Letter www.acsami.org
Protein-Based Multifunctional Nanocarriers for Imaging, Photothermal Therapy, and Anticancer Drug Delivery Uday Narayan Pan,† Rumi Khandelia,† Pallab Sanpui,‡ Subhojit Das,† Anumita Paul,*,† and Arun Chattopadhyay*,†,‡ †
Department of Chemistry and India
‡
Centre for Nanotechnology, Indian Institute of Technology Guwahati, Guwahati 781039, Assam,
S Supporting Information *
ABSTRACT: We report a simple approach for fabricating plasmonic and magneto-luminescent multifunctional nanocarriers (MFNCs) by assembling gold nanorods, iron oxide nanoparticles, and gold nanoclusters within BSA nanoparticles. The MFNCs showed self-tracking capability through singleand two-photon imaging, and the potential for magnetic targeting in vitro. Appreciable T2-relaxivity exhibited by the MFNCs indicated favorable conditions for magnetic resonance imaging. In addition to successful plasmonic-photothermal therapy of cancer cells (HeLa) in vitro, the MFNCs demonstrated efficient loading and delivery of doxorubicin to HeLa cells leading to significant cell death. The present MFNCs with their multimodal imaging and therapeutic capabilities could be eminent candidates for cancer theranostics. KEYWORDS: multifunctional nanocarriers, photoluminescence, magnetic targeting, photothermal therapy, two photon imaging, cancer theranostics
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phobicity).8−11 Serum albumin (SA), in this regard, has a wellestablished profile as a successful carrier of therapeutic drugs and imaging agents.10−16 For example, Abraxane, a SA NPbased formulation of anticancer drug paclitaxel, is approved by the United States Food and Drug Administration (FDA) for treating metastatic breast cancer and clinical trials for metastatic pancreatic and nonsmall cell lung cancer have also been pursued.12,17 Fortunately, the protein matrices of bovine serum albumin (BSA) NPs have been observed not to significantly affect the physicochemical properties of embedded inorganic NPs and drugs,13,15 underscoring their suitability as an effective platform for assembling multiple theranostic functionalities. Fluorescence imaging has gained particular interest as an inexpensive, sensitive, and noninvasive optical imaging modality in biomedical applications.18 Because of their stable photoluminescence (PL), nontoxicity, and chemical inertness, gold nanoclusters (Au nanoclusters) have emerged as fluorescent probes superior to photobleachable organic dyes or toxic quantum dots.13,19 Moreover, Au nanoclusters show potential for in vivo imaging because of their emission and two-photon excitation lying within the near-infrared (NIR) “biological window” of 650−900 nm.13,19 Magnetic resonance imaging (MRI), on the other hand, is one of the extensively used and
n incipient challenge in nanomedicine is to develop colloidal multifunctional nanoparticles (NPs) for theranostics. An ideal particle is deemed to consist of designated modalities for diagnosis of disease, targeted delivery, controlled release and monitoring of therapeutics, all in a single platform.1 In this regard, various inorganic NPs are known to show great potential as the “functional components” because of their fascinating and tunable physicochemical properties and possibility of multiple theranostic modalities from individual NPs.1−3 Magnetic, luminescent, and plasmonic properties are the three most extensively studied and, more importantly, biomedically exploitable properties of inorganic NPs.3−7 Although successful attempts of combining any two of the above-mentioned functionalities have been made, integrating all the three in one system has remained a challenge.4 Regarding the construction of multifunctional NPs, assembling individual magnetic, plasmonic and luminescent NPs on a common platform is desirable over the “hybrid NP” approach unifying discrete domains of different materials within a single hybrid nanostructure as the latter often leads to impairment of one property by the other because of close proximity.4 The choice of the unifying platform is crucial with respect to the ‘degree of multifunctionality’ and theranostic efficiency of the resultant system. Protein-based NPs could be important candidates for this purpose because of their inherent biodegradability, nontoxicity, and the availability of hydrophilic as well as hydrophobic patches (due to constituent amino acids) in the same system, thus facilitating incorporation of theranostic entities with varying hydrophilicity (or hydro© XXXX American Chemical Society
Special Issue: Focus on India Received: May 22, 2016 Accepted: August 1, 2016
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DOI: 10.1021/acsami.6b06099 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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Scheme 1. Schematic Depiction of Preparing MFNCs, Their Capacity for in Vitro MRI Contrasting and Magnetic Targeting, Two-Photon Imaging, Plasmonic Photothermal Therapy, and Inducing Cell Death in Cancer Cells (HeLa), Following Successful Loading and Delivery of Anticancer Drug Dox
spatially precise methods of disease diagnosis.20 The potential of superparamagnetic iron oxide NPs (IONPs) as MRI contrast agent in theranostic applications is well-established.5 The superparamagnetic nature with high magnetic saturation of IONPs also makes them suitable for magnetic targeting and hyperthermia.21 Bimodal imaging, combining fluorescent probe and MRI contrasting agent, is expected to demonstrate superior diagnostic capability. Multimodal therapy, coupled with multimodal diagnosis, holds the key to success of future cancer theranostics, as conventional chemotherapeutic drugs are not always capable of complete eradication of tumor due to inadequate efficacy and/or drug resistance.14 Plasmonic photothermal therapy (PPTT) is a promising approach of cancer treatment in which plasmonic nanomaterials, following extinction of light in the NIR region, efficiently generate heat to kill cancer cells, leading to either complete ablation of tumor or its increased susceptibility to other chemotherapeutic drugs.22−24 Gold nanorods (Au NRs) are ideal for PPTT because of ease of preparation and surface modification, biocompatibility, and having significant extinction cross-section in the “biological window” (650−900 nm) because of longitudinal localized surface plasmon resonance (LSPR).23,24 Moreover, two photon luminescence (TPL) of Au NRs could be exploited as an additional imaging modality.25,26 Herein, we report the creation of protein-based multifunctional nanocarriers (MFNCs) by incorporating Au NRs, IONPs and Au nanoclusters in BSA, thus with simultaneous integration of corresponding plasmonic, magnetic and luminescence properties (Scheme 1). The PL exhibited by the MFNCs was probed for their cellular internalization in vitro through single photon as well as two-photon imaging. The plasmonic nature of the MFNCs was pursued for in vitro PPTT while the associated magnetic property, in addition to the demonstration of
appreciable T2-weighted MR contrast, was exploited for magnetic targeting in vitro. Moreover, anticancer drug doxorubicin (Dox)-loaded MFNCs successfully delivered the drug to the cervical cancer cells (HeLa) resulting in efficient killing of cancer cells and allowing simultaneous probing of the release of the drug from the NCs. Individual building blocks for the MFNCs, namely, Au NRs (length, 34.7 ± 5.1 nm; aspect ratio, 3.3 ± 0.6 nm), IONPs (size, 8.2 ± 2.2 nm), and BSA-stabilized Au nanoclusters (size, 1.6 ± 0.2 nm), were synthesized by following previously reported methods (Figures S1−S3).13,27−29 The MFNCs were formed by adopting desolvation-based approach employed in the preparation of Au nanocluster-embedded BSA NPs in our previous study.13 As schematically depicted in Scheme 1, Au NRs, IONPs, and BSA-stabilized Au nanoclusters were first mixed thoroughly and then acetone was added drop by drop to the mixture to form the MFNCs (with details in the Experimental Section in the Supporting Information). Asprepared MFNCs were collected by centrifugation (20 000 rcf) and then redispersed in water for further studies. Field emission scanning electron microscopic (FESEM) and transmission electron microscopic (TEM) images (Figure 1A, B and Figure S4) demonstrated that the MFNCs were nearly spherical in shape with an average size of 120.5 ± 27.6 nm (from FESEM). The average hydrodynamic diameter (dH) of water-dispersed MFNCs was estimated to be 203.1 nm through DLS measurements (Figure 1C). The apparent increase in dH, as compared to the size observed in FESEM and TEM, could be due to the hydration layer formed at the nanocarrier surface in water.2 Since the optimal size-range for effective tumoraccumulation through ‘enhanced permeation and retention’ (EPR) effect is reported to be 10−200 nm,30 the present MFNCs (∼120 nm in size and dH ∼203 nm) show the B
DOI: 10.1021/acsami.6b06099 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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Figure 1. Representative (A) FESEM and (B) TEM images of MFNCs (scale bar: A, 300 nm; B, 100 nm). (C) Size-distribution of the MFNCs dispersed in water as measured by DLS. TEM image of (D) a typical MFNC and (E) at higher magnification showing component Au NR, IONPs, and ultrasmall Au nanoclusters (some of them marked with yellow circles for guidance) (scale bar: 20 nm). HRTEM images, focused at different regions of the same MFNC, demonstrating the presence of (F) individual Au nanoclusters (scale bar: 2 nm), (G) lattice fringes of Au NR (scale bar: 2 nm) with (H) corresponding IFFT pattern and (I) lattice fringes of IONPs (scale bar: 5 nm) with (J) corresponding IFFT pattern.
potential for “passive targeting” in vivo. As is evident in TEM images at higher magnifications (Figure 1D−F), individual MFNCs comprised of − in addition to the easily identifiable Au NRs − IONPs and Au nanoclusters, which could be distinguished based on the significant difference in their respective sizes. Furthermore, high resolution TEM (HRTEM) images of MFNCs and corresponding inverse fast Fourier transform (IFFT) patterns (Figure 1G−J) confirmed the presence of Au NRs and IONPs by revealing the characteristic lattice fringes due to (111) plane of face-centered cubic (fcc) Au NRs and (311) plane of inverse spinel Fe3O4 NPs (IONPs), respectively.28,31 It may be mentioned here that the distribution of AuNRs and IONPs among the MFNCs (Figure 1B and Figure S4) was not uniform. This could be due to the possible heterogenity of interaction of AuNRs and IONPs with BSA during the preparartion of MFNCs. The brown-colored colloidal dispersion of MFNCs exhibited bright red PL (Figure 2A and Figure S5) when irradiated with UV light (λex = 365 nm). Moreover, because of the presence of the IONPs, the MFNCs could easily be separated from the colloidal dispersion by an external magnet as shown in Figure 2A (Figure S5). The vibrating sample magnetometric (VSM) measurement revealed superparamagnetism in the MFNCs with a saturation magnetization of 4.66 emu g−1 (at room
temperature) (Figure 2B). However, this value was lower than that observed with free IONPs (27.88 emu g−1, Figure S6). This could be a result of incorporating nonmagnetic components namely Au nanoclusters, Au NRs, and BSA within the MFNCs. The extinction spectrum of MFNCs (Figure 2C) showed distinct band around 800 nm due to the characteristic longitudinal LSPR of the component Au NRs.27 However, the extinction maximum of the Au NRs was observed to be redshifted by 50 nm in MFNCs as compared to that of CTABstabilized free Au NRs (750 nm). The frequency-dependent extinction coefficients of metallic nanomaterials including Au NRs are highly sensitive to dielectric functions of the medium and the surface coating material(s) as well as the volumefraction of the latter.9,32 Thus, the shift in extinction maximum of Au NRs could be due to a change in local environment surrounding the Au NRs within MFNCs. Nonetheless, this was all the more advantageous as the red-shift of the Au NRassociated LSPR band allowed the MFNCs to better match the wavelength of the NIR laser (808 nm) employed for PPTT of cancer cells in the present study. The PL excitation spectrum of BSA-stabilized Au nanoclusters showed a weak shoulder around 365 nm and a broad band with maximum at 505 nm, which did not change significantly following the formation of MFNCs (Figure 2D). C
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with either conventional one-photon or two-photon imaging. Most importantly, successful two-photon imaging of MFNCs within cancer cells, with excitation and emission falling within the biological window (650−900 nm), is expected to expand their applicability for effective in vivo imaging. Moreover, the presence of IONPs within the MFNCs supports the possibility of their application as contrasting agent in MRI. The T2relaxivity of the MFNCs was calculated to be 448.04 mM−1 s−1 (Figure 3A, B). The exceptional PL properties coupled with appreciable T2-relaxivity of the MFNCs, indeed, demonstrated their potential in multimodal in vivo imaging.
Figure 2. (A) Digital photographs of aqueous dispersion of MFNCs in daylight (upper panel) and UV light (lower panel) in absence (left panel) or presence (right panel) of external magnet. (B) Hysteresis loop (magnetic moment, M, versus applied magnetic field, H) of MFNCs measured in VSM. (C) Normalized extinction spectra of (a) Au NRs, (b) Au nanoclusters, (c) IONPs, and (d) MFNCs. (D) Normalized excitation spectra of (a) BSA-stabilized Au nanoclusters (λem = 663 nm) and (b) MFNCs (λem = 650 nm). (E) Normalized emission spectra of (a) BSA-stabilized Au nanoclusters and (b) MFNCs. (F) Photostability of (a) BSA-stabilized Au nanoclusters, (b) MFNCs, and (c) FITC. CLSM images of HeLa cells, following incubation with MFNCs for 4 h, under (G) one-photon (488 nm laser; 1, 2, and 3 denote fluorescence, DIC, and merged image, respectively) and (H) two-photon (730 nm multiphoton laser) excitation (scale bar: 20 μm).
Figure 3. (A) T2-weighted MR images of MFNCs at various dilutions with corresponding concentrations of Fe mentioned adjacent to them (TR = 2000 ms and TE = 7.8 ms). (B) T2 relaxivity of MFNCs calculated from the plot of relaxation rate versus Fe concentration. (C) In vitro magnetic targeting of MFNCs: Experimental setup (middle) and CLSM images of HeLa cells on coverslips kept near (left) and away (right) from the magnet, respectively. (D) Changes in temperatures of aqueous dispersions of MFNCs (with extinction of 0.966, 0.695, and 0.35 at 808 nm), free Au NRs (with extinction of 0.665 at 808 nm), MFNCs without Au NRs and water under NIR laser (808 nm) irradiation. (E) Viability of HeLa cells after treatment with MFNCs or MFNCs without Au NRs and subsequent NIR laser (808 nm) irradiation for 10 min. HeLa cells without any nanomaterial treatment were used as control. The MTT assay was performed at 24 h postirradiation.
However, when excited at 505 nm, the PL emission maximum of MFNCs (λem = 650 nm) was observed to be blue-shifted by ∼13 nm from that of as-synthesized Au nanoclusters (λem = 663 nm) (Figure 2E). A similar blue-shift was also noticed upon excitation at 365 nm (Figure S7). The shift in the emission maximum, as demonstrated in our previous study,13 was due to the change in pH of the aqueous dispersion of MFNCs (pH 8.8) as compared to that of the Au nanoclusters (pH 12.0). Interestingly, the MFNCs were found to be much superior to FITC (commonly used organic dye for bioimaging) in terms of photostability and did not exhibit significant photobleaching or blinking (Figure 2F and Figure S8) indicating their potential in bioimaging applications, especially as self-tracking delivery vehicles. To investigate the self-tracking capability of the MFNCs following their internalization by cancer cells in vitro, we incubated HeLa cells with the nanocarriers for 4 h and subsequently observed under confocal laser scanning microscope (CLSM) with both one-photon (argon ion laser, excitation at 488 nm) and two-photon (Ti-sapphire laser, excitation at 730 nm) excitation sources. The red PL of MFNCs was successfully observed inside HeLa cells, following their uptake, under one-photon (Figure 2G and Figure S9) as well as two-photon excitation (Figure 2H). The control (nontreated) HeLa cells did not exhibit any fluorescence under similar experimental conditions (Figure S10). The results demonstrated that our MFNCs could be probed intracellularly
Magnetic targeting has drawn much attention in therapeutic applications as drug-loaded magnetic nanocarriers could be directed to the diseased area using external magnetic field with reduced side effects caused by nonspecific drug release.21,33 Possible magnetic targeting of the present MFNCs was explored in vitro by growing HeLa cells on two separate coverslips inside a cell culture dish and subsequently incubating them with MFNCs while keeping an external magnet just beneath one of the coverslips as shown in Figure 3C. After 2 h of incubation, when the coverslips were observed under CLSM, the cells on the coverslip just above the magnet were found to show more red PL because of the MFNCs as compared to the ones on the other coverslip (kept away from the magnet) (Figure 3C). This revealed that the MFNCs were effectively attracted toward the external magnet resulting in higher uptake by HeLa cells near to the magnet. As mentioned earlier, the MFNCs with extinction maxima at 800 nm are expected to exhibit PPTT of cancer cells. To pursue D
DOI: 10.1021/acsami.6b06099 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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ACS Applied Materials & Interfaces this, the MFNCs were first dispersed in water at room temperature and the dispersions having different concentrations (with extinction of 0.35, 0.695, and 0.966 at 808 nm) were irradiated with 808 nm NIR laser (Figure 3D). The temperatures of all the three dispersions containing MFNCs were increased by ∼20 °C within 10 min of irradiation, with only marginal increase in temperature later on. However, control experiment with MFNCs devoid of Au NRs (TEM image in Figure S11) resulted in an increment of ∼3 °C only under similar conditions, indicating the importance of incorporated Au NRs (within MFNCs) in generating heat under NIR irradiation. Interestingly, when comparable amount of Au NRs was considered, the MFNCs (extinction 0.695) were found to be as efficient as free Au NRs (extinction 0.665) in generation of heat (with temperature increase of 19.7 and 20.8 °C; respectively, by 10 min). The PPTT-efficiency of the MFNCs in vitro was assessed by irradiating MFNCs-treated HeLa cells for 10 min and determining the cell viability at 24 h postirradiation. As is evident in Figure 3D, 55% or more HeLa cells were killed as a result of NIR irradiation in the presence of MFNCs. Control experiments with HeLa cells in absence of MFNCs eliminated the possibility of cell death due to laser irradiation only. Moreover, similar experiment with MFNCs without Au NRs did not show any change in cell viability following NIR irradiation signifying the importance of plasmonic nature of the MFNCs for PPTT as an additional therapeutic modality. The results of in vitro PPTT by the MFNCs are of particular interest from in vivo viewpoint as the 808 nm NIR-laser has been shown to provide deep-tissue penetration with minimum attenuation of light during PPTT in animal models thus improving the overall therapeutic efficacy.24 Finally, the ability of the MFNCs to deliver chemotherapeutic drugs and eventually kill cancer cells was demonstrated in vitro using Dox as the model anticancer drug. Dox was loaded into the MFNCs by incubating 147.5 μg mL−1 MFNCs with 3.28 μg mL−1 Dox under gentle stirring for 2 h (with details in Experimental Section). The successful loading of Dox into the MFNCs was confirmed by fluorescence spectroscopy which revealed an additional peak at ∼590 nm, characteristic of Dox fluorescence, in the emission spectrum of Dox-loaded MFNCs (Dox-MFNCs) (Figure S12C). The drugloading efficiency of the MFNCs for Dox was estimated to be ∼92%. The efficient loading of Dox by MFNCs, as reported previously,13,34 could be due to the interaction of Dox with BSA through electrostatic interaction as well as hydrogen bonding. Zeta potential (ζ) of Dox-MFNCs (ζ = −20.8 ± 0.8 mV) at physiological pH was found to be increased when compared to unloaded MFNCs (ζ= −36.8 ± 0.7 mV), possibly because of the incorporation of positively charged Dox molecules (pKa 8.4). However, loading Dox into the MFNCs did not result in significant alteration of the morphological, magnetic, extinction and PL properties of the MFNCs (Figure S12). The dH of the Dox-MFNCs, i.e., 206 nm as measured by DLS (Figure S12F), was well-suited for possible passive targeting as mentioned earlier. Release kinetics of Dox from the Dox-MFNCs in vitro was studied in PBS (pH 7.4) at 37 °C. The results (Figure S12G) essentially showed linear increase in cumulative Dox release in first 5 h accompanied by slow release in later time points. Approximately 21% Dox was found to be released from the Dox-MFNCs within 24 h. The stability of the theranostic nanocarriers in the biological media is critical for their intended biomedical applications. The stability of the Dox-MFNCs was checked by following their emission spectra and it was observed
that they were stable over a period of 24 h in human blood serum and 7 days in water (Figure S13). Moreover, TEM analysis (Figure S13C) revealed that the overall morphology of the Dox-MFNCs did not change significantly after incubation in PBS (pH 7.4) for 7 days. The sizes of the constituent Au NRs and IONPs were found to be intact too. Following their incubation for 4 h, the uptake of DoxMFNCs by HeLa cells was followed under CLSM with single photon (Figures S14 and 15) and two-photon (Figure 4A)
Figure 4. CLSM images of (A) Dox-MFNCs and (B) Dox-treated HeLa cells recorded with two photon excitation. Scale bar: 10 μm. (C) Viability of HeLa cells treated with varying concentrations of MFNCs and Dox-MFNCs for 36 h.
excitations. The MFNCs, as already demonstrated in Figure 2G, H, tended to remain in the cytoplasm of HeLa cells following their internalization, whereas free Dox was observed to accumulate in the nucleus of the HeLa cells (bright red PL in Figure 4B). Careful observation of the two-photon CLSM images of HeLa cells treated with Dox-MFNCs (Figure 4A) and in comparison to those with MFNCs (Figure 2H) and free Dox (Figure 4B) indicated successful internalization of DoxMFNCs and subsequent release of Dox (exhibiting strong PL in the nucleus) inside HeLa cells. As a result of successful delivery of Dox by MFNCs to HeLa cells, Dox-MFNCs were also found to efficiently kill the cancer cells in a dose-dependent manner (Figure 4C). The IC50 value of the Dox-MFNCs for HeLa cells, as determined by the cell viability assay (Figure 4C), was found to be 119 μg mL−1, which corresponded to 2.3 E
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μg mL−1 Dox. However, slow/incomplete release of Dox from MFNCs could lead to the apparent increase in IC50 values as compared to free Dox (IC50 = 0.5 μg mL−1, Figure S16).11,13 Induction of “apoptosis” or programmed cell death in HeLa cells, following the treatment with Dox-MFNCs, was monitored from the characteristic apoptotic bodies observed under FESEM as shown in Figure S17. Interestingly, the MFNCs themselves did not exhibit significant cytotoxicity against HeLa cells (less than 14% cell death even at as high concentration as 161.7 μg mL−1) indicating their suitability in theranostic applications. In summary, we have successfully developed BSA-based plasmonic magneto-luminescent nanocarriers (herein referred as MFNCs) by assembling Au NRs, IONPs and Au nanoclusters in protein, without affecting their individual properties. The potential of the MFNCs as self-tracking luminescent probes in theranostic applications was validated in vitro by single as well as two photon fluorescence imaging. Moreover, the MFNCs showed considerable T2-relaxivity and capability of being magnetically guided, appropriate for MRI application and targeted drug delivery, respectively. Significant extinction of the MFNCs in the NIR window, owing to the characteristic plasmonic nature of the nanocarriers, led to successful PPTT of HeLa cells in vitro. Following efficient loading of Dox, the MFNCs were observed to successfully deliver the anticancer drugs to HeLa cells, leading to significant cell death, while the PL of the nanocarriers helped in visualizing the delivery through fluorescence microscopy. With the proven serum stability, the present MFNCs could be ideal candidates for cancer theranostics wherein they are expected to, following their accumulation in tumor through size-dependent passive targeting and/or magnetic guidance, provide multimodal therapy and imaging of cancer cells simultaneously.
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REFERENCES
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b06099. Detailed Experimental Section, synthesis and characterization of Au NRs, IONPs and Au nanoclusters, DoxMFNCs, additional TEM images of MFNCs, z-stacking of CLSM images of MFNCs, Dox-MFNCs and Doxtreated HeLa cells, and Figure S1−S17 (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank the Department of Electronics and Information Technology, Government of India (Grant 5(9)/2012-NANO (Vol II)) for funds. P.S. thanks the Department of Science and Technology, New Delhi, for a fund (Award Letter SB/FTP/ ETA-0122/2014). We thank the Central Instruments Facility, IIT Guwahati for measurements. We also thank Dr. Baijayanta Saharia, Jayanta Mazumder, Rishi Kant, Shilaj Roy, Satyapriya Bhandari, and Sabyasachi Pramanik for their help. F
DOI: 10.1021/acsami.6b06099 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Letter
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DOI: 10.1021/acsami.6b06099 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
