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Loading of 5-fluorouracil onto Halloysite nanotubes for targeted drug delivery using a subcritical gas antisolvent process (GAS)
J. of Supercritical Fluids 159 (2020) 104756
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Loading of 5-fluorouracil onto Halloysite nanotubes for targeted drug delivery using a subcritical gas antisolvent process (GAS) Sekar Harikrishnan a , Rossen Sedev a,b,∗ , Chau Chun Beh a , Craig Priest b , Neil R. Foster a,∗ a b
Western Australian School of Mines: Minerals, Energy & Chemical Engineering, Curtin University, Perth, WA 6102, Australia Future Industries Institute, University of South Australia, Adelaide, SA 5095, Australia
h i g h l i g h t s
g r a p h i c a l
a b s t r a c t
• Halloysite • • • •
clay nanotubes are biocompatible and efficient drug nanocarriers. A gas antisolvent process with subcritical CO2 achieves a high drug loading (43 %). Distinction between tightly bound (adsorbed) and loosely bound (coprecipitated) drug. 5-Fluorouracil release is complete at pH = 7.4 but only 30 % in pH = 1.2. 5-Fluorouracil release follows firstorder kinetics with a characteristic time of 2 h.
a r t i c l e
i n f o
Article history: Received 12 September 2019 Received in revised form 13 January 2020 Accepted 14 January 2020 Available online 21 January 2020 Keywords: Gas antisolvent process Halloysite nanotubes 5-fluorouracil Drug loading Drug release
a b s t r a c t Halloysite clay nanotubes are efficient biocompatible nanocarriers for pharmaceutical products. A gas antisolvent (GAS) process employing subcritical carbon dioxide delivers a product with high loading (43 %) and free of residual solvent. An anticancer agent, 5-fluorouracil, was loaded on nanoclay particles. A quantitative distinction is made between tightly-bound (adsorbed) and loosely-bound (co-precipitated) drug. The physical stages of the dense gas process are clearly outlined. The strongly attached drug is released completely at pH = 7.4 but only up to 30 % in pH = 1.2. The release follows first-order kinetics with a characteristic time of 2 h. The mass ratio of clay nanotubes to 5-fluorouracil affects the loading with adsorbed drug as well as the ratio of loose to tightly attached drug molecules. The system Halloysite5-fluorouracil can be implemented in drug delivery systems to achieve a desired release profile. The GAS process is a one-pot green process with a low solvent consumption and high-purity product. © 2020 Elsevier B.V. All rights reserved.
Abbreviations: ATR, attenuated total reflection; API, active pharmaceutical ingredient; EDS, energy dispersive X-Ray spectroscopy; FTIR, fourier-transform infrared spectroscopy; 5FU, 5-fluorouracil; GAS, gas antisolvent process; HNT, halloysite clay nanotubes; HPLC, high performance liquid chromatography; MWCO, molecular weight cut-off; NMR, nuclear magnetic resonance; PBS, phosphate-buffered saline; PLA, poly(d,l-lactide); PLGA, poly(d,l-lactide-co-glycolide); SEM, scanning electron microscopy; TEM, transmission electron microscopy; TGA, thermal gravimetric analysis; UV-vis, ultraviolet-visible spectroscopy. ∗ Corresponding authors at: Western Australian School of Mines: Minerals, Energy & Chemical Engineering, Curtin University, Perth, WA 6102, Australia. E-mail addresses: [email protected] (R. Sedev), [email protected] (N.R. Foster). https://doi.org/10.1016/j.supflu.2020.104756 0896-8446/© 2020 Elsevier B.V. All rights reserved.
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S. Harikrishnan, R. Sedev, C.C. Beh et al. / J. of Supercritical Fluids 159 (2020) 104756
Nomenclature m0 m1 mA mfree mtot mHNT E L M NA R RD RD ∞ Vm ˛ ˛F ˇ ε *
mass of washed precipitate before heat treatment, kg mass of washed precipitate after heat treatment, kg mass of drug loaded (= m0 – m1 ), kg mass of loosely attached drug, kg total mass of API used in the experiment, kg total mass of HNT used in the experiment, kg drug encapsulation, = (m0 – m1 )/mtot drug loading, = (m0 – m1 )/m0 molecular mass, kg/mol Avogadro constant, mol−1 mass ratio of HNT and API (=mHNT /mtot ) cumulative release (mass of drug released over mass of drug loaded) final cumulative release (RD at t = ∞) molecular volume, m3 /mol hydrogen bond donor parameter (Kamlet-Taft parameter) mass ratio of loosely attached drug and total drug (=mfree /mtot ) hydrogen bond acceptor parameter (Kamlet-Taft parameter) relative volumetric expansion, (V1 -V0 )/V0 ; V0 is initial volume, V1 is expanded volume polarizability (Kamlet-Taft parameter) viscosity, Pa.s density, kg/m3 characteristic time of release, s
1. Introduction Even the most medically suitable active pharmaceutical ingredient (API), if administered directly, is likely to distribute unevenly throughout the body and therefore influence both target and healthy cells. Thus, inefficient delivery reduces the desired therapeutic effect and also increases the risk of adversely affecting normal cells. The use of drug carriers (as vehicles loaded with the API) aims to engineer higher API concentrations where needed and lower concentrations elsewhere. Nanocarriers carry relatively large amounts of drug that can be transported safely to a target destination and released locally in the most effective manner. They can travel naturally, e.g. down the gastrointestinal tract, or be guided externally, e.g. by using a magnetic field. The size and surface properties can be manipulated to regulate their ability to penetrate the tissues in the body and target specific locations. Release can be triggered by a change in pH or temperature but also using ultrasound or near-infrared laser irradiation. Nanocarriers are a subject of intense research [1–6]. A common problem in the design of nanocarriers for drug delivery is that drug loadings are rather low (typically below 10 % [7]). Dense gases with their liquid-like solvation power and gas-like mass-transfer properties [8] offer a number of advantages when dealing with pharmaceuticals. The products are generated at moderate temperatures and are completely free of residual solvent [9]; drug extraction, separation, synthesis, sterilization, and particle formation are all readily achievable [9]. The Gas Antisolvent (GAS) process is a dense gas process in which active pharmaceutical ingredients are precipitated by using carbon dioxide as antisolvent [10–12]. The API is dissolved in a suitable solvent and then the API-solvent system is rapidly pressurized with carbon dioxide. Carbon dioxide is miscible with most organic solvents but a poor solvent for many drugs. As the solvent mixes
with carbon dioxide, its volume expands and its solvent power reduces [13]. At a certain point the drug solution becomes supersaturated and precipitation occurs [14]. The precipitate is then washed with carbon dioxide and retrieved as a dry powder. Thus using the GAS process, micronisation and/or encapsulation are achieved in a single step; the amount of organic solvent used is reduced and the levels of residual solvent are extremely low [9,15,16]. The prodrug 5-fluorouracil (5-fluoro-2,4(1H,3H)pyrimidinedione or 5FU) is an antimetabolite used clinically for the treatment of colorectal, breast, and head and neck cancers [17]. It is administered intravenously or orally [18,19]. Conventional intravenous injection increases its bioavailability but treatment is often ineffective due to the drug not reaching the target site in sufficient amounts [20]. There are also side effects due to the cytotoxicity of 5FU to normal cells [21]. Oral administration is considered most effective in treating colon cancer as it is simple and allows targeted delivery in the colon. Halloysite clay nanotubes have recently emerged as a cheap, natural and biocompatible nanocarrier for drug delivery [22,23]. The aluminosilicate sheets are rolled into tubes, in a carpet-like manner, exposing silica on the outer surface and alumina on the inner one. Because of this geometry, the interior volume is rather small and rather inaccessible. Successful drug loading necessitates acidic etching to increase the inner diameter [22]. Acid activation is a common procedure in the clay industry [24] and the conditions are harsh. We propose that acid treatment can be avoided by using the GAS process because dense gas processes, in general, provide a better molecular access to the adsorption sites on the solid surface. The micronization of 5FU using a GAS process at 40 ◦ C and 100 bar or 150 bar has been reported by Kalantarian et al. [25]. The product, precipitated from a 50:50 methanol-dichloromethane mixture, showed uniform spherical morphology with an average particle size of 248 nm. Esfandiari & Ghoreishi [26] also micronized 5FU using a GAS process after dissolving the API in dimethyl sulfoxide (DMSO). Finer particles were produced when increasing the pressure (500 nm at 90 bar, 260 nm at 150 bar) or the flow rate of CO2 (480 nm at 1.6 ml/min, 360 nm at 2.4 ml/min). Larger particles were obtained when increasing the temperature (400 nm at 34 ◦ C, 600 nm at 46 ◦ C) or the initial 5FU concentration (310 nm at 20 mg/ml, 520 nm at 100 mg/ml). Cabezas et al. [27] employed a GAS process to micronize 5FU and also to impregnate polymer scaffolds with it. Poly (d, l-lactide) (PLA) and poly (d, l-lactide-co-glycolide) (PLGA) were used as encapsulating agents with a polymer:drug ratio of 4. A relatively high drug loading (17 %) was achieved with PLGA at 40 ◦ C and 180 bar. To the best of our knowledge loading of Halloysite nanotubes with an API using a GAS process has not been reported. In this work a version of the GAS technique using subcritical carbon dioxide is used to load 5-fluorouracil onto Halloysite nanotubes. The work examines the role of the drug/nanocarrier mass ratio. Drug loadings for chemically unmodified clay nanotubes are higher than those achieved by conventional methods. A quantitative distinction is made between tightly bound and loosely attached drug molecules. Drug release in aqueous media is pH-dependent and can be used in developing pH-triggered delivery systems.
2. Materials & methods Halloysite nanoclay (CAS Number 1332-58-7), 5-fluorouracil 99 %, HPLC (CAS Number: 51-21-8), and Methanol for HPLC ≥ 99.9 % (CAS Number: 67-56-1) were obtained from Sigma-Aldrich and used as received. The solubility of 5FU in methanol was determined to be 7.7 g/L at 25 ◦ C. Nearly saturated solutions were prepared by dissolving 70 mg of 5FU in 10 ml methanol. The Halloysite powder was added to the 5FU solution and stirred at 1000 rpm for 2 h at 25 ◦ C. The suspension was further sonicated for 30 min (ultra-
S. Harikrishnan, R. Sedev, C.C. Beh et al. / J. of Supercritical Fluids 159 (2020) 104756 Table 1 Amounts of drug, mtot , and nanocaly, mHNT , used and their mass ratio, R = mHNT /mtot . mtot [mg]
mHNT [mg]
R
70 70 70 70
70 105 210 420
1.0 1.5 3.0 6.0
sonic bath Unisonics, FXP-10 M) to ensure maximum nanoclay dispersion before use. Drug loading tests were performed using this suspension. The ratio of Halloysite nanoclay and 5FU in the initial suspension was varied. The amounts are listed in Table 1. 2.1. GAS process A schematic of the GAS rig used for loading the nanoclay with 5-fluorouracil is shown in Fig. 1a. Ten milliliters of a nanoclay and drug suspension were loaded into a 60 ml high-pressure cell with a viewing window (Jerguson Gauge R-32 Size 13). The Jerguson cell and relevant connections were immersed in a water bath and maintained at 25 ◦ C throughout the experiment. The cell was gradually pressurized by pumping CO2 from the bottom using a syringe pump (Teledyne ISCO 260D). The GAS process was performed in two stages: (i) Gas expansion of the suspension, and (ii) Flushing the precipitated product with gaseous CO2 . In the gas expanding stage, the suspension was pressurized to 120 bar using a CO2 supply flow rate of 20 ml/min to ensure rapid expansion and achieve high supersaturation. The suspension volume reached 5 times its initial volume at a pressure of 76 bar. At higher pressure the drug-loaded nanoclay precipitated (Fig. 1b–e) and gradually settled at the bottom of the chamber (Fig. 1d). The maximum pressure of 120 bar was maintained for a further 30 min to achieve complete precipitation. In the flushing stage, CO2 was fed from top of the Jerguson cell and released from the bottom at a constant flow rate of 10 ml/min for 12 min. Flushing the precipitate with CO2 removes the residual methanol very efficiently. The mass ratio of carbon dioxide to methanol was larger than 12 implying that solvent content was reduced down to tens of ppm or less, i.e. well below pharmacopeia set limits [16]. Finally, the Jerguson cell was depressurized and the precipitate was retrieved for further analysis. The precipitated product contained both fluorouracil-loaded nanoclay and co-precipitated fluorouracil. To remove the loosely attached drug, the precipitate was gently washed with 1 ml methanol for 1 min using an orbital shaker (Ratek OM1) and then centrifuged at 4500 rpm for 10 min. UV–vis spectrometry (Shimadzu, UV-1800) at 266 nm [28] was used to determine the concentration of 5FU in the supernatant and therefore the amount of free drug washed from the product. The filtered solids (washed product) were dried in an oven at 60 ◦ C overnight. The stability of the product was confirmed with FTIR spectroscopy. 2.2. Particle characterization Electron microscopy was used to image particle morphology and confirm the mean particle size. A small amount of the drug-loaded nanocarrier was suspended in methanol, placed on top of a standard aluminum stub (Ted Pella, 12.7 mm × 8 mm) and left to evaporate. The samples were coated with a 5 nm layer of platinum using (Sputterer coater, Cressington 208HR). Images were acquired using an SEM (Zeiss, Neon 40 EsB CrossBeam) at 5 kV using combined InLens and Secondary electron detectors. Back scatter detection was used for Energy Dispersion X-ray spectroscopy (EDS). Holey carbon grids (Ted Pella) were used for Transmission Electron Microscopy (TEM)
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imaging. The TEM imaging was performed on a FEI Talos FS200X at 10 kV input voltage. Dynamic light scattering experiments were performed with a Zetasizer Nano ZS (Malvern) equipped with a folded capillary zeta cell (DTS10170) to obtain the zeta potential of the Halloysite particles. Particles were dispersed in water with the pH adjusted by adding 0.1 M HCl or 0.1 M NaOH. The dispersions were sonicated for 30 min prior to the measurement.
2.3. FTIR characterization Infrared spectra of pristine Halloysite nanoclay, 5-fluorouracil and the drug-loaded nanocarrier were obtained with a Fourier Transform Infrared spectrometer (Perkin Elmer, Spectrum 400 FTIR). Dry samples were placed on the crystal and compressed with the diamond tipped probe. Four scans between 500 cm−1 and 4000 cm−1 were recorded with a resolution of 2 cm−1 . The spectrum was recorded and analyzed after background subtraction and attenuated total reflection (ATR) correction. The ATR correction ensures low noise level by compensating the shift in the absorption peak due to anomalous dispersion [29].
2.4. Thermogravimetric analysis The drug loading, L, of the washed product was determined by using TGA (Mettler Toledo, V1.03). A dry sample of about 20 mg was placed in a platinum crucible which was subjected to the following thermal treatment (under Argon flowing at 20 ml/min): increasing the temperature from 35 ◦ C to 100 ◦ C (at 25 ◦ C/min); 15 min at 100 ◦ C; increase from 100 ◦ C to 800 ◦ C at 10 ◦ C/min and, finally, hold for 5 min at 800 ◦ C. The peak decomposition temperatures of 5-fluorouracil (340 ◦ C) and Halloysite (510 ◦ C) were determined experimentally. These values are in agreement with published results [30–33]. For the drug loaded samples, temperature was maintained at 340 ◦ C for 30 min to ensure complete decomposition of the 5FU. The drug loading, L, was determined using the initial mass of the sample, m0 , and the mass after 5FU decomposition, m1 : L=
m0 − m1 m0
(1)
The encapsulation efficiency, E, was calculated as the mass of drug loaded, mA (= m0 – m1 ) to the total mass of drug used, mtot : E=
mA mtot
(2)
2.5. In vitro drug release In vitro release was performed using a modified Franz diffusion cell [34]. A Slide-A-Lyzer dialysis cassette (ThermoFisher, 2000 MWCO) was filled with 25 mg of product dispersed in 12 ml of PBS buffer mimicking the osmotic conditions inside the human body. The loaded cassette (donor compartment) was placed in a 500 ml beaker (acceptor compartment) filled with PBS buffer and stirred at 100 rpm to maintain uniform concentration on the outer side of the membrane. The release of 5-fluorouracil was monitored over several hours at 37.5 ◦ C. Aliquots of 4 ml were retrieved at regular time intervals and replaced with fresh medium to avoid depletion effects. The concentration of 5FU was determined using UV spectrometry and the characteristic peak at 266 nm. Measurements were performed every 20 min for the first 2 h and again after 24 h, to assess the complete (maximum) release.
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Fig. 1. (a) Schematic of the rig used to perform the GAS process. Precipitation of Halloysite clay loaded with 5-fluorouracil: (b) suspension loaded in the Jerguson cell at atmospheric pressure; (c) early expansion stage; (d) late expansion state; (e) final precipitated product.
3. Results and discussion During the GAS process, the 5-fluorouracil was loaded onto the nanoclay by adsorption. Previous studies, using kaolinite as a carrier, have shown that drug molecules adsorb mostly through hydrogen bonding and van der Waals interactions [35]. Halloysite has the same composition as kaolinite but has a tubular, rather than platy, morphology [36]. Therefore the same type of forces should be responsible for 5FU adsorption. The product of the GAS process contains clay nanoparticles with adsorbed drug molecules but also an excess of co-precipitated drug. In order to discriminate between the loosely attached drug and the adsorbed API, the product was gently washed with methanol. The outcome is presented in Fig. 2. The mass fraction of the loosely attached drug, ˛F (=mfree /mtot , where mfree is the mass of the free drug and mtot is the total mass of drug used), rapidly decreased as the mass ratio R (=mHNT /mtot , where mHNT is the mass of the Halloysite nanoclay) increased. Since the total amount of 5-fluorouracil, mtot , used in each test was kept constant, the amount of drug firmly adsorbed on the nanoclay carrier should increase with the number of adsorption sites on the Halloysite surface and, therefore, with the mass ratio R. At the same time the excess amount of 5-fluorouracil removable by methanol washing, mfree , should decrease as clearly seen in Fig. 2. Drug molecules can either adsorb or co-precipitate as free drug and the total amount is constant. The amount of free drug decreases with R because of the increasing amount of adsorbing nanoclay.
Fig. 2. Mass fraction of free drug, ˛F (=mfree /mtot , where mfree is the mass of the free drug and mtot is the total mass of drug used), washed with methanol as a function of the mass ratio R (=mHNT /mtot , where mHNT is the mass of the Halloysite nanoclay). Inset: Mass fraction of free drug as a function of the inverse mass ratio.
The inverse of R can be interpreted as an increasing amount of drug at a fixed amount of nanoclay. In that case, the mass fraction of free drug, ˛F , would be expected to increase proportionally to 1/R – as illustrated in the inset in Fig. 2.
S. Harikrishnan, R. Sedev, C.C. Beh et al. / J. of Supercritical Fluids 159 (2020) 104756
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Fig. 3. TEM images of Halloysite nanotubes: (a) pristine and (b) loaded with 5-fluorouracil. EDS spectra and SEM images of (c) pristine Halloysite nanotubes, (d) 5-fluorouracil, and (e) Halloysite nanotubes loaded with 5-fluorouracil. (f) FTIR Spectra of pristine Halloysite clay (HNT), 5-fluorouracil (5FU), and HNT loaded with 5FU.
The fact that loosely attached drug was washed away for all levels of R studied is consistent with the fact that an excess amount of drug was used. The molecular volume of 5-fluorouracil, V m , can be reliably estimated by using its molecular mass, M, and density, , as V 2/3 = m =M/(NA .). The area per molecule is then estimated as V m 2 28 Å . Given that the specific surface area of Halloysite is 64 m2 /g (BET method), we estimate that sub-monolayer adsorption of fluorouracil could only happen at mass ratios larger than R = 20. We emphasize the washing step as it discriminates between loosely and tightly attached drug molecules. It is sometimes ignored in the literature and reported drug loadings can be misleading. In general, the loosely attached drug does not have to be removed from the product as it may be useful in providing a burst release ahead of a slower release of the adsorbed drug. The tubular morphology of the nanoclay was confirmed by SEM/TEM imaging (Fig. 3a, b). The inner space (lumen) is clearly seen in the TEM image. The external diameter ranges from 50 nm to 80 nm and the lumen width is 10 nm–15 nm. The length of the nanotubes varies between about 500 nm and 2 m. These numbers are consistent with published reports [21,37]. It should be noted that images of individual clay nanotubes can be misleading as the quality and behavior of the total clay sample is determined by the average characteristics. The adsorption of 5FU was confirmed using Energy Dispersive X-Ray Spectroscopy (EDS) – Fig. 3c–e. The peaks of aluminum (1.5 keV), silicon (1.7 keV) and oxygen (0.5 keV) are a signature of the nanoclay (Fig. 3c) reflecting its chemical composition. Similarly, the peaks of nitrogen (0.4 keV) and fluorine (0.7 keV) reveal the elements present in the 5FU molecule (Fig. 3d). The carbon and platinum peaks are due to the carbon mounting tape and the thin platinum coating needed for correct SEM imaging. All peaks specific to the clay material and the drug were reliably detected in the precipitation product of the GAS process – Fig. 3e. Thus EDS imaging of the washed product confirmed that a certain
amount of 5-fluorouracil was adsorbed (firmly attached) onto the nanoclay carrier. This fact was further confirmed using FTIR spectroscopy. The IR spectra of the individual materials as well as the washed product of the GAS process are presented in Fig. 3f. The characteristic wavenumbers of the chemical bonds encountered in Halloysite are 910 cm−1 (Al−OH bending [38]), 1007 cm−1 (Si–O–Si symmetric stretching [38]), 1634 cm−1 (water deformation), 1724 cm−1 (stretching vibration of water interlayer), 3623 cm−1 and 3698 cm−1 (OH stretching of inner hydroxyls [39]). For 5-fluoruracil we have 795 cm−1 (surface hydroxyl translation [38]), 1242 cm−1 (CF stretching), 1630 cm−1 (CO stretching) and 3000 cm −1 (NH stretching). The presence of NH, carbonyl and CF stretching bands at 3000, 1630 and 1242 cm−1 in the GAS-treated nanoclay confirms the presence of 5-fluorouracil in the product. The absorbance peaks of Halloysite at 910 cm−1 and 1007 cm−1 , corresponding to Al−OH and in-plane SiOSi stretching, retained their position after the GAS process. Similarly, the inner OH stretching vibration corresponding to peaks at 3623 cm −1 and 3698 cm−1 is also retained indicating that the chemical signature of nanoclay is not altered during the GAS process. It has been previously shown that the structure of 5-fluorouracil is unaffected by the temperature and pressure conditions used during the GAS process [26]. Thus FTIR results confirm the loading of the Halloysite with 5-fluorouracil and the physical nature of the adsorption. The drug loading achieved with the GAS process is shown in Fig. 4a as a function of the mass ratio R. The loading, L, gradually decreases with the mass ratio of Halloysite to 5-fluorouracil. Since this precipitation product was washed with methanol, the loading represents the adsorbed (tightly attached) drug molecules. A treatment of the Halloysite (on its own) with the GAS process did not change its properties and, in particular, its specific surface area (64 m2 /g BET method). If adsorption is the loading mechanism and it is in turn decided
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Fig. 4. (a) Drug loading, L (mass of loaded drug relative to total mass of the precipitation product), of the washed product as a function of the mass ratio R (=mHNT /mtot , where mHNT is the mass of the Halloysite nanoclay and mtot is the total mass of drug used). Inset: Adsorption, (mass of drug adsorbed per mass nanoclay), as a function of the solution concentration of free drug, cF [g/L]. (b) Encapsulating efficiency, E (mass of loaded drug relative to the total mass of drug used), as a function of the mass ratio R.
by the inherent properties of the drug-carrier combination, then one may expect the loading to be independent of the mass ratio R. However, the fraction of the adsorbed drug is significant and this causes a depletion of the solution to which the nanoclay is exposed. The adsorption, (in g 5FU per g nanoclay), is a linear function of the concentration of the free drug in the solution, cF – inset in Fig. 4a. This trend is similar to the Henry adsorption isotherm which applies under equilibrium conditions and explains the decrease in loading with the mass ratio R. The encapsulation efficiency, E, as a function of the ratio R, is shown in Fig. 4b. It is practically constant and averages at about 40 %. This value could possibly be improved given that an excess amount of drug was used in these experiments – see Eq. (2). Rao et al. [21] achieved a better efficiency (E = 52 %) of encapsulating 5FU but their system was chemically different – the Halloysite nanotubes were embedded in a hydrogel. The observations made in this work lead to the schematic representation of the GAS process shown in Fig. 5a. In stage AB a drug solution of fixed concentration is prepared (red line in Fig. 5a). When the nanoclay is added at point B, some of the drug molecules adsorb on the clay surface. This creates a small depletion in the amount of 5FU in the dispersion. Stage BC is a spontaneous “mechanical” loading as practiced by many authors (e.g. [22]). The GAS process starts in earnest at point C when carbon dioxide is pumped into the system at a constant mass rate (the amount of CO2 is shown with a black line in Fig. 5a). In the stage CD, solvent expansion is small but the increase in diffusivity is significant and that increases the overall loading as drug molecules reach previously unavailable adsorption sites. As expansion proceeds beyond point D, there is further decrease in viscosity but the dominant effect is decline in solvent power and the ensuing precipitation of the
Fig. 5. (a) Qualitative sketch of the stages of the GAS process: amount of dissolved drug (5FU) – red line, amount of Halloysite clay (HNT) – blue line, and amount of carbon dioxide – black line. See text for description. (b) – Decrease of the relative viscosity, ε /0 (ε is viscosity at expansion ε and 0 is viscosity at expansion ε = 0), and – Decrease of the relative polarity parameter, *ε /*0 , (*ε is the polarity parameter at expansion ε and *0 is the polarity parameter at expansion ε = 0), as functions of the volumetric expansion, ε. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
drug. At point E, the precipitation is complete and the precipitate is then backwashed with carbon dioxide (EF). At point F the system is depressurized and the product is retrieved as a dry powder. At all times the amount of Halloysite is constant (blue line in Fig. 5a) and the nanotubes are uniformly dispersed throughout the liquid by the incoming CO2 flux. This scheme shows that the diminished viscosity of the gas-expanded solvent (stage CD) is the main source of improvement to the mobility of the drug molecules and therefore the final drug loading. In support of the above scheme we compare the viscosity (representing molecular mobility) and the polarity (representing solvent power) of the CO2 -expanded methanol in Fig. 5b. The data from Sih et al. [40] show a sharp decrease in viscosity, , at very low volumetric expansion, ε (the viscosity dependence was normalised by the viscosity value at ε = 0). Since diffusivity is inversely proportional to viscosity (Stokes-Einstein equation) a significant improvement in diffusive transport is achieved in the CD stage of the GAS process. The polarity of the gas-expanded liquid determines the solubility of the 5FU and we discuss the KamletTaft parameters (˛, ˇ and *) reported by Wyatt et al. [41] for methanol−CO2 mixtures. The hydrogen bond donor parameter (˛ = 0.98 ± 0.03) and the hydrogen bond acceptor parameter (ˇ = 0.74 ± 0.05) are practically constant over the whole range of CO2 mole fractions [41]. Most of the decrease in polarity of the gas-expanded solvent with increasing CO2 concentration is due to decreasing polarity parameter, *. Its value (normalized by the value of the pure methanol) is graphed in Fig. 5b. Initially, * decreases slower than the viscosity and this corresponds to stage CD in Fig. 5a. With further expansion (beyond ε = 0.7), the polarity parameter
S. Harikrishnan, R. Sedev, C.C. Beh et al. / J. of Supercritical Fluids 159 (2020) 104756
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Fig. 6. Cumulative drug release, RD (ratio of amount of drug released and total amount of loaded drug), from the 5FU-loaded HNT in aqueous environment as a function of time at pH = 7.4 (䊐) and pH = 1.2 ().
decreases at a faster rate than viscosity and precipitation becomes dominant. This correspond to the stage DE in Fig. 5a. The maximum drug loading achieved in the present work using the GAS process is about 45 % (Fig. 4a). Such a high loading has not been matched by other methods used to load 5FU onto various carriers. For instance: 8 % on montmonrillonite [42], 7 % on porous clay heterostructures [43] and 22 % on kaolinite [44], when using the soaking method, and 12%–28% on chitosan via solvent evaporation emulsification [20]. A higher loading of 5FU (55 %) has been reported [44] but the kaolinite carrier was methoxylated prior to drug loading. Thus we conclude that the GAS process provides the highest drug loading onto natural (chemically unaltered) Halloysite nanotubes. The benefit of a drug carrier is largely determined by the type of drug release that it enables. The gastrointestinal tract has a varying pH from about 1.2 in the stomach to 7.4 in the intestine. It is therefore of key interest to examine drug release at these two pH values. The time release profiles of 5FU from the washed product, in simulated gastric and intestinal pH, are presented in Fig. 6. The release at intestinal pH is faster (about 80 % after the first 3 h) and practically complete (RD = 1) after 24 h. At gastric pH the release is slower (25 % after the first 3 h) and even after 24 h remains just under 30 %. The release profiles shown in Fig. 6 are well described with an exponential increase of RD in time using only two parameters RD ∞ and (RD ∞ is RD at t=∞, i.e. maximum release, and is characteristic time and more precisely the point of RD = 1 – e–1 ≈ 0.63):
Fig. 7. Dependence of (a) the final cumulative release, RD ∞ , at pH = 7.4 (䊐) and pH = 1.2 (), and (b) characteristic time, [min], at pH = 7.4 (䊐) and pH = 1.2 (), on the mass ratio R (=mHNT /mtot , where mHNT is the mass of the Halloysite nanoclay and mtot is the total mass of drug used).
(3)
Fig. 8. Electrokinetic potential, [mV], of Halloysite nanoclay dispersed in water (䊐 – Ref. [48]; 䊉 – this work) as a function of pH of the aqueous solution. The pK of 5-fluorouracil is shown with a vertical dashed line.
Eq. (3) describes a first-order kinetics of formation which is encountered in many practical situations including the release of a drug loaded onto a carrier [45]. The variations in maximum release, RD ∞ , and characteristic time, , with the mass ratio, R, are graphed in Fig. 7. At the higher pH, the maximum release is consistently high (above 90 %). At the lower pH, it steadily increases with R (Fig. 7a). The characteristic time does not depend on pH (Fig. 7b) and, within the scatter, remains approximately constant at a value of about 2 h. The characteristic time of 5FU release is in the range between gastric half emptying (1.2 ± 0.3) h and time to reach the colon (2.8 ± 1.5) h [46]. Therefore the 5FU-HNT drug-carrier combination is relevant to the development of oral delivery systems. The clear similarity between the two release profiles shown in Fig. 6 together with the fact that the characteristic time did not change with pH or mass ratio (Fig. 7b) suggest that the mechanism
of desorption and dissolution is the same. Given that 5-fluorouracil is a water soluble drug (11.1 g/L [47]) we attribute the complete release at pH = 7.4 (Fig. 8) to electrostatic interactions between the 5FU molecules and the nanoclay. At pH = 2 the zeta potential of Halloysite particles is practically zero [48] – Fig. 8. Therefore the nanoclay surface carries a very small surface charge, i.e. it is practically neutral. A similar situation is observed with both silica [49] (the outer surface of the Halloysite nanotubes is mostly composed of silica) and with kaolinite [50] (a clay very similar to Halloysite). At the higher pH = 7.4, the zeta potential is −40 mV (Fig. 8) and therefore the nanoclay surface is charged negatively. The 5-flurouracil behaves like a weak acid [51], i.e. at low pH the molecule is neutral and at high pH it deprotonates to become an anion. The pK of 5FU is 8.0 [52] and therefore at pH = 7.4 a significant portion of the molecules (exactly 50 % at pK) should be in their
∞
RD = RD
1−e
−t/
8
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anionic form. A recent NMR study concluded that 5FU is actually completely ionized at physiological conditions [51]. At such pH, the electrostatic repulsion between the negatively charged nanoclay surface and the 5FU anions ensures complete drug release. At acidic pH, electrostatic interactions are insignificant and drug release is only partial. The dissolution profiles of pure 5-fluorouracil at gastric and colonic pH are very similar. The release, RD , after 6 h is 98 % at pH = 7.4 and 88 % at pH = 1.2. This difference reflects the increased ionization of the 5FU molecule under alkaline conditions [53]. Both profiles match well the dissolution profile of HNT-5FU at pH = 7.4 (Fig. 6). This confirms the dominant role of electrostatic interactions in the formation of the 5FU-HNT complex. 4. Conclusion The Gas Antisolvent Process (GAS) can be used to load Halloysite nanotubes with 5-fluorouracil. For chemically untreated nanotubes, the drug loading (43 %) is better than what was achieved by other methods. The fraction of tightly attached drug (via physical adsorption) is compared quantitatively to the fraction of loosely attached (co-precipitated) drug. The later can be used to deliver a burst release. The mass ratio between clay nanotubes and 5fluorouracil affects both the drug loading due to adsorption and the ratio of loose to tightly attached drug molecules. The GAS process proceeds in two key stages and during the first one improved mass transport creates an increased drug loading. On the whole it is a one-pot green process with a low consumption of solvent and high-purity of the precipitated product. The strongly attached drug is released completely at physiological pH but only up to 30 % in acidic pH. In both cases the release follows first-order kinetics with a characteristic time of about 2 h. The differences are attributed to variations in electrostatic interactions with pH. We have described an efficient method to load Halloysite with 5-fluorouracil in a single step. The mass ratio R can be used to tailor the amounts of readily available and sustainably released drug. Declaration of Competing Interest
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Contents lists available at ScienceDirect
The Journal of Supercritical Fluids journal homepage: www.elsevier.com/locate/supflu
Loading of 5-fluorouracil onto Halloysite nanotubes for targeted drug delivery using a subcritical gas antisolvent process (GAS) Sekar Harikrishnan a , Rossen Sedev a,b,∗ , Chau Chun Beh a , Craig Priest b , Neil R. Foster a,∗ a b
Western Australian School of Mines: Minerals, Energy & Chemical Engineering, Curtin University, Perth, WA 6102, Australia Future Industries Institute, University of South Australia, Adelaide, SA 5095, Australia
h i g h l i g h t s
g r a p h i c a l
a b s t r a c t
• Halloysite • • • •
clay nanotubes are biocompatible and efficient drug nanocarriers. A gas antisolvent process with subcritical CO2 achieves a high drug loading (43 %). Distinction between tightly bound (adsorbed) and loosely bound (coprecipitated) drug. 5-Fluorouracil release is complete at pH = 7.4 but only 30 % in pH = 1.2. 5-Fluorouracil release follows firstorder kinetics with a characteristic time of 2 h.
a r t i c l e
i n f o
Article history: Received 12 September 2019 Received in revised form 13 January 2020 Accepted 14 January 2020 Available online 21 January 2020 Keywords: Gas antisolvent process Halloysite nanotubes 5-fluorouracil Drug loading Drug release
a b s t r a c t Halloysite clay nanotubes are efficient biocompatible nanocarriers for pharmaceutical products. A gas antisolvent (GAS) process employing subcritical carbon dioxide delivers a product with high loading (43 %) and free of residual solvent. An anticancer agent, 5-fluorouracil, was loaded on nanoclay particles. A quantitative distinction is made between tightly-bound (adsorbed) and loosely-bound (co-precipitated) drug. The physical stages of the dense gas process are clearly outlined. The strongly attached drug is released completely at pH = 7.4 but only up to 30 % in pH = 1.2. The release follows first-order kinetics with a characteristic time of 2 h. The mass ratio of clay nanotubes to 5-fluorouracil affects the loading with adsorbed drug as well as the ratio of loose to tightly attached drug molecules. The system Halloysite5-fluorouracil can be implemented in drug delivery systems to achieve a desired release profile. The GAS process is a one-pot green process with a low solvent consumption and high-purity product. © 2020 Elsevier B.V. All rights reserved.
Abbreviations: ATR, attenuated total reflection; API, active pharmaceutical ingredient; EDS, energy dispersive X-Ray spectroscopy; FTIR, fourier-transform infrared spectroscopy; 5FU, 5-fluorouracil; GAS, gas antisolvent process; HNT, halloysite clay nanotubes; HPLC, high performance liquid chromatography; MWCO, molecular weight cut-off; NMR, nuclear magnetic resonance; PBS, phosphate-buffered saline; PLA, poly(d,l-lactide); PLGA, poly(d,l-lactide-co-glycolide); SEM, scanning electron microscopy; TEM, transmission electron microscopy; TGA, thermal gravimetric analysis; UV-vis, ultraviolet-visible spectroscopy. ∗ Corresponding authors at: Western Australian School of Mines: Minerals, Energy & Chemical Engineering, Curtin University, Perth, WA 6102, Australia. E-mail addresses: [email protected] (R. Sedev), [email protected] (N.R. Foster). https://doi.org/10.1016/j.supflu.2020.104756 0896-8446/© 2020 Elsevier B.V. All rights reserved.
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Nomenclature m0 m1 mA mfree mtot mHNT E L M NA R RD RD ∞ Vm ˛ ˛F ˇ ε *
mass of washed precipitate before heat treatment, kg mass of washed precipitate after heat treatment, kg mass of drug loaded (= m0 – m1 ), kg mass of loosely attached drug, kg total mass of API used in the experiment, kg total mass of HNT used in the experiment, kg drug encapsulation, = (m0 – m1 )/mtot drug loading, = (m0 – m1 )/m0 molecular mass, kg/mol Avogadro constant, mol−1 mass ratio of HNT and API (=mHNT /mtot ) cumulative release (mass of drug released over mass of drug loaded) final cumulative release (RD at t = ∞) molecular volume, m3 /mol hydrogen bond donor parameter (Kamlet-Taft parameter) mass ratio of loosely attached drug and total drug (=mfree /mtot ) hydrogen bond acceptor parameter (Kamlet-Taft parameter) relative volumetric expansion, (V1 -V0 )/V0 ; V0 is initial volume, V1 is expanded volume polarizability (Kamlet-Taft parameter) viscosity, Pa.s density, kg/m3 characteristic time of release, s
1. Introduction Even the most medically suitable active pharmaceutical ingredient (API), if administered directly, is likely to distribute unevenly throughout the body and therefore influence both target and healthy cells. Thus, inefficient delivery reduces the desired therapeutic effect and also increases the risk of adversely affecting normal cells. The use of drug carriers (as vehicles loaded with the API) aims to engineer higher API concentrations where needed and lower concentrations elsewhere. Nanocarriers carry relatively large amounts of drug that can be transported safely to a target destination and released locally in the most effective manner. They can travel naturally, e.g. down the gastrointestinal tract, or be guided externally, e.g. by using a magnetic field. The size and surface properties can be manipulated to regulate their ability to penetrate the tissues in the body and target specific locations. Release can be triggered by a change in pH or temperature but also using ultrasound or near-infrared laser irradiation. Nanocarriers are a subject of intense research [1–6]. A common problem in the design of nanocarriers for drug delivery is that drug loadings are rather low (typically below 10 % [7]). Dense gases with their liquid-like solvation power and gas-like mass-transfer properties [8] offer a number of advantages when dealing with pharmaceuticals. The products are generated at moderate temperatures and are completely free of residual solvent [9]; drug extraction, separation, synthesis, sterilization, and particle formation are all readily achievable [9]. The Gas Antisolvent (GAS) process is a dense gas process in which active pharmaceutical ingredients are precipitated by using carbon dioxide as antisolvent [10–12]. The API is dissolved in a suitable solvent and then the API-solvent system is rapidly pressurized with carbon dioxide. Carbon dioxide is miscible with most organic solvents but a poor solvent for many drugs. As the solvent mixes
with carbon dioxide, its volume expands and its solvent power reduces [13]. At a certain point the drug solution becomes supersaturated and precipitation occurs [14]. The precipitate is then washed with carbon dioxide and retrieved as a dry powder. Thus using the GAS process, micronisation and/or encapsulation are achieved in a single step; the amount of organic solvent used is reduced and the levels of residual solvent are extremely low [9,15,16]. The prodrug 5-fluorouracil (5-fluoro-2,4(1H,3H)pyrimidinedione or 5FU) is an antimetabolite used clinically for the treatment of colorectal, breast, and head and neck cancers [17]. It is administered intravenously or orally [18,19]. Conventional intravenous injection increases its bioavailability but treatment is often ineffective due to the drug not reaching the target site in sufficient amounts [20]. There are also side effects due to the cytotoxicity of 5FU to normal cells [21]. Oral administration is considered most effective in treating colon cancer as it is simple and allows targeted delivery in the colon. Halloysite clay nanotubes have recently emerged as a cheap, natural and biocompatible nanocarrier for drug delivery [22,23]. The aluminosilicate sheets are rolled into tubes, in a carpet-like manner, exposing silica on the outer surface and alumina on the inner one. Because of this geometry, the interior volume is rather small and rather inaccessible. Successful drug loading necessitates acidic etching to increase the inner diameter [22]. Acid activation is a common procedure in the clay industry [24] and the conditions are harsh. We propose that acid treatment can be avoided by using the GAS process because dense gas processes, in general, provide a better molecular access to the adsorption sites on the solid surface. The micronization of 5FU using a GAS process at 40 ◦ C and 100 bar or 150 bar has been reported by Kalantarian et al. [25]. The product, precipitated from a 50:50 methanol-dichloromethane mixture, showed uniform spherical morphology with an average particle size of 248 nm. Esfandiari & Ghoreishi [26] also micronized 5FU using a GAS process after dissolving the API in dimethyl sulfoxide (DMSO). Finer particles were produced when increasing the pressure (500 nm at 90 bar, 260 nm at 150 bar) or the flow rate of CO2 (480 nm at 1.6 ml/min, 360 nm at 2.4 ml/min). Larger particles were obtained when increasing the temperature (400 nm at 34 ◦ C, 600 nm at 46 ◦ C) or the initial 5FU concentration (310 nm at 20 mg/ml, 520 nm at 100 mg/ml). Cabezas et al. [27] employed a GAS process to micronize 5FU and also to impregnate polymer scaffolds with it. Poly (d, l-lactide) (PLA) and poly (d, l-lactide-co-glycolide) (PLGA) were used as encapsulating agents with a polymer:drug ratio of 4. A relatively high drug loading (17 %) was achieved with PLGA at 40 ◦ C and 180 bar. To the best of our knowledge loading of Halloysite nanotubes with an API using a GAS process has not been reported. In this work a version of the GAS technique using subcritical carbon dioxide is used to load 5-fluorouracil onto Halloysite nanotubes. The work examines the role of the drug/nanocarrier mass ratio. Drug loadings for chemically unmodified clay nanotubes are higher than those achieved by conventional methods. A quantitative distinction is made between tightly bound and loosely attached drug molecules. Drug release in aqueous media is pH-dependent and can be used in developing pH-triggered delivery systems.
2. Materials & methods Halloysite nanoclay (CAS Number 1332-58-7), 5-fluorouracil 99 %, HPLC (CAS Number: 51-21-8), and Methanol for HPLC ≥ 99.9 % (CAS Number: 67-56-1) were obtained from Sigma-Aldrich and used as received. The solubility of 5FU in methanol was determined to be 7.7 g/L at 25 ◦ C. Nearly saturated solutions were prepared by dissolving 70 mg of 5FU in 10 ml methanol. The Halloysite powder was added to the 5FU solution and stirred at 1000 rpm for 2 h at 25 ◦ C. The suspension was further sonicated for 30 min (ultra-
S. Harikrishnan, R. Sedev, C.C. Beh et al. / J. of Supercritical Fluids 159 (2020) 104756 Table 1 Amounts of drug, mtot , and nanocaly, mHNT , used and their mass ratio, R = mHNT /mtot . mtot [mg]
mHNT [mg]
R
70 70 70 70
70 105 210 420
1.0 1.5 3.0 6.0
sonic bath Unisonics, FXP-10 M) to ensure maximum nanoclay dispersion before use. Drug loading tests were performed using this suspension. The ratio of Halloysite nanoclay and 5FU in the initial suspension was varied. The amounts are listed in Table 1. 2.1. GAS process A schematic of the GAS rig used for loading the nanoclay with 5-fluorouracil is shown in Fig. 1a. Ten milliliters of a nanoclay and drug suspension were loaded into a 60 ml high-pressure cell with a viewing window (Jerguson Gauge R-32 Size 13). The Jerguson cell and relevant connections were immersed in a water bath and maintained at 25 ◦ C throughout the experiment. The cell was gradually pressurized by pumping CO2 from the bottom using a syringe pump (Teledyne ISCO 260D). The GAS process was performed in two stages: (i) Gas expansion of the suspension, and (ii) Flushing the precipitated product with gaseous CO2 . In the gas expanding stage, the suspension was pressurized to 120 bar using a CO2 supply flow rate of 20 ml/min to ensure rapid expansion and achieve high supersaturation. The suspension volume reached 5 times its initial volume at a pressure of 76 bar. At higher pressure the drug-loaded nanoclay precipitated (Fig. 1b–e) and gradually settled at the bottom of the chamber (Fig. 1d). The maximum pressure of 120 bar was maintained for a further 30 min to achieve complete precipitation. In the flushing stage, CO2 was fed from top of the Jerguson cell and released from the bottom at a constant flow rate of 10 ml/min for 12 min. Flushing the precipitate with CO2 removes the residual methanol very efficiently. The mass ratio of carbon dioxide to methanol was larger than 12 implying that solvent content was reduced down to tens of ppm or less, i.e. well below pharmacopeia set limits [16]. Finally, the Jerguson cell was depressurized and the precipitate was retrieved for further analysis. The precipitated product contained both fluorouracil-loaded nanoclay and co-precipitated fluorouracil. To remove the loosely attached drug, the precipitate was gently washed with 1 ml methanol for 1 min using an orbital shaker (Ratek OM1) and then centrifuged at 4500 rpm for 10 min. UV–vis spectrometry (Shimadzu, UV-1800) at 266 nm [28] was used to determine the concentration of 5FU in the supernatant and therefore the amount of free drug washed from the product. The filtered solids (washed product) were dried in an oven at 60 ◦ C overnight. The stability of the product was confirmed with FTIR spectroscopy. 2.2. Particle characterization Electron microscopy was used to image particle morphology and confirm the mean particle size. A small amount of the drug-loaded nanocarrier was suspended in methanol, placed on top of a standard aluminum stub (Ted Pella, 12.7 mm × 8 mm) and left to evaporate. The samples were coated with a 5 nm layer of platinum using (Sputterer coater, Cressington 208HR). Images were acquired using an SEM (Zeiss, Neon 40 EsB CrossBeam) at 5 kV using combined InLens and Secondary electron detectors. Back scatter detection was used for Energy Dispersion X-ray spectroscopy (EDS). Holey carbon grids (Ted Pella) were used for Transmission Electron Microscopy (TEM)
3
imaging. The TEM imaging was performed on a FEI Talos FS200X at 10 kV input voltage. Dynamic light scattering experiments were performed with a Zetasizer Nano ZS (Malvern) equipped with a folded capillary zeta cell (DTS10170) to obtain the zeta potential of the Halloysite particles. Particles were dispersed in water with the pH adjusted by adding 0.1 M HCl or 0.1 M NaOH. The dispersions were sonicated for 30 min prior to the measurement.
2.3. FTIR characterization Infrared spectra of pristine Halloysite nanoclay, 5-fluorouracil and the drug-loaded nanocarrier were obtained with a Fourier Transform Infrared spectrometer (Perkin Elmer, Spectrum 400 FTIR). Dry samples were placed on the crystal and compressed with the diamond tipped probe. Four scans between 500 cm−1 and 4000 cm−1 were recorded with a resolution of 2 cm−1 . The spectrum was recorded and analyzed after background subtraction and attenuated total reflection (ATR) correction. The ATR correction ensures low noise level by compensating the shift in the absorption peak due to anomalous dispersion [29].
2.4. Thermogravimetric analysis The drug loading, L, of the washed product was determined by using TGA (Mettler Toledo, V1.03). A dry sample of about 20 mg was placed in a platinum crucible which was subjected to the following thermal treatment (under Argon flowing at 20 ml/min): increasing the temperature from 35 ◦ C to 100 ◦ C (at 25 ◦ C/min); 15 min at 100 ◦ C; increase from 100 ◦ C to 800 ◦ C at 10 ◦ C/min and, finally, hold for 5 min at 800 ◦ C. The peak decomposition temperatures of 5-fluorouracil (340 ◦ C) and Halloysite (510 ◦ C) were determined experimentally. These values are in agreement with published results [30–33]. For the drug loaded samples, temperature was maintained at 340 ◦ C for 30 min to ensure complete decomposition of the 5FU. The drug loading, L, was determined using the initial mass of the sample, m0 , and the mass after 5FU decomposition, m1 : L=
m0 − m1 m0
(1)
The encapsulation efficiency, E, was calculated as the mass of drug loaded, mA (= m0 – m1 ) to the total mass of drug used, mtot : E=
mA mtot
(2)
2.5. In vitro drug release In vitro release was performed using a modified Franz diffusion cell [34]. A Slide-A-Lyzer dialysis cassette (ThermoFisher, 2000 MWCO) was filled with 25 mg of product dispersed in 12 ml of PBS buffer mimicking the osmotic conditions inside the human body. The loaded cassette (donor compartment) was placed in a 500 ml beaker (acceptor compartment) filled with PBS buffer and stirred at 100 rpm to maintain uniform concentration on the outer side of the membrane. The release of 5-fluorouracil was monitored over several hours at 37.5 ◦ C. Aliquots of 4 ml were retrieved at regular time intervals and replaced with fresh medium to avoid depletion effects. The concentration of 5FU was determined using UV spectrometry and the characteristic peak at 266 nm. Measurements were performed every 20 min for the first 2 h and again after 24 h, to assess the complete (maximum) release.
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Fig. 1. (a) Schematic of the rig used to perform the GAS process. Precipitation of Halloysite clay loaded with 5-fluorouracil: (b) suspension loaded in the Jerguson cell at atmospheric pressure; (c) early expansion stage; (d) late expansion state; (e) final precipitated product.
3. Results and discussion During the GAS process, the 5-fluorouracil was loaded onto the nanoclay by adsorption. Previous studies, using kaolinite as a carrier, have shown that drug molecules adsorb mostly through hydrogen bonding and van der Waals interactions [35]. Halloysite has the same composition as kaolinite but has a tubular, rather than platy, morphology [36]. Therefore the same type of forces should be responsible for 5FU adsorption. The product of the GAS process contains clay nanoparticles with adsorbed drug molecules but also an excess of co-precipitated drug. In order to discriminate between the loosely attached drug and the adsorbed API, the product was gently washed with methanol. The outcome is presented in Fig. 2. The mass fraction of the loosely attached drug, ˛F (=mfree /mtot , where mfree is the mass of the free drug and mtot is the total mass of drug used), rapidly decreased as the mass ratio R (=mHNT /mtot , where mHNT is the mass of the Halloysite nanoclay) increased. Since the total amount of 5-fluorouracil, mtot , used in each test was kept constant, the amount of drug firmly adsorbed on the nanoclay carrier should increase with the number of adsorption sites on the Halloysite surface and, therefore, with the mass ratio R. At the same time the excess amount of 5-fluorouracil removable by methanol washing, mfree , should decrease as clearly seen in Fig. 2. Drug molecules can either adsorb or co-precipitate as free drug and the total amount is constant. The amount of free drug decreases with R because of the increasing amount of adsorbing nanoclay.
Fig. 2. Mass fraction of free drug, ˛F (=mfree /mtot , where mfree is the mass of the free drug and mtot is the total mass of drug used), washed with methanol as a function of the mass ratio R (=mHNT /mtot , where mHNT is the mass of the Halloysite nanoclay). Inset: Mass fraction of free drug as a function of the inverse mass ratio.
The inverse of R can be interpreted as an increasing amount of drug at a fixed amount of nanoclay. In that case, the mass fraction of free drug, ˛F , would be expected to increase proportionally to 1/R – as illustrated in the inset in Fig. 2.
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5
Fig. 3. TEM images of Halloysite nanotubes: (a) pristine and (b) loaded with 5-fluorouracil. EDS spectra and SEM images of (c) pristine Halloysite nanotubes, (d) 5-fluorouracil, and (e) Halloysite nanotubes loaded with 5-fluorouracil. (f) FTIR Spectra of pristine Halloysite clay (HNT), 5-fluorouracil (5FU), and HNT loaded with 5FU.
The fact that loosely attached drug was washed away for all levels of R studied is consistent with the fact that an excess amount of drug was used. The molecular volume of 5-fluorouracil, V m , can be reliably estimated by using its molecular mass, M, and density, , as V 2/3 = m =M/(NA .). The area per molecule is then estimated as V m 2 28 Å . Given that the specific surface area of Halloysite is 64 m2 /g (BET method), we estimate that sub-monolayer adsorption of fluorouracil could only happen at mass ratios larger than R = 20. We emphasize the washing step as it discriminates between loosely and tightly attached drug molecules. It is sometimes ignored in the literature and reported drug loadings can be misleading. In general, the loosely attached drug does not have to be removed from the product as it may be useful in providing a burst release ahead of a slower release of the adsorbed drug. The tubular morphology of the nanoclay was confirmed by SEM/TEM imaging (Fig. 3a, b). The inner space (lumen) is clearly seen in the TEM image. The external diameter ranges from 50 nm to 80 nm and the lumen width is 10 nm–15 nm. The length of the nanotubes varies between about 500 nm and 2 m. These numbers are consistent with published reports [21,37]. It should be noted that images of individual clay nanotubes can be misleading as the quality and behavior of the total clay sample is determined by the average characteristics. The adsorption of 5FU was confirmed using Energy Dispersive X-Ray Spectroscopy (EDS) – Fig. 3c–e. The peaks of aluminum (1.5 keV), silicon (1.7 keV) and oxygen (0.5 keV) are a signature of the nanoclay (Fig. 3c) reflecting its chemical composition. Similarly, the peaks of nitrogen (0.4 keV) and fluorine (0.7 keV) reveal the elements present in the 5FU molecule (Fig. 3d). The carbon and platinum peaks are due to the carbon mounting tape and the thin platinum coating needed for correct SEM imaging. All peaks specific to the clay material and the drug were reliably detected in the precipitation product of the GAS process – Fig. 3e. Thus EDS imaging of the washed product confirmed that a certain
amount of 5-fluorouracil was adsorbed (firmly attached) onto the nanoclay carrier. This fact was further confirmed using FTIR spectroscopy. The IR spectra of the individual materials as well as the washed product of the GAS process are presented in Fig. 3f. The characteristic wavenumbers of the chemical bonds encountered in Halloysite are 910 cm−1 (Al−OH bending [38]), 1007 cm−1 (Si–O–Si symmetric stretching [38]), 1634 cm−1 (water deformation), 1724 cm−1 (stretching vibration of water interlayer), 3623 cm−1 and 3698 cm−1 (OH stretching of inner hydroxyls [39]). For 5-fluoruracil we have 795 cm−1 (surface hydroxyl translation [38]), 1242 cm−1 (CF stretching), 1630 cm−1 (CO stretching) and 3000 cm −1 (NH stretching). The presence of NH, carbonyl and CF stretching bands at 3000, 1630 and 1242 cm−1 in the GAS-treated nanoclay confirms the presence of 5-fluorouracil in the product. The absorbance peaks of Halloysite at 910 cm−1 and 1007 cm−1 , corresponding to Al−OH and in-plane SiOSi stretching, retained their position after the GAS process. Similarly, the inner OH stretching vibration corresponding to peaks at 3623 cm −1 and 3698 cm−1 is also retained indicating that the chemical signature of nanoclay is not altered during the GAS process. It has been previously shown that the structure of 5-fluorouracil is unaffected by the temperature and pressure conditions used during the GAS process [26]. Thus FTIR results confirm the loading of the Halloysite with 5-fluorouracil and the physical nature of the adsorption. The drug loading achieved with the GAS process is shown in Fig. 4a as a function of the mass ratio R. The loading, L, gradually decreases with the mass ratio of Halloysite to 5-fluorouracil. Since this precipitation product was washed with methanol, the loading represents the adsorbed (tightly attached) drug molecules. A treatment of the Halloysite (on its own) with the GAS process did not change its properties and, in particular, its specific surface area (64 m2 /g BET method). If adsorption is the loading mechanism and it is in turn decided
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Fig. 4. (a) Drug loading, L (mass of loaded drug relative to total mass of the precipitation product), of the washed product as a function of the mass ratio R (=mHNT /mtot , where mHNT is the mass of the Halloysite nanoclay and mtot is the total mass of drug used). Inset: Adsorption, (mass of drug adsorbed per mass nanoclay), as a function of the solution concentration of free drug, cF [g/L]. (b) Encapsulating efficiency, E (mass of loaded drug relative to the total mass of drug used), as a function of the mass ratio R.
by the inherent properties of the drug-carrier combination, then one may expect the loading to be independent of the mass ratio R. However, the fraction of the adsorbed drug is significant and this causes a depletion of the solution to which the nanoclay is exposed. The adsorption, (in g 5FU per g nanoclay), is a linear function of the concentration of the free drug in the solution, cF – inset in Fig. 4a. This trend is similar to the Henry adsorption isotherm which applies under equilibrium conditions and explains the decrease in loading with the mass ratio R. The encapsulation efficiency, E, as a function of the ratio R, is shown in Fig. 4b. It is practically constant and averages at about 40 %. This value could possibly be improved given that an excess amount of drug was used in these experiments – see Eq. (2). Rao et al. [21] achieved a better efficiency (E = 52 %) of encapsulating 5FU but their system was chemically different – the Halloysite nanotubes were embedded in a hydrogel. The observations made in this work lead to the schematic representation of the GAS process shown in Fig. 5a. In stage AB a drug solution of fixed concentration is prepared (red line in Fig. 5a). When the nanoclay is added at point B, some of the drug molecules adsorb on the clay surface. This creates a small depletion in the amount of 5FU in the dispersion. Stage BC is a spontaneous “mechanical” loading as practiced by many authors (e.g. [22]). The GAS process starts in earnest at point C when carbon dioxide is pumped into the system at a constant mass rate (the amount of CO2 is shown with a black line in Fig. 5a). In the stage CD, solvent expansion is small but the increase in diffusivity is significant and that increases the overall loading as drug molecules reach previously unavailable adsorption sites. As expansion proceeds beyond point D, there is further decrease in viscosity but the dominant effect is decline in solvent power and the ensuing precipitation of the
Fig. 5. (a) Qualitative sketch of the stages of the GAS process: amount of dissolved drug (5FU) – red line, amount of Halloysite clay (HNT) – blue line, and amount of carbon dioxide – black line. See text for description. (b) – Decrease of the relative viscosity, ε /0 (ε is viscosity at expansion ε and 0 is viscosity at expansion ε = 0), and – Decrease of the relative polarity parameter, *ε /*0 , (*ε is the polarity parameter at expansion ε and *0 is the polarity parameter at expansion ε = 0), as functions of the volumetric expansion, ε. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
drug. At point E, the precipitation is complete and the precipitate is then backwashed with carbon dioxide (EF). At point F the system is depressurized and the product is retrieved as a dry powder. At all times the amount of Halloysite is constant (blue line in Fig. 5a) and the nanotubes are uniformly dispersed throughout the liquid by the incoming CO2 flux. This scheme shows that the diminished viscosity of the gas-expanded solvent (stage CD) is the main source of improvement to the mobility of the drug molecules and therefore the final drug loading. In support of the above scheme we compare the viscosity (representing molecular mobility) and the polarity (representing solvent power) of the CO2 -expanded methanol in Fig. 5b. The data from Sih et al. [40] show a sharp decrease in viscosity, , at very low volumetric expansion, ε (the viscosity dependence was normalised by the viscosity value at ε = 0). Since diffusivity is inversely proportional to viscosity (Stokes-Einstein equation) a significant improvement in diffusive transport is achieved in the CD stage of the GAS process. The polarity of the gas-expanded liquid determines the solubility of the 5FU and we discuss the KamletTaft parameters (˛, ˇ and *) reported by Wyatt et al. [41] for methanol−CO2 mixtures. The hydrogen bond donor parameter (˛ = 0.98 ± 0.03) and the hydrogen bond acceptor parameter (ˇ = 0.74 ± 0.05) are practically constant over the whole range of CO2 mole fractions [41]. Most of the decrease in polarity of the gas-expanded solvent with increasing CO2 concentration is due to decreasing polarity parameter, *. Its value (normalized by the value of the pure methanol) is graphed in Fig. 5b. Initially, * decreases slower than the viscosity and this corresponds to stage CD in Fig. 5a. With further expansion (beyond ε = 0.7), the polarity parameter
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7
Fig. 6. Cumulative drug release, RD (ratio of amount of drug released and total amount of loaded drug), from the 5FU-loaded HNT in aqueous environment as a function of time at pH = 7.4 (䊐) and pH = 1.2 ().
decreases at a faster rate than viscosity and precipitation becomes dominant. This correspond to the stage DE in Fig. 5a. The maximum drug loading achieved in the present work using the GAS process is about 45 % (Fig. 4a). Such a high loading has not been matched by other methods used to load 5FU onto various carriers. For instance: 8 % on montmonrillonite [42], 7 % on porous clay heterostructures [43] and 22 % on kaolinite [44], when using the soaking method, and 12%–28% on chitosan via solvent evaporation emulsification [20]. A higher loading of 5FU (55 %) has been reported [44] but the kaolinite carrier was methoxylated prior to drug loading. Thus we conclude that the GAS process provides the highest drug loading onto natural (chemically unaltered) Halloysite nanotubes. The benefit of a drug carrier is largely determined by the type of drug release that it enables. The gastrointestinal tract has a varying pH from about 1.2 in the stomach to 7.4 in the intestine. It is therefore of key interest to examine drug release at these two pH values. The time release profiles of 5FU from the washed product, in simulated gastric and intestinal pH, are presented in Fig. 6. The release at intestinal pH is faster (about 80 % after the first 3 h) and practically complete (RD = 1) after 24 h. At gastric pH the release is slower (25 % after the first 3 h) and even after 24 h remains just under 30 %. The release profiles shown in Fig. 6 are well described with an exponential increase of RD in time using only two parameters RD ∞ and (RD ∞ is RD at t=∞, i.e. maximum release, and is characteristic time and more precisely the point of RD = 1 – e–1 ≈ 0.63):
Fig. 7. Dependence of (a) the final cumulative release, RD ∞ , at pH = 7.4 (䊐) and pH = 1.2 (), and (b) characteristic time, [min], at pH = 7.4 (䊐) and pH = 1.2 (), on the mass ratio R (=mHNT /mtot , where mHNT is the mass of the Halloysite nanoclay and mtot is the total mass of drug used).
(3)
Fig. 8. Electrokinetic potential, [mV], of Halloysite nanoclay dispersed in water (䊐 – Ref. [48]; 䊉 – this work) as a function of pH of the aqueous solution. The pK of 5-fluorouracil is shown with a vertical dashed line.
Eq. (3) describes a first-order kinetics of formation which is encountered in many practical situations including the release of a drug loaded onto a carrier [45]. The variations in maximum release, RD ∞ , and characteristic time, , with the mass ratio, R, are graphed in Fig. 7. At the higher pH, the maximum release is consistently high (above 90 %). At the lower pH, it steadily increases with R (Fig. 7a). The characteristic time does not depend on pH (Fig. 7b) and, within the scatter, remains approximately constant at a value of about 2 h. The characteristic time of 5FU release is in the range between gastric half emptying (1.2 ± 0.3) h and time to reach the colon (2.8 ± 1.5) h [46]. Therefore the 5FU-HNT drug-carrier combination is relevant to the development of oral delivery systems. The clear similarity between the two release profiles shown in Fig. 6 together with the fact that the characteristic time did not change with pH or mass ratio (Fig. 7b) suggest that the mechanism
of desorption and dissolution is the same. Given that 5-fluorouracil is a water soluble drug (11.1 g/L [47]) we attribute the complete release at pH = 7.4 (Fig. 8) to electrostatic interactions between the 5FU molecules and the nanoclay. At pH = 2 the zeta potential of Halloysite particles is practically zero [48] – Fig. 8. Therefore the nanoclay surface carries a very small surface charge, i.e. it is practically neutral. A similar situation is observed with both silica [49] (the outer surface of the Halloysite nanotubes is mostly composed of silica) and with kaolinite [50] (a clay very similar to Halloysite). At the higher pH = 7.4, the zeta potential is −40 mV (Fig. 8) and therefore the nanoclay surface is charged negatively. The 5-flurouracil behaves like a weak acid [51], i.e. at low pH the molecule is neutral and at high pH it deprotonates to become an anion. The pK of 5FU is 8.0 [52] and therefore at pH = 7.4 a significant portion of the molecules (exactly 50 % at pK) should be in their
∞
RD = RD
1−e
−t/
8
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anionic form. A recent NMR study concluded that 5FU is actually completely ionized at physiological conditions [51]. At such pH, the electrostatic repulsion between the negatively charged nanoclay surface and the 5FU anions ensures complete drug release. At acidic pH, electrostatic interactions are insignificant and drug release is only partial. The dissolution profiles of pure 5-fluorouracil at gastric and colonic pH are very similar. The release, RD , after 6 h is 98 % at pH = 7.4 and 88 % at pH = 1.2. This difference reflects the increased ionization of the 5FU molecule under alkaline conditions [53]. Both profiles match well the dissolution profile of HNT-5FU at pH = 7.4 (Fig. 6). This confirms the dominant role of electrostatic interactions in the formation of the 5FU-HNT complex. 4. Conclusion The Gas Antisolvent Process (GAS) can be used to load Halloysite nanotubes with 5-fluorouracil. For chemically untreated nanotubes, the drug loading (43 %) is better than what was achieved by other methods. The fraction of tightly attached drug (via physical adsorption) is compared quantitatively to the fraction of loosely attached (co-precipitated) drug. The later can be used to deliver a burst release. The mass ratio between clay nanotubes and 5fluorouracil affects both the drug loading due to adsorption and the ratio of loose to tightly attached drug molecules. The GAS process proceeds in two key stages and during the first one improved mass transport creates an increased drug loading. On the whole it is a one-pot green process with a low consumption of solvent and high-purity of the precipitated product. The strongly attached drug is released completely at physiological pH but only up to 30 % in acidic pH. In both cases the release follows first-order kinetics with a characteristic time of about 2 h. The differences are attributed to variations in electrostatic interactions with pH. We have described an efficient method to load Halloysite with 5-fluorouracil in a single step. The mass ratio R can be used to tailor the amounts of readily available and sustainably released drug. Declaration of Competing Interest
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