Multifunctional Liposome Nanocarriers Combining Upconverting

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Multifunctional Liposome Nanocarriers Combining Upconverting Nanoparticles and Anticancer Drugs Yue Huang, Eva Hemmer, Federico Rosei, and Fiorenzo Vetrone J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b02013 • Publication Date (Web): 02 May 2016 Downloaded from http://pubs.acs.org on May 16, 2016

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The Journal of Physical Chemistry

Multifunctional Liposome Nanocarriers Combining Upconverting Nanoparticles and Anticancer Drugs Yue Huang†, Eva Hemmer†, Federico Rosei†,#,‡, Fiorenzo Vetrone†,#,‡,* †

Institut National de la Recherche Scientifique - Énergie, Matériaux et Télécommunications,

Université du Québec, Varennes, Québec J3X 1S2, Canada #

Institute for Fundamental and Frontier Science, University of Electronic Science and

Technology of China, Chengdu, PR China ‡

Centre for Self-Assembled Chemical Structures, McGill University, Montreal, Québec H3A

2K6, Canada

ABSTRACT Lanthanide-doped upconverting nanoparticles (UCNPs) are well known for their inherent ability to convert low energy near-infrared (NIR) excitation wavelengths into higher energy emission wavelengths covering the ultraviolet (UV) to NIR regions. This optical feature makes UCNPs highly attractive for a broad range of applications including (bio)imaging and the biomedical use of light-triggered processes such as drug release. In the quest for novel theranostic approaches, the combination of multiple modalities on a single nanoscale platform,

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for example combining optical imaging and drug delivery, is very desirable. In this context, liposomes, artificially prepared constructs composed of a lamellar phase lipid bilayer, have been introduced as suitable nanocarriers for UCNPs. Here, we developed a hybrid nanocarrier consisting of Er3+ and Yb3+ co-doped NaGdF4 UCNPs that were encapsulated in the aqueous core of the liposomes and the potential of the obtained nanocarriers for drug delivery was shown by co-loading the model anticancer drug doxorubicin (DOX). Under 980 nm excitation, a decrease of the green upconversion emission of the NaGdF4:Er3+, Yb3+ UCNPs was observed when DOX was co-loaded with the UCNPs in the liposome nanocarrier. This quenching effect is assigned to the energy transfer between the donor UCNP and the acceptor DOX and is most significant since it allows for the spectral monitoring of the DOX loading and release from the liposome nanocarriers. Thus, the drug loading, release and spectral monitoring properties of the obtained liposome nanocarriers were thoroughly characterized allowing us to assess their future potential as theranostic nanocarriers.


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INTRODUCTION Various optical imaging techniques have been developed over the past few decades, resulting in powerful modalities used to image biological environments and to study diverse biological processes.1-3 However, many of these techniques employ fluorophores or luminophores such as organic dyes or semiconductor quantum dots (QDs) that require excitation with high-energy radiation in the ultraviolet (UV) or visible (VIS) region.4-5 Excitation at these wavelengths incurs a set of drawbacks including low penetration depth into biological tissues due to diverse absorption and scattering processes, the risk of photo-induced tissue damage due to the phototoxicity of UV light, photobleaching restricting their temporal use (in organic dyes) and autofluorescence of the background resulting in low contrast.6 Consequently, there is specific need to develop novel optical bioprobes that overcome these limitations and eventually allow for reliable and efficient optical deep tissue in vivo bioimaging. In this context, the use of nearinfrared (NIR) radiation as an excitation source has attracted significant attention for in vitro and in vivo applications, due to several advantages, which mitigate the shortcomings of high-energy excitation.6 These include larger tissue penetration depths (up to cm penetration has been reported),7 weak autofluorescence (since typically the NIR excitation light is specific to the optical probe), and low phototoxicity of the NIR light when compared with conventional highenergy light excitation.8 The deeper penetration of NIR compared to UV-VIS wavelengths results from the reduced scattering and absorption of light by water, blood and biological tissue in the wavelength range from 700 to 1870 nm, spectrally overlapping with the so-called three biological windows (first biological window, NIR-I: 700-950 nm; second biological window, NIR-II: 1000-1350 nm; and third biological window, NIR-III: 1550-1870 nm).9 Seeking to develop novel optical probes for biomedical applications, the recognition of these advantages of


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NIR light over UV-VIS light induced a clear trend for a shift in the excitation wavelengths of choice from the UV-VIS to the NIR regions of the spectrum. Due to their unique optical and chemical properties, luminescent lanthanide (Ln3+)-doped upconverting nanoparticles (UCNPs) are considered as promising optical nanoprobes that may overcome the various aforementioned problems associated with conventional fluorophores.10-11 UCNPs are well known for their ability to convert low energy NIR excitation light to higher energies, covering a broad wavelength region from the UV to the VIS to the NIR via a process commonly referred to as upconversion and have been explored for manifold biomedical applications (see Supporting Information (Figure S1) for a detailed description of the upconversion process).12,13 Moreover, in the search for multifunctional nanomaterials, much effort has been expended in the development of hybrid systems containing UCNPs. Various groups around the world have reported diverse examples of the intelligent combination of UCNPs with other materials or moieties to form multifunctional nanoplatforms, typically applied to address biomedical challenges. A prime example involves combining UCNPs with mesoporous silica (SiO2) where the latter is used as a carrier for assorted functional moieties including for example, photodynamic therapy (PDT) drugs, which can convert the upconverted light to toxic reactive oxygen species 14 or for photocaged molecules, such as siRNA, that can be released upon absorption of the upconverted light

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. The UCNP/SiO2 nanoplatform can also

be used for targeted and controlled delivery of anticancer drugs (i.e., doxorubicin (DOX)), for combination of cancer imaging and therapeutics.17 In addition, UCNPs have been functionalized with a number of different polymer and block co-polymer systems also for applications in NIRtriggered PDT, controlled drug delivery, nanothermometry as well as multimodal imaging involving both upconversion and magnetic resonance imaging.18-23 A number of recent studies


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have also focused on developing hybrid UCNP/plasmonic systems, which were not only used to investigate the plasmonic upconversion enhancement but have also been studied as multifunctional nanocarriers for combined drug release, upconversion imaging and photothermal therapy applications.24-27 UCNPs are suitable energy donors for Förster resonance energy transfer (FRET, or luminescence resonance energy transfer (LRET)) based systems that are of particular interest for application in biological assays.28-32 Compared to other organic and inorganic fluorophores that have been employed in LRET-based systems (such as fluorescent bio-molecules5, 33, silver or gold nanoparticles34-35, silica nanoparticles36, graphene oxide37, and quantum dots5, 38-40), UCNPs have several benefits based on the aforementioned advantages including elimination of autofluorescence (due to probe specific excitation), photodegradation (allowing for dynamic studies) and phototoxicity towards the biological samples under investigation due to excitation with NIR light. In addition, they have very narrow absorption and emission lines with large Stokes shift. This implies that the excitation and emission wavelengths are far apart and the risk of cross-contamination of the bands is minimal. Also contrary to heavy metal based nanostructures, UCNPs are promising candidates when seeking to minimize toxic effects as shown in recent in vitro and in vivo studies.41-42 Related to LRET-based systems, designing UCNPs with different emissions (i.e. different colors) allows for multiplexing (optical barcoding) where a number of analytes can be detected simultaneously. Another promising strategy to assemble multifunctional nanoplatforms employs liposomes, which are artificially prepared nanocarriers composed of a lamellar phase lipid bilayer (Scheme 1). They are considered as good candidates for drug delivery since their structure is similar to that of cell membranes.43 Improved therapeutics has been reported for liposome-based drug


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delivery due to its great potential to dramatically enhance the biocompatibility and reduce the side effects of potentially toxic drugs.44 In addition, further studies showed the selective trapping of liposomes by tumor tissues due to the high permeability of tumor vasculature towards liposomes in combination with the lack of proper lymphatic drainage.45-46 This phenomenon of trapping large structures or assemblies ranging from 100-200 nm is commonly referred to as the enhanced permeability and retention (EPR) effect.47 Recently, some liposomal formulations, such as Doxil and Ambisome, have received approval from the US Food and Drug Administration (FDA) and have been used for drug delivery in clinical cancer therapy.48-50 In the context of liposome-based nanocarriers, research reports have shown that the encapsulation of nanostructures, such as QDs, metallic nanoparticles or iron oxide within liposomes provides a multitude of innovative approaches for pharmaceutical and biomedical applications.51-54 However, their use as delivery systems for various types of nanoparticles is still in the research stage.44 Furthermore, Soga et al. reported on the encapsulation of UCNPs and NIR-emitting optical nanoprobes in liposomes for bioimaging applications.55-56 Despite these highly promising results, data on liposome encapsulated UCNPs are still scarce, and further studies on the design, characterization and application of liposome encapsulated UCNPs are required to develop their application in biomedicine. Here, we report on the co-encapsulation of NaGdF4:Er3+, Yb3+ UCNPs with the anticancer drug DOX in liposomes, to construct a NIR excited nanocarrier with combined optical imaging and therapeutic modalities. Scheme 1 summarizes the liposome structure and different encapsulations of NaGdF4:Er3+, Yb3+ UCNPs and DOX in liposome. The UCNPs and DOX are distributed in the internal aqueous phase of the liposomes because of their hydrophilic nature. The obtained liposome nanocarriers were thoroughly characterized with regard to their physico-


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chemical properties as well as potential for drug loading, release and their spectral monitoring. Our work lays the foundation for future applications of these UCNP/liposome nanocarriers for multimodal biomedical use, involving the simultaneous detection and therapy of disease.

Scheme 1. Schematic representation showing the structures of the blank liposome, Lipo-UCNPs, Lipo-DOX and Lipo-UCNPs-DOX. When UCNPs and DOX were incorporated into the liposome, UCNPs and DOX were located in the inner core of the liposome. Upon laser irradiation (980 nm), energy transfer occurred from UCNPs to DOX, where the upconverted green emission is partially absorbed by the DOX.


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EXPERIMENTAL DETAILS Materials. Lanthanide oxides (Ln2O3 (99.99%), Ln = Gd, Yb, Er), trifluoroacetic acid (99%), sodium trifluoroacetate (98%), oleic acid (90%), 1-octadecene (90%), and doxorubicin hydrochloride (DOX) were purchased from Alfa Aesar. Oleylamine (70%), cholesterol (≥ 99%), fetal bovine serum (FBS) and phosphate buffered saline (PBS, pH = 7.4) were obtained from Sigma Aldrich. 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, > 99%) was acquired from Avanti Polar Lipids. All starting chemicals were used without further purification. Ultrapure deionized water (Millipore system) was used for all experiments. Synthesis of (CF3COO)3Ln (Ln = Gd, Yb, Er) Precursors. In the typical synthesis of the lanthanide trifluoroacetate precursors, 0.975 mmol (353.4 mg) Gd2O3, 0.025 mmol (9.6 mg, 2 mol% doping rate) Er2O3, and 0.25 mmol (98.5 mg, 20 mol% doping rate) Yb2O3 were mixed in a 100 mL three-neck round-bottom flask with 5 mL distilled water and 5 mL trifluoroacetic acid, followed by refluxing under magnetic stirring at 80 °C until a clear solution was obtained. Subsequently, the temperature was reduced to 60 °C, to evaporate the residual trifluoroacetic acid and water. The dried precursor was isolated in the form of a colorless powder. Synthesis of NaGdF4:Er3+, Yb3+ Upconverting Nanoparticles (UCNPs). The synthesis of the UCNPs was carried out in a well-ventilated fume hood as described previously.57 Typically, for the synthesis of NaGdF4:Er3+, Yb3+ UCNPs, 2.5 mmol (340 mg) sodium trifluoroacetate as well as 20 mL 1-octadecene,10 mL oleic acid and 10 mL oleylamine were added to the obtained (CF3COO)3Ln precursor. Subsequently, the mixture was degassed for 30 min at 100 °C under vacuum and magnetic stirring. The reaction mixture was then heated to 240 °C under stirring in the presence of a gentle argon flow and maintained at 240 °C for 2 h. Subsequently, the solution


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was left to cool to room temperature and the resultant oleate-capped UCNPs were precipitated with ethanol and collected by centrifugation. The obtained product was washed three times with hexane/ethanol (4:1) and dispersed in 5 mL hexane as stock solution. Oleate-Citrate Ligand Exchange. To obtain water dispersible UCNPs, ligand exchange was carried out as previously described in the literature.58 60 mg of oleate-capped NaGdF4:Er3+, Yb3+ UCNPs obtained from the previous step were dispersed in 5 mL of hexane and mixed with 5 mL of a 0.2 M trisodium citrate buffer (adjusted to pH 4). The mixture was kept on a shaker for 3 h, and subsequently transferred into a separation funnel, from which the aqueous phase containing the citrate-coated NaGdF4:Er3+, Yb3+ UCNPs was collected. The UCNPs were precipitated with acetone (1:5 aqueous:organic ratio), collected by centrifugation and redispersed in 5 mL of trisodium citrate buffer (adjusted to pH 7) for removal of residual oleic acid. The dispersion was placed on a shaker for an additional 2 h. Finally, the citrate-coated UCNPs were precipitated with acetone, washed three times with acetone and water, collected by centrifugation and dispersed in 2 mL water for further experiments. Preparation of NaGdF4:Er3+, Yb3+ UCNPs Encapsulated in Liposomes (Lipo-UCNPs). Blank liposomes composed of DOPC and cholesterol, in the molar ratio of 2:1, were prepared by a thin-film hydration method.59 In a typical experiment, a mixture of 0.025 mmol DOPC and 0.0125 mmol cholesterol was dissolved in a 5 mL solution of chloroform and methanol (4:1 v/v). In the subsequent drying step (30 min at 40 °C), which was carried out in a rotary evaporator (Büchi, Switzerland), the lipid film was formed. The obtained lipid film was flushed with argon and maintained overnight in a desiccator to remove any residual traces of chloroform. In the following step, the lipid film was hydrated by use of 2 mL PBS, followed by vortexing and sonication at 30 °C for 10 min resulting in a homogeneous suspension of blank liposomes.


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To encapsulate NaGdF4:Er3+, Yb3+ UCNPs in liposomes, the lipid film was hydrated with 60 mg NaGdF4:Er3+, Yb3+ in 2 mL PBS instead of pure PBS, followed by vortexing and sonication as described earlier. The final suspension (UCNPs encapsulated in liposomes) was further purified by centrifuging at 4,000 rpm for 10 min to remove non-entrapped NaGdF4:Er3+, Yb3+ UCNPs followed by filtering through a 0.22-µm pore size membrane to discard any larger residues. Loading of the Doxorubicin (DOX) into the Liposome (Lipo-DOX and Lipo-UCNPsDOX). DOX was loaded into the liposome nanocarrier via an ammonium sulfate gradient method.60 Briefly, the previously prepared lipid film was hydrated by adding 250 mM of ammonium sulphate with or without 60 mg NaGdF4:Er3+, Yb3+, sonicated with a bath type sonicator at 30 °C, followed by passing it through a Sephadex G-50 column for desalting. The sample was then mixed with 0.005 mmol DOX (5:1 lipid:DOX molar ratio) and incubated at 30 °C for 30 min. Non-entrapped DOX was removed by a Sephadex G-50 column, while nonentrapped NaGdF4:Er3+, Yb3+ was removed by slow centrifugation. Finally, the loading efficiency of DOX was determined by using absorption spectroscopy at a wavelength of 480 nm after lysis of the liposome using 1% Triton X-100. The loading efficiency (%) was calculated following equation 1:61

Loading efficiency (%) =

Encapsulated drug in liposomes × 100% Amount of total drug added

(Eq. 1)

DOX Release Study. The DOX release from the liposome nanocarrier was monitored using the dialysis method.62 The dialysis was conducted in two release media, namely PBS (pH 7.4) and 50% FBS in PBS. DOX-loaded liposomes, at a concentration of 0.5 mM DOPC diluted in the media, were transferred into a dialysis bag (molecular weight cut-off of 6000 Da) and then


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immersed in 100 mL of release media under continuous gentle stirring for 12 h at 37 °C. At predetermined time intervals, aliquots were taken from the release media and equal volume of fresh medium was added. The leakage of DOX was determined by the increase of the DOX related absorption peak at 480 nm. DOX release studies were carried out in triplet. Error bars in Figure 5 represent the calculated standard derivation. Characterization. The crystalline phase of all nanostructures under investigation was analyzed by X-ray diffraction (XRD, Bruker D8 Advanced Diffractometer, Cu Kα radiation). The morphology and size distribution of the UCNPs were determined with a Philips CM200 high-resolution transmission electron microscope (HRTEM), equipped with an energy-dispersive X-ray (EDX) unit. Dynamic light scattering (DLS, Malvern, Zetasizer Nano S90) was used to determine the hydrodynamic radius of the liposomes. For Fourier transform infrared (FTIR) spectroscopy of DOX-modified citrate-capped UCNPs, citrate-capped UCNPs as well as pure DOX, dried samples were mixed with potassium bromide (KBr, FTIR grade, Alfa Aesar, Ward Hill, USA), and the spectra of the powders were recorded using a Nicolet 6700 FTIR spectrometer from Thermo Electron. The UV-VIS absorption spectra were obtained using a UVVIS spectrometer (Varian 5000). Luminescent measurements were carried out under 980 nm excitation using a Thorlabs fiber-coupled laser diode (maximum power 330 mW). The laser beam was focused on the sample using a lens to obtain a spot with a Gaussian intensity distribution with a 0.4 mm diameter. The emitted light was collected by a lens in a 90° configuration, and then transferred to a spectrophotometer (Avaspec - 2048L - USB2) using an optical fiber.


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RESULTS AND DISCUSSION Preparation and Characterization of NaGdF4:Er3+, Yb3+ UCNPs Encapsulated in Liposomes (Lipo-UCNPs). Oleate-capped NaGdF4 UCNPs doped with 2 mol% Er3+ and 20 mol% Yb3+ were prepared by the previously described thermal decomposition method.57 This method involves the thermal breakdown of metal trifluoroacetates in the presence of coordinating (oleic acid and oleylamine) and non-coordinating (octadecene) ligands. As can be observed in Figures 1A and S2, the particle size distribution of the resultant well-dispersed, spherical UCNPs was determined to be 6.44 ± 0.07 nm. XRD measurements (Figure 1B) confirmed that the UCNPs crystallized in the cubic (α-) phase, and no impurity crystalline phase was found. The particular host material (NaGdF4) and dopant pair (Er3+/Yb3+) were chosen for several reasons. First, fluorides such as NaGdF4 are well-known for their suitability as a host material for upconversion.57 The upconversion process is critically dependent on the vibrational energy of the host crystal (phonon energy). Hence, if the vibrational energy is too large (e.g. in some oxide materials), then the excited states are depopulated non-radiatively and the upconversion luminescence suffers. Thus, fluoride materials are the current state-of the-art since they have low phonon energies. The majority of work in the literature focuses on NaYF4, however, NaGdF4 contains the paramagnetic gadolinium ion (Gd3+), which can be used as a magnetic resonance contrast agent allowing for future multi-modal applications (e.g., optomagnetic imaging, cell tracking, controlled and monitored drug release).58,

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Moreover, the

dopant pair Er3+/Yb3+ is one of the most intensively studied in UCNPs, and it is characterized by its bright emission in the green wavelength region providing a good overlap with the absorption spectrum of DOX (as shown later in the discussion). Liposomes composed of DOPC and cholesterol, in the molar ratio of 2:1 were synthesized


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following a well-established and optimized thin-film hydration procedure reported in literature.59 The ratio between DOPC and cholesterol is critical for the formation of well-ordered lipid layers. In essence, cholesterol has an ordering effect on saturated and monounsaturated phosphatidylcholine bilayers, where lipid chain ordering could be increased through the interactions with the rigid cholesterol molecule. It has been reported that the lipid phase shows lipid disorder when the ratio of DOPC and cholesterol is less than approximately 4:1.64 The DOPC:cholesterol molar ratio of 2:1 used here fulfills the requirement for the formation of stable liposome nanocarriers. The encapsulation of the NaGdF4:Er3+, Yb3+ UCNPs in the liposomes (Lipo-UCNPs) was accomplished using the same above mentioned thin film hydration method,59 and the encapsulation (along with the blank liposomes, for comparison) was confirmed via TEM imaging (Figures 2A and 2B). The TEM images of both the blank liposomes and the LipoUCNPs demonstrated that their sizes were of the order of 130 nm. In addition, these images clearly showed the uniform spherical structure of the blank liposomes and Lipo-UCNPs. TEM images of the Lipo-UCNPs (in Figure 2B) further confirm the successful encapsulation of the UCNPs (several per liposome) in the inner aqueous compartment of the liposomes. In previous work, QDs of 4-5 nm diameter have been found incorporated into the lipid bilayer of liposome nanocarriers.65 In contrast, the TEM image in Figure 2B provides evidence that the UCNPs are mainly located in the aqueous core of the liposome and that the interaction with the lipid membrane is minimal. This is also supported by the fact that the lipid bilayer is hydrophobic, thus the hydrophilic UCNPs will want to avoid this area. To obtain further evidence for the encapsulation of NaGdF4:Er3+, Yb3+ UCNPs in the liposome, energy-dispersive X-ray microanalysis (EDX) was performed on the Lipo-UCNPs sample and the results, shown in


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Figure 2C, indicated that the Na, Gd, F, Yb, Er elements were present in the nanocarrier liposome.

Figure 1. (A) TEM image of the oleate-capped NaGdF4:Er3+, Yb3+ UCNPs, (B) XRD pattern of the NaGdF4:Er3+, Yb3+ UCNPs (Reference: cubic (α-) NaGdF4, JCPDS file No. 27-0697).


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Figure 2. TEM images of (A) blank liposomes and (B) liposomes with encapsulated NaGdF4:Er3+, Yb3+ UCNPs (Lipo-UCNPs). The white arrows indicate the presence of the UCNPs. (C) Elemental analysis of Lipo-UCNPs by EDX (peaks assigned to carbon and copper are due to the TEM copper grid used as the sample holder).

Loading of the Anticancer Drug Doxorubicin (DOX) in the Lipo-UCNPs (Lipo-UCNPsDOX). The goal of this study was to develop a Lipo-UCNPs hybrid nanocarrier with the potential for therapeutic capabilities. Thus, to endow the Lipo-UCNPs with a therapeutic modality, DOX was selected as the model anticancer drug to be co-encapsulated with the UCNPs using the ammonium sulphate technique.60 To confirm the sizes of the various liposome nanocarriers, blank liposomes, those loaded solely with DOX (Lipo-DOX), the Lipo-UCNPs, as well as the Lipo-UCNPs-DOX were studied via dynamic light scattering (DLS) and the results are presented in Figure 3. It is shown that the mean size of the various liposomes ranged from 150 to 160 nm with a polydispersity index of less than 0.20, demonstrating the narrow size distribution of all samples under investigation. We observed no significant difference (P > 0.05) in the size of all liposomes under investigation. In addition, the sizes of the liposomes measured using DLS were determined to be larger than those obtained using TEM (~150 nm versus 130 nm, respectively). This is due to the fact that DLS provides information on the hydrodynamic diameter of the nanostructures when dispersed in a solvent,66 while TEM images provide


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information about the diameter in the dry state.

Figure 3. Average diameter of (A) blank liposome (average diameter, d = 156.8 nm ± 2.19 nm; polydispersity index = 1.58 nm ± 0.02 nm), (B) Lipo-UCNPs (d = 159.6 nm ± 2.37 nm; polydispersity index = 1.87 nm ± 0.03 nm), (C) Lipo-DOX (d = 152.2 nm ± 1.98 nm; polydispersity index = 1.25 nm ± 0.02 nm) and (D) Lipo-UCNPs-DOX (d = 157.9 nm ± 2.58 nm; polydispersity index = 1.98 nm ± 0.04 nm) characterized by DLS. Note: Mean ± standard deviation; n = 3.


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Upconversion Luminescence of NaGdF4:Er3+, Yb3+ and Liposome Encapsulated NaGdF4:Er3+, Yb3+ (Lipo-UCNPs). It is well known that NaGdF4:Er3+, Yb3+ UCNPs possess strong two-photon excited VIS upconversion emission (Figure S1).21 In a typical upconversion process involving the Er3+/Yb3+ ion pair, the 980 nm laser excites the Yb3+ ion from the 2F7/2 ground state to its single excited state (2F5/2). Since Yb3+ is doped in significantly larger quantities than Er3+, subsequent energy transfer from the excited Yb3+ ions to Er3+ ions in close proximity excites the Er3+ ions from the 4I15/2 ground state to the first intermediate excited state (4I11/2). Following a second energy transfer from excited Yb3+ ions in their close vicinity, the Er3+ ions are then promoted from the first excited intermediate state, 4I11/2, to the upper 4F7/2 excited state. After multiphonon relaxation to the lower 2H11/2 and 4S3/2 excited states, green emission can be observed and is caused by radiative decay from the 2H11/2 and 4S3/2 excited states to the 4

I15/2 Er3+ ground state. In addition, red emission from the 4F9/2 state can arise from two different

mechanisms occurring simultaneously. First, the 4F9/2 Er3+ excited state can be populated via multiphonon relaxation from the upper 4S3/2 excited state. Second, the 4F9/2 Er3+ state can be directly populated via energy transfer from an excited Yb3+ ion. This happens after some of the excited Er3+ ions decay non-radiatively from the 4I11/2 intermediate state (populated by the first energy transfer from an Yb3+ ion) to the lower 4I13/2 excited state. For a thorough explanation of the upconversion process in Er3+ and Yb3+ co-doped materials, please refer to Figure S1. Figure 4A shows the upconverted luminescence spectra of the Lipo-UCNPs and LipoUCNPs-DOX nanocarriers following excitation with 980 nm. As expected, two green emission bands in the region of 515 to 535 nm and from 535 to 570 nm as well as one red emission band in the region of 640 to 680 nm were observed. As previously described, these peaks are characteristic of the Er3+ ions and can be ascribed to the 2H11/2 4I15/2 (520 nm), 4S3/2 4I15/2


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(540 nm) and 4F9/2 4I15/2 (655 nm) f-f transitions. Interestingly, the intensity of the green emission in the region of 515 to 570 nm was reduced by approximately 42% after DOX loading, while the red emission was essentially unchanged in the Lipo-UCNPs-DOX nanocarriers compared to Lipo-UCNPs (in the absence of DOX loading). This is confirmed by considering the green:red ratios between the Lipo-UCNPs and Lipo-UCNPs-DOX, which change from 1.72:1 for the Lipo-UCNPs to 1.14:1 for the Lipo-UCNPs-DOX. To clarify the origin of this quenching effect, the absorbance of DOX was analyzed by UV-VIS spectroscopy. As shown in Figure 4B, there is a partial spectral overlap between the absorption spectrum of DOX and the upconverted green emission of the UCNPs in the wavelength region from 510 to 570 nm. Consequently, the UCNPs and DOX molecules act as energy donors and energy acceptors, respectively, where the upconverted green emission is partially absorbed by the DOX (note that the DOX has no absorption in the NIR).67 This energy transfer from the UCNPs to the DOX molecules can only happen if both the donor and the acceptor moieties are in sufficiently close proximity, which eventually results in the observed quenching of the upconverted green emission. Generally speaking, there are different potential mechanisms for energy transfer that can take place between the donor moiety (UCNPs) and the acceptor moiety (DOX). These include luminescence resonance energy transfer (LRET, requiring a donor-acceptor distance of typically less than 10 nm since the probability for LRET follows an R-6 relation (R being the distance between the donor (UCNPs) and the acceptor (DOX)) and radiative energy transfer mechanisms such as reabsorption, with a combination of the different processes occurring simultaneously being likely.68-69 When UCNPs and DOX are simultaneously encapsulated in the core of the liposome (Lipo-UCNPs-DOX), the loading efficiency of DOX reached a maximum of 72% (refer to the following section for details about the DOX loading efficiency), indicating


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that DOX molecules filled the empty space of the central aqueous core. This can be observed by comparing to DOX loading in the absence of UCNPs (Lipo-DOX) revealing a loading efficiency of 98%. It must be taken into account that DOX is present in excess compared to the UCNPs (see Supporting Information for an estimation of the UCNP:DOX ratio). Hence, there is an abundant amount of energy acceptors (DOX molecules) in the vicinity of the donor UCNPs allowing for energy transfer and consequently quenching of the green emission. Moreover, some of the DOX molecules are expected to interact with the citrate capping ligand on the surface of the UCNPs. This citrate capping results in a net negative surface charge (cit-) while, doxorubicin hydrochloride is a weak chemical base.60, 70 Consequently, some electrostatic interaction between the UCNPs with a negative surface charge and the positively charged DOX molecules can occur.71 For clarification, FTIR and UV-VIS absorption spectroscopy was performed on citratecapped UCNPs after incubation with DOX overnight, followed by three washing steps in order to remove unbound DOX molecules. The obtained spectra (Figure S3) provide clear evidence for the presence of DOX on the UCNPs’ surface. Thus, a non-radiative LRET can be expected since some of the DOX molecules (those being electrostatically bound to the UCNP surface) are likely to be located within LRET distance, while radiative energy transfer processes, e.g., by reabsorption, can take place between the UCNPs and those DOX molecules that are not bound to the UCNP surface but that are statistically distributed in the aqueous core of the liposome.


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Figure 4. (A) Upconversion emission spectra of Lipo-UCNPs and Lipo-UCNPs-DOX nanocarriers under 980 nm excitation (emission intensities normalized to the red emission peak). (B) Upconversion emission spectrum of LipoUCNPs (green) and UV-VIS absorption spectrum of DOX (red) indicating the partial spectral overlap between the DOX absorption and the UCNPs emission.

DOX Loading and Release in Lipo-UCNPs-DOX Nanocarriers. To determine the efficacy of the therapeutic modality in this hybrid nanocarrier, we investigated the drug loading and release behavior using our model drug (DOX). The UV-VIS absorption spectrum of LipoUCNPs shows no absorption in the VIS region, while a strong absorption band centered at 480 nm is observed for DOX (Figure 5A). Therefore, the loading efficiency of DOX into the


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liposomes could be investigated by monitoring the absorbance A at 480 nm following lysis of the DOX-loaded liposomes with 1% Triton X-100 (A(Lipo-DOX)480nm = 0.53, A(Lipo-UCNPsDOX)480nm = 0.37). The loading efficiency of the blank liposomes with DOX (Lipo-DOX) reached up to 98%, while in contrast, a value of only 72% DOX loading efficiency was achieved in case of Lipo-UCNPs-DOX, a decrease of 26% compared with the blank liposome control (refer to Figure S4 for further details regarding the determination of the DOX loading rate). This decrease in loading efficiency can be attributed to the fact that parts of the central aqueous core space in the liposomes are occupied by UCNPs resulting in less volume available for DOX thus influencing the final DOX loading efficiency in the Lipo-UCNPs-DOX nanocarriers. To study the drug release from the liposome nanocarriers (Lipo-UCNPs-DOX), release studies were performed in different media, namely PBS and 50% FBS in PBS. PBS was used to mimic the in vitro condition, while 50% FBS was employed to mimic the in vivo conditions. Figure 5B demonstrates the drug release profiles of the Lipo-UCNPs-DOX nanocarriers in the two different media studied. A very limited overall leakage of DOX of approximately 6% into the PBS was observed upon maintaining the dispersion at 37 °C for 12 h. This indicated a good stability of the Lipo-UCNPs-DOX in PBS solvent. In contrast to this, 39% of the DOX molecules were released from the Lipo-UCNPs-DOX nanocarriers over a time span of 12 h when dispersed in 50% FBS in PBS. This faster release may be ascribed to the interaction between serum proteins and the lipid inducing the destabilization of the lipid bilayers in the Lipo-UCNPsDOX nanocarriers and resulting in the observed accelerated DOX release.72 In addition, upconversion emission spectra were recorded on Lipo-UCNPs-DOX in 50% FBS in PBS over a time span of 12 h (Figure 5C). As previously mentioned, the DOX molecules can quench the green UCNP emission. Upon release of DOX from the liposome into the dispersion medium


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(most easily those DOX molecules that are not electrostatically bound to the UCNP, however, since the electrostatic interaction is a weak bonding, release from the UCNP surface may also happen), the distance between the UCNPs and DOX significantly increases. However, as mentioned above, efficient energy transfer between the UCNPs and the DOX can only happen if both units are close to each other. Consequently, the quenching effect of DOX on the green upconversion emission becomes significantly reduced upon release of DOX from the liposomes, and an increase in the green emission intensity is expected upon DOX release. Thus, monitoring the spectral changes in the green emission intensity upon increased incubation time allows for the estimation of the drug release from the nanocarriers. The recovery of the green emission band is obvious for the Lipo-UCNPs-DOX dispersed in 50% FBS in PBS over 12 h (Figure 5C). We also observe here a strong correlation between the green upconversion emission intensity and the degree of drug release, which allows for the plotting of a DOX release calibration curve (Figure 5D), which can be exploited to spectrally monitor the release of DOX. Thus, these findings present the potential opportunity to use the developed Lipo-UCNPs-DOX nanocarriers as an optical probe to monitor the drug release efficacy in possible future therapeutic applications.


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Figure 5. (A) UV-VIS absorbance spectra of free DOX, Lipo-UCNPs and Lipo-UCNPs-DOX. (B) Drug release profile of Lipo-UCNPs-DOX nanocarriers in different media (PBS and 50% FBS) at 37 °C. (C) Upconversion emission spectra of Lipo-UCNPs-DOX nanocarriers obtained at two different times upon DOX release in 50% FBS in PBS. (D) The upconverted green emission intensity of the Er3+ ions in NaGdF4:Er3+, Yb3+ UCNPs encapsulated in the Lipo-UCNPs-DOX nanocarriers as a function of the cumulatively released DOX.


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CONCLUSIONS In summary, we developed a novel nanocarrier with potential for diagnostic and therapeutic applications, which is based on the co-encapsulation of Er3+ and Yb3+ co-doped NaGdF4 UCNPs and the model anticancer drug DOX in liposomes (Lipo-UCNPs-DOX). Under 980 nm laser excitation, the Lipo-UCNPs (without DOX co-encapsulation) showed strong green emission. Conversely, after loading the Lipo-UCNPs with DOX (Lipo-UCNPs-DOX), quenching of the upconverted green emission was observed, due to energy transfer between the UCNPs (donor) and DOX molecules (acceptor). Such energy transfer is facilitated by the partial spectral overlap between the absorption of DOX and the green emission of the UCNPs. DOX release studies further demonstrated a recovery of the green upconversion emission intensity of the UCNPs, which correlated well with the extent of DOX release. Thus, the nanocarrier presented here, a liposome co-encapsulated with NaGdF4:Er3+, Yb3+ UCNPs and the anticancer drug DOX, provides a promising theranostics platform for simultaneous bioimaging and therapy. Future work will focus on the surface functionalization of these nanocarriers with targeting moieties to endow them with selectivity and specificity relevant for targeted diagnostics and therapeutics for diseases such as cancer. ASSOCIATED CONTENT Supporting Information Scheme for upconversion mechanism of Er3+, Yb3+ co-doped UCNPs; particle size distribution of oleate-capped UCNPs; estimation of the UCNP:DOX ratio; FTIR and UV-VIS absorption spectra on citrate-capped UCNPs incubated with DOX, citrate-capped UCNPs, and pure DOX; UV-VIS absorption spectra of aqueous DOX solutions at various DOX concentration used to determine the calibration curve of DOX in PBS. This material is available free of charge via the


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Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Phone: +1 (514) 228-6847 Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS F.V. and F.R. acknowledge funding from the Natural Sciences and Engineering Research Council (NSERC) of Canada and Fonds de Recherche du Québec - Nature et technologies (FRQNT) for supporting this research. F.R. acknowledges NSERC for partial funding and salary support through an EWR Steacie Memorial Fellowship. F.V. also wholeheartedly thanks the Fondation Sibylla Hesse for funding. Y.H. acknowledges financial support from the Merit Scholarship Program for Foreign Students from the Ministère de l'Éducation, du Loisir et du Sport du Québec. E.H. is thankful to the Alexander von Humboldt Foundation for financial support in the frame of the Feodor Lynen Research Fellowship. REFERENCES (1) Zhang, J.; Campbell, R. E.; Ting, A. Y.; Tsien, R. Y. Creating New Fluorescent Probes for Cell Biology. Nat. Rev. Mol. Cell Biology 2002, 3, 906-918. (2) Kikuchi, K. Design, Synthesis and Biological Application of Chemical Probes for BioImaging. Chem. Soc. Rev. 2010, 39, 2048-2053.


25


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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(3) Peng, H. S.; Chiu, D. T. Soft Fluorescent Nanomaterials for Biological and Biomedical Imaging. Chem. Soc. Rev. 2014, 44, 4699-4722. (4) Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.; Weiss, S. Quantum Dots for Live Cells, in vivo Imaging, and Diagnostics. Science 2005, 307, 538-544. (5) Resch-Genger, U.; Grabolle, M.; Cavaliere-Jaricot, S.; Nitschke, R.; Nann, T. Quantum Dots versus Organic Dyes as Fluorescent Labels. Nat. Methods 2008, 5, 763-775. (6) Hemmer, E.; Benayas, A.; Legare, F.; Vetrone, F. Exploiting the Biological Windows: Current Perspectives on Fluorescent Bioprobes Emitting Above 1000 nm. Nanoscale Horiz. 2016, 1, 168-184. (7) Jalani, G.; Naccache, R.; Rosenzweig, D. H.; Lerouge, S.; Haglund, L.; Vetrone, F.; Cerruti, M. Real-Time, Non-Invasive Monitoring of Hydrogel Degradation Using LiYF4:Yb3+/Tm3+ NIRto-NIR Upconverting Nanoparticles. Nanoscale 2015, 7, 11255-11262. (8) Wolfbeis, O. S. An Overview of Nanoparticles Commonly Used in Fluorescent Bioimaging. Chem. Soc. Rev. 2015, 44, 4743-4768. (9) Smith, A. M.; Mancini, M. C.; Nie, S. Bioimaging: Second Window for in vivo Imaging. Nat. Nanotechnol. 2009, 4, 710-711. (10) Zheng, W.; Huang, P.; Tu, D. T.; Ma, E.; Zhu, H. M.; Chen, X. Y. Lanthanide-Doped Upconversion Nano-Bioprobes: Electronic Structures, Optical Properties, and Biodetection. Chem. Soc. Rev. 2015, 44, 1379-1415. (11) Wang, M.; Abbineni, G.; Clevenger, A.; Mao, C. B.; Xu, S. K. Upconversion Nanoparticles: Synthesis, Surface Modification and Biological Applications. Nanomed.-Nanotechnol. 2011, 7, 710-729.


26

Page 27 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(12) Auzel, F. Upconversion and Anti-Stokes Processes with f and d Ions in Solids. Chem. Rev. 2004, 104, 139-173. (13) Zhou, J.; Liu, Z.; Li, F. Upconversion Nanophosphors for Small-Animal Imaging. Chem. Soc. Rev. 2012, 41, 1323-1349. (14) Idris, N. M.; Gnanasammandhan, M. K.; Zhang, J.; Ho, P. C.; Mahendran, R.; Zhang, Y. In vivo Photodynamic Therapy using Upconversion Nanoparticles as Remote-Controlled Nanotransducers. Nat. Med. 2012, 18, 1580-1585. (15) Jayakumar, M. K. G.; Idris, N. M.; Zhang, Y. Remote Activation of Biomolecules in Deep Tissues using Near-Infrared-to-UV Upconversion Nanotransducers. PNAS 2012, 109, 8483-8488. (16) Yang, Y. M.; Liu, F.; Liu, X. G.; Xing, B. G. NIR Light Controlled Photorelease of siRNA and its Targeted Intracellular Delivery Based on Upconversion Nanoparticles. Nanoscale 2013, 5, 231-238. (17) Yang, D. M.; Kang, X. J.; Ma, P. A.; Dai, Y. L.; Hou, Z. Y.; Cheng, Z. Y.; Li, C. X.; Lin, J. Hollow Structured Upconversion Luminescent NaYF4:Yb3+, Er3+ Nanospheres for Cell Imaging and Targeted Anti-Cancer Drug Delivery. Biomaterials 2013, 34, 1601-1612. (18) Xu, H.; Cheng, L.; Wang, C.; Ma, X. X.; Li, Y. G.; Liu, Z. Polymer Encapsulated Upconversion Nanoparticle/Iron Oxide Nanocomposites for Multimodal Imaging and Magnetic Targeted Drug Delivery. Biomaterials 2011, 32, 9364-9373. (19) Jin, J. F.; Gu, Y. J.; Man, C. W. Y.; Cheng, J. P.; Xu, Z. H.; Zhang, Y.; Wang, H. S.; Lee, V. H. Y.; Cheng, S. H.; Wong, W. T. Polymer-Coated NaYF4:Yb3+, Er3+ Upconversion Nanoparticles for Charge-Dependent Cellular Imaging. ACS Nano 2011, 5, 7838-7847.


27


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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(20) Ryu, J.; Park, H. Y.; Kim, K.; Kim, H.; Yoo, J. H.; Kang, M.; Im, K.; Grailhe, R.; Song, R. Facile Synthesis of Ultrasmall and Hexagonal NaGdF4: Yb3+, Er3+ Nanoparticles with Magnetic and Upconversion Imaging Properties. J. Phys. Chem. C 2010, 114, 21077-21082. (21) Hemmer, E.; Quintanilla, M.; Légaré, F.; Vetrone, F. Temperature-Induced Energy Transfer in Dye-Conjugated Upconverting Nanoparticles: A New Candidate for Nanothermometry. Chem. Mater. 2015, 27, 235-244. (22) Cheng, T.; Ortiz, R. F.; Vedantham, K.; Naccache, R.; Vetrone, F.; Marks, R. S.; Steele, T. W. J. Tunable Chemical Release from Polyester Thin Film by Photocatalytic Zinc Oxide and Doped LiYF4 Upconverting Nanoparticles. Biomacromolecules 2015, 16, 364-373. (23) Jalani, G.; Naccache, R.; Rosenzweig, D. H.; Haglund, L.; Vetrone, F.; Cerruti, M. Photocleavable Hydrogel-Coated Upconverting Nanoparticles: A Multifunctional Theranostic Platform for NIR Imaging and On-Demand Macromolecular Delivery. J. Am. Chem. Soc. 2016, 138, 1078-1083. (24) Huang, Y.; Rosei, F.; Vetrone, F. A Single Multifunctional Nanoplatform Based on Upconversion Luminescence and Gold Nanorods. Nanoscale 2015, 7, 5178-5185. (25) Chen, C. W.; Lee, P. H.; Chan, Y. C.; Hsiao, M.; Chen, C. H.; Wu, P. C.; Wu, P. R.; Tsai, D. P.; Tu, D.; Chen, X. Y.; Liu, R. S. Plasmon-Induced Hyperthermia: Hybrid Upconversion NaYF4:Yb/Er and Gold Nanomaterials for Oral Cancer Photothermal Therapy. J. Mat. Chem. B 2015, 3, 8293-8302. (26) Cheng, L.; Yang, K.; Li, Y. G.; Chen, J. H.; Wang, C.; Shao, M. W.; Lee, S. T.; Liu, Z. Facile Preparation of Multifunctional Upconversion Nanoprobes for Multimodal Imaging and Dual-Targeted Photothermal Therapy. Angew. Chem. Int. Edit. 2011, 50, 7385-7390.


28

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(27) Dong, B. A.; Xu, S.; Sun, J. A.; Bi, S.; Li, D.; Bai, X.; Wang, Y.; Wang, L. P.; Song, H. W. Multifunctional

NaYF4:

Yb3+,

Er3+@Ag

Core/Shell

Nanocomposites:

Integration

of

Upconversion Imaging and Photothermal Therapy. J. Mat. Chem. B 2011, 21, 6193-6200. (28) Chen, N. T.; Cheng, S. H.; Liu, C. P.; Souris, J. S.; Chen, C. T.; Mou, C. Y.; Lo, L. W. Recent Advances in Nanoparticle-Based Förster Resonance Energy Transfer for Biosensing, Molecular Imaging and Drug Release Profiling. Int. J. Mol. Sci. 2012, 13, 16598-16623. (29) Hwang, S. H.; Im, S. G.; Sung, H.; Hah, S. S.; Cong, V. T.; Lee, D. H.; Son, S. J.; Oh, H. B. Upconversion Nanoparticle-based Förster Resonance Energy Transfer for Detecting the IS6110 Sequence of Mycobacterium Tuberculosis Complex in Sputum. Biosens. Bioelectron. 2014, 53, 112-116. (30) Zhang, S.; Wang, J.; Xu, W.; Chen, B. T.; Yu, W.; Xu, L.; Song, H. W. Fluorescence Resonance Energy Transfer between NaYF4:Yb,Tm Upconversion Nanoparticles and Gold Nanorods: Near-infrared Responsive Biosensor for Streptavidin. J. Lumin. 2014, 147, 278-283. (31) Mattsson, L.; Wegner, K. D.; Hildebrandt, N.; Soukka, T. Upconverting Nanoparticle to Quantum Dot FRET for Homogeneous Double-Nano Biosensors. RSC Adv. 2015, 5, 1327013277. (32) Wu, S. J.; Duan, N.; Li, X. L.; Tan, G. L.; Ma, X. Y.; Xia, Y.; Wang, Z. P.; Wang, H. X. Homogenous Detection of Fumonisin B1 with a Molecular Beacon Based on Fluorescence Resonance Energy Transfer between NaYF4: Yb, Ho Upconversion Nanoparticles and Gold Nanoparticles. Talanta 2013, 116, 611-618. (33) Feng, Y. S.; Liu, L. W.; Hu, S. Y.; Zou, P.; Zhang, J. Q.; Huang, C.; Wang, Y.; Wang, S. H.; Zhang, X. H. Efficient Fluorescence Energy Transfer System between Fluorescein


29


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 46

Isothiocyanate and CdTe Quantum Dots for the Detection of Silver Ions. Luminescence 2016, 31, 356-363. (34) Ghosh, D.; Chattopadhyay, N. Gold and Silver Nanoparticles Based Superquenching of Fluorescence: A Review. J. Lumin. 2015, 160, 223-232. (35) Ren, D. H.; Wang, J.; Wang, B.; You, Z. Probes for Biomolecules Detection Based on RETEnhanced Fluorescence Polarization. Biosens. Bioelectron. 2016, 79, 802-809. (36) Wang, L.; Tan, W. H. Multicolor FRET Silica Nanoparticles by Single Wavelength Excitation. Nano Lett. 2006, 6, 84-88. (37) Shi, J. Y.; Tian, F.; Lyu, J.; Yang, M. Nanoparticle Based Fluorescence Resonance Energy Transfer (FRET) for Biosensing Applications. J. Mat. Chem. B 2015, 3, 6989-7005. (38) Medintz, I. L.; Clapp, A. R.; Mattoussi, H.; Goldman, E. R.; Fisher, B.; Mauro, J. M. SelfAssembled Nanoscale Biosensors Based on Quantum Dot FRET Donors. Nat. Mater. 2003, 2, 630-638. (39) Stanisavljevic, M.; Krizkova, S.; Vaculovicova, M.; Kizek, R.; Adam, V. Quantum DotsFluorescence Resonance Energy Transfer-Based Nanosensors and their Application. Biosens. Bioelectron. 2015, 74, 562-574. (40) Shamirian, A.; Ghai, A.; Snee, P. T. QD-Based FRET Probes at a Glance. Sensors 2015, 15, 13028-13051. (41) Gnach, A.; Lipinski, T.; Bednarkiewicz, A.; Rybka, J.; Capobianco, J. A. Upconverting Nanoparticles: Assessing the Toxicity. Chem. Soc. Rev. 2015, 44, 1561-1584. (42) Hemmer, E.; Vetrone, F.; Soga, K. Lanthanide-Based Nanostructures for Optical Bioimaging: Small Particles with Large Promise. MRS Bull. 2014, 39, 960-964.


30

Page 31 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(43) Peetla, C.; Stine, A.; Labhasetwar, V. Biophysical Interactions with Model Lipid Membranes: Applications in Drug Discovery and Drug Delivery. Mol. Pharm. 2009, 6, 12641276. (44) Al-Jamal, W. T.; Kostarelos, K. Liposomes: From a Clinically Established Drug Delivery System to a Nanoparticle Platform for Theranostic Nanomedicine. Accounts Chem. Res. 2011, 44, 1094-1104. (45) Stapleton, S.; Milosevic, M.; Allen, C.; Zheng, J.; Dunne, M.; Yeung, I.; Jaffray, D. A. A Mathematical Model of the Enhanced Permeability and Retention Effect for Liposome Transport in Solid Tumors. PloS one 2013, 8. (46) Ozturk, D.; Yonucu, S.; Yilmaz, D.; Unlu, M. B. Influence of Vascular Normalization on Interstitial Flow and Delivery of Liposomes in Tumors. Phys. Med. Biol. 2015, 60, 1477-1496. (47) Maruyama, K. Intracellular Targeting Delivery of Liposomal Drugs to Solid Tumors Based on EPR Effects. Adv. Drug Deliv. Rev. 2011, 63, 161-169. (48) Chang, H. I.; Yeh, M. K. Clinical Development of Liposome-Based Drugs: Formulation, Characterization, and Therapeutic Efficacy. Int. J. Nanomedicine 2012, 7, 49-60. (49) Allen, T. M.; Cullis, P. R. Liposomal Drug Delivery Systems: From Concept to Clinical Applications. Adv. Drug Deliv. Rev. 2013, 65, 36-48. (50) Immordino, M. L.; Dosio, F.; Cattel, L. Stealth Liposomes: Review of the Basic Science, Rationale, and Clinical Applications, Existing and Potential. Int. J. Nanomedicine 2006, 1, 297315. (51) Al-Jamal, W. T.; Al-Jamal, K. T.; Cakebread, A.; Halket, J. M.; Kostarelos, K. Blood Circulation and Tissue Biodistribution of Lipid-Quantum Dot (L-QD) Hybrid Vesicles Intravenously Administered in Mice. Bioconjug. Chem. 2009, 20, 1696-1702.


31


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 46

(52) Al-Jamal, W. T.; Al-Jamal, K. T.; Tian, B.; Cakebread, A.; Halket, J. M.; Kostarelos, K. Tumor Targeting of Functionalized Quantum Dot-Liposome Hybrids by Intravenous Administration. Mol. Pharm. 2009, 6, 520-530. (53) Chithrani, D. B.; Dunne, M.; Stewart, J.; Allen, C.; Jaffray, D. A. Cellular Uptake and Transport of Gold Nanoparticles Incorporated in a Liposomal Carrier. Nanomed. Nanotech. Biol. Med. 2010, 6, 161-169. (54) He, Y. N.; Zhang, L. H.; Zhu, D. W.; Song, C. X. Design of Multifunctional Magnetic Iron Oxide Nanoparticles/Mitoxantrone-Loaded Liposomes for Both Magnetic Resonance Imaging and Targeted Cancer Therapy. Int. J. Nanomed. 2014, 9, 4055-4066. (55) Soga, K.; Tokuzen, K.; Tsuji, K.; Yamano, T.; Hyodo, H.; Kishimoto, H. NIR Bioimaging: Development of Liposome-Encapsulated, Rare-Earth-Doped Y2O3 Nanoparticles as Fluorescent Probes. Eur. J. Inorg. Chem. 2010, 2010, 2673-2677. (56) Soga, K.; Tokuzen, K.; Fukuda, K.; Hyodo, H.; Hemmer, E.; Venkatachalm, N.; Kishimoto, H. Application of Ceramic/Polymer Conjugate Materials for Near Infrared Biophotonics. J. Photopolym. Sci. Tech. 2012, 25, 57-62. (57) Mai, H. X.; Zhang, Y. W.; Si, R.; Yan, Z. G.; Sun, L. D.; You, L. P.; Yan, C. H. HighQuality Sodium Rare-Earth Fluoride Nanocrystals: Controlled Synthesis and Optical Properties. J. Am. Chem. Soc. 2006, 128, 6426-6436. (58) Naccache, R.; Chevallier, P.; Lagueux, J.; Gossuin, Y.; Laurent, S.; Vander Elst, L.; Chilian, C.; Capobianco, J. A.; Fortin, M. A. High Relaxivities and Strong Vascular Signal Enhancement for NaGdF4 Nanoparticles Designed for Dual MR/Optical Imaging. Adv. Healthc. Mater. 2013, 2, 1478-1488.


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(59) Al-Jamal, W. T.; Al-Jamal, K. T.; Bomans, P. H.; Frederik, P. M.; Kostarelos, K. Functionalized-Quantum-Dot-Liposome Hybrids as Multimodal Nanoparticles for Cancer. Small 2008, 4, 1406-1415. (60) Haran, G.; Cohen, R.; Bar, L. K.; Barenholz, Y. Transmembrane Ammonium-Sulfate Gradients in Liposomes Produce Efficient and Stable Entrapment of Amphipathic Weak Bases. Biochim. Biophys. Acta 1993, 1151, 201-215. (61) Nie, Y.; Ji, L.; Ding, H.; Xie, L.; Li, L.; He, B.; Wu, Y.; Gu, Z. W. Cholesterol Derivatives Based Charged Liposomes for Doxorubicin Delivery: Preparation, in vitro and in vivo Characterization. Theranostics 2012, 2, 1092-1103. (62) Xiong, X. B.; Huang, Y.; Lu, W. L.; Zhang, X.; Zhang, H.; Nagai, T.; Zhang, Q. Enhanced Intracellular Delivery and Improved Antitumor Efficacy of Doxorubicin by Sterically Stabilized Liposomes Modified with a Synthetic RGD Mimetic. J. Control. Release 2005, 107, 262-275. (63) Park, Y. I.; Lee, K. T.; Suh, Y. D.; Hyeon, T. Upconverting Nanoparticles: A Versatile Platform for Wide-Field Two-Photon Microscopy and Multi-Modal in vivo Imaging. Chem. Soc. Rev. 2015, 44, 1302-1317. (64) Zheng, H.; Liu, W. R.; Anderson, L. Y.; Jiang, Q. X. Lipid-Dependent Gating of a VoltageGated Potassium Channel. Nat. Commun. 2011, 2, 250. (65) Tian, B.; Al-Jamal, W. T.; Al-Jamal, K. T.; Kostarelos, K. Doxorubicin-Loaded LipidQuantum Dot Hybrids: Surface Topography and Release Properties. Int. J. Pharm. 2011, 416, 443-447. (66) Larson, D. R.; Zipfel, W. R.; Williams, R. M.; Clark, S. W.; Bruchez, M. P.; Wise, F. W.; Webb, W. W. Water-Soluble Quantum Dots For Multiphoton Fluorescence Imaging in vivo. Science 2003, 300, 1434-1436.


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(67) Li, K.; Su, Q. Q.; Yuan, W.; Tian, B.; Shen, B.; Li, Y. H.; Feng, W.; Li, F. Y. Ratiometric Monitoring of Intracellular Drug Release by an Upconversion Drug Delivery Nanosystem. ACS App. Mater. Interfaces 2015, 7, 12278-12286. (68) Forster, T. Zwischenmolekulare Energiewanderung und Fluoreszenz. Annal. Phys. 1948, 437, 55-75. (69) Mednitz, I.; Hildebrandt, N., FRET - Förster resonance energy transfer, from theory to applications. Wiley-VCH: Weinheim, Germany, 2014. (70) Bolotin, E. M.; Cohen, R.; Bar, L. K.; Emanuel, N.; Ninio, S.; Lasic, D. D.; Barenholz, Y. Ammonium Sulfate Gradients for Efficient and Stable Remote Loading of Amphipathic Weak Bases into Liposomes and Ligandoliposomes. J. Liposome Res. 1994, 4, 455-479. (71) Liu, B.; Li, C.; Xie, Z.; Hou, Z.; Cheng, Z.; Jin, D.; Lin, J. 808 nm photocontrolled UCL imaging guided chemo/photothermal synergistic therapy with single UCNPs-CuS@PAA nanocomposite. Dalton Trans. 2016, Advance Article. (72) Ravily, V.; Santaella, C.; Vierling, P. Membrane Permeability and Stability in Buffer and in Human Serum of Fluorinated di-O-Alkylglycerophosphocholine-Based Liposomes. Biochim. Biophys. Acta 1996, 1285, 79-90.


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TABLE OF CONTENTS

Multifunctional liposome nanocarriers combining lanthanide-doped upconverting nanoparticles (UCNPs) and the anticancer drug doxorubicin (DOX), suitable for spectral monitoring of drug release.


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Scheme 1. Schematic representation showing the structures of the blank liposome, Lipo-UCNPs, Lipo-DOX and Lipo-UCNPs-DOX. When UCNPs and DOX were incorporated into the liposome, UCNPs and DOX were located in the inner core of the liposome. Upon laser irradiation (980 nm), energy transfer occurred from UCNPs to DOX, where the upconverted green emission is partially absorbed by the DOX. 261x190mm (150 x 150 DPI)


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Figure 1. (A) TEM image of the oleate-capped NaGdF4:Er3+, Yb3+ UCNPs, (B) XRD pattern of the NaGdF4:Er3+, Yb3+ UCNPs (Reference: cubic (α-) NaGdF4, JCPDS file No. 27-0697). 87x162mm (150 x 150 DPI)



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Figure 2. TEM images of (A) blank liposomes and (B) liposomes with encapsulated NaGdF4:Er3+, Yb3+ UCNPs (Lipo-UCNPs). The white arrows indicate the presence of the UCNPs. (C) Elemental analysis of Lipo-UCNPs by EDX (peaks assigned to carbon and copper are due to the TEM copper grid used as the sample holder). 296x86mm (150 x 150 DPI)


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Figure 3. Average diameter of (A) blank liposome (average diameter, d = 156.8 nm ± 2.19 nm; polydispersity index = 1.58 nm ± 0.02 nm), (B) Lipo-UCNPs (d = 159.6 nm ± 2.37 nm; polydispersity index = 1.87 nm ± 0.03 nm), (C) Lipo-DOX (d = 152.2 nm ± 1.98 nm; polydispersity index = 1.25 nm ± 0.02 nm) and (D) Lipo-UCNPs-DOX (d = 157.9 nm ± 2.58 nm; polydispersity index = 1.98 nm ± 0.04 nm) characterized by DLS. Note: Mean ± standard deviation; n = 3. 86x140mm (150 x 150 DPI)



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Figure 4. (A) Upconversion emission spectra of Lipo-UCNPs and Lipo-UCNPs-DOX nanocarriers under 980 nm excitation (emission intensities normalized to the red emission peak). (B) Upconversion emission spectrum of Lipo-UCNPs (green) and UV-VIS absorption spectrum of DOX (red) indicating the partial spectral overlap between the DOX absorption and the UCNPs emission. 68x100mm (150 x 150 DPI)


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Figure 5. (A) UV-VIS absorbance spectra of free DOX, Lipo-UCNPs and Lipo-UCNPs-DOX. (B) Drug release profile of Lipo-UCNPs-DOX nanocarriers in different media (PBS and 50% FBS) at 37 °C. (C) Upconversion emission spectra of Lipo-UCNPs-DOX nanocarriers obtained at two different times upon DOX release in 50% FBS in PBS. (D) The upconverted green emission intensity of the Er3+ ions in NaGdF4:Er3+, Yb3+ UCNPs encapsulated in the Lipo-UCNPs-DOX nanocarriers as a function of the cumulatively released DOX. 184x151mm (150 x 150 DPI)



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TOC. Multifunctional liposome nanocarriers combining lanthanide-doped upconverting nanoparticles (UCNPs) and the anticancer drug doxorubicin (DOX), suitable for spectral monitoring of drug release. 228x148mm (150 x 150 DPI)


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Figure S1. Scheme showing the upconversion mechanism of Er3+ and Yb3+ co-doped UCNPs. 254x190mm (150 x 150 DPI)



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Figure S2. Particle size distribution of the oleate-capped NaGdF4:Er3+, Yb3+ UCNPs. 87x68mm (150 x 150 DPI)


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Figure S3. (A) FTIR spectra of citrate-capped UCNPs after incubation with DOX (UCNPs-cit-DOX, black curve) as well as reference spectra for pure DOX (red curve) and citrate-capped UCNPs (UCNPs-cit, blue curve). (B) UV-VIS absorption spectra of UCNPs-cit-DOX. 186x73mm (150 x 150 DPI)



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Figure S4. (A) UV-VIS absorption spectra of aqueous DOX solutions at various DOX concentrations used to determine the (B) calibration curve of DOX in PBS. 184x77mm (150 x 150 DPI)


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Multifunctional Liposome Nanocarriers Combining Upconverting

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