poly(d ,l -lactice) nanoparticles for sustained release by supercritical assisted atomization technique

J. of Supercritical Fluids 95 (2014) 106–117

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Preparation of rifampicin/poly(d,l-lactice) nanoparticles for sustained release by supercritical assisted atomization technique P.W. Labuschagne a,∗ , R. Adami b,∗ , S. Liparoti b , S. Naidoo a , H. Swai a , E. Reverchon b,c a b c

Polymers & Composites, Council for Scientific & Industrial Research (CSIR), PO Box 395, Pretoria, South Africa Department of Industrial Engineering, University of Salerno, Via Giovanni Paolo II, 132, 84084 Salerno, Italy Research Centre for Nanomaterials and Nanotechnology (NANOMATES), Salerno University, Salerno, Italy

a r t i c l e

i n f o

Article history: Received 16 April 2014 Received in revised form 5 August 2014 Accepted 8 August 2014 Available online 18 August 2014 Keywords: Rifampicin Poly(d,l-lactide) Supercritical assisted atomization (SAA) Nano-encapsulation

a b s t r a c t In this work supercritical assisted atomization (SAA) process was used for the co-precipitation of poly(d,llactide) (PDLLA) and rifampicin (RIF) as nanoparticles for sustained release applications. The effect of the variation of PDLLA/RIF ratio on co-precipitate characteristics was mainly investigated. The precipitated particles were analyzed in terms of their morphological, thermodynamic and crystallographic properties. In addition, loading efficiency and in-vitro release studies were conducted. Spherical PDLLA/RIF nanoparticles with mean diameter ranging from 123 to 148 nm were prepared. Loading efficiency was greater than 100% resulting in RIF loadings of 28.8 to 50.5%. X-ray diffraction revealed that the encapsulated RIF is in an amorphous state, while NMR spectra indicated no structural modifications after the SAA process. In-vitro release studies showed an initial burst release of 80–87% of total RIF loaded, necessary to suppress the generation of resistance by the microorganism, followed by first-order sustained release between 0.4 and 0.8 mg/L RIF per day over a period of 17 days. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Tuberculosis (TB) is the second largest cause of death in the world from a single infectious agent. The World Health Organization (WHO) estimates that, due to its infectious nature, approximately one third of the world population is latently infected by its causative agent, Mycobacterium tuberculosis and approximately 9 million new cases are reported each year [1]. Rifampicin (RIF) is one of the most powerful antibiotics against bacterial pathogens [2] since it can diffuse easily into tissue, living cells and bacteria, making it highly effective against pathogens such as M. tuberculosis. The bactericidal activity of RIF is due to its binding to the bacterial DNA-dependant RNA polymerase and its inhibition [3]. However, despite being an effective drug for TB, there are some challenges in treating TB with RIF. First, its bactericidal activity is directly proportional to its concentration at the target site [4]. Due to its poor water solubility, dissolution in biological liquids is

∗ Corresponding authors at: CSIR, Polymers & Ceramics, Meiring naude Ave., Brummeria, Gauteng 0084, South Africa.; University of Salerno, via Giovanni Paolo II 132, 84084 Fisciano (SA), Italy. Tel.: +27 128412149. E-mail addresses: [email protected] (P.W. Labuschagne), [email protected] (R. Adami). http://dx.doi.org/10.1016/j.supflu.2014.08.004 0896-8446/© 2014 Elsevier B.V. All rights reserved.

low and can limit its ability to reach the required concentration. Second, long term continuous therapy leads to the risk of hepatoxicity in many patients [5]. Third, anti-TB therapy is complex and prolonged, usually 6–9 months, with a high pill burden [6,7]. This leads to patient non-compliance and the subsequent emergence of multidrug-resistant TB [8]. The current strategy for limiting adverse side-effects and enhancing therapeutic activity of anti-TB drugs is via encapsulation within a carrier, from where it is released in a controlled way over an extended period of time. This technique results in improved patient compliance, improved bioavailability, lower dose, lower cost and lower toxic side effects [9]. Currently, the most popular techniques for the preparation of encapsulated RIF in polymeric carriers are the double emulsion solvent evaporation and spray drying. These techniques are able to produce particles of RIF encapsulated in biodegradable polymeric carriers, such as: poly(lactideco-glycolide) (PLG) [10,11], poly(lactide) (PLA), poly(nbutylcyanoacrylate) (PBCA) [12], poly(isobutylcyanoacrylate) (PIBCA) [12], alginate [13] and gelatine [14] with particle sizes ranging between 0.2 and 5 ␮m and encapsulation efficiencies between 10 and 70%. However, these processes have some drawbacks: for example, the double-emulsion solvent-evaporation technique is complex and involves multiple steps, requires the use of surfactants and solvent removal usually takes up to 12 h [15–17].

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Fig. 1. Schematic representation of the SAA apparatus.

Spray drying often requires high temperatures; it is difficult to obtain spherical particles [18] and a secondary drying step can be required to reduce residual solvent to acceptable levels [19,20]. Alternative processing techniques in which supercritical carbon dioxide (sc-CO2 ) plays a key role in the preparation of pharmaceutical products have become well established over the last years [21,22]. The unique properties of supercritical carbon dioxide such as, tunable solvent power, liquid-like density, gas-like diffusivity, together with its non-toxicity and low cost, allowed this technology to be successfully proposed in several pharmaceutical processes such as micronization [23,24], modification [25,26] and encapsulation [26,27] of drugs. Typical sc-CO2 based processes include rapid expansion of supercritical solutions (RESS) [28], rapid expansion of supercritical solutions into liquid solvents (RESOLV) [29], particles from gas saturated solutions (PGSS) [30] and several antisolvent techniques such as supercritical anti-solvent (SAS) [31], gas anti-solvent (GAS) [32] and solution-enhanced dispersion by supercritical fluids (SEDS) [33]. While many of these processes have been applied for preparation of controlled release particles, few have been able to produce particles in the nanosize range [29,34]. However, the most attractive micronization technique using scCO2 is the Supercritical Assisted Atomization (SAA). It is based on the formation of an expanded liquid solution formed by an organic solvent, one or more solutes and sc-CO2 . The expanded liquid is, then, atomized in a precipitation vessel. When compared to ordinary atomization techniques, SAA has the advantage of operating on a reduced viscosity and low surface tension medium, due to the specific characteristic of expanded liquids and, thus, produces an enhanced atomization due to reduced cohesive forces. SAA has been successfully applied to the micronization of several active principles [35–38] and has been particularly successful in the production of polymer + drug micrometric co-precipitates [39–41]. It is also possible to operate at reduced pressure to produce particles of thermo-sensitive compounds [42–44]. SAA technique was previously used to produce micrometric RIF particles [24,45] but, at the best of our knowledge, has never been used to produce polymer-RIF co-precipitates. Therefore, the aim of this work was to evaluate whether the SAA process can be applied for the preparation of RIF-loaded polymer nanoparticles. poly(d,l-lactide) (PDLLA) was used as matrix

for RIF. The effect of the variation of polymer/drug ratio on the co-precipitate characteristics was investigated. The precipitated particles were analyzed in terms of morphological, thermodynamic and crystallographic properties. In addition, loading efficiency and in-vitro release studies were conducted.

2. Materials and methods 2.1. Materials The PDLLA homopolymer (Resomer® R 203 H, Mw : 28k) was supplied by Evonik, Germany. Rifampicin (823 g/mol) was purchased from Sigma-Aldrich (South Africa). Dichloromethane (DCM) and acetone were supplied by Sigma Aldrich (Milan, Italy). CO2 (purity 99.9%) was purchased from SON (Naples, Italy). All material was used as received.

2.2. SAA apparatus SAA apparatus (Fig. 1) consists of two high pressure pumps (mod. 305, Gilson) delivering liquid solution and CO2 to the saturator. The saturator is a high pressure vessel (25 cm3 internal volume) loaded with stainless steel perforated saddles, which assured a large contact surface between the liquid solution and CO2 . The expanded liquid obtained in the saturator is sprayed through a thin wall injection nozzle (80 ␮m internal diameter) into the precipitator (3 dm3 internal volume). A controlled flow of N2 is taken from a cylinder, heated in an electric heat exchanger (mod. CBEN 24G6, Watlow) and sent to the precipitator to induce droplet evaporation. The saturator and the precipitator are electrically heated using thin band heaters (Watlow, mod. STB3EA10). A stainless steel filter, located at the bottom of the precipitator, allowed powder collection and the gaseous stream flow out. To operate below the atmospheric pressure, the plant is equipped with a vacuum system, formed by two vacuum pumps (DVP mod. ZA100P) operating downstream of the precipitator. The system is completed by a condenser, which separates liquids from the gas stream.

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2.3. Analytical methods 2.3.1. Scanning electron microscopy (SEM) Samples were analyzed by a JEOL 7500 F field-emission SEM (FESEM) at an accelerating voltage of 2 kV. The sample was mounted on an Aluminium stub using a carbon tape. The samples were sputter coated by Carbon to avoid charging. 2.3.2. Particle size (PS) and particle size distribution (PSD) PS and PSD were calculated from SEM images using the Image Pro Premier software (Media Cybernetics, Bethesda, MD, USA). Approximately 500 particles of each formulation were measured to calculate PSD. 2.3.3. Viscosity The kinematic viscosities of the RIF/PDLLA solutions were measured at 25 ◦ C using an Ubbelohde viscometer (K = 0.00101). 2.3.4. Differential scanning calorimetry (DSC) A DSC, calibrated with indium and zinc, was used to perform the thermal analysis on the samples (DSC Q2000, TA Instruments, USA). Samples were run in triplicate. A heating rate of 10 ◦ C/min was used in a nitrogen atmosphere, with a flow rate of 40 mL/min. The temperature range was 20 ◦ C to 300 ◦ C. Aluminium sample pans were used. The sample masses, which were accurately determined on an analytical balance, ranged between 2 and 5 mg. 2.3.5. X-ray diffraction (XRD) Crystallographic analysis of samples were performed on a powder X-ray diffractometer (XPERT PRO PANanalytical, Netherlands). The measurement conditions were a Cu K␣ radiation generated at 45 kV and 40 mA as X-ray source in the range 2–50◦ (2) and step angle 0.0131◦ /s. 2.3.6. Drug loading (DL) and loading efficiency (LE) The amount of RIF loaded into the particles was determined by accurately weighing a sample of the particles and dissolving in 10 mL of DCM. The absorbance of the solution was measured with a UV/VIS spectrometer (Lamda 35, Perkin Elmer) at a wavelength of 474 nm. The RIF content was measured by using the calibration curve, which was prepared by plotting concentration versus absorbance data. Samples were run in triplicate. Drug loading (% w/w) was calculated according to the following equation: DL% =

mass of RIF loaded × 100% total mass of particles

(1)

Loading efficiency (%) was expressed as actual drug loading versus the amount of drug added to the process as represented by the following equation: LE% =

mass of RIF loaded × 100% total mass of RIB added in the solution

(2)

2.3.7. In-vitro release studies Into 50 mL Nalgene® centrifuge tubes were placed 25 mL of a 0.01 M phosphate buffered saline (PBS) (pH = 7.4) with 0.2 mg/mL ascorbic acid added as an antioxidant and 1 mg/mL Tween 80. A known mass of particles was suspended in the media after which the tubes were sealed and placed in a shaker bath at 100 RPM and 37 ◦ C. At given time intervals, the tubes were centrifuged at 5000 rpm for 15 min and 5 mL of the supernatant removed for analysis. The remainder of the supernatant was removed and the microsphere pellets were re-suspended in fresh PBS (25 mL), similar to the method used by Liggens and Burt [46]. The amount of RIF in 5 mL of the supernatant was determined with UV/vis analysis at 474 nm, according to the standard curve of RIF. Dissolution curves were determined from triplicate runs.

2.3.8. Nuclear magnetic resonance spectroscopy (NMR) The chemical structure change between pure and released RIF from the PDLLA/RIF co-precipitate was analyzed with nuclear magnetic resonance (NMR) spectroscopy which was run in deuterated chloroform (CDCl3) on a Bruker 400 MHz instrument. 2.3.9. Drug-release kinetics Mathematical modelling of the release kinetics can offer important information of the manner in which a drug is released from the drug-loaded polymer matrices. According to Arifin, Lee and Wang [47], there are three main the mechanisms that can govern drug release from a polymer matrix, namely, diffusion from the nondegraded polymer, enhanced diffusion due to polymer swelling, and release due to polymer degradation and erosion. The kinetics and mechanism of RIF release from the SAA produced particles were evaluated by fitting the in vitro drug release data to four commonly used kinetics models, namely, zero order, first order, Higuchi and Korsmeyer–Peppas models. The equations for the abovementioned kinetics are [48]: Zero order equation: Mt = M0 + K0 t

(3)

where Mt is the cumulative amount of drug released at any specified time t, M0 is the initial amount of drug in the formulation, and K0 is the zero order release rate constant. First order equation: ln Mt = ln M0 + K1 t

(4)

where Mt is the cumulative amount of drug released at any specified time t, M0 is the initial amount of drug in the formulation, and K1 is the first order release rate constant. Higuchi equation: Mt = M0 + KH t 1/2

(5)

where Mt is the cumulative amount of drug released at any specified time t, M0 is the initial amount of drug in the formulation, and KH is the Higuchi release rate constant. The Higuchi equation describes the mechanism of drug release as a diffusion process, based on Fick’s law [49]. Korsmeyer–Peppas equation: Mt = Kk t n M˛

(6)

where Mt is the cumulative amount of drug released at any specified time t, M˛ is the amount of drug release at time, Kk is the Korsmeyer–Peppas release rate constant, n is the diffusion exponent and Mt /M˛ is the fraction of the drug release at time t. The Korsmeyer–Peppas model predicts whether the mechanism of release follows Fick’s Law or an anomalous behaviour [49]. The zero order and first orders kinetics are used to describe the order of the drug release while the Higuchi and the Korsemeyer– Peppas equations are used to determine the mechanism of drug release from the systems. The release data was used to plot the zero order equation as %cumulative release vs time, the first order equation as log of the % drug remaining vs time, Higuchi equation as %cumulative release vs square root of time and the Korsmeyer–Peppas equation as log of the cumulative release % vs log of time [50,51]. Regression analysis (using Microsoft Excel 2010) was performed on these four plots to obtain the R2 (coefficient of correlation) values of the linear curves, the rate constants and n-values. The n-values are obtained from the slope of the Korsmeyer–Peppas plots, and represent the different release mechanisms as described in Table 1 [52]:

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Table 1 Diffusion exponent and the solute release mechanism. Diffusion exponent (n)

Overall solute diffusion mechanism

0.43 0.43 < n < 0.85 0.85

Fickian diffusion Anomalous (non-Fickian) diffusion Case II transport

2.4. Statistical analysis The data obtained were expressed as mean ± standard deviation and analyzed statistically by the one-way analysis of variance (ANOVA) method, followed by unpaired Student’s t-test to compare the difference between the sample groups. A difference with P < 0.05 was considered statistically significant. In addition the skewness (Sk ), which is a measure of the asymmetry of the PSD curve, was also determined. Data analysis was performed using the statistical analysis package of Microsoft Excel 2010. 3. Results and discussion SAA process is very versatile and it is possible to micronize several kinds of compounds, pharmaceuticals and polymers, and the same compound could be processed using different solvents and different operating conditions depending on the target of the product needed [24,31]. sc-CO2 and the liquid solution (formed by the solvent and the solid solute) are continuously fed at constant flow rates and fixed ratio in the saturator (at fixed temperature and pressure), before the atomization in the injector, allowing a constant concentration of the compounds. Both PDLLA and RIF have been micronized individually in previous studies using the SAA process in different configurations: performing the precipitation under atmospheric and reduced pressure [24,42]. These studies were used as a guide and the choice of the plant configuration, the solvent used and the operating conditions were optimized to perform the co-precipitation of PDLLA and RIF (Table 2). The plant configuration and the solvent were chosen considering the characteristic of PDLLA: the low glass transition temperature (Tg ) required a reduced precipitation pressure to use lower temperatures. The SAA micronization of PDLLA and RIF separately was successful and the optimized process conditions were kept constant for the co-precipitation experiments, while only the PDLLA/RIF ratio was varied. 3.1. Particle morphology SEM images of unprocessed PDLLA and RIF are shown in Fig. 2(a) and (b). The unprocessed RIF consists of rod-like crystalline particles with lengths between 10 and 200 ␮m and thickness between 2 and 90 ␮m. The unprocessed PDLLA consists mostly of fibres with lengths from 120 to 350 ␮m and diameter from 30 to 100 ␮m. Each fibre is made up of smaller fibres with diameters from 0.5 to 1 ␮m.

Table 2 Process operating conditions used for the co-precipitation of PDLLA and RIF. Variable

Setting

Solvent RIF concentration (mg/mL) Saturator temperature (◦ C) Saturator pressure (bar) Precipitator temperature (◦ C) Precipitator pressure (bar) CO2 /solution flow ratio PDLLA/RIF ratio (wt/wt)

Acetone 10 80 85 40 0.7 0.7 6/1; 4/1; 2/1

Fig. 2. SEM micrographs of unprocessed RIF (a) and unprocessed PDLLA (b).

After SAA processing, according to the process conditions shown in Table 2, RIF particles showed smooth spherical shape with diameters ranging from 200 to 400 nm, with some exhibiting a collapsed morphology. This phenomenon has been reported before and is explained as the formation of “deflated balloons” when solvent evaporates very fast resulting in the solid phase collapsing [24]. In this study, the SAA processed PDLLA particles were also smooth and spherical with size range between 100 and 200 nm. Mixtures of PDLLA and RIF at different weight ratios (2:1, 4:1, 6:1) were processed at the operating conditions reported in Table 2. The dry powder obtained showed a pale reddish colour, suggesting the presence of RIF, which has a characteristic dark red colour, diluted by the presence of the white polymer. The particles observed at the SEM, showed a uniform spherical morphology at all weight ratios of PDLLA/RIF investigated, as reported in Fig. 3. Due to similarities in the morphology of SAA processed PDLLA and RIF particles, it is difficult to conclude only with the support of SEM images whether PDLLA/RIF co-precipitation has occurred. However, there is no evidence of the large rod-like crystalline RIF particles, which suggests that the RIF is precipitated either separate or in mixture with PDLLA as spherical particles. Moreover, the precipitation process that characterizes SAA, is droplet formation and drying; it supports the hypothesis of co-precipitation of PDLLA and RIF together inside the droplets during drying. The SEM images were taken at the same magnification (40k), therefore they allow for a qualitative evaluation of polydispersity.

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Fig. 3. SEM micrographs of PDLLA/RIF co-precipitates at ratio 2:1 (a), 4:1 (b) and 6:1 (c).

3.2. Particle size, size distribution and skewness Particle size distributions (PSD) of the PDLLA/RIF co-precipitates at all weight ratios are shown in Fig. 4a–c, while the mean particles size (PS) and skewness (Sk ) are summarized in Table 3: PSD is non-symmetrical in all cases with a relatively long tail in the 300 to 500 nm range, which is confirmed by the positive Sk values. The mean particle sizes for all ratios are in the nano-range with the PDLLA/RIF 6:1 co-precipitate showing a slightly larger mean particle size. The result of the one-way ANOVA indicates that the differences in particle size means are significant (P < 0.05). Further unpaired Student-t test analysis between the sample groups showed that the mean of PDLLA/RIF 6:1 is statistically significantly different from both PDLLA/RIF 2:1 and PDLLA/RIF 4:1 (P < 0.05); but, that there is not a statistically significant difference between PDLLA/RIF 2:1 and PDLLA/RIF 4:1 (P > 0.05). The larger mean particle size of PDLLA/RIF 6:1 can be explained as follows: one of the factors that affect particles sizes in the SAA process is solution viscosity, which can be influenced by polymer molecular weight [43], solute concentration [43] or operating temperature [42]. The kinematic

viscosity of the solution containing PDLLA:RIF 6:1 was measured as 0.687 mm2 /s, while the viscosity of the solution with lower PDLLA concentration (PDLLA:RIF = 2:1) was measured as 0.489 mm2 /s. At increased solution viscosities, the efficiency of the atomization mechanism decreases, leading to larger particles [53]. Polydispersity is an indication of the broadness of the PSD. For all PDLLA:RIF ratios the qualitative polydispersity evaluation indicated a relatively narrow PSD. This is an indication that relatively homogenous mixing occurred in the SAA saturator [54] and an efficient jet break up was obtained at the nozzle. A small amount of particles with the “deflated balloon” morphology were also detected across all formulations. 3.3. Drug loading and loading efficiency RIF loading and loading efficiency in the different PDLLA/RIF coprecipitates were measured in order to confirm the presence of RIF in the obtained particles, and to compare the final drug loading versus the amount of drug added to the process. The initial RIF content (wt% of total solids in the solution) before the SAA process

Table 3 Particle characteristics and dimensions of PDLLA/RIF co-precipitates formed by SAA. PDLLA:RIF

Surface morphology

D10%; D50%; D90%a (nm)

Mean particle diameter (nm)

Skewness (Sk )

6:1 4:1 2:1

Smooth and spherical Smooth and spherical Smooth and spherical

76.1; 127.8; 246.3 57.9; 110.8; 202.2 65.2; 106.8; 214.7

148 ± 78 123 ± 64 128 ± 68

1.67 1.81 1.80

a

Percentile distributions.

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Fig. 4. Particle size distributions of PDLLA/RIF co-precipitates formed by SAA with different PDLLA/RIF ratios: (a) 6:1, (b) 4:1 and (c) 2:1.

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Fig. 5. DSC thermograms of PDLLA/RIF co-precipitates at different ratios. Tg (PDLLA) 59.9 ◦ C, Tg (PDLLA:RIF 2:1) 55.7 ◦ C, Tg (PDLLA:RIF 4:1) 58.9 ◦ C, Tg (PDLLA:RIF 6:1) 57.8 ◦ C.

Table 4 Comparison of RIF content in solution versus RIF loading in the PDLLA/RIF coprecipitates formed in SAA. PDLLA:RIF 6:1 4:1 2:1

RIF content (wt%)

RIF loading (wt%)

14.3 20.0 33.3

28.8 ± 1.66 35.5 ± 0.37 50.5 ± 1.73

and the subsequent RIF loadings (wt% in product) derived from Eq. (1) are shown in Table 4. As expected, the RIF loading decreases with increasing PDLLA:RIF ratio. However, in all cases the RIF loading in the particles

after SAA processing is greater than the one initially added to the formulation. This is reflected in the loading efficiencies which were calculated as being much higher than 100%. This result has occurred previously when the SAA process was used to encapsulate gentamicin sulphate in bovine serum albumin (BSA) [40]. In that study it was explained that part of the BSA precipitated in the saturator, leading to higher drug/polymer ratios in the precipitation vessel. This effect is known as the antisolvent effect of sc-CO2 and was also noted during SAA experiments on polylactide which precipitated in the saturator at higher gas to liquid ratios [28]. Therefore, a similar behaviour could be expected for the system PDLLA/RIF, with some of the polymer precipitated in the saturator due to the antisolvent effect.

Fig. 6. X-ray diffraction patterns of PDLLA/RIF co-precipitates at different ratios.

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Fig. 7. In-vitro release profiles from PDLLA/RIF co-precipitates at different ratios. Each point represents the mean ± standard deviation of the samples.

3.4. Differential scanning calorimetry DSC thermograms were done to study the thermal behaviour of pure PDLLA and RIF together with the co-precipitated PDLLA/RIF particles at different ratios (Fig. 5). The unprocessed RIF (as received) shows a melting endotherm in the range 178–202 ◦ C, immediately followed by an exothermal recrystallisation at 200.8 ◦ C, and an exothermal decomposition in the range from 228 to 293 ◦ C. This is the characteristic behaviour of RIF form II (RIF-II) where immediately after melting it recrystallizes to form I (RIF-I) and, then, decomposes [55]. The unprocessed

PDLLA (as received) shows a Tg at 59.5 ◦ C and the absence of a melting endotherm, indicating that its grade is naturally amorphous. In all the PDLLA/RIF co-precipitates the addition of RIF resulted in a decrease in the Tg of PDLLA, in the range 55.7–58.9 ◦ C. Tg behaviour in polymer–drug systems are mainly determined by intermolecular interactions and the molecular environments [56]. A reduced Tg is usually associated with positive excess mixing volume, which is brought about by poor interaction and/or conformational and free volume changes. Fourier-Transform Infrared (FTIR) analysis has shown no peak shifts in the PDLLA/RIF

Fig. 8. Proton NMR spectra overlay of non-encapsulated (bottom) and encapsulated (top) rifampicin (CDCl3, 400 MHz) after digestion of PLLA and release of the drug.

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Fig. 9. First-order plots of RIF remaining versus time in PDLLA/RIF co-precipitates with PDLLA/RIF 2:1 (a); PDLLA/RIF 4:1 (b); PDLLA/RIF 6:1 (c).

co-precipitate spectra, suggesting no specific interaction between PDLLA and RIF (data not shown), thus confirming the hypothesis that each particle is a physical mixture of PDLLA and RIF. In addition, the amorphous nature of the PDLLA and the relatively large molecular volume of RIF could contribute to a positive excess mixing volume, thereby lowering the Tg of the PDLLA. The Tg s of these compositions are associated with an increased hysteresis peak, which is indicative of built-in molecular stress which can be attributed to the rapid vitrification of the PDLLA in the SAA process. No RIF-II melting endotherm is observed, confirming that the RIF is in an amorphous state (RIF-a). The amorphous state of the RIF can be explained by very rapid solidification in the SAA process which occurs too fast for crystallisation to occur [57]. Exotherms are observed from 170 to 175 ◦ C which corresponds to the Tg of RIF-a [58] and is followed by RIF decomposition at ±250 ◦ C. 3.5. X-ray diffraction XRD scans of the unprocessed RIF were compared with the SAA co-precipitates at different PDLLA/RIF ratios to further analyse

the crystalline behaviour (Fig. 6). The unprocessed RIF spectrum shows the characteristic peaks of RIF-I at 13.65 and 14.35 ◦ . These peaks are not present in any of the PDLLA/RIF co-precipitates, further confirming that the obtained particles are completely amorphous. 3.6. In-vitro release studies Drug release studies were carried out on the different PDLLA/RIF co-precipitates prepared with SAA. All compositions showed a bimodal release pattern with a high initial burst release followed by a sustained release pattern (Fig. 7). The co-precipitates at all the PDLLA/RIF ratios studied showed an initial rapid release of 80–87% of total loading over the first 5 h, corresponding to 16.7 and 23.3 mg h/L of RIF which is within the typical RIF concentration found in humans after being administered a dose of 600 mg [59]. The initial rapid release shown here is similar to the results reported in literature for RIF sustained release in different formulations [58,60,61]. It is an important result, since a starting high Cmax (peak concentration) of RIF is needed to suppress

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Fig. 10. Higuchi plots of RIF cumulative release versus time in PDLLA/RIF co-precipitates with PDLLA/RIF 2:1 (a); PDLLA/RIF 4:1 (b); PDLLA/RIF 6:1 (c).

the generation of resistance by the microorganism and in this case, the burst release was found to vary as a function of initial drug loading with the composition containing the highest RIF loading (PDLLA:RIF = 2:1) showing the largest burst release. After the burst release, sustained RIF release of between 0.4 and 0.8 mg/L per day over a period of 17 days is observed, which is above the minimum inhibitory concentration (MIC) for RIF of 0.2 mg/L [62]. A slower release lasted until around day 4 at which point the rate increased up to day 8, followed by another continuous slow release over the remaining 7 days, which ensure the maintaining of the MIC. At the end, between 87 and 93% of the RIF was released. The high initial burst release can be attributed to a particle formation mechanism similar to spray drying [18]. As solvent and CO2 diffuses outward from the centre of the droplets, the smaller RIF molecules tend to move along while the polymer chains move slower. This leads to higher drug concentration around the edge of the particles once they have solidified, leading to burst release. The release pattern obtained with PDLLA/RIF nanoparticles obtained by SAA is significant and very promising since for non-encapsulated RIF a daily dose is required to maintain the MIC [10].

3.7. Nuclear magnetic resonance (NMR) spectroscopy The molecular integrity of the released rifampicin was investigated by NMR spectroscopy (Fig. 8) 1H NMR spectra of both non-encapsulated (bottom spectrum) and encapsulated (top spectrum) rifampicin after the release were identical. The four proton groups indicated in the structure (a–d) were used as diagnostic signals to confirm the similarity of both structures. The neighbouring functional groups of these protons are relatively more labile than the rest of the molecule. They would have been expected to experience significant chemical shifts if the encapsulation process had compromised the integrity of the molecule. The single imino proton (a) was observed at 8.3 ppm for both drug samples. Similarly, both spectra presented the alkene proton (b) as a doublet integrating for one proton at 6.4 ppm. The methoxy and methyl groups (c) and (d) were observed as sharp singlets at 2.2 ppm and 2.1 ppm, respectively. Each signal integrated for three protons. The difference in intensities of the two NMR spectra is due to the relatively much lower drug concentration present in the crude drug release sample.

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3.8. Drug release kinetics Kinetic studies revealed that the R2 values for the first order models were highest for all three PDLLA/RIF ratios, indicating that drug release followed first order kinetics (Fig. 9a–c). PDLLA/RIF 2:1 and 4:1 follow Higuchi equation (R2 > 0.9), indicating the drug release is governed by diffusion (Fig. 10a–c). The n-values ranged from 0.0148 to 0.218 indicating that the drug release from all studies demonstrated Fickian diffusion (n < 0.43). The R2 value of Higuchi for PDLLA/RIF 6:1 was 0.87, showing fairly good linearity, indicating that the system follows Fick’s law. This was confirmed with the n-value <0.43. 4. Conclusions In this work we showed that the SAA process can be successfully applied for the preparation of PDLLA/RIF nanoparticles. They are smooth and spherical with mean particle size below 150 nm and a narrow particle size distribution for all PDLLA/RIF ratios. The formulation with the highest PDLLA/RIF ratio showed the largest mean particle size. DSC and XRD results suggested that in the nanoparticles obtained by SAA the RIF is amorphously dispersed in the PDLLA matrix. (NMR indicated no RIF degradation after encapsulation). FTIR analysis suggested limited interaction between PDLLA and RIF which could have contributed to positive excess mixing volumes as supported by DSC thermographs. In-vitro release studies showed that the formulation has a high initial burst release followed by a sustained drug release corresponding to first order kinetics. Burst effect is greater at higher RIF loadings. Drug release mechanism was shown to correspond with Fickian diffusion. Acknowledgements This research was supported by a grant from the Department of Science and Technology, Republic of South Africa. We thank Dr Mohammed Balogun (Materials Science and Manufacturing, CSIR, South Africa) for carrying out the NMR studies. References [1] W.H. Organization, Tuberculosis Fact Sheet, W.H. Organization, Geneva, 2010. [2] C. Becker, J.B. Dressman, H.E. Junginger, S. Kopp, K.K. Midha, V.P. Shah, et al., Biowaiver monographs for immediate release solid oral dosage forms: rifampicin, J. Pharmaceutical Sciences 98 (2009) 2252–2267. [3] G. Hartmann, K.O. Honikel, F. Knüsel, J. Nüesch, The specific inhibition of the DNA-directed RNA synthesis by rifamycin, Biochimica et Biophysica Acta, Nucleic Acids and Protein Synthesis 145 (1967) 843–844. [4] T. Gumbo, A. Louie, M.R. Deziel, W. Liu, L.M. Parsons, M. Salfinger, et al., Concentration-dependent Mycobacterium tuberculosis killing and prevention of resistance by rifampin, Antimicrobial Agents and Chemotherapy 51 (2007) 3781–3788. [5] M.A. Steele, R.F. Burk, R.M. DesPrez, Toxic hepatitis with isoniazid and rifampin: a meta-analysis, Chest 99 (1991) 465–471. [6] D. Dube, G.P. Agrawal, S.P. Vyas, Tuberculosis, From molecular pathogenesis to effective drug carrier design, Drug Discovery Today 17 (2012) 760–773. [7] S.K. Mehta, N. Jindal, Formulation of Tyloxapol niosomes for encapsulation, stabilization and dissolution of anti-tubercular drugs, Colloids and Surfaces B: Biointerfaces 101 (2013) 434–441. [8] R. Pandey, Z. Ahmad, Nanomedicine and experimental tuberculosis: facts, flaws, and future, Nanomedicine: Nanotechnology, Biology and Medicine 7 (2011) 259–272. [9] D. Dube, G.P. Agrawal, S.P. Vyas, Tuberculosis: from molecular pathogenesis to effective drug carrier design, Drug Discovery Today 17 (2012) 760–773. [10] M. Dutt, G.K. Khuller, Chemotherapy of Mycobacterium tuberculosis infections in mice with a combination of isoniazid and rifampicin entrapped in poly(dllactide-co-glycolide) microparticles, J. Antimicrobial Chemotherapy 47 (2001) 829–835. [11] E.L.W. Barrow, G.A. Winchester, J.K. Staas, D.C. Quenelle, J.A.Y.K. Staas, Use of microsphere technology for targeted delivery of rifampin to Mycobacterium tuberculosis—infected macrophages, Antimicrobial Agents and Chemotherapy 42 (1998) 2682–2689.

[12] Y.V. Anisimova, S.I. Gelperina, C.A. Peloquin, L.B. Heifets, Nanoparticles as antituberculosis drugs carriers: effect on activity against Mycobacterium tuberculosis in human monocyte-derived macrophages, J. Nanoparticle Research 2 (2000) 165–171. [13] Z. Ahmad, a. Zahoor, S. Sharma, G.K. Khuller, Inhalable alginate nanoparticles as antitubercular drug carriers against experimental tuberculosis, International J. Antimicrobial Agents 26 (2005) 298–303. [14] G.K. Saraogi, P. Gupta, U.D. Gupta, N.K. Jain, G.P. Agrawal, Gelatin nanocarriers as potential vectors for effective management of tuberculosis, International J. Pharmaceutics 385 (2010) 143–149. [15] Q. Ain, S. Sharma, S.K. Garg, G.K. Khuller, Role of poly[dl-lactide-co-glycolide] in development of a sustained oral delivery system for antitubercular drug(s), International J. Pharmaceutics 239 (2002) 37–46. [16] M. Dutt, G.K. Khuller, Sustained release of isoniazid from a single injectable dose of poly(dl-lactide-co-glycolide) microparticles as a therapeutic approach towards tuberculosis, International J. Antimicrobial Agents 17 (2001) 115–122. [17] R. Pandey, A. Zahoor, S. Sharma, G. Khuller, Nanoparticle encapsulated antitubercular drugs as a potential oral drug delivery system against murine tuberculosis, Tuberculosis 83 (2003) 373–378. [18] P. O’Hara, J. Hickey, Respirable PLGA microspheres containing rifampicin for the treatment of tuberculosis: manufacture and characterization, Pharmaceutical Research 17 (2000) 955–961. [19] A. Paudel, Z.A. Worku, J. Meeus, S. Guns, G. Van den Mooter, Manufacturing of solids dispersions of poorly water soluble drugs by spray drying: formulation and process considerations, International J. Pharmaceutics 453 (2012) 254–284. [20] S. Ohtake, R.A. Martin, L. Yee, D. Chen, D.D. Kristensen, D. Lechuga-Ballesteros, et al., Heat-stable measles vaccine produced by spray drying, Vaccine 28 (2010) 1275–1284. [21] M.J. Cocero, Á. Martín, F. Mattea, S. Varona, Encapsulation and co-precipitation processes with supercritical fluids: fundamentals and applications, J. Supercritical Fluids 47 (2009) 546–555. [22] A. Naylor, A.L. Lewis, L. Illum, Supercritical fluid-mediated methods to encapsulate drugs: recent advances and new opportunities, Therapeutic Delivery 2 (2011) 1551–1565. [23] J. Kerˇc, S. Srˇciˇc, Z. Knez, P. Senˇcar-Boˇziˇc, Micronization of drugs using supercritical carbon dioxide, International J. Pharmaceutics 182 (1999) 33–39. [24] E. Reverchon, G. Della Porta, Micronization of antibiotics by supercritical assisted atomization, J. Supercritical Fluids 26 (2003) 243–252. [25] A. Kordikowski, T. Shekunov, P. York, Polymorph control of sulfathiazole in supercritical CO2 , Pharmaceutical Research 18 (2001) 682–688. [26] K. Gong, R. Viboonkiat, I.U. Rehman, G. Buckton, J. Darr, Formation and characterization of porous indomethacin-PVP coprecipitates prepared using solvent-free supercritical fluid processing, J. Pharmaceutical Sciences 94 (2005) 2583–2590. [27] M. Türk, G. Upper, P. Hils, Formation of composite drug–polymer particles by co-precipitation during the rapid expansion of supercritical fluids, J. Supercritical Fluids 39 (2006) 253–263. [28] P.G. Debenedetti, J.W. Tom, X. Kwauk, S.-D. Yeo, Rapid expansion of supercritical solutions (RESS): fundamentals and applications, Fluid Phase Equilibria 82 (1993) 311–321. [29] A. Sane, J. Limtrakul, Formation of retinyl palmitate-loaded poly(l-lactide) nanoparticles using rapid expansion of supercritical solutions into liquid solvents (RESOLV), J. Supercritical Fluids 51 (2009) 230–237. [30] Z. Mandˇzuka, Zˇ . Knez, Influence of temperature and pressure during PGSSTM micronization and storage time on degree of crystallinity and crystal forms of monostearate and tristearate, J. Supercritical Fluids 45 (2008) 102–111. [31] E. Reverchon, R. Adami, G. Caputo, I. De Marco, Spherical microparticles production by supercritical antisolvent precipitation: interpretation of results, J. Supercritical Fluids 47 (2008) 70–84. [32] M. Charoenchaitrakool, C. Polchiangdee, P. Srinophakun, Production of theophylline and polyethylene glycol 4000 composites using gas anti-solvent (GAS) process, Materials Letters 63 (2009) 136–138. [33] E. Franceschi, a.M. De Cesaro, S.R.S. Ferreira, J. Vladimir Oliveira, Precipitation of ␤-carotene microparticles from SEDS technique using supercritical CO2 , J. Food Engineering 95 (2009) 656–663. [34] H. Jin, F. Xia, C. Jiang, Y. Zhao, L. He, Nanoencapsulation of lutein with hydroxypropylmethyl cellulose phthalate by supercritical antisolvent, Chinese J. Chemical Engineering 17 (2009) 672–677. [35] E. Reverchon, R. Adami, G. Caputo, Production of cromolyn sodium microparticles for aerosol delivery by supercritical assisted atomization, AAPS PharmSciTech 8 (2007). [36] E. Reverchon, R. Adami, M. Scognamiglio, G. Fortunato, G. Della Porta, Beclomethasone microparticles for wet inhalation, produced by supercritical assisted atomization, Industrial & Engineering Chemical Research 49 (2010) 12747–12755. [37] Z. Du, C. Tang, Y.X. Guan, S.J. Yao, Z.Q. Zhu, Bioactive insulin microparticles produced by supercritical fluid assisted atomization with an enhanced mixer, International J. Pharmaceutics 454 (2013) 174–182. [38] M.Q. Cai, Y.X. Guan, S.J. Yao, Z.Q. Zhu, Supercritical fluid assisted atomization introduced by hydrodynamic cavitation mixer (SAA–HCM) for micronization of levofloxacin hydrochloride, J. Supercritical Fluids 43 (2008) 524–534. [39] S. Liparoti, R. Adami, G. Caputo, E. Reverchon, Supercritical assisted atomization: polyvinylpyrrolidone as carrier for drugs with poor solubility in water, J. Chemistry (2013).

P.W. Labuschagne et al. / J. of Supercritical Fluids 95 (2014) 106–117 [40] G. Della Porta, R. Adami, P.D.E.L. Gaudio, L. Prota, R. Aquino, E. Reverchon, Albumin/gentamicin microspheres produced by supercritical assisted atomization: optimization of size, Drug Loading and Release 99 (2010) 4720–4729. [41] E. Reverchon, A. Antonacci, I. Chimica, Drug–polymer microparticles produced by supercritical assisted atomization, J. Supercritical Fluids 97 (2007) 1626–1637. [42] R. Adami, S. Liparoti, E. Reverchon, A new supercritical assisted atomization configuration, for the micronization of thermolabile compounds, Chemical Engineering J. 173 (2011) 55–61. [43] R. Adami, S. Liparoti, L. Izzo, D. Pappalardo, E. Reverchon, PLA–PEG copolymers micronization by supercritical assisted atomization, J. of Supercritical Fluids 72 (2012) 15–21. [44] S. Liparoti, R. Adami, E. Reverchon, PEG micronization by supercritical assisted atomization, operated under reduced pressure, J. Supercritical Fluids 72 (2012) 46–51. [45] E. Reverchon, R. Adami, G. Caputo, Supercritical assisted atomization: performance comparison between laboratory and pilot scale, J. Supercritical Fluids 37 (2006) 298–306. [46] R.T. Liggins, H.M. Burt, Paclitaxel loaded poly(l-lactic acid) microspheres: properties of microspheres made with low molecular weight polymers, International J. Pharmaceutics 222 (2001) 19–33. [47] D.Y. Arifin, L.Y. Lee, C.H. Wang, Mathematical modeling and simulation of drug release from microspheres: implications to drug delivery systems, Advanced Drug Delivery Reviews 58 (2006) 1274–1325. [48] G. Murtaza, M. Ahamd, N. Akhtar, F. Rasool, A comparative study of various microencapsulation techniques: effect of polymer viscosity on microcapsule characteristics, Pakistan J. Pharmaceutical Sciences 22 (2009) 291–300. [49] K. Pagar, P. Vavia, Rivastigmine-loaded l-lactide-depsipeptide polymeric nanoparticles: decisive formulation variable optimization, Scientia Pharmaceutica 81 (2013) 865–885. [50] J. Vijay, J. Sahadevan, R. Prabhakaran, R. Gilhotra, Formulation and evaluation of cephalexin extended-release matrix tablets using hydroxy propyl methyl cellulose as rate-controlling polymer, J. Young Pharmacists 4 (2012) 3–12.

117

[51] J.V.V. Naga Phani, M. Mohan Varma, Development and evaluation of novel mucoadhesive multipartculate drug delivery system of simvastatin, Indian J. Pharmaceutica Education and Research 47 (2013) 62–70. [52] J. Siepmann, N.A. Peppas, Higuchi equation: derivation, applications, use and misuse, International J. Pharmaceutics 418 (2011) 6–12. [53] E. Reverchon, G. Lamberti, A. Antonacci, Supercritical fluid assisted production of HPMC composite microparticles, J. Supercritical Fluids 46 (2008) 185–196. [54] A. Martín, M.J. Cocero, Micronization processes with supercritical fluids: fundamentals and mechanisms, Advanced Drug Delivery Reviews 60 (2008) 339–350. [55] S. Agrawal, Y. Ashokraj, P.V. Bharatam, O. Pillai, R. Panchagnula, Solid-state characterization of rifampicin samples and its biopharmaceutic relevance, European J. Pharmaceutical Sciences 22 (2004) 127–144. [56] I.M. Kalogeras, A novel approach for analyzing glass-transition temperature vs. composition patterns: application to pharmaceutical compound + polymer systems, European J. Pharmaceutical Sciences 42 (2011) 470–483. [57] E. Reverchon, I. De Marco, G. Della Porta, Rifampicin microparticles production by supercritical antisolvent precipitation, International J. Pharmaceutics 243 (2002) 83–91. [58] Y.J. Son, J.T. McConville, A new respirable form of rifampicin, European J. Pharmaceutics and Biopharmaceutics 78 (2011) 366–376. [59] A.H. Diacon, R.F. Patientia, A. Venter, P.D. Van Helden, P.J. Smith, H. McIlleron, et al., Early bactericidal activity of high-dose rifampin in patients with pulmonary tuberculosis evidenced by positive sputum smears, Antimicrobial Agents and Chemotherapy 51 (2007) 2994–2996. [60] V. Patomchaiviwat, O. Paeratakul, P. Kulvanich, Formation of inhalable rifampicin-poly(l-lactide) microparticles by supercritical anti-solvent process, AAPS PharmSciTech 9 (2008) 1119–1129. [61] B.S. Rao, K.V.R. Murthy, Studies on rifampicin release from ethylcellulose coated nonpareil beads, International J. Pharmaceutics 231 (2002) 97–106. [62] R. Pandey, G.K. Khuller, Oral nanoparticle-based antituberculosis drug delivery to the brain in an experimental model, J. Antimicrobial Chemotherapy 57 (2006) 1146–1152.