Hollow silver alginate microspheres for drug delivery and surface enhanced Raman scattering detection

Abstract

Multifunctional silver alginate hydrogel microspheres are assembled via a template assisted approach using calcium carbonate cores. Sodium alginate is immobilized into the highly porous structure of calcium carbonate microspheres followed by cross-linking in the presence of silver ions. The simultaneous processes of the growth of silver nanoparticles in the alginate matrix and the removal of the calcium carbonate template are triggered by ascorbic acid. The abundance of silver nanoparticles and their interparticular junctions in the alginate network allow for the detection of solutes using Raman spectroscopy using the surface of the plasmonic microspheres. Rhodamine B was used to illustrate the potential applications of such multifunctional plasmonic alginate hydrogel microspheres for sensing at low concentrations. A proof of principle for using such particles for the quick identification of microorganisms is then demonstrated using the Escherichia coli bacterium.

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Introduction

The development of theranostic micro- and nanoconstructs at high spatial resolution, enables fast and cheap diagnosis and treatment of diseases, and thus facilitates the advancement of personalized medicine. However, there are a limited number of structures enabling the combination of drug delivery as well as sensing functions. To date, the potency of such hybrid hydrogel systems has been shown in applications such as targeted drug delivery,¹ biosensors,^2,3 wound dressing,^4–6 sorbent agents,^7,8 and as antimicrobial structures.⁹ The tremendous interest in alginate based hydrogel materials for the fabrication of functional systems is particularly driven due to their biocompatibility, relatively low cost and low toxicity.¹⁰ Hierarchical systems composed of alginate polymer as a matrix constituent in which inorganic nanoparticles (NPs) are distributed, are commonly achieved by controlling the physicochemical properties of alginate,¹¹ the surface chemistry of the NPs as well as the process of production, either triggered by external stimuli or by following a self-assembly technique.¹²

A very versatile and powerful technique for rapid identification and sensing of different analytes, such as drugs, pollutants, biomolecules, microorganisms, and cancerous cells is surface enhanced Raman scattering (SERS). As SERS strongly depends on material, morphology and the incident light for the excitation of surface plasmons it is highly desirable to make reliable, sensitive and reproducible SERS substrates. Usually silver and gold nanoparticles are the most commonly used materials for SERS substrates. This is due their unique optical properties, in particular localized plasmon resonance and the capability to concentrate an electromagnetic field at the interparticular junctions. In comparison with gold, silver is much more favourable for SERS applications due to a stronger resonance and smaller imaginary part of the refractive index in the resonance frequency.¹³ Several techniques have been proposed for the fabrication of highly effective SERS structures ranging from self-assembly on surfaces, drop casting, spin coating, and deposition on substrates to laser deposition¹⁴ and lithographic approaches. Among them the growth of plasmonic nanoparticles directly on substrates such as glass,¹⁵ silicon,¹⁶ silica nanospheres,¹⁷ or polymer films¹⁸ has received much attention. Using this approach, well-ordered and uniformly spaced metallic nanoparticles can be achieved in a simple fashion even within a hydrogel matrix¹⁹ and alginate beads.²⁰

However, the large size of alginate beads limit their application as a drug delivery system, especially for IV administration. Hence, a template assisted approach by using artificial colloidal microparticles has been developed as a simple and versatile method for the construction of functional microspheres ranging from hollow polyelectrolyte microcapsules^21–23 to plasmonic microspheres.^24–28 Amongst numerous solid templates, calcium carbonate (CaCO₃) microparticles^29–31 deserve special attention due to their particular features, such as inexpense, biocompatibility,³² mild decomposition conditions³³ and porous structure.^34,35 The porosity is a valuable characteristic of CaCO₃ microparticles, which enables the incorporation of a variety of molecules, such as insulin,³⁶ oxidised glucose,³⁷ therapeutic enzymes,³⁸ photodynamic dyes,³³ as well as metallic nanoparticles without the assistance of additional functionalization.^39,40

However, using CaCO₃ alone is disadvantageous for use as a carrier due to its instability in water based solutions and weak protection of encapsulated materials.³³ To protect such carriers the encapsulation of the CaCO₃ core is currently achieved primarily using electrostatic deposition of a number of polyelectrolytes,⁴¹ which is a rather expensive and time consuming technique.

Therefore, to combine two functionalities such as template assisted, alginate based drug delivery and a NP based sensing system, our specific aims were (i) to develop a template assisted synthesis approach for hollow alginate beads for drug delivery purposes based on a CaCO₃ template, (ii) since we used Ag for cross linking of the alginate polymers we wished to demonstrate that our approach enabled growth of Ag NPs inside the alginate matrix, aiming to obtain SERS signals on the same beads and (iii) we wished to demonstrate the use of these microparticles for the detection of molecules and analysis of the composition of bacterial cells, using a SERS approach.

Experimental

Materials

Chemicals: sodium alginate (ALG) (C₆H₈O₆), calcium chloride (CaCl₂), silver nitrate (AgNO₃), sodium carbonate (Na₂CO₃), ascorbic acid (C₆H₈O₆), Rhodamine B, and tetramethylrhodamine isothiocyanate–dextran (TRITC–Dex, 35 kDa) were purchased from Sigma-Aldrich. In all experiments ultra-pure water with a resistivity higher than 18.2 MΩ cm was used.

Particle synthesis

Calcium carbonate microparticle synthesis: spherical calcium carbonate microparticles with a mean diameter of 3.6 ± 0.5 μm were synthesized according to the Volodkin protocol³⁵ as follow: 1 mL of Na₂CO₃ (0.33 M) and 1 mL of CaCl₂ (0.33 M) were rapidly mixed in a glass vessel and stirred at 500 rpm for 1 min. Precipitated microparticles of calcium carbonate (CaCO₃) were collected using centrifugation (2000 rpm, 2 min) and subsequently washed with pure ethanol. This procedure was repeated three times. After the washing procedure was completed microparticles were dried in an oven at 60 °C for 30 minutes.

Fabrication of silver alginate hydrogel microspheres: to produce silver alginate hydrogel microspheres, 25 mg of powdered CaCO₃ microparticles was placed in a 2 mL Eppendorf tube. 1 mL of 2% (w/v) sodium alginate was injected into the tube and left under intensive agitation for 10 min in a shaker producing sodium alginate containing CaCO₃ microparticles (AlgCaCO₃). Then, the AlgCaCO₃ particles were precipitated via centrifugation (3000 rpm, 3 minutes) and washed in pure water. The washing procedure was repeated 3 times. Further injection of 0.1 mL of 0.75 M AgNO₃ to AlgCaCO₃ initiated cross-linking of the sodium alginate. The mixture was agitated for five minutes in a shaker and then separated via centrifugation (3000 rpm, 3 minutes) and washed with pure water. Addition of 0.5 mL of 0.1 M ascorbic acid initiated the growth of silver nanoparticles from the Ag in the alginate as well as dissolving the calcium carbonate structure. The formed microspheres with Ag particles were collected using centrifugation (3000 rpm, 3 minutes) and thoroughly washed. Such prepared microspheres were stored in water.

Loading of CaCO₃ particles with TRITC–Dex

The entrapment of TRITC–Dex macromolecules was performed as follow: 25 mg of CaCO₃ microparticles as a powder was placed in a 2 mL plastic tube and 1 mL of TRITC–Dex solution (1 mg mL⁻¹) was added and intensively shaken for 30 min. The suspension containing the microparticles was centrifuged (3000 rpm, 3 min) and washed once with water. The collected microparticles loaded with TRITC–Dex were then ready to be used for the fabrication of silver alginate microspheres as mentioned above.

Particle characterization

Particle morphology was characterised using scanning electron microscopy (SEM) with MIRA II LMU (Tescan) at the operating voltage of 15 kV, in secondary electron and back scattering electron mode. In our study we used magnifications between 100 and 40.000 times. Samples were prepared by air drying a drop of an aqueous suspension of the microparticles on a silicon wafer at room temperature.

An analysis of the silver nanoparticle distribution in samples was performed using transmission electron microscopy (TEM) with a Libra-120 (Carl Zeiss, Germany).

The size distribution of the calcium carbonate particles was obtained by post processing and image analysis of SEM micrographs using Image J software (NIH, http://rsb.info.nih.gov/ij/). At least 100 measurements per sample were performed.

The X-ray powder diffraction patterns were obtained using a Xcalibur/Gemini diffractometer using Cu-Ka radiation. The accelerating voltage and the applied current were 40 kV and 40 mA, respectively. The theoretical CaCO₃ and Ag spectra were taken from the works of Le Bail A.⁴² and Saha²⁰ for calcite and vaterite, respectively, and from Owen⁴³ for Ag.

The zeta-potential of the nanoparticles suspension was measured by averaging the data from 10 replicates analysed using a Zetasizer Nano-Z (Malvern Instruments Ltd, UK).

Optical microscopy analysis was performed using a Leica TCS SP inverted confocal scanning microscope (Leica, Germany) equipped with a 100/1.4–0.7-oil immersion objective.

Raman studies

A confocal Raman microscope (Renishaw inVia, UK) equipped with a diode-pumped 785 nm NIR laser excitation (60 mW) controlled by a neutral optical density filter was used. The laser beam was focused through a 50× (Leica N PLAN, NA = 0.5) microscope objective. The spectra were acquired using a thermoelectrically cooled CCD detector optimized for near IR (spectral resolution of 1 cm⁻¹). All spectra were collected using WiRE software V4.1 (Renishaw, U.K.) and SynchroScan was applied for processing of the spectra. The laser power in all SERS measurements was 0.03 mW and an integration time of 10 s was used. The laser power was measured using a power meter (Newport – Optical Power Meter 1830-C) before the microscope objective.

Bacteria studies

A bacterial culture of Escherichia coli strain K-12 was cultivated overnight in liquid GRM medium (15 g and 10 g of pancreatic hydrolysate of fish flour and casein, respectively, 2 g of yeast extract, 3.5 g of NaCl and 1 g of glucose in distilled water and adjusted to the final volume of 1 L). The 4 mL of overnight bacterial culture of an optical density of about 1.2 measured at 600 nm wavelength (OD₆₀₀) was centrifuged for 5 min at 5000 g at room temperature. The obtained pellet was washed three times with a sterile 0.9% NaCl solution through suspension of the pellet and centrifugation at 5000 g for 5 min. The suspension was prepared in such a way that it contained 1.5 × 10⁹ bacterial cells per mL.

Results and discussion

We prepared the silver alginate whole particles decorated with silver nanoparticles with a narrow size distribution. To achieve this a natural porous template of calcium carbonate was employed.

Sodium alginate (SA) was directly immobilized onto porous CaCO₃ microparticles following ionic crosslinking in silver nitrate solution. At this stage silver ions penetrated the alginate matrix and conjugated with the hydroxylic and carboxylic groups in the hydrogel network. Plenty of hydroxylic and carboxylic groups in the alginate chain can adsorb Ag⁺ and thus initiate cross-linking between the 1,4-linked β-D-mannuronic (M) and α-L-guluronic (G) acid residues of alginate.⁴⁴

According to our results, the sodium alginate (SA) impregnated the pores of the vaterite microparticles following ionic cross-linking in the silver nitrate solution. This mechanism of hydrogel formation usually occurs in the presence of divalent ions, such as Ca²⁺, Mn²⁺, Co²⁺, Cu²⁺, Cd²⁺.⁴⁵ The influence of Ca²⁺, which might appear from the CaCO₃ microparticles upon the formation of the hydrogel can be neglected due to stability of CaCO₃ for short periods in water. In addition, this can be excluded because the entire procedure for the fabrication of the silver alginate hydrogel microspheres takes several minutes, which is not enough to saturate the solution with Ca²⁺. In the case of Ag⁺ both carboxylic and hydroxylic groups participate in ion uptake. It is likely that carboxylic groups facilitate the adsorption of silver ions, whereas hydroxylic groups can participate in the reduction to metal⁴⁵ nanoparticles. Moreover, hydroxylic groups have the ability to coordinate Ag⁺ ions providing their capping and furthering the growth of the nanoparticles. Scanning electron microscopy images show that the microspheres possess a smooth surface with relatively low contrast between the CaCO₃ and CaCO₃ covered by SA (Fig. 1a and b). In contrast, we observed a high contrast and rough surface in the SEM images after adding silver nitrate, which can indicate the uptake of metal ions and their reduction (Fig. 1c). After the treatment with silver nitrate, relatively small seeds in the order of a few nanometers nucleate in the alginate matrix.

Fig. 1 Scheme of preparation of silver alginate hydrogel microspheres by impregnation of sodium alginate (2 mg mL⁻¹) in the porous structure of calcium carbonate microparticles. The addition of silver nitrate initiates the gelling of the sodium alginate and populates the sodium alginate with silver ions. The growth of the silver nanoparticles is accomplished using ascorbic acid, which also removes calcium carbonate yielding the hollow structure of the silver alginate hydrogel microspheres. SEM images corresponding to (a) calcium carbonate; (b) calcium carbonate covered with sodium alginate; (c) calcium carbonate covered with sodium alginate after the injection of silver nitrate; (d) silver alginate hydrogel microspheres. The scale bars correspond to 1 μm.

Subsequent growth of the silver nanoparticles was initiated immediately by adding ascorbic acid to the solution containing modified calcium carbonate microparticles. At this stage, a few possible processes can take place: (1) ascorbic acid initiates the growth of silver nanoparticles in the hydrogel network structure; (2) the acidic environment dissolves the calcium carbonate microparticles; (3) calcium ions that appeared during the dissolution of the calcium carbonate might additionally cross-link the sodium alginate and thus increase the stability of the microspheres. The X-ray diffraction (XRD) measurements revealed five diffraction peaks (111), (200), (220), (311) and (222) that can be assigned to a face-centered cubic (fcc) Ag lattice. No peaks from either the silver chloride (AgCl) crystals or from CaCO₃ were found in the X-ray spectra of the silver alginate hydrogel microspheres. This indicates the formation of silver nanoparticles preferentially on the carboxylic/hydroxylic groups instead of the silver chloride residues. This satisfies the prediction of the hollow structure of the silver alginate hydrogel microspheres. The magnified SEM and TEM images of one individual silver alginate microsphere indicate the hollow structure of the microspheres (Fig. 2c) containing mostly non transparent silver nanoparticles (Fig. 2d). The SEM images also show that the silver alginate hydrogel microspheres maintain their size (3 ± 1 μm) in agreement with the initial CaCO₃ template. The surface charge of the silver alginate microsphere was determined by measuring the electrophoretic mobility. The zeta-potential of the plasmonic microspheres was determined to be −22 mV, which is higher than the CaCO₃ microparticles (−10 mV). This means that the Ag ions in the alginate matrix provide a better charge distribution in the particles. Such colloidal stability of the particles prevented their aggregation. To demonstrate the functionality of the alginate microspheres, dye labelled dextran molecules (TRITC–Dex) were loaded in their cavities. This was achieved by taking advantage of the porous structure of the CaCO₃ microparticles. The confocal scanning laser microscopy (CLSM) image reveals that the TRITC–Dex was successfully embedded in the cavity of the alginate microspheres (Fig. 2e). To estimate the loading efficiency of the embedded dye molecules spectrofluorometric measurements were performed. The loading capacity was determined to be 10 wt%, which is consistent with the data in a previous study.^32,39 A high content of silver nanoparticles in the alginate microspheres allowed detection of a low concentration of RhoB by acquiring the Raman spectra from the microspheres.

Fig. 2 (a) XRD pattern of silver alginate microspheres (top) and calcium carbonate microparticles (bottom). (b) SEM image of silver alginate hydrogel microspheres. (c) Magnified SEM image of silver alginate microspheres. Back scattered electron mode was used for the SEM images. (d) Transmission electron microscopy image of silver alginate particles (e) confocal fluorescence microscopy image of silver alginate microspheres loaded with TRITC–dextran molecules (red color). The scale bars on b, c, d and e images correspond to 5, 1, 2 and 20 μm respectively.

The SERS capability of the silver alginate microspheres was tested by measuring RhoB as a model analyte. Prior to the SERS measurements, RhoB was dissolved in water at a concentration of 10⁻³ M and further diluted with a factor of 10², 10³, 10⁵, and 10⁶. Silver alginate microspheres were incubated with a selected concentration of RhoB for 1 hour to ensure adsorption of the chromophore molecules onto the surface of the microspheres. The typical reference Raman and SERS spectra of RhoB at selected concentration are plotted in Fig. 3. The SERS measurements were acquired from a minimum of 10 microspheres. The peak at 1203 cm⁻¹ is due to C–C stretching, and C–O–C stretching can be seen at 1281 cm⁻¹. Strong peaks located at 1358 cm⁻¹, 1507 cm⁻¹, 1530 cm⁻¹, and 1648 cm⁻¹ represent aromatic stretching of the dye.

Fig. 3 SERS spectra acquired from the surface of the silver alginate microspheres upon adsorption of RhoB (measured concentrations are included in each SERS spectra). Raman reference spectra of RhoB (10–3 M) and blank silver alginate microspheres are shown for reference. All spectra are shifted for clarity. All SERS spectra were acquired with a laser wavelength of 785 nm through a 50× air objective at a power of 0.03 mW.

The effect of the concentration of Rhodamine B on the SERS sensitivity of the silver microspheres was also measured. As shown in Fig. 3 the microspheres allow detection of Rhodamine molecules at concentrations as low as 10–8 M. A reliable SERS signal and peaks, which are attributed to Rhodamine B were detected for 10–5 M, 10–6 M and 10–8 M concentrations of dye. The SERS spectrum acquired at a low concentration of RhoB was not sufficient, and alginate peaks dominated. In fact, bands for the alginate matrix are also present in spectra for the samples that were measured at higher concentrations of dye molecules, however those peak do not overlapped with the dye peaks between 1200 cm⁻¹ and 1600 cm⁻¹ (Fig. S1†). No peaks attributed to calcium carbonate microparticles were measured, which indicates the decomposition of the CaCO₃ microparticles. To evaluate the efficiency of the silver alginate microspheres an analytical enhancement factor (EF) was calculated using an intensity at 1358 cm⁻¹, which corresponds to the aromatic C–C stretching vibrational mode. The enhancement factor was found to be equal to 1.7106, which is in agreements with the EF obtained for silver based structures.^46,47

In order to show the versatility of the silver alginate microspheres, the detection of microorganisms in solution was performed by detecting E. coli. The SERS spectra showed the chemical signature of the bacteria (Fig. 4). The bands in the SERS spectra are assigned to bacterial features, which are frequently found in the Raman spectra of E. coli strains.^48,49 Two intense bands at 733 cm⁻¹ and 1334 cm⁻¹ are assigned either to flavin derivates or other adenine containing molecules. The peak centred at 1373 cm⁻¹ originated from DNA, while bands at 625 cm⁻¹ are assigned to COO-chemical groups. The peak at 1450 cm⁻¹ originates from lipids.

Fig. 4 SERS spectra of E. coli (a) obtained from randomly selected silver alginate microspheres by resuspension of those colloids in solution containing E. coli (curves 3–6) and a normal Raman spectrum of E. coli in solution (curve 1). Curve 2 corresponds to the Raman signal generated from silver alginate microspheres in PBS buffer. Optical image of two silver alginate microspheres (b). The inset shows a Raman reconstructed image of a silver alginate microsphere, which is plotted for adenine containing molecules at 733 cm⁻¹ (dotted square in optical image). The Raman signal of the adenine containing molecules is shown in red. The scale bar in the optical image corresponds to 10 μm.

The mixing of silver alginate microspheres and bacterial cells might induce their aggregation, and thereby bacteria are taken in between a few microspheres. Another possibility for the interaction of bacteria with the SERS microspheres relies on the porous morphology of the silver alginate microspheres, which facilities coupling between the beads and cells. All these factors can contribute to the fast and label-free detection of molecular signatures of the surface of the bacterial cells. In addition, the Raman imaging also shows the presence of adenine signatures (731 cm⁻¹) on the surface of the bacterial cells (Fig. 4b).

Conclusions

In conclusion, we showed a simple fabrication method for plasmonic hollow microspheres based on the template assisted fabrication of organic–inorganic hybrids composed of a biocompatible alginate matrix and silver nanoparticles. This conjugation takes advantage of porous calcium carbonate microparticles, with respect to filling their pores with sodium alginate. The gelling of the sodium alginate was then achieved in the presence of silver ions that were further grown in situ by adding ascorbic acid. Both silver nanoparticle expansion and calcium carbonate dissolution were initiated by the ascorbic acid yielding hollow plasmonic microspheres. A high content of silver nanoparticles in the alginate microspheres with small interparticular junctions enabled a strong enhancement of Raman scattering. The elaborated structure was demonstrated to allow the detection of a low concentration of Rhodamine B with an analytical EF up to 10⁶. The possibility of using silver alginate particles as a SERS platform for quick bacterial identification was demonstrated. We believe that such silver alginate microspheres can be used for the SERS based detection of molecules, as well as a reservoir for the loading of a payload. The silver alginate hydrogel microspheres possess a dual functionality. They can be used for theranostics according to a concept described in ref. 49 and 50.

Acknowledgements

The study was supported by the Government of the Russian Federation (grant No. 14.Z50.31.0004 to support scientific research projects implemented under the supervision of leading scientists at Russian institutions and Russian institutions of higher education). The study was partly supported by RFBR, research project No. 15-29-01172 ofi_m. B. P. acknowledges the FWO for postdoctoral scholarship. We would like to thank B. Khlebtsov (Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, Saratov 410049, Russia) for TEM measurements, and A. Skaptsov (Saratov State University, Saratov, Russia) for XRD measurements.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra02019d

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