Jeffcoat, R.A. Zweindinger, A. Schindler, Sustained drug delivery systems. II. Factors affecting release rates from poly(e-caprolactone) and related biodegradable polyesters, J. Pharm. Sci. 68 (1979) 1534– 1538. M. Miyajima, A. Koshika, J. Okada, M. Ikeda, K. Nishimura, Effect of polymer crystallinity on papaverine release from poly( L-lactid acid) matrix, J. Control. Rel. 49 (1997) 207–215. M. Miyajima, A. Koshika, J. Okada, A. Kusai, M. Ikeda, Factors influencing the diffusion-controlled release of papaverine from poly( L-lactid acid) matrix, J. Control. Rel. 56 (1998) 85–94. R. Falk, T.W. Randolph, J.D. Meyer, R.M. Kelly, M.C. Manning, Controlled release of ionic compounds from poly( L-lactide) microspheres produced by precipitation with a compressed antisolvent, J. Control. Rel. 44 (1997) 77–85. T. Higushi, Mechanism of sustained-action medication. Theoretical analysis of rate of release of solid drugs dispersed in solid matrices, J. Pharm. Sci. 52 (1963) 1145– 1149.