Volume 6, Issue 11 e202200158
Research Article
Open Access

Compartmentalization Accelerates Photosensitized NADH to NAD+ Conversion

Roland E. P. Nau

Roland E. P. Nau

Institute of Inorganic Chemistry I, Universität Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany

These authors contributed equally to this work.

Contribution: Data curation (equal), Formal analysis (equal), ​Investigation (equal), Methodology (equal), Writing - original draft (equal), Writing - review & editing (equal)

Search for more papers by this author
Julian Bösking

Julian Bösking

Institute of Inorganic Chemistry I, Universität Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany

These authors contributed equally to this work.

Contribution: Data curation (equal), Formal analysis (equal), ​Investigation (equal), Methodology (equal), Writing - original draft (equal), Writing - review & editing (equal)

Search for more papers by this author
Jun.-Prof. Dr. Andrea Pannwitz

Corresponding Author

Jun.-Prof. Dr. Andrea Pannwitz

Institute of Inorganic Chemistry I, Universität Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany

Contribution: Conceptualization (lead), Funding acquisition (lead), Resources (lead), Supervision (lead), Writing - original draft (equal), Writing - review & editing (equal)

Search for more papers by this author
First published: 01 August 2022
Citations: 1

Graphical Abstract

Top speed: Acceleration of reaction rates by one order of magnitude was achieved through compartmentalization of the photosensitizer and the substrate nicotinamide adenine dinucleotide (NADH) in liposomes. Differences in reaction dynamics were found for two types of photosensitizer incorporation, probably due to local concentrations and the diffusion behavior of singlet oxygen. These findings might be relevant in the context of reaction engineering and artificial cellular compartments.


Confinement of reaction spaces was achieved in a biomimetic manner by using liposome vesicles that are based on phospholipid bilayer membranes, similar to cellular compartments. Encapsulation of photosensitizer (PS) and substrate within the inner aqueous compartment of liposomes accelerated the photosensitized model reaction of nicotinamide adenine dinucleotide (NADH) conversion to its oxidized form (NAD+) by one order of magnitude compared to classical homogeneous reaction conditions. Furthermore, it was found that the reaction proceeds around 40 % faster when the photosensitizer is dissolved in the inner aqueous compartment instead of being embedded within the phospholipid bilayer which is attributed to the diffusion behavior of singlet oxygen which acts as oxidant in this reaction. These experimental findings will allow for reaction and molecular systems design for photochemical and catalytic conversions and will be relevant in the context of creating artificial cellular compartments such as fully artificial chloroplasts.


In nature, biochemical and chemical reactions take place in spatially confined cellular compartments, whereby compartmentalization of reaction spaces is achieved by lipid bilayers forming membranes that span up intracellular organelles, vesicles and cellular membranes.1-3 Such compartmentalization is advantageous for biochemical cellular processes as it allows for local fine tuning of reaction conditions and reagent concentrations as well as the encapsulation of toxic species to protect the organism. Furthermore, enclosing chemical reagents in a confined reaction space generally accelerates diffusion-based processes due to increased rates of collisions in confined environment, especially at high local concentrations. Translating the concept of compartmentalization for artificial systems can be highly beneficial as demonstrated by light-independent chemical reactions including enzymatic conversions,4-7 and some light-driven reactions in compartmentalizing water droplets,8 polymersomes,9 light-responsive micelles,10 and at the gas-liquid interphase of bubbles in water.11 Compartmentalization within phospholipid based liposome vesicles has also been investigated for photochemical conversions at the membrane-water interface,12-17 but systems that employ light driven reactions within the inner aqueous compartment are scarce.18-20 However, light-driven reactions are typically collision-based and therefore oftentimes limited by the excited state lifetime of the photoactive species as effective encounters need to happen within the excited state lifetime typically ranging from picoseconds to microseconds in solution and within liposomal membranes.16, 21, 22

We hypothesized, that by enclosing the light absorbing photosensitizer (PS) and the model substrate NADH in the inner aqueous compartment of phospholipid-based vesicles (liposomes) we would accelerate the reaction rate of light-driven reactions. The model reaction of choice was the conversion of NADH to NAD+ via the reactive oxygen species singlet oxygen (1O2) generated by photosensitization with the PS tris(2,2’-bipyridine)-ruthenium(II) chloride (RuC0) or bis(2,2’-bipyridine)-(4,4’-dinonyl-2,2’-bipyridine)-ruthenium(II) hexafluorophosphate (RuC9). RuC0 as a chloride salt is water soluble and was previously used as a PS in photochemical singlet oxygen sensitization reactions under typical homogeneous reaction conditions.24 RuC9 is an amphiphilic compound which integrates into the phospholipid bilayer of liposomes (see Scheme 1).22 By testing these two PS we assessed the differences of photochemical reaction dynamics in the inner aqueous phase (RuC0) as well as the interface of lipid bilayer and water (RuC9). Furthermore, we anticipated that the poorly water-soluble oxygen and singlet oxygen required for the reaction diffuses across the vesicle membrane and is available for sensitization and the follow-up NADH oxidation reaction within the vesicle.

Details are in the caption following the image

The investigated photochemical model reaction: Photosensitized singlet oxygen generation leads to NADH to NAD+ conversion. Under classical bulk conditions this photochemical conversion is slow, while it is fast under compartmentalized conditions in liposome vesicles (representation is not scaled: the lipid bilayer is around 3 nm thick23 while the diameter is around 145 nm according to DLS data in this study). PS=RuC0 and RuC9.

Results and Discussion

The here employed liposomes consisted of a 100 : 1 mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and the stabilizing lipid 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] ammonium salt (14:0 PEG 2000 PE) and were prepared by lipid film deposition and follow-up hydration with 10 mM phosphate buffer. If applicable, the hydrophobic RuC9 was added during lipid film generation, while the water-soluble RuC0 and NADH were dissolved in buffer and added during the hydration step. Extrusion and purification via size exclusion chromatography yielded monodisperse liposomes with hydrodynamic diameter of around 145 nm, as determined via dynamic light scattering (DLS). The purification via size exclusion was necessary to remove excess NADH and, if applicable, RuC0 outside of the liposomes but yielded different absolute amounts of liposomes within each sample batch. To be able to compare the performance of photosensitized NADH conversion under confinement with the classical bulk experiments the relative amounts of RuCn and NADH (RuCn : NADH ratio) were calculated for each liposome and bulk sample as described in the Supporting Information on page S7–S9 along with detailed sample preparation procedures. The light-driven reaction of NADH → NAD+ was performed by irradiation with an LED light source at 465±26 nm (39.1 mW/cm2 average power at the sample). DLS measurements prior and after the photochemical experiments yielded similar size distributions for each reaction, which indicated that the liposomes stayed stable during the photochemical reaction (representative data are shown in the Supporting Information on page S10). The generation of singlet oxygen by the photosensitizers was proven via NIR-emission spectroscopy (vide infra and Supporting Information on page S21–22) and the proposed generation of the H2O2 as an additional product (Scheme 1) was verified via UV-vis absorption spectroscopy and is reported in the Supporting Information page S23. NADH conversion was monitored via emission and UV-vis absorption spectroscopy. In most experiments the light-induced oxidation of NADH to NAD+ was close to complete within the investigated time and according to emission spectroscopy. Pseudo-turn over numbers (pseudo-TON) based on the photosensitizer, similar to catalysis experiments, can be directly retrieved from the respective inverted RuCn : NADH ratio.

The reaction kinetics of the overall reaction was monitored via temporal evolution of the NADH fluorescence emission band at 462 nm (Figure 1A) which was in agreement with the vanishing of the individual NADH absorption band at 337 nm (exemplary data see Supporting Information, Figure S11). We want to mention here, that the emission spectra have the advantage, that scattering does not affect the spectrum as much as the absorption spectrum (see Figure 1 and Figure S11 and references22, 26). All decays were fitted monoexponentially (see Figure 1B and Supporting Information page S12–20) using eq. 1 with the preexponential factor A, the rate constant k and the baseline adjustment y0.
Details are in the caption following the image

(A) Emission spectra of RuC9-liposomes with encapsulated 56 mM NADH solution at RuC9 : NADH=1 : 100 (λex=300 nm) after irradiation at 465 nm for different time intervals. (B) NADH conversion as function of irradiation time under different conditions for RuCn : NADH=1 : 100 including experiments with NADH in absence of a photosensitizer, the RuCn-liposomes and bulk experiments using RuC0 as photosensitizer. (C) NADH conversion rate constants k as a function of RuCn : NADH ratio and linear fits (please note that the x-axis is logarithmic to properly display small Ru : NADH ratios). Experimental conditions: Irradiation was performed with an LED at 465 nm. Local NADH concentration within RuCn-liposome was 56 mM and overall concentration of NADH in RuCn-liposome experiments was about 0.1 mM. Bulk experiments were always conducted in the presence of RuC0.

The emission band at around 630 nm in Figure 1A is attributed to the phosphorescence emission of the PS and stayed constant during all irradiation experiments, indicating the photostability of the PS during the experiments.

To elucidate the role of compartmentalization, we tested several reaction conditions at several NADH concentrations and with varying RuCn : NADH ratios in classical bulk conditions as well as in RuC0- and RuC9-liposomes. It was found that the encapsulation of the PS and the NADH substrate yielded consistently one order of magnitude faster reaction rates compared to the classical bulk experiments which is visually summarized in Figure 1 and Table 1 for the representative data sets RuCn : NADH ratios 1 : 100 and 1 : 25 and resolved in in the following.

Table 1. Reaction rates k of light-driven NADH conversion under various conditions with exemplary data of a RuCn : NADH ratios of 1 : 100 and 1 : 25, as well as the change of k depending on the RuCn : NADH ratio urn:x-wiley:23670932:media:cptc202200158:cptc202200158-math-0002 .




RuCn : NADH=1 : 100

RuCn : NADH=1 : 25

urn:x-wiley:23670932:media:cptc202200158:cptc202200158-math-0003 /min−1












RuC0 in bulk, 0.1 mM NADH[a]





RuC0 in bulk, 1 mM NADH[b]





RuC0 in bulk, 56 mM NADH[c]



  • [a] Same overall conc. of NADH as in liposome experiments; [b] Same NADH bulk concentration as reported in bulk experiments25; [c] Same NADH bulk concentration as within the liposomes.

There are the following explanations for such large acceleration under compartmentalization conditions: (i) High local concentrations of the substrate and photosensitizer, (ii) possible follow-up oxidation reactions involving the reactive oxygen species H2O2 which is generated during oxidation with singlet oxygen (see scheme 1), and (iii) elimination of reaction bottle necks such as inner filter effects and oxygen solubility (vide infra) which are limiting in the bulk experiment and are discussed in the following.

To benchmark our NADH conversion experiments with reported bulk experiments we performed the photoinduced NADH conversion under homogeneous conditions as reported by Mengele et.al. who worked with a PS : NADH ratio of 1 : 500 using PS=Ru(tbbpy)32+ (tbbpy=4,4’-di-tert-butyl-2,2’-bipyridine) at 1 mM NADH concentration.25 While the reported rate constant is 0.032±0.002 min−1,25 we obtained a slightly smaller rate constant of k=0.021±0.003 min−1 using PS=RuC0 under otherwise identical conditions which we attribute to the electronic differences in PS properties.

Increasing the RuC0 : NADH ratio in this bulk experiment at constant NADH concentration (1 mM NADH) accelerated the reaction rate constant k to a minor extent according to the linear relationship k=m ⋅ (ratio RuCn : NADH) with a slope m=urn:x-wiley:23670932:media:cptc202200158:cptc202200158-math-0004 0.8±0.6 min−1 (Figure 1C, Table 1 entry 4). Working at lower NADH concentration in the bulk experiment (0.1 mM NADH) yielded overall concentrations comparable to the liposome experiments. Increasing the RuC0 : NADH ratio accelerated the reaction rate according to urn:x-wiley:23670932:media:cptc202200158:cptc202200158-math-0005 3.1±0.1 min−1 (Figure 1C, Table 1 entry 3), making it twice as fast at RuC0 : NADH=1 : 25 compared to 1 mM NADH conditions. This relationship is counterintuitive, as encounter-dependent reactions typically are faster (and not slower) at increased substrate concentration. We attribute this inverted effect to higher concentrations of PS at the same PS : NADH ratio which entails inner filter effect of the colored PS.27 Furthermore, oxygen concentration in the aqueous bulk solution is only 0.28 mM at 20 °C and normal pressure28 which is an excess for the 0.1 mM bulk experiment and indicates for the 1 mM experiment that additional oxygen needs to diffuse into the solution.

Adding ‘naked’ liposomes to the 0.1 mM NADH solution and RuC0 in the bulk at Ru : NADH=1 : 25 yielded a conversion rate constant of 0.16±0.02 min−1 which is similar to the purely homogeneous experiments (see Supporting Information page S20). On the other hand, encapsulation of NADH into liposomes at an overall NADH concentration of 0.1 mM yielded a 21- and 15-times faster reaction rate constant for RuC0 and RuC9-liposomes respectively at RuC0 : NADH=1 : 25 (Table 1, Figure 1C). This significant acceleration in both liposome systems can only be explained by the high local concentration of the NADH and the PS within the inner aqueous liposome compartment (cNADH,local=56 mM) which increases the chance of encounters between freshly generated singlet oxygen molecules and NADH molecules. However, by translating these local reaction condition to respective bulk experiments with cNADH=56 mM we obtained extremely low reaction rates of 0.3±0.3 min−1 which is comparable with the photodegradation of NADH in absence of a PS, underlining the limiting effect of oxygen due to its low solubility. It also highlights the beneficial effect of compartmentalization at high local concentration within liposomes, especially when working with a poorly water-soluble gas as co-reagent.

Despite their similarly beneficial effects, the consistently faster reaction rates within RuC0 vs. RuC9-liposomes are astonishing: Upon incorporation of the PS via the two hydrophobic C9 alkyl chains into the lipid bilayer in RuC9-liposomes the reaction rate constant k dropped by factor 1.5±0.2 compared to similar experiments to the water soluble RuC0 in RuC0-liposomes. Furthermore, by increasing the RuCn : NADH ratio the reaction rate constant for RuC0-liposomes accelerates by factor 1.4±0.2 as compared to RuC9-liposomes with membrane anchored PS (urn:x-wiley:23670932:media:cptc202200158:cptc202200158-math-0006 is 64±2 min−1 vs. 46±3 min-1, see table 1). These results were counterintuitive, because RuC9 had a 10 % higher quantum yield for singlet oxygen generation compared to RuC0 in deuterated organic solvent (Φ1O2,RuC9=0.80±0.03). Both compounds produced singlet oxygen in liposomes with deuterated buffer (see Supporting Information page S22), and singlet oxygen is reported to be more stable in DPPC as compared to water. The latter was anticipated from the reported and much longer mean free diffusion pathlength of singlet oxygen of 900 nm in DPPC as compared to 209 nm in water, or generally 10–155 nm in biological environments.29-33 Expanding the diameter of the liposomes by about 50 % for RuC0 liposomes shows no effect to the conversion rate vs. the Ru : NADH ratio (see Supporting Information page S18–19). We consider the following explanations for such different reaction dynamics of RuC9-liposomes vs. RuC0-liposomes: (i) Anchoring of large amounts of PS in a lipid bilayer membrane can lead to unproductive deactivation pathways of the photoexcited states. These include inner filter effects and triplet-triplet annihilation deactivation.16, 34 However, the amounts of singlet oxygen produced in both liposomes seems to be similar, with no significant differences according to our measurements (see Supporting Information page S22). (ii) The solubility of oxygen within the hydrophobic DPPC-bilayers at ambient temperature is reported to be factor 3 lower compared to water,32, 35 which might reduce the local concentration of oxygen around the PS in the case of RuC9-liposomes where the PS is located at the interface of membrane to water. However, we did not observe significant differences in singlet oxygen production in liposomes. However, we did observe that singlet oxygen can be generated though photosensitization within the respective other aqueous compartment and can diffuse across the membrane. This was observed in an experiment where we encapsulated only NADH within the liposomes and added water soluble RuC0 to the outer aqueous bulk of the solution and observed NADH conversion (see Supporting Information page S17). This leaves the last explanation as the main contribution to the differences in reaction dynamics: (iii) RuC9 is located directly within the membrane, which is the very outmost location of the encapsulated reaction space. Approximately half of the singlet oxygen generated at close proximity to the membrane will therefore diffuse into the bulk and is hence unavailable for the chemical reaction within the liposome.


In summary, we demonstrated the acceleration of the photosensitized oxidation of NADH to NAD+ by one order of magnitude (factor 21 or 15) through encapsulation of NADH and the singlet oxygen producing PS into liposomes. Furthermore, we have found, that the reaction proceeds 1.4±0.2 times faster when the PS is dissolved in the inner aqueous compartment as compared to the PS being anchored within the liposome membrane. As a major benefit to both systems we identified the high local concentration of the PS and NADH within the vesicles, as well as the availability of the poorly water-soluble oxygen gas which acts as a reagent in this reaction and can diffuse across the membrane from the aqueous bulk into the inner part of the liposomes. All in all, these experiments show that the confinement of reactants into the inner aqueous compartments of liposome is highly beneficial for accelerating reaction rates of light-driven multicentered chemical reactions. Based on these results we propose as a design principle for future molecular systems engineering to compartmentalize the water-soluble substrate and photosensitizer at high local concentration. Anchoring of photosensitizer in the compartmentalizing matrix (here lipid bilayer) can yield less high reaction rates but holds all advantages of compartmentalization in addition to the benefits of an immobilized active component which can be easily recovered and recycled. In addition, these biomimetic compartments provide a model system for an artificial cellular compartment which might be relevant in the context of artificial cellular compartments such as artificial chloroplasts.


This research was funded by the Deutsche Forschungsgemeinschaft (DFG, project TRR234 “CataLight” project number 364549901, project B8). The authors thank Alexander Mengele for scientific discussion and Sven Rau for access to the spectrometers. Open Access funding enabled and organized by Projekt DEAL.

    Conflict of interest

    The authors declare no conflict of interest.

    Data Availability Statement

    The data that support the findings of this study are available in the supplementary material of this article.