ProteinâNanoparticle Agglomerates as a Plasmonic Magneto

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Cite This: ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

Protein−Nanoparticle Agglomerates as a Plasmonic MagnetoLuminescent Multifunctional Nanocarrier for Imaging and Combination Therapy Uday Narayan Pan,† Pallab Sanpui,‡ Anumita Paul,*,† and Arun Chattopadhyay*,†,§ †

Department of Chemistry, Indian Institute of Technology Guwahati, Guwahati, Assam 781039, India Department of Biotechnology, BITS Pilani, Dubai Campus, P.O. Box 345055, Dubai International Academic City, Dubai, United Arab Emirates § Centre for Nanotechnology, Indian Institute of Technology Guwahati, Guwahati, Assam 781039, India

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‡

S Supporting Information *

ABSTRACT: We report the fabrication of a plasmonic magneto-luminescent multifunctional nanocarrier (PML-MF nanocarrier) by lysozyme-mediated agglomeration of goldcoated iron oxide nanoparticles (IO@AuNPs) and subsequent coating of these agglomerates with BSA-stabilized gold nanoclusters (BSA-AuNCs). The agglomeration-mediated enhancement of plasmonic absorbance at the NIR biological window helped in plasmonic photothermal therapy (PPTT) by PML-MF nanocarriers. PML-MF nanocarriers demonstrated excellent in vitro bioimaging and magnetic targeting capabilities due to the strong photoluminance and superparamagnetism of the constituent AuNCs and IO@AuNPs, respectively. Moreover, these nanocarriers showed the successful loading and delivery of doxorubicin to cancer cells with a significant killing efficiency that could be synergistically improved by combining with PPTT. KEYWORDS: protein−nanoparticles agglomeration, plasmonic magneto-luminescent multifunctional nanocarrier, plasmonic photothermal therapy, bioimaging, cancer theranostics

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Surface plasmon, superparamagnetism, and luminescence are, arguably, the three most important properties of nanoparticles that have been broadly investigated for different biomedical applications including cancer theranostics.6 Thus, development of an ideal plasmonic magneto-luminescent multifunctional nanocarrier (PML-MF nanocarrier) by smart manipulation of protein-mediated agglomeration of nanoscale gold particles seems a challenge worth addressing.1−6 Herein, we propose the fabrication of a PML-MF nanocarrier in a twostep process. First, agglomeration of iron oxide core gold shell nanoparticles (IO@Au NPs) was pursued in the presence of a lysozyme (Lyz) in an aqueous medium. Thereafter, the coating of these nanoscale agglomerates was carried out with a luminescent BSA-Au nanocluster (AuNC-BSA) matrix (Scheme 1). The IO@Au NPs exhibited both magnetic as well as plasmonic characteristics originating from the iron oxide core and Au shell, respectively. Additionally, the plasmonic Au shell provided the IO@Au NPs with a better chemical stability, biocompatibility, and helped in protein-

rotein-mediated agglomeration of metal and metal oxide nanoparticles, especially nanoscale gold particles, offers potential biomedical implications.1−3 To this end, protein− gold nanoparticle (AuNP) agglomerates have been studied for drug delivery applications.1−3 However, to the best of our knowledge, protein-based agglomeration of AuNPs has not been exploited extensively for the construction of multifunctional theranostic nanoparticles (MNTPs) capable of simultaneous targeting, therapy, and imaging of cancer cells. The protein matrix of stable protein−AuNP agglomerates provides a hydrophilic as well as a hydrophobic environment suitable for encapsulation of a wide array of drugs with varying polarity. It can also act as a unifying platform for assembling individual diagnostically or therapeutically relevant nanomaterials, thus offering the option of targeting, multimodal imaging and multiple therapeutic avenues often desired of an ideal MTNP.1−4 Besides, the protein component of the protein− AuNP agglomerate-based MNTPs may facilitate smooth internalization of the MTNPs by cancer cells. It may also help in gradual disintegration of MTNPs in the intracellular environment, by various proteolytic enzymes following completion of the assigned theranostic task, into smaller nanoscale components easily removable by renal clearance.1−5 © XXXX American Chemical Society

Received: March 12, 2019 Accepted: June 27, 2019 Published: June 27, 2019 A

DOI: 10.1021/acsabm.9b00210 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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ACS Applied Bio Materials Scheme 1. Schematic Representation of the Fabrication of the PML-MF Nanocarrier and Its Capacity for Plasmonic Photothermal Therapy, Drug Delivery, Bioimaging, and in Vitro Magnetic Targeting

mediated agglomeration.1−4,7,8 The broadening and redshifting of the plasmonic absorbance peak of IO@Au NPs toward the NIR biological window (due to Lyz-mediated agglomeration) could be exploited for potential plasmonic photothermal therapy (PPTT).1−3 On the other hand, the magnetic properties of IO@Au NPs presented the possibility of additional diagnostic and therapeutic tools including magnetic resonance imaging (MRI), magnetic hyperthermia, and magnetic targeting.4,9−12 The photoluminescence (PL) of AuNCs, being superior to that of semiconductor quantum dots and organic dyes with respect to photostability, a large Stokes shift, and biocompatibility, offered the option of low-cost, sensitive, and noninvasive optical imaging of cancer cells.4,13 Additionally, BSA-stabilized AuNCs facilitated the coating of Lyz-IO@AuNP agglomerates, leading to an improved stability of the PML-MF nanocarriers.1−3 It may be noted here that AuNC and the Au shell around the IONP, in spite of both being gold-based nanomaterials, served two distinct purposes in the final PML-MF nanocarriers. The Au nanoshell imparted PPTT-relevant plasmonic characteristics to the PML-MF nanocarriers, while the PL of the nanocarriers was contributed by the AuNCs. The PML-MF nanocarrier, thus produced in the present study, incorporated plasmonic, magnetic, and luminescence modalities in a single system. The strong PL characteristic of the PML-MF nanocarrier was explored for imaging of cancer cells including HeLa (cervical cancer), HepG2 (liver cancer), and A375 (malignant melanoma). The superparamagnetic nature demonstrated by the PML-MF nanocarrier was also exploited for in vitro magnetic targeting. Furthermore, doxorubicin (Dox, a well-known chemotherapeutic drug) was successfully loaded into the PML-MF nanocarrier and the Doxloaded PML-MF nanocarriers (DPML-MF nanocarriers) were found to be efficiently delivering the chemotherapeutic drug to

cancer cells with a significant antiproliferation efficacy. Finally, the synergistic therapeutic effect was also observed when DPML-MF-nanocarrier-treated cancer cells were subjected to laser irradiation. In order to construct the PML-MF nanocarrier, first oleicacid-stabilized iron oxide nanoparticles (IONPs) were prepared by thermal decomposition of iron−oleate complexes and then phase-transferred to water using tetramethylammonium hydroxide (TMAOH) (details in the experimental section, Supporting Information).4,14 The average size of the IONPs produced was calculated to be 6.7 ± 1.2 nm from TEM images (Figure S1A−C). The selected area diffraction pattern (SAED) and inverse fast Fourier transform (IFFT) of highresolution TEM (HRTEM) image of IONPs showed the presence of the characteristic 311 plane of iron oxides (Figure S1D−F).4,14,15 Moreover, the powder XRD pattern of IONPs showed characteristic peaks (2θ) at 30.4°, 35.6°, 43.4°, 53.8°, 57.3°, 63.1°, and 74.5° from (220), (311), (400), (422), (511), (440), and (533) planes (Figure S1G) of cubic inverse spinel iron oxides.4,14,15 As-prepared IONPs were found to be superparamagnetic (with a saturation magnetization of 27.6 emu g−1, Figure S1H). Digital images (Figure S1I) showed IONPs could easily be separate from their dispersion using an external magnet. IO@Au NPs (comprising of the Au shell around the IONP core) were prepared in an aqueous medium by reducing Au3+ onto IONPs using boiling citrate method (details in the experimental section, Supporting Information).16−20 Following the reduction of Au3+, the color of the black IONP dispersion turned red, indicating the formation of a gold shell (Figure S2). The formation of the Au shell around IONPs resulted in a distinct surface plasmon resonance (SPR) peak at 521 nm as observed in the extinction spectrum of IO@Au NPs, which was otherwise absent in IONPs (Figure 1A).16−20 The average B


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ACS Applied Bio Materials

Figure 1. (A) Extinction spectra of (a) IONPs, (b) IO@Au NPs, (c) Lyz-IO@Au agglomerates, and (d) PML-MF nanocarrier. (B) Powder XRD pattern of IO@AuNPs showing presence of both the peaks of Au and IO. (C) Normalized emission spectra of (a) AuNCs-BSA and (b) the PMLMF nanocarrier at an excitation of 505 nm. (D) Normalized emission spectra of (a) AuNCs-BSA and (b) the PML-MF nanocarrier at an excitation of 365 nm. (E) Excitation spectra of (a) AuNCs-BSA and the (b) PML-MF nanocarrier with emission maxima fixed at 666 and 658 nm, respectively. (F−H, J) TEM images of the PML-MF nanocarrier. (I) Magnified TEM images of “J” showing the presence of IO@Au NPs and AuNCs-BSA, some of the AuNCs are marked in yellow circles. (K, L) Magnified TEM images of PML-MF nanocarriers showing the presence of IO@Au NPs and AuNCs-BSA; some of the AuNCs are marked in yellow circles. (M) HRTEM of one IO@Au NP present inside the PML-MF nanocarrier and (N) the corresponding IFFT pattern showing lattice fringes of both Au and IO. (O) Elemental mapping of the PML-MF nanocarrier, showing the presence of both Au (green) and Fe (red). (O1) Scanning transmission electron microscopic image (STEM) of the PMLMF nanocarrier, (O2) showing the mapping of Au, (O3) showing the mapping of Fe, and (O4) the merged image of O1, O2, and O3. (P) SAED pattern of the PML-MF nanocarrier showing diffraction patterns of both Au and IO. (Q) Digital images of aqueous dispersion of the PML-MF nanocarrier, (Q1, Q2) in day light and (Q3, Q4) under UV light showing their easy magnetic separation and magnetofluorescent nature. (R) VSM hysteresis loop of the PML-MF nanocarrier showing the superparamagnetic nature of it.

size of IO@Au NPs was estimated (form TEM images) to be 9.3 ± 2.6 nm, which was larger than that of IONPs, thus indicating a 2.6 nm thick Au shell formed around the IO core (Figure S3). Analyses of the SAED and IFFT (from HRTEM image) patterns revealed the presence of both Au (111) and

IO (311) planes in a single IO@Au NP (Figure S3C−E). Elemental mapping also showed the presence of both Fe and Au in the same particle (Figure S3F). The powder XRD pattern of IO@Au (Figure 1B) demonstrated the peaks (2θ) at 30.4°, 35.5°, 44.4°, 53.8°, 56.6°, and 75.4°, characteristic of C


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Figure 2. (A) Temperature changes occurring under 650 nm laser irradiation with time of (a) PML-MF nanocarriers and (b) IO@Au NPs dispersed in water. (B) MTT-based viability assay of HeLa cells treated with (a) PML-MF nanocarriers and (b) PML-MF nanocarriers followed by 650 nm laser irradiation for 10 min. (C) CLSM images of HeLa, HepG2, A375, and HEK cells treated with the PML-MF nanocarrier for 2 h; images were recorded with a 488 nm excitation laser.

(220), (311), (400), (422), (511), and (533) planes of iron oxide and (2θ) at 38.42°, 45.5°, 64.8°, and 77.7°,

corresponding to (111), (200), (220), and (311) planes of Au.16−21 Vibrating sample magneto-metric (VSM) analyses D


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having enough number of IONPs to have been attracted by the external magnet employed in the present experiment. Nonetheless, the observation, as made in Figure 1Q4, was not due to the instability of the PML-MF nanocarriers as supported by additional stability data described later (Figure S18). Analysis of extinction spectra showed slight broadening following coating of AuNCs-BSA, this is due to further agglomeration of IO@Au NPs in the presence of acetone and AuNCs-BSA (Figure 1A). VSM analysis showed PML-MF nanocarriers are superparamagnetic in nature with a saturation magnetization of 2.3 emu g−1 (Figure 1R). The excitation spectrum of AuNCs-BSA showed a broad band around 505 nm and a hump at 365 nm (Figure 1E).4,22 As is evident from Figure 1E, the excitation spectrum did not change significantly following the formation of the PML-MF nanocarrier. However, the emission spectra of the PML-MF nanocarrier, at both the excitation wavelength of 505 and 365 nm, showed a blue shift of 8 nm in emission maxima (λem = 658 nm) compared to those of as-synthesized AuNCs-BSA (λem = 666 nm) (Figure 1C,D). This shift in λem was due to the change in pH of the medium.4,22 Moreover, the PML-MF nanocarrier showed a higher photostability (Figure S9, experimental details) than FITC indicating its superiority over the organic dyes in bioimaging applications.4,22 As mentioned earlier, the Lyz-mediated agglomeration of IO@Au NPs caused broadening and red-shifting of the SPR peak. Consequently, the PML-MF nanocarrier showed a 3.3fold increase, compared to IO@Au, in the absorbance at 650 nm (Figure 1A) making the former a potential candidate for PPTT of cancer cells. Indeed, the aqueous dispersion of the PML-MF nanocarrier, when irradiated with a 650 nm laser, showed a rapid increase of temperature of 5.3 °C within 10 min followed by further increase of 0.83 °C over the next 10 min of time (Figure 2A). However, the control experiment with as-synthesize IO@Au showed only a slight increase in temperature (1.5 °C over 20 min) under a similar set of conditions (Figure 2A), indicating the importance of Lyzmediated agglomeration to enhance the PPTT ability of IO@ Au NPs in the PML-MF nanocarrier. Furthermore, PPTT efficacy of the PML-MF nanocarrier in vitro was tested by first incubating the HeLa cells with the PML-MF nanocarrier for 6 h followed by laser irradiation (with 0.5 W 650 nm laser) for 10 min. Finally, an MTT-based viability assay was carried out after 24 h of irradiation to estimate the cell death (Figure 2B). Results showed that 24% of the HeLa cells were killed when laser irradiation was applied after PML-MF nanocarrier treatment at 200 μg/mL (Figure 2B). PML-MF-nanocarriertreated cells (without laser irradiation) did not show any killing after 30 h of incubation (Figure 2B). The control experiment with nontreated HeLa cells, on the other hand, recorded no cell death as a result of laser irradiation only (Figure S10A). Moreover, IO@Au NP-treated HeLa cells when irradiated with a laser also did not show significant killing; only 5% of the cells were killed when laser irradiation was applied after treatment of 200 μg/mL IO@Au NPs (Figure S10B, details in the Supporting Information). Thus, the above results established the PML-MF nanocarrier as an efficient PPTT agent. The bioimaging capability of the PML-MF nanocarrier in vitro was investigated on three different types of cancer cells, HeLa, HepG2 and A375, and one normal cell, HEK. Cells were first grown on a coverslip, subsequently treated with the PML-MF nanocarrier (150 μg/mL) for 2 h and then fixed using formaldehyde (details in the experimental section,

showed that IO@Au was superparamagnetic with a saturation magnetization of 12.5 emu g−1 (Figure S3G). Digital images (Figure S3H) also showed the magnetic nature and easy separation of IO@Au NPs from an aqueous dispersion. Taken together, the experimental data mentioned above confirmed the formation of IO@Au NPs. Lyz-mediated formation of stable nanoscale agglomerates was carried out by adding Lyz solution to the aqueous dispersion of IO@Au NPs followed by gentle mixing (details in the experimental section, Supporting Information).1−3 Color of the dispersion immediately turned purple-blue, indicating the completion of the agglomeration (Figure S2).1−3 Absorption spectroscopy showed that addition of Lyz to the aqueous dispersion of IO@Au NPs caused the broadening and red-shifting of the SPR peak (λabs at 600 nm) of IO@Au NPs due to Lyz-mediated agglomeration (Figure 1A, Figure S4G).1−3 TEM images, HRTEM and the corresponding IFFT pattern, SAED, and elemental mapping confirmed the formation of agglomerates of the IO@Au NPs (Lyz-IO@Au agglomerates) (Figure S4).1−3,16−20 Luminescent BSA-stabilized gold nanoclusters (AuNCsBSA) were synthesized following a previously reported protocol.4,22 TEM images, digital images (Figure S5), emission, and excitation spectra (Figure 1C−E) confirmed the formation of AuNCs-BSA. The emission spectra of AuNCs-BSA (at 505 nm as well as 365 nm excitation) showed a strong emission peak at 666 nm due to the core nanoclusters (Figure 1C,D). In the emission spectrum of AuNCs-BSA (at 365 nm excitation), an additional emission peak appeared at ∼450 nm (Figure 1D), due to BSA as reported earlier.4,22 However, the control experiment with an aqueous BSA solution demonstrated a strong emission maximum at 345 nm (Figure S6), confirming that the emission around 666 nm in AuNCs-BSA originated from core AuNCs but not from BSA. Finally, the PML nanocarriers were fabricated by coating Lyz-IO@Au agglomerates with the AuNCs-BSA matrix in an acetone-mediated desolvation method followed by heat stabilization (detailed method in the Supporting Information).4,22 The BSA coating around Lyz-IO@Au agglomerates was observed in the TEM images of the PML-MF nanocarrier (Figure 1F−H). Further investigation of TEM images, HRTEM, corresponding IFFT pattern, SAED, and elemental mapping (Figure 1I−P, Figure S7) confirmed the presence of both IO@Au NPs and AuNCs-BSA in the final PML-MF nanocarrier construct. However, maintaining the same number of IO@Au NPs and AuNCs in each PML-MF nanocarrier was difficult, and the numbers might vary slightly from particle to particle. Nonetheless, this did not affect the overall characteristic of these nanocarriers collectively, which was confirmed by the reproducibility of the results over several batches of PMLMF nanocarrier preparations. The average size of the nanocarriers calculated from TEM images was found to be 620 ± 53 nm. The average hydrodynamic diameter of the PML-MF nanocarriers was found to be 711 nm in DLS measurements (Figure S8). The digital images in Figure 1Q showed that the PML-MF nanocarrier could be easily separated by an external magnet, clearly demonstrating their plasmonic magneto-fluorescent nature. It may be mentioned here that, as observed in Figure 1Q4, the external magnet was not able to attract all of the PML-MF nanocarriers, leaving a tiny fraction remaining suspended in the medium. This could be due to the possibility of these PML-MF nanocarriers not E


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Figure 3. In vitro magnetic targeting: CLSM images of HeLa cells treated with the PML-MF nanocarrier. The top panel represents cells close to the magnet, and the bottom panel represents cells away from the magnet.

albumin nanoparticle, inside which IO@Au NPs, AuNCs, and Dox are present. Thus, PML-MF nanocarriers are expected to follow a similar endocytosis pathway as albumin nanocarriers. Targeting of disease sites is another important aspect of modern day cancer theranostic.23−25 Magnetic nanocarriers (MNs) could be guided by a peripheral magnetic field toward the disease site to avoid collateral damages. As discussed above, vibrating sample magnetometric (VSM) analysis showed that PML-MF nanocarriers were superparamagnetic in nature with saturation magnetization 2.3 emu g−1. Although the value is lower compared to that of IO NPs (27.6 emu g−1) and IO@Au NPs (12.5 emu g−1), due to the contribution of a diamagnetic gold shell and AuNCs-BSA, it is still high enough for magnetic targeting.4,26 In this regard, a model experiment was performed in vitro where HeLa cells were grown on two coverslips placed opposite to each other in a 60 mm Petri dish.4,11,26 A powerful rare earth magnet was then placed at the bottom of one of the coverslips, and the cells were incubated with the PML-MF nanocarrier for 2 h (Figure 3, details in the experimental section, Supporting Information). Finally, following fixation, the cells were visualized under CLSM (488 nm laser excitation). It was observed that cells present on the coverslip placed just above the magnet showed a much higher PL emission intensity compared to those present on the coverslip away from the magnet (Figure 3), indicating the potential of the PML-MF nanocarrier for magnetic targeting.

Supporting Information). Fixed cells were then imaged under a confocal laser scanning microscope (CLSM) with 488 and 355 nm laser excitations. Bright red PL of PML-MF nanocarriers were observed inside all four kinds of cells at both excitations (Figure 2C, Figure S11). Moreover, the Z-stacking images in both depth and orthogonal projection for all cell lines confirmed the internalization of the PML-MF nanocarrier by the cancer cells (Figure S12). The nontreated (control) cells of all different cell lines did not show any such PL under similar sets of experimental conditions (Figures S13 and S14), thus establishing the PML-MF nanocarrier as a bioimaging probe. In order to get insight into uptake of PML-MF nanocarriers, internalization studies were performed in the presence of sodium azide (NaN3), a well-known inhibitor of the endocytosis pathway.11 When HeLa cells were incubated with the PML-MF nanocarrier in the presence of NaN3, an 82% decrease in the uptake of PML-MF nanocarriers was estimated from the CLSM images (Figure S15). This indicated the endocytosis being the preferred pathway for uptake of PML-MF nanocarriers. Among four types of endocytosis pathways (phagocytosis, micropinocytosis, clathrin-mediated endocytosis, and caveolae-mediated endocytosis), the albumin nanocarrier is known to preferentially follow micropinocytosis along with a slight propensity of clathrin-mediated endocytosis and caveolae-mediated endocytosis for some specific cancers.11 As in the present study, we have essentially designed an F


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Figure 4. (A, B) MTT-based cell viability of HePG2 and A375 cells treated with varying concentrations of the PML-MF nanocarrier and DPMLMF nanocarrier. (C) MTT-based cell viability of HeLa cells treated with the (a) PML-MF nanocarrier, (b) DPML-MF nanocarrier, (c) PML-MF nanocarrier then irradiated with a 650 nm laser, and (d) DPML-MF nanocarrier then irradiated with a 650 nm laser. (D) Isobologram plots showing a synergistic effect.

As is evident from previous discussions, Lyz (helped in the agglomeration of IO@Au) and BSA of the AuNCs-BSA composite (helped in coating the Lyz-IO@Au agglomerates) together provided significant amount of protein matrices within PML-MF nanocarriers. Considering the ability of proteins to efficiently carry hydrophilic and hydrophobic drug molecules, the potential of the present PML-MF nanocarrier as a drug carrier was evaluated by loading a chemotherapeutic drug, Dox. Loading of the drug was carried out by incubating 3.28 μg mL−1 of Dox with 162.3 μg mL−1 of the PML-MF nanocarrier under stirring for 2 h (experimental details in the Supporting Information). The PL emission spectrum of the Dox-loaded PML-MF nanocarrier (DPMLMF nanocarrier) exhibited an additional peak at 591 nm (characteristic of Dox), confirming the loading of the drug into the PML-MF nanocarrier (Figure S16). Encapsulation efficiency for Dox was calculated to be 82% (details in the Supporting Information). The kinetics of Dox release from the DPML-MF nanocarrier was also studied in two different pH buffers, i.e., PBS buffer (pH 7.4) and acetate buffer (pH 4.4). Results showed that 30% of Dox was being released in PBS buffer (pH = 7.4) and 37% in acetate buffer (pH = 4.4) upon 24 h of incubation at 37 °C (Figure S17). A comparatively fast release of Dox was noticed up to 6 h, and thereafter, a slow

release up to 24 h was observed in both buffers. The loading of Dox in PML-MF nanocarriers was mainly due to the hydrophobic, hydrophilic, hydrogen boding, and van der Waals interaction with BSA.27 The release profile observed in the present study did not demonstrate significant effect of pH on the release kinetics of Dox, indicating diffusion-driven drug release from the PML-MF nanocarrier being the possible mechanism.28 The DPML-MF nanocarrier was also found to be stable in human blood serum (HBS) up to 24 h as tested by monitoring the PL emission of the DPML-MF nanocarrier with time (Figure S18). The internalization of the DPML-MF nanocarrier by cancer cells was confirmed from the CLSM images (Figure S19) of cells treated with the DPML-MF nanocarrier for 2 h (details in the experimental section, Supporting Information). In order to observe the Dox emission from the DPML-MF-nanocarriertreated cells, they were examined under CLSM by irradiation with a 488 nm laser of 0.2 mW power, since preliminary experiments (data not shown) established that the laser power of less than 10 mW did not result in detectable emission from the PML-MF-treated cells. When irradiated with a 488 nm laser, the bright red emission of Dox was observed predominantly from the nuclei of the DPML-MF-nanocarrier-treated cells (Figure S19), which was absent in PMLG


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potential of the DPML-MF nanocarrier along with PPTT and the established DPML-MF nanocarrier as a multimodal therapeutic material. The reason for synergistic action, although not completely elucidated in the present study, could be the release of additional Dox due to laser irradiation.29 In conclusion, we have developed an advanced plasmonic magneto-luminescent multifunctional nanocarrier (PML-MF nanocarrier) using the Lyz-mediated agglomeration of IO@Au NPs followed by successful coating of the agglomerates with luminescent AuNCs-BSA. Agglomeration significantly enhanced the plasmonic absorbance of the IO@Au NPs in the NIR biological window and thus facilitated in vitro plasmonic photothermal therapy. The luminescent nature of AuNCs-BSA remained unaffected in the PML-MF nanocarrier and thus acted as a probe for bioimaging for different kinds of cancer cells including HeLa, HepG2, and A375 cells. The superparamagnetic nature of the PML-MF nanocarrier was utilized for in vitro magnetic targeting, indicating the possibility of targeted drug delivery. The presence of proteins in the PMLMF nanocarrier helped to efficiently load and deliver the chemotherapeutic drug Dox to cancer cells with a significant killing effect. Finally, the DPML-MF nanocarriers showed a synergistic therapeutic ability in vitro, when combined with laser irradiation of nanocarrier-treated HeLa cells. Serum albumin-based nanocarriers have shown promise with respect to in vivo therapeutic potential with Abraxane being one of the prominent examples.1,6,22,30 Although the allogenic BSA, used as a model protein to fabricate the present nanocarrier system, might raise immunological concerns, the issue of the immunogenicity can easily be mitigated by replacing it with human serum albumin (HSA) for in vivo applications.30 Moreover, unlike a free drug, albumin-based nanoparticles have been demonstrated to help in prolonged blood circulation for encapsulated drugs, maintaining a high concentration of the drug in blood over a relatively longer period of time.30 On the other hand, the presence of lysozyme is also expected not to affect the potentials of the present nanocarrier system in vivo as lysozyme-based nanocarriers have also been widely tested for applications in vivo.1 Moreover, the nontoxicity of gold and iron-based nanoparticles used for the construction of the PMLMF nanocarriers is well-demonstrated for applications in vivo.1,6,10,13 Thus, combining therapeutic modalities like PPTT and anticancer drug delivery with magnetic targeting, luminescence-based imaging capability and appreciable blood serum stability, and PML-MF nanocarrier, developed in the present form could be a potential candidate for cancer theranostics.

MF-nanocarrier-treated cells. As Dox is known to bind to the DNA of the cells, thus, CLSM images in Figure S19 essentially confirmed the release of Dox within cells.4,22 Z-stacking images of DPML-MF-nanocarrier-treated cells also confirmed the internalization of the DPML-MF nanocarriers by the cells (Figure S20).4,22 Successful uptake of DPML-MF nanocarriers by cancer cells was expected to result in a considerable antiproliferative efficacy, which was pursued by an MTT-based cell viability assay (Figure 4A−C). Results of an MTT assay showed that the PML-MF nanocarriers did not possess a considerable killing effect as it was only able to kill 1.2% of HeLa, 2.6% of Hep G2, and 3.1% of A375 cells even at a high concentration such as 200 μg/mL (for HeLa) and 150 μg/mL (HepG2 and A375). However, the DPML-MF nanocarrier showed a considerable killing effect in a dose-dependent manner with IC50 values 154.4, 121.6, and 93.4 μg/mL for HeLa, HePG2, and A375 cells, respectively, which corresponded to 3.1, 2.4, and 1.9 μg/mL of Dox as calculated from the encapsulation efficiency. In a parallel set of experiments, IC50 values of free Dox was found to be 0.65, 0.57, and 0.47 μg/mL for HeLa, HePG2, and A375 cells, respectively (Figure S21). This increase in IC50 values in terms of Dox amount for the DPML-MF nanocarrier, as compared to free Dox, could be due to an incomplete or slow release of Dox from the DPMLMF nanocarriers as mentioned in previous studies.4,22 Interestingly, when HeLa cells were cotreated with varying concentrations of free Dox and 200 μg/mL PML-MF nanocarriers, followed by laser irradiation for 10 min, an increase in killing efficiency was observed with an IC50 value of 0.46 μg/mL. This increased killing efficiency might be due to the contribution of photothermal effect of the nanocarriers (Figure S22A). The MTT assay was also performed in normal cells (HEK) as a control experiment, and the results (Figure S22B) showed that PML-MF nanocarriers did not kill HEK cells significantly. Nonetheless, DPML-MF nanocarriers showed a significant antiproliferative effect in HEK cells with an IC50 value of 211 μg/mL, similar to the cancerous cells. Although the selective anticancer response of the DPML-MF nanocarriers was not observed in the present study, suitable active targeting strategies coupled with magnetic targeting can be employed to enable these nanocarriers for the preferential eradication of cancer in vivo. Moreover, an increase in IC50 values in PML-MF nanocarriers (in terms of amount of Dox) apparently may contribute negatively to the potential of the present nanocarrier. However, considering the targeting ability of the nanocarriers, possible PPTT ability, image guided delivery option, and side effects caused by chemotherapeutic drugs, the present nanocarrier holds more potential compared to free Dox regarding the cancer theranostics. Considering the therapeutic potential of the DPML-MF nanocarrier both as an antiproliferative and PPTT agent, the combination therapeutic efficacy of these nanocarriers was evaluated by irradiating DPML-MF-nanocarrier-treated HeLa cells with a 650 nm laser (details in the experimental section, Supporting Information). The result of the MTT-based cell viability assay, presented in Figure 4C, clearly demonstrated that the combined therapeutic module comprised of 650 nm laser-mediated PPTT of DPML-MF-nanocarrier-treated HeLa cells was superior in killing the cancer cells when compared to those of either DPML-MF-treated or PPTT of PML-MFnanocarrier-treated HeLa cells. Moreover, the isobologram plot (Figure 4D) further revealed the synergistic therapeutic

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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/acsabm.9b00210.

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TEM images, HRTEM, SAED, XRD, VSM, and elemental mapping of IO@Au and Lyz-IO@Au agglomerates and CLSM and Z-stacking images of PML-MF- and DPML-MF-nanocarrier-treated cancer cells (PDF)

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ACS Applied Bio Materials *E-mail: [email protected].

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ORCID

Uday Narayan Pan: 0000-0002-8545-7223 Pallab Sanpui: 0000-0002-2785-0115 Anumita Paul: 0000-0002-2999-9749 Arun Chattopadhyay: 0000-0001-5095-6463 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the Central Instruments Facility, IIT Guwahati, for providing the instrumental facility. The department of Electronics and Information Technology, Government of India (no. 5(9)/2012-NANO, vol. III), is acknowledged for financial assistance. We also acknowledge Anil P. Bidkar, Satyapriya Bhandari, Sabyasachi Pramanik, Shilaj Roy, Ayan Pal, and Aditi Banerjee for their help.

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