Intracellular Fate of the Photosensitizer Chlorin e4 with Different Carriers and Induced Metabolic Changes Studied by 1H NMR Spectroscopy

1. Introduction

The chlorophyll-based porphyrinic derivatives of chlorin e6 (Ce6) are efficient photosensitizers (PSs) in photodynamic therapy (PDT) of cancer and non-cancerous diseases. PDT relies on the activation of PS by light irradiation, upon which the excited PS transfers energy to molecular oxygen, which leads to the formation of cytotoxic singlet oxygen and reactive oxygen species (ROS) in its immediate surroundings [1]. Among the favorable properties of Ce6 derivatives is their intense light absorption in the wavelength region of 600–700 nm [2,3], where tissue penetration of light is deeper than at shorter wavelengths [4]. Several Ce6 derivatives have received approval for specific applications of cancer [2,5], and the sodium salt of chlorin e4 (Ce4, Figure 1A) has been recently patented for use in PDT of different cancers, including ovarian cancer under the trademark Photosoft [6,7].

Figure 1. (A) Structure of the photosensitizer chlorin e4 (Ce4); (B) building block of the polymeric network formed by polyvinylpyrrolidone (PVP, MW_av 10 kDa), *: repeated building blocks [ ]_n; (C) polyethylene glycol (PEG)—polypropylene glycol (PPG) triblock copolymer PEG-PPG-PEG Kolliphor P 188 (KP) and a sketch of a KP-micelle.

With the development of so-called third-generation PSs [5], the use of nano-platforms for drug delivery has become an essential part of PDT to improve PS efficiency [8]. Nanoparticle (NP)-based carriers can improve the solubility and bioavailability of the often lipophilic and aggregating porphyrinic PSs [8]. Further, NP surface modifications like polyethylene glycol (PEG) can improve PS stability during transport in the blood by reducing PS removal through macrophages or the reticuloendothelial system [9,10]. Based on their large size, NPs can accumulate in tumor tissue via the enhanced permeability and retention (EPR) effect of the tumor vasculature that is more permeable for macromolecules than normal tissue [11]. Finally, the NP surface offers possibilities for chemical modifications aiming at specific targeting [12]. Cell entry of NPs normally takes place via endocytic pathways, where the specific route is determined by the cell type and by the nature of the NP [13]. Physicochemical properties of NPs, like size, shape, chemical composition, and surface charge, determine cell uptake and intracellular trafficking [13,14,15,16]. Among the various materials applied for nano-sized delivery systems, polyvinylpyrrolidone (PVP, Figure 1B) [17,18,19] and block copolymer micelles (BCMs, Figure 1C) [20,21,22] consisting of polymers with PEG and polypropylene glycol (PPG) blocks of different sizes and molecular weights (PEG-PPG-PEG, Pluronics) have gained much interest as carriers for PSs [8,23,24,25,26,27]. Recently, we have shown that Ce6-derived PSs, including Ce4, are well-encapsulated into PVP and into biodegradable BCMs formed by Kolliphor P188 (KP, Figure 1C) [28,29,30,31]. Both carriers stabilize the chlorin in an aqueous solution by preventing aggregation and maintaining its photophysical properties, and the uptake of Ce4 into HeLa cells could be proved by fluorescence microscopy [31].

Both PS (Ce4) and NP (PVP, KP) are biocompatible and have very low dark toxicity [17,20,31,32], which is required to avoid side effects while at the same time ensuring a controlled cytotoxic effect of the PS that is directed by light irradiation for localized therapy. Typically, the dark toxicity of PSs is assessed under the exclusion of light by common cytotoxicity assays like the Alamar Blue [33,34] or MTT test (using a methyl-thiazol-tetrazolium dye) [34,35] that are based on the activity of cellular enzymes. However, the perturbation of the physiologic state of the cells, i.e., their metabolic response toward treatment with PSs in the dark, is rarely studied. Similarly, the role of the nano-sized polymeric PS-delivery system must be evaluated and better understood: How does the carrier affect the biocompatibility, overall cellular homeostasis, and molecular processes triggered by the PS? How does the nature of the carrier modulate these processes? In vitro model studies using cultured human cells are suitable and easily accessible to address the intracellular fate of PS in the dark and its polymeric carrier [36,37]. Interactions of drug delivery systems with cells encompass their cell uptake pathways, intracellular distribution, and induction of specific cell metabolite alterations. Understanding these key processes allows us to assess their in vivo tolerability, toxicity, and efficacy in transporting their payloads to the target [36].

High-resolution magic angle spinning (HR-MAS) NMR spectroscopy can be used to study intracellular small molecules giving rise to ¹H NMR signals with atomic resolution. Moreover, the metabolic response of cultured living cells to interventions like drug treatment or specific growth conditions can be addressed [38,39,40]. Whole-cell spectra are evaluated for qualitative alterations and quantitative, altered metabolite ratios based on multivariate analysis. This metabolomic approach is an emerging technique applied in nanotoxicological studies to identify alterations in endogenous small metabolites induced by NPs [41]. It has already been proven as an efficient tool for nanotoxicological investigations employing NMR analysis of cell extracts [42,43] and culture media to determine the extracellular metabolites [44] or HR-MAS NMR of whole cells [45].

PVP and KP micelles represent two delivery systems with different chemical compositions and structures, which in turn modify the dynamic properties of the corresponding systems, including Ce4 release and reactivity toward proteins [31]. In this study, we aim to address the question of how PVP and KP-micelles affect the intracellular fate of Ce4 and the metabolic response of cancer cells following treatment with Ce4 alone or encapsulated into the carriers. For this, HR-MAS NMR spectroscopy was applied to cultured HeLa cells following incubation with the different test media in the dark. The results allow us to assess the extent of physiological interference of the PVP and BCM-based carriers as well as the porphyrinic PSs, that are non-toxic in the dark, on a molecular or even atomic level. Knowledge of the metabolic perturbation will help predict the in vivo tolerability of Ce4 or related PSs, including the impact of PVP or BCM carriers in the dark during the accumulation period preceding PDT.

2. Materials and Methods

2.1. Chemicals

Ce4 was purchased from Frontier Scientific (Logan, UT, USA). PVP (average MW = 10 kDa) and Kolliphor P188 (KP, average MW = 8.4 kDa) were obtained from Sigma-Aldrich (Buchs, Switzerland). The deuterated solvents DMSO-d6 (99.95%) and D₂O (99.9%) were purchased from Cambridge Isotopes Laboratories, Inc. (Andover, MA, USA). Phosphate-buffered saline (PBS, 50 mM, pH = 7.3) was prepared by mixing aliquots of 50 mM solutions of KH₂PO₄ and Na₂HPO₄ (provided by Sigma-Aldrich, Buchs, Switzerland) in H₂O or D₂O containing 0.9% NaCl.

2.2. Cell Culture

The human HeLa cervical cancer cell line was kindly provided by the group of Prof. Mühlemann, University of Bern. For cell culture, phenol red-free Dulbecco’s Modified Eagle Medium (DMEM) was prepared by diluting DMEM powder (D5030-1L, Sigma) in 1 L distilled water and adding L-glutamine, sodium bicarbonate, D-glucose, and sodium pyruvate (all Sigma-Aldrich, Buchs, Switzerland). The growth medium was supplemented with 10% fetal calf serum (FCS), 100 μg/mL streptomycin (S), and 100 IU penicillin (P, DMEM+/+). The cells were grown at 37 °C in 5% CO₂ under a humid atmosphere. Trypsin-EDTA (T/E) with 0.5 g/L trypsin (1:250) and 0.2 g/L EDTA·4Na, FCS, P, and S was obtained from Amimed (BioConcept, Allschwil, Switzerland). The culture medium was sterile-filtered through 0.1 µm polyethersulfone (PES) membranes (Nalgene™ Rapid-Flow™, Thermo Fisher Scientific, Fisher Scientific AG, Reinach, Switzerland).

2.3. Cellular Uptake by Flow Cytometry

Relative quantitative uptake of Ce4 alone and combined with PVP and KP into HeLa cells was measured by applying flow cytometry.

Time-dependent uptake curves were recorded using the Amnis Image Stream^®X Mark II imaging flow cytometer (Luminex Corporate, Austin, TX, USA). HeLa cells were incubated with loading media containing either no additives (controls) or 5 μM Ce4 with and without PVP (16.7 μM) or KP (3 mM) for 0.5 h, 1 h, 2 h, and 3 h. The incubation was stopped by removing the loading media, followed by washing, trypsinization, and resuspension of the cells in DMEM+/+. After the addition of PSs, all sample preparation steps were performed under the exclusion of light. Laser excitation was performed at λ = 405 nm (2 mW) and fluorescence detection at λ = 642–745 nm in addition to bright-field and side-scattering detection. Data were processed using IDEAS^® 6.2 software (Amnis Corporation, Seattle, WA, USA). On a subpopulation of single spherical cells, the normalized (by number of pixels) mean fluorescence intensity per cell was determined, and for all sample classes, the mean fluorescence intensity of all events (8000–10,000 cells) for each class and incubation time was plotted as a function of time.

Dose-dependent uptake was recorded using a SORP LSR II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and FACSDiva software, Version 8.0.1 (BD Biosciences). HeLa cells were incubated with loading media containing either no additives (controls) or Ce4 at concentrations of 10 μM, 25 μM, and 62.5 μM each with and without PVP (208.3 μM) or KP (3 mM) for 2 h. All samples were prepared in triplicate. After incubation, the cells were excited as described above for the time-dependent uptake curves. A laser with a wavelength of 407 nm was used for excitation, and the Brilliant Violet filter (660/20 nm, BV650) was used to measure fluorescence emission. Ten thousand events were recorded and replicated for each sample condition. Data analysis was performed using Flowing Software (version 2.5.1, Turku Bioscience, Turku, Finland). Side- and forward-scattering areas were measured to identify intact single cells.

2.4. Sample Classes for 1H HR-MAS NMR Spectroscopy

For the ¹H HR-MAS NMR spectroscopic study, HeLa cells were incubated with “loading media”, i.e., FCS- and P/S-free culture media (DMEM−/−) containing (i) Ce4 (10 μM), (ii) Ce4 (25 μM), (iii) Ce4 (62.5 μM), (iv) Ce4-PVP (10 μM, 208.3 μM), (v) Ce4-PVP (25 μM, 208.3 μM), (vi) Ce4-PVP (62.5 μM, 208.3 μM), (vii) only PVP (208.3 μM), (viii) Ce4-KP (10 μM, 3 mM), (ix) Ce4-KP (25 μM, 3 mM), (x) Ce4-KP (62.5 μM, 3 mM), (xi) only KP (3 mM), and (xii) and (xiii) pure PBS as controls (two times). Each of the samples (i–xiii) was individually prepared from one culture flask in triplicate, resulting in 39 (3 × 13) cell samples in total. The sample groups and their labels are summarized in .

2.5. Preparation of the Loading Media i–xiii

For the loading media, the following stock solutions were prepared: 2 mM Ce4 in PBS (H₂O) containing 10% DMSO, 60 mM KP in PBS (H₂O), and 20.83 mM PVP in PBS (H₂O). Aliquots of these stock solutions were mixed with DMEM−/− culture medium to yield final concentrations of Ce4 (10 µM, 25 µM, 62.5 µM), KP (3 mM), and PVP (208.3 µM). Since Ce4 required 10% DMSO for dissolution to prepare the stock solution, the same amount of DMSO was also added to all other samples. Each loading medium contained the same final amount of 10.9% PBS and 0.3% DMSO, below the toxic DMSO concentration for HeLa cell cultures [46,47]. The final concentration of KP (3 mM) had to be chosen such that it was above the critical micelle concentration (cmc) of KP to ensure the existence of KP-micelles (0.45 mM in PBS [29]). In the case of PVP, the concentration of 208.3 µM was selected to ensure sufficient loading capacity for the highest Ce4 concentration of 62.5 µM corresponding to a molar ratio of 3:10 (Ce4:PVP) [31]. The loading media were sterile-filtered through 0.1 µm PES membranes (Nalgene™ Rapid-Flow™, Thermo Fisher Scientific) before incubation.

2.6. Preparation of Cell Samples for 1H HR-MAS NMR Spectroscopy

Initially, 3 batches of HeLa cells (labelled “a”, “b”, and “c”) were grown in six 75 cm² culture flasks in 10 mL DMEM+/+ to approximately 80% confluence to provide enough cell material for 13 samples (i–xiii) per batch. For each sample, HeLa cells were seeded at ~0.75 × 10⁶ cells/mL in 25 cm² flasks using 5 mL (~3.75 × 10⁶ cells per sample) DMEM+/+ and allowed to grow for 24 h. Subsequently, the culture medium was removed, and the cells were washed with PBS (H₂O). Beginning with the addition of the loading media, the cell culture samples and all work-up processes were strictly excluded from light exposure to avoid phototoxic reactions of the PS. For this, the culture flasks and Falcon tubes were wrapped with alumina foil and the light was turned off in the laboratory during cell handling. After adding 5 mL of loading medium, the cells were incubated for 2 h at 37 °C in a 5% CO₂ atmosphere. Then, the loading medium was removed, the cells were washed with PBS (H₂O), and they were harvested by trypsinization. The cells were transferred to 15 mL Falcon tubes and centrifuged at 2000 rcf for 5 min. The supernatant was removed, and the cell pellet was washed three times with PBS (H₂O), finally resuspended in 20 µL D₂O-based PBS, and transferred to a black Eppendorf vial. To stop the metabolism, cells were lysed by three alternating cycles of ultrasonication (45 s) and submersion into liquid nitrogen (15 s) [48]. Frozen samples were stored at −80 °C until measurement.

On the day of use, the cell sample was thawed within 2 min and heated for 20 min at 70 °C to inactivate enzymes and ensure stability during the measurement time [48]. Finally, the lysed and heated cell suspension (~5 × 10⁶ cells in PBS-D₂O, ~50 mg) was transferred to a 50 μL zirconium oxide MAS rotor and the rotor was briefly centrifuged (~5 s) to avoid any air bubbles before closing it with a PTFE Teflon insert and KelF rotor cap. The amount of sample inside the rotor was controlled by weight.

2.7. 1H HR-MAS NMR Spectroscopy–Data Acquisition

All HR-MAS NMR experiments were performed on a Bruker Avance II spectrometer (Bruker BioSpin, Fällanden, Switzerland) operating at a resonance frequency of 500.13 MHz for ¹H nuclei. The instrument was equipped with a 4 mm HR-MAS dual inverse ¹H/¹³C probe (Bruker BioSpin) with a magic angle gradient. The samples were inclined around the magic angle (54.7°), spun at 5000 Hz, and the temperature was set to 273 K (nominal). Data acquisition was performed with the Bruker software TopSpin (version 3.2, patch level 5). All samples were measured in random order, and the time between thawing and the start of the acquisition was kept constant (47 min ± 8 min). For each sample, the following spectra were recorded: (i) T₂-edited ¹H spectra using the 1D PROJECT (Periodic Refocusing Of J Evolution by Coherence Transfer [49]) pulse sequence with water presaturation (“projectpr1d”) and a T₂ filter of 400 ms to suppress broad components with short T₂ relaxation times. In total, 512 transients were accumulated for each 1D ¹H spectrum, applying a spectral width of 6009.6 Hz, a data size of 32 k points, an acquisition time of 2.73 s, and a relaxation delay of 4 s. (ii) ¹H spectra using a 1D NOESY (Nuclear Overhauser Enhancement SpectroscopY) pulse sequence (“noesygppr1d” from the Bruker pulse-program library) with spoil gradients for water suppression. In total, 256 transients were acquired over a spectral width of 5000 Hz with a data size of 32 k points, a mixing time of 10 ms, an acquisition time of 3.28 s, and a relaxation delay of 4 s. (iii) 1D ¹H diffusion edited spectra applying the 1D DOSY (Diffusion Ordered SpectroscopY) stimulated echo pulse sequence (“ledbpgppr2s1d” from the Bruker pulse-program library) with longitudinal eddy current delay of 5 ms, bipolar gradients for diffusion, 2 spoil gradients, and presaturation of the water resonance. Each spectrum was recorded with 256 transients, a spectral width of 6009.6 Hz, a data size of 32 k points, and a relaxation delay of 2 s. To suppress small fast diffusing metabolites, a gradient was applied at 40% of the maximal gradient strength corresponding to 13.5 G/cm, applying a sine-shaped gradient. The diffusion time Δ was set to 50 ms and the gradient length δ to 6 ms.

On selected samples, 2D DOSY spectra (“ledbpgppr2s” from the Bruker pulse-program library) were acquired using the same parameters as for the 1D DOSY spectra applying an incremented linear gradient ramp from 5 to 95% over 64 steps with 16 scans per increment.

To support resonance assignments, 2D ¹H¹H TOCSY (TOtal Correlation SpectroscopY) spectra were acquired on a representative subset of samples applying the DIPSI2 pulse sequence (“dipsi2phpr” from the Bruker pulse program library) with presaturation of the water resonance. For the TOCSY experiment, 128 transients were acquired with a data size of 2048 × 256 (F2, F1) over a spectral width of 5000 Hz in both dimensions (F2, F1). The relaxation delay was set to 1.2 s, and the TOCSY mixing time to 74.8 ms.

2.8. Processing of 1H HR-MAS NMR Spectra

Spectral processing was performed using Bruker Topspin software (version 3.5b, patch level 7). For the 1D PROJECT and 1D NOESY spectra, exponential multiplication of the FIDs with a line-broadening factor of 1.0 Hz was performed, followed by phasing and baseline correction. The baseline offset was corrected (zero-order polynomial) for the whole spectral range, and for the 1D PROJECT spectra, a 5th-order polynomial baseline correction was also applied to the spectral region between 5.1 ppm and 10 ppm. Chemical shifts were referenced to the -CH₃ resonance of creatine (Cre) at δ = 3.03 ppm. For the 1D DOSY spectra, an exponential multiplication of the FIDs with a line broadening factor of 5 Hz was applied. The spectra were phased, baseline corrected (zero-order polynomial over the whole spectral range), and referenced to the -CH₃ resonance of choline (Cho) at δ = 3.208 ppm. 2D DOSY spectra were processed using the Bruker software “Dynamics Center” (version 2.6.2) by fitting the peak intensities of selected peaks as a function of gradient strength with a bi-exponential fitting function. The TOCSY data were processed by zero filling (to 512) in F1 and using shifted squared sine window functions in both dimensions prior to 2D Fourier transformation.

Assignment of the resonances was based on ¹H¹H TOCSY spectra , spectral databases (Human Metabolome Database, HMDB [50] and in-house database), and literature data [45,51,52,53,54].

2.9. Data Analysis

Multivariate analyses of the 1D PROJECT spectra were performed using MATLAB R2019b (The MathWorks, Inc., Natick, MA, USA), PLS Toolbox (version 8.8.1, Eigenvector Research, Inc., Manson, WA, USA), and Excel (Microsoft Office 365 ProPlus, Redmond, WA, USA).

The 39 ¹H HR-MAS NMR spectra were subdivided in the spectral range between 0.8 and 9.5 ppm into 259 individually sized buckets (integral regions), excluding regions with noise, with the residual water resonance (4.8–5.25 ppm), ethanol resonance, and PEG-CH₂ and PPG-CH₃ resonances of KP (1.11–1.2 ppm and 3.62–3.74 ppm). Bucket selection was performed on an overlay of all spectra, followed by integration in the corresponding regions using TopSpin software. The integrals were imported into PLS-Toolbox for further analysis of the 39 × 259 data matrix. To account for differences in sample amounts, probabilistic quotient normalization (PQN) [55] was applied to the integrals of each sample. Furthermore, preprocessing included mean centering and Pareto scaling. The data were subdivided into 12 classes according to (i)–(xii) (see Section 2.4), with the six control samples (xii, xiii) belonging to one class. Unsupervised principal component analysis (PCA) was performed to probe for sample clustering. Partial least squares discriminant analysis (PLS-DA) was used to calculate a model for distinguishing the spectra based on the assignment to 12 classes. Orthogonal partial least squares analysis (oPLS) was applied to calculate a regression model for predicting concentration-dependent metabolite alterations. Cross-validation for PCA, PLS-DA, and oPLS was performed by Venetian blinds with 10 splits and a blind size of 1. Model statistics (root mean square error of calibration/cross-validation, RMSEC/RMSECV) and classification errors were calculated for PCA and PLS-DA. Validation metrics R² and Q² were calculated for the linear oPLS regression model. Univariate analysis was performed on single metabolites (representative buckets/integrals) in Excel.

3. Results and Discussion

3.1. Cell Uptake of Ce4 and the Polymeric Carriers PVP and KP

To quantify the cellular uptake of Ce4 depending on its formulation, i.e., either in its free form or encapsulated into PVP or KP micelles, HeLa cells were measured by flow cytometry following treatment with the corresponding samples added to the culture medium. For this, the intrinsic fluorescence intensity of Ce4 associated with the HeLa cells was recorded. Both time-dependent uptake for a fixed Ce4 concentration of 5 μM (Figure 2A) and concentration-dependent uptake during a fixed time of 2 h (same Ce4 concentrations as applied in the HR-MAS NMR study, Figure 2B) were measured. Ce4 uptake by HeLa cells was time-dependent and marginally leveled off after approximately 2–3 h. It was slightly reduced when Ce4 was encapsulated in PVP and distinctly reduced in the presence of KP micelles (Figure 2A). This reduction was also found for the Ce4 concentration range of 10–62.5 μM when applied with carriers (Figure 2B). In the presence of PVP or KP, cell uptake increased with Ce4 concentration without reaching saturation and was always lower than Ce4 in its free form, except for Ce4-PVP at 62.5 μM Ce4.

Figure 2. Chlorin e4 (Ce4) uptake into HeLa cells measured as intrinsic fluorescence following addition of Ce4 alone (blue), encapsulated into polyvinylpyrrolidone (PVP, green), or Kolliphor P188 (KP, red): (A) as function of incubation time (5 μM Ce4, 16.7 μM PVP, 3 mM KP) and (B) as function of Ce4 concentration (208.3 μM PVP, 3 mM KP, 2h incubation).

In the absence of carriers, a fluorescence drop was observed at the highest Ce4 level (62.5 μM). At this concentration, chlorin aggregation becomes prevalent, leading to pronounced fluorescence quenching [31] so that the measured fluorescence intensity no longer correlates with the actual intracellular Ce4 concentration. In contrast, these aggregation and quenching effects are prevented through the encapsulation of Ce4 into PVP or KP, where it exists in the monomeric state [31].

Reduced cellular drug uptake from Pluronic micelles has been previously reported [31,56] and discussed in detail with respect to a single incubation time [31]. Possible causes for a reduced uptake of PEGylated lipid NPs by tumor cells have been related to steric hindrance and blocking of ligands at the cell surface through PEG chains. These drawbacks accompanying the advantages of PEGylated lipid NPs were denoted as the “PEG-dilemma” [57,58]. Under in vivo conditions, the disadvantage of reduced cell uptake is partly compensated by the enhanced stability and prolonged lifetime of the drug-loaded NPs during the transport in the bloodstream. This is mainly due to the stealth effect of the PEG corona preventing the opsonization process [59] and the EPR-mediated accumulation of PEGylated NPs in tumor tissue [11]. However, these effects are not reflected in the in vitro cell culture model.

3.2. The Metabolic Profile of Treated and Untreated HeLa Cells: T2-Edited 1H HR-MAS NMR Spectra

In Figure 3, a summed T₂-filtered (PROJECT) ¹H HR-MAS NMR spectrum of all lysed HeLa cell suspensions in PBS is shown. Typically, with the relaxation filter, underlying broad peaks deriving from large molecules like proteins, lipids, or polysaccharides are suppressed, facilitating the analysis of small molecules that give rise to sharp resonances. Each single spectrum provides a metabolic snapshot of the cells following their different exposures to the various test media (i–xiii). A total of 53 compounds could be identified, which are summarized in , along with their abbreviations, chemical shifts, and multiplicities. The most prominent peaks originate from cell metabolites such as Lac, mobile lipids (Lip), the choline-containing compounds Cho, PC, and GPC, Cre, and several amino acids like Gln, Leu, Ile, and Val. The spectral region between 5 ppm and 10 ppm that typically exhibits resonances of much lower intensity is dominated by contributions from nucleosides, nucleotides, and related components such as UDP- and UTP derivatives. Among those, the nucleotide sugars UNGlc, UNGal, and UGlcA (most likely overlapping with UGlc) are assigned based on the resonances deriving from their anomeric protons around 5.6 ppm with the characteristic multiplet structure [52]. The assignment of the purine-derived metabolites Ino and Ado remains ambiguous since the chemical shift values of the two single purine resonances are hard to distinguish (annotation Ino/Ado). In addition, there is a pronounced pH dependence in the chemical shifts of adenine-derived nucleotides (AXP), as previously discussed [53]. In the current study, AXP, most likely AMP [53,60], and UMP were only observed in the spectra of samples treated with Ce4. Furthermore, among the compounds listed in , some were only detectable in the presence of KP or without applying a T₂ filter (see Section 3.3 below).

Figure 3. ¹H high-resolution magic angle spinning NMR summed spectrum (T₂-filtered) of lysed HeLa cell suspension in phosphate-buffered saline with resonance assignments. Spectral regions of 5.2–7.0 ppm and 7.0–9.6 ppm were scaled up by a factor of 4 and 8, respectively. Ac: acetate; Ala: alanine; β-Ala: β-alanine; Asp: aspartate; AXP: adenosine phosphate; Cho: choline; Cit: citrate; Cre: creatine; Eth: ethanol; FA: fatty acids; For: formic acid; Fum: fumarate; Gln: glutamine; Glu: glutamate; Gly: glycine; GPC: glycerophosphocholine; GSH: glutathione; His: histidine; Hxn: hypoxanthine; Ile: isoleucine; Ino/Ado: inosine/adenosine; Lac: lactate; Leu: leucine; Lip: lipids; Lys: lysine; 1-MNA: 1-Methylnicotinamide; NA: nicotinamide; PC: phosphocholine; PEG: polyethylene glycol; Phe: phenylalanine; Pro: proline; R5P: ribose-5-phosphate; sFA: fatty acids (saturated); Suc: succinate; Tau: taurine; Thr: threonine; Tyr: tyrosine; TyrP: tyrosine-containing peptide; UDP: uridine diphosphate; UGlc(A): UDP-glucose/UDP-glucuronic acid; UNGal: UDP-N-acetyl-galactosamine; UNGlc: UDP-N-acetyl-glucosamine; Ura: uracil; Urd: uridine; UTP: uridine triphosphate; Val: valine.

3.3. The Metabolic Profile of Treated and Untreated HeLa Cells: Diffusion-Edited 1H HR-MAS NMR Spectra

In 1D ¹H DOSY, small metabolites with a larger diffusion coefficient can be suppressed by adjusting the diffusion time and gradient strength during acquisition so that only the broad resonances of slowly diffusing macromolecular components remain visible. The effects of T₂ relaxation and diffusion filters on the ¹H NMR spectra are visualized in Figure 4 and and contrasted to unfiltered 1D NOESY spectra covering both sharp and broad components .

Figure 4. (A) HR-MAS ¹H 1D diffusion-edited NMR spectra (scaled up by a factor of 16) and (B) HR-MAS ¹H-T₂-edited (PROJECT) NMR spectra of lysed HeLa cell suspensions in phosphate-buffered saline (PBS). Cells incubated with PBS as controls (Ctrl, black), Kolliphor P188 (KP, red), and polyvinylpyrrolidone (PVP, green). The polyethylene glycol (PEG)-CH₂ resonance was only visible in cells incubated with KP and is highlighted in red. (C) HR-MAS ¹H 2D DOSY spectrum of a cell suspension incubated with KP; 1D diffusion- and 1D T₂-edited projections are shown in red. The PEG-CH₂ resonance corresponds to a slow and a fast diffusing component (highlighted in red). Cho: choline; Cre: creatine; GPC: glycerophosphocholine; LysP: lysine-containing peptide; PC: phosphocholine; PEG: polyethylene glycol; PLC: phosphatidylcholine.

3.4. Metabolic Analysis of Cell Spectra: Impact of Ce4, PVP, and KP

To probe the impact of Ce4 alone or encapsulated into PVP or KP micelles onto the metabolic profile of cultured HeLa cells in the dark, cells were incubated under the exclusion of light for 2 h with three different Ce4 concentrations (10 μM, 25 μM, and 62.5 μM) in pure PBS, PVP, or KP each at a constant polymer concentration in PBS. The polymers without Ce4 were also included, and cells incubated with pure PBS added to the culture media served as controls. This resulted in 12 sample classes measured in triplicates and labeled as Ce4-10, Ce4-25, Ce4-62.5, KP, KP-Ce4-10, KP-Ce4-25, KP-Ce4-62.5, PVP, PVP-Ce4-10, PVP-Ce4-25, PVP-Ce4-62.5, and Ctrl . The spectra were subdivided into individually sized bucket regions covering single resonances as much as possible.

3.5. Limitations

While HR-MAS NMR has the potential to study a wide range of semi-solid materials with restricted mobility and short T₂ relaxation times, larger structures entering a more solid-like phase are no longer observable by this technique. For example, most cellular proteins give rise to very broad lines, rendering them NMR-invisible under HR-MAS conditions. In the current study, the lack of PVP resonances in the cell HR-MAS NMR spectra indicates that PVP becomes part of a larger complex, possibly endosomal membranes, most likely leading to severe line broadening, thus causing its NMR invisibility. Nevertheless, the presence of intracellular PVP was indicated indirectly by its attenuating impact on Ce4 effects on the cell metabolite spectra.

Cell uptake pathways, intracellular fate, and metabolic responses of PS-carrier systems may turn out differently depending on the cell type [15]. Therefore, it will be of interest in future studies if the observed metabolic alterations described here for HeLa cells will be similarly transferable to other cell lines. Further, the application of serum-free incubation media, a commonly applied method in metabolomic cell intervention studies [88], excludes the impact of proteins during cell uptake. However, serum proteins may bind to the NP outer surface, possibly resulting in modifications of cell uptake and response. Finally, the complete exclusion of light may not be guaranteed at a 100% level, so subtle effects may be due to oxidative processes triggered by light. However, in the current experimental setup, dead cells were excluded, and the different incubation media were all applied simultaneously under the same conditions. In addition, metabolic antioxidant cell defense mechanisms in response to oxidative stress mainly involve alterations in the GSH/GSSG and NAD/NADH or NADP/NADPH levels [89], which was not observed in the current study.

4. Conclusions

In the current study, HR-MAS NMR was applied to cells exposed to different PS concentrations with and without two different carriers, namely block copolymer micelles and PVP, under the exclusion of light.

Addressing the intracellular fate of the components, the NMR data allow the following conclusions: (i) based on induced chemical shift changes of the cell-PLC resonance, the PS localizes in or near cellular membranes when applied in its free form and when associated with carriers. (ii) Once taken into the cells, the PS remains largely associated with PVP and is less tightly bound to the micelles. This was deduced from the observation that PS-induced effects were more strongly attenuated by PVP than by the KP-micelles. (iii) The micellar carrier material is cell-internalized, as was evidenced by the appearance of a PEG resonance in the cell spectra. (iv) The micellar components partly disassemble inside the cell since the PEG resonances arose from components with different diffusion properties. (iv) Intracellular PVP could only indirectly be traced via its attenuating impact on the PS-induced changes in cell metabolites. The current approach thus has offered the unique possibility to trace both the PS and the block copolymer inside the cell on a molecular and even atomic level. To our knowledge, this is the first time intracellular sub-molecular PS membrane localization has been detected via ring current-induced chemical shift perturbation of the phospholipid resonances. At the same time, examination of the small metabolite cell spectra and their multi- and univariate analyses indicated the metabolic response to the PS and carrier material under the exclusion of light.

Addressing the metabolic response of the cells, the NMR data allow the following conclusions: (i) the pure carrier material, PVP and KP-micelles, exhibits low toxicity based on the largely unchanged cell metabolic profile. (ii) The physiologic state of the cells was noticeably perturbed when exposed to the pure PS and correlated with the PS dose. (iii) The metabolic response to PS treatment was clearly attenuated when the PS was associated with KP-micelles and even more with PVP. (iv) Comparing PVP and KP-micelles, it can be delineated that, in general, carriers with a stronger binding capacity for the drug molecule are capable of reducing unwanted physiological perturbations more efficiently, thus maintaining cell homeostasis. This is an important aspect that may be extrapolated to other drugs that are likewise applied with comparable nano-platforms. In particular, for porphyrinic PSs, it is crucial to keep any activity or correspondence with their cellular environment as low as possible during the dark incubation and accumulation period before the selective phototoxic reaction is triggered by light irradiation. In this sense, the carrier not only fulfills the task of protecting the PS during its transport in the bloodstream and thus enhances PS accumulation in the diseased tissue but also protects the intracellular environment from the PS. Thus, potential side effects and dark toxicity can be further lowered. For the development of PS-based therapies, the presented data underline the importance of using carriers of physiologically inert material with high drug affinity to prevent premature PS release, reduce unwanted side effects, and thus improve patient compliance. Ideally, a PS remains inactive by its protecting carrier envelope in non-target tissue until cleared from the body. At the same time, photoactivity testing is crucial to obtain systems optimized for both dark conditions and upon light irradiation.

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