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Silver nanoparticles modified nanocapsules for ultrasonically activated drug delivery
Materials Science and Engineering C 32 (2012) 2349–2355
Contents lists available at SciVerse ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Silver nanoparticles modified nanocapsules for ultrasonically activated drug delivery S. Anandhakumar a, V. Mahalakshmi a, Ashok M. Raichur a, b,⁎ a b
Department of Materials Engineering, Indian Institute of Science, Bangalore 560012, India Department of Applied Chemistry, University of Johannesburg, Doornfontein, 2028, South Africa
a r t i c l e
i n f o
Article history: Received 31 January 2012 Received in revised form 22 June 2012 Accepted 4 July 2012 Available online 10 July 2012 Keywords: Silver NPs In-situ synthesis Ultrasonication Remote activated drug delivery
a b s t r a c t Novel ultrasound-sensitive nanocapsules were designed via layer-by-layer assembly (LbL) of polyelectrolytes for remote activated release of biomolecules/drug. Nanocapsules embedded with silver nanoparticles in the walls were synthesized by alternate assembly of poly(allylamine hydrochloride) (PAH) and dextran sulfate (DS) on silica template followed by nanoparticle synthesis and subsequent template removal thus yielding nanocapsules. The silver NPs were synthesized in situ within the capsule walls under controlled conditions. The nanocapsules were found to be well dispersed and the silver NPs were evenly distributed within the shell. FITC-dextran permeated easily into the capsules containing silver NP's due to the pores generated during the formation of NP's. When the loaded nanocapsules were sonicated, the presence of the silver NPs in the shell structure led to rupturing of the shell into smaller fragments thus releasing the FITC-dextran. Such nanocapsules have the potential to be used as drug delivery vehicles and offer the scope for further development in the areas of modern medicine, material science, and biochemistry. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Externally activated and targeted release of drugs has a high priority for further development in nanomedicine and drug delivery [1–5]. They provide many advantages over other conventional systems such as increased bioavailability, efficacy and reduction of systemic toxicity. In order to meet these requirements, various drug delivery systems based on polymer and other inorganic nanostructures have been designed to increase the efficacy of drugs [6–11]. Among them, polyelectrolyte capsules (PECs) have received considerable attention, as they offer the added advantage of surface functionalization by several methods [12,13]. As a result, there is a continuing research interest in designing PECs with different functions which are responsive to external (modern) triggers such as magnetic, enzymatic, light and ultrasound [14–17]. The presence of NPs/enzymes in the shell structure allows the development of nanopores within the shell structure leading to final rupture of the shell under a given stimulus. Hence, such modified capsules allow tunable permeability for loaded molecules, from relatively slow release to burst like release based on a predesigned mechanism. Modified capsules are prepared by either fabricating capsules with enzyme degradable polyelectrolytes or incorporating metal nanoparticles (NPs) in the capsule wall, which on treatment with enzymes or metal NP based triggers (light, magnetic and ultrasound) release their contents.
Previously, metal NPs have been incorporated into PECs by either using them as one of the shell components or by introducing them into the previously-formed capsule suspension [16,17]. This often leads to inhomogeneous deposition of NPs on the capsule surface due to aggregation of capsules in the suspension. The limitations of these methods can be partially solved by in-situ synthesis of Ag NPs in the capsule wall having a more uniform distribution [18,19]. However, size distribution of in-situ synthesized NPs is quite often not narrow. Also, since the NPs are embedded in an insoluble complex gel-like structure, it causes the NPs to aggregate after synthesis in the wall which in turn can affect the encapsulation process. Further, the methods developed so far are quite complex making them unsuitable for practical applications. Hence it is important to develop a new method to produce nanoscale vehicles (nanocapsules) for externally activated drug delivery. An ideal triggerable nanocapsule system should be easy to prepare from readily available layer components, exhibit good responsiveness and should be compatible with more than one trigger. Here we extend our polyol route of synthesizing Ag particles to nanocapsules and demonstrate its applicability as an externally triggerable drug delivery system [20]. In this study we provide a proof-of-concept towards designing the “remote-rupturing nanocapsules” for release of active substance through external ultrasonic treatment. To the best of our knowledge, fabrication of NPs incorporated nanocapsules by in-situ synthesis for remote activated drug release has not been reported yet. 2. Materials and methods
⁎ Corresponding author at: Department of Materials Engineering, Indian Institute of Science, Bangalore 560012, India. Tel.: +91 80 22933238; fax: +91 80 23600472. E-mail address: [email protected] (A.M. Raichur). 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2012.07.006
Dextran sulfate (DS) (MW = 500 kDa), poly(allylamine hydrochloride) (PAH) (MW = 70 kDa), poly(ethylene glycol) (PEG) (MW =
2350
S. Anandhakumar et al. / Materials Science and Engineering C 32 (2012) 2349–2355
6 kDa), tetraethyl orthosilicate (TEOS), ethanol, ammonium hydroxide, hydrofluoric acid (HF), FITC-dextran (Mw = 70 kDa), and ammonium fluoride (NH4F) (Sigma-Aldrich) and silver nitrate (Ranbaxy, India) were used without any further purification. Milli-Q water with resistivity greater than 18 MΩ cm was used in this study. All pH adjustments were done with 0.1 M HCl or 0.1 M NaOH. 2.1. Synthesis of silica nanoparticles Silica nanoparticles were prepared by hydrolysis of TEOS in ethanol medium in the presence of ammonium hydroxide [21]. Briefly, 0.1 M of TEOS in ethanol medium was ultrasonicated in a bath. After 20 min, 28% ammonium hydroxide was added as a catalyst to promote condensation reaction. Sonication was continued for another 60 min to get a white turbid suspension of silica nanoparticles. Then the particles were washed with water and used for LbL assembly. 2.2. Capsule preparation Capsules were prepared by the LbL technique using silica nanoparticles as sacrificial templates. The silica particles (1% w/w) were alternatively coated at pH 5 by incubating them in 1 mg/ml PAH and DS polymer solution respectively prepared with 0.2 M NaCl. After adsorption for 15 min, the particles were separated by centrifugation and the residual unadsorbed polyelectrolyte was removed by washing thrice with pH 5 water. Even with charges on their surfaces, silica particles in the nanoscale have a tendency to aggregate due to their large surface area and high interfacial energy. In order to avoid this, centrifugation during assembly and washing steps were carried out at 7000 rpm for 15 min. These parameters were obtained after conducting ‘n’ number of trial experiments by changing parameters such as particle concentration, centrifugation speed and time. After four bilayers were deposited, the silica core was dissolved in HF buffer (0.1 M HF: 0.2 M NH4F) and the obtained capsules were washed and stored in water.
2.5. Ultrasonic irradiation The effect of sonication on the stability of capsules was investigated by sonicating the capsule suspension in a bath reactor. Irradiation was performed with an ultrasonic generator operating at 170 W and 50 Hz (Soniclean 160HT, Australia), which works at an ultrasonic power comparable to those of ultrasonic generators used in medicine and in pharmaceutical field. Before and after irradiation, the capsules were analyzed by atomic force microscopy (AFM) to visualize the morphological changes that occurred on the capsule surface. For release studies, the capsules loaded with FITC-dextran were exposed to ultrasonic treatment and fluorescence spectra for FITC-dextran in the supernatant were recorded at 490 nm (excitation wave length of FITC-dextran). After sonication, each sample was sedimented by centrifugation for 30 min at 7000 rpm and the supernatant was subjected to fluorimetry. It was observed that at times, only centrifugation did not effectively settle down the broken fragments. In such cases, the sample was gently left to stand in the fridge for several days and the fluorescence of supernatant was then measured to get much more reliable data. 2.6. Characterization Confocal images of polyelectrolyte capsules in water were observed using a Zeiss LSM 510 META confocal scanning system (Zeiss, Germany) equipped with a 100×/1.4–1.7 oil immersion objective. To visualize the capsules, FITC-dextran was used as model probe molecule. Fluorescence spectra of the released dextran were recorded using a Jobin Yvon Fluorolog3 spectrofluorimeter (Horiba Scientific Instruments, USA). For AFM analysis, a drop of capsule suspension was placed on silicon wafer and air dried overnight. Then the samples were characterized using a Nanosurf Easy Scan2 AFM (Nanoscience Instruments Inc., USA) in air at room temperature by contact mode. FE-TEM measurements were performed on a Tecnai F30 (FEI, Eindhoven, Netherlands) microscope operating at 200 kV. 3. Results and discussion
2.3. Fabrication of hybrid nanocapsules 3.1. Preparation of silica NPs and nanocapsules The fabrication of silver NP modified microcapsules has been reported earlier [20]. The same procedure was used here to produce nanocapsules and parameters such as centrifugation speed, time etc., were optimized to prevent aggregation during LbL assembly. Fabrication of nanocapsules involved the sequential adsorption of three layers of PAH and DS on silica nanoparticles followed by silver NP synthesis. AgNO3 was used as precursor for the formation of silver NPs and reduction was performed at 50 °C in the presence of PEG. After the synthesis of silver NPs, the coated particles were washed and deposited with another bilayer of PAH and DS. Then the silica core was dissolved with HF buffer to yield silver NP incorporated hollow capsules. Finally the capsules were washed three times with water and used for ultrasonic experiments.
Silica nanoparticles were prepared by Stober's method and used as templates for capsule fabrication. TEM investigation revealed that the silica particles had a mean diameter of 500 ± 100 nm. The particles were found to be spherical, smooth and non-aggregated as shown in Fig. 1. Alternate deposition of PAH and DS on to these silica particles
2.4. Encapsulation of FITC-dextran FITC-dextran was encapsulated inside the polyelectrolyte capsules by thermal encapsulation method [22]. The silver NP synthesized capsules were incubated with FITC-dextran at room temperature for 1 h followed by incubation at 70 °C for 1 h. After encapsulation, the capsule suspension was washed three times with water to remove the excess dextran present in the supernatant solution. Pure polyelectrolyte capsules (without silver NPs) filled with FITC-dextran was used as control capsules. For control capsules, the encapsulation was carried out by mixing capsule suspension with FITC-dextran at pH 3.5 in 0.2 M NaCl solution for 1 h and incubated at 70 °C for another 1 h as mentioned previously.
Fig. 1. TEM image of the prepared silica nanoparticles.
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Fig. 2. Scheme demonstrating the preparation of silver NP modified hybrid nanocapsules and encapsulation and release of dextran using ultrasonic irradiation. (a—Prepared silica template; a–b → LbL assembly of PAH and DS; b–c → Silver NP synthesis and dissolution of template; c–d → Incubation of capsules in dextran solution; d–e → Thermal encapsulation of dextran at 70 °C; e–f → Washing of loaded capsules to remove excess dextran present in supernatant solution; f–g →Release of dextran after ultrasonic irradiation).
followed by dissolution of silica core with HF yielded hollow capsules. Two types of capsules were prepared, the first one was solely composed of polyelectrolytes (PAH/DS)4, while the second one had a hybrid shell composition [(PAH/DS)3/AgNPs/(PAH/DS)] consisting of Ag NPs and PAH/DS polyelectrolytes. A schematic of the procedure for fabrication of hybrid nanocapsules is shown in Fig. 2. Stable capsules were produced with and without embedded silver nanoparticles and the capsules were not damaged during core dissolution as shown in Fig. 3. Both the capsules showed that collapsed structure and the spherical structure of nanocapsules were retained. The presence of silver NPs in the wall did not seem to alter the shape and stability of capsules significantly when compared to pure polyelectrolyte capsules. The diameter of the hybrid capsules is about 500 ± 100 nm with a thickness range of 30 ± 5 nm (AFM data not shown here). The capsules were also examined by TEM. As seen in Fig. 4, both the capsules retained their spherical morphology and were well dispersed without any sign of aggregation. Hybrid capsules appeared darker than the pure polyelectrolyte capsules due to the presence of silver NPs. It can be seen in Fig. 4c, that silver NPs are uniformly distributed within the walls of the capsule. Averaging the size of NPs present in 20 capsules yielded a mean diameter of 25 ± 5 nm which is consistent with AFM measurements.
3.2. Capsule permeability and encapsulation FITC-dextran was used as a model biomolecule to study the permeability of the nanocapsules. Both pure capsules and silver NP embedded capsules were incubated with FITC-dextran (1:1 volume ratio) for 1 h and visualized under CLSM. Fig. 5a and b shows confocal images of FITC-dextran stained polyelectrolyte and hybrid capsules in aqueous solution. Pure polyelectrolyte capsules were impermeable whereas hybrid capsules were easily permeable to dextran molecules which can be attributed to pore formation during NPs synthesis. Therefore, it is believed that the formation of new NPs during synthesis creates defects and discontinuities in the form of voids, pores and crevices or small breakage in the polymeric shell network thus allowing the penetration of macromolecules [23,24]. For encapsulation studies, thermal encapsulation method was used to load FITC-dextran in the capsules. This method involves the heat treatment of capsule suspension in the presence of low molecular weight compounds, by exploiting the decrease in permeability for encapsulation [22]. Fig. 5c shows the CLSM image of the hybrid nanocapsules loaded with FITC-dextran at 70 °C. The shell shrinkage and densification induced by heat treatment prevents the FITC-dextran movement from interior to bulk. However, due to the limited optical resolution of CLSM, it
Fig. 3. AFM images of nanocapsules. (a) Pure polyelectrolyte capsule and (b) hybrid capsule with synthesized silver NPs. After synthesizing the silver NPs, the thickness of the capsules is increased from 15 ± 3 nm to 30 ± 5 nm. The average size of the nanocapsules prepared was 500 ± 100 nm.
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Fig. 4. TEM images of nanocapsules. (a) Pure polyelectrolyte capsules, (b) Silver NP modified capsules at low magnification and (c) at high magnification. Insets show zoomed cross section of the marked area in the corresponding images. The size of NPs present in the capsule shell is 25 ± 5 nm.
was difficult to conclude from this image as to whether the FITC-dextran was inside the capsule or just adsorbed on the surface of the capsules. In order to confirm the encapsulation, the morphological changes of capsules prior and after encapsulation were monitored by AFM investigations. The AFM image of empty and loaded capsules showed different morphologies. The control capsules were completely collapsed and flat (Fig. 3a) whereas the loaded capsules exhibited a much higher average vertical height than control capsules as shown in Fig. 6. The typical double wall thickness of a pure polyelectrolyte capsule (obtained from AFM) was about 15±3 nm which increased to 90±10 nm after FITC-dextran encapsulation. It is believed that this increase in thickness is contributed from both encapsulation and temperature enhanced swelling of the capsules. It is well known that wall densification and swelling of the capsules occur at the expense of reduction in capsule diameter when they are heat treated at elevated temperature [22]. It is difficult to assess the contribution of encapsulation and temperature treatment towards swelling of the
Fig. 5. Investigation of permeability and encapsulation of FITC-dextran by CLSM. (a) Pure polyelectrolyte capsules, (b) Silver NP modified hybrid capsules and (c) thermally encapsulated hybrid capsules. Scale bar=2 μm.
capsules. However, the strong fluorescence signal from the capsule interior confirmed that significant amount of FITC-dextran has been loaded in the capsules. 3.3. Ultrasonic irradiation With the perspective of using these capsules as drug delivery vehicles for remote-activated release, we investigated the rupture of the capsules under ultrasonic irradiation. It has been mentioned in the literature that ultrasound intensity of the range of 1000–10,000 W/cm 2 is used for different diseases like uterine fibroid treatment [25,26]. This intensity can induce lesions, or tissue necrosis at a small location deep in the tissue while leaving the tissue between the ultrasound source and focus unharmed. However, this occurs only when the focal temperature exceeds 70 °C [25]. In order to avoid this issue, we have used ultrasonic irradiation of maximum power of 170 W
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Fig. 8. Fluorescence spectrum of released FITC-dextran from silver NP modified nanocapsules at various time intervals.
the shells heavy and increases the rigidity of the capsules thus it favors the rupture of the capsules.
3.4. Release studies
Fig. 6. AFM image of FITC-dextran loaded (silver) capsule and its height profile. After thermal encapsulation, the average height of the capsule increased from 15 ± 3 nm to 90 ± 10 nm at the expense of diameter reduction from 500 ± 100 nm to 350 ± 50 nm.
for our experiments. Fig. 7 shows the morphological changes of the capsules when exposed to ultrasonic irradiation for various time intervals. During ultrasonication, acoustic cavitation bubbles are formed which collapse with greater energy in the suspension causing mechanical damage to the capsules. Due to this, the capsules gets ruptured and deformed after ultrasonic irradiation. It should be noted that the capsule surface is no longer continuous and is broken into smaller fragments. As shown in Fig. 7a and c, the rupturing of the capsules occurs at even less than 5 s of ultrasonic exposure and there were significant size differences in the fragments of ruptured capsules when the exposure time was increased to 15 s. It is understood that the presence of silver NPs in the wall creates a density contrast in the polymeric network and reduces the elasticity of the capsule wall, which makes them more prone to rupture by acoustic cavitation [17,27]. It can be concluded that presence of NPs makes
Fluorescence spectrometry was used to investigate the release of dextran from the loaded capsules as shown in Fig. 8. For release studies, thermally encapsulated capsules were irradiated for different time interval (up to 15 s). After irradiation, the capsule suspension was centrifuged and FITC-dextran fluorescence emission from the supernatant was recorded. The fluorescence signal of high intensity at 515 nm corresponding to FITC-dextran was observed in all the samples. The fluorescence spectra obtained from the samples showed that there is no significant difference in fluorescence intensity of released FITC-dextran. It confirmed that the rupture of capsules is immediate leading to the burst release of dextran. This result corroborates well with the rupturing study investigated by AFM. On the contrary, no such fluorescence signal was observed from the control capsules (pure polyelectrolyte nanocapsules) even after 1 min of ultrasonic irradiation as shown in Fig. 8. When the exposure time was increased beyond 2 min, very low intensity of the fluorescence signal was observed corresponding to low amount of release of the loaded FITC-dextran (data not shown). This might be caused by diffusion of FITC-dextran from capsule interior to bulk due to concentration gradient. This proves our hypothesis that hybrid capsules rupture faster than the control capsules thus releasing the encapsulated dextran.
Fig. 7. AFM images to show the rupturing behavior of nanocapsules at various time intervals. (a) 5 s, (b) 10 s and (d) 15 s.
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Fig. 9. Release of FITC-dextran with various exposure time.
3.5. Quantification of release Release of FITC-dextran after ultrasonic irradiation was investigated in water at pH 7.4. In order to avoid the diffusion based release (release by concentration gradient), experiments were performed within 15 min at a time interval of 5 min. Hybrid and control capsules showed different release profile as shown in Fig. 9. Hybrid capsules showed burst release of around 70% due to instantaneous rupture of
the capsules and there was no significant release after 5 min. On the other hand, in control capsules the release was almost linear. The amount of dextran released increased up to 15% which can be attributed to the large imbalance of dextran concentration between capsule interior and bulk. The relationship between the ultrasonic power (intensity) and temperature of medium on release was investigated. As shown in Fig. 10a, there was no significant difference in release observed when the power increased from 50 to 170 W. So, it is believed that small nanopores formed during the ultrasonication at 50 W were enough to allow the dextran to move from the capsule interior to bulk. For temperature experiments (Fig. 10b), the release was almost constant when the temperature was increased from 30° to 70 °C. This is due to the fact that capsules gets ruptured faster (with in 1 min) in the presence of silver NPs. Similarly, pure polyelectrolyte capsules (control capsules) did not show any change in release when the release was performed at different power of ultrasonication and temperature of the medium (data not shown). These results demonstrate that the nanocapsules reported here do have the potential to be used for drug delivery and can be easily activated externally by exposing them to ultrasonic irradiation. 4. Conclusion We have demonstrated the fabrication of hybrid nanocapsules containing Ag NPs in their shell for ultrasonically activated drug delivery. The pores formed due to in-situ synthesized NPs in the capsule shell could be effectively exploited for the encapsulation of FITCdextran by utilizing thermal encapsulation method. The capsules containing NPs ruptured quickly when subjected to sonication. The release studies showed that these capsules were ruptured within 5 s resulting in the release of loaded dextran molecules. These capsules can also be further engineered to be biocompatible and susceptible to remotely induced release using various triggers such as ultrasonication and light due to the presence of silver NPs in the shell. Thus drug delivery vehicles with performance metrics like the one presented here have the potential to be studied under in vivo conditions. Acknowledgments We thank the Institute Nanoscience Initiative, Indian Institute of Science for microscopy facility and Prof. S. Ramakrishnan, Department of Inorganic and Physical Chemistry for fluorescence spectrometer. This work is supported by Dow Chemical International Pvt. Ltd., India. References
Fig. 10. Influence of sonication power (a) and temperature (b) on release of dextran.
[1] A.G. Skirtach, A.M. Yashchenok, H. Möhwald, Chem. Commun. 47 (2011) 12736. [2] A.P. Esser-Kahn, S.A. Odom, N.R. Sottos, S.R. White, J.S. Moore, Macromolecules 44 (2011) 5539. [3] G.B. Sukhorukov, in: S. Svenson (Ed.), Carrier-Based Drug Delivery, ACS Symposium Book Series, 879, ACS, D. C. Washington, 2004, pp. 249–266. [4] S. Anandhakumar, V. Nagaraja, A.M. Raichur, Colloids Surf. B: Biointerfaces 78 (2010) 266. [5] S. Anandhakumar, S.P. Vijayalakshmi, G. Jagadeesh, Ashok M. Raichur, ACS Appl. Mater. Interfaces 3 (9) (2011) 3419. [6] S. De Koker, R. Hoogenboom, B.G. De Geest, Chem. Soc. Rev. 41 (2012) 2867–2884. [7] K. Ariga, Y.M. Lvov, K. Kawakami, Q. Ji, J.P. Hill, Adv. Drug Deliv. Rev. 63 (2011) 762–771. [8] Y. Kaneda, Adv. Drug Deliv. Rev. 64 (2012) 730–738. [9] K. Ariga, A. Vinu, Y. Yamauchi, Q. Ji, J.P. Hill, Bull. Chem. Soc. Jpn. 85 (1) (2012) 1–32. [10] T.K. Dash, V.B. Konkimalla, J. Control Release 158 (2012) 15–33. [11] K. Ariga, Q. Ji, M.J. McShane, Y.M. Lvov, A. Vinu, J.P. Hill, Chem. Mater. 24 (2012) 728. [12] G. Decher, Science 277 (1997) 1232. [13] E. Donath, G.B. Sukhorukov, F. Caruso, S.A. Davis, H. Möhwald, Angew. Chem. Int. Ed. 37 (16) (1998) 2201.
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Contents lists available at SciVerse ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Silver nanoparticles modified nanocapsules for ultrasonically activated drug delivery S. Anandhakumar a, V. Mahalakshmi a, Ashok M. Raichur a, b,⁎ a b
Department of Materials Engineering, Indian Institute of Science, Bangalore 560012, India Department of Applied Chemistry, University of Johannesburg, Doornfontein, 2028, South Africa
a r t i c l e
i n f o
Article history: Received 31 January 2012 Received in revised form 22 June 2012 Accepted 4 July 2012 Available online 10 July 2012 Keywords: Silver NPs In-situ synthesis Ultrasonication Remote activated drug delivery
a b s t r a c t Novel ultrasound-sensitive nanocapsules were designed via layer-by-layer assembly (LbL) of polyelectrolytes for remote activated release of biomolecules/drug. Nanocapsules embedded with silver nanoparticles in the walls were synthesized by alternate assembly of poly(allylamine hydrochloride) (PAH) and dextran sulfate (DS) on silica template followed by nanoparticle synthesis and subsequent template removal thus yielding nanocapsules. The silver NPs were synthesized in situ within the capsule walls under controlled conditions. The nanocapsules were found to be well dispersed and the silver NPs were evenly distributed within the shell. FITC-dextran permeated easily into the capsules containing silver NP's due to the pores generated during the formation of NP's. When the loaded nanocapsules were sonicated, the presence of the silver NPs in the shell structure led to rupturing of the shell into smaller fragments thus releasing the FITC-dextran. Such nanocapsules have the potential to be used as drug delivery vehicles and offer the scope for further development in the areas of modern medicine, material science, and biochemistry. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Externally activated and targeted release of drugs has a high priority for further development in nanomedicine and drug delivery [1–5]. They provide many advantages over other conventional systems such as increased bioavailability, efficacy and reduction of systemic toxicity. In order to meet these requirements, various drug delivery systems based on polymer and other inorganic nanostructures have been designed to increase the efficacy of drugs [6–11]. Among them, polyelectrolyte capsules (PECs) have received considerable attention, as they offer the added advantage of surface functionalization by several methods [12,13]. As a result, there is a continuing research interest in designing PECs with different functions which are responsive to external (modern) triggers such as magnetic, enzymatic, light and ultrasound [14–17]. The presence of NPs/enzymes in the shell structure allows the development of nanopores within the shell structure leading to final rupture of the shell under a given stimulus. Hence, such modified capsules allow tunable permeability for loaded molecules, from relatively slow release to burst like release based on a predesigned mechanism. Modified capsules are prepared by either fabricating capsules with enzyme degradable polyelectrolytes or incorporating metal nanoparticles (NPs) in the capsule wall, which on treatment with enzymes or metal NP based triggers (light, magnetic and ultrasound) release their contents.
Previously, metal NPs have been incorporated into PECs by either using them as one of the shell components or by introducing them into the previously-formed capsule suspension [16,17]. This often leads to inhomogeneous deposition of NPs on the capsule surface due to aggregation of capsules in the suspension. The limitations of these methods can be partially solved by in-situ synthesis of Ag NPs in the capsule wall having a more uniform distribution [18,19]. However, size distribution of in-situ synthesized NPs is quite often not narrow. Also, since the NPs are embedded in an insoluble complex gel-like structure, it causes the NPs to aggregate after synthesis in the wall which in turn can affect the encapsulation process. Further, the methods developed so far are quite complex making them unsuitable for practical applications. Hence it is important to develop a new method to produce nanoscale vehicles (nanocapsules) for externally activated drug delivery. An ideal triggerable nanocapsule system should be easy to prepare from readily available layer components, exhibit good responsiveness and should be compatible with more than one trigger. Here we extend our polyol route of synthesizing Ag particles to nanocapsules and demonstrate its applicability as an externally triggerable drug delivery system [20]. In this study we provide a proof-of-concept towards designing the “remote-rupturing nanocapsules” for release of active substance through external ultrasonic treatment. To the best of our knowledge, fabrication of NPs incorporated nanocapsules by in-situ synthesis for remote activated drug release has not been reported yet. 2. Materials and methods
⁎ Corresponding author at: Department of Materials Engineering, Indian Institute of Science, Bangalore 560012, India. Tel.: +91 80 22933238; fax: +91 80 23600472. E-mail address: [email protected] (A.M. Raichur). 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2012.07.006
Dextran sulfate (DS) (MW = 500 kDa), poly(allylamine hydrochloride) (PAH) (MW = 70 kDa), poly(ethylene glycol) (PEG) (MW =
2350
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6 kDa), tetraethyl orthosilicate (TEOS), ethanol, ammonium hydroxide, hydrofluoric acid (HF), FITC-dextran (Mw = 70 kDa), and ammonium fluoride (NH4F) (Sigma-Aldrich) and silver nitrate (Ranbaxy, India) were used without any further purification. Milli-Q water with resistivity greater than 18 MΩ cm was used in this study. All pH adjustments were done with 0.1 M HCl or 0.1 M NaOH. 2.1. Synthesis of silica nanoparticles Silica nanoparticles were prepared by hydrolysis of TEOS in ethanol medium in the presence of ammonium hydroxide [21]. Briefly, 0.1 M of TEOS in ethanol medium was ultrasonicated in a bath. After 20 min, 28% ammonium hydroxide was added as a catalyst to promote condensation reaction. Sonication was continued for another 60 min to get a white turbid suspension of silica nanoparticles. Then the particles were washed with water and used for LbL assembly. 2.2. Capsule preparation Capsules were prepared by the LbL technique using silica nanoparticles as sacrificial templates. The silica particles (1% w/w) were alternatively coated at pH 5 by incubating them in 1 mg/ml PAH and DS polymer solution respectively prepared with 0.2 M NaCl. After adsorption for 15 min, the particles were separated by centrifugation and the residual unadsorbed polyelectrolyte was removed by washing thrice with pH 5 water. Even with charges on their surfaces, silica particles in the nanoscale have a tendency to aggregate due to their large surface area and high interfacial energy. In order to avoid this, centrifugation during assembly and washing steps were carried out at 7000 rpm for 15 min. These parameters were obtained after conducting ‘n’ number of trial experiments by changing parameters such as particle concentration, centrifugation speed and time. After four bilayers were deposited, the silica core was dissolved in HF buffer (0.1 M HF: 0.2 M NH4F) and the obtained capsules were washed and stored in water.
2.5. Ultrasonic irradiation The effect of sonication on the stability of capsules was investigated by sonicating the capsule suspension in a bath reactor. Irradiation was performed with an ultrasonic generator operating at 170 W and 50 Hz (Soniclean 160HT, Australia), which works at an ultrasonic power comparable to those of ultrasonic generators used in medicine and in pharmaceutical field. Before and after irradiation, the capsules were analyzed by atomic force microscopy (AFM) to visualize the morphological changes that occurred on the capsule surface. For release studies, the capsules loaded with FITC-dextran were exposed to ultrasonic treatment and fluorescence spectra for FITC-dextran in the supernatant were recorded at 490 nm (excitation wave length of FITC-dextran). After sonication, each sample was sedimented by centrifugation for 30 min at 7000 rpm and the supernatant was subjected to fluorimetry. It was observed that at times, only centrifugation did not effectively settle down the broken fragments. In such cases, the sample was gently left to stand in the fridge for several days and the fluorescence of supernatant was then measured to get much more reliable data. 2.6. Characterization Confocal images of polyelectrolyte capsules in water were observed using a Zeiss LSM 510 META confocal scanning system (Zeiss, Germany) equipped with a 100×/1.4–1.7 oil immersion objective. To visualize the capsules, FITC-dextran was used as model probe molecule. Fluorescence spectra of the released dextran were recorded using a Jobin Yvon Fluorolog3 spectrofluorimeter (Horiba Scientific Instruments, USA). For AFM analysis, a drop of capsule suspension was placed on silicon wafer and air dried overnight. Then the samples were characterized using a Nanosurf Easy Scan2 AFM (Nanoscience Instruments Inc., USA) in air at room temperature by contact mode. FE-TEM measurements were performed on a Tecnai F30 (FEI, Eindhoven, Netherlands) microscope operating at 200 kV. 3. Results and discussion
2.3. Fabrication of hybrid nanocapsules 3.1. Preparation of silica NPs and nanocapsules The fabrication of silver NP modified microcapsules has been reported earlier [20]. The same procedure was used here to produce nanocapsules and parameters such as centrifugation speed, time etc., were optimized to prevent aggregation during LbL assembly. Fabrication of nanocapsules involved the sequential adsorption of three layers of PAH and DS on silica nanoparticles followed by silver NP synthesis. AgNO3 was used as precursor for the formation of silver NPs and reduction was performed at 50 °C in the presence of PEG. After the synthesis of silver NPs, the coated particles were washed and deposited with another bilayer of PAH and DS. Then the silica core was dissolved with HF buffer to yield silver NP incorporated hollow capsules. Finally the capsules were washed three times with water and used for ultrasonic experiments.
Silica nanoparticles were prepared by Stober's method and used as templates for capsule fabrication. TEM investigation revealed that the silica particles had a mean diameter of 500 ± 100 nm. The particles were found to be spherical, smooth and non-aggregated as shown in Fig. 1. Alternate deposition of PAH and DS on to these silica particles
2.4. Encapsulation of FITC-dextran FITC-dextran was encapsulated inside the polyelectrolyte capsules by thermal encapsulation method [22]. The silver NP synthesized capsules were incubated with FITC-dextran at room temperature for 1 h followed by incubation at 70 °C for 1 h. After encapsulation, the capsule suspension was washed three times with water to remove the excess dextran present in the supernatant solution. Pure polyelectrolyte capsules (without silver NPs) filled with FITC-dextran was used as control capsules. For control capsules, the encapsulation was carried out by mixing capsule suspension with FITC-dextran at pH 3.5 in 0.2 M NaCl solution for 1 h and incubated at 70 °C for another 1 h as mentioned previously.
Fig. 1. TEM image of the prepared silica nanoparticles.
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Fig. 2. Scheme demonstrating the preparation of silver NP modified hybrid nanocapsules and encapsulation and release of dextran using ultrasonic irradiation. (a—Prepared silica template; a–b → LbL assembly of PAH and DS; b–c → Silver NP synthesis and dissolution of template; c–d → Incubation of capsules in dextran solution; d–e → Thermal encapsulation of dextran at 70 °C; e–f → Washing of loaded capsules to remove excess dextran present in supernatant solution; f–g →Release of dextran after ultrasonic irradiation).
followed by dissolution of silica core with HF yielded hollow capsules. Two types of capsules were prepared, the first one was solely composed of polyelectrolytes (PAH/DS)4, while the second one had a hybrid shell composition [(PAH/DS)3/AgNPs/(PAH/DS)] consisting of Ag NPs and PAH/DS polyelectrolytes. A schematic of the procedure for fabrication of hybrid nanocapsules is shown in Fig. 2. Stable capsules were produced with and without embedded silver nanoparticles and the capsules were not damaged during core dissolution as shown in Fig. 3. Both the capsules showed that collapsed structure and the spherical structure of nanocapsules were retained. The presence of silver NPs in the wall did not seem to alter the shape and stability of capsules significantly when compared to pure polyelectrolyte capsules. The diameter of the hybrid capsules is about 500 ± 100 nm with a thickness range of 30 ± 5 nm (AFM data not shown here). The capsules were also examined by TEM. As seen in Fig. 4, both the capsules retained their spherical morphology and were well dispersed without any sign of aggregation. Hybrid capsules appeared darker than the pure polyelectrolyte capsules due to the presence of silver NPs. It can be seen in Fig. 4c, that silver NPs are uniformly distributed within the walls of the capsule. Averaging the size of NPs present in 20 capsules yielded a mean diameter of 25 ± 5 nm which is consistent with AFM measurements.
3.2. Capsule permeability and encapsulation FITC-dextran was used as a model biomolecule to study the permeability of the nanocapsules. Both pure capsules and silver NP embedded capsules were incubated with FITC-dextran (1:1 volume ratio) for 1 h and visualized under CLSM. Fig. 5a and b shows confocal images of FITC-dextran stained polyelectrolyte and hybrid capsules in aqueous solution. Pure polyelectrolyte capsules were impermeable whereas hybrid capsules were easily permeable to dextran molecules which can be attributed to pore formation during NPs synthesis. Therefore, it is believed that the formation of new NPs during synthesis creates defects and discontinuities in the form of voids, pores and crevices or small breakage in the polymeric shell network thus allowing the penetration of macromolecules [23,24]. For encapsulation studies, thermal encapsulation method was used to load FITC-dextran in the capsules. This method involves the heat treatment of capsule suspension in the presence of low molecular weight compounds, by exploiting the decrease in permeability for encapsulation [22]. Fig. 5c shows the CLSM image of the hybrid nanocapsules loaded with FITC-dextran at 70 °C. The shell shrinkage and densification induced by heat treatment prevents the FITC-dextran movement from interior to bulk. However, due to the limited optical resolution of CLSM, it
Fig. 3. AFM images of nanocapsules. (a) Pure polyelectrolyte capsule and (b) hybrid capsule with synthesized silver NPs. After synthesizing the silver NPs, the thickness of the capsules is increased from 15 ± 3 nm to 30 ± 5 nm. The average size of the nanocapsules prepared was 500 ± 100 nm.
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Fig. 4. TEM images of nanocapsules. (a) Pure polyelectrolyte capsules, (b) Silver NP modified capsules at low magnification and (c) at high magnification. Insets show zoomed cross section of the marked area in the corresponding images. The size of NPs present in the capsule shell is 25 ± 5 nm.
was difficult to conclude from this image as to whether the FITC-dextran was inside the capsule or just adsorbed on the surface of the capsules. In order to confirm the encapsulation, the morphological changes of capsules prior and after encapsulation were monitored by AFM investigations. The AFM image of empty and loaded capsules showed different morphologies. The control capsules were completely collapsed and flat (Fig. 3a) whereas the loaded capsules exhibited a much higher average vertical height than control capsules as shown in Fig. 6. The typical double wall thickness of a pure polyelectrolyte capsule (obtained from AFM) was about 15±3 nm which increased to 90±10 nm after FITC-dextran encapsulation. It is believed that this increase in thickness is contributed from both encapsulation and temperature enhanced swelling of the capsules. It is well known that wall densification and swelling of the capsules occur at the expense of reduction in capsule diameter when they are heat treated at elevated temperature [22]. It is difficult to assess the contribution of encapsulation and temperature treatment towards swelling of the
Fig. 5. Investigation of permeability and encapsulation of FITC-dextran by CLSM. (a) Pure polyelectrolyte capsules, (b) Silver NP modified hybrid capsules and (c) thermally encapsulated hybrid capsules. Scale bar=2 μm.
capsules. However, the strong fluorescence signal from the capsule interior confirmed that significant amount of FITC-dextran has been loaded in the capsules. 3.3. Ultrasonic irradiation With the perspective of using these capsules as drug delivery vehicles for remote-activated release, we investigated the rupture of the capsules under ultrasonic irradiation. It has been mentioned in the literature that ultrasound intensity of the range of 1000–10,000 W/cm 2 is used for different diseases like uterine fibroid treatment [25,26]. This intensity can induce lesions, or tissue necrosis at a small location deep in the tissue while leaving the tissue between the ultrasound source and focus unharmed. However, this occurs only when the focal temperature exceeds 70 °C [25]. In order to avoid this issue, we have used ultrasonic irradiation of maximum power of 170 W
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Fig. 8. Fluorescence spectrum of released FITC-dextran from silver NP modified nanocapsules at various time intervals.
the shells heavy and increases the rigidity of the capsules thus it favors the rupture of the capsules.
3.4. Release studies
Fig. 6. AFM image of FITC-dextran loaded (silver) capsule and its height profile. After thermal encapsulation, the average height of the capsule increased from 15 ± 3 nm to 90 ± 10 nm at the expense of diameter reduction from 500 ± 100 nm to 350 ± 50 nm.
for our experiments. Fig. 7 shows the morphological changes of the capsules when exposed to ultrasonic irradiation for various time intervals. During ultrasonication, acoustic cavitation bubbles are formed which collapse with greater energy in the suspension causing mechanical damage to the capsules. Due to this, the capsules gets ruptured and deformed after ultrasonic irradiation. It should be noted that the capsule surface is no longer continuous and is broken into smaller fragments. As shown in Fig. 7a and c, the rupturing of the capsules occurs at even less than 5 s of ultrasonic exposure and there were significant size differences in the fragments of ruptured capsules when the exposure time was increased to 15 s. It is understood that the presence of silver NPs in the wall creates a density contrast in the polymeric network and reduces the elasticity of the capsule wall, which makes them more prone to rupture by acoustic cavitation [17,27]. It can be concluded that presence of NPs makes
Fluorescence spectrometry was used to investigate the release of dextran from the loaded capsules as shown in Fig. 8. For release studies, thermally encapsulated capsules were irradiated for different time interval (up to 15 s). After irradiation, the capsule suspension was centrifuged and FITC-dextran fluorescence emission from the supernatant was recorded. The fluorescence signal of high intensity at 515 nm corresponding to FITC-dextran was observed in all the samples. The fluorescence spectra obtained from the samples showed that there is no significant difference in fluorescence intensity of released FITC-dextran. It confirmed that the rupture of capsules is immediate leading to the burst release of dextran. This result corroborates well with the rupturing study investigated by AFM. On the contrary, no such fluorescence signal was observed from the control capsules (pure polyelectrolyte nanocapsules) even after 1 min of ultrasonic irradiation as shown in Fig. 8. When the exposure time was increased beyond 2 min, very low intensity of the fluorescence signal was observed corresponding to low amount of release of the loaded FITC-dextran (data not shown). This might be caused by diffusion of FITC-dextran from capsule interior to bulk due to concentration gradient. This proves our hypothesis that hybrid capsules rupture faster than the control capsules thus releasing the encapsulated dextran.
Fig. 7. AFM images to show the rupturing behavior of nanocapsules at various time intervals. (a) 5 s, (b) 10 s and (d) 15 s.
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Fig. 9. Release of FITC-dextran with various exposure time.
3.5. Quantification of release Release of FITC-dextran after ultrasonic irradiation was investigated in water at pH 7.4. In order to avoid the diffusion based release (release by concentration gradient), experiments were performed within 15 min at a time interval of 5 min. Hybrid and control capsules showed different release profile as shown in Fig. 9. Hybrid capsules showed burst release of around 70% due to instantaneous rupture of
the capsules and there was no significant release after 5 min. On the other hand, in control capsules the release was almost linear. The amount of dextran released increased up to 15% which can be attributed to the large imbalance of dextran concentration between capsule interior and bulk. The relationship between the ultrasonic power (intensity) and temperature of medium on release was investigated. As shown in Fig. 10a, there was no significant difference in release observed when the power increased from 50 to 170 W. So, it is believed that small nanopores formed during the ultrasonication at 50 W were enough to allow the dextran to move from the capsule interior to bulk. For temperature experiments (Fig. 10b), the release was almost constant when the temperature was increased from 30° to 70 °C. This is due to the fact that capsules gets ruptured faster (with in 1 min) in the presence of silver NPs. Similarly, pure polyelectrolyte capsules (control capsules) did not show any change in release when the release was performed at different power of ultrasonication and temperature of the medium (data not shown). These results demonstrate that the nanocapsules reported here do have the potential to be used for drug delivery and can be easily activated externally by exposing them to ultrasonic irradiation. 4. Conclusion We have demonstrated the fabrication of hybrid nanocapsules containing Ag NPs in their shell for ultrasonically activated drug delivery. The pores formed due to in-situ synthesized NPs in the capsule shell could be effectively exploited for the encapsulation of FITCdextran by utilizing thermal encapsulation method. The capsules containing NPs ruptured quickly when subjected to sonication. The release studies showed that these capsules were ruptured within 5 s resulting in the release of loaded dextran molecules. These capsules can also be further engineered to be biocompatible and susceptible to remotely induced release using various triggers such as ultrasonication and light due to the presence of silver NPs in the shell. Thus drug delivery vehicles with performance metrics like the one presented here have the potential to be studied under in vivo conditions. Acknowledgments We thank the Institute Nanoscience Initiative, Indian Institute of Science for microscopy facility and Prof. S. Ramakrishnan, Department of Inorganic and Physical Chemistry for fluorescence spectrometer. This work is supported by Dow Chemical International Pvt. Ltd., India. References
Fig. 10. Influence of sonication power (a) and temperature (b) on release of dextran.
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