Upconversion Colloid for Tracking Cellular Uptake of Nanoparticles

TL;DR

Researchers developed a new type of upconverting nanoparticle colloid that can be taken up by cells, opening possibilities for biomedical and environmental applications.

Contribution

A novel PEGylated upconversion colloid with enhanced colloidal stability and emission for in vitro cell uptake studies.

Findings

The UCC@Ale-PEG was successfully internalized by epithelial cells and macrophages.

Surface modification improved colloidal stability and upconversion emission.

The method combines high-temperature coprecipitation and hydrothermal treatment for nanoparticle synthesis.

Abstract

Upconverting nanoparticles, which transform low-energy infrared radiation into high-energy visible or UV light, show great potential in today’s technology. High-quality upconversion colloid (UCC) consisting of lanthanide-based nanoparticles with a diameter of ~10 nm was obtained using a combination of two processes: high-temperature coprecipitation and hydrothermal treatment in an autoclave. The UCC was then PEGylated with PEG-alendronate (PEG-Ale) to facilitate its dispersion in aqueous cell culture media intended for in vitro cell uptake assays. The surface modification of the nanoparticles increased both the colloidal stability in water and the upconversion emission by mitigating surface quenching. UCC@Ale-PEG was characterized by transmission and scanning electron microscopy, dynamic light scattering, and fluorescence microscopy detecting upconversion photoluminescence emission. The results of an in vitro assay revealed that this new generation of UCC can be internalized by various cell types, including epithelial cells and macrophages, upon several hours of exposure, suggesting broad application potential of this type of UCC in biomedicine, bioengineering, and environmental sciences.

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Full text

1. Introduction

Advances in the development of nanoparticle-based technologies currently often exploit photon upconversion, in which excitation by low-energy near-infrared radiation (NIR) results in the emission of high-energy visible or ultraviolet light due to anti-Stokes shift, offering great potential for many applications [1,2]. The mechanism of upconversion luminescence is quite complex and, depending on the type of particles, involves at least three basic processes: absorption of the excited state, photon avalanche, and energy transfer upconversion [3]. In this regard, lanthanide-based upconversion colloid (UCC) is particularly interesting. It consists of an inert host matrix (typically NaYF_4_) doped with a sensitizer (usually Yb^3+^, Nd^3+^) and an activator (e.g., Tm^3+^, Er^3+^, Ho^3+^). UCC has many advantages, the major one being that it has no background in biological tissues, and NIR light can penetrate relatively deep into the body [4]. In addition, it has no photobleaching, non-blinking, sharp emission bands, long photoluminescence lifetime, low photodamage to surrounding tissues, and tunable emission color [5].

Various approaches are being used for the synthesis of the UCC with controllable morphology of nanoparticles (i.e., shape and size), specific chemical composition, crystal structure, and optical properties [6]. In terms of size, the prevailing trend in manufacturing is to reduce it in order to facilitate internalization and diffusion of the nanoparticles into cells and tissues. In addition, if the size is <10 nm, the particles can be easily biodegraded or excreted from the body, but at the same time, they can be prone to surface quenching, which reduces the efficiency of upconversion luminescence [7,8]. Nevertheless, both biodegradation and excretion of particles also depend on their composition, surface charge, hydrophilic properties, etc. [9]. Common methods of UCC synthesis include thermal decomposition or coprecipitation of metal salts in high-boiling solvents in the presence of compounds with polar groups and long hydrocarbon chains (e.g., oleylamine and oleic acid), hydrothermal/solvothermal techniques, microwave heating, etc. [10,11,12]. Other techniques take advantage of the growth of a lanthanide shell around the seed [13]. However, particles prepared in organic solvents in this way are hydrophobic, undispersible in water, and therefore not suitable for biological applications. Moreover, despite their promising optical properties, bioapplications of UCC are limited by other challenges such as chemical and colloidal stability, resistance to aggregation and non-specific interactions, biocompatibility, reproducibility, and functionalization [14]. A significant concern relates to the dissolution of UCC nanoparticles in physiological aqueous environments, which is accompanied by the release of potentially cytotoxic fluoride and lanthanide ions [15]. For example, fluoride ions can inhibit mitochondrial activity, cell growth, protein synthesis, and proliferation in cultured human pulp cells [16].

It is therefore necessary to choose a suitable strategy for transferring hydrophobic UCC nanoparticles from the organic to the aqueous phase and to modify their surface using various ligands and polymer coatings of sufficient thickness [17]. Here, ligand exchange and conjugation with a number of different low- and high-molecular weight hydrophilic molecules and chelating agents, such as citric acid, poly(ethylene glycol) (PEG), poly(maleic anhydride-alt-1-octadecene)-PEG, poly(isobutylene-alt-maleic) anhydride-PEG, polyvinylpyrrolidone, poly(acrylic acid), polyethyleneimine, and encapsulation in a silica shell, have been described [18,19,20]. At the same time, it is important that the coating is not washed off the surface of the particles. Therefore, the polymer should contain effective anchoring groups that attach to the surface of UCC nanoparticles through non-covalent or covalent interactions; examples include bisphosphonate functional groups of PEG-alendronate [21].

Due to their remarkable physicochemical properties, upconversion nanoparticles are used in several industrial applications, as well as in the life sciences [22]. In the former case, these include photovoltaics (solar cells), displays, photocatalysis, security (anti-counterfeiting, fingerprinting), and barcoding. In nanomedicine, they include biosensing, theranostics, in vitro/in vivo bioimaging, drug delivery (photodynamic and photothermal therapy), and optogenetics [5]. Inspired by the potential of future biological applications, the aim of this study was to design, prepare, and characterize new UCC based on small (~10 nm) PEGylated NaYF_4_:Yb,Er nanoparticles and to monitor their internalization into different cell lines that differ in the extent to which they use specific endocytic pathways for the internalization of biological or nanoparticle cargo [23]. Another objective was to assess the detection limit, i.e., the lowest functional concentration of nanoparticles in UCC that can be determined from the upconversion luminescence signal. This approach should determine efficient uptake of the UCC in cultured cells as a key step towards assessment of future in vivo studies and applications of UCC.

2. Results

2.1. UCC

NaYF_4_:Yb,Er-based UCC was prepared using our newly developed hybrid strategy integrating high-temperature coprecipitation of lanthanide chlorides in a high-boiling solvent (octadec-1-en) with subsequent hydrothermal treatment in an autoclave at 310 °C. In order to significantly reduce the particle size, a controlled amount of water was added to the reaction mixture, with completion of the reaction in the autoclave. This approach ensured that water was retained in the feed even at a high temperature (310 °C) and elevated pressure and promoted the nucleation of primary particles at the expense of their growth. Ultrastructural analysis of UCC dried from hexane using TEM showed relatively well-dispersed nanoparticles of an approximately spherical shape with a high concentration in a monolayer with Dn = 9 nm and a dispersity Ð = 1.09 (Figure 1a,d), indicating a confined particle size distribution. The crystal structure of the UCC was analyzed by powder X-ray diffraction (PXRD) and the experimental pattern was compared with reference data from the International Center for Diffraction Data (ICDD) database (Figure 2). NaYF_4_, NaYbF_4_, and NaErF_4_ are known to crystallize in two polymorphs: the cubic α-phase (space group Fm–3m) and the hexagonal β-phase (space group P6_3_/m). These compounds are isostructural within each polymorphic modification, resulting in essentially identical PXRD peak positions (differences arise only from minor variations in lattice parameters due to ionic radius differences). For clarity, only the reference patterns of NaYF_4_ were shown. Comparison of the experimental pattern with the reference data indicated the coexistence of both polymorphs in the sample, with the cubic α-phase, which exhibits a lower upconversion signal than the hexagonal phase, being the predominant crystalline component. Dynamic light scattering (DLS) measurement of the UCC in hexane showed a hydrodynamic diameter Dh = 18 nm with polydispersity PD = 0.17 (Table 1). The relatively small hydrodynamic diameter of UCC in hexane confirmed the positive role of the residual oleic acid on the particle surface, which makes them well-dispersed in a nonpolar solvent, as the hydrophobic interactions minimize aggregation. The somewhat larger Dh value of the particles compared to Dn was ascribed to the fact that TEM measured the particles in a dry state, whereas DLS characterized them in a solvated state, which increased their size. DLS then revealed a pronounced difference in the hydrodynamic behavior of UCC depending on the dispersion medium. UCC in water exhibited Dh = 105 nm, which was significantly more than in hexane, indicating the tendency of unmodified nanoparticles to form aggregates in aqueous environments. Such aggregation arises from high surface energy and insufficient colloidal stabilization. Polydispersity PD = 0.19 then remained practically unchanged from that of UCC in hexane.

The upconversion luminescence spectrum of UCC excited by a 980 nm laser exhibited a small Er^3+^ emission peak at 408 nm attributed to the ^2^H_9/2_ → ^4^I_15/2_ electron transition (Figure 3). The incident photons were absorbed by Yb^3+^ ions at ^2^F_7/2_ ground state, pushing them to ^2^F_5/2_ excited state. After that, the energy was transferred to the neighboring Er^3+^ ions via energy transfer upconversion (ETU), populating ^4^I_11/2_ state due to matching excited energy states. This Er^3+^ level could also be populated by direct excitation from its ^4^I_15/2_ state by ground state absorption process. Additional energy transfer occurred from another Yb^3+^ to the Er^3+^, exciting higher ^4^F_7/2_ level of Er^3+^. The higher energy states ^4^Gj (J = 7/2, 9/2, and 11/2) of Er^3+^ were excited by multiphonon relaxation and energy transfer from Yb to Er. The red emission (^4^F_9/2_ of Er^3+^) was then produced by the back-energy transfer between ^4^Gj (Er^3+^) and a ground state of Yb^3+^ and Er^3+^ ions. The Er^3+^ ions finally decayed non-radiatively into luminescent states ^2^H_9/2_, ^2^H_11/2_, ^4^S_3/2_, and ^4^F_9/2_, which emitted at 408, 520, and 545 (both green), and 650 nm (red) [24]. The spectra clearly showed that UCC in hexane exhibited high emission intensity due to the fact that hexane is a nonpolar solvent without high-energy O–H vibrational modes. In contrast, in water, strong O–H stretching vibrations (at ~3400 cm^−1^) could effectively couple with Er^3+^ excited states, inducing multiphoton non-radiative relaxation and significantly quenching luminescence. In addition, in water, the residues of oleic acid on the surface of particles were removed, and the surface was susceptible to quenching and surface defects. In contrast, in hexane, oleic acid remained intact, preserving surface passivation and ensuring colloidal stability, which enhanced emission.

2.2. PEGylation of UCC

To render UCC colloidally stable in aqueous environments over a long-term period, it was PEGylated using PEG-Ale. TEM micrograph of UCC@Ale-PEG dried from water showed the dispersed nanoparticles with Dn = 11 nm and Ð = 1.13, confirming a relatively narrow particle size distribution (Table 1; Figure 1b,e). Based on particle size measurements, the PEGylated particles appeared to be ~2 nm larger than the starting particles.

Upon PEGylation of UCC, the Dh decreased to 90 nm with a low PD = 0.14, reflecting improved colloidal stability and reduced aggregation in water compared to unmodified UCC (Table 1). This can be ascribed to the PEG chains acting as a hydrophilic steric barrier that prevents close particle–particle contact, thereby narrowing the particle size distribution. Also, when UCC@Ale-PEG was transferred to the DMEM medium, the nanoparticles remained dispersed, which is important for further biological utilization (Figure 1c,f). The dispersibility of UCC@Ale-PEG also enabled analysis of these particles using HRTEM and selected area electron diffraction (SAED; Figure 1g–i), which confirmed that they retain a single-crystalline nature with a characteristic continuous lattice structure of each nanoparticle, similarly to previously reported particles [25]. Reduction in surface charge from 25 mV for neat UCC to 14 mV for UCC@Ale-PEG observed during ζ-potential measurements was consistent with partial shielding of surface lanthanide ions by conjugated PEG chains, which decreases the contribution of positively charged lanthanides to the electro-kinetic potential. Importantly, despite this reduction, the ζ-potential of PEGylated particles accompanied by steric stabilization contributed to preserving colloidal stability. A small difference in the emission intensity of uncoated and PEG-coated particles in water could be explained by the fact that the PEG-Ale shell partially shielded the UCC surface from contacts with water, thereby reducing non-radiative quenching by O–H vibrations. Moreover, PEG contributed to improved colloidal stability, preventing aggregation and excessive scattering.

2.3. Cell Uptake Assay of the UCC@Ale-PEG

In order to assess whether, and to what extent, the UCC@Ale-PEG is internalized into cells, we performed an in vitro cell uptake assay evaluated by fluorescence microscopy. Two complementary in vitro models were employed to investigate cellular interactions with UCC@Ale-PEG: human epithelial Caco-2 cells and mouse monocyte/macrophage RAW 264.7 cells. The cells were incubated with UCC nanoparticles for a given period of time prior to subsequent fixation, fluorescent labeling, and microscopy imaging to detect and localize UCC using their upconversion luminescence. Orthogonal views of confocal z-stacks of both UCC@Ale-PEG and uncoated UCC incubated with Caco-2 cells and Raw264.7 macrophages discriminated intracellular (internalized) particles from particles still adhered to the cell surface (extracellular; Figure 4 and Figure 5). The results confirmed that both types of cells internalized both PEGylated and uncoated UCC, although to various extents and with varying efficiency. A substantial proportion of particles remained on the cell surface after 16–24 h of incubation, whereby the pattern of the fluorescence signal, namely the size and intensity of the fluorescent spots, suggested that most of them were aggregates that were poorly internalized by endocytosis.

2.4. Detection Limit for Upconversion Emission

In order to assess the sensitivity of detection of UCC@Ale-PEG and determine whether upconversion emission can be tracked from single particles or only from their smaller aggregates or from intracellular endosomes—where they naturally accumulate upon internalization—the UCC@Ale-PEG detection limit assay was designed. A series of dilutions of UCC@Ale-PEG was prepared from the stock solution (4 mg/mL) and transferred in 1 µL drops onto glass coverslips, one uncoated for fluorescence measurement and the other coated with carbon for SEM analysis. The goal was to determine the dilution of UCC@Ale-PEG that provides the distribution of single particles on a coverslip with a spacing between particles > 1 µm (using SEM with a back-scattered electron detector), and then to assess whether single dots of emission can be detected in the same dilution using fluorescence microscopy. The results showed that a higher concentration of UCC@Ale-PEG formed an almost continuous monolayer of particles after drying on the coverslip surface, which was confirmed by SEM. The emission signal from this monolayer was significantly higher than the non-specific background signal from the regions of interest (ROI) on the coverslip that did not contain UCC used as a negative control (Figure 6).

Upconversion fluorescence emission spots were detected in each of the tested dilutions yet with intensities (peak gray values in the histogram) decreasing proportionally with the dilution and with the size of the spots (aggregates) until dilution of 1:1000 (4 µg/mL), where little upconversion signal (if any) was detected. The smallest discrete spots distinguishable by fluorescence had a diameter between 6 and 10 pixels (pixel size 86 nm) (Figure 6).

Analysis of corresponding dilutions by SEM revealed that separation of individual nanoparticles to ~1 μm spacing between particles was achieved in the sample diluted 1:1000 (4 µg/mL), resulting in ~3–4 nanoparticles per μm^2^. When the same dilution was analyzed by fluorescence (upconversion emission) within the ROI area comparable to the SEM image (6 μm × 8 μm rectangle), signal from individual UCC was not detected (Figure 6). We suppose that the bright spots detected by the fluorescence microscopy originated from small aggregates rather than from individual nanoparticles, but it was not possible to discriminate whether these aggregates formed during drying of the colloid on the glass surface or whether they were already present in the colloid.

3. Discussion

In this study, a new approach to the synthesis of very small upconversion particles was proposed, consisting of a combination of two synthetic procedures that allow control of particle size, dispersity, and crystallinity. Considerable attention was paid mainly to the significant influence of water added to the reaction mixture on particle size. It is known that under normal synthesis conditions at atmospheric pressure in a reaction vessel containing undried lanthanide precursors, an uncontrollable amount of water is present, originating from erbium chloride hexahydrate and a methanolic solution of NaOH and NH_4_F; the size of the resulting particles is then typically in the range of 20–30 nm [26]. In contrast, if the reactants are rigorously dried, particles > 100 nm are formed. The particles prepared by the hybrid method of high-temperature coprecipitation and hydrothermal treatment in an autoclave were thus much smaller than the upconversion nanoparticles obtained by common techniques, such as coprecipitation of lanthanide chlorides, thermal decomposition of lanthanide oleates, or microwave-assisted synthesis. The small size of particles obtained by this new hybrid method can be explained by the nucleation mechanism of particle formation. The nuclei formed during the above-mentioned hybrid approach were then in greater quantity, but smaller in size than when using conventional coprecipitation. Moreover, in the latter method, nuclei rapidly grew into mature particles at the expense of small ones due to Ostwald ripening.

An important aspect of the design of upconversion nanoparticles is their surface engineering, which must minimize the release of lanthanide ions into the surrounding environment, reduce toxicity, ensure biocompatibility, increase colloidal stability, and, last but not least, enable future binding of target biomolecules (drugs). Here, we decided to employ the widely used PEG-Ale, whose bisphosphonate groups readily complex with the lanthanide ions of the particles, thereby firmly anchoring the PEG to their surface [26]. The colloidal stability of UCC@Ale-PEG in water was dominated by PEG-induced steric effects rather than electrostatic repulsions. The resulting UCC@Ale-PEG conjugate was then used to monitor cellular uptake employing upconversion luminescence. The nature of the UCC@Ale-PEG and the method of its detection in cells by measuring emission from photon upconversion have important advantages. First, the emission is not dependent on pH, which drops significantly in the endosomal compartment when the late endosome (with internalized cargo) fuses with the lysosome; second, the particles do not undergo fast enzymatic degradation in endo-lysosomal compartments (due to their composition), nor proteolytic cleavage in the cytosol, and thus have essentially unlimited lifetime and fluorescence quantum yield.

Internalization of UCC@Ale-PEG into the cells is a prerequisite for its potential use as a long-term in vivo tracer and/or marker, as the particles must first cross one of the epithelial barriers to enter the body and are then spread by body fluids. Cells performing specialized functions, such as gut epithelial cells and macrophages, have exceptionally high uptake rates due to their role in nutrient absorption and immune defense, respectively. In this study, we used (i) Caco-2 cells, which are human gut tissue-derived epithelial cells undergoing differentiation into enterocytes and forming polarized monolayer in vitro mimicking the small intestine in the gastrointestinal system, and (ii) AW264.7 cells, which are mouse monocyte/macrophages commonly used as a model of inflammation in mice. Both cell types have also been recently utilized for internalization of nanoparticles and therapeutics [27,28,29]. However, they vary in the rate and mode of endocytosis critical for the efficiency of particle internalization, with Caco-2 cells being mainly effective in clathrin-coated endocytosis and macropinocytosis, while macrophages are characteristic for their phagocytic activity. Caco-2 cells were grown to high confluency and left to differentiate into stratified epithelium with the formation of an apical brush border, which mimics the intestinal epithelial barrier. Based on the efficient internalization of UCC@Ale-PEG by Caco-2 cells, it can thus be assumed that in vivo, where the intestinal barrier is the primary entry point into the body, UCC would be internalized to a similar extent. On the other hand, macrophages, which can cross the blood–brain barrier under certain conditions, represent a convenient model to study foreign particle transfer throughout the body and their potential accumulation in target organs (including the brain). RAW macrophages are very potent in phagocytosis, namely, the internalization (and digestion) of large solid objects, usually pathogens, foreign particles, or apoptotic bodies. This allows the internalization of even very large UCC@Ale-PEG aggregates to be monitored, which would otherwise not be internalized by classical endocytosis. Cell uptake assays confirmed that UCC@Ale-PEG can be internalized by various cell types and through different endocytic mechanisms used by these cells, opening up the possibility for future applications in more complex in vivo environments.

4. Methods and Materials

4.1. Materials

YCl_3_, YbCl_3_, ErCl_3_∙6H_2_O, octadec-1-ene, oleic acid, paraformaldehyde, Hoechst 33342 stain, and fetal bovine serum (FBS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). DMEM medium was obtained from Merck (Darmstadt, Germany), and N-hydro-xylsuccinimide-functionalized methoxy poly(ethylene glycol) (MeO-PEG-NHS; Mn = 5000 g/mol) was from Rapp Polymere (Tübingen, Germany). The sodium salt of 4-amino-1-hydroxy-1-phosphonobutyl phosphonic acid trihydrate (alendronate; Ale) was purchased from TCI (Tokyo, Japan). 4′,6-Diamidino-2-phenylindole (DAPI) and CellMask deep red were purchased from Thermo Fisher Scientific (St. Louis, MO, USA). Fetal bovine serum (FBS) was purchased from Biosera (Cholet, France). All other chemicals and solvents were from Lachema (Brno, Czech Republic). Deionized water was prepared using a Milli-Q IQ7000 system (Merck).

4.2. Synthesis of PEG-Alendronate (PEG-Ale)

PEG-Ale was obtained by modifying a previously published method [25]. A solution of sodium alendronate (0.64 g; 2.0 mmol) in phosphate-buffered saline (PBS; pH = 7.4) was cooled to 5 °C. MeO-PEG-NHS (1.0 g; 0.2 mmol NHS groups) was added to the solution with stirring, and the reaction mixture was maintained at 5 °C for 5 h to facilitate reaction between the NHS ester and the amino group of alendronate under formation of an amide bond. The resulting PEG-Ale was then purified by size exclusion chromatography using a Sephadex G-25 (Sigma-Aldrich) column with water as eluent and lyophilized.

4.3. Synthesis of UCC

The synthesis of UCC was performed in two stages. First, in a 100 mL three-necked flask, lanthanide chlorides (YCl_3_, YbCl_3_, and ErCl_3_·6H_2_O in the molar ratio of 0.78:0.20:0.02, respectively) were dissolved in octadec-1-ene (30 mL) and oleic acid (12 mL) at 160 °C for 30 min with stirring (350 rpm) under an Ar atmosphere. After cooling the reaction mixture to 40 °C, a methanolic solution (12 mL) of NaOH (2.5 mmol) and NH_4_F (4 mmol) was added dropwise. To evaporate the methanol and residual water, the temperature was gradually increased to 120 °C and then maintained for 30 min with stirring in an Ar atmosphere. In the second step, the mixture was cooled to 60 °C in the above atmosphere, transferred to an autoclave equipped with a magnetic stirrer, and 0.75 mL of water was added. The sealed autoclave was subsequently heated in the sand bath at 310 °C for 1.5 h with stirring at 350 rpm. After cooling to room temperature (RT), ethanol was added to the resulting UCC, the particles were separated by centrifugation (3460 rcf) for 30 min, and then redispersed in hexane.

For biological experiments, it was necessary to transfer UCC into water. This was achieved by gradually replacing hexane with ethanol (ethanol/hexane 1:3 and 2:3 v/v, and then ethanol alone) with centrifugation (3460 rcf) for 30 min after each step. The ethanol was then gradually replaced with deionized water using the same gradient procedure. Finally, the particles were washed twice with deionized water.

4.4. PEGylation of UCC

PEG-Ale (30 mg) was added to an aqueous UCC (1 mL; 22.2 mg particles/mL), the mixture was stirred at RT for 12 h, and dialyzed for 48 h against deionized water using a cellulose Spectra/Por^®^ membrane (MWCO = 100 kDa; Spectrum Laboratories; Rancho Dominquez, CA, USA) to remove unbound components. The resulting PEG-functionalized nanoparticles (UCC@Ale-PEG) were collected by centrifugation (3460 rcf) and resuspended in water.

4.5. Characterization Methods

Ultrastructural analysis of UCC by transmission electron microscopy (TEM) has been done as follows: first, the surface of grids for electron microscopy (carbon-coated copper 400 mesh—EMS #215-412-8400) was hydrophilized by glow discharge using Plasma HPT-100 cleaner (Henniker; Runcorn, UK), grids were then floated on a drop of a sample for 3 min, blotted and air-dried. TEM and HRTEM micrographs were acquired on a JEM2100-Plus transmission electron microscope (JEOL; Tokyo, Japan) operated at 200 kV using TemCam XF416 camera (TVIPS; Gauting, Germany). Selected area electron diffraction (SAED) was performed at 200 kV accelerating voltage, 10 μm SA aperture, and camera length set at 600 mm. The number-average (Dn), weight-average diameter (Dw), and dispersity (Ð) of at least 300 particles measured by ImageJ 1.52a software (National Institutes of Health; Bethesda, MD, USA) were defined as:

$$[eqn]$$ $$[eqn]$$ $$[eqn]$$

where ni and Di are the number and diameter of the particle, respectively.

Powder X-ray diffraction (PXRD) pattern was collected at RT using a high-resolution Anton Paar XRDynamic 500 diffractometer (Graz, Austria) with Advacam Pixos 2000 pixel detector (Prague, Czech Republic) utilizing Bragg–Brentano beam geometry under CuKα radiation (λ = 1.54 Å) in the 2θ range of 3–140° with a step of 0.01° and 300 s counting time at each step. The scattering vector q was defined by the following equation:

$$[eqn]$$

where λ = 1.54 Å is the radiation wavelength and 2θ is the scattering angle.

Ultrastructural analysis of UCC by scanning electron microscopy (SEM) was done as follows: first, a serial dilution of a sample was done in Milli-Q water, and 1 µL drops from each dilution were applied on a carbon-coated glass coverslip, air-dried, and mounted on SEM pins. SEM micrographs were acquired on Helios NanoLab 660 G3 UC apparatus (Thermo Fischer Scientific; Waltham, MA, USA) using a retractable high contrast solid-state backscatter electron detector.

Dynamic light scattering (DLS) using a ZSU 5700 Zetasizer Ultra (Malvern Instruments, Malvern, UK) provided hydrodynamic diameter Dh, polydispersity PD values, and ζ-potential for the nanoparticles.

Photoluminescence spectra were recorded using an FS5 spectrofluorometer (Edinburgh Instruments; Edinburgh, UK) equipped with a 980 nm CW laser and 2 W output power.

4.6. Cell Experiments

4.6.1. Cell Culture and Labeling

Cell lines Caco-2 (ATCC #HTB-37, Manassas, VA, USA), RAW264.7 (ATCC #TIB71, Manassas, VA, USA), and C6 (ATCC #CCL-107, Manassas, VA, USA) were cultured in DMEM medium enriched with 10% FBS supplemented with non-essential amino acids (Thermo Fisher Scientific), L-glutamine, and a mixture of antibiotics (penicillin, streptomycin, and amphotericin B from Merck). Cells were grown in a thermostat at 37 °C under 5% CO_2_ with medium changed twice a week.

For the cell uptake assay, Caco-2 cells were seeded at low density in 35 mm µ-dishes with a glass bottom coated with collagen. The cells were then grown to full confluence and cultivated for another two weeks to differentiate into enterocytes, forming a polarized monolayer. After that, cells were starved overnight in serum-free medium for synchronization of the cell cycle (G0/G1 phase arrest). The UCC@Ale-PEG was added in FBS-containing medium to trigger the uptake and incubated for 16 and 24 h. Cells were then washed with PBS, plasma membrane stained with CellMask deep red, DNA (cell nuclei) stained with Hoechst 33342, and finally fixed with 4% paraformaldehyde in PBS.

RAW264.7 cells were seeded in 8-well glass-bottom chamber (Ibidi µ-Slide) without coating (Gräfelfing, Germany). The UCC@Ale-PEG was added to the cells after media exchange and incubated for 16 and 24 h. Cells were then washed with PBS, stained with CellMask deep red (1 µg/mL), fixed with 4% paraformaldehyde in PBS, washed in PBS, and subjected to DNA (cell nuclei) staining by Hoechst 33342 (20 µM) followed by microscopy imaging.

4.6.2. Microscopy

Samples prepared from all three cell types were analyzed on Carl Zeiss LSM 880 NLO fluorescence optical microscope (Oberkochen, Germany) equipped with a tunable femtosecond laser Chameleon Ultra II (Coherent, Saxonburg, PA, USA). The UCC@Ale-PEG was excited with a focused 966 nm laser beam, pulse frequency 80 MHz, pulse width 320 fs, and average power 1 mW at the sample plane, using 40 × 1.1 NA water immersion objective. The bidirectional scanning was set to the lowest possible speed of 66 µs per pixel and 140 nm pixel size, with the pinhole fully open to allow the upconversion signal, emitted up to milliseconds after the excitation, to reach the confocal detector. The spectral emission profile of the upconversion signal was verified by lambda detection on 32 channels (8.8 nm per channel) in 410–690 nm range of GaAsP spectral detector operated in photon counting mode. For standard UCC@Ale-PEG detection, two-channel settings (540–570 nm and 640–670 nm), using the same detector in photon counting mode, were used. Due to the open pinhole, the axial resolution for UCC@Ale-PEG imaging was similar to a wide-field type of acquisition, which is several µm. CellMask deep red and Hoechst 33342 signals were one-photon excited with 633 or 405 nm lasers, respectively, and detected also on a spectral Zeiss GaAsP detector (Oberkochen, Germany) using channel mode, with a confocal pinhole size set to 1 Airy unit. The axial resolution for DAPI and CellMask deep red corresponded to the confocal mode, which was ~1 µm. UCC@Ale-PEG and DAPI with CellMask deep red images were acquired in frame (2D experiment) or stack (3D experiment) sequential scanning mode. Orthogonal projections from confocal z-stacks were used to determine the precise localization of UCC@Ale-PEG within cells and to discriminate them from surface-bound particles.

5. Conclusions

Previous studies have pointed to limitations in the size control of the prepared UCC, which was typically in the range of 30–160 nm [30]. In this report, upconversion colloid based on small PEGylated NaYF_4_:Yb,Er nanoparticles (~10 nm) was designed using a newly developed method combining coprecipitation and hydrothermal treatment in the presence of water in an autoclave. The small size resulted from increased nucleation during the particle formation process. The reduction in size of these nanoparticles, together with their high dispersibility in aqueous media, facilitated endocytosis by cells. The narrow particle size distribution ensured identical and reproducible physicochemical properties, including improved colloidal stability. The colloid was characterized not only in terms of particle size and size distribution using TEM and DLS, but also by characteristic upconversion fluorescence. Modification of the UCC surface with PEG-Ale improved upconversion emission in water by reducing surface quenching. The results then demonstrated that the dispersion medium and surface functionalization critically affected the colloidal stability of UCC. While UCC containing oleic acid residues on the particle surface remained well dispersed in nonpolar hexane, it tended to aggregate in polar water. PEGylation mitigated this problem by providing hydrophilic steric stabilization, reducing both hydrodynamic size and polydispersity, and enabled the use of such particles for biological applications that mostly require an aqueous environment.

Finally, two model cell lines were selected to characterize UCC–cell interactions and the internalization of nanoparticles in a context mimicking the intestinal epithelial barrier (Caco-2) and tissue-patrolling immune cells (Raw264.7). Differences between epithelial cells and macrophages reflected distinct endocytic and phagocytic mechanisms, which are highly relevant for the rational design of nanoparticle-based diagnostic or therapeutic platforms. In particular, uptake by macrophages highlighted the relevance of this system for modeling interactions with the innate immune system, including nanoparticle clearance and intracellular fate, which is critical for assessing biocompatibility and translational potential of nanomaterials.

In a prospective study, it will be interesting to assess whether co-incubation and/or internalization of UCC@Ale-PEG by cells has any long-term impact on their viability or growth rate. It will also be important to determine the fate of intracellular UCC after its internalization. Does it simply accumulate in lysosomes, undergo some form of degradation, or is it expelled from cells by exocytosis as an undegradable substance? Ultimately, experiments should be transferred to in vivo models in order to assess the mechanism, kinetics, and efficiency of nanoparticle internalization, transport, and accumulation in tissues and organs, as well as their long-term fate in the organism. It is the unique nature of UCC@Ale-PEG, namely its colloidal stability, fluorescent properties, high photostability, and NIR excitation, which can significantly facilitate these studies by enabling sustained cell labeling and signal tracking with minimal background fluorescence. We anticipate that colloids with these characteristics will be particularly suitable for applications in nanomedicine, such as in vivo bioimaging, sensing, NIR-activated photodynamic therapy, or intracellular drug delivery.