Controlled Release of the Anti-cancer Drug Paclitaxel from Bioresorbable Poly(ester-ether-ester) Microspheres

G. D. GUERRA1, M. GAGLIARDI2, N. BARBANI2, C. CRISTALLINI1

1Institute for Biomedical and Composite Materials, U.O.S. of Pisa; 2Department of Chemical Engineering 1Italian National Research Council (CNR), 2University of Pisa

1,2Largo Lucio Lazzarino, I-56122 Pisa ITALY

giuliodante.guerra@diccism.unipi.it http://www.imcb.cnr.it/home.htm

Abstract: - The release of the anti-cancer drug paclitaxel (PTX) from microspheres of the bioresorbable poly(ε-caprolactone-oxyethylene-ε-caprolactone) tri-block copolymer was studied. The microspheres, both loaded and not with PTX, were prepared by emulsion-evaporation technique, then characterized by SEM, AFM, total reflection and spotlight FT-IR spectroscopy, and DSC. The quantities of PTX released were measured by HPLC. The results showed a slow and very regular release, which fits very well the Peppas equation, Mt/M∞ = k · tn, where Mt is the amount of solute released at the time t, M∞ is the amount of drug released at the plateau condition, k represents the Peppas kinetic constant and n the diffusion order. Key-Words: - Poly(ester-ether-ester), Paclitaxel, Microspheres, Bioresorbable Material, Characterization, Controlled Release 1 Introduction Poly(ester-ether-ester) tri-block copolymers have been proposed as bioresorbable materials since many years [1,2]. The development of the non-catalyzed synthesis of these copolymers [3-5] allowed to avoid the use, as a catalyst, of 2-ethylhexanoic acid, tin(II) salt, commonly known as stannous octoate, which was found to be cytotoxic [6]. The copolymers having poly(ε-caprolactone) chains as the polyester blocks (PCL-POE-PCL) were tested for cell adhesion and proliferation and cytotoxicity [7], as well as for cytocompatibility and hemocompatibility [8]. All the copolymers were found to be biocompatible with respect to the tests carried out. The degradation in vitro of the copolymers both in the absence and in the presence of cells was also tested [9], and the degradation product, 6-hydroxyhexanoic acid, were found to not alter the endothelial metabolism [10], and to modulate the endothelin release by human umbilical vein endothelial cells, with no significant alteration of the vasoconstrictor-vasodilator balance [11]. PCL-POE-PCL was melt-spun to make fibers to be used as bioresorbable suture threads [12]. Composites of hydroxyapatite and copolymers with the same monomer composition were prepared both by direct copolymer synthesis [13] and by blending; the latter material was employed to make periodontal membranes, which were successfully tested for the bioresorption in vitro and in vivo [14]. The release of the anti-cancer drug 5-fluorouracil by thin sheets of PCL-POE-PCL was also investigated [15]; the dependence of the release kinetics on the interactions between the copolymers and the drug was evaluated with ab initio calculations at the Hartree-Fock and second order Møller-Plesser levels [16]. This paper regards the use of PCL-POE-PCL for the release of a different anti-cancer

drug, the 5β,20-epoxy-1,2α,4,7β,10β,13α-hexahydroxytax-11-en-9-one-4,10-diacetate-2-benzoate 13-ester with (2R, 3S)-N-benzoyl-3-phenylisoserine (paclitaxel, PTX).

Fig.1 Structural formula of paclitaxel. PTX (see Fig.1) is a diterpenoid first isolated from the bark of the western yew Taxus brevifolia. It aids the polymerization of tubulin dimers to form microtubules, which are not only very stable, but also dysfunctional, leading to cell death; this property makes PTX a very effective anti-tumor agent [17]. The use of PTX in cancer therapy gives rise to the problem of its administration to the patients. Direct intravenous administration was found to have undesirable side effects on healthy cells [17-20], so that different PTX administration techniques were needed. Films of chitosan-poly(vinyl alcohol) blends made by casting, both as such and cross-linked with glutaraldehyde, released a very scarce percentage of the PTX initially put into them [21]. Release of PTX from microspheres of

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biodegradable macromolecular materials was tested successfully [22-28]. The controlled release of PTX from PCL-POE-PCL microspheres is now reported. 2 Experimental 2.1 Materials The PCL-POE-PCL copolymer was the previously synthesized C27 sample [7], having an ester to ether units 66:34 molar ratio, a central polyether chain with a mean Mn value of 3.5 × 104, and a total mean Mn value of 20.37 × 104. PTX (Sorin, Italy) was used as supplied. 2.2 Microspheres Fabrication The PCL-POE-PCL microspheres loaded with PTX were prepared adding to 5 mL of copolymer solutions 60 g/L in CH2Cl2 either 15 or 60 mg of PTX, in order to obtain drug/polymer percentages of 5% and 20% w/w, respec-tively. Each solution was rapidly poured into 100 mL of 25 g/L solution of poly(vinyl alcohol) (Aldrich) in a three-neck flask; the two-phases liquid was stirred at 800 rpm for 2 h, then the so obtained micro-emulsion was stirred at 250 rpm overnight, keeping the flask opened to allow CH2Cl2 evaporation. The microspheres were separated by centrifugation at 14000 rpm, washed tenfold with H2O and lyophilized. “Placebo microspheres” without PTX were also prepared by the same procedure. 2.3 Characterization 2.3.1 Microscopy Scanning Electron Microscopy (SEM) was carried out, using a JEOL T-300 instrument, on samples coated with 24 carat gold in a vacuum chamber. Atomic Force Microscopy (AFM) was carried out by means of an Auto Probe CP apparatus (Park Scientific Instruments), operating in “non-contact” mode (NC-AFM), to minimize damages of the sample by the tip. The apparatus was equipped with a probe tip by etched silicon (2 µm thick silicon Ultralever, spring constant 10-20 N m-1) and with a 5 µm piezoelectric scanner. The images were acquired at the rate of 1 scanning line per second. 2.3.2 FT-IR spectroscopy Total reflection and spotlight Fourier-transform infrared (FT-IR) spectra and maps were carried out by means of a Perkin Elmer Spectrum One FT-IR Spectrometer, equipped with a Perkin Elmer Universal ATR Sampling Accessory and a Perkin Elmer Spectrum Spotlight 300 FT-IR Imaging System, using the “image” mode of the instrument. All

spectra were recorded in the mid infrared region (4000–700 cm 1) at 32 scans/pixel and a spectral resolution of 4 cm 1. 2.3.3 Differential scanning calorimetry Differential scanning calorimetry (DSC) was carried out in triplicate with a Perkin Elmer DSC7 apparatus, on 5 to 7 mg of both PTX loaded and placebo microspheres in Al pans, from 40.00 to 240.00°C at 20.00°C per min under N2 flux. 2.3.4 Liquid chromatography High performance liquid chromatography (HPLC) was carried out using the following apparatus: a Perkin Elmer 410 LC Pump, an Alltech C8 5U Alltima column having 4.6 mm diameter and 10 cm length, and a Perkin Elmer LC 90 UV Spectrophotometric Detector. The moving phase was a 58:42 v/v CH3CN-H2O solution; the flux speed was 1 mL/min; the injected volume was 100 µL; the detector wavelength was kept at 230 nm; the PTX retention time was 3.04 min. 2.3.5 PTX release 10 mg of PLX-loaded microparticles, both containing 5% and 20% in weight of drug, were weighted and immersed in 5 ml of the delivery medium composed of a phosphate buffered solution (PBS) at pH 7.4 containing 0.05% (w/v) in sodium dodecyl sulphate (SDS, Sigma®). In order to avoid the tendency of the microparticles to aggregate, samples were sonicated after the immersion in the delivery medium. The use of the SDS surfactant was finalized to increase the solubility of the PLX in water and also to reproduce a condition more similar to the in vivo one. Samples were maintained in a lightly stirred bath at a fixed temperature (37°C ± 1°C) through all the testing period (34 days). Withdraws from the delivery medium were carried out at established times and the delivery solution was replaced by fresh solution after each withdraw, in order to maintain a sink condition. The sink condition occurs when the amount of solute delivered is lower than 10-20% or even 30% of the maximum solubility of the solute in the dissolution medium [29]. For this reason, the solubility limit of PLX in the delivery solution at 37°C has to be known. This value was gathered from literature [30] and used to verify if the sink condition was always verified. Withdraws from the delivery medium were analyzed using HPLC at room temperature. The quantities of released PTX calculated by a previously made calibration curve. The solute delivery kinetics was studied using two different mathematical models, expressed by the equations of Peppas (1) and Hopfenberg (2), respectively [31]:

(1)

(2)

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Mt represents the amount of solute released at the generic time t, M∞ is the amount of drug released when the system reaches the plateau condition, k and n represent the Peppas kinetic constant and the diffusion order respectively, k0 is the Hopfenberg kinetic constant, c0 is the uniform starting drug concentration, a is the mean radius of the microparti-cles, m is a “shape factor” and was assumed to be 3 because of the spherical geometry of the delivery platform [31]. In the present work, the value of M∞ was considered equal to M0, considering that polymer used to obtain microparticles is biodegradable and it ensures that the entire amount of drug contained in the delivery platform is released at infinite times. Values of M0 were calculated considering the percentage encapsulation efficiency (%EE) [32]. Mean values of %EE were found to be 86.9% (± 1.7%) for samples loaded with 5% in PTX and 82.6% (± 1.3%) for samples loaded with 20%. Using %EE the actual amount of drug loaded within samples was estimated. Eq. 1 and Eq. 2 are only valid for the first 60% of the total released drug [33]. Release orders allowed establishing the regime governing the release process. In particular, for spherical swellable samples, three different controlled release mechanisms can be identified [34]: n = 0.43 (Fickian diffusion, concentration gradient-controlled regime), 0.43 < n < 0.85 (anomalous non-Fickian transport) and n = 0.85 (case-II transport, swelling-controlled regime). A not very different behaviour can be supposed for the rather hydrophilic C27 copolymer. 3 Results and Discussion SEM analysis (Fig.2) showed microspheres of regular shape, having average diameters ranging between 1.2 µm and 3.5 µm. No aggregation was observed for each sample.

Fig.2 SEM image of PCL-POE-PCL microspheres, loaded with 5% PTX. Fig.3 shows the AFM image of a microsphere with a diameter of about 1.7 µm. The particle appears to have a quite smooth and non-porous surface. The morphological analysis, carried out by both scanning electron and atomic

force microscopy, indicates that the technique used is very suitable for the fabrication of microspheres able to be injected, in the form of a liquid suspension, into the cancerous tissue, with the aim to release in situ the anti-cancer drug PTX.

Fig.3 AFM image of a PCL-POE-PCL microsphere, loaded with 5% PTX. Spotlight FT-IR Chemical Imaging Analysis was used first to confirm that the microspheres were full solid and not hollow ones. Indeed, the group of particles shown in Fig.4 has the maximum absorbance in the central part, owing to the greatest density of matter present there.

Fig.4 Transmission chemical map of a small group of microspheres, loaded with 5% PTX. Moreover, spotlight FT-IR gives useful information about the presence of PTX in the copolymer structure. The spectrum of the drug (Fig.5) shows the wide absorption band at 3339 cm-1, due to the stretching of the —NH and —

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OH groups [35]. The same band is absent from the spectrum of the pure copolymer, shown in Fig.6.

Fig.5 FT-IR spectrum of pure PTX. The absorption band at 3339 cm-1 is visible in the left region of the spectrum.

Fig.6 FT-IR spectrum of the pure PCL-POE-PCL. No absorption band is visible at 3339 cm-1. The chemical maps and the correlation maps, evaluated by generating in real time thousands of spectra, allow an accurate analysis of the surface. The chemical map, taken on a 1-mm2 area of grouped microspheres is shown in Fig.7. Fig.8 shows the spectrum taken at a medium point of the chemical map, having the abscissa of -300 µm and the ordinate of 100 µm in the map. The presence of the absorption band at 3339 cm-1 is the most evident sign of the presence of the drug within the microspheres. The correlation between the chemical map in Fig.7 and the medium spectrum in Fig. 8 is shown in Fig.9. The presence a high correlation in the most part of the map is the greatest evidence of the uniform distribution of the drug within the particles. This fact is very important for the use of the microspheres as a device for the controlled release in situ of the anti-cancer drug. Indeed, the uniform distribution

avoids the risk that the release may depend on the region of the microparticle, more or less rich in PTX, in contact with the cancerous tissue.

Fig.7 Chemical map of a group of microspheres loaded with 5% PTX.

Fig.8 Medium spectrum taken at the signed point of the chemical map. The DSC traces of pure PTX and of the microspheres are shown in Fig.10. The DSC trace of the pure PTX shows a single melting endotherm at 223°C, according to the result of Liggins et al. [36]. Conversely, in the PTX loaded microspheres, no melting peak of the PTX crystals at the same temperature is visible, indicating that in the particles the drug is present in an amorphous form rather than in a crystalline one. Moreover, the melting signal of the loaded microspheres is split in two peaks, and the main one has its maximum at a lower temperature than the melting peak of the placebo microspheres visible in the curve (b), a signal that in the curve (a) appears only as a weak shoulder; the melting temperature and enthalpy values calculated from the curves are listed in Table 1. This fact is correlated to the

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presence of less perfect copolymer crystals in the loaded microspheres.

Fig.9 Correlation of the map of the microspheres with the medium spectrum.

Fig.10 DSC traces of pure PTX (upper graph) and of the microspheres (lower graph), loaded (a) and not loaded (b) with 20% PTX. Table 1. Melting temperature (Tm) and enthalpy (∆H) values of the microspheres loaded with PTX (20% w/w) and of the placebo ones.

Microspheres Tm (°C) ∆H (J/g) PTX-loaded 56.3 66.3

Placebo 60.3 84.9

In Fig.11 the values of Mt/M∞ versus time are reported. From literature, solubility limit of PTX in the PBS solution containing SDS was found to be 0.08±0.01 mg/ml at 37°C. Then, the perfect sink condition is satisfied if the concentration of PTX detected in the delivery medium is smaller than 2.4·10-3 mg/ml, that represent the upper limit of 30% in respect of the maximum solubility. This condition was verified for all tested samples in each withdraw. Releasing profiles obtained showed that the fraction of drug released from samples loaded with a smaller amount of PTX was greater than the fraction eluted from microparti-cles loaded with 20% in PTX. It could be reasonably attributed to greater interactions established between drug molecules in the samples loaded with a greater amount of active principle in respect to the samples loaded with a smaller amount of drug, where drug molecules are more dispersed in the polymeric matrix. In addition, a contained burst effect was detected in the first days (< 1% for microspheres loaded with 20% in PTX and < 1.5% for 5%) and a linear trend after the first week of test. This behaviour is desirable in the case of the release of drugs that could show toxic effects, such as Paclitaxel [37].

Fig.11 Profiles of the PTX released from microparticles loaded with both 5% (■) and 20% () PTX. Table 2. Kinetic parameters evaluated using Peppas and Hopfenberg models.

Peppas model Hopfenberg model PTX loaded k (d-n) n k0 (µm/d)

5% 0.012 (± 0.003) 0.523

(± 0.004) 7.30·10-5

(± 1.59·10-5)

20% 0.005 (± 0.002) 0.567

(± 0.011) 1.47·10-4

(± 4.05·10-5) Kinetic parameters of releasing profiles were evaluated using equations (1) and (2) and obtained results are summarised in Table 2. For the Peppas model, kinetic constant decreased with the increase in PTX and it could be attributed to the slower kinetic release of the drug in the presence of a greater amount of active principle, according as previously stated. Concerning drug release orders, values

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are comprised in the range from 0.43 to 0.85, indicating an anomalous non-Fickian transport mechanism for swellable spheres. This transport mechanism is typical of polymeric systems not showing a solute transport controlled by the concentration gradient nor the macromolecular relaxation rate but the superposition of both diffusion and relaxation phenomena influence the delivery kinetics. Concerning the Hopfenberg model, kinetic constants k0 increased with the increase of the starting drug loading.

Fig.12. Comparison between experimental and theoretical data obtained through the Peppas (upper) and the Hopfenberg (lower) models for the PTX released from microparticles loaded with both 5% () and 20% () PTX. In Fig.12 the comparison between the experimental releasing profiles and theoretical trends evaluated using kinetic parameters obtained through the theoretical models are reported. Correlations between data and Peppas equation are 0.989 for samples loaded with 5% and 0.992 for samples loaded with 20 % PTX. From correlation values theoretical models evaluated can be considered accurate and the Peppas equation can be used to describe the release behaviour of these systems. On the contrary, Hopfenberg model is less accurate for the description of releasing profiles. It could be justified considering that Hopfenberg model was developed for surface-eroding polymer matrices while copolymer CL27 could be supposed to undergo a bulk hydration, considering that its macromolecules contain a fraction (~0.34) of POE that is highly hydrophilic, and then tend to absorb water within the whole structure of the microparti cle. However, a further investigation of the degradation

mechanism is necessary to validate this hypothesis. The particulate structure of the tested samples seems to overcome one of the major limits related to the release of drugs from biodegradable matrices, represented by a delivery kinetics showing a discontinuous trend. This characteristic was highlighted, for poly(L-lactide)-block- poly(oxyethylene)-block-poly(L-lactide) copolymers, in a paper reporting the analysis of tetracycline release from sintered tablets [38]. In the case of the PTX release from PCL-POE-PCL microparticles, two distinct release mechanisms were not shown, likely since the degradation of the C27 is still very scarce after 35 days of dipping [9], and then only the PTX extraction by absorbed water occurs; this behaviour offers the advantage of more controlled delivery kinetics. 4 Conclusion In the present work a preliminary morphological, chemical and functional characterization of a micro-particulate delivery platform, obtained using PCL-POE-PCL copolymer, was reported. Morphological analysis, carried out by SEM, showed that the obtained particles are spherical, not aggregated and with a sharp radius distribution. AFM analysis confirmed these results, giving additional information about the surface characteristics of the particles, which resulted smooth and non-porous. Physicochemical analysis, carried out by FT-IR Chemical Imaging and DSC, confirmed the presence of the PTX drug within the microparticles, as well as that the drug interferes with the crystalline structure of the copolymer. Drug delivery tests showed that the starting drug payload, contained within the sample, affects the release kinetics; in particular, a faster release was detected in the samples loaded with the smaller amount of PTX. However, both systems showed the tendency to release the drug slowly, and it may portend that the delivery of the overall amount of PTX occurs with a vey sustained kinetics. It is a desirable characteristic of anti-mitotic delivery systems, because it provides a very prolonged treatment, so that it is not necessary to administer the drug subjecting the patient to several administrations. References: 1. D. Cohn, H. Younes, Biodegradable PEO/PLA Block

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