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Cite this: DOI: 10.1039/c3nr04448c
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Multifunctional gold coated thermo-sensitive liposomes for multimodal imaging and photothermal therapy of breast cancer cells† Aravind Kumar Rengan,a Madhura Jagtap,b Abhijit De,b Rinti Banerjeea and Rohit Srivastava*a Plasmon resonant gold nanoparticles of various sizes and shapes have been extensively researched for their applications in imaging, drug delivery and photothermal therapy (PTT). However, their ability to degrade after performing the required function is essential for their application in healthcare. When combined with biodegradable liposomes, they appear to have better degradation capabilities. They degrade into smaller particles of around 5 nm that are eligible candidates for renal clearance. Distearoyl phosphatidyl choline : cholesterol (DSPC : CHOL, 8 : 2 wt%) liposomes have been synthesized and coated with gold by in situ reduction of chloro-auric acid. These particles of size 150–200 nm are analyzed for their stability, degradation capacity, model drug-release profile, biocompatibility and photothermal effects on cancer cells. It is observed that when these particles are subjected to low power continuous wave near
Received 21st August 2013 Accepted 18th October 2013
infra-red (NIR) laser for more than 10 min, they degrade into small gold nanoparticles of size 5 nm. Also,
DOI: 10.1039/c3nr04448c
the gold coated liposomes appear to have excellent biocompatibility and high efficiency to kill cancer cells through photothermal transduction. These novel materials are also useful in imaging using specific
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NIR dyes, thus exhibiting multifunctional properties for theranostics of cancer.
1. Introduction Gold nanoparticles have been extensively researched for their efficiency in photo-thermal therapy.1 Gold nanocages,2–4 nanoshells,5–7 nanorods,8–10 etc., in particular have been explored for their feasibility in the treatment of cancer. Gold is biocompatible but not biodegradable.11 In general, the foremost disadvantage of nanoparticles is their prolonged retention in the physiological system that can hinder diagnosis and prognosis.12 It has been found that in the case of inorganic nanoparticles, if the hydrodynamic diameter can be minimized to less than 5.5 nm,13 then such particles may be easily cleared through the renal system. However, almost all the nanoparticles used in the current strategy of photothermal treatment (PTT) have $20 nm dimensions,14,15 that make them unsuitable for renal clearance. Romanowski et al. developed a drug delivery system with dipalmitoyl phosphatidyl choline (DPPC) and gold.16 They have shown that by incorporating gold on to the surface of DPPC, a core–shell effect is attained thereby creating an absorbance in the near infra-red (NIR) region suitable for photothermal
a
Department of Bioscience and Bioengineering, IIT Bombay, Powai, Mumbai, 400076, India. E-mail: [email protected]
b
Molecular Functional Imaging Lab, KS-325, 326, ACTREC, Tata Memorial Centre, Kharghar, Sector 22, Navi Mumbai, 410210, India
† Electronic supplementary information (ESI) available: Additional gures. See DOI: 10.1039/c3nr04448c
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mediated drug release. They have also shown that such DPPC based liposomes are sensitive and specic to the NIR laser wavelengths that correspond to their absorbance wavelengths.17 NIR absorbance can also be achieved by silica gold nanostructures, but most of the reported literature describe sizes >100 nm which are non-biodegradable.11 On the other hand, in the case of gold coated liposomes (liposome core–Au shell), the core is made of biodegradable lipid.16 Aer reaching the phase transition temperature, the lipid destabilizes (and hence the liposome core), releasing the drug. The size of the system (liposome–Au) is also expected to reduce, as the gold seeds on the surface will split apart. The phase transition temperature of DPPC is 41 C, which can accommodate a drug and release it above body temperature (37 C). Here, the temperature increment (causing drug release) is indirectly responsible for cell death. If the phase transition temperature of the liposome can be raised to 42–43 C, by a lipid combination then the heat generated can by itself kill the cancer cells and at the same time degrade the particles into small structures capable of getting cleared through the renal system. DPPC based liposomes have been prepared by Pallab et al. which have a phase transition temperature of 43 C and were used for magnetic targeting and drug release.18 The idea of using a unique lipid combination of 1,2-distearoyl-sn-glycero-3phosphocholine : cholesterol (DSPC : CHOL) with Au for PTT of cancer has not been explored yet. Towards this aim, during this study we have prepared these DSPC : CHOL liposomes and then
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coated them with Au to achieve a novel nanostructure (hence forth shall be represented as Lipos Au NPs) that can undergo phase transition at 42–43 C. We are reporting the degradation process of the gold coated liposome particles into individual gold seeds of size less than 5.5 nm on varying the laser irradiation time. To the best of our knowledge, this is the rst report demonstrating such bio-degradable liposome–Au particles. Apart from its role in PTT, gold can also be used in imaging, exhibiting multifunctional capabilities.19–22 In general, optical imaging is more of a functional type with high sensitivity enabling identication of molecular interactions at the cellular level and X-ray based imaging, with its high resolution has a better role in anatomical rather than functional imaging.23–26 A combination of both anatomical and functional imaging would serve as a better imaging modality having all the benets of tracking and localization of particles as well as understanding molecular interactions. Though the scattering ability of gold itself may nd use in optical imaging, it renders a negative effect on the absorption ability that is required for PTT. Moreover, due to the poor penetration depth of scattered light (that is not in the NIR range) in tissue-scale imaging, in vivo optical imaging using this property of Au is difficult. Hence, we have attempted to coat the Lipos Au NPs with indo cyanine green (ICG – an FDA approved NIR dye)27 to confer optical imaging functionality to the particles. We have also checked the CT (X-ray based) signalling ability of Lipos Au NPs. Thus, in addition to PTT of breast cancer cells exhibited by these novel particles, we have also explored the multifunctional capabilities of these structures in terms of optical and CT (X-ray) imaging capabilities using the same.
2.
Materials and methods
2.1. Materials Tetrachloroauric acid trihydrate (HAuCl4$3H2O) was purchased from Acros Organics (Thermo Fisher Scientic Inc.). Distearoyl phosphatidylcholine (DSPC) with >99% purity was obtained from Lipoid (Germany) and used without further purication. Cholesterol (CH) and risazurin sodium salt (Alamar blue) were purchased from Sigma Aldrich Company (St. Louis, USA). L-Ascorbic acid (AA) was purchased from SRL Pvt. Ltd, India. Dulbecco's modied Eagle medium (DMEM), RPMI-1640, fetal bovine serum (FBS), antibiotic antimycotic solution, phosphate buffered saline (PBS) and trypsin–EDTA solution were purchased from Himedia Laboratories, Mumbai (India). All other reagents were purchased from Spectrochem India Pvt. Ltd. All chemicals were reagent grade and used as received. All glassware was cleaned with freshly prepared aqua-regia and rinsed with water before use. A Milli-Q water system (Millipore, Bedford, MA, USA), supplied with distilled water, provided high purity water for these experiments. 2.2. Preparation of thermosensitive liposomes The liposomes were prepared by the thin lm hydration method. Briey, mixture of lipids DSPC and cholesterol with different molar ratio was dissolved in a 2 : 1
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chloroform : methanol mixture. The solution was then dried in a rotary evaporator under vacuum to obtain a thin lipid lm at 40 C. The lipid lm was hydrated with a phosphate buffer saline solution by rotating the round bottom ask at about 180 rpm at 60 C until the lipid lm was completely hydrated and a homogeneous dispersion was formed. The liposome suspension was then sonicated with 50% intensity using a probe sonicator to obtain small unilamellar liposomes, which were further characterized by Dynamic Light Scattering (DLS) – (BI 200SM, Brookhaven Instruments Corporation, USA) Transmission Electron Microscopy TEM (JEOL 2100F – 200 kV, FEI Technai 12 BioTwin-120 kV) and Scanning Electron Microscopy, SEM (JSM-7600F). 2.3. Preparation of gold coated liposomes Liposomes made up of DSPC : CHOL with molar ratio 8 : 2 (our proposed thermo-sensitive liposome formulation) with concentration 2 mg ml1 were prepared with the thin lm hydration method. Aer preparing multi lamellar vesicles (MLVs), they were sonicated for 4.5 min to obtain unilamellar liposomes. Different molar ratios of chloroauric acid : ascorbic acid AA were added to 200 ml of liposome solution (2 mg ml1 lipid concentration), which produced an abrupt color change from the characteristic translucent white of liposomes to a violet, blue or green color depending on the quantity of gold reduced. 2.4. Stability study The Lipos Au particles were prepared as per the above mentioned protocol for its absorption to peak at 630 nm. Au nanoparticles were prepared using the same concentrations of HAuCl4 and AA without the liposome templates or any stabilizing agent. Both the samples were kept for incubation at room temperature and intermittent UV-Vis readings were obtained at 0, 2, 5, 10, 15, 20 and 30 min along with photographic images of the respective samples. 2.5. Degradation study The Lipos Au samples (5) concentration was subjected to varying time periods of laser irradiation with 750 nm continuous wave laser (650 mW power, PMC, India) for 5, 10, 15, 20 and 30 min. These samples were then analysed for their UV-Vis absorbance. Some of the samples that were irradiated with varying time periods with laser were taken up for SEM/TEM analysis to understand the changes in shape and surface morphology. 2.6. Photothermal transduction experiment The photothermal effect of the prepared nanostructures was studied with the help of a 750 nm NIR laser (650 mW-PMC, India). In a typical experiment, 200 ml (triplets) of the prepared Lipos Au NPs (100 mg ml1 gold concentration) and control solutions (water and blank liposome samples) were taken in a 96 well plate and allowed to oat on a water bath at 37 C. The laser is allowed to pass through the wells containing test and
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control solutions that are separated from each other to avoid heat transfer. The temperature increment was recorded with the help of a digital infra-red thermometer (oakton mini-infrapro). The temperature was recorded at 0, 2, 5, 10 and 15 min. All the experiments were performed in triplets and the subsequent rise in temperature was plotted as an increment from the baseline (37 C). 2.7. Cell uptake study MDA-MB-231 cells were seeded onto a 96 well plate with a density of 1 104 cells per well. Cells were washed three times with PBS the following day, and then the cells were incubated with Lipos Au NPs at 37 C for 8 and 24 hours (at 0.5 and 1 mg ml1 lipid concentrations that were equivalent to 70 and 140 mg ml1 of gold). Thereaer, the cells were washed three times with PBS to remove unbound particles. Cells were then trypsinized by adding 40 ml trypsin EDTA. These trypsinized cells containing gold nanostructures were then dissolved in freshly prepared aqua regia (1 ml) and made up to 12 ml by diluting with MilliQ water. They were then subjected to Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES) analysis to understand the percentage of elemental gold taken up by the cancer cells. In order to check the particles uptake in a different breast carcinoma cell line and to visually conrm it, TEM analysis was performed in MDA MB 468 cancer cells. The cancer cells were seeded in two 60 mm plates with a density of 5 105 cells per plate and grown until 80% conuence. They were incubated with Lipos Au NPs for 24 hours at 37 C. The culture medium was discarded and the cells were collected, washed with PBS buffer and xed with 2.5% glutaraldehyde in 0.2 M phosphate buffer for 12 h at 4 C. Cells were post xed with osmium tetroxide, washed with sodium cacodylate buffer and dehydrated with different concentrations of alcohol. Cells were then embedded and polymerized using araldite A and B and sectioned. Sections were mounted onto 200-mesh copper grids and stained with uranyl acetate for visualization under TEM.
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irradiation experiment. Cells were washed three times with PBS. The following order of treatment was performed: no treatment, NIR laser alone (7.5 min irradiation), Lipos Au NPs alone and Lipos Au NPs with laser (7.5 min irradiation). For treatment with Lipos Au NPs, cells were incubated with particles at 37 C for 5 hours (100 mg ml1 gold concentration). Thereaer, cells were washed three times with PBS to remove unbound particles. Cells were then resupplied with DMEM containing 10% FBS. Cells were irradiated with a NIR laser (750 nm) and then incubated at 37 C for 12 hours. Then the cells were washed with PBS and subjected to Alamar Blue assay. For qualitative examination, 24 well plates were used which were seeded with a density of 1 105 cells per well. The remaining protocol was similar to the one followed for the above mentioned alamar blue assay, except that at the end of 5 hours incubation, cells were washed with PBS and stained with propidium iodide (for visualization of dead cells). Cells were examined using a Olympus model IX81 laser scanning confocal microscope equipped with lters set for excitation/emission wavelengths at 488/617 nm for propidium iodide.
2.9. Photothermal mediated model drug (calcein) release Thermosensitive liposomes loaded with calcein were prepared by previously reported methods with slight modication.18 First the calcein, which was not encapsulated, was removed from the liposome suspension by centrifugation at 20 000g, 4 C for 20 min and then resuspended in PBS to make the total lipid concentration 2 mg ml1. 200 ml of the calcein loaded liposomes were added to 5 mM HAuCl4 solution that was reduced to Au by the addition of ascorbic acid (20 mM). Aer vortexing, 20 ml of the sample was added to 2 ml of PBS and mixed well. The sample was then subjected to NIR laser irradiation (750 nm) and uorescence intensity (485 nm ex and 520 nm em) was measured in a spectrouorimeter intermittently at various time periods. For 100% release, liposomes were treated with 1% Triton X. Percentage release of calcein was calculated by the following formula,
2.8. Biocompatibility and in vitro photothermal therapy In vitro biocompatibility studies were done using the Alamar Blue assay in L929 cell-line. Exponentially growing cells were dispensed into a 96-well at bottom plate at a concentration of 1 104 cells per well. Aer allowing 24 hours for cell attachment, Lipos Au NP solutions were diluted appropriately in fresh media and added (200 mL) in the lipid concentration ranging from 100 mg ml1 to 1 mg ml1 (3 wells per sample concentration). The media was not changed during the incubation of 48 h. Following incubation, cell viability was determined by the addition of Alamar Blue (20 mL, 1 mg ml1 dye in sterile PBS). The plate was incubated for an additional 4 hours at 37 C and 5% CO2,28 allowing viable cells to convert the blue solution into pink dye. Absorbance values at 560 nm and 620 nm were collected and cell viability was calculated as a percentage compared to untreated control cells. For the photothermal cytotoxicity study, MDA-MB-231 cells were seeded onto a 96 well plate with a density of 1 104 cells per well one day before the This journal is © The Royal Society of Chemistry 2014
% release ¼ [(Ft Fi)/(Ff Fi)] 100 where Ft ¼ uorescence at time t, Fi ¼ initial uorescence, Ff ¼ uorescence with 100% release, i.e. Triton-X treatment.
2.10.
Optical imaging experiment
The preformed Lipos Au NPs were mixed with ICG (50 ml of 1 mM per ml of Lipos Au solution) and incubated for an hour in dark. The solution was then centrifuged (20 000g, 20 min) to remove unbound ICG and the pellet was resuspended in milli Q water. This ICG coated lipos–Au sample was then subjected to zeta potential analysis for conrmation of the coating. Optical imaging experiment was performed in phantom samples using Spectrocam Multispectral imaging instrument (USA). The excitation source was 750 nm (continuous wave) diode laser with 650 mV power (PMC, India). The emission lter was 830 nm with 10 nm band pass. The acquired images were pseudo
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coloured according to the emission range by the Spectrocam soware.
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2.11.
X-Ray CT imaging experiment
Commercially available gold seeds (15 nm and 40 nm) were purchased from Nanocs. A tri-modality microPET-CT-SPECT (model GE FLEX™ Triumph™) pre-clinical imaging system was used for performing the phantom CT scan. Plastic microcentrifuge tubes containing various AuNPs including Lipos Au nanoparticles, along with tubes containing water as negative control and Lopamidol 300 iodine contrast agent as positive control. Triumph XO soware was used for acquiring the scan and image was reconstructed using Access COBRA XXM soware.
3.
Results and discussion
DSPC : CHOL were prepared by the thin lm hydration method followed by coating with Au (in the presence of ascorbic acid (AA)) and charge based ICG coating. On irradiation with laser light, the entire structure destabilizes causing drug release and simultaneous rise of temperature (Fig. 1). The Lipos Au coating has been optimised to be at 400 mg ml1 lipid concentration (ESI, Fig. S1†). Lipos Au NPs have been characterized for their size, shape and morphology. They are found to have broad NIR peaks as shown in Fig. 2a. The hydrodynamic size of these particles was found to be around 200 nm (Fig. 2c). The SEM and TEM images obtained indicated the size to be around 100– 150 nm (Fig. 2d–f). Considering the DLS measurement of the bare liposomes (ESI, Fig. S2a†) with that of the Lipos Au NPs (Fig. 2c), it was observed that the sizes are comparable both before and aer Au coating, indicating the compact self-
Fig. 1
Fig. 2 (a) UV-Vis absorbance of lipos au samples with varying molarity ratios of ascorbic acid and HAuCl4 (b) corresponding photographic images of Lipos Au NPs solution (c) DLS measurement of Lipos Au NPs (d) FEG-TEM (e) FEG-SEM (f) STEM (scanning transmission electron microscopy) image of Lipos Au NP showing the hollow central region with dark contrast. (Bare liposomes were used as controls in (a) and (b); scale bars: (d) 20 nm, (e) 10 nm, (f) 50 nm).
assembly of Au on to the liposomal surface. The liposome core has been characterized for its thermo-sensitivity and model drug release (ESI, Fig. S3†). It has been observed that once 43 C was attained, the liposome destabilizes to release the model drug. More than threefold of calcein (65.49 2.8%) was released at 43 C in comparison to just 14.9 2.4% at 37 C.
Multifunctional gold coated liposome nanoparticle (Lipos Au NP) – role in imaging, drug delivery and photothermal therapy PTT.
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It was observed that only in the presence of the liposome template, the gold coating was stable and was tunable to the NIR region. When the liposome templates were not included, the wavelengths of the samples remained xed in the 500– 600 nm region and the intensity of the solution gradually reduced as shown in Fig. 3a. In the absence of a liposome template, the gold structures that were formed by reducing HAuCl4 in the presence of AA were not stable. Though nanostructures were prepared by the addition of AA to HAuCl4 previously by Wang et al., a stabilising agent (sodium benzene
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sulphonate) was required to retain the morphology, shape and size of the formed structures.29 In the case of Lipos Au NPs, the liposome template itself acted as a stabilizer. It retained the in situ reduced gold on its surface without undergoing any aggregation. An experiment to understand the stabilizing effect of the liposome template was performed as shown in Fig. 3. To maintain uniformity in color and absorbance, the Lipos Au NPs were tuned to give an absorbance at 600 nm similar to the solution that lacked a liposome template. Even aer 72 hours, the solution containing liposome templates showed little variation in their absorbance or color, whereas the solution containing only HAuCl4 and AA (without liposomes) began to fade (in color) and absorbance intensity reduced within a few minutes aer preparation (Fig. 3a(i), see ESI, Fig. S5† performed with the actual NIR absorbing Lipos Au NPs). The control solution (i.e. without liposome template) precipitated and began to settle down making the solution transparent as discussed above. Now to understand drug release from the Lipos Au NPs, the particles were subjected to laser irradiation for varying time duration. Drug release can happen only when the Lipos Au structure destabilizes through the heat generated by the photothermal effect. To further understand this process, we subjected the solution containing Lipos Au NPs (5 concentration) to graded time periods of laser irradiation (5, 10, 15, 20 and 30 min) with 2 min interval between every 5 min of irradiation. It was observed that immediately aer 5–10 min of laser irradiation, the particle morphology began to change (Fig. 4b(i–iv)). This was accompanied by reduction of the UV-Vis absorbance intensity as shown in Fig. 4a. This degradation phenomenon
Fig. 4 Lipos Au degradation studies (a) UV-Vis absorbance of Lipos Au
Stability of Lipos Au solution compared with Au solution without liposome template, UV-Vis absorbance of (a) HAuCl4 sol. reduced by AA without liposome template, (b) with liposome template (c) photographic images at 0 min [(i) without liposome template, (ii) with liposome template], (d) at 2 min (e) at 4 min (f) at 6 min (g) at 8 min (h) at 10 min (i) at 30 min. Fig. 3
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NPs treated with NIR laser for varying time periods. (b(i–iv)) Representative FEG-SEM images of Lipos Au NPs treated with NIR laser for varying time periods, (c–e) representative FEG-TEM images of Lipos Au NPs treated with NIR laser for varying time periods, (f) FEG-TEM HR image of Lipos Au NPs treated with NIR laser for 30 min showing particles of size 5 nm (inset scale bar: 5 nm). (Scale bars: (b) 100 nm, (c–e) 50 nm, (f) 5 nm).
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was conrmed by TEM analysis (Fig. 4c–e). At the end of 30 min, almost the entire morphology of the particle was lost. The particles were degraded into small gold particles of size around 5 nm as shown in Fig. 4f. This experiment gave insight that the maximum time period of laser irradiation was permissible to be less than 10 min. As the NIR absorbance changes beyond 10 min of irradiation, the time scale was limited to 7.5 min for further PTT experiments. The degradability studies also revealed that the Lipos Au system is capable of degrading into smaller gold particles of size 5 nm that has an implication in getting cleared through the renal system. The Lipos Au NPs were synthesized with AA : Au in the ratio 3 : 2, so that they were able to absorb NIR light around 750 nm (Fig. 5a). A photothermal transduction experiment was performed to understand the temperature increment that can be achieved by Lipos Au NPs. It was observed that the Lipos Au NPs
(a) Representative absorbance of Lipos Au NPs 750 nm sample that was utilized for further photothermal related experiments, (b) photothermal transduction experiment showing a significant temperature increment for Lipos Au NPs.
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reached a temperature of 43 C in 5 to 10 min. The control solutions i.e. water and just liposomes (without Au) reached only 39 C during the same time period (Fig. 5b). The bio compatibility test performed with L929 cells suggested that these Lipos Au NPs were biocompatible even at 1 mg ml1 of Lipos Au (lipid concentration). When tested from 10 mg ml1 to 1 mg ml1, the cell viability was more than 90% as shown in Fig. 6a. As the nanoparticles can undergo different routes of uptake, a TEM analysis of cell uptake employing Lipos Au NPs was performed. These particles were incubated with breast cancer cell lines (MDA MB 468). At the end of 24 hours, it was found that the nanoparticles were well spread in the cytosol region of the cancer cells without aggregation or being engulfed by endosomes (Fig. 6c and d). Though previous studies have shown that gold nanoparticles when delivered via liposomes can circumvent endosome mediated uptake,30 the current analysis differs in that the liposomes (core) are getting delivered through gold nanoparticles (shell) covering them, whereas in the previous reported studies gold nanoparticles occupied the core region.30 As the gold coating is not contiguous over the liposome core, the exposed lipid membranes might interact with the cell membranes to enhance the uptake of nanoparticles. However, the current analysis could not explain the
Fig. 5
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Fig. 6 (a) Biocompatibility (b) cell uptake of Lipos Au in MDA MB 231 cancer cells based on ICP AES analysis of Au, (c and d) cell uptake of Lipos Au (TEM) in MDA MB 468 cancer cells.
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intact particle morphology even aer the expected lipid cellular interactions, for which further detailed analysis is required. In the photothermal mediated cytotoxicity experiment, the cells incubated with Lipos Au NPs and irradiated with 750 nm NIR laser for 7.5 min, lost their viability in comparison to the controls. This was conrmed both quantitatively and qualitatively (Fig. 7). A NIR Laser mediated model drug release study was performed using calcein dye loaded Lipos Au NPs (Fig. 7e). Within 5 min of NIR laser irradiation, almost 50% of the model drug was released. As already observed in the photothermal transduction experiment, the Lipos Au solution temperature was able to reach above 43 C within 5 min of laser irradiation (Fig. 5b). Hence, it is very much evident that the destabilization of the Lipos Au NPs (due to the photothermal effect) was responsible for drug release. By the end of 10–15 min almost 85% of the model drug was released, whereas in the control solution (liposome loaded with calcein – without Au coating), the release was limited to around 10% due to the absence of photothermal effect. The Lipos Au NPs were also subjected to CT phantom analysis at 70 and 90 kVp. In both the cases, they gave good CT contrast equivalent to the iodine control (Fig. 8a(i and ii)). The signal generated by Lipos Au NPs was much better than that obtained from commercially available gold seeds (15 nm and 40 nm Au NPs). We hypothesize that the presence of liposome enables a scattered accumulation of gold atoms within the lipid system that can provide a larger area to enhance X-ray
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Fig. 8 (a) X-ray CT imaging (Lipos AuNPs) at (i) 70 kVp and (ii) 90 kVp, (b) variation of zeta potential with Au and ICG coating on the liposome. Bright field image of Lipos Au ICG solution (c-(i)) and corresponding optical image (c-(ii)), bright field image of free ICG solution (d-(i)) and corresponding optical image (d-(ii)). (ex: 750 nm, em: 830 nm.)
attenuation more efficiently than the gold seeds. The Lipos Au NPs were coated further with ICG (50 ml of 1 mM per ml of Lipos Au NP solution). ICG coating on the lipos Au surface was
(a–c) Photothermal cytotoxicity (qualitative analysis) (i) differential scanning images of (a) cells incubated with Lipos Au NPs only (b) cells irradiated for 7.5 min with 750 nm laser (c) cells incubated with Lipos Au NPs and irradiated for 7.5 min with 750 nm laser (a–c), (ii) corresponding CLSM images exhibiting PI stain (dead cells) in red, (c(iii)) merged images of (c(i and ii)) showing dead cells due to PTT, (d) photothermal cytotoxicity (quantitative analysis – Alamar Blue assay) (e) photothermal mediated calcein (model drug) release from Lipos Au NPs. (Scale bars: (a–c) 200 mm.)
Fig. 7
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performed to make the Lipos Au NPs uorescent when irradiated with the same NIR laser (750 nm) that was actually meant for inducing the photothermal effect, thus enhancing the multifunctional capabilities of the nanoparticle system. Both the Au coating and the ICG coating were conrmed by zeta analysis as shown in Fig. 8b. It was found that the Lipos Au ICG solution gave good contrast equivalent to that of free ICG solution (Fig. 8c and d). The Lipos Au NPs were coated with ICG without compromising the uorescence of the dye. There was no obvious quenching observed due to the addition of Lipos Au NPs. This showed that there is a likely possibility of tagging the Lipos Au NPs with NIR dye to enhance the multifunctional properties of the system acquiring dual imaging modality (i.e. anatomical and functional).
4. Conclusion It is observed that Lipos Au NPs can be used for photothermal therapy of cancer cells. They can also be efficiently deployed for drug delivery application using NIR laser irradiation. The same laser light may be useful in optical imaging of the particles when coated with appropriate NIR dye. In addition to PTT, drug delivery and optical imaging, the Lipos Au NPs exhibited its true multifunctional ability by emitting good signals in CT X-ray analysis. We have shown the degradability of the system, by achieving 5 nm size particles assuring the capability of renal clearance. Further in vivo studies are to be performed to understand the complete efficacy of these novel biodegradable agents in cancer research.
Conflict of interest The authors declare no competing nancial interest.
Acknowledgements The authors would like to acknowledge TMC Seed-in-Air Intramural funding to A. D. and Ms Renu Malhotra for assisting with some of the biology experiments at ACTREC; Spectrocam Instruments for demonstrating the optical imaging experiments; IIT B Healthcare initiative for funding the project and SAIF-IITB for characterization studies.
References 1 X. Huang, P. K. Jain, I. H. El-Sayed and M. A. El-Sayed, Lasers in Medical Science, 2008, 23, 217. 2 J. Chen, D. Wang, J. Xi, L. Au, A. Siekkinen, A. Warsen, Z.-Y. Li, H. Zhang, Y. Xia and X. Li, Nano Lett., 2007, 7, 1318. 3 L. Au, D. Zheng, F. Zhou, Z.-Y. Li, X. Li and Y. Xia, ACS Nano, 2008, 2, 1645. 4 J. Chen, C. Glaus, R. Laforest, Q. Zhang, M. Yang, M. Gidding, M. J. Welch and Y. Xia, Small, 2010, 6, 811.
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5 A. R. Lowery, A. M. Gobin, E. S. Day, N. J. Halas and J. L. West, Int. J. Nanomed., 2006, 1, 149. 6 A. M. Gobin, J. J. Moon and J. L. West, Int. J. Nanomed., 2008, 3, 351. 7 J. A. Schwartz, A. M. Shetty, R. E. Price, R. J. Stafford, J. C. Wang, R. K. Uthamanthil, K. Pham, R. J. McNichols, C. L. Coleman and J. D. Payne, Cancer Res., 2009, 69, 1659. 8 H. Jin, P. Yang, J. Cai, J. Wang and M. Liu, Appl. Microbiol. Biotechnol., 2012, 94, 1199. 9 E. B. Dickerson, E. C. Dreaden, X. Huang, I. H. El-Sayed, H. Chu, S. Pushpanketh, J. F. McDonald and M. A. ElSayed, Cancer Lett., 2008, 269, 57. 10 G. P. Goodrich, L. Bao, K. Gill-Sharp, K. L. Sang, J. Wang and J. D. Payne, J. Biomed. Opt., 2010, 15, 18001. 11 D. Bechet, P. Couleaud, C. Frochot, M.-L. Viriot, F. Guillemin and M. Barberi-Heyob, Trends Biotechnol., 2008, 26, 612. 12 M. Longmire, P. L. Choyke and H. Kobayashi, Nanomedicine, 2008, 3, 703. 13 H. S. Choi, W. Liu, P. Misra, E. Tanaka, J. P. Zimmer, B. Itty Ipe, M. G. Bawendi and J. V. Frangioni, Nat. Biotechnol., 2007, 25, 1165. 14 J. H. Ryu, H. Koo, I.-C. Sun, S. H. Yuk, K. Choi, K. Kim and I. C. Kwon, Adv. Drug Delivery Rev., 2012, 64, 1447. 15 X. Huang and M. A. El-Sayed, Journal of Advanced Research, 2010, 1, 13. 16 T. S. Troutman, J. K. Barton and M. Romanowski, Adv. Mater., 2008, 20, 2604. 17 S. J. Leung, X. M. Kachur, M. C. Bobnick and M. Romanowski, Adv. Funct. Mater., 2011, 21, 1113. 18 P. Pradhan, J. Giri, F. Rieken, C. Koch, O. Mykhaylyk, M. D¨ oblinger, R. Banerjee, D. Bahadur and C. Plank, J. Controlled Release, 2010, 142, 108. 19 A. M. Gobin, M. H. Lee, N. J. Halas, W. D. James, R. A. Drezek and J. L. West, Nano Lett., 2007, 7, 1929. 20 J.-L. Li, L. Wang, X.-Y. Liu, Z.-P. Zhang, H.-C. Guo, W.-M. Liu and S.-H. Tang, Cancer Lett., 2009, 274, 319. 21 X. Huang, I. H. El-Sayed, W. Qian and M. A. El-Sayed, J. Am. Chem. Soc., 2006, 128, 2115. 22 S. E. Skrabalak, J. Chen, L. Au, X. Lu, X. Li and Y. Xia, Adv. Mater., 2007, 19, 3177. 23 T. F. Massoud and S. S. Gambhir, Genes Dev., 2003, 17, 545. 24 V. Ntziachristos, Nat. Methods, 2010, 7, 603. 25 M. J. Paulus, S. S. Gleason, S. J. Kennel, P. R. Hunsicker and D. K. Johnson, Neoplasia, 2000, 2, 62. 26 J. V. Frangioni, J. Clin. Oncol., 2008, 26, 4012. 27 J. T. Alander, I. Kaartinen, A. Laakso, T. P¨ atil¨ a, T. Spillmann, V. V. Tuchin, M. Venermo and P. V¨ alisuo, Int. J. Biomed. Imaging, 2012, 2012, 1. 28 S. T. Selvan, T. T. Tan and J. Y. Ying, Adv. Mater., 2005, 17, 1620. 29 S. Wang, K. Qian, X. Bi and W. Huang, J. Phys. Chem. C, 2009, 113, 6505. 30 P. Nativo, I. A. Prior and M. Brust, ACS Nano, 2008, 2, 1639.
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Cite this: DOI: 10.1039/c3nr04448c
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Multifunctional gold coated thermo-sensitive liposomes for multimodal imaging and photothermal therapy of breast cancer cells† Aravind Kumar Rengan,a Madhura Jagtap,b Abhijit De,b Rinti Banerjeea and Rohit Srivastava*a Plasmon resonant gold nanoparticles of various sizes and shapes have been extensively researched for their applications in imaging, drug delivery and photothermal therapy (PTT). However, their ability to degrade after performing the required function is essential for their application in healthcare. When combined with biodegradable liposomes, they appear to have better degradation capabilities. They degrade into smaller particles of around 5 nm that are eligible candidates for renal clearance. Distearoyl phosphatidyl choline : cholesterol (DSPC : CHOL, 8 : 2 wt%) liposomes have been synthesized and coated with gold by in situ reduction of chloro-auric acid. These particles of size 150–200 nm are analyzed for their stability, degradation capacity, model drug-release profile, biocompatibility and photothermal effects on cancer cells. It is observed that when these particles are subjected to low power continuous wave near
Received 21st August 2013 Accepted 18th October 2013
infra-red (NIR) laser for more than 10 min, they degrade into small gold nanoparticles of size 5 nm. Also,
DOI: 10.1039/c3nr04448c
the gold coated liposomes appear to have excellent biocompatibility and high efficiency to kill cancer cells through photothermal transduction. These novel materials are also useful in imaging using specific
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NIR dyes, thus exhibiting multifunctional properties for theranostics of cancer.
1. Introduction Gold nanoparticles have been extensively researched for their efficiency in photo-thermal therapy.1 Gold nanocages,2–4 nanoshells,5–7 nanorods,8–10 etc., in particular have been explored for their feasibility in the treatment of cancer. Gold is biocompatible but not biodegradable.11 In general, the foremost disadvantage of nanoparticles is their prolonged retention in the physiological system that can hinder diagnosis and prognosis.12 It has been found that in the case of inorganic nanoparticles, if the hydrodynamic diameter can be minimized to less than 5.5 nm,13 then such particles may be easily cleared through the renal system. However, almost all the nanoparticles used in the current strategy of photothermal treatment (PTT) have $20 nm dimensions,14,15 that make them unsuitable for renal clearance. Romanowski et al. developed a drug delivery system with dipalmitoyl phosphatidyl choline (DPPC) and gold.16 They have shown that by incorporating gold on to the surface of DPPC, a core–shell effect is attained thereby creating an absorbance in the near infra-red (NIR) region suitable for photothermal
a
Department of Bioscience and Bioengineering, IIT Bombay, Powai, Mumbai, 400076, India. E-mail: [email protected]
b
Molecular Functional Imaging Lab, KS-325, 326, ACTREC, Tata Memorial Centre, Kharghar, Sector 22, Navi Mumbai, 410210, India
† Electronic supplementary information (ESI) available: Additional gures. See DOI: 10.1039/c3nr04448c
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mediated drug release. They have also shown that such DPPC based liposomes are sensitive and specic to the NIR laser wavelengths that correspond to their absorbance wavelengths.17 NIR absorbance can also be achieved by silica gold nanostructures, but most of the reported literature describe sizes >100 nm which are non-biodegradable.11 On the other hand, in the case of gold coated liposomes (liposome core–Au shell), the core is made of biodegradable lipid.16 Aer reaching the phase transition temperature, the lipid destabilizes (and hence the liposome core), releasing the drug. The size of the system (liposome–Au) is also expected to reduce, as the gold seeds on the surface will split apart. The phase transition temperature of DPPC is 41 C, which can accommodate a drug and release it above body temperature (37 C). Here, the temperature increment (causing drug release) is indirectly responsible for cell death. If the phase transition temperature of the liposome can be raised to 42–43 C, by a lipid combination then the heat generated can by itself kill the cancer cells and at the same time degrade the particles into small structures capable of getting cleared through the renal system. DPPC based liposomes have been prepared by Pallab et al. which have a phase transition temperature of 43 C and were used for magnetic targeting and drug release.18 The idea of using a unique lipid combination of 1,2-distearoyl-sn-glycero-3phosphocholine : cholesterol (DSPC : CHOL) with Au for PTT of cancer has not been explored yet. Towards this aim, during this study we have prepared these DSPC : CHOL liposomes and then
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coated them with Au to achieve a novel nanostructure (hence forth shall be represented as Lipos Au NPs) that can undergo phase transition at 42–43 C. We are reporting the degradation process of the gold coated liposome particles into individual gold seeds of size less than 5.5 nm on varying the laser irradiation time. To the best of our knowledge, this is the rst report demonstrating such bio-degradable liposome–Au particles. Apart from its role in PTT, gold can also be used in imaging, exhibiting multifunctional capabilities.19–22 In general, optical imaging is more of a functional type with high sensitivity enabling identication of molecular interactions at the cellular level and X-ray based imaging, with its high resolution has a better role in anatomical rather than functional imaging.23–26 A combination of both anatomical and functional imaging would serve as a better imaging modality having all the benets of tracking and localization of particles as well as understanding molecular interactions. Though the scattering ability of gold itself may nd use in optical imaging, it renders a negative effect on the absorption ability that is required for PTT. Moreover, due to the poor penetration depth of scattered light (that is not in the NIR range) in tissue-scale imaging, in vivo optical imaging using this property of Au is difficult. Hence, we have attempted to coat the Lipos Au NPs with indo cyanine green (ICG – an FDA approved NIR dye)27 to confer optical imaging functionality to the particles. We have also checked the CT (X-ray based) signalling ability of Lipos Au NPs. Thus, in addition to PTT of breast cancer cells exhibited by these novel particles, we have also explored the multifunctional capabilities of these structures in terms of optical and CT (X-ray) imaging capabilities using the same.
2.
Materials and methods
2.1. Materials Tetrachloroauric acid trihydrate (HAuCl4$3H2O) was purchased from Acros Organics (Thermo Fisher Scientic Inc.). Distearoyl phosphatidylcholine (DSPC) with >99% purity was obtained from Lipoid (Germany) and used without further purication. Cholesterol (CH) and risazurin sodium salt (Alamar blue) were purchased from Sigma Aldrich Company (St. Louis, USA). L-Ascorbic acid (AA) was purchased from SRL Pvt. Ltd, India. Dulbecco's modied Eagle medium (DMEM), RPMI-1640, fetal bovine serum (FBS), antibiotic antimycotic solution, phosphate buffered saline (PBS) and trypsin–EDTA solution were purchased from Himedia Laboratories, Mumbai (India). All other reagents were purchased from Spectrochem India Pvt. Ltd. All chemicals were reagent grade and used as received. All glassware was cleaned with freshly prepared aqua-regia and rinsed with water before use. A Milli-Q water system (Millipore, Bedford, MA, USA), supplied with distilled water, provided high purity water for these experiments. 2.2. Preparation of thermosensitive liposomes The liposomes were prepared by the thin lm hydration method. Briey, mixture of lipids DSPC and cholesterol with different molar ratio was dissolved in a 2 : 1
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chloroform : methanol mixture. The solution was then dried in a rotary evaporator under vacuum to obtain a thin lipid lm at 40 C. The lipid lm was hydrated with a phosphate buffer saline solution by rotating the round bottom ask at about 180 rpm at 60 C until the lipid lm was completely hydrated and a homogeneous dispersion was formed. The liposome suspension was then sonicated with 50% intensity using a probe sonicator to obtain small unilamellar liposomes, which were further characterized by Dynamic Light Scattering (DLS) – (BI 200SM, Brookhaven Instruments Corporation, USA) Transmission Electron Microscopy TEM (JEOL 2100F – 200 kV, FEI Technai 12 BioTwin-120 kV) and Scanning Electron Microscopy, SEM (JSM-7600F). 2.3. Preparation of gold coated liposomes Liposomes made up of DSPC : CHOL with molar ratio 8 : 2 (our proposed thermo-sensitive liposome formulation) with concentration 2 mg ml1 were prepared with the thin lm hydration method. Aer preparing multi lamellar vesicles (MLVs), they were sonicated for 4.5 min to obtain unilamellar liposomes. Different molar ratios of chloroauric acid : ascorbic acid AA were added to 200 ml of liposome solution (2 mg ml1 lipid concentration), which produced an abrupt color change from the characteristic translucent white of liposomes to a violet, blue or green color depending on the quantity of gold reduced. 2.4. Stability study The Lipos Au particles were prepared as per the above mentioned protocol for its absorption to peak at 630 nm. Au nanoparticles were prepared using the same concentrations of HAuCl4 and AA without the liposome templates or any stabilizing agent. Both the samples were kept for incubation at room temperature and intermittent UV-Vis readings were obtained at 0, 2, 5, 10, 15, 20 and 30 min along with photographic images of the respective samples. 2.5. Degradation study The Lipos Au samples (5) concentration was subjected to varying time periods of laser irradiation with 750 nm continuous wave laser (650 mW power, PMC, India) for 5, 10, 15, 20 and 30 min. These samples were then analysed for their UV-Vis absorbance. Some of the samples that were irradiated with varying time periods with laser were taken up for SEM/TEM analysis to understand the changes in shape and surface morphology. 2.6. Photothermal transduction experiment The photothermal effect of the prepared nanostructures was studied with the help of a 750 nm NIR laser (650 mW-PMC, India). In a typical experiment, 200 ml (triplets) of the prepared Lipos Au NPs (100 mg ml1 gold concentration) and control solutions (water and blank liposome samples) were taken in a 96 well plate and allowed to oat on a water bath at 37 C. The laser is allowed to pass through the wells containing test and
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control solutions that are separated from each other to avoid heat transfer. The temperature increment was recorded with the help of a digital infra-red thermometer (oakton mini-infrapro). The temperature was recorded at 0, 2, 5, 10 and 15 min. All the experiments were performed in triplets and the subsequent rise in temperature was plotted as an increment from the baseline (37 C). 2.7. Cell uptake study MDA-MB-231 cells were seeded onto a 96 well plate with a density of 1 104 cells per well. Cells were washed three times with PBS the following day, and then the cells were incubated with Lipos Au NPs at 37 C for 8 and 24 hours (at 0.5 and 1 mg ml1 lipid concentrations that were equivalent to 70 and 140 mg ml1 of gold). Thereaer, the cells were washed three times with PBS to remove unbound particles. Cells were then trypsinized by adding 40 ml trypsin EDTA. These trypsinized cells containing gold nanostructures were then dissolved in freshly prepared aqua regia (1 ml) and made up to 12 ml by diluting with MilliQ water. They were then subjected to Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES) analysis to understand the percentage of elemental gold taken up by the cancer cells. In order to check the particles uptake in a different breast carcinoma cell line and to visually conrm it, TEM analysis was performed in MDA MB 468 cancer cells. The cancer cells were seeded in two 60 mm plates with a density of 5 105 cells per plate and grown until 80% conuence. They were incubated with Lipos Au NPs for 24 hours at 37 C. The culture medium was discarded and the cells were collected, washed with PBS buffer and xed with 2.5% glutaraldehyde in 0.2 M phosphate buffer for 12 h at 4 C. Cells were post xed with osmium tetroxide, washed with sodium cacodylate buffer and dehydrated with different concentrations of alcohol. Cells were then embedded and polymerized using araldite A and B and sectioned. Sections were mounted onto 200-mesh copper grids and stained with uranyl acetate for visualization under TEM.
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irradiation experiment. Cells were washed three times with PBS. The following order of treatment was performed: no treatment, NIR laser alone (7.5 min irradiation), Lipos Au NPs alone and Lipos Au NPs with laser (7.5 min irradiation). For treatment with Lipos Au NPs, cells were incubated with particles at 37 C for 5 hours (100 mg ml1 gold concentration). Thereaer, cells were washed three times with PBS to remove unbound particles. Cells were then resupplied with DMEM containing 10% FBS. Cells were irradiated with a NIR laser (750 nm) and then incubated at 37 C for 12 hours. Then the cells were washed with PBS and subjected to Alamar Blue assay. For qualitative examination, 24 well plates were used which were seeded with a density of 1 105 cells per well. The remaining protocol was similar to the one followed for the above mentioned alamar blue assay, except that at the end of 5 hours incubation, cells were washed with PBS and stained with propidium iodide (for visualization of dead cells). Cells were examined using a Olympus model IX81 laser scanning confocal microscope equipped with lters set for excitation/emission wavelengths at 488/617 nm for propidium iodide.
2.9. Photothermal mediated model drug (calcein) release Thermosensitive liposomes loaded with calcein were prepared by previously reported methods with slight modication.18 First the calcein, which was not encapsulated, was removed from the liposome suspension by centrifugation at 20 000g, 4 C for 20 min and then resuspended in PBS to make the total lipid concentration 2 mg ml1. 200 ml of the calcein loaded liposomes were added to 5 mM HAuCl4 solution that was reduced to Au by the addition of ascorbic acid (20 mM). Aer vortexing, 20 ml of the sample was added to 2 ml of PBS and mixed well. The sample was then subjected to NIR laser irradiation (750 nm) and uorescence intensity (485 nm ex and 520 nm em) was measured in a spectrouorimeter intermittently at various time periods. For 100% release, liposomes were treated with 1% Triton X. Percentage release of calcein was calculated by the following formula,
2.8. Biocompatibility and in vitro photothermal therapy In vitro biocompatibility studies were done using the Alamar Blue assay in L929 cell-line. Exponentially growing cells were dispensed into a 96-well at bottom plate at a concentration of 1 104 cells per well. Aer allowing 24 hours for cell attachment, Lipos Au NP solutions were diluted appropriately in fresh media and added (200 mL) in the lipid concentration ranging from 100 mg ml1 to 1 mg ml1 (3 wells per sample concentration). The media was not changed during the incubation of 48 h. Following incubation, cell viability was determined by the addition of Alamar Blue (20 mL, 1 mg ml1 dye in sterile PBS). The plate was incubated for an additional 4 hours at 37 C and 5% CO2,28 allowing viable cells to convert the blue solution into pink dye. Absorbance values at 560 nm and 620 nm were collected and cell viability was calculated as a percentage compared to untreated control cells. For the photothermal cytotoxicity study, MDA-MB-231 cells were seeded onto a 96 well plate with a density of 1 104 cells per well one day before the This journal is © The Royal Society of Chemistry 2014
% release ¼ [(Ft Fi)/(Ff Fi)] 100 where Ft ¼ uorescence at time t, Fi ¼ initial uorescence, Ff ¼ uorescence with 100% release, i.e. Triton-X treatment.
2.10.
Optical imaging experiment
The preformed Lipos Au NPs were mixed with ICG (50 ml of 1 mM per ml of Lipos Au solution) and incubated for an hour in dark. The solution was then centrifuged (20 000g, 20 min) to remove unbound ICG and the pellet was resuspended in milli Q water. This ICG coated lipos–Au sample was then subjected to zeta potential analysis for conrmation of the coating. Optical imaging experiment was performed in phantom samples using Spectrocam Multispectral imaging instrument (USA). The excitation source was 750 nm (continuous wave) diode laser with 650 mV power (PMC, India). The emission lter was 830 nm with 10 nm band pass. The acquired images were pseudo
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coloured according to the emission range by the Spectrocam soware.
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2.11.
X-Ray CT imaging experiment
Commercially available gold seeds (15 nm and 40 nm) were purchased from Nanocs. A tri-modality microPET-CT-SPECT (model GE FLEX™ Triumph™) pre-clinical imaging system was used for performing the phantom CT scan. Plastic microcentrifuge tubes containing various AuNPs including Lipos Au nanoparticles, along with tubes containing water as negative control and Lopamidol 300 iodine contrast agent as positive control. Triumph XO soware was used for acquiring the scan and image was reconstructed using Access COBRA XXM soware.
3.
Results and discussion
DSPC : CHOL were prepared by the thin lm hydration method followed by coating with Au (in the presence of ascorbic acid (AA)) and charge based ICG coating. On irradiation with laser light, the entire structure destabilizes causing drug release and simultaneous rise of temperature (Fig. 1). The Lipos Au coating has been optimised to be at 400 mg ml1 lipid concentration (ESI, Fig. S1†). Lipos Au NPs have been characterized for their size, shape and morphology. They are found to have broad NIR peaks as shown in Fig. 2a. The hydrodynamic size of these particles was found to be around 200 nm (Fig. 2c). The SEM and TEM images obtained indicated the size to be around 100– 150 nm (Fig. 2d–f). Considering the DLS measurement of the bare liposomes (ESI, Fig. S2a†) with that of the Lipos Au NPs (Fig. 2c), it was observed that the sizes are comparable both before and aer Au coating, indicating the compact self-
Fig. 1
Fig. 2 (a) UV-Vis absorbance of lipos au samples with varying molarity ratios of ascorbic acid and HAuCl4 (b) corresponding photographic images of Lipos Au NPs solution (c) DLS measurement of Lipos Au NPs (d) FEG-TEM (e) FEG-SEM (f) STEM (scanning transmission electron microscopy) image of Lipos Au NP showing the hollow central region with dark contrast. (Bare liposomes were used as controls in (a) and (b); scale bars: (d) 20 nm, (e) 10 nm, (f) 50 nm).
assembly of Au on to the liposomal surface. The liposome core has been characterized for its thermo-sensitivity and model drug release (ESI, Fig. S3†). It has been observed that once 43 C was attained, the liposome destabilizes to release the model drug. More than threefold of calcein (65.49 2.8%) was released at 43 C in comparison to just 14.9 2.4% at 37 C.
Multifunctional gold coated liposome nanoparticle (Lipos Au NP) – role in imaging, drug delivery and photothermal therapy PTT.
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It was observed that only in the presence of the liposome template, the gold coating was stable and was tunable to the NIR region. When the liposome templates were not included, the wavelengths of the samples remained xed in the 500– 600 nm region and the intensity of the solution gradually reduced as shown in Fig. 3a. In the absence of a liposome template, the gold structures that were formed by reducing HAuCl4 in the presence of AA were not stable. Though nanostructures were prepared by the addition of AA to HAuCl4 previously by Wang et al., a stabilising agent (sodium benzene
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sulphonate) was required to retain the morphology, shape and size of the formed structures.29 In the case of Lipos Au NPs, the liposome template itself acted as a stabilizer. It retained the in situ reduced gold on its surface without undergoing any aggregation. An experiment to understand the stabilizing effect of the liposome template was performed as shown in Fig. 3. To maintain uniformity in color and absorbance, the Lipos Au NPs were tuned to give an absorbance at 600 nm similar to the solution that lacked a liposome template. Even aer 72 hours, the solution containing liposome templates showed little variation in their absorbance or color, whereas the solution containing only HAuCl4 and AA (without liposomes) began to fade (in color) and absorbance intensity reduced within a few minutes aer preparation (Fig. 3a(i), see ESI, Fig. S5† performed with the actual NIR absorbing Lipos Au NPs). The control solution (i.e. without liposome template) precipitated and began to settle down making the solution transparent as discussed above. Now to understand drug release from the Lipos Au NPs, the particles were subjected to laser irradiation for varying time duration. Drug release can happen only when the Lipos Au structure destabilizes through the heat generated by the photothermal effect. To further understand this process, we subjected the solution containing Lipos Au NPs (5 concentration) to graded time periods of laser irradiation (5, 10, 15, 20 and 30 min) with 2 min interval between every 5 min of irradiation. It was observed that immediately aer 5–10 min of laser irradiation, the particle morphology began to change (Fig. 4b(i–iv)). This was accompanied by reduction of the UV-Vis absorbance intensity as shown in Fig. 4a. This degradation phenomenon
Fig. 4 Lipos Au degradation studies (a) UV-Vis absorbance of Lipos Au
Stability of Lipos Au solution compared with Au solution without liposome template, UV-Vis absorbance of (a) HAuCl4 sol. reduced by AA without liposome template, (b) with liposome template (c) photographic images at 0 min [(i) without liposome template, (ii) with liposome template], (d) at 2 min (e) at 4 min (f) at 6 min (g) at 8 min (h) at 10 min (i) at 30 min. Fig. 3
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NPs treated with NIR laser for varying time periods. (b(i–iv)) Representative FEG-SEM images of Lipos Au NPs treated with NIR laser for varying time periods, (c–e) representative FEG-TEM images of Lipos Au NPs treated with NIR laser for varying time periods, (f) FEG-TEM HR image of Lipos Au NPs treated with NIR laser for 30 min showing particles of size 5 nm (inset scale bar: 5 nm). (Scale bars: (b) 100 nm, (c–e) 50 nm, (f) 5 nm).
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was conrmed by TEM analysis (Fig. 4c–e). At the end of 30 min, almost the entire morphology of the particle was lost. The particles were degraded into small gold particles of size around 5 nm as shown in Fig. 4f. This experiment gave insight that the maximum time period of laser irradiation was permissible to be less than 10 min. As the NIR absorbance changes beyond 10 min of irradiation, the time scale was limited to 7.5 min for further PTT experiments. The degradability studies also revealed that the Lipos Au system is capable of degrading into smaller gold particles of size 5 nm that has an implication in getting cleared through the renal system. The Lipos Au NPs were synthesized with AA : Au in the ratio 3 : 2, so that they were able to absorb NIR light around 750 nm (Fig. 5a). A photothermal transduction experiment was performed to understand the temperature increment that can be achieved by Lipos Au NPs. It was observed that the Lipos Au NPs
(a) Representative absorbance of Lipos Au NPs 750 nm sample that was utilized for further photothermal related experiments, (b) photothermal transduction experiment showing a significant temperature increment for Lipos Au NPs.
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reached a temperature of 43 C in 5 to 10 min. The control solutions i.e. water and just liposomes (without Au) reached only 39 C during the same time period (Fig. 5b). The bio compatibility test performed with L929 cells suggested that these Lipos Au NPs were biocompatible even at 1 mg ml1 of Lipos Au (lipid concentration). When tested from 10 mg ml1 to 1 mg ml1, the cell viability was more than 90% as shown in Fig. 6a. As the nanoparticles can undergo different routes of uptake, a TEM analysis of cell uptake employing Lipos Au NPs was performed. These particles were incubated with breast cancer cell lines (MDA MB 468). At the end of 24 hours, it was found that the nanoparticles were well spread in the cytosol region of the cancer cells without aggregation or being engulfed by endosomes (Fig. 6c and d). Though previous studies have shown that gold nanoparticles when delivered via liposomes can circumvent endosome mediated uptake,30 the current analysis differs in that the liposomes (core) are getting delivered through gold nanoparticles (shell) covering them, whereas in the previous reported studies gold nanoparticles occupied the core region.30 As the gold coating is not contiguous over the liposome core, the exposed lipid membranes might interact with the cell membranes to enhance the uptake of nanoparticles. However, the current analysis could not explain the
Fig. 5
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Fig. 6 (a) Biocompatibility (b) cell uptake of Lipos Au in MDA MB 231 cancer cells based on ICP AES analysis of Au, (c and d) cell uptake of Lipos Au (TEM) in MDA MB 468 cancer cells.
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intact particle morphology even aer the expected lipid cellular interactions, for which further detailed analysis is required. In the photothermal mediated cytotoxicity experiment, the cells incubated with Lipos Au NPs and irradiated with 750 nm NIR laser for 7.5 min, lost their viability in comparison to the controls. This was conrmed both quantitatively and qualitatively (Fig. 7). A NIR Laser mediated model drug release study was performed using calcein dye loaded Lipos Au NPs (Fig. 7e). Within 5 min of NIR laser irradiation, almost 50% of the model drug was released. As already observed in the photothermal transduction experiment, the Lipos Au solution temperature was able to reach above 43 C within 5 min of laser irradiation (Fig. 5b). Hence, it is very much evident that the destabilization of the Lipos Au NPs (due to the photothermal effect) was responsible for drug release. By the end of 10–15 min almost 85% of the model drug was released, whereas in the control solution (liposome loaded with calcein – without Au coating), the release was limited to around 10% due to the absence of photothermal effect. The Lipos Au NPs were also subjected to CT phantom analysis at 70 and 90 kVp. In both the cases, they gave good CT contrast equivalent to the iodine control (Fig. 8a(i and ii)). The signal generated by Lipos Au NPs was much better than that obtained from commercially available gold seeds (15 nm and 40 nm Au NPs). We hypothesize that the presence of liposome enables a scattered accumulation of gold atoms within the lipid system that can provide a larger area to enhance X-ray
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Fig. 8 (a) X-ray CT imaging (Lipos AuNPs) at (i) 70 kVp and (ii) 90 kVp, (b) variation of zeta potential with Au and ICG coating on the liposome. Bright field image of Lipos Au ICG solution (c-(i)) and corresponding optical image (c-(ii)), bright field image of free ICG solution (d-(i)) and corresponding optical image (d-(ii)). (ex: 750 nm, em: 830 nm.)
attenuation more efficiently than the gold seeds. The Lipos Au NPs were coated further with ICG (50 ml of 1 mM per ml of Lipos Au NP solution). ICG coating on the lipos Au surface was
(a–c) Photothermal cytotoxicity (qualitative analysis) (i) differential scanning images of (a) cells incubated with Lipos Au NPs only (b) cells irradiated for 7.5 min with 750 nm laser (c) cells incubated with Lipos Au NPs and irradiated for 7.5 min with 750 nm laser (a–c), (ii) corresponding CLSM images exhibiting PI stain (dead cells) in red, (c(iii)) merged images of (c(i and ii)) showing dead cells due to PTT, (d) photothermal cytotoxicity (quantitative analysis – Alamar Blue assay) (e) photothermal mediated calcein (model drug) release from Lipos Au NPs. (Scale bars: (a–c) 200 mm.)
Fig. 7
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performed to make the Lipos Au NPs uorescent when irradiated with the same NIR laser (750 nm) that was actually meant for inducing the photothermal effect, thus enhancing the multifunctional capabilities of the nanoparticle system. Both the Au coating and the ICG coating were conrmed by zeta analysis as shown in Fig. 8b. It was found that the Lipos Au ICG solution gave good contrast equivalent to that of free ICG solution (Fig. 8c and d). The Lipos Au NPs were coated with ICG without compromising the uorescence of the dye. There was no obvious quenching observed due to the addition of Lipos Au NPs. This showed that there is a likely possibility of tagging the Lipos Au NPs with NIR dye to enhance the multifunctional properties of the system acquiring dual imaging modality (i.e. anatomical and functional).
4. Conclusion It is observed that Lipos Au NPs can be used for photothermal therapy of cancer cells. They can also be efficiently deployed for drug delivery application using NIR laser irradiation. The same laser light may be useful in optical imaging of the particles when coated with appropriate NIR dye. In addition to PTT, drug delivery and optical imaging, the Lipos Au NPs exhibited its true multifunctional ability by emitting good signals in CT X-ray analysis. We have shown the degradability of the system, by achieving 5 nm size particles assuring the capability of renal clearance. Further in vivo studies are to be performed to understand the complete efficacy of these novel biodegradable agents in cancer research.
Conflict of interest The authors declare no competing nancial interest.
Acknowledgements The authors would like to acknowledge TMC Seed-in-Air Intramural funding to A. D. and Ms Renu Malhotra for assisting with some of the biology experiments at ACTREC; Spectrocam Instruments for demonstrating the optical imaging experiments; IIT B Healthcare initiative for funding the project and SAIF-IITB for characterization studies.
References 1 X. Huang, P. K. Jain, I. H. El-Sayed and M. A. El-Sayed, Lasers in Medical Science, 2008, 23, 217. 2 J. Chen, D. Wang, J. Xi, L. Au, A. Siekkinen, A. Warsen, Z.-Y. Li, H. Zhang, Y. Xia and X. Li, Nano Lett., 2007, 7, 1318. 3 L. Au, D. Zheng, F. Zhou, Z.-Y. Li, X. Li and Y. Xia, ACS Nano, 2008, 2, 1645. 4 J. Chen, C. Glaus, R. Laforest, Q. Zhang, M. Yang, M. Gidding, M. J. Welch and Y. Xia, Small, 2010, 6, 811.
Nanoscale
Paper
5 A. R. Lowery, A. M. Gobin, E. S. Day, N. J. Halas and J. L. West, Int. J. Nanomed., 2006, 1, 149. 6 A. M. Gobin, J. J. Moon and J. L. West, Int. J. Nanomed., 2008, 3, 351. 7 J. A. Schwartz, A. M. Shetty, R. E. Price, R. J. Stafford, J. C. Wang, R. K. Uthamanthil, K. Pham, R. J. McNichols, C. L. Coleman and J. D. Payne, Cancer Res., 2009, 69, 1659. 8 H. Jin, P. Yang, J. Cai, J. Wang and M. Liu, Appl. Microbiol. Biotechnol., 2012, 94, 1199. 9 E. B. Dickerson, E. C. Dreaden, X. Huang, I. H. El-Sayed, H. Chu, S. Pushpanketh, J. F. McDonald and M. A. ElSayed, Cancer Lett., 2008, 269, 57. 10 G. P. Goodrich, L. Bao, K. Gill-Sharp, K. L. Sang, J. Wang and J. D. Payne, J. Biomed. Opt., 2010, 15, 18001. 11 D. Bechet, P. Couleaud, C. Frochot, M.-L. Viriot, F. Guillemin and M. Barberi-Heyob, Trends Biotechnol., 2008, 26, 612. 12 M. Longmire, P. L. Choyke and H. Kobayashi, Nanomedicine, 2008, 3, 703. 13 H. S. Choi, W. Liu, P. Misra, E. Tanaka, J. P. Zimmer, B. Itty Ipe, M. G. Bawendi and J. V. Frangioni, Nat. Biotechnol., 2007, 25, 1165. 14 J. H. Ryu, H. Koo, I.-C. Sun, S. H. Yuk, K. Choi, K. Kim and I. C. Kwon, Adv. Drug Delivery Rev., 2012, 64, 1447. 15 X. Huang and M. A. El-Sayed, Journal of Advanced Research, 2010, 1, 13. 16 T. S. Troutman, J. K. Barton and M. Romanowski, Adv. Mater., 2008, 20, 2604. 17 S. J. Leung, X. M. Kachur, M. C. Bobnick and M. Romanowski, Adv. Funct. Mater., 2011, 21, 1113. 18 P. Pradhan, J. Giri, F. Rieken, C. Koch, O. Mykhaylyk, M. D¨ oblinger, R. Banerjee, D. Bahadur and C. Plank, J. Controlled Release, 2010, 142, 108. 19 A. M. Gobin, M. H. Lee, N. J. Halas, W. D. James, R. A. Drezek and J. L. West, Nano Lett., 2007, 7, 1929. 20 J.-L. Li, L. Wang, X.-Y. Liu, Z.-P. Zhang, H.-C. Guo, W.-M. Liu and S.-H. Tang, Cancer Lett., 2009, 274, 319. 21 X. Huang, I. H. El-Sayed, W. Qian and M. A. El-Sayed, J. Am. Chem. Soc., 2006, 128, 2115. 22 S. E. Skrabalak, J. Chen, L. Au, X. Lu, X. Li and Y. Xia, Adv. Mater., 2007, 19, 3177. 23 T. F. Massoud and S. S. Gambhir, Genes Dev., 2003, 17, 545. 24 V. Ntziachristos, Nat. Methods, 2010, 7, 603. 25 M. J. Paulus, S. S. Gleason, S. J. Kennel, P. R. Hunsicker and D. K. Johnson, Neoplasia, 2000, 2, 62. 26 J. V. Frangioni, J. Clin. Oncol., 2008, 26, 4012. 27 J. T. Alander, I. Kaartinen, A. Laakso, T. P¨ atil¨ a, T. Spillmann, V. V. Tuchin, M. Venermo and P. V¨ alisuo, Int. J. Biomed. Imaging, 2012, 2012, 1. 28 S. T. Selvan, T. T. Tan and J. Y. Ying, Adv. Mater., 2005, 17, 1620. 29 S. Wang, K. Qian, X. Bi and W. Huang, J. Phys. Chem. C, 2009, 113, 6505. 30 P. Nativo, I. A. Prior and M. Brust, ACS Nano, 2008, 2, 1639.
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