Near-Infrared-Triggered Photodynamic, Photothermal, and on

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B: Biomaterials and Membranes

Near Infrared-Triggered Photodynamic, Photothermal and On-Demand Chemotherapy by Multifunctional Upconversion Nanocomposite Balmiki Kumar, Aparna Murali, Irshad Mattan, and Supratim Giri J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.9b01870 • Publication Date (Web): 10 Apr 2019 Downloaded from http://pubs.acs.org on April 11, 2019

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The Journal of Physical Chemistry

Near Infrared-Triggered Photodynamic, Photothermal and

On-demand

Chemotherapy

by

Multifunctional

Upconversion Nanocomposite Balmiki Kumar, † Aparna Murali, ‡ Irshad Mattan, † and Supratim Giri*† †

Department of Chemistry, National Institute of Technology, Rourkela. Odisha- 769008, India

*E-mail: [email protected] ‡Department

of Biotechnology and Medical Engineering, National Institute of Technology,

Rourkela. Odisha- 769008, India ABSTRACT In an attempt to integrate photodynamic therapy (PDT) with photothermal therapy (PTT) and chemotherapy for enhanced anti-cancer activity, we have rationally synthesized a multifunctional upconversion nanoplatform using NaYF4: Yb/Tm/Er/Fe nanoparticles (NP) as core and NaYbF4: 1% Tm as a shell. The as-synthesised core-shell upconversion (CSU) NPs exhibited a diverse and enhanced photoluminescence emissions in wide range (UV to NIR) consequent upon Fe3+ doping in the core and fabrication of an active shell. Subsequently, CSU was first decorated with titania NPs as photosensitizer. Next, mesoporous silica (MS) shell loaded with doxorubicin (DOX) via photo-cleavable Ru-complex as gating was developed around titania containing CSU. Finally, gold nanorods (GNRs) with localized surface plasmon resonance (LSPR) at 800 nm were incorporated around MS layer to obtain the multifunctional nanoplatform. We demonstrated that the UV, blue and NIR emissions from the CSU produced ROS mediated PDT through titania activation, induced DOX release 1 ACS Paragon Plus Environment

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through photo-cleavage of Ru-complex and generated hyperthermia by LSPR activity of GNRs, respectively upon a single NIR excitation through FRET. The therapeutic efficacy was validated on HeLa cell lines in vitro by various microscopic and biochemical studies under a significantly milder NIR irradiation and lower dosage of the nanoplatforms, which have been further demonstrated as diagnostic nanoprobe for cell-imaging.

1. Introduction The toxicity and adverse side effects associated with large dosage of anti-cancer drugs have compelled the researchers to focus on the synthesis of multifunctional nanoplatforms as potential nanomedicine that could supersede the conventional chemotherapy.1-5 A nanomedicine that could effectively invoke several therapeutic modalities by a single trigger and show diagnostic attributes in low dosage would be deemed as an ideal system in a clinical setup.2,

4-7

Especially, in the cases where the cancers exhibit resistance towards certain

therapeutic routes over others; such multimodal nanoplatform is expected to show a more effective therapeutic output.1-2, 4, 6 In pursuit of developing such multimodal nanomedicine, researchers have so far developed few bimodal therapeutic nanoplatforms by combining any two of the three commonly used non-invasive therapeutic strategies, which are photodynamic therapy (PDT), photothermal therapy (PTT) and chemotherapy. For example, there have been reported therapeutic nanoplatforms demonstrating a combination of either PDT and PTT, or PTT and chemotherapy,

13-17

or PDT and chemotherapy.

3, 6-7, 18-20

8-12

.Although, incorporating

more than two therapeutic modalities is theoretically expected to increase the efficacy of cancer treatment,1,

4, 6, 21-25

it is a significant challenge to integrate them in a single

nanoplatform. Herein, we have successfully integrated the three different therapeutic modalities mentioned above in a single nanoplatform through precisely controlled activation mechanism.

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Utilization of low intensity non-invasive trigger that causes no significant damage to normal tissues and application of minimal dosage of nanomaterials constitute the two most desired objectives in the development of an effective nanomedicine for cancer therapeutics.1, 4 Recent literature demonstrated that near infra-red (NIR) laser can serve as one such viable noninvasive trigger.1, 4, 6 In order to achieve a therapeutic output triggered by NIR excitation, the majority of the studies have reported the utilization of upconversion nanoparticle (UCNP), which converts lower energy NIR (980 nm) signal into a photoluminescence (PL) involving higher energy photons in general.1,

26-28

The emitted photons resulting from upconversion

(UC) process were generally utilized in photosensitizer (PS) mediated PDT20,

29-37

,

photochemical reaction induced chemotherapy5, 7, 37-41 and nanotransducer induced PTT.12, 37, 42-43

However, only few UCNP based systems have been reported exhibiting therapeutic

property in dual modes within a single nanoplatform.3, 6, 8-12, 17-18 The limitations are attributed to the lack of a diverse UC emission profile and lower intensity PL output of the concerned UC materials, usually.44 Interestingly, the optimum NIR power deemed safe for skin penetration is 0.726 W/cm2.45-47 At a lower power, it is difficult to generate a UC mediated PL strong enough to induce any photophysical changes required for inducing multimodal therapeutic effects.28, 48-49 The use of a higher power NIR laser to enhance the PL output for multimodal therapeutic effects could be detrimental to the normal tissues by local heating effect.2, 4, 40 Therefore, in order to integrate several therapeutic modalities within a single UC based nanoplatform, capable of showing the multimodal therapeutic outcomes in the abovementioned conditions, it is imperative to diversify the PL profile and enhance the PL intensities of the concerned UCNP by attempting rational manipulation at the nanoscale level. In this article, through transition metal doping and epitaxial growth of active shell, we have engineered core-shell upconversion (CSU) nanocrystals to successfully meet the abovementioned challenges of achieving a versatile and enhanced PL output. Next, in the direction 3 ACS Paragon Plus Environment


of integrating the three different therapeutic modalities within a single nanoplatform, we adopted three different strategies to utilize different emission peaks of the concerned PL profile for a combined therapeutic output. In this context, the surface of CSU was first decorated with anatase form of titania NPs that are capable of being activated by the UV emission of CSU to generate reactive oxygen species (ROS) for PDT effects.32, 50-52 In the next level, a layer of mesoporous silica (MS) was grown over the TiO2 decorated CSU to facilitate loading of the chemotherapeutic agents and introduce additional functionalities at the surface. Chemotherapeutic drug doxorubicin (DOX) was encapsulated within the pores of the mesoporous silica layer and chemically gated with blue light activable Ru-complex acting as gate-keeper molecules to restrict the leakage of loaded cargo in the absence of any stimuli.53 The uncaging process of the Ru-complex leading to the opening of pores was facilitated by the blue emission from CSU. In the final step, gold nanorods (GNR) with localized surface plasmon resonance (LSPR) along longitudinal axis at 800 nm were decorated on the surface of nanoplatform to attain hyperthermia effects upon activation by the intense NIR emission (800 nm) of CSU at NIR.11 Also, we intend to show that the power density of excitation laser source was used at such a low value that it did not have any heating effects in the absence of GNRs. Through proof-of-concept experiments, we aimed to demonstrate that the CSU nanoparticles with a diverse and strong emissions were able to induce PDT by activation of titania NPs, chemotherapy by uncaging of Ru-complex gatekeeper molecules to release DOX and PTT mediated by the strong SPR activity in GNRs synchronously through the FRET mechanism between the CSU and the incorporated photoactive components in different wavelength regimes upon a single 980 nm laser excitation. We validated the FRET based energy transfer process from CSU to the respective photoactive components by UCL life-time analysis. Furthermore, through extensive in-vitro studies on HeLa cell lines using various biochemical 4 ACS Paragon Plus Environment

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and microscopic analyses, we also attempted to validate the therapeutic efficacy of the nanoplatform being synergistic in nature i.e. the overall therapeutic outcome is greater than the combined contributions of each of three different modalities when present separately. Additionally, the other unutilized intense PL emissions in green and red regions from CSU have been shown to aid in imaging the same HeLa cell lines acting as a potential diagnostic probe. Herein, we demonstrate the successful integration of three different therapeutic modalities, i.e., PDT, chemotherapy and PTT, in a single nanoplatform, where all the photoactive agents are activated on-demand by a single NIR wavelength for a synergistically enhanced therapeutic effects in vitro through precise trigger mechanisms.The overall concept of the system has been depicted by a schematic diagram in Figure 1.



Fig. 1. Schematic representation of (A) overall synthetic step and (B) schematic representation of the concept of trimodal therapeutic (photodynamic, photothermal and chemotherapy) and imaging application of the nanoplatform.

2. Results and discussion Synthesis, Morphology, Phase, and Luminescence Properties of core and core-shell upconversion (CSU) nanoparticles First, the oleic acid capped core material (NaYF4: Yb/Tm/Er/Fe) with the ratios of Y/Yb/Tm/Er/Fe as 44.3/35/0.5/0.2/20 mol% was synthesised by thermal decomposition 6 ACS Paragon Plus Environment

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method using trifluoroacetates precursors at an elevated temperature.54 The doping of the core material with 35 mol% Yb3+ and 20 mol% Fe3+ was carried out to enhance the overall intensity of UC emissions.55 In the next level, the core-shell nanoparticles were designed to further tune the UC emission and negate the surface-mediated quenching effects,

48, 56

by

epitaxial growth of a shell of NaYbF4 with 1 mol% of Tm3+ doping around the core (NaYF4: Yb/Tm/Er/Fe).57-58 The relatively higher doping concentration of Tm3+ in the shell accounted for enhancing the emission maxima at 800 nm.59-60 The TEM analysis revealed that the core upconversion material was comprised of nanoparticles with an average diameter of 26.4 ± 2.7 nm (Fig. 2A) as obtained from size distribution plot (Fig. S1A, Supporting Information). The corresponding SAED pattern indicated the polycrystalline nature of the core materials (Fig. 2A, inset). The EDS spectra of the same material confirmed the elemental composition (Fig. 2B). The TEM analysis (Fig. 2C) of core-shell upconversion nanoparticles (CSU) indicated an increase of the average diameter of the particles to 32.1 ± 3.2 nm (Fig. S1B, Supporting Information). The HRTEM image (Fig. 2D) of CSU showed the presence of a shell over the surface of the core upconversion particles. The identical nature of the host matrix of both the core and shell made it difficult to mark the distinction between core and the shell in CSU nanoparticles through HRTEM. However, the difference in size distribution between core and CSU clearly indicated the synthesis of shell on Fe-doped core upconversion nanoparticles. The SAED study of CSU showed the polycrystalline nature as evident from the bright concentric rings in the electron diffraction pattern (Fig. 2D, inset). The PXRD analysis (Fig. 2E) clearly established the existence of pure crystalline hexagonal or β-phase for both the core (red curve) and the CSU (green curve).54 The FTIR studies revealed the characteristic stretching frequencies of aliphatic hydrogen and C=O for carboxylic groups at 2928 cm−1 and 1710 cm−1, respectively (Fig. S2, Supporting Information) for the non-polar oleic acid coating on as-synthesized CSU nanoparticles.



The upconversion PL spectrum of the core material under 980 nm laser excitation has been shown in Fig. 2F (red curve). The core nanoparticles doped with Er3+ and Tm3+ led to a diverse emission in wider range from UV to NIR.36, 59 The Er3+ doping resulted in prominent green and red emissions, respectively. The two green emissions ranging from 517 to 532 nm and from 532 to 551 nm were attributed to the 2H11/2 to 4I15/2 and 4S3/2 to 4I15/2 transitions, respectively.59, 61 The red emission from 635 to 670 nm arose from 4F9/2 to 4I15/2 transition.59 The Tm3+ doping resulted prominent emission peaks in UV region (350 nm) and blue region (450 and 470 nm) due to 1D2 to 3H6 (for UV) and 1D2 to 3F4 along with 1G4 to 3H6 electronic transitions, respectively.59-60 Additionally, an NIR emission at 800 nm due to 3H4 to 3H6 transitions59, 62 was also observed. The concentration of Tm3+ was kept at 0.5 mol% in the core owing to the fact that at low concentration transitions in the UV range are more facilitated.50,

60

Interestingly, the addition of 20 mol% Fe3+ doping augmented the overall

upconversion emission in significant manner (Fig. S3, Supporting Information) as compared to that of a non-Fe3+ doped system reported earlier.55 This strategy enabled the achievement of first level of intensity enhancement in our as-synthesised core material. The upconversion life-time decay for Fe-doped core nanoparticles at various emissions has also been shown in Fig. S4 (Supporting Information). The mechanism of upconversion luminescence in Fe3+ doped core UCNP has been represented in Fig. S5 (Supporting Information). An appropriate shell is known to enhance the emission of the UC core either by suppressing surface-mediated quenching or by harvesting more energy from the shell.56 The second level of the enhancement of PL emission intensity exhibited by CSU was evident in Fig. 2F (black curve). Such striking enhancement was attributed to the introduction of the active shell of NaYbF4 doped with 1% Tm3+. The rationale behind using 99 mol% sensitizer (Yb3+) in the host along with 1 mol% of Tm3+ as dopant in the shell material was to generate more intense emissions61, 63 in blue (470 nm) and NIR region (800 nm),59-60, 62 respectively. Interestingly, 8 ACS Paragon Plus Environment

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the relatively lower concentration of Tm3+ along with Fe3+ in core led to the intense emissions in UV region, as expected rationally.50,

60, 62, 64

At higher concentration of Tm3+, cross-

relaxation is expected to predominate leading to 1G4 to 3H6 and 3H4 to 3H6 transitions based emissions in the blue and NIR region, respectively.59-60, 62 Thus, epitaxial growth of the active shell was responsible for the increase in the overall intensities of emission by several folds compared to that achieved through the first level of enhancement (Fig. 2F). The CIE1931 plot (Fig. 2G), indicated a bluish-white emission with x = 0.2 and y= 0.26 colour coordinates by CSU as confirmed visually (Fig. 2G, inset). Thus, the adopted strategy of core-shell based fabrication of UCNP to generate a diverse and enhanced emission provided suitable options to activate multiple photo-transducers absorbing different λmax in the UV-NIR range by a single laser (980 nm) excitation.



Fig. 2. TEM analysis of (A) oleic acid capped core upconversion nanoparticles (compo) used as core material with corresponding SAED pattern (inset). (B) EDS spectra for elemental composition of core upconversion nanoparticles. (C-D) TEM analysis of core-shell upconversion (CSU) nanoparticles with SAED pattern (inset). (E) XRD analysis of core (red curve) and CSU nanoparticles (green curve) to confirm the β-phase of the particles. (F) A comparison studies on the florescence behaviour of core (red curve) and core-shell (black curve) nanoparticles. (G) CIE-1931 chromaticity plot of CSU nanoparticles, the corresponding PL image of OA-CSU in CHCl3 is shown in inset.

Modifications and characterizations of titania and mesoporous silica (MS) layer coated CSU nanoparticles (CSUT-MS) i) Titania modification on CSU. The emissions in the near UV region obtained from the wide range of UC emissions from the as-synthesized CSU were utilized for photoactivation of titania (TiO2) NPs for PDT mediated therapeutic outcome. Titania, generally known to exhibit 10 ACS Paragon Plus Environment

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photocatalytic ability, has been reported in the earlier studies to be capable of causing photodynamic killing of various types of cancers both in vitro and in vivo upon irradiation with UV light.65-67 However, since UV based therapy suffers from the limited penetration ability (few millimetres under skin surface), the direct excitation of titania nanoparticles using UV appears mainly suitable to treat the superficial tumours.66, 68 Some efforts to improve the penetration depth of the excitation source and thereby enhancing the PDT effect of titania has been reported recently by using UCNP in the design of nanoplatforms.32, 51-52, 69 Herein, the CSU was first made free of its oleic acid coating by dispersing the particles into the acidic solution prior to the TiO2 decoration step by sol-gel method. The decorated TiO2 were crystallized towards more photoactive anatase phase by a novel and milder method using simple hot water treatment.70 The present work also reports a first instance of fabricating anatase form of TiO2 on upconversion NPs through milder conditions. The anatase nature of decorated TiO2 on the surface of CSU was clearly evident from wide-angle PXRD analysis of TiO2 decorated CSU (CSUT) in Fig. S6, blue curve (Supporting Information). TEM analysis of CSUT is shown in (Fig. S7, Supporting Information). Apart from the characteristic peaks of the ‘β’ phase of CSU; the additional diffraction peaks of CSUT that appeared at 2θ values of 25.21, 37.81, 47.71, 54.21 and 62.51 degree, corresponding to the (101), (004), (200), (105) and (204) planes, respectively, were indexed for anatase phase of TiO2 (JCPDS 21– 1272).70-71 The FTIR analysis of CSUT has been given in Fig. S8, Supporting Information. The UV-Vis diffuse reflectance spectrum (DRS) of the CSUT (post hot water treatment) indicated no significant absorbance of visible light, instead it showed an effective optical response to UV light Fig. S9A (Supporting Information). The optical band-gap of 3.05 eV was calculated by Tauc-plot analysis Fig. S9B (Supporting Information) indicating the photoactivity of the as-synthesised CSUT in UV region.



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ii) Mesoporous silica layer functionalization on CSUT. In the next step of modification, a layer of amine functionalized mesoporous silica (MS) was grown over the surface of CSUT to obtain CSUT-MS. Additionally, MS layer functions to protect the brightness of CSU intact by acting as an inert shell to avoid quenching of UC based PL at the surface of CSU from external conditions.48, 56, 63 This modification allowed us to load the chemotherapeutic agent doxorubicin (DOX) to achieve the gated delivery system on application of an appropriate light trigger. The TEM analysis evidenced a layer of MS shell around the CSUT (Fig. S10, Supporting Information). The EDAX analysis of CSUT-MS confirmed the presence of Ti, Si and O along with all the other elements of CSU (Fig. S11, Supporting Information). The wide-angle PXRD exhibited the presence of all the peaks for CSU and the crystalline peaks of coated titania along with a characteristic broad peak of amorphous silica (Fig. S6, red curve, Supporting Information). The low angle PXRD showed a sharp peak at 2.7 degree (2θ), characteristic of MCM-41 type of material (Fig. S12, Supporting Information). sorption studies provided an isotherm characteristic to

72-73

The N2

MCM-41 type of material, further

confirming the mesoporous nature of the external silica layer (Fig. S13, Supporting Information). The BET surface area of CSUT-MS was calculated to be 754 m2/g and the BJH pore size was found to be around 3.14 nm (Fig. S13, Supporting Information). The presence of primary amine functionality in CSUT-MS was confirmed by FTIR studies, (Fig. S14, green curve, Supporting Information). Additionally, the band at 940 cm–1 was assigned to Si-O-Ti and the band at 1050 cm–1 was assigned to Si-O bonds, respectively.74 iii) Conjugation of blue light activable Ru-complex gate-keeper molecules. The free-amine functional groups on the outer MS layer served as the site for modifications by photocleavable gate-keeper Ru-complex molecules (Ru2). These molecules can effectively block the pores to restrict the diffusion of the loaded drug inside MS layer.53 Upon absorbing the blue UC emission from CSU, Ru2 is expected to get cleaved40, 53, 75-76 and thereby possible 12 ACS Paragon Plus Environment

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unblocking of the pores and release of loaded drug would likely to take place. The Rucomplex was synthesised and analysed by 1H-NMR (Fig. S15, Supporting Information) to confirm the structure of the complex. The ESI-MS analysis of Ru-complex showed a % [M+] ion peak for the complex at m/z 759.23, corresponding to the molecular weight of the asprepared Ru-complex (Fig. S16, Supporting information). The FTIR analysis further established the functional groups in the Ru-complex (Fig. S14, blue curve, Supporting Information). Prior to Ru2 conjugation, the folic acid (FA) conjugation was carried out to facilitate internalization of the nanostructures by the cancer cells, a commonly reported cancer cell targeting approach.77 The UV-vis analysis of the Ru-complex showed a broad band at 360-480 nm (approx.), which was attributed to metal-to-ligand charge transfer (MLCT) band of the Ru complex (Fig. S17, Supporting Information). Additionally, the band at 290 nm is assigned to π-π* transition of the phenanthroline ligand in the Ru complex.53, 78 This clearly established that Ru-complex can get activated by absorbing the 1D2-3H4 and 1G4-3H6 emission (emission in blue region) from the upconversion core-shell nanoparticles to trigger the targeted application. The in situ loading of DOX in the MS layer of CSUT-MS followed by the conjugation of the Ru-complex gatekeepers yielded DOX loaded CSUT-MS gated by Ru complex (CSUT-MSDG) and the details has been discussed in the subsequent section. The conjugation of Ru-complex to CSUT-MS was supported by FTIR (Fig. S14, red curve, Supporting Information) and UV-vis (Fig. S18, Supporting information) analyses. The FTIR spectrum of Ru2 conjugated CSUT-MS clearly exhibited the characteristic peaks of both CSUT-MS and Ru-complex. FTIR spectrum of free Ru2 molecules has been shown in (Fig. S14, blue curve, Supporting Information). The conjugation of FA to CSU-MS was confirmed by UV-vis analysis, where the peak at 285 nm was attributed to the pterin ring of FA.79 These results established the successful modification of NH2 containing MS surface of the nanoplatform with FA and Ru2 moieties. The reaction involving conjugation of FA to CSUTMS has been shown in Fig. S19, Supporting Information. 13 ACS Paragon Plus Environment


iv) GNR decoration on CSUT-MSDG. In the final step, bovine serum albumin (BSA) modified gold nanorods were decorated on the surface of CSUT-MS through physisorption to achieve the hyperthermia effects.80 The BSA coating on the GNRs was carried out to reduce the toxicity due to CTAB and facilitate their physical adsorption onto the nanoplatform.81-84 The GNRs were synthesised by seed-mediated method and the associated LSPR band was adjusted to 800 nm by tuning the concentration of L-ascorbic acid, AgNO3 and the seed solution. Such modification enabled the GNRs to absorb the 800 nm (3H4 to 3H6) emission from CSU for hyperthermia activity SPR. The GNRs were separately characterized by TEM, UV-vis-NIR and PXRD analyses. The TEM analysis clearly showed the rod-shaped nanostructures with an average length of 40.8 ± 5.1 nm (Fig. 3A). The HRTEM image displayed prominent homogenous fringes with d-spacing value of 0.20 nm. The SAED pattern exhibited the crystalline nature of the GNRs (Fig. 3B, inset), which was further confirmed by PXRD analysis (Fig. S20A, Supporting Information). The UV-vis-NIR analysis (Fig. S20B, Supporting Information) of GNR showed the two absorbance maxima; a more intense one at 800 nm and a weaker one at 530 nm corresponding to the surface plasmon activity of longer and shorter axes, respectively. The dark field HAADF and EDAX mapping of the GNRs clearly showed the rod-shaped morphology along with the distribution of elements in the particles (Fig. S21, Supporting Information). Also, the UV-vis-NIR studies of BSA coated GNR confirmed that the BSA coating did not affect the absorbance maxima of the GNRs (Fig. S22, Supporting information) indicating a stable coating of BSA on GNRs surface.82 The TEM analysis of the fabricated nanoplatform containing CSUT-MSDG decorated with GNR (CSUT-MSDGG) showed the presence of nanorods on the surface of core-shell nanoparticles (Fig. 3C). The HAADF image of the final ensemble (Fig. 3D) clearly showed the overall modifications of the system with GNRs decoration. The elemental mapping has been shown in Fig. 3D, inset. The EDAX spectral analysis showing presence of Au, Ag along with the other elemental constituents of GNR-decorated CSUT-MS further established the 14 ACS Paragon Plus Environment

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composition of the nanoplatform (Fig. S23, Supporting Information). The overall schematic diagram for the synthesis of CSUT-MSDGG is shown in Figure 1.

Fig. 3. TEM analysis of as-synthesised GNRs (A) and HRTEM of GNRs (B) showing clear lattice fringes; the corresponding SAED pattern (in inset). (C) TEM image of CSUT-MSDGG at lower and (D) HAADF image of CSUT-MSDGG with elemental mapping (inset).

In-vitro assessment of 1O2 generation, photothermal effects and chemotherapeutic efficiency by 980 nm NIR laser trigger The choice of TiO2, Ru2 gatekeeper molecules and GNRs with LSPR at 800 nm in the overall fabrication can be rationally established by the fact that the as-synthesised anatase form of titania decorated on CSU, the Ru2 cages and LSPR tuned GNRs exhibit an effective overlap of their absorbance maxima with the various intense emissions from UC core in UV (< 350 nm), blue region and NIR (800 nm) region, respectively (Fig. 4A). The steady state PL analysis of CSU particles after all the fabrication steps clearly showed the quenching of UC 15 ACS Paragon Plus Environment


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emissions in UV, blue and NIR regions as compared to the unmodified ones (Fig. 4B). Also, steady-state fluorescence analysis after each modification step has been provided in (Fig. S24, Supporting Information). The energy-transfer efficiency (Eo) can be calculated by quantifying the UC luminescence intensities for various emissions before and after quenching as a result of chemical modifications. The calculated Eo for the emissions of interest have been enlisted in Table 1. Table 1. Energy transfer efficiency (E), estimated from the quenching of upconversion emissions at 350 nm, 470 nm, 540 nm and 800 nm. UC emission wavelengths

E = (If-Ii)/If *100

350 nm 470 nm 540 nm 800 nm

~84 % ~65 % ~18% ~52 %

Furthermore, the upconversion luminescence (UCL) lifetime at various emissions, i.e., 350 nm, 470 nm, 540 nm and 800 nm further showed marked decrease in the lifetime for the final modification than the unmodified system, whereas the lifetime of decay at 650 nm remained significantly unchanged in both the systems (Table 2), shown in Fig. 4C-G. All these observations clearly established selective energy transfer between the core-shell upconversion nanoparticles to the photoactive counterparts (decorated TiO2, Ru2 and GNRs) incorporated in the nanoplatform. Table 2. Upconversion luminescence (UCL) lifetime studies of core-shell nanoparticles before and after over all chemical modifications at various emissions

CSU-MS CSUT-MSGG

350 nm

470 nm

540 nm

650 nm

800 nm

0.43 ms 0.31 ms

0.45 ms 0.30 ms

0.30 ms 0.23 ms

0.46 ms 0.44 ms

0.60 ms 0.45 ms

***The lifetime for each emission is determined by fitting the decay with a single exponential function.


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To demonstrate in situ generation of 1O2 due to energy transfer between CSU and decorated TiO2 nanoparticles, the DPBF (1,3-diphenylisobenzofuran) based chemical assay was performed (Fig. 4H-I).31 The gradual decrease of DPBF concentration over time in presence of as-synthesised nanoplatform upon NIR irradiation indicated proportionate increase in 1O2 generation (Fig. 4H). On the other hand, all the other control experiments (NPs + no NIR, DPBF + NIR, no NPs) did not show any appreciable decrease in DPBF concentration even over a period of 20 min. The gradual decrease of DPBF absorption peak (at 413 nm) due to the generation of 1O2 was studied by UV-vis analysis (Fig. 4I).

Fig. 4. Overlap spectra of UC emission of core-shell upconversion nanoparticles and absorbance spectra of TiO2, GNRs and Ru-complex (Ru2). (B) Comparison of PL spectra of core-shell nanoparticles before and after final chemical modifications. Upconversion luminescence (UCL) decay lifetimes of core-shell nanoparticles at (C) 350 nm, (D) 470 nm, (E) 540 nm (F) 650 nm and (G) 800 nm, before and after chemical modifications. (H) DPBF based 1O2 generation assay. (I) Gradual decrease in DPBF concentration with NIR irradiation time.



Subsequently, the on-demand drug release from CSUT-MSDGG was assessed by NIR laser mediated trigger. Prior to the drug release experiment; the amount of Ru-complex conjugated to the nanoplatform was evaluated by UV-vis analysis of the supernatant post conjugation experiment (Fig. S25, Supporting information). The total amount Ru-complex that was conjugated to CSUT-MS was found to be approximately 44.1 µg/mg of CSUT-MS. Next, in order to ensure that the blue emissions from CSU triggered the uncaging process via cleaving the Ru-complex; we performed UV-vis analysis, where a gradual increase in the absorbance of Ru complex along with a small red-shift in the absorption band was detected upon NIR irradiation for a period of 4h indicating the uncaging process due to the cleavage of the complex from the nanoplatform (Fig. 5A). This pattern of red-shift in Ru-complex, when exposed to blue light to induce photo-trigger was coherent to the earlier reported observations;53 whereas the same Ru-complex solution did not show any shift in the absence of blue light and under 980 nm laser exposure without the upconversion nanoparticles (Fig. S26, Supporting Information). A schematic representation of DOX release from the nanoplatform in response to the cleavage of Ru-complex by blue emission from CSU has been shown in Fig. S27B, Supporting Information. The loading % of DOX was determined from the collected supernatant after the loading experiment through UV-Vis analysis (Fig. S28, Supporting Information). The unknown concentration of DOX was determined by the calibration curve (Fig. S29, Supporting Information). The loading % of DOX on nanoplatform was calculated to be around 17% (approx.), i.e., 0.51 mg on the total amount of nanoplatform taken for loading (i.e., 30 mg). The loading of DOX and subsequent conjugation of Rucomplex to block the channels was further evident from the N2-sorption analysis, (Fig. S30, Supporting Information), wherein CSUT-MSDG showed a flattening of isotherm with surface area of 158.4 m2/g and BJH pore-size of 2.23 nm, i.e., lower than CSUT-MS. Subsequently, the NIR triggered DOX release study due to the uncaging of Ru2 (in PBS buffer at ~37 ⁰C) revealed that nearly 83% loaded DOX was released within 8 h when CSUT-MSDGG was 18 ACS Paragon Plus Environment

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exposed to NIR laser (980 nm) set at a power density of 0.7 W/cm2 for duration of 30 min (Fig. 5B). In the absence of NIR exposure, a separate control experiment was also carried out under similar conditions. The results of the control experiments showed a negligible release of drug i.e. less than 2% of the loaded DOX (Fig. 5B), indicating that the DOX release was indeed photochemically triggered by uncaging of Ru2 from the mesoporous channels by NIR exposure and not by premature leakage. Also, to rule out the possible DOX release in response to NIR and decorated GNR mediated heating effect, DOX release from CSUTMSDG showed no significant difference (approx. 85 %) as compared to CSUT-MSDGG, indicating GNRs induced hyperthermia based local heating did not affect the DOX release (Fig. S31, Supporting Information). Eventually, the strong surface plasmon activities of GNRs decorated on the CSUT-MSDGG surface generated localized heating upon NIR irradiation of CSU. Since LSPR of the synthesised GNRs show appreciable overlap with the emission at 800 nm from CSU (3H4 to 3

H6 transition), the energy transfer through FRET process is expected to effectively induce

hyperthermic effects upon NIR irradiation. The photothermal performance of CSUT-MSDGG was studied by monitoring the thermal response of NIR irradiation on the nanoplatform dispersion with a 980 nm laser at 0.7 W/cm2 for 20 min (Fig. 5C). A set of control experiments, i.e. water + NIR, CSUT-MSDG + NIR and CSUT-MSDGG + no NIR was carried out to establish the energy transfer between GNRs and CSU for generating photothermal effects. In the presence of NIR, the temperature of the solution reached up to 50.7 °C in case of CSUT-MSDGG, whereas the temperature of the solution in case of control experiments barely crossed above 33 °C; indicating that CSUT-MSDGG could be used as an effective formulation for photothermal killing of cancer cells (Fig. 5C). The photothermal conversion efficiency of GNR decorated nanoplatform was calculated as 20.8%,85-86 which is



generally reported for GNR induced photothermal effects. A detailed information and corresponding calculation steps has been given in (Fig. S32, Supporting Information). Assessment of therapeutic efficacy of CSUT-MSDGG nanoplatform in cancer cells invitro Prior to exposing the fabricated nanoplatform to the complex biological environment, it is important to consider the stability, surface property and non-specific interaction with biomolecules, particularly proteins of the concerned nanoplatform. A detailed study on colloidal stability through aggregation studies in different dispersion media, surface charge analysis and protein binding assay has been provided in Fig. S33 (Supporting Information). The studies indicated that the fabricated nanoplatform exhibited favourable attributes to be applicable in biological system. The therapeutic potential of CSUT-MSDGG was evaluated in vitro on HeLa cells for desired photodynamic, chemo and photothermal effects by various biochemical and microscopic analyses. Before the NIR mediated experiments, we checked the cytocompatibility of FA conjugated CSUT-MS, CSUT-MSDG and CSUT-MSDGG seeded with HeLa cells by MTT assay at various concentrations of 25, 50, 100 and 200 µg/mL, respectively for a period of 48 h (Fig. 5D). The results indicated that none of the materials showed cytotoxicity up to a concentration of 200 μg mL-1 in the absence of NIR exposure. However, the viability decreased slightly at highest particle concentration. Alongside, we also optimized the dosage of NIR laser by subjecting the HeLa cells to NIR laser (980 nm) exposure at a power density of 0.7 W/cm2. The choice of this power for all our in vitro cell studies was based on the optimum limit of NIR exposure deemed safe for skin exposure is set as 0.726 W/cm 2 by American National Standards for Safe use of Lasers.45-47 The results (Fig. 5E) established that the NIR laser exposure at such lower power did not affect the viability HeLa cells for over 20 min. The phase-contrast microscopic images of HeLa cells post irradiation (at 0.7 W/cm2 for 20 ACS Paragon Plus Environment

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20 min) did not show any change in morphology (Fig. 5F) and the cells looked as viable as that of the unirradiated cells (Fig. 5F, inset), justifying the use of the mentioned power density for all the subsequent experiments. In order to categorically establish the role of each therapeutic component integrated in CSUTMSDGG, a concentration of 50 µg/mL particles dosage was chosen for all the further cellbased experiments. We did not choose the particle dose of 25 µg/mL since; it was difficult to attribute the therapeutic contribution of each modality integrated in the nanoplatform at this dose. Also, to track the localization of particles within the cells, FITC tagged CSUT-MSDGG was incubated with HeLa cells for a period of 24 h and studied subsequently under confocal microscopy. The obtained results clearly indicated intracellular localization of the particles within the cytoplasm as a result of uptake by HeLa cells (Fig. 5G-I). To justify the role of FA in facilitating cellular uptake, HeLa cells were incubated with FA devoid nanoplatform. The results evidently showed that most of the particles remained outside the cell, thereby, establishing the role of FA (Fig. S34, Supporting Information).



Fig. 5. UV-vis analysis of Ru-complex (A) post NIR mediated disassociation from nanoplatform during pore unblocking mechanism. The arrow indicates the red-shift of Ru-complex post NIR triggered dissociation from the nanoplatform. (B) NIR triggered doxorubicin (DOX) release as a result for uncaging of nanoplatform in PBS buffer at ~37 ⁰C (cartoon shown below). (C) Hyperthermic response of nanoplatform as a result of NIR irradiation. (D) Concentration dependent cytotoxicity studies of nanoparticles through MTT assay. (E) Evaluation of effects of NIR irradiation on HeLa cells (F) The phase contrast microscopic image showing the effect of NIR irradiation on HeLa cells viability up to a period of 20 mins (Scale 100 µm), TCP is shown in inset. Confocal images of FITCtagged nanoparticles (green) in HeLa cells post 12 h of incubation (G-I); cell skeleton was stained with TRITC-phalloidin (red) and nucleus with Hoechst. (G) the confocal image in x-y plane; (H) images taken under dark field showing green emission from FITC tagged nanoconjugates (I) the image as seen from z-axis clearly shows FITC-tagged nanoconjugates existing in the same focal plane as that of the HeLa cells cytoplasm. (Fig. G-I, Scale 50 µm)

Besides, the fact that the integrity of nanoplatform is crucial for the proposed multimodal therapy; therefore, to ensure that our assembled nanostructure remained intact and did not disassemble while or after entering into the HeLa cells, we have carried out confocal studies using the nanoplatform with CSUT-MS tagged with Rhodamine B dye and BSA capped GNRs with FITC tagging in the final GNR decorated CSUT-MS. The confocal studies clearly showed that both green emission from FITC (Fig. 6B) and red emission from Rhodamine B 22 ACS Paragon Plus Environment

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(Fig. 6C) originated from the tagged nanoplatform. Also, the merged image (Fig. 6D) clearly exhibited both red and green emission from the same zone of nanoconjugates (shown in white arrows), thus, proving the integrity of the assembled structure within the cells (Fig. 6A-D). Finally, an optimal condition of exposure time 20 min at 0.7 W/cm2 and particle concentration of 50 µg/mL were fixed for all the experiments to avoid any undesirable false positive outcomes.

Fig. 6. Confocal studies using the nanoplatform with CSUT-MS tagged with Rhodamine B dye and BSA capped GNRs with FITC tagging in the final GNR decorated CSUT-MS, i.e., CSUT-MSGG. (A) Bright field image. The confocal studies clearly showed that both green emission from FITC (B) and red emission from Rhodamine B (C) originated from CSUT-MSGG. Also, the merged image (D) clearly exhibited both red and green emission from the same zone of nanoconjugates (shown in white arrows). (Scale 50 µm for A-D).

To confirm and compare the extent of intracellular 1O2 generation by CSUT-MS, CSUTMSDG and CSUT-MSDGG, HeLa cells were first incubated with each of the particle variants separately for 12 h to allow them to internalize. After incubation, the cells were subjected to 23 ACS Paragon Plus Environment


ROS detection reagent (H2DCFDA, 2’,7’-dichlorofluorescindiacetate) and were subsequently exposed to NIR laser. The flow-cytometry based analysis using the H2DCFDA dye was carried out to precisely evaluate the cell-population statistics involved in ROS generation in response to the different particles variants upon activation by NIR laser (Fig. 7A1-A4). The histogram for ROS exhibited greater shift (demarcated by green dashed arrows) in the FL1-A intensity (for DCFDA) for cell population treated with CSUT-MSDGG nanoplatform as compared to CSUT-MS, CSUT-MSDG and control. The extent of intracellular generation of 1

O2 for CSUT-MSDGG treated cells was additionally confirmed by the confocal analyses

(Fig. 7B1-B4), where the amount of ROS generation was seen to be the highest in case of CSUT-MSDGG, intermediate in case of CSUT-MSDG and the least in case of CSUT-MS (Fig. 7C). The greatest ROS generation in case of CSUT-MSDGG as compared to the other two systems was attributed to the cumulative effect of direct stress caused to the cells due to the combination of titania based photosensitization, release of DOX87 and an additional effect of stress caused by GNR induced hyperthermic activity.88-89


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Fig. 7. Flow cytometry based analysis of 1O2 (ROS) generation within the HeLa cells: (A1) TCP (A2) CSUT-MS (A3) CSUT-MSDG and (A4) CSUT-MSDGG. The analysis of histogram for each group showed significant shift of FL1-A intensity (for DCFDA) in case CSUT-MSDGG compared to the other systems in the group, validating high ROS generation in CSUT-MSDGG. Confocal microscopic studies on HeLa cells treated with DCFDA dye for intracellular ROS generation: (B1) TCP (B2) CSUT-MS (B3) CSUT-MSDG and (B4) CSUT-MSDGG (Scale 100 µm for B1-B4). The analysis of the fold increase in intensities from each variant have been validated in a box-plot (C) using ImageJ. A dosage concentration of 50 µg/ml and 0.7 W/cm2 NIR irradiation power was used for the studies.

The efficiency of CSUT-MS, CSUT-MSDG and CSUT-MSDGG samples in NIR mediated combined therapeutic effect was clearly demonstrated by flow cytometry-based PI (Propidium Iodide) live-dead assay (Fig. 8A1-A4) and apoptosis/necrosis (Fig. 8B1-B4) biochemical assay. The results of PI studies revealed that among all the treatment variants, CSUTMSDGG showed maximum dead cells population (89.9 %) as compared to CSUT-MS (22.4 25 ACS Paragon Plus Environment


%) and CSUT-MSDG (59.4%), respectively. The MTT assay for the therapeutic sets showed the cell viability trend similar to the PI based analysis, displaying maximum cell death in case of CSUT-MSDGG upon NIR treatment (Fig. S35, Supporting Information). The live-dead assay was further corroborated by trypan blue based dye exclusion assay (Fig. S35, Supporting Information). Furthermore, apoptotis/necrosis assay was performed to establish whether the cell killing was performed through the apoptotic or necrotic pathways. The results showed that the CSUT-MS mostly led to the accumulation of apoptotic cells; wherein the early apoptotic (EAp) cell population was found to be 30.7 % (Fig. 8B2) at the investigated particle concentration and NIR irradiation dosage. The result established that the PDT effects by upconversion-titania system mostly resorted to apoptotic pathway of cell killing, consistent with the earlier reports.50,

52

On the other hand, CSUT-MSDG (Fig. 8B3) showed the

population distribution pattern of 19.5 % for EAp, 3.5 % of late apoptotic (LAp) and 32.2 % of necrotic cells; whereas in case of CSUT-MSDGG system, 12.6 % of EAp, 6.8% of LAp and a necrotic population as high as 63.5 % were observed (Fig. 8B3). The highest accumulation of necrotic cells in case of CSUT-MSDGG could be due a possible outcome of hyperthermia based effects generated by GNRs.1, 88, 90 Finally, to check the overall therapeutic effects of the treatment groups, the cells treated with particles were further imaged under phase contrast microscope post NIR exposure (Fig. 8C1-8C4). The phase-contrast microscopic images clearly showed that the cells completely lost their cytoplasmic integrity and viability in the case of the CSUT-MSDGG treated system. Thus, colony formation was highly impended (Fig. 8C4) as compared with CSUT-MS (Fig. 8C2) and CSUT-MSDG (Fig. 8C2), where few live cells can also be seen along with the dead cell population. Also, the immunocytochemistry-based analysis using TRITC-Phalloidin to stain cytoskeletal F-actin filaments and Hoechst stained nucleus further established that the cells under CSUT-MSDGG mediated therapeutic activity completely lost their actin cytoskeleton and their characteristic morphological features (Fig. 8D1-D4). Furthermore, in order to validate whether the observed 26 ACS Paragon Plus Environment

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therapeutic output in our proposed trimodal system is synergistic in nature, we evaluated the therapeutic efficacy of monomodal modifications, i.e., ‘only PDT’ (with TiO2 component, no DOX and GNRs); ‘only chemo’ (with DOX component, no TiO2 and GNRs) and ‘only PTT’ (with GNRs component, no TiO2 and DOX), by PI based live-dead assay. From the PI based live dead assay results, it can be clearly observed that the monomodal systems did not elicit significant cell-death in response to the therapeutic treatment when present individually and even the sum total of therapeutic contributions of each monomodal systems (i.e., only PDT, only chemo and only PTT) was observed to be lesser as compared to the therapeutic output of the trimodal system in discussion (i.e., 89.9 %, Fig. 8A4) under the identical experimental conditions. The total dead cell population for the sets with ‘only PDT’, ‘only chemo’ and ‘only PTT’ modality was found to be 19.9 %, 16.5 % and 16.7 %, respectively (Fig. 8E1-E4), which when additively combined, makes the total therapeutic contribution of 53.1 %, much lower than our proposed multimodal nanoplatform (CSUT-MSDGG) with all the 3 therapeutic modalities integrated in a single platform under identical dosage and NIR exposure conditions. This result demonstrates the appreciably high therapeutic efficacy of CSUT-MSDGG at remarkably milder conditions being a consequence of three different therapeutic modalities activated synchronously through the single NIR wavelength (980 nm) trigger to yield a synergistically enhanced therapeutic outcome. Eventually, the approach of NIR induced trimodal therapeutic demonstrated herein, not only exhibited a synergistic output but also helped to lower down the dosage of nanoparticles and at the same time allowed to confine the power of the laser exposure at a clinically acceptable optimum level. Although, the integration of multiple therapeutic modalities in a single nanoplatform poses a great challenge and the efficacy of such nanoformulations can be critically limited by the possible interference between the different functionalities, a rational approach in the design could provide a way to supersede those limitations.4 Therefore, in pursuit of developing such 27 ACS Paragon Plus Environment


trimodal therapeutic nanoplatform we have demonstrated an effective strategy to enable the nanoplatform exhibit significant oncolytic outcomes in synergistic manner even at lower particle dosage and milder NIR power of irradiation. This current strategy, backed up with detailed physical characterisations and cellular studies, ruled out any notable interferences from each other between the integrated therapeutic components. Lastly, given the superior chemical inertness and proven biocompatible nature of silica layer, we expect our nanoformulation to be stable against immediate biodegradation.91-92 Also, based on the knowledge of the in vivo behaviour of silica based systems studied, we expect such formulations not to interfere with the natural clearance mechanism.91, 93


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Fig. 8. Flow cytometry based evaluation of live-dead HeLa cell population analysis through propidium iodide (PI) dye staining in: (A1) TCP (A2) CSUT-MS (A3) CSUT-MSDG and (A4) CSUT-MSDGG. Apoptosis analysis for each particle variants through Annexin V (for early apoptosis) and 7AAD (for necrosis) by flow cytometry studies on: (B1) TCP (B2) CSUT-MS (B3) CSUT-MSDG and (B4) CSUT-MSDGG. Phase-contrast microscopic images of HeLa cells after: (C1) TCP (C2) CSUT-MS (C3) CSUT-MSDG and (C4) CSUT-MSDGG (Scale 100 µm for C1-C4). Confocal microscopic studies to study the effects of cytoskeleton organisation on HeLa cells post NIR irradiation stained with TRITC-phalloidin (red) for cytoskeletal actin filaments and Hoechst (blue) for nucleus: (D1) TCP (D2) CSUT-MS (D3) CSUT-MSDG and (D4) CSUT-MSDGG (Scale 75 µm for D1-D4). The decrease in cell number in case of CSUT-MSDGG was attributed to extensive cell death; most of the dead cells were removed during staining process. (E1-E4) PI-based live-dead analysis after NIR exposure for monomodal sets with each therapeutic component present separately, i.e., ‘only PDT’ (E2), ‘only chemo’ (E3) and ‘only PTT’ (E4); TCP (E1); The results clearly established that monomodal systems when individually present, yielded a significantly lower therapeutic outcome when combined additively than when integrated in a trimodal single nanoplatform (in case of CSUT-



MSDGG, total dead cell population 89.9 %) for synergistic outcome at a dosage concentration of 50 µg/ml and 0.7 W/cm2 NIR irradiation power.

HeLa cell imaging using CSUT-MSDGG nanoplatform Eventually, in order to exploit the strong UC emission from the nanoplatform for imaging application, CSUT-MSDGG treated HeLa cells were subjected to imaging under NIR excitation using a normal phase-contrast microscope, the eyepiece of which was attached with an external digital camera.36 Since, the internalization and further localization of FITC tagged CSUT-MSDGG within HeLa cells was confirmed through confocal microscopic studies, any luminescent signal originating as a result of NIR induced UC process was thus ensured to come from inside the cells. In order to acquire the image showing the UC based PL coming from the HeLa cells upon NIR irradiation, we initially captured an image of the cells with our setup under the normal illumination of the microscope without NIR irradiation (Fig. 9A). This fixed the positioning of the cells under the microscope field of vision. In the second step, without changing the field of vision, NIR irradiation was turned on with an external laser focused onto the cells at a fixed power density of 0.7 W/cm2 while maintaining a dark background by turning off the visible illumination from the microscope (Fig. 9B). The irradiation with NIR laser resulted in the PL emission of the CSUT-MSDGG present inside the HeLa cells. In the final step, an overlay image was generated to track the photoluminescent particles within the cells (Fig. 9C). Since, the major emission in blue region present in CSU’s visible PL spectra was used in chemotherapeutic application; the nanoplatform appeared green photoluminescent due to the left over major PL emission peak in the visible region (Fig. 5A). Notably, the enhanced brightness of this green PL achieved through double set of fabrication approach (Fig. 2F and Fig. S3) allowed us to image the nanoplatform in absence of any sophisticated photon detectors like photomultiplier tube (PMT) in spite of being subjected under a lower application dose (50 µg/mL) and a lower NIR excitation power density (0.7 W/cm2) suitable for effective therapeutic application. This result 30 ACS Paragon Plus Environment

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indicated that such nanoplatform possessed a strong potential to be used as a cost-effective bioimaging probe for diagnostics. Thus, the overall merit of the nanoplatform reported herein lies in the fact that the system demonstrated a strong theranostic potential in vitro at a significantly milder condition, triggered by a single excitation source via a rational engineering at nanoscale level.

Fig. 9. Upconversion imaging of HeLa cells in vitro. (9A) Image of the cells under microscope when viewed through camera integrated setup under the normal illumination without NIR irradiation. (9B) NIR illumination of the HeLa cells with CSUT-MSDGG (using 50 µg/ml of CSUT-MSDGG and NIR irradiation power density of 0.7 W/cm2) using in a dark background by turning off the visible illumination from the microscope. (9C) Overlay image showing the emission from CSUT-MSDGG under NIR illumination (marked by yellow arrows).

3. Experimental Synthesis of Core-shell nanocrystals (CSU) Synthesis of ytterbium, erbium, thulium, iron co-doped (Y/Yb/Tm/Er/Fe = 44.3/35/0.5/0.2/20 mol %, overall 1.32 mmoles) was carried out by modified thermal decomposition method.55, 58, 94-95

In a typical synthesis of NaYF4: Yb/Tm/Er/Fe with the ratios of Y/Yb/Tm/Er/Fe as

44.3/35/0.5/0.2/20 mol %, firstly, Yb2O3 (0.462 mmoles, 182.06 mg),

Tm2O3 (0.0066

mmoles, 2.546 mg), Er2O3 (0.00264 mmoles, 1.01 mg), Y2O3 (0.584 mmoles, 132.04 mg) and anhydrous FeCl3 (0.264 mmoles, 42.84 mg) were taken in a double-necked round bottom flask dissolved in 10 ml 50% Trifluoroacetic acid (TFA). The mixture was heated at 80 °C under vigorous stirring in an oil bath. The solution first turned cloudy and then colorless after 1h, the stirring was continued at the same temperature under vacuum conditions to facilitate the evaporation of residual TFA until a dry product is obtained. The residue obtained was 31 ACS Paragon Plus Environment


dispersed in 2 mL of millipore water under sonication followed by the addition of 448 mg of CF3COONa, 9 mL of oleic acid (OA) and 9 mL of 1-octadecene (OD) in the same flask. The mixture was sonicated for about 20 min to homogeneously disperse the content before subjecting it to thermal decomposition step. At this point, the flask was completely evacuated using vacuum pump. The temperature of the reaction was then gradually raised to 110 °C and held for 30 min under vacuum to remove any air or vapor formed from the reaction mixture in the flask. The temperature further increased at a slow rate to 300-320 oC while maintaining a highly inert conditions by passing argon gas into the reaction set-up. The heating was continued for 1 h, meanwhile the vapor was periodically removed by loosening the septum and then tightly plugging it back. Finally, the reaction was cooled to r.t., yielding a colloidal dispersion of particles. Next, 20 mL of hexane-acetone mixture (1:4 v/v ratio) was added to precipitate the nanocrystals, which was then isolated by centrifugation the contents at 3000 rpm. The residue was air-dried to obtain the core nanocrystals (NC). Subsequently, the NaYbF4 with 1% Tm3+ epitaxial shell was grown over the core nanoparticles. The following formula was used to calculate the amount of shell precursor to be used for shell growth, Y/X = [π(r + n)2 h πr2h]/πr2h (where n relates to the shell thickness, r for NC radius, and h was related to the NC length, X is the calculated amount of upconversion core material and Y is the amount of shell precursor required). Prior to the upconversion core-shell synthesis, the trifluoroacetate precursor of the shell constituents was synthesized by method same as that of the described earlier during the synthesis of the core nanocrystals. The trifluoroacetate precursor for shell was synthesized by dissolving 800 µmoles of overall oxides of the starting materials, 99 mol% of Yb2O3 (792 µmoles, 312 mg) and 1 mol% of Tm2O3 (8 µmoles, 3.08 mg), in 10 ml of 50 % TFA. In the next step, to the dried residue from previous step, 1.8Y CF3COONa (272 mg), 8 ml OA and 7 ml OD were added and the mixture was sonicated for 30 mins, solution (B). In the meantime, the core 32 ACS Paragon Plus Environment

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solution (A) was prepared by dispersing 1 mmol of core particles in 1 ml of cyclohexane, 2 ml OA and 3 ml OD in a 10 ml glass vial under sonication for about 30 mins. Subsequently, the dispersion of core nanoparticles, solution (A), was added into the dispersion of shell, solution (B), in dropwise manner under constant stirring (mantle maintained at 450 rpm). Next, similar to the steps described during the core UC synthesis, the thermal decomposition of the mixture was carried out under inert conditions to obtain the final product. Removal of OA coat from the CSU The OA coat from the surface of CSU nanocrystals was removed following a previously reported protocol with slight modifications.96 In a typical process, OA coated CSU NPs (300 mg) were dispersed in a 30 mL aqueous solution with the pH of the solution adjusted to 4 by addition of 0.1 M HCL. The mixture was stirred vigorously for 2h to facilitate protonation of the carboxylate groups in OA and subsequent detachment from the CSU surface. At the completion, the aqueous solution was mixed with diethyl ether to extract the OA out of the solution. The extraction cycle was repeated 3 times to ensure complete removal of OA. Finally, all the water layers containing the water dispersible fraction with hydrophilic CSU were isolated by centrifugation and precipitation with acetone. The final product was dispersed in water for further use. Synthesis of TiO2 decorated CSU (CSUT) The TiO2 nanoparticles decoration on the surface of CSU was carried out be hydrolysis of titania precursor by a reported sol-gel method.52 The OA-free upconversion nanoparticles (240 mg) were added to 25 mL of water-free ethanol and the mixture was sonicated for 15 min to completely disperse the particles in the solution. Next, 75 μL of concentrated ammonia solution (28 wt%) was added under constant stirring. Subsequently, 120 μL of titania precursor, tetrabutyl titanate (TBOT), dispersed in 5 mL of water-free ethanol was added carefully using a syringe pump set at a flow rate of 1 mL min-1. The reaction was left for 24 h 33 ACS Paragon Plus Environment


at 45 oC under constant but gentle stirring. The final product was isolated by centrifugation and carefully washed with DI water and then ethanol, 3 times each, to remove any unreacted precursor and ammonia from the final product. The residue was dried in oven at 100oC to completely remove any solvent from the product. Finally, the product (CSUT) with amorphous titania coat was redispersed in DI water for the next step of crystallization. Hot water induced crystallization of TiO2 in CSUT The phase transformation to generate photocatalytically active form was carried out following a previously reported literature.70 To induce crystallinity and phase transformation of amorphous TiO2 on the surface of CSU, the product obtained in the previous step was subjected to a hot water treatment by immersing it into DI water (~25 ml) maintained at 92 oC for 35 h. Finally, heat-treated content was isolated by centrifugation and air-dried to obtain the final product (CSUT). Synthesis of CSUT-MS Following a previously reported literature for mesoporous silica shell growth,39, 97 typically, to a aq. dispersion of CTAB (0.1 g) in 30 mL DI water, 20 mg of CSUT, 3 mL of EtOH and 150 µL of NaOH (2 mol mL−1) was added. The resultant mixture was heated to 70 °C under constant stirring. Next, 150 µL of TEOS followed by 50 µL of 3-APTES was added to the mixture in a dropwise manner and the reaction was continued for another 10 min. The product was isolated via centrifugation at 12, 000 rpm for 8 min and repeatedly washed with EtOH for at least 3 times. Finally, the product was redispersed in methanol (20 mL) containing 1 wt% NaCl and the content was stirred vigorously at r.t. for 8 h to completely remove the CTAB surfactant from the pores. The mesoporous silica (MS) layer coated CSUT (CSUT-MS) product was isolated by centrifugation and washed thoroughly with water-ethanol mixture for at least 3 times to obtain the final product.


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Synthesis of FA conjugated CSUT-MS The EDC coupling method was used to conjugate folic acid (FA) molecules to the nanoplatform.93 In a typical method, 6 mg of FA (0.0065 mmol) was allowed to react with 30 mg of CSUT-MS in the presence of 4.5 mg of EDC.HCl (0.0045 mmol) and 20 mg of NHS (0.17 mmol) in 10 mL anhydrous DMSO solvent. The content was stirred in dark conditions at r.t. for 12h. The final product was collected by centrifugation at 8000 rpm for 5 mins. The product was washed with DI water for at least thrice to remove any unconjugated FA molecules. Synthesis of [Ru(Phen)2PPh3Cl] complex (Ru2) The synthesis of Ru-complex was carried out by two steps process reported previously with slight modifications.40, 53, 78 Step-1: Synthesis of Ru(Phen)2Cl2 RuCl3.3H2O (1.000 g, 8.824 mmol), LiCl (1.077 g) and phenanthroline (1.195 g, 7.649 mmol) were added into reagent 7 mL of grade DMF. The mixture was refluxed for 8 h under constant stirring. After the completion, the reaction was cooled down to r.t. and reagent grade cold acetone (32 ml) was added into it. This mixture was kept at 0°C for overnight. The content of the mixture was then filtered to obtain a black crystalline product and a red-violet filtrate. The filtered product was further carefully washed with 10 ml cold water followed by 10 ml of cold diethyl ether at least thrice. The residue was air-dried to obtain the final product. Step-2: Synthesis of Ru(Phen)2PPh3Cl Ru(Phen)2Cl2. 2H2O (521 mg, 1 mmol), obtained in the previous step, and 1 mmol of the PPh3 ligand were added into ethanol-water mixture (100 mL, 80% ethanol, 20% water) in inert conditions. The content was then refluxed for 4-6 h under constant stirring. Upon completion of the reaction, the solution was evaporated using rotary evaporator until dryness. In the next 35 ACS Paragon Plus Environment


step, 50 mL of cold water was added to the reaction vessel followed by the addition of 2 mL of 70% perchloric acid to precipitate the complex as perchlorate salt. The product (red-orange color) was filtered, washed gently with cold water and dried under air. Chromatographic purification was performed using CHCl3-MeOH (9:1 by volume) as eluent. The product was purified further using chilled acetone followed by reprecipitation with ether resulting fine crystalline solid. (Detailed synthetic scheme given in Fig. S16, Supporting Information) Dark red solid (95% yield);1H NMR (400 MHz, DMSO-D6) δ 9.49 (dd, 2H, J1 = 12.8 Hz, J2 = 5.2 Hz), 8.82(d, 1H, J = 8 Hz), 8.70 (d, 1H, J = 8 Hz),8.59 (d, 1H, J = 8 Hz), 8.36-8.20 (m, 5H),), 8.13(d, 1H, J = 8.8 Hz),7.93 (dd, 1H, J1 = 8.4 Hz, J2 = 5.2 Hz), 7.86 (dd, 1H, J1 = 8.4 Hz, J2 = 5.6 Hz),7.62-7.52 (m, 2H), 7.42(d, 1H, J = 5.2 Hz), 7.34-7.03 (m, 15H); MS (ESI, +ve) m/z (relative intensity) 759 ([M]+, 100%). Doxorubicin (DOX) loading and capping with photo-cleavable gate-keeper (Ru2) Step- 1: DOX loading To load DOX, CSUT-MS nanoparticles (30 mg) were added to 3 ml ethanolic solution of (1 mg/mL) doxorubicin. The mixture was first sonicated to open the pores and then kept for stirring in dark conditions at room temperature for 24 hours. The DOX loaded CSUT-MS nanoparticles (CSUT-MSD) were separated by centrifugation at 7000 rpm and washed several times to remove the excess of physisorbed drugs. The loading % of DOX loaded on CSUTMS nanoparticles was calculated by UV/Vis spectroscopy. The unknown concentrations of DOX were determined by standard calibration curve. Step- 2: capping of DOX loaded CSUT-MS with Ru2 The gatekeeper molecule was conjugated following a slightly modified strategy discussed previously.40, 53Prior to the final conjugation, the synthesized Ru-complex was converted into


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aquo complex. For this, 9.0 mg of Ru-complex was dissolved in 3 ml of 1:2 ethanol-water mixture. The mixture was sonicated for 20 mins to make a clear solution. It was then subjected to heating at 70ᵒC in oil bath in dark conditions for 1h to obtain the aquo complex form of the caging molecules. Subsequently, 1 ml of the solution was directly added to̴ 30 mg of DOX loaded CSUT-MS, i.e., CSUT-MSD. The mixture was stirred in dark conditions for 12h. CSUT-MSD gated with Ru2 (CSUT-MSDG) nanoparticles were collected by centrifugation at 3000 rpm and washed twice with ethanol before further use. Synthesis and BSA modifications of gold nanorods (GNRs) with 800 nm LSPR GNRs with 800 nm LSPR were prepared following a seed-mediated method previously reported.98-99 HAuCl4 (50 µL, 50 mM) was added into the 10 ml aqueous dispersion of CTAB (0.1 M) followed by addition of 600 µL ice-cold NaBH4 (10 mM), resulting into a brownishyellow seed. The seed solution was aged for at least 2 h at r.t. before used for GNR synthesis. To prepare the growth solution, 100 µL of HAuCl4 (50 mM) was added into 10 ml of CTAB solution (0.1 M) under gentle shaking of the contents in tube. Next, 58 µL of AgNO3 (10 mM) was added first followed by sequential addition of 200 µL of HCl (1.0 M) and 80 µL of ascorbic acid (0.1 M). At this point the solution turned colorless upon gentle shaking by tilting the tube for 10 secs. 12 µL of the prepared seed solution was then added by gently dipping the micropipette tip into the growth solution and the solution was left undisturbed for overnight to allow the growth of GNRs. The GNRs were purified from the CTAB solution by washing with DI water (2 cycles of centrifugation and re-dispersion) and the product was finally redispersed in DI water for further step of BSA coating. Next, the BSA coated GNRs were synthesised by following the method described in previous literature with slight modifications.82-83 In a typical procedure, to the concentrated solution of GNR in 1 ml DI water add 1 ml of 10 mg/ml BSA solution (0.25mM) in PBS buffer (pH 7.4) under sonication. Subsequently, the solution was vortexed and incubated for 8 h. The excess 37 ACS Paragon Plus Environment


of unbound BSA was removed by centrifugation; the BSA coated GNR was stored in 1 ml PBS buffer for further use. Synthesis of GNR decorated CSUT-MSDG To decorate GNRs on CSUT-MSDG, 500 µl of BSA coated GNR was added to 5 mg of CSUT-MSDG taken in 1 ml of PBS buffer (pH 7.4). The mixture was sonicated for 5 mins and then stirred at r.t. for 6h. The final product was isolated by centrifugation at 4000 rpm for 3 mins and was gently washed with 1 ml PBS buffer at least thrice by repeated centrifugation and redispersion. The product was stored in 1 ml PBS buffer for further use. Synthesis of nanoplatforms with monomodal therapeutic components The synthesis of monomodal nanoplatforms with each therapeutic component present separately, i.e., ‘only PDT’ (with TiO2 component alone, and no DOX or GNR), ‘only chemo’ (with Ru2 gated DOX alone, and no TiO2 component or GNR) and ‘only PTT’ (with GNR alone, and no TiO2 component or DOX) is carried out by following the same procedure mentioned before in the previous sections except for the characteristic omission of the steps required during particular monomodal sets. DPBF assay for 1O2 generation To assess the 1O2 generation efficiency of the system, the chemical assay using DPBF was performed.31 The increase in 1O2 leads to the decrease in DPBF concentration at 413 nm that can be easily monitored using UV-vis spectrophotometer. For this, from the stock of 8 mM DPBF solution prepared in acetonitrile, 20 µL was taken and diluted further in 1 ml of acetonitrile taken in a cuvette. Next, 1.0 mg of CSUT-MS nanoparticles was added. The dispersion was subjected to NIR irradiation (980 nm) with a laser for 20 mins and corresponding decrease in DPBF concentration was recorded after every 5 min. Simultaneously, control experiments were carried out for the comparison purpose. 38 ACS Paragon Plus Environment

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In vitro evaluation of Photothermal effects under NIR excitation To study the photothermal effect induced by indirect excitation of GNRs by 800 nm emission from core-shell upconversion nanoparticles, the aqueous solution of 200 µg/mL (0.2 mL) CSUT-MSDGG was irradiated using a 980 nm NIR laser for 20 mins at a power density of 0.7 W/cm2. The temperature of the solution was measured using a digital thermometer. As a control experiment, DI water and CSUT-MS without GNRs were irradiated under similar conditions. In vitro upconversion emission triggered un-caging and DOX release To study the on-demand triggered release of DOX in response to uncaging of Ru2 from the channels, typically, 5 mg of CSUT-MSDGG nanoparticles in 500 µL PBS buffer were placed in a dialysis membrane fitted on top of the cuvette containing 2.5 ml PBS buffer as release medium. The nanoparticles were irradiated from top by a 980 nm NIR laser at a power density of approx. 0.7 W/cm2 nm. Post exposure for about 20 min duration (5 min on and 1 min off cycle), the release was recorded after almost 1 hour to allow the diffusion of DOX into the cuvette. The temperature was maintained at ~37 ⁰C during the release experiment. The amount of released doxorubicin is determined by UV-vis spectroscopy. The release experiments were performed in triplicates and the data obtained was presented as mean ± S.D. Colloidal stability and protein-binding studies The detailed protocol pertaining to the colloidal stability studies through DLS experiments and protein binding assay has been explained in Supporting Information (Sec. 2.1). In-vitro evaluation of nanoplatform efficacy for cancer cells killings The in-vitro cell studies were performed out to evaluate the NIR induced synchronously operational photodynamic, chemo-, and hyperthermic activities of the materials on human cervical adenocarcinoma (HeLa) cells through standard MTT assay, trypan blue assay; FACS based propidium iodide (PI) for live-dead cell assay, apoptotic assay and intracellular ROS 39 ACS Paragon Plus Environment


detection assay. Microscopic analyses were also performed to substantiate the theranostic abilities of the reported multifunctional nanoplatform. The experiments were carried out in triplicates and data was presented as mean ± S.D. The detailed protocols for in vitro cell studies have been explained in Supporting Information (in Sec. 2.2). Imaging of HeLa cells under NIR excitation The HeLa cells were seeded at a concentration of 103 cells/mL on a coverslip was treated with 50 μg/mL of CSUT-MSDGG nanoparticles. The particles treated cells were incubated for 12 h at 37°C. Prior to imaging, the cells were washed several times carefully with PBS buffer to wash-out any loosely adhered nanoconjugates from the cells surface. The NIR laser, set at a power density of 0.7 W/cm2 was then irradiated onto the coverslip containing the cells incubated with the particles to obtain the upconversion emissions from particles inside the cells. The images were taken with a 12.2-m.p. (megapixel) digital single-lens reflex camera (Canon, EOS Rebel XSi at ISO 1600, 82 mm focal length, f/11 aperture, zero exposure bias with shutter speed 20s) fitted to the eyepiece of a phase-contrast microscope (Primovert, Carl Zeiss, Germany). An overlay of the images was generated by ImageJ software. Characterizations The TEM analysis was carried out on FEI, Technai, F30 G2 supertwin (USA) operational at 300 kV. The samples for the analysis were prepared by dispersing the particles in appropriate solvents at a concentration of 100 µg mL-1. The dispersion was drop-casted on a 300 mesh carbon-coated copper grid and was allowed to dry under vacuum conditions for overnight before the analysis. The Energy Dispersive X-ray (EDX) analysis was performed on (Oxford Instruments, U.K). The Fourier transform infrared analysis (FTIR) was done on Perkin-Elmer Spectrometer. The powder XRD in wide and small angle was performed on X-Ray diffractometer (RIGAKU JAPAN/ULTIMA-IV) using CuKα radiation (λ=0.154 nm). To record the PXRD pattern in low angle, a continuous mode with the full open detector operated 40 ACS Paragon Plus Environment

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in 2Ɵ range of 2°-10° and a scan-rate of 2° with a step-size of 0.05° was maintained. The DLS and zeta-potential analysis was performed on a Malvern Zetasizer (Malvern/ NANO-ZS90). The UV-Vis spectroscopic analysis was carried out on Agilent, Cary 100 Spectrophotometer. The N2 adsorption-desorption studies was performed on BET analyser (Quanta chrome) and BJH method was used for pore-size analysis. The steady-state fluorescence analysis for the materials were carried out using Horiba Jobin Yvon, USA/Fluoromax 4P fitted with an external 980 nm NIR laser source (particle concentration of 0.5 mg/mL in appropriate solvent was maintained). The lifetime analysis for upconversion emissions were performed in Edinburgh UV-VIS-NIR (FLS-980) Spectrometer (USA). The 1

H-NMR studies were recorded on a 400 MHz NMR spectrometer. The multiplicities of the

proton signals are indicated as ‘s’ (singlet), ‘d’ (doublet), ‘t’ (triplet), ‘q’ (quartet) and ‘m’ (multiplet). The coupling constants or J values were reported in Hz. The mass-spectrometry (ESI-MS) was carried out on (Perkin-Elmer) to determine the molecular weight of Rucomplex. The confocal analysis was carried out on Leica TCS SP8 (Germany). 4. Conclusions In essence, we demonstrated an engineered strategy to integrate several therapeutic modalities, i.e. photodynamic, chemo- and photothermal therapy in a single nanoplatform by rationally incorporating the photo-activation effect of TiO2, Ru-complex gated on demand DOX release mechanism and GNR assisted hyperthermia effect; all triggered by a single 980 nm NIR excitation source to produce a synergistically enhanced therapeutic output. An attempt has also been made herein to diversify and enhance the upconversion PL emission by several folds in the wide wavelength range of 300-800 nm. For this, the transition metal ion Fe3+ has been doped to incur the first level of enhancement in PL intensity in the core nanoparticles. Additionally, introduction of an active shell of NaYbF4: 1%Tm3+ led to the second level of enhancement as well as diversification of the PL profile, making the system a 41 ACS Paragon Plus Environment


versatile platform for exciting multiple photo-active agents simultaneously. The construct has been demonstrated to exhibit a significant in vitro therapeutic outcome along with bioimaging application validated with 2D cell culture even at lower NIR irradiation (0.7 W/cm2) and lower dosage (50 µg/mL), the combination of which appeared to be a remarkably milder one in the context of NIR induced cancer therapeutic research in vitro. Through several controlled experiments, we conclusively proved that the excitation laser source was used at such an optimum value that it did not exhibit any observable heating effect in the absence of GNR. Such lower excitation and dosage administration is expected to largely cut down the possibilities of damage to the normal tissues. More importantly, in the cases where the cancers show resistance towards certain therapeutic modalities over the others, this reported strategy is expected to offer more therapeutic options. The results of the current proof-of concept study performed in vitro at the remarkably milder conditions invoke further validation in vivo to help this trimodal nanoplatform to perform as a comprehensive and efficiency driven cancer therapeutic. Associated Content Supporting Information. The detailed protocols and associated supporting figures associating with the manuscript have been given in the Supporting Information. “This material is available free of charge via the Internet at http://pubs.acs.org.” Author Information Corresponding Author Supratim Giri (e-mail: [email protected]) Present Addresses Department of Chemistry, National Institute of Technology, Rourkela, Odisha- 769008, India. Notes The authors declare no competing financial interest 42 ACS Paragon Plus Environment

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Acknowledgements The authors acknowledge MoU-DAE-BRNS Project (No. 2009/34/36/BRNS/3174), Department of Physics, S.V. University, Tirupati, India for extending the experimental facility with the kind permission from Prof. C. K. Jayasankar to utilize the lifetime facility for UCL life time analyses. The authors acknowledge and thank the financial supports obtained from DBT, India (Grant No. BT/PR6230/GBD/27/391/2012) and DST, India (Grant No.SB/FT/CS191/2011). We thank Utpal K. Swain for the TEM analysis.

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LIST OF FIGURES

Fig. 1. Schematic representation of (A) overall synthetic step and (B) schematic representation of the concept of trimodal therapeutic (photodynamic, photothermal and chemotherapy) and imaging application of the nanoplatform.

ACS Paragon Plus Environment


Fig. 2. TEM analysis of (A) oleic acid capped core upconversion nanoparticles (compo) used as core material with corresponding SAED pattern (inset). (B) EDS spectra for elemental composition of core upconversion nanoparticles. (C-D) TEM analysis of core-shell upconversion (CSU) nanoparticles with SAED pattern (inset). (E) XRD analysis of core (red curve) and CSU nanoparticles (green curve) to confirm the β-phase of the particles. (F) A comparison studies on the florescence behaviour of core (red curve) and core-shell (black curve) nanoparticles. (G) CIE1931 chromaticity plot of CSU nanoparticles, the corresponding PL image of OA-CSU in CHCl3 is shown in inset.


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Fig. 3. TEM analysis of as-synthesised GNRs (A) and HRTEM of GNRs (B) showing clear lattice fringes; the corresponding SAED pattern (in inset). (C) TEM image of CSUT-MSDGG at lower and (D) HAADF image of CSUT-MSDGG with elemental mapping (inset).



Fig. 4. Overlap spectra of UC emission of core-shell upconversion nanoparticles and absorbance spectra of TiO2, GNRs and Ru-complex (Ru2). (B) Comparison of PL spectra of core-shell nanoparticles before and after final chemical modifications. Upconversion luminescence (UCL) decay lifetimes of core-shell nanoparticles at (C) 350 nm, (D) 470 nm, (E) 540 nm (F) 650 nm and (G) 800 nm, before and after chemical modifications. (H) DPBF based 1O2 generation assay. (I) Gradual decrease in DPBF concentration with NIR irradiation time.


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Fig. 5. UV-vis analysis of Ru-complex (A) post NIR mediated disassociation from nanoplatform during pore unblocking mechanism. The arrow indicates the red-shift of Ru-complex post NIR triggered dissociation from the nanoplatform. (B) NIR triggered doxorubicin (DOX) release as a result for uncaging of nanoplatform in PBS buffer at ~37 ⁰C (cartoon shown below). (C) Hyperthermic response of nanoplatform as a result of NIR irradiation. (D) Concentration dependent cytotoxicity studies of nanoparticles through MTT assay. (E) Evaluation of effects of NIR irradiation on HeLa cells (F) The phase contrast microscopic image showing the effect of NIR irradiation on HeLa cells viability up to a period of 20 mins (Scale 100 µm), TCP is shown in inset. Confocal images of FITC-tagged nanoparticles (green) in HeLa cells post 12 h of incubation (G-I); cell skeleton was stained with TRITC-phalloidin (red) and nucleus with Hoechst. (G) the confocal image in x-y plane; (H) images taken under dark field showing green emission from FITC tagged nanoconjugates (I) the image as seen from z-axis clearly shows FITC-tagged nanoconjugates existing in the same focal plane as that of the HeLa cells cytoplasm. (Fig. G-I, Scale 50 µm)



Fig. 6. Confocal studies using the nanoplatform with CSUT-MS tagged with Rhodamine B dye and BSA capped GNRs with FITC tagging in the final GNR decorated CSUT-MS, i.e., CSUTMSGG. (A) Bright field image. The confocal studies clearly showed that both green emission from FITC (B) and red emission from Rhodamine B (C) originated from CSUT-MSGG. Also, the merged image (D) clearly exhibited both red and green emission from the same zone of nanoconjugates (shown in white arrows). (Scale 50 µm for A-D).


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A1

TCP

A2 CSUT-MS

B2B1 TCP

B2

B3 CSUT-MSDG

B4 CSUT-MSDGG

CSUT-MS

A3

CSUT-MSDG

C

A4

CSUT-MSDGG

Fig. 7. Flow cytometry based analysis of 1O2 (ROS) generation within the HeLa cells: (A1) TCP (A2) CSUT-MS (A3) CSUT-MSDG and (A4) CSUT-MSDGG. The analysis of histogram for each group showed significant shift of FL1-A intensity (for DCFDA) in case CSUT-MSDGG compared to the other systems in the group, validating high ROS generation in CSUT-MSDGG. Confocal microscopic studies on HeLa cells treated with DCFDA dye for intracellular ROS generation: (B1) TCP (B2) CSUT-MS (B3) CSUT-MSDG and (B4) CSUT-MSDGG (Scale 100 µm for B1-B4). The analysis of the fold increase in intensities from each variant have been validated in a box-plot (C) using ImageJ. A dosage concentration of 50 µg/ml and 0.7 W/cm2 NIR irradiation power was used for the studies.



Fig. 8. Flow cytometry based evaluation of live-dead HeLa cell population analysis through propidium iodide (PI) dye staining in: (A1) TCP (A2) CSUT-MS (A3) CSUT-MSDG and (A4) CSUT-MSDGG. Apoptosis analysis for each particle variants through Annexin V (for early apoptosis) and 7AAD (for necrosis) by flow cytometry studies on: (B1) TCP (B2) CSUT-MS (B3) CSUT-MSDG and (B4) CSUT-MSDGG. Phase-contrast microscopic images of HeLa cells after: (C1) TCP (C2) CSUT-MS (C3) CSUT-MSDG and (C4) CSUT-MSDGG (Scale 100 µm for C1C4). Confocal microscopic studies to study the effects of cytoskeleton organisation on HeLa cells post NIR irradiation stained with TRITC-phalloidin (red) for cytoskeletal actin filaments and Hoechst (blue) for nucleus: (D1) TCP (D2) CSUT-MS (D3) CSUT-MSDG and (D4) CSUTMSDGG (Scale 75 µm for D1-D4). The decrease in cell number in case of CSUT-MSDGG was


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attributed to extensive cell death; most of the dead cells were removed during staining process. (E1-E4) PI-based live-dead analysis after NIR exposure for monomodal sets with each therapeutic component present separately, i.e., ‘only PDT’ (E2), ‘only chemo’ (E3) and ‘only PTT’ (E4); TCP (E1); The results clearly established that monomodal systems when individually present, yielded a significantly lower therapeutic outcome when combined additively than when integrated in a trimodal single nanoplatform (in case of CSUT-MSDGG, total dead cell population 89.9 %) for synergistic outcome at a dosage concentration of 50 µg/ml and 0.7 W/cm2 NIR irradiation power.

A

Fig. 9. Upconversion imaging of HeLa cells in vitro. (9A) Image of the cells under microscope when viewed through camera integrated setup under the normal illumination without NIR irradiation. (9B) NIR illumination of the HeLa cells with CSUT-MSDGG (using 50 µg/ml of CSUT-MSDGG and NIR irradiation power density of 0.7 W/cm2) using in a dark background by turning off the visible illumination from the microscope. (9C) Overlay image showing the emission from CSUT-MSDGG under NIR illumination (marked by yellow arrows).



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LIST OF TABLES:

Table 1. Energy transfer efficiency (E), estimated from the quenching of upconversion emissions at 350 nm, 470 nm, 540 nm and 800 nm. UC emission wavelengths

350 nm 470 nm 540 nm 800 nm

E = (If-Ii)/If *100

~84 % ~65 % ~18% ~52 %

Table 2. Upconversion luminescence (UCL) lifetime studies of core-shell nanoparticles before and after over all chemical modifications at various emissions.

CSU-MS CSUT-MSGG

350 nm

470 nm

540 nm

650 nm

800 nm

0.43 ms 0.31 ms

0.45 ms 0.30 ms

0.30 ms 0.23 ms

0.46 ms 0.44 ms

0.60 ms 0.45 ms

*** The lifetime for each emission is determined by fitting the decay with a single exponential function.


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TOC Graphic


Near-Infrared-Triggered Photodynamic, Photothermal, and on

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