Research Article pubs.acs.org/journal/ascecg
Coordination Complexes and One-Step Assembly of Lignin for Versatile Nanocapsule Engineering Elisavet D. Bartzoka,† Heiko Lange,† Karsten Thiel,‡ and Claudia Crestini*,† †
Department of Chemical Sciences and Technologies, University of Rome Tor Vergata, Via della Ricerca Scientifica, 00133 Rome, Italy ‡ Fraunhofer Institute for Manufacturing Technology and Advanced Material, IFAM, Wiener Straße 12, 28359 Bremen, Germany ABSTRACT: Nanoencapsulation of active substances with controlled release in harmless matrices has been the subject of numerous scientific efforts mainly due to the significant biomedical potential of such endeavors. Lignin, the environmentally sustainable byproduct of the pulp and paper industry, contains a multitude of phenolic hydroxyl groups, some of which, are known to readily and strongly chelate with iron ions. In this effort we demonstrated that the concerted use of chelation chemistry, oil in water emulsion principles, and low energy sonication, offers a facile, one-pot strategy to assemble lignin nanocapsules (LNCs) of a controlled architecture. Under these conditions capsules are shown to rapidly assemble utilizing two driving forces, the π-stacking propensity of lignin and its metal chelating ability at alkaline pH. Detailed size exclusion chromatographic evidence validates that the formation of capsules is driven mainly by the enumerated physical interactions with no significant chemical modification of the lignin. The developed process was systematically optimized so as to create the foundations for the morphology and the yield of the capsules being modulated as a function of sonication time, power, and surface contact area. Both pure LNCs and Fe-LNCs were synthesized in high yields with size distributions varying from 0.3 to 6 μm and their release efficiencies were evaluated in detail. As anticipated, the complexation effects of the phenolic OH groups offered to the Fe-LNCs, increased stability, reduced shell thickness (allowing for greater loading efficiencies), and lower release kinetics, compared to LNCs. KEYWORDS: Lignin, Fe(III), Coordination complex, Ultrasound, Nanocapsules, One-step assembly, Delivery
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essential to much of modern science and technology.12,13 Thus, the development of engineering strategies for particles and capsules are at the base for advance in materials design. Nanoencapsulation involves the incorporation of an active substance within a coating material and has gained enormous interest in the recent years, since it provides a cost-effective way to enclose and deliver diverse actives, including drugs, cells, pesticides and flavors in the cavity formed within the shell walls and the internal phase by achieving a controlled release rate of the ingredients.14 Metal−organic capsules or particles are specifically interesting for the development of stimuli responsive materials, due to the dynamic nature of the supramolecular architecture; they possess hybrid physiochemical properties of the metals and the organic matrix and allow for the development of controlled structures and functionalities by tuning the different building blocks.15,16
INTRODUCTION Nowadays, there is an important and urgent need to efficiently use all available biomass components for achieving a sustainable use of renewable resources.1 Agricultural and forestry residues constitute an invaluable renewable source of Lignocellulosic materials. Despite the fact that the use of biopolymers, primarily cellulose, is now considerable, lignin, the second most abundant biopolymer which accounts for 15−30% of biomass, is a vastly underutilized source of aromatic compounds.2 Besides its polyphenolic character, lignin offers a wide range of additional features, such as aggregation properties,3−5 high biocompatibility, ability to absorb UV light,6 and antioxidant activity7 that render it ideal for the production of novel, high-performance concepts and products using the lignin,8 coming either as byproduct of current processes or from emerging platforms such as the biorefinery.9,10 Nanostructures of natural polymers have received steadily growing interest as a result of their peculiar properties and applications superior to their bulk counterparts.11 Moreover, their preparation methods involve easy handling in aqueous medium, avoiding the use of environmentally impacting organic solvents. The ability to generate such minuscule structures is © XXXX American Chemical Society
Special Issue: Lignin Refining, Functionalization, and Utilization Received: April 28, 2016 Revised: June 1, 2016
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DOI: 10.1021/acssuschemeng.6b00904 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
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General Procedure for the Ultrasound (US) One-Step Assembly of LNCs. Emulsions of olive oil in polyphenol solutions were prepared by dispersing 100 μL of olive oil in 1000 μL of 5% (w/ v) lignin aqueous solution and sonicated as described above. One-Step Assembly of Fe(III) Coordination Complexes on Lignin and Nanocapsule Formation. Fe-LNCs were obtained by adding 200 μL of FeCl3 aqueous solution (10 mg/mL) in 600 μL of the 5% w/v lignin solution in MOPS (pH 7.4) and sonicating the o/w emulsion, in order to keep the molar ratio between aromatic hydroxyl (OH) groups (quantified via31P NMR data) and FeCl3 1:6. Lower amounts of the initial lignin solution (final amount of lignin is 30 mg) than in the general procedure (50 mg) are used, to keep the same initial solutions and the volume of the sonicated dispersion constant. LNCs and Fe-LNCs Isolation. Separation and washing of the capsules was realized by centrifugation of the sonicated samples at 5000 rpm for 15 min after having left the systems equilibrate for 12 h. The lower part corresponding to the lignin residues was removed and the foamy supernatant consisting in nanocapsules was washed twice with 1 mL of distilled water. The different washing solutions were reunited for further analysis. Encapsulation of Actives. This step was performed using the fluorescent probe Coumarin 6. For the emulsion preparation, 100 μL of a solution of Coumarin 6 in olive oil (0.2 mg/mL) were added in 1000 μL of 5% w/v lignin aqueous solution and the resulting o/w suspension was sonicated as previously described. Characterization of Lignin Nanocapsules. In order to evaluate the morphological characteristics and dimensions of the colloidal systems, we analyzed the isolated supernatant via optical and fluorescent microscopy imaging using the excitation line of λ = 488 nm for FITC and via cryogenic scanning electron microscopy (cryoSEM). Optical and Fluorescent Microscopy Analysis. A 10 μL portion of the isolated capsules were added in 90 μL of distilled water to yield a suspension of capsules in water in a final volume of 100 μL. A 5 μL portion of the above-mentioned suspension was transferred on the microscope glass slide that was covered with a simple coverslip prior to microscopy analysis. In case the sample was too dense and the capsules overlapped and impaired the subsequent image analysis, the sample was further diluted. A Ziess microscope was used, and all images were obtained with 10×, 40×, 63×, and 100× objective lens magnification. Quantitative Analysis of Capsules. The microscopy images were processed using the image analysis software ImageJ and subsequent Excel based analyses to obtain a statistical summary of yields (expressed in number of nanocapsules/mL), mean diameters incl. standard deviations and polydispersion indices. In order to have statistical relevant results in terms of nanocapsule numbers, we chose the three best images for the Software-based ImageJ analysis. Error analysis was performed according to this type of measurement protocol using five identical samples prepared on different days by different experimentalists. A comparison between a manual counting and an ImageJ Software-based counting has been done, screening different settings in software. Shell Size Analysis. This was carried out as follows: lignin was labeled with fluorescein isothiocyanate (FITC) by reacting 10% of the free aromatic hydroxyl groups of the starting lignin (determined via quantitative 31P NMR data) prior to capsule formation. The reaction was performed by dissolving 100 mg of initial KL in a total volume of 4 mL of DMF in the presence of catalytic amounts of DMAP, at 30 °C and was left in agitation for 24 h. The workup consisted in acidification of the reaction with hydrochloric acid (HCl) to pH 3 and isolation via centrifugation at 5000 rpm and 15 min. Washing of the resulting FITC-functionalized lignin with distilled water was performed several times until the characteristic yellow color of FITC was no longer evident in the residual aqueous solutions. Capsules were formed following the general procedure by sonicating an o/w emulsion consisting in 1000 μL of a 5% w/v aqueous solution of the labeled lignin and 100 μL of a nonfluorescent mineral oil as the encapsulated hydrophobic compound. The shell thickness was estimated from the fluorescent contour of the capsules.
The encapsulation of active substances can be achieved by a variety of techniques.17−19 Capsules can be filled by either using the encapsulating material directly as template or by incorporating the material into the hollow capsule. The active can also be adsorbed on the surface of the capsule.20 The choice of a suitable encapsulation method is dependent on the nature of the specific core and shell materials, as well as of the desirable features and the end-use of the generated colloidal system.21 Both valorization of lignin and microencapsulation can benefit from sonochemistry.22 The ultrasound technique is vastly applied in the generation of novel materials in the micro and nanoscale23 and facilitates their encapsulation with active agents. It is proved to be an essential tool, since it offers simplified ways to achieve reactions that would otherwise require extreme conditions or the use of toxic reagents (e.g., for template-based synthesis or workup) combined with multistep generation and removal processes.24 Low power sonochemical treatments allow for easy emulsion formation. The operating shear forces favor stacking of products that display a significant tendency to associate.25,26 We report here a rapid, simple, and robust method of one step assembly of polyphenols and of coordination complexes on polyphenols to prepare nanocapsules. The polyphenol of choice as the organic matrix was kraft lignin, while the inorganic cross-linker was Fe(III). The propensity of lignin to associate together with its multifunctionality has been exploited into the development of a simple procedure for the synthesis of lignin nanocapsules. It has been recently discovered that Kraft lignin can be readily used to create microcapsules that according to preliminary studies are non toxic as such.27 With the aim at elucidating the ability of lignin to generate supramolecular architectures by stacking and/or complex formation, in view of the development of new materials, we investigated the different parameters affecting the release and stability of lignin capsules. Lignin contains significant amounts of phenolic OH groups. Such moieties are in principle prone to the formation of complexes with metal ions. In order to obtain more stable and versatile capsules we used Fe (III) as templates for the formation of nanocapsules based on lignin-iron coordination complexes.
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EXPERIMENTAL SECTION
Materials. Kraft lignin (KL) (alkali lignin, low sulfonate content, CAS number 8068-05-1), Coumarin 6, fluorescein isothiocyanate (FITC), 4- morpholinepropanesulfonic acid (MOPS), dimethylformamide (DMF), hydrochloric acid (HCl), 4-dimethylaminopyridine (DMAP), sodium dodecyl sulfate (SDS), n-hexane, acetic acid glacial, acetyl bromide, THF inhibitor-free, CHROMASOLV Plus for HPLC, FeCl3, olive oil highly refined of low acidity and mineral oil were purchased by Sigma-Aldrich and used as received. Distilled water was used. One-Step Assembly of Lignin Nanocapsules (LNCs) by Sonication. The generation of the different types of nanocapsules was realized by sonication of oil in water (o/w) emulsions applying in principal an acoustic power of 160 W, for 40 s, and using a 3 mm diameter microtip connected to a 20 kHz Branson probe (Branson Digital Sonifier by Ultrasonic Corporation, Model 450L). Different conditions were applied during the optimization study of the method, including time of sonication (20, 40, 80 s, 5, 10, 15, 30 min), 10% and 40% amplitudes (40 and 160 W, respectively), cavitation region (various sonication vessels incl. Eppendorf, small and big Falcon centrifuge tubes), and types of sonication probe (3 mm tip and 10 mm horn). B
DOI: 10.1021/acssuschemeng.6b00904 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
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6 loaded LNCs and Fe-LNCs (∼2 × 1010 capsules/mL) of five different sets of preparations were disassembled upon CHCl3 addition and incubation in an ultrasonication bath. The organic phase was removed every 10 min and fresh solvent was added until the foam was discolored. The organic phases of each set were reunited and left to dry overnight. The dry sample was redissolved in the exact volume of 5 mL of CHCl3 and the amount of the extracted Coumarin 6 was quantified based on a standard curve of UV absorbance versus concentration calibrated with a set of different solutions of Coumarin 6 in CHCl3. Indirect loading studies were performed by analyzing the washing aqueous solutions. The aqueous suspension was centrifuged at 5000 rpm for 15 min after having added CHCl3 in excess (5 mL). The bottom phase was collected and left to dry overnight. The dry sample was redissolved in 2 mL of the solvent before the quantitative analysis via UV−vis. The quantities of Coumarin 6 quantified by direct and indirect study are expressed in percentages found in the capsules or the washes, respectively, with respect to the initial concentration of Coumarin 6 and the mass return shall reach the total initial amount of active added. The loading amount of the Coumarin 6 is finally expressed as femtograms per capsule. Release Kinetics Determination. In vitro release of the encapsulated hydrophobic active was performed in sodium dodecyl sulfate (SDS) 10% w/v aqueous solution, applying a protocol that consisted in analyzing the amounts of the active released at designated time points via UV−vis and quantifying them on the basis of the respective calibration curves at an absorbance maximum of 471nm wavelength. The aliquots were lyophilized and the resulting samples were redissolved in an exact volume of 1 mL of distilled H2O and filtered through a 0.45 μm filter before the UV−vis measurement. The analysis was performed as follows: 1 mL of SDS (10% w/v) aqueous solution was added to the entire volume of one set of Coumarin 6 filled LNCs (∼100 μL), and the resulting suspension was left under gentle agitation at 25 °C. At fixed intervals (10, 30 min, 1, 2, and 3 h) the sample was centrifuged at 5000 rpm for 5 min and intake amounts of Coumarin 6 in the aqueous loaded LNCs were isolated from the supernatant foam. The remaining foam after each separation was redispersed in 1 mL of SDS and left under gentle shaking. The procedure was continued until no peak attributable to Coumarin 6 was detected in the UV spectrum. The results of this type of analysis were processed based on the absorbance of the aliquots at the λmax of Coumarin 6 and the quantified amount was added to the amount of Coumarin 6 found in the analysis of the precedent time point yielding to the release percentage that was normalized to the maximum amount released and calculated in the following way:
Cryo-SEM. For the cryo-SEM experiments a QuorumPP2000T cryo-preparation system installed at a FEI Helios 600 Dualbeam machine was used. The sample preparation and measurement were performed as follows: First a rivet was mounted on a cryo shuttle and the shuttle attached on a transfer rod. A drop of the sample was subsequently placed on the rivet and then plunge frozen in “slush” nitrogen. The use of nitrogen slush avoids the “Leiden” effect which allows an insulating gaseous layer to form around the sample preventing rapid cooling rates. Hence, freezing in nitrogen slush gives a faster cooling rate than can be achieved by plunging into normal liquid nitrogen. The frozen sample was then transferred to the cryo preparation chamber in a small sealed vacuum container inside the transfer device and mounted on the cryo-preparation stage. The stage in the prep chamber was cooled down with l-N2 but heated to −130 °C. The rest of the prep chamber had a temperature of l-N2 (−196 °C). This ensures that possible contamination is kept away from the sample and ice build-up is prevented on the cryo sample. The sample has then been fractured with a knife. To reduce water on the fractured surface the temperature of the preparation stage was increased to −90 °C for about 5 min. After that platinum was sputtered on the fractured surface in the preparation chamber. Afterward, the shuttle was inserted in the SEM with the transfer rod. The cryo stage inside the SEM was again at −130 °C while a cryo shield inside the SEM was cooled down to −196 °C, again to prevent contamination of the sample. Lignin Characterization. The lignin characterization consisted in determination of the distribution of molecular weights by means of gel permeation chromatography (GPC) analysis. The treatment for the isolation of both types of lignin (the lignin of the capsule shell and the fraction found in the washing solution) involved lyophilization and subsequent extraction of the samples with n-hexane. Gel Permeation Chromatography (GPC). Approximately, 5 mg lignin was suspended in 1 mL glacial acetic acid/acetyl bromide (9:1 (v/v)) for 2 h in accordance with the method provided by Asikkala et al.28 The solvent was then removed under reduced pressure, and the residue was dissolved in HPLC-grade THF and filtered over a 0.45 μm syringe filter prior to injection. GPC-analyses were performed using a Shimadzu instrument consisting of a controller unit (CBM-20A), a pumping unit (LC 20AT) equipped with a 20 μL sample loop, a degasser unit (DGU-20A3), a column oven (CTO-20AC), a diode array detector (SPD-M20A), and a refractive index detector (RID10A); the instrumental setup was controlled using the Shimadzu Lab Solution software package (Version 5.42 SP3). Three analytical GPC columns (each 7.5 mm × 30 mm) were connected in series for analyses: Agilent PLgel 5 μm 10 000 Å, followed by Agilent PLgel 5 μm 1000 Å and Agilent PLgel 5 μm 500 Å. HPLC-grade THF (Chromasolv, Sigma-Aldrich) was used as eluent (isocratic at 0.75 mL/min, at 40 °C). Calibration was performed with polystyrene standards (Sigma-Aldrich, MW range 162−5 × 106 g/mol). Final analyses of each sample was performed using the intensities of the UV signal at λ = 280 nm employing a tailor-made MS Excel-based table calculation, in which the number-average molecular weight (Mn) and the weight-average molecular weight (Mw) were calculated based on the measured absorption (au) at a given time (min) after corrections for baseline drift and THF-stemming artifacts.29 Cyclic Voltammetry. Isolated lignin capsules were tested at room temperature using an Autolab potentiostat (EcoChemie, Utrecht, The Netherlands). Cyclic voltammetry was recorded from −0.5 to +0.7 V versus an external Ag/AgCl reference electrode with a scan rate of 50 mV/s in 1 M KCl supporting electrode solution and a frequency of 100 Hz. Once the sensor’s signal was stable, the desired sample solution was added to the solution and the resulting signal was evaluated. Active Loading Measurement−Entrapment Efficiency (EE). Drug entrapment capacity of the LNCs was determined spectrophotometrically via UV−vis analysis at an absorbance maximum 463 nm, applying a direct loading study of the capsules and an indirect study of the washing solutions. Spectra were obtained using a Shimadzu UV− vis spectrophotometer UV 1800. Direct loading studies on LNCs and Fe-LNCs have been performed as follows: A total of 100 μL Coumarin
release (%) =
mass of Coumarin 6 released × 100 mass of efficiently encapsulated Coumarin 6
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RESULTS AND DISCUSSION One-Step Self-assembly of Lignin Stacked Nanocapsules by Means of Ultrasound Treatment. Lignin is the most abundant natural biopolyphenol characterized by an amphiphilic backbone of hydroxyl (OH) groups bound to hydrophobic aromatic rings with numerous possibilities for functionalization and structural modification. Hence, it shall constitute an ideal shell component for systems of transportation of poorly soluble actives that will have a high affinity with the hydrophobic core of the lignin, whereas at the same time the hydroxyl groups will eventually render the whole system water dispersible. Lignin is known to possess intriguing intrinsic π-stacking and complexing properties, as well as to present various hydrophobic interactions.4,5 These interactions within lignin subunits are fundamental for the generation of nanocapsules via the sonochemical method that is applied in our study. Overall, this approach leads to the formation of nanocapsules by means of sonication of an oil in water (o/w) C
DOI: 10.1021/acssuschemeng.6b00904 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
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Figure 1. Macroscopic image of KL LNCs and KL Fe-LNCs (left), cryo SEM image of the capsule (center), and possible Fe-LNCs architectures of the capsule shell based on stable Fe−O−lignin complexes (right).
obtained in the absence of Fe(III). The color change can be attributed to the complex formation. Cyclic Voltammetry. The presence of iron in the ligniniron capsules was confirmed by using cyclic voltammetry. As shown in the Figure 2, the lignin based nanocapsules have a different electronic configuration from the nanocapsules based on the lignin−Fe(III) complex. Both the anodic and cathodic currents of the lignin based nanocapsules show similar behavior to the starting lignin, whereas the iron-lignin composite exhibits
emulsion. The oil, in which the potential organic encapsulant shall be incorporated, is used as a template that will form the organic core and the aqueous phase in which the natural polymer is dissolved will form the shell, that will be eventually hardened by cross-linking reactions caused by the high ultrasound frequencies. As a hydrophobic template, we use the biocompatible olive oil, or mineral oil. The ultrasound power is used to promote the existing hydrophobic interactions of the biopolymer within the oil phase in such a way that lignin will reorient itself and result in the desired shell−core structures.30 The mechanisms that lie behind this method take advantage of two main ultrasound induced phenomena, emulsification and radical generation.31 Sonochemistry is known in general to enhance physical and chemical processes that arise from the main effect of the sonication, termed cavitation.32 The cavitation occurs when moderate and high sound intensities are applied in a liquid and leads in the formation of localized hotspots with extreme temperatures and pressures despite the normal conditions in the bulk solution. As a result, this method gives the opportunity to achieve reactions that would otherwise require hours in the frame of a few minutes or even seconds. More specifically in our case, emulsification comes as a result of the intense physical mixing of the two phases.33 One-Step Fe(III) Coordination Complex Formation and Nanocapsule Assembly by Means of Ultrasound Treatments. In an attempt to exploit both the stacking tendency of lignins and their ability to form stable complex with metal systems, ultrasound treatment was used to induce self assembly of nanocapsules comprised of lignin and Fe(III). Fe(III) was used as a facile and inexpensive inorganic crosslinker to chelate the phenolic moieties of lignin chains and promote the formation of a stable complex network between the metal and the polyphenol, prior to sonication (Figure 1). The generated complex consisted the aqueous phase of the o/w emulsion and was subsequently submitted to the standard ultrasound treatment. In order to obtain uniform capsules and avoid their disassembly due to destabilization of the complex, slightly alkaline conditions were selected in which lignin was found soluble and the tris-complex state between the polyphenol and the metal was predominant.34 For this purpose we used a MOPS buffer at pH 7.4 for the preparation of the mother solutions containing the formed stable tris-complex. As demonstrated in Figure 1, the color in the final product obtained is darker as compared to the white lignin capsules
Figure 2. Cyclic voltammograms of Kraft lignin, LNCs, and Fe-LNCs. The scan rate is 50 mV/s in 1 M KCl supporting electrolyte solution. D
DOI: 10.1021/acssuschemeng.6b00904 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
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Figure 3. (A) Optical and fluorescent microscopy images obtained with 10×, 40×, 63×, and 100× objective lens magnification: (i) optical microscopy image of LNCs 40×, (ii) optical and (iii) fluorescent microscopy image of FITC labeled LNCs 100×, (iv and vi) optical (10×, 63×, respectively) and (v) fluorescent microscopy image of Fe-LNCs (10×) showing the aggregation properties of the systems. (B) Cryo-SEM images of lignin based nanocapsules: (i and ii) LNCs and (iii and iv) Fe-LNCs demonstrating the distinct core−shell nature of the systems.
Table 1. Size, Shell Thickness, Yield, and Loading of LNCs and Fe-LNCs entry
power (W)
1 2
40 40
3 4
160 160
sample LNCs FeLNCs LNCs FeLNCs
polydispersity
min−max dimensions (μm)
average shell size ± stdv (μm)
yield (capsules/mL)a
2 2
0.4 0.5
0.6−5 0.5−6
0.59 ± 0.01 0.48 ± 0.01
4 × 109 4.5 × 109
1.2 1.3
0.4 0.5
0.4−3 0.6−4
0.31 ± 0.01 0.26 ± 0.01
2 × 1010 2.2 × 1010
average diameter (μm)a
average % loading ± stdv
70 ± 10 90 ± 10
Average error is 0.01 μm in the diameter of the capsules and 1.5 × 109 capsules/mL for the yield (5% error), whereas between different experimentalists the error can reach up to 0.1 μm and 6.9 × 109 capsules/mL, respectively (28%).
a
an irreversible redox process with a reduction current peak in the potential between −0.4 and −0.2 V, which is in agreement with literature results for the electrochemical analysis of the FeCl3 solutions.35 This finding further supports the presence of Fe(III) in the KL-Fe-LNCs and confirms the different architecture of the capsules formed in the presence of Fe(III) salts. Characterization of Nanocapsules. Among the different methods for the preparation of nanocapsules the ultrasound assisted approach favors the encapsulation of actives in a simple and rapid one-step coating process.36,37 This method generally provides the possibility to obtain nanoparticles.14 This term encompasses both intact particles and capsules with shell−core morphology38 and the selective generation of one of them mainly depends on the composition of the solution that undergoes the sonication treatment. Hence, taking into consideration that our initial dispersions contain an organic oil phase, this synthetic approach shall in principle lead to the formation of oil-filled nanocapsules. In order to confirm our hypothesis, we used FITC as the fluorescent probe to label the lignin and a mineral oil as the nonfluorescent core and analyzed the products obtained from the ultrasound assisted synthesis via fluorescent microscopy. As shown in the Figure 3Aiii, the systems are spherical nanocapsules consisting of a fluorescent shell that covers a black core, which overall confirms the capsule nature of the systems. Further verification of the capsule nature of our systems was acquired by means of cryo- scanning electron microscopy (SEM) of the capsules. The cryogenic technique was preferred due to the delicate nature of our samples that retain their native and original conformation in the fully hydrated state that would be otherwise compromised under the vacuum environment of
the normal SEM experiments. As shown in the Figure 3B, spherical capsules were obtained with a distinction between the shell and core. The dimensions of the capsules were found to be in total agreement with the optical microscopy imaging analysis, with capsule sizes in the range of 300 nm−6 μm. In the case of the Fe-LNCs, samples imaged via both cryoSEM and optical microscopy showed spherical capsule structures that were part of a larger aggregation network (Figure 3Aiv, v, vi, Biii, iv). Capsule aggregation occurring at FeCl3 addition with an increase in the maximum capsule dimension (Table 1, entries 2, 4) are in accordance with the DLVO theory, which explains the decrease in the range of double layer repulsion between particles when a salt is added to the dispersion.39,40 DLVO theory is the classical explanation of the stability of colloids in suspension. It looks at the balance between two opposing forces, electrostatic repulsion and Van der Waals attraction to explain why some colloidal systems coagulate while others do not. Addition of the salt causes in principal the initiation of the aggregation process of the colloid. Figure 3Biii and iv shows in particular the formation of a neck due to the intercapsule collision under ultrasonic irradiation.41 The encapsulated fluorescent hydrophobic molecule Coumarin 6 can be seen in Figure 3Av to be uniformly distributed within the nanospheres. The drug distribution is a critical parameter that would help in uniform drug release after an eventual in vivo administration of the capsules. Size and Morphology of the Colloidal Systems. The amphiphilic character and the continuous movement of our systems impaired the application of standard methods like dynamic light scattering for size and size distribution analysis. In order to evaluate the size of the capsules obtained, we developed a new protocol based on the analysis of microscopy E
DOI: 10.1021/acssuschemeng.6b00904 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
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Table 2. Nanocapsule Dimensions and Yield as a Function of Surface Contact Area, Power, Sonication Probe, and Time of Sonication entry
surface contact areab
power (W)
sonication probec
time of sonication
mean diameter (μm)a
min−max dimension (μm)
polydispersity
1 2 3 4 5 6 7 8 9 10 11 12 13 14
8% 25% 8% 25% 25% 25% 25% 25% 25% 25% 25% 25% 25% 25%
40 40 40 40 40 40 160 160 40 40 40 40 40 40
tip tip tip tip tip horn tip horn tip tip tip tip tip tip
20 s 20 s 40 s 40 s 40 s 40 s 40 s 40 s 20 s 40 s 5 min 10 min 15 min 30 min
1.8 1.9 2.2 2 1.7 1.8 1.1 1 2 1.8 2 2.1 1 0.8
0.5−5 0.5−5 0.8−5 0.5−6 0.6−6 0.9−3 0.4−3 0.5−3 0.8−5 0.6−6 0.5−5 0.5−4 0.4−2 0.4−1.5
0.6 0.5 0.5 0.4 0.4 0.3 0.4 0.5 0.4 0.4 0.3 0.3 0.2 0.2
yield (capsules/mL)a 2.6 2.2 4.2 4.7 4.8 1.6 8.8 1.6 3.6 4.3 4.7 5.6 9.6 1.5
× × × × × × × × × × × × × ×
109 109 109 109 109 109 109 1010 109 109 109 109 109 1010
Average error is 0.01 μm in the diameter of the capsules and 1.5 × 109 capsules/mL for the yield (5% error), whereas between different experimentalists the error can reach up to 0.1 μm and 6.9 × 109 capsules/mL, respectively (28%). bSurface contact area calculated as a ratio between the surface of the sonication probe and the small vessel; 8% for the tip/Eppendorf, or 25% for tip/Falcon tube. cTip (3 mm), horn (1 cm)
a
of the method, since less lignin is required for the formation of the same number of capsules. Since, better and more desirable capsule features were obtained from the use of the high amplitude (40% = 160 W), the experiments and analyses regarding the loading and release behavior have been performed only on these systems and with this setup. As shown in the Table 1, an increased average loading efficiency of the Fe-LNCs of 90% compared to the 70% of the LNCs was noticed during the loading studies of three different samples, leading to the assumption that, even though thinner, the shell comprised of lignin and iron constitutes a more efficient encapsulating system. Tailoring of the Properties of Nanocapsules. Once the capsule nature of the generated systems was confirmed and a well-defined and robust method for their evaluation and analysis was established, a systematic study on the assembly process parameters was carried out. One of the main challenges of the ultrasound mediated self-assembly methodology is the difficulty to handle and carefully control the different parameters that take place, including amplitude, time, temperature, cavitation region, that are interdependent, as well as the scaling up process.43,44 Table 2 shows the different experimental conditions explored. The results follow the anticipated trends based on the theory of sonication.45 More specifically, we chose as model system the standard KL LNCs and studied the effect on the various capsule features provoked by the changes of the values of one parameter while keeping constant the others. The standard conditions for the generation and isolation of capsules included: the use of the ultrasonic tip, 40 W, 40 s of sonication, and 15 min of centrifugation at 5000 rpm, unless otherwise stated. Different areas of direct contact of the molecules in the bulk solution with the probe were studied at two different time values (20 and 40 s of sonication). The surface contact area was expressed as a ratio of surfaces (8 or 25%) depending on the free sonication surface of the small vessel used in the experiment (Eppendorf or Falcon tube) and the surface of the tip. As demonstrated in Table 2 and entries 1−4, the change in the surface contact area does not significantly affect
images via the software ImageJ. In an attempt to exploit to the maximum the microscopy imaged capsules, we processed the ImageJ data via Excel which yielded statistical medium for dimensions, polydispersity and number of capsules among three images. Manual and software counting and size analysis of the capsules has been applied in parallel and showed a small deviation of 1−8 capsules, depending on the resolution of the image as well as on the circularity parameters set as default in the software ImageJ. Since better correspondences were found using more flexible circularity criteria, i.e., 0.80−1.00, this setting was generally chosen as “default” setting. Based on capsule behavior, adjustment of this value might be nonetheless necessary. The same protocol was used for the analysis of the shell and the determination of the average radius of the capsules. The shell size analysis has been performed manually. Spherical capsules with diverse dimensions and shells were studied, depending on the shell components and preparation procedure. As demonstrated in the Table 1 that summarizes the quantitative analysis results of the different lignin based nanocapsules, the addition of Fe (III) salt does not affect the average capsule dimensions, the yield or polydispersion of the system; in fact the deviation in the values is found within the error of the method. The average polydispersity indices range from 0.4 to 0.5 on a scale 0 to 1; the higher the polydispersity index, the more polydisperse the capsules are. There is a significant decrease in size and increase in number of capsules generated when higher sonication amplitude is applied (entries 3, 4), where the capsule sizes range from 400 nm to 3 μm.The average shell size of the capsules is in the range of 0.1−0.6 μm which represents a thick shell wall. The increase in the sonication power led to decreased shell wall dimensions, a trend that corresponds to the expected one from literature.42,43 Interestingly, the addition of Fe(III) leads to further decrease in the shell dimension whereas the number and size of capsules remain similar. That finding indicates that even using smaller lignin quantities, the complexation with iron leads to the formation of capsules with comparable size features and slightly increased yields but thinner shells, overall increasing the yield F
DOI: 10.1021/acssuschemeng.6b00904 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
Research Article
ACS Sustainable Chemistry & Engineering
analysis and that the distribution of the molecular weights shown is relative to the lignin and not to the capsule unit i.e., the capsule components. This is specifically relevant in the case of Fe-LNCs where iron(III) acts as a cross-linker. Figure 4
the size or yield of capsules, when a small reaction vessel is used. The different parameters used are not independent, as shown in Table 2 entries 5−8, where different sonication probes are studied with respect to different sonication amplitudes. The use of increased cavitation region (horn 10 mm) in combination with increased power contributes to an overall increase in the number of smaller and less polydisperse capsules. When the horn is used at 40 W, the system becomes less polydisperse compared to the capsules formed by the tip, as shown by the difference between the maximum and minimum dimensions found at the microscopy analysis (0.9−3 μm for the horn based generation of capsules (entry 6) whereas the capsule size can reach up to 6 μm when using the ultrasonication tip (entry 5)). The same effect of formation of capsules with sizes in a smaller range is noticed when increased power is used independently of the sonication probe (entries 7, 8). Other than that, these findings indicate that the scaling up process using the ultrasonication horn for the sonication of an eventual larger volume is not totally equivalent to the simple batch experiment and does not lead directly to the obtention of the same type of capsules. In principle, the increase of the yields and the decrease of the size of the capsules can be modulated by the increase of sonication power (comparing entries 5 to 7 and 6 to 8 that correspond to the same probe used for sonication) and/or sonication time up to a plateau. Entries 9−14 in Table 2 show the effect of increasing sonication time. In all the cases above, the increase in yield can be attributed to the increased exposure to the ultrasound irradiation that led to the prolonged main effect of the acoustic cavitation and the enhancement of the physicochemical reactions. The increase in the sonication time also led to the generation of more monodisperse capsules as shown by the decreased polydispersion indices (e.g., 0.2 at 30 min, entry 14), hence providing a way to control the polydispersity of the systems depending on eventual needs. Interestingly, lignin capsules were formed even at low powers of sonication used in this study, where no cross-linking reactions are expected; this indicates the strong associative properties of lignin and further confirms that lignin is an ideal polyphenol for self-assembly. All in all, this method was proved to be tunable with respect to the required features of each eventual application. Lignin Characterization and Evaluation of Structural Variations. In order to detect the structural changes caused in the lignin backbone due to the ultrasonic irradiation, lignin that remained in the residual and washing solutions after the sonication and lignin that built up the capsule were analyzed. The samples have been extracted with n-hexane in order to remove the remaining olive oil residues and avoid as much as possible the oil−lignin overlap in the various types of analysis. All samples of lignin were analyzed via GPC. In an attempt to capture the differences between the fractions of lignin that participate in the capsule formation and the lignin that precipitates in the residual solutions, we proceeded in a series of analysis of both capsules and residues and compared it to the starting material that was analyzed under the same conditions. The GPC analysis of Kraft lignin is not straightforward. This specific analysis protocol has been chosen on the basis of a study carried out on the referencing and parametrization of GPC of lignins.29 It is worth recalling that capsules were destroyed by solvent treatment before GPC
Figure 4. GPC analysis of sonicated lignin in residues and capsules at 160 W showing the overlays of the molecular weight distribution of the different lignin fractions onto the starting lignin material of (A) LNCs and (B) Fe-LNCs.
shows the GPC profiles of the starting lignin, the lignin that constitutes the capsules and the residual lignin present in solution. Apparently the capsules are composed of a higher molecular weight fraction of the original lignin. This finding can be rationalized taking into consideration that the driving force for capsule formation is at low sonication intensity the πstacking effect that is supposed to be more pronounced in higher molecular weight fractions. This is further confirmed by the fact that the GPC profile of the residual lignin after capsule formation shows on the contrary a reduced molecular weight distribution profile. This leads to the assumption that a higher molecular weight fraction is responsible for building up the capsule shell. In Figure 4A, the curves that correspond to the lignin of the LNCs show a small shift in higher molecular weights for the lignin isolated from the LNCs, indicating indeed the need of a higher molecular weight fraction that together with the π-stacking reactions within the lignin backbone lead to the capsule shell formation. In the case of the of Fe-LNCs (Figure 4B), the molecular weight distribution of the starting lignin and the lignin that actually constitutes the capsules is not significantly different, possibly indicating that in this case the complexation reaction of lignin with iron allows also efficient stacking of shorter lignin oligomers, while when only stacking is occurring a higher molecular weight fraction is necessary to obtain stable capsules. Preparation of Coumarin 6 Loaded LNCs. In order to evaluate the encapsulation capacity of LNCs and Fe-LNCs, Coumarin 6, a fluorescent dye, was used as a hydrophobic active probe. Since Coumarin 6 shows an intense color, its eventual adsorption on the capsule surface rather than inside would have been immediately evident. Interestingly, the capsules filled with Coumarin 6 retained their standard color and Coumarin 6 was only evident after the disassembly of the capsules. This clearly indicates that the acitive is efficiently G
DOI: 10.1021/acssuschemeng.6b00904 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
Research Article
ACS Sustainable Chemistry & Engineering
LNCs released 100% of Coumarin 6 after the first 30 min. The release pattern of the Coumarin 6 shows a burst release in the first 10 min (Figure 6). Fe-LNCs showed a slower release
incorporated inside the capsules and not actually adsorbed on their surface. (Figure 5B)
Figure 5. (A) Fluorescence microscopy of Coumarin 6 filled LNCs. (B) Fe-LNCs loaded with Coumarin 6 (filled capsules, left; capsules destroyed by solvent CHCl 3, center; and isolated for UV quantification, right).
Entrapment Efficiency (EE). The encapsulation of bioactive molecules into the capsules is of great importance for active delivery. Successful encapsulation of the fluorescent probe was demonstrated by fluorescence microscopy, which showed homogeneous distribution within the capsules (Figure 5A). The loading capacity was determined by the evaluation of the dye released after destruction of the nanospheres upon addition of CHCl3 and ultrasound treatment (Figure 5B). The released dye was monitored by UV analysis. The effective loading was evaluated via the quantification of the free Coumarin 6 released upon capsule breaking. It was further verified by double checking of the residual Coumarin that remained into the solution after sonication. As revealed by the UV loading studies, 70% of the initial amount of Coumarin 6 added (20 μg) was efficiently loaded in the LNCs. About 20% (±13%) of the initial active is found in the washes and not in the capsules, thus confirming the 70% of active that was found to be efficiently entrapped in the capsules; the other 20−30% is not encapsulated and is released during the washing steps, hence found in the analysis of washing residues. The nearly quantitative mass balance obtained in the two different measurements (Coumarin 6 released by the capsules vs Coumarin 6 washed away after capsules synthesis) allows us to use the results from the quantification of Coumarin 6 in the washings as an indirect methodology for the efficient and accurate determination of the effective loading into the capsules. This approach is specifically significant and useful for the determination of the release behavior of the capsules. The Coumarin 6 average loading per capsule can be subsequently determined based on the number of the loaded capsules and the total amount of Coumarin 6 loaded in the capsules, as revealed by the optical microscopy and the abovementioned loading analysis, respectively. The 70% of EE (entrapment efficiency) corresponds to 14 μg of Coumarin 6 loaded in 2 × 109 capsules that were formed in 100 μL by an approximate final amount of 0.25 mg of lignin. Thus, the amount of Coumarin 6 was 7 fg per nanocapsule. Fe-LNCs were able to encapsulate 90% of the Coumarin 6, which corresponds to 18 μg efficiently loaded Coumarin 6 overall in 2.2 × 109 capsules and 8.2 fg per Fe-LNC. The increased EE can be attributed to the increased volume for encapsulation in the case of Fe-LNCs, since the capsules were found to have increased dimensions and decreased shell thicknesses (Table 1). Active Release Studies. The active release studies have been carried out in 10% SDS by the determination of the amount of Coumarin 6 released as a function of time.
Figure 6. Coumarin 6 release profile from LNCs and Fe-LNCs.
pattern compared to the LNCs. More specifically, only 19% was out at the first 10 min in Fe-LNCs compared to the 70% of the LNCs case, indicating improved shell strength of the capsule in Fe-LNCs. In both cases Coumarin 6 was found to be completely released after 2 h. The encapsulated and released amount highly depends on the shell density. Noteworthy, the thinner shell of the Fe-LNCs appears to be stronger than the simple lignin shell of the LNCs. This reveals that the lignin−Fe nanocontainers have a denser network and are deconstructed more slowly. These experiments prove that the linkages in FeLNCs are sufficiently strong for high resistance of thin walls but do not hamper the deconstruction of the lignin capsule in a surfactant medium, making them useful for future applications. Depending on the concentration of the surfactant release medium and the shell composition, the release properties of our LNCs and Fe-LNCs appear to be flexibly tunable. Role of Fe(III) Coordination Complexes in Lignin Ultrasound Assembled Capsules. Overall, the contribution of the Fe(III) self-assembled complexes in the shell architecture has shown to be of pivotal relevance in order to tune shell thickness. The decrease in shell thickness associated with Fe(III)−lignin complexes leads noteworthy to increase of shell strength and reduced release kinetics of hydrophobic actives. Moreover, the presence of iron coordination complexes into the nanospheres can eventually enlarge the field of potential applications. It has been reported that magnetic microspheres possess a strong magnetic moment and can successfully deliver nonmagnetic substances like cells and drugs to a magnetic field.46 They are small in size (
