Rational Surface Design of Upconversion Nanoparticles with

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Characterization, Synthesis, and Modifications

Rational surface design of upconversion nanoparticles with polyethylenimine (PEI) coating for biomedical applications: better safe than brighter? Anna Guller, Annemarie Nadort, A Generalova, Evgeny Valerievich Khaydukov, Andrey V Nechaev, Inna A Kornienko, Elena V Petersen, Liuen Liang, Anatoly B Shekhter, Yi Qian, Ewa M Goldys, and Andrei V Zvyagin ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00633 • Publication Date (Web): 08 Aug 2018 Downloaded from http://pubs.acs.org on August 10, 2018

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ACS Biomaterials Science & Engineering

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Rational surface design of upconversion nanoparticles with polyethylenimine (PEI) coating for biomedical applications: better safe than brighter? Anna E. Guller1-3,9,*, Annemarie Nadort1,3,* (), Alla N. Generalova4,7*, Evgeny V. Khaydukov2,7, Andrey V. Nechaev5, Inna A. Kornienko6, Elena V. Petersen6, Liuen Liang1,3, Anatoly B. Shekhter2, Yi Qian1, Ewa M. Goldys1,3,9, and Andrei V. Zvyagin1-3, 8 1

Macquarie University, North Ryde 2109 NSW, Australia,

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Sechenov First Moscow State Medical University, Russia,

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The ARC Centre of Excellence for Nanoscale BioPhotonics, Australia,

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Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry of the RAS, Russia,

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Institute of Fine Chemical Technologies, Moscow Technological University, Moscow, Russia,

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Moscow Institute of Physics and Technology, Russia,

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Scientific Research Centre “Crystallography and Photonics” Russian Academy of Sciences, Russia,

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Lobachevsky Nizhniy Novgorod State University, Russia;

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University of New South Wales, Sydney 2032 NSW, Australia

*Equally contributing co-authors Corresponding author: Annemarie Nadort, PhD, NHMRC Early Career Research Fellow E-mail: [email protected]

Abstract Upconversion nanoparticles (UCNPs) coated with polyethylenimine (PEI) are popular background-free optical contrast probes and efficient drug and gene delivery agents attracting attention in science, industry and medicine. Their unique optical properties are especially useful for subsurface nanotheranostics applications, in particular, in skin. However, high cytotoxicity of PEI limits safe use of UCNP@PEI and this represents a major barrier for clinical translation of UCNP@PEI-based technologies. Our study aims to address this problem by exploring additional surface modifications to UCNP@PEI to create less toxic and functional nanotheranostic materials. We designed and synthesized six types of layered polymer

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coatings that envelop the original UCNP@PEI surface, five of which reduced the cytotoxicity to human skin keratinocytes under acute (24h) and subacute (120h) exposure. In parallel, we examined the photoluminescence spectra and lifetime of the surface modified UCNP@PEI. To quantify their brightness, we developed original methodology to precisely measure the colloidal concentration to normalize the photoluminescence signal using a non-digesting mass spectrometry protocol. Our results, specified for the individual coatings, show that despite beneficial effect on biocompatibility decreasing the cytotoxicty, the external polymer coatings of UCNP@PEI quench the upconversion photoluminescence in biologically relevant aqueous environments. This trade-off between cytotoxicity and brightness for surface-coated UCNPs emphasizes the need for the combined assessment of the viability of normal cells exposed to the nanoparticles and the photophysical properties of postmodification UCNPs. We present an optimised methodology for rational surface design of UCNP@PEI in biologically relevant conditions, which is essential to facilitate the translation of such nanoparticles to the clinical applications. KEYWORDS Upconversion nanoparticles; photoluminescence; surface modification; cytotoxicity; skin. Introduction Nanoparticles doped with lanthanide ions (Ln3+) such as Er3+ or Tm3+ are capable of “upconversion”, that is transforming near-infrared (NIR) excitation light (λ ≈ 980 nm) into higher energy emission photons in the visible and NIR spectral range (λ = 450 - 850 nm)1-2. The most common upconversion nanoparticles (UCNPs), NaYF4: Yb:Er , are excited at 980 nm and emit light in the green and red spectral bands3. . There is a growing body of literature on the use of UCNPs in various research and industrial areas (for review, see2, 4-5, and references therein), in particular in bioimaging and nanotheranostics6-8. The particular advantage of UCNPs is that their excitation and luminescence emission occur in the “tissue transparency window”, providing a possibility of deep probing of biological matter up to a few mm’s9. However, the luminescence brightness in all upconversion materials is low, with a typical range 0.1 – 5% conversion


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efficiencies4, 10-11. In order to be used in biological applications, as-synthesized UCNP nanocrystals need to be surface-modified (e.g., coated with polymers) to make the resulting nanoparticles stable in aqueous environment and suitable for further functionalization. However, this may lead to undesirable quenching of luminescence by attached molecules4, 12-13. Even more importantly, surface modification also tends to affect the UCNP cytotoxicity14 , and therefore, a careful consideration of the effects of various surface coatings on the cytocompatibility of UCNPs is urgently required. This investigation must be done in nonmalignant cells, as healthy tissues must be protected against any harmful action of therapeutic or diagnostic UCNP use. The potential damaging effects of lanthanide-doped UCNPs on non-malignant cells have only recently come to light14-16, contradicting the commonly held view, based on experiments in linear cancer cells, that UCNPs are nontoxic (for review see17-18). Additionally, the details of surface functionalization, such as functional groups and charge properties induce different biological interactions, leading to, for example, differences in protein corona formation19-20. This in turn has a strong effect on the cellular uptake of UCNPs and on their blood circulation times21-23: two events which are critically important for biomedical applications of nanoprobes and nanodrugs. Polyethylenimine (PEI) is commonly used for surface modification of nanoparticles, including UCNPs, due to its advantageous amphiphilic nature and ease of further binding15, 24-29. PEI is a polycationic linear or branched amphiphilic polymer (AP) with ample amine groups (NH2) and a high positive charge24. Due to its ability to permeate through cellular membranes and form stable complexes with nucleic acids, this AP is well known as an efficient agent for the intracellular delivery of genes and molecules of biological significance30-31. The oleate moieties that reside on the NaYF4: Yb:Er nanocrystal surface following the last stages of synthesis, form stable bonds with hydrophobic sites of PEI by physical adsorption with partially removed oleic acid, resulting in a dense layer of polymer enveloping the particle (Figure 1). This capping layer stabilizes UCNPs in aquatic solutions14-15, 24, 32 and is expected to prevent water quenching of upconversion luminescence33-34. Surface modification of UCNPs with PEI contributes to intensive



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internalization of the particles27, 29, 35, which is thought to result from clathrin-mediated endocytosis27, and may be desirable for biomedical applications.

Figure 1. Surface modification of as-synthesized oleate-capped nanocrystals UCNP to UCNP@PEI. Oleic acid (pink) residues on the surface of UCNP are partly replaced with branched PEI (blue) via physical absorption.

However, significant toxicity of PEI due to high polycationic charge density 36 remains a problem for its application in normal living cells and tissues30-31, necrotic or apoptotic processes36,

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. PEI causes severe membrane damage inducing

. When PEI is used as a surface modifier of nanoparticles, its

damaging effects become three times less prominent in comparison to unbound branched PEI of the same molecular weight (possibly because of less cationic ligands interacting with cells)40, but still notable14-15. The viability of normal cells in the presence of PEI-coated UCNPs significantly depends on the concentration and exposure time. For example, the viability loss in murine bone marrow mesenchymal stem cells of about 5-15% was reported upon exposure to UCNP@PEI at a concentrations of 1-25 µg/ml during 1-2 days, while under higher doses combined with longer incubation time (100 µg/ml; 24 h) the survival rate of the cells decreased dramatically with only 63% viability29, 35. Our recent experiments on human skin epidermal keratinocytes (HaCaT) and primary hippocampal cell cultures provided evidence in favor of the theory that cytotoxic effects of UCNP@PEI originate from PEI coating and not from βNaYF4: Yb3+,Er3+ nanocrystals14-15, while this is not the case for some other types of UCNPs16. The UCNP@PEI demonstrated the highest uptake by keratinocytes and at the same time the highest cytotoxicity, in comparison with the effects of the same nanocrystals modified with 7 other types of coatings14.


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These findings motivated us to search for a rational way to exploit the intracellular delivery potential of UCNP@PEI for skin-related applications, while avoiding the toxic effects and undesirable quenching of photoluminescence by surface ligands. To this end, we explored and juxtaposed the effects of additional one and double-layer coatings composed of various biocompatible polymers on the photoluminescence properties of UCNP@PEI and on the cytotoxicity of these particles not only in standard (24 h), but also in longitudinal (120 h) in vitro cultures of human skin keratinocytes. Materials and Methods Materials The following materials were purchased from Sigma-Aldrich and used without further purification: sodium chloride, phosphate buffered saline, pH 7.0 (PBS), tetramethylammonium hydroxide pentahydrate (TMAH), hexane, branched polyethylenimine (PEI, Mw 25000), poly (acrylic acid-co- maleic acid) (PAMA, Mw 70000), Na-salt dextran sulphate (DxS, Mw 50000), dextran (Dx, Mw 70000), sulfochitosan (SCh, 800-2000cP), chitosan (Ch, 800-2000cP). Ethanol, propanol-2, chloroform were of analytical grade and purchased from Aldrich. Synthesis of upconversion nanoparticles Hydrophobic monodisperse hexagonal phase β-NaY0.78Yb0.2Er0.02F4 nanoparticles were synthesized as described elsewhere14, 41. Briefly, a mixture of rare-earth trifluoroacetates, obtained by evaporation of suspension of Y2O3 (0.78 mM), Yb2O3 (0.2 mM), Er2O3 (0.02 mM) in 70% trifluoroacetic acid and subsequent milling and vacuum drying, was mixed with sodium trifluoroacetate (2 mM), oleic acid and 1octadecene and thoroughly stirred. Then, the obtained mix was heated in oxygen-free (argon) atmosphere. Next, the product was cooled in the excessive volumes of 1-octadecene and isopropanol, centrifuged, washed with 100% ethanol and dried. The resulting oleate-capped nanocrystals (OA-UCNPs) were dissolved in chloroform, precipitated with isopropanol, centrifuged again and dried at room temperature.



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Surface modification of UCNPs In order to evaluate the effects of surface modifications on the UCL properties and cytotoxicity of UCNPs the following strategies were applied and resulted in preparation of eight individual types of aqueous colloids of the nanoparticles with different polymer coatings (Figure 2). The list of the polymers used for UCNP@PEI modification included PAMA, Sch, DxS, Dx and Ch. These polymers are biocompatible, non-cytotoxic, water-soluble and commonly used for the surface modification of nanoparticles, especially by the layer-by-layer technique3, 42. In addition, these polymers can generate a different zeta-potential of the UCNPs, which enables the study of particle charge on cell interactions. The details of coating procedures are given below.

Figure 2. Surface modification of UCNPs. (1) Modification of oleic acid-capped as-synthesized UCNPs (OAUCNPs) with TMAH allowed preparation of bare hydrophilic particles (TMAH-UCNP). (2) Single layer surface coating of UCNPs with branched PEI was used as a basic approach to get stable aqueous colloid dispersions of the particles (UCNP@PEI). (3) Sequential modification of UCNP@PEI resulted in particles with double-layered


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coatings, UCNP@PEI@PAMA, UCNP@PEI@Sch, UCNP@PEI@DxS and UCNP@PEI@Dx. (4) Double-coated UCNP@PEI@PAMA are further modified with the third layer of Dx or Ch, resulting in UCNP@PEI@PAMA@Dx and UCNP@PEI@PAMA@Ch. The layers of PAMA, DxS, SCh and Ch are bound to the preceding coating (PEI or PAMA) by ionic bonds. The external layer of Dx is bound to PEI or PAMA by hydrogen bonds.

Bare hydrophilized UCNPs: surface modification of UCNPs with tetramethylammonium hydroxide pentahydrate (TMAH-UCNPs) As-synthesized oleate-capped UCNPs (OA-UCNPs) were modified with TMAH as we described previously14-15 to obtain reference bare particles stable in aqueous solutions. TMAH is a low molecular weight phase transition agent, which absorbs on the surface of NaYF4:Yb3+:Er3+ nanocrystals to partially displace the oleate tails, but it does not form a monolayer coating14. As a result, the nanocrystals remain exposed to the environment and can directly interact with biological buffers and cellular membranes. In short, 1 mL of 1% aqueous solution of TMAH was added dropwise to the 20 µL of UCNP dispersion (10 mg/mL) in chloroform, resulting in in two immiscible phases. Then the emulsion was thoroughly shaken, and chloroform was evaporated to transfer the UCNPs to water phase. To remove an excess of TMAH, the UCNP aqueous suspension was washed three times with water by consecutive centrifugation at 13400 rpm for 10 min. The pellet was then dispersed in 1 mL of PBS, pH 7.2. These particles, TMAH-UCNPs, were used as reference material to evaluate the effects of mono- and multilayer surface coatings on cytotoxic potential and photoluminescent properties of UCNPs. Preparation of UCNP@PEI Basic surface modification of as-synthesized NaYF4:Yb3+:Er3+ nanocrystals with PEI, resulting in singlelayer coated UCNP@PEI particles, was performed by the widely accepted solvent evaporation method described elsewhere14. Briefly, 0.25 ml of a solution of branched PEI in chloroform (15 mg/ml) was added to 0.02 ml of the dispersion of OA-UCNPs (4 mg/ml) in chloroform. The mixture was sonicated in an ultrasonic bath for 5 minutes and then stirred for 1.5 hours at room temperature. After evaporation of the



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solvent, the resulting residue was added with 1 ml of distilled water. After further sonication the resulting dispersion was kept under vigorous stirring for 1 hour at room temperature. Coating of the UCNP@PEI with the second layer (UCNP@PEI@L1) UCNP@PEI were sequentially modified with a single additional coating layer L1, to result in particles with a structure of UCNP@PEI@L1, where L1 was formed by one of the following polymers such as PAMA, Dx, DxS or SCh (See Fig. 2). For the preparation of the secondary PAMA, DxS, Dx or Sch coating, respectively an 0.2 mL of aqueous solution of PAMA (2 mg/ml), 0.4 mL of aqueous solution of DxS (1 mg/mL), Dx (1 mg/ml) or Sch (1 mg/ml) was added to 0.2 mL of UCNP@PEI (0.8 mg/ml). The mixture was sonicated for 5 min and incubated for 30 min, while stirring at room temperature. Then the solution was centrifuged at 13400 rpm for 10 min with addition of water (this procedure was repeated three times to remove free polymer). The pellet was then dispersed in 0.4 mL PBS, pH 7.2. Coating the UCNP@PEI@PAMA with the third layer (UCNP@PEI@PAMA@L2) The second layer on the surface of UCNP@PEI was prepared from PAMA as described above, and the third, the most external coating (L2), was formed by Dx or Ch (See Fig. 2). For preparation of UCNP@PEI@PAMA@Dx, 0.4 mL of Dx aqueous solution (1 mg/mL) was added to 0.2 mL of UCNP@PEI@PAMA (0.8 mg/ml). For preparation of UCNP@PEI@PAMA@Ch, 0.04 mL of 1% chitosan solution in 1% acetic acid (2 mg/ml) was added to 0.4 mL of UCNP@PEI@PAMA (0.8 mg/ml). Both mixtures were sonicated for 5 min and incubated for 30 min, while stirring at room temperature. Then the solutions were centrifuged as described above for UCNP@PEI@L1 to remove free Dx and Ch, and the pellets were dispersed in 0.4 mL PBS, pH 7.2. Characterization of UCNPs Morphology and surface charge of the particles The shape and size of as-synthesized UCNPs were defined by transmission electron microscopy (TEM). A dispersion of OA-UCNPs in chloroform was dropped on a TEM copper grid and examined under a Philips CM10 TEM (Philips, The Netherlands) after drying of the sample. The morphology of the


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nanocrystals was assessed by TEM image analysis (ImageJ freeware). Dynamic light scattering (DLS) examination was performed by a sub-micron particle analyzer (Coulter N4-MD, Coultronics, France) at a scattering angle of 90°. The ζ-potential of the modified UCNPs was analyzed by a zeta-potental analyzer (Brookhaven Corporation Instruments T90Plus, Brookhaven, USA). Absolute conversion efficiency measurement The absolute conversion efficiency (ηUC) measurement set-up was carefully prepared and calibrated, as detailed elsewhere11. In short, the OA-UCNP sample was dried, and the powder was confined in a custom-made sample holder (glass plate with circular hole of 1.5 mm diameter × 0.45 mm depth) and placed against one of the exit ports of an integrating sphere. The sample was excited by a CW 980 nm laser with intensity from 3 to 3×102 W/cm2. The photoluminescence signal was detected by a photodiode (Thorlabs, PDA-55) placed at the detection port, using lock-in amplification (Stanford Research Systems, model SR830). In addition, the non-absorbed excitation power was measured by using the appropriate filter sets and the ηUC was calculated as the ratio of the emitted power and absorbed power in (W/W). Upconversion photoluminescence spectra and lifetime measurements The fluorimeter Fluorolog-3 HJY (Horiba Jobin Yvon, France), equipped with the photomultiplier tube Hamamatsu R929P (Hamamatsu Photonics, Japan), was used to define the optical spectra and kinetics of the nanoparticles’ UCL. The UCNP samples, as prepared in PBS, were transferred to a quartz cuvette and placed in the cuvette chamber of the fluorimeter, with the exception of the OA-UCNP sample, which was dissolved in chloroform. As excitation source, a semiconductor laser ATC (Semiconductor devices, Russia) emitting at 980 nm was used, which was fiber coupled and focused on the sample by a lens. The power density was fixed during UCL measurements at 2·101 W/cm2, continuous-wave (CW). To measure the UCL lifetime, the PMT signal was recorded with the high-speed oscilloscope Tektronix TDS 6804B (Tektronix Inc, USA). During the measurements of the luminescence lifetime, a monochromator was fixed at 540 nm with the slit width of 5 nm. Excitation pulse duration was 1 ms with a constant repetition rate 50 Hz.



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Photoluminescence intensity measurements To quantify the UCL brightness as a dependence of additional surface coatings, we measured the UCL signal for each sample directly followed by its concentration measurement using inductively coupled plasma mass spectrometry (ICP-MS). ICP-MS study was performed using an Agilent 7500 ICP-mass spectrometer (Agilent Technologies, USA) according to the manufacturer’s protocol. ICP-MS is known as a highly sensitive method to measure the ionized atom concentrations (as low as parts per quadrillion), which has been applied to UCNPs43. To prevent ionic interference from the medium during ICPMS we transferred all samples from PBS to water (milliQ) by washing and re-dispersing 3 times. We firstly validated the linearity of the ICP-MS method to quantify UCNP colloidal concentration. Yttrium standard solutions and UCNP@PEI aqueous dispersions (1 mg/mL) were serially diluted in a mild acid, 2% HNO3, and the yttrium concentration versus yttrium count rate (r2>0.99) was examined, as well as the particle relative concentration versus yttrium count rate (r2>0.99) (see Figure S1 in Electronic Supplementary Information, ESI). Secondly we confirmed the repeatability of the ICP-MS method. The yttrium count rates of a colloidal UCNP solution were measured over different time points and days during the period of two weeks of the same sample gave an average standard deviation of ±1.4% from the mean value (see Figure S2, ESI). Since the relative sample concentrations rather than absolute concentrations were required for the current study, the aqueous dispersions of UCNP@ PEI, UCNP@PEI@L1 and UCNP@PEI@L2 were diluted 100 times in 2% HNO3, and analyzed by ICP-MS without further digestion. The yttrium counts for each sample were collected 3 times and averaged. The UCL spectra of the undiluted colloidal solutions in water were acquired in a 10 mm optical path quartz cuvette using a fluorimeter Fluorolog-3 HJY (Horiba Jobin Yvon, France), equipped with an external 980 nm CW diode laser (power density in quartz cuvette 1·101 W/cm2). The total UCL intensity was calculated as the integrated area under the green and red emission peaks (expressed in counts), after background and noise correction of the UCL spectra. To quantitatively compare the UCL intensity versus type of surface modification of the particles, independent of concentration, the total UCL intensity was divided by the yttrium counts per sample, and normalized to the emission signal from the UCNP@PEI sample. Due to


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the low concentration of UCNPs, the UCL was not expected to be significantly influenced by reabsorption of emitted light by particles or by large attenuation due to scattering. Cell culture and viability assay The HaCaT cell line was kindly donated by Prof. N. Onischenko (Moscow, Russia). These cells are nontumorigenic spontaneously immortalized human keratinocytes, preserving a differentiated phenotype similar to normal epidermal cells of the human skin, but exhibiting sustainable proliferation in vitro44-45. The cells were seeded onto 96-well plates (Greiner CELLSTAR®) at the density of 1.5×103 cells per well, and grown at 37 °C and 5% CO2 in complete culture medium prepared from Dulbecco’s Modified Eagle’s Medium (DMEM/F12/Ham medium, #D8437, Sigma Aldrich) supplemented with 10% Fetal Bovine serum (#12003C, Sigma Aldrich) and 1% Penicillin-Streptomycin (10,000 U/mL; #15140122, Gibco). The dispersions of the particles in complete media were prepared at the concentration of 125 µg/mL and sonicated for 30 minutes immediately before adding to the wells. After 24 h of the initial growth, the culture medium was removed from the wells, and the cells were washed twice with sterile PBS solution. Then the cells were exposed to the dispersions of TMAH-UCNPs, UCNP@PEI, UCNP@PEI@L1 and UCNP@PEI@PAMA@L2 nanoparticles (100 µL/per well) and incubated in unfed culture for the next 24 or 120 h under the standard conditions. To test the cytotoxicity, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT) reagent (#M2128, Sigma-Aldrich) reduction and conversion into a blue formazan salt was used as a measure of activity of the cellular mitochondrial dehydrogenase46. The UCNP dispersions and control cell culture medium were removed from the wells, then the cultures were washed three times with PBS and 100 µL of MTT reagent (0.5 mg/mL in the phenol red free cell culture medium) (DMEM/F12; #D6434, SigmaAldrich) was added to each well. The cells were incubated at 37 °C for 1 h to allow precipitation of insoluble formazan crystals. Then, the supernatant was carefully collected, and 100 µL dimethyl sulfoxide was added to the wells and left for 10 min in the dark at room temperature to dissolve purple formazan



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crystals. The absorbance of the resulting dye solution was evaluated in comparison with the control culture by measuring the optical density at 570 nm by a multi-well UNIPLAN photometer (Pikon Ltd, Russia). Since the absorbance is proportional to the number of viable cells, we expressed the mean cell viability as a percentage of the absorbance values of the control culture assays. Confocal microscopy UCNP@PEI at a concentration of 125 µg/mL were incubated with HaCaT keratinocytes grown on coverslips at 37 °C for 48 h under 5% CO2. After washing with PBS three times, cells were fixed in 4% formaldehyde, and coverslips were sealed. Internalization of UCNP@PEI into HaCaT cells was assessed by confocal microscopy (Olympus, Fluoview FV1200, USA), modified to allow illumination with a 978 nm laser (Leo Photonics, China) and low-pass filtering of the confocal UCL signal. The image containing the UCNP signal was overlaid with a bright field image, obtained by confocal laser scanning of a green laser and detecting the back-reflected light. Statistical methods The cell viability data was expressed as means ± standard deviations (SD) and 95% confidence intervals (CI95%) for means. The differences between the means were evaluated by applying CI95% boundary comparisons and post-hoc analysis following the ANOVA test. The level of statistical significance was set at p < 0.05 and 2-side tests were used in all cases. The data were analyzed using SPSS 13.0 for Windows (SPSS Ink, Chicago, USA).

Results Characterization of as-synthesized UCNPs Results of the TEM study of OA-UCNPs (β-NaY0.78Yb0.2Er0.02F4) are presented in Fig. 3(a) and 3(b). The average size of the nanocrystals was evaluated as 93±8 nm. The majority of the nanocrystals exhibited hexagonal morphology. The absolute conversion efficiency of OA-UCNPs was measured approximately


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0.1% at laser excitation density of 10 W/cm2, with a maximum conversion efficiency of 1.5% measured at 300 W/cm2 (see Fig. 3(c)).

Figure 3. (a) Transmission electron microscopy (TEM) image of as-synthesized β-NaY0.78Yb0.2Er0.02F4 nanoparticles, showing a hexagonal morphology. Note that some particles are positioned on their narrow side exposing the rectangular facet to the electron beam. (b) Histogram of OA-UCNPs size distribution, obtained from the TEM images. The nanoparticles possess a mean particle size of approximately 93±8 nm (derived from a Gaussian fit to the histogram with mean 93 nm and full-width at half maximum of 16 nm). (c) Absolute conversion efficiency of OA-UCNPs (powder) measured with an integrating sphere as described in11.

Characterization of surface modified UCNPs

The as-synthesized particles were subsequently used to prepare 8 types of aqueous dispersions of surface modified UCNPs. As it is shown in Fig. 4, bare UCNPs hydrophilized with TMAH had the smallest hydrodynamic size (94±8 nm) according to DLS measurements, while the size of other surface modified UCNPs varied between 142±9 nm (UCNP@PEI@PAMA) and 175±14 nm (UCNP@PEI@SCh), as the smallest and largest particles, respectively. The hydrodynamic size of UCNPs coated with PEI, as well as the size of UCNP@PEI@L1 and UCNP@PEI@L2, increased in comparison with the TEM-measured size of the as-synthesized particles due to the hydration of the polymer coatings and formation of "swollen” polymer shells47 . Two types of surface modified UCNPs had positive zeta-potential, ranging



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from approximately 12 mV (UCNP@PEI@Dx) to 42 mV (UCNP@PEI). All other studied particles were charged negatively, exhibiting the zeta-potential of -9 to -21 mV (see Fig. 4). The obtained aqueous colloid dispersions of surface modified UCNPs were stable in electrolytes (PBS) over at least two weeks.

We recorded the UCL spectra for all samples in PBS (except for OA-UCNP in chloroform), as shown in Fig. 5(a)-(i). Each sample exhibited the typical emission spectrum for Er3+-doped UCNPs with green (510–560 nm) and red (640–680 nm) luminescent bands under 980 nm CW laser excitation. The inset (j) shows the UCL-lifetimes as measured for the green UCL peak. Next, we quantified the relative UCL intensity as a dependency of coating of the surface-modified UCNP colloids, by correcting the integrated UCL signal measured for colloidal samples in water for the sample concentration. Surface coating of UCNPs with PEI resulted in the brightest photoluminescence among all the nanomaterials examined in this experiment. The results are shown in Fig. 6, together with the results from the cytotoxicity tests (next section) for the direct comparison of both functional parameters. The addition of one or more extra surface layers to UCNP@PEI decreased the brightness, ranging from 10 to 80% of the UCL from UCNP@PEI. At the same time, bare TMAH-UCNPs also demonstrated a decrease in UCL, to 27% of the signal from UCNP@PEI.


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Figure 4. Hydrodynamic diameter and zeta-potential of surface-modified UCNPs, measured thrice at room temperature (error bar represents standard deviation derived from the DLS size distribution).

Figure 5. Upconversion photoluminescence spectra of as-synthesized OA-UCNPs in chloroform (a), bare TMAHUCNPs (b) and surface modified UCNP@PEI (c), UCNP@PEI@Dx (d), UCNP@PEI@DxS (e), UCNP@PEI@Sch (f), UCNP@PEI@PAMA (g), UCNP@PEI@PAMA@Dx (h) and UCNP@PEI@PAMA@Ch (i) under CW 980 nm laser excitation (20 W/cm2), (b) – (i) in PBS. Bottom left panel shows the UCL lifetime measured as the temporal full width at half maximum intensity at 540 nm. The lifetime of UCNP@PEI@PAMA@Dx (h) could not be reliably determined.

The effects of surface modification of UCNPs on viability of human skin keratinocytes The results of the MTT viability test of human skin keratinocytes exposed to the surface-modified UCNPs for 24 h or 120 h are presented in Figure 6, and Table S1 (ESI) shows the detailed statistical analysis. All surface modified UCNPs examined in this study decreased the viability of HaCaT keratinocytes at the



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concentration of 125 µg/mL, in comparison with the control, for more than 20% (and up to 87%), under 24 h and 120 h exposures, respectively. The individual effects of the surface coatings are particularly noticeable when considering the development of acute (24 h) and subacute (120 h) cytotoxic reactions. In particular, UCNP@PEI and all the particles with the structure UCNP@PEI@L1 demonstrated enhanced toxic effects with longer exposures, while the bare TMAH-UCNPs and UCNP@PEI@PAMA@L2 induced only negligible reduction of cellular viability in 120 h culture, in comparison with 24 h tests. The toxic potential of bare TMAH-UCNPs can be considered moderate as the loss of the keratinocytes’ population reached approximately 25-30% under 24 h and 120 h exposure to these particles in vitro. The lowest survival rates of HaCaT cells were observed in the presence of UCNP@PEI. These positively charged particles induced significant damage to almost 50% of the cells after 24 h incubation and more that 85% to the cells in the cultures maintained for 120 hours. The difference between the effects of UCNP@PEI and hydrophilized bare TMAH-UCNP was statistically significant for both exposure times (see Table S1, ESI). In particular, a 24 h incubation of keratinocytes in the presence of UCNP@PEI resulted in 23% lower survival rate than in the cultures with TMAH-UCNP (CI95% for difference: 7; 39%), and under 120-hour exposure this difference reached 58% (CI95% for the difference: 31; 85%). Except for UCNP@PEI@Dx, all studied further surface modifications of UCNP@PEI resulted in better survival rates of the skin epithelial cells, with a 25-45% higher viability in 24 h cultures and an impressive 4 to 5 times higher viability in the 120hour tests, as compared with UCNP@PEI. The inset in Figure 6 shows the internalization of the UCNP@PEI by HaCaT cells in a representative confocal z-plane. To confirm that the UCNPs are within the cells, several z-planes are shown in the SI, Fig. S3.


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Figure 6. Cytotoxicity and brightness parameters for UCNP@PEI and UCNP@PEI with further surface coatings. Cytotoxicity is represented by the grey and blue bars: Average viability of HaCaT keratinocytes co-cultured with UCNPs at concentrations of 125 µg/mL or without nanoparticles (control) for 24 h (grey bars) or 120 h (blue bars). The control sample viability was set to 100%. The brightness (measured for colloidal samples in water) is represented by the yellow bars: Relative UCL emission, corrected for sample concentration and normalized to the UCL emission of UCNP@PEI. Error bars represent CI95% for means. Inset: Uptake of UCNP@PEI by HaCaT keratinocytes after 48 h exposure in vitro as imaged using confocal microscopy, showing an overlay of bright field and UCL (green colour) images. The UCL signal outside the cells is due to non-internalized and/or exterenalized UCNPs.

Discussion The rapid development of upconverting nanomaterials will expectedly result in an increasing number of suggested biomedical applications for their use. UCNPs attract the particular interest of dermatological researchers and clinicians because of the exceptional potential of these particles as a background-free



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contrasting probe, with the simultaneous ability to deliver therapeutics to the skin cells: i.e. to perform as a multifunctional nanotheranostic agent, having special advantages for skin cancer treatment. At the same time, the accumulation of evidence on the cytotoxic action of UCNPs on normal cells, and specifically on keratinocytes14, raises concerns regarding the safety of their use in human skin. The current study was performed in order to find reasonable strategies to develop the optimal composition and structure of UCNPs, which could reconcile the colloidal stability of the particles’ in aqueous environments; their optical brightness; and reduce the cytotoxic damage to normal epidermal cells. In order to reach this goal, β-NaY0.78Yb0.2Er0.02F4 UCNPs coordinated with oleic acid were synthesized inhouse by the solvothermal method, resulting in hydrophobic particles . To prepare the particles for use in aqueous biological environments, two approaches for further surface modification were applied to make the particles hydrophilic. We used the ligand exchange reaction between oleic acid residuals persisting on the surface of as-synthesized UCNPs and low molecular weight phase transition agent, tetramethylammonium hydroxide (TMAH) to obtain reference bare hydrophilic UCNPs. The second approach relied on the formation of one, two or three additional polymer coatings on the surface of the OA-UCNPs. In this study we focused on coating the UCNPs with the amphiphilic compound polyethylenimine (PEI), because of its wide use in cell biology as a gene transfection vehicle, though at the expense of significant cytotoxicity, which can be detrimental when it interacts with healthy cells. Several methods to modify UCNPs with PEI were proposed24-25, 27, 48-52. Of special interest is the one-pot procedure to simultaneously synthesize NaYF4:Yb,Er/Tm nanocrystals and coat them with PEI, as demonstrated by Wang et al.24. In our study we applied synthesis and surface modification sequentially, in order to study the effects of every modification step on the cytotoxicity and photophysical properties of the particles. With the intention to limit the cytotoxic effects of PEI, six additional surface modifications of the particles were performed to create single (L1) or double (L1, L2) external polymer layers on the surface of the UCNP@PEI particles (see Figure 2). Multilayer-structured nanomaterials are known in the field of polymeric nanoparticles53,


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however, this attractive technique is not fully recognized in the area of inorganic nanoparticles, and only a few relevant reports are available for PEI-modified inorganic nanomaterials54. Likewise, the reports on the L-b-L assembling of polymer surface coatings on UCNPs are scarce. For example, (Gd3+)-doped UCNPs coated with polyethylene glycol and successively coated with one or two layers of PEI, were synthesized in a study where PEI was applied for cell transfection55. In another publication, a multilayer coating of Mn-doped UCNPs with clorin E6 dimethyl maleic acid and polyethylene glycol was shown to enhance the efficiency of photodynamic therapy56. Recently, Wang et al.57 applied the L-b-L strategy to prepare advanced nanotheranostic agents as a nanocomposite containing mesoporous bioactive glass, UCNPs and silica for controlling the fate of bone marrow stromal cells.

In our study, the obtained particles were characterized in terms of their size, surface charge, UCL emission spectra and lifetime, relative UCL intensity, and cytotoxicity towards human skin keratinocytes. According to the results of DLS measurements, surface modification of OA-UCNPs with TMAH, led to formation of hydrophilized bare particles with the diameter close to the original size of as-synthesized nanocrystals and a negative surface charge (see Fig. 4). The UCNP@PEI demonstrated an increased hydrodynamic diameter (~160 nm) and high positive surface potential (~ +42 mV). Formation of a single layer of dextran of the surface of UCNP@PEI induced some shrinkage of the size of the particles (to ~145 nm), which was not statistically significant, and partially neutralized the cationic charge (~ +12mV). All other external capping of UCNP@PEI induced inversion of the zeta-potential to negative values in the range from - 9 mV to -16 mV, while the diameter of the particles with structures like UCNP@PEI@L1 and UCNP@PEI@PAMA@L2 statistically did not change, in comparison with UCNP@PEI. Taking this data into account, we assume that the observed changes in photophysical properties and cytotoxicity of the particles were mainly the effect of surface modifications. The photophysical properties of the UCNP samples were also influenced by the surface modifications. Figure 5 shows that the different surfaces (TMAH, PEI, PEI@L1, PEI@L1@L2) have a moderate effect on the UCL spectral signature, i.e. the ratio of the green to red emission peak, and a moderate effect on the UCL lifetime, while the influence of surface modification on the total UCL intensity emitted by the



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particles is much larger (Figure 6). The radiative rates from the red and green emission states of the Erbium ion govern the green/red ratio and the total UCL intensity. In turn, the population of these radiative states is a result of a complex interplay of 2 and 3-photon processes, involving energy absorption, energy transfer between sensitizers, activators and sensitizer-activator pairs (phonon-assisted or resonant) and non-radiative relaxation (phonon-assisted or cross-relaxation). The surface coating can influence this in two ways: by introducing high-energy oscillators on the surface (molecular groups that are present in the coating) on the one hand, and by removing the high-energy oscillators in the solvent (OH groups in aqueous solutions) further away from the surface on the other hand13,

58-59

. The former

increases surface quenching, the latter reduces surface quenching. The interplay of these two processes can result in reduced or enhanced radiative emission, including different proportions of the green and/or red bands, as our results show. The UCNP@PEI show the highest relative total UCL brightness among all the studied particles, while the bare TMAH-UCNPs demonstrated only a third of this signal intensity. This indicates the prominent beneficial role of PEI as a protective coating, shielding the UCNP nanocrystal surfaces from the quenching by water27,24 . On the other hand, adding the external polymer layers to UCNP@PEI resulted in decrease of relative UCL, of up to 10-80% of the intensity of the signal in UCNP@PEI. There are possible explanations for this decrease in the UCL signal: a) the charged polymers introduce irregularities in the tight PEI coating allowing water molecules to penetrate the coating; and b) the polymers introduce their own –OH groups within the PEI chains, Indeed, surface quenching due to irregular coatings has been reported60. Additional coating thus warrants an additional check of the UCL signal for an optimized design of biologically applied UCNPs.

We note that the lifetime measurements seem contradictory to the UCL intensity measurements, i.e. a longer lifetime does not necessarily correlate with an increased UCL signal, which we explain as follows. Several recent studies have found that the main mechanism of surface quenching is multiphonon deactivation of the sensitizers (Ytterbium (Yb)-ions), instead of the activators61-62. The fast energy transfer between Yb-ions throughout the whole nanocrystal makes that both core and surface Yb-ions are influenced by surface quenching. Together with the nonlinear nature of the UCL process, surface


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quenching results in a higher order effect on the UCL brightness, which explains the large reductions in UCL intensity seen in Figure 6. The multiphonon deactivation of Yb-ions will also majorly reduce the Yb excited state lifetime, but may have a smaller effect on the green or red radiative lifetimes of the Erbium ions, as seen in Figure 5. In addition, we also consider the difference in medium (PBS for lifetime versus water for brightness measurements) and the substantial time delay between measurements of lifetime and brightness, which were performed at different locations, as potential additional sources for the lifetime/brightness discrepancy. The exact mechanism of the observed effects requires further studies. However, these findings show that the photophysical properties are highly sensitive to environmental changes, which is important to consider for the design of bioimaging applications of surface-coated UCNPs. The parallel study of the cytotoxicity of surface modified particles showed notable cytotoxicity induced on human epidermal cells (HaCaT) by all the currently studied particles (TMAH, PEI, PEI@L1, and PEI@L1@L2). Our previously reported data revealed good cytocompatibility of various surface modified UCNPs with dermal fibroblasts, while the cytotoxic effect on keratinocytes during 24 h in vitro culture was similar to the current results14. In this report, the first evidence of subacute (120 h) toxicity of lanthanide-doped UCNPs in human keratinocytes’ monolayers is presented. In Figure 6 and Table E1 (ESI) we show that all further surface modifications of UCNP@PEI, except UCNP@PEI@Dx, significantly reduced the cytotoxicity of UCNP@PEI. Since both UCNP@PEI and UCNP@PEI@Dx are the only particles with positive zeta-potential, it is reasonable to conclude that the cationic charge of the particles increased their toxicity. The further surface modifications of UCNP@PEI shielded the positive charge resulting in a negative zeta-potential, which allowed reduced toxicity of the particles with human skin keratinocytes in both 24 h and 120 h cultures. This result is in accordance with the well-known cytotoxic effect of polycations31, which is attributed mainly to the irreversible damage of plasma membranes and release of reactive oxygen species and lysosomal enzymes. The cytotoxicity results further indicate the critical role of surface coatings in acute (24 h) and subacute (120 h) cytotoxic



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effects of UCNPs. The survival rate for cells exposed to bare TMAH-UCNP was statistically not different after exposure for 1 or 5 days (Table E1, ESI). This was also the case for the double-layer coated UCNP@PEI, e.g. UCNP@PEI@PAMA@Dx and UCNP@PEI@PAMA@Ch, indicating the advantage of multilayer surface wrapping of UNCP@PEI for safer long-term interactions with the epidermal cells. For the other studied particles there was a 5-15% reduction of viability of HaCaT cells in 120 h, in comparison to 24 h culture conditions. Conclusion and recommendations In summary, we have demonstrated that the photophysical and biological properties of PEI-coated NaYF4:Yb3+:Er3+ nanoparticles (UCNP@PEI) are significantly changed by additional wrapping with single or double layers of poly(acrylic acid-co-maleic acid) sodium salt (PAMA) and/or various polysaccharides, such as dextran, chitosan, Na-salt dextran sulphate or sulfochitosan. We report on the Lb-L assembly of these novel-design particles, as well as the details of a new protocol to quantitatively compare the colloidal UCL brightness after surface modifications, and validate the use of ICP-MS for the precise measurement of the concentration of colloidal UCNPs. In our experiment, all additional coatings of UCNP@PEI decrease the relative intensity of the photoluminescence of the particles. At the same time, shielding of UCNP@PEI with additional surface coatings can be useful to tame the toxic effects of PEI on human epidermal cells, especially under long exposure times. Generally, the viability of the keratinocytes can be effectively protected for up to 120 hours by all additional external coatings studied in the current work, which shield or inverse the high cationic charge of UCNP@PEI. Thus, a compromise between acceptable brightness and cytocompatibility of UCNP@PEI is necessary, where UCNP@PEI coated with single additional layers of PAMA or sulfochitosan might be a good candidate. UCNP@PEI@PAMA and UCNP@PEI@Sch exhibit a high photoluminescence intensity in comparison to the other additional coating materials on the surface of UCNP@PEI, and increase the viable cell count for approximately 2040% as compared with UCNP@PEI under 24 h and 120 h exposures in a monolayer keratinocytes culture, respectively. Therefore the demonstrated strategies of surface modification of UCNP@PEI may


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be considered as efficient approaches to keep normal keratinocytes alive during the course of UCNPbased anti-cancer cytotoxic treatment (e,g., photodynamic therapy) in the nearby vicinity to the lesion. Further study of the subsequent layers L1 and L2 on the effectiveness of the particles as a transfection or drug delivery agent is recommended, in addition to the extent and the dynamics of internalization of the designed particles. We suggest that these experiments should take into account another important effect on the interaction of the nanomaterials with the cells, which is the formation of surface protein corona. Our preliminary research indicate a very strong and time-dependent interaction between UCNP@PEI and serum proteins, leading to a dense protein layer around the particles and a two-times accelerated nonspecific uptake63. The present study reveals the exceptional importance of rational surface design of UCNPs by careful exploring of their functional properties in the relevant biomedical context, including the cell type, the hydrophilicity of the environment and the exposure terms. We determined that each specific surface modification of as-synthesized UCNPs uniquely defines the intensity of photoluminescence signal as well as the degree of cytotoxicity of the particles. Our results show that increasing the biological safety of UCNPs may result in an accompanying loss in upconversion brightness, thereby decreasing the unique optical functionality of this probe. The extent to which the photoluminescent signal can be sacrificed will depend on the detection conditions of the desired application, therefore, a tailored trade-off between cytotoxicity and brightness is essential for successful clinical translation of the UCNP nanotechnology. Supporting information An electronic supporting information file accompanies this manuscript and contains further details on the ICP-MS validation study, confocal images and a table containing the detailed statistical analysis of the viability tests. Conflict of interest The authors declare that there is no conflict of interest regarding the publication of this paper.



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Acknowledgement The authors thank to Prof. N. Onischenko (V.I. Shumakov Federal Research Center of Transplantation and Artificial Organs, Russia) for providing us HaCaT keratinocytes. We gratefully thank Peter Wieland and the Geochemical Analysis Unit in Earth and Planetary Sciences (Macquarie University) for ICP-MS usage and support and Dr. Yiqing Lu for confocal imaging support. We acknowledge the Macquarie University Research Infrastructure Block Grants that supported the confocal system used in this work. E.G. thanks the Australian Research Council for funding (CE14010003 and DP140104458). A.N. thanks the NHMRC for funding (NHMRC ECF APP1124160). A.N.G. and E. K. acknowledge financial support from Russian Foundation for Basic Research [RFBR grant №17-03-01033]. A.V.N. would like to acknowledge Russian Science Foundation [RSF grant № 16-13-10528]. Authors’ contribution Anna E. Guller, Annemarie Nadort and Alla N. Generalova are equal first authors. References 1. Haase, M.; Schafer, H., Upconverting nanoparticles. Angew. Chem. Int. Ed. Engl. 2011, 50 (26), 5808-29. DOI: 10.1002/anie.201005159. 2. Zhou, J.; Liu, Q.; Feng, W.; Sun, Y.; Li, F., Upconversion luminescent materials: advances and applications. Chem. Rev. 2015, 115 (1), 395-465. DOI: 10.1021/cr400478f. 3. Chen, G.; Qiu, H.; Prasad, P. N.; Chen, X., Upconversion nanoparticles: design, nanochemistry, and applications in theranostics. Chem. Rev. 2014, 114 (10), 5161-214. DOI: 10.1021/cr400425h. 4. Nadort, A.; Zhao, J.; Goldys, E. M., Lanthanide upconversion luminescence at a nanoscale: fundamentals and optical properties. Nanoscale 2016. DOI: 10.1039/c5nr08477f. 5. Generalova, A. N.; Chichkov, B. N.; Khaydukov, E. V., Multicomponent nanocrystals with anti-Stokes luminescence as contrast agents for modern imaging techniques. Advances in Colloid and Interface Science 2017. DOI: 10.1016/j.cis.2017.05.006. 6. Xu, C. T.; Zhan, Q. Q.; Liu, H. C.; Somesfalean, G.; Qian, J.; He, S. L.; Andersson-Engels, S., Upconverting nanoparticles for pre-clinical diffuse optical imaging, microscopy and sensing: Current trends and future challenges. Laser & Photonics Reviews 2013, 7 (5), 663-697. DOI: 10.1002/lpor.201200052. 7. Hemmer, E.; Venkatachalam, N.; Hyodo, H.; Hattori, A.; Ebina, Y.; Kishimoto, H.; Soga, K., Upconverting and NIR emitting rare earth based nanostructures for NIR-bioimaging. Nanoscale 2013, 5 (23), 11339-61. DOI: 10.1039/c3nr02286b. 8. Wang, F.; Liu, X., Recent advances in the chemistry of lanthanide-doped upconversion nanocrystals. Chem. Soc. Rev. 2009, 38 (4), 976-89. DOI: 10.1039/b809132n. 9. Grebenik, E. A.; Nadort, A.; Generalova, A. N.; Nechaev, A. V.; Sreenivasan, V. K.; Khaydukov, E. V.; Semchishen, V. A.; Popov, A. P.; Sokolov, V. I.; Akhmanov, A. S.; Zubov, V. P.; Klinov, D. V.; Panchenko, V. Y.; Deyev, S. M.; Zvyagin, A. V., Feasibility study of the optical imaging of a breast cancer lesion labeled with upconversion nanoparticle biocomplexes. J Biomed Opt 2013, 18 (7), 76004. DOI: 10.1117/1.JBO.18.7.076004. 10. Chen, G.; Shen, J.; Ohulchanskyy, T. Y.; Patel, N. J.; Kutikov, A.; Li, Z.; Song, J.; Pandey, R. K.; Agren, H.; Prasad, P. N.; Han, G., (alpha-NaYbF4:Tm(3+))/CaF2 core/shell nanoparticles with efficient near-infrared to near-


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For table of content use only Title: Rational surface design of upconversion nanoparticles with polyethylenimine (PEI) coating for biomedical applications: better safe than brighter? Table of contents graphic

The study of additional surface modifications to polyethylenimine-coated upconversion nanoparticles reveals a compromise between their cytotoxicity and brightness.

Authors: Anna E. Guller1-3,9,*, Annemarie Nadort1,3,* (), Alla N. Generalova4,7*, Evgeny V. Khaydukov2,7, Andrey V. Nechaev5, Inna A. Kornienko6, Elena V. Petersen6, Liuen Liang1,3, Anatoly B. Shekhter2, Yi Qian1, Ewa M. Goldys1,3,9, and Andrei V. Zvyagin1-3, 8 1

Macquarie University, North Ryde 2109 NSW, Australia, 2Sechenov First Moscow State Medical

University, Russia, 3The ARC Centre of Excellence for Nanoscale BioPhotonics, Australia, 4Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry of the RAS, Russia, 5Institute of Fine Chemical Technologies, Moscow Technological University, Moscow, Russia, 6Moscow Institute of Physics and Technology, Russia,7Scientific Research Centre “Crystallography and Photonics” Russian Academy of Sciences, Russia, 8Lobachevsky Nizhniy Novgorod State University, Russia; 9University of New South Wales, Sydney 2032 NSW, Australia *Equally contributing co-authors Corresponding author: Annemarie Nadort, PhD, NHMRC Early Career Research Fellow E-mail: [email protected]


Rational Surface Design of Upconversion Nanoparticles with

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