2.1.

Chemicals

The cationic diporphyrin-based 5,15-bis-[4-(3-Trimethylammonio-propyloxy)-phenyl]-porphyrin (also referred to herein as XF73) with a molar mass of $M=765.81\mathrm{g}/\mathrm{mol}$, including the counter ion, was synthesized by Xiangdong Feng (Solvias Company, Basel, Switzerland) and kindly provided by Destiny Pharma Ltd. (Brighton, United Kingdom).

The 5,10,15,20-Tetrakis(1-methyl-4-pyridinio)-porphyrin tetra(p-toluenesulfonate) (also referred to herein as TMPyP) with a molar mass of $M=1363.63\mathrm{g}/\mathrm{mol}$, purity 97%, ${\mathrm{NaN}}_3$ sodium azide, Mannitol, NaCl, KCl, ${\mathrm{Na}}_2{\mathrm{HPO}}_4$, ${\mathrm{KH}}_2{\mathrm{PO}}_4$, and ${\mathrm{D}}_2\mathrm{O}$ have been purchased by Sigma Aldrich (Taufkirchen, Germany), and were used as received. The photosensitizers (PSs) were dissolved in bi-distilled water at a stock concentration of 1 mM and stored at 4°C until use. Figure 1(a) shows the chemical structure of XF73 and TMPyP.

Fig. 1

(a) Chemical structures of the porphyrins XF73 and TMPyP. (b) Normalized emission spectrum of the Waldmann-UV236 lamp. Absorption spectrum of XF73 and TMPyP with a concentration of ${10}^{-5}\mathrm{M}$ each.

Phosphate-buffered saline (PBS; PAA Laboratories GmbH, Pasching, Austria) at pH 7.4 has been used for aggregation experiments and contains NaCl (0.14 M), KCl ($2.7\times {10}^{-3}\mathrm{M}$), ${\mathrm{Na}}_2{\mathrm{HPO}}_4$ ($1.0\times {10}^{-2}\mathrm{M}$), and ${\mathrm{KH}}_2{\mathrm{PO}}_4$ ($1.8\times {10}^{-3}\mathrm{M}$). For the NMR spectroscopy, a parent solution of the PSs dissolved in ${\mathrm{D}}_2\mathrm{O}$ was made and a PBS solution for dilution containing ${\mathrm{D}}_2\mathrm{O}$ has been prepared by adding NaCl, KCl, ${\mathrm{Na}}_2{\mathrm{HPO}}_4$, and ${\mathrm{KH}}_2{\mathrm{PO}}_4$ with the accordant concentrations.

2.2.

Absorption Spectrum

Absorption spectra were recorded at room temperature with a spectrophotometer (DU640, Beckman Instruments GmbH, Munich, Germany) in a concentration range of $1\times {10}^{-6}\mathrm{M}$ to $2\times {10}^{-3}\mathrm{M}$. The percentaged transmission has been measured and the absorption cross-section $\sigma ({\mathrm{cm}}^2)$ was calculated according to Eq. (1):

2.3.

Photostability

The PSs were irradiated with an incoherent broadband lamp (UV236; emission $\lambda =380$ to 480 nm) provided by Waldmann Medizintechnik (Villingen-Schwenningen, Germany). The maximal light intensity was $15.2\mathrm{mW}{\mathrm{cm}}^{-2}$ at the level of the irradiated samples. The samples were irradiated for either 15 min ($13.7\mathrm{J}{\mathrm{cm}}^{-2}$) or 60 min ($54.8\mathrm{J}{\mathrm{cm}}^{-2}$). The emitted spectrum of the light source was recorded with a spectrometer (270 M, Jobin Yvon, Longjumeau, France) with 300 grid lines/mm and a spectral resolution of approximately 0.4 nm [Fig. 1(b)]. The detection range was 350 to 650 nm. The recorded spectral data were corrected regarding the spectral sensitivity of the spectrometer. The emission spectrum of the Waldmann UV lamp was normalized to its maximum between 400 and 450 nm.

2.4.

Cell Experiments

The C. albicans strain ATCC-MYA-273 was used for the experiments. The planktonic cells of C. albicans were diluted to a number of ${10}^6$. For the incubation of C. albicans, the PSs stock solution has been diluted with ${\mathrm{H}}_2\mathrm{O}$. The cells were incubated with a PS concentration of ${10}^{-4}\mathrm{M}$ in the dark for 15 min in ${\mathrm{H}}_2\mathrm{O}$ plus 50% PBS in falcons at slow rotation. The cells were rinsed twice with PBS to remove the not included or nonadherent PSs and afterward dissolved in pure ${\mathrm{H}}_2\mathrm{O}$. For the singlet oxygen luminescence experiments, the planktonic cells were excited with a frequency doubled Nd:YAG-Laser (PhotonEnergy, Ottensoos, Germany).

2.5.

Fluorescence Spectrophotometer

The localization of XF73 in C. albicans was examined by fluorescence microscopy (Zeiss Vario-AxioTech, Goettingen, Germany) with an appropriate dual-band filter set for excitation and emission (Omega Optical, Brattleboro, Vermont) and a $63\times$ magnification. Planktonic C. albicans were incubated 2 h with ${10}^{-4}\mathrm{M}$ XF73 in PBS and were rinsed twice with PBS.

2.6.

Singlet Oxygen Luminescence and Quantum Yield of ${}^1{\mathrm{O}}_2$ Formation (${\mathrm{\Phi }}_{\mathrm{\Delta }}$)

Solutions with PSs were filled in a cuvette (QS-101, Hellma Optik, Jena, Germany) and solutions of the planktonic cell suspension were investigated in acrylic cuvettes (SARSTEDT, Nümbrecht, Germany), both during magnetic stirring. The PSs were excited with a frequency doubled Nd:YAG-laser (PhotonEnergy, Ottensoos, Germany) with a wavelength $\lambda =532\mathrm{nm}$, power output $P=50\mathrm{mW}$, frequency of $f=2\mathrm{kHz}$, and therefore, energy per pulse of $E=2.5\times {10}^{-5}\mathrm{J}$. Every sample was irradiated with 40,000 pulses. The C. albicans planktonic cells were excited with a frequency doubled Nd:YAG-laser (PhotonEnergy, Ottensoos, Germany) with a wavelength $\lambda =532\mathrm{nm}$, power output $P=60\mathrm{mW}$, frequency of $f=5\mathrm{kHz}$, and therefore, energy per pulse of $E=1.2\times {10}^{-5}\mathrm{J}$. Every sample was irradiated with 100,000 pulses.

Direct detection as described in previous papers^18–20 was done by time resolved measurements at 1270 nm (30 nm full width half maximum filter) in near-backward direction with respect to the exciting beam using an infrared-sensitive photomultiplier (R5509-42, Hamamatsu Photonics Deutschland GmbH, Herrsching, Germany) with using an additional 950 nm cut-off-filter. The luminescence intensity is given by

For the determination of ${\mathrm{\Phi }}_{\mathrm{\Delta }}$ of XF73 in ${\mathrm{H}}_2\mathrm{O}$, it is compared with the ${\mathrm{\Phi }}_{\mathrm{\Delta }}$ of TMPyP, which is reported in literature being 0.74²¹ and $0.77\pm 0.04$¹² in aqueous solution. Therefore, five probes of each PS of different concentrations (between 30% and 70% absorption at a wavelength of $\lambda =532\mathrm{nm}$) are irradiated and the emitted ${}^1{\mathrm{O}}_2$-photons are determined with the integral over the luminescence curve, given with the fit routine mentioned.

3.1.

Absorption Spectroscopy in Aqueous PS Solution

Changes in the $\pi$-electron-system of porphyrin molecules can lead to the change of absorption cross-section $\sigma$ and hence may affect ${}^1{\mathrm{O}}_2$ generation. TMPyP showed a constant absorption cross-section in the range from ${10}^{-6}$ to ${10}^{-3}\mathrm{M}$ (data not shown). In contrast to TMPyP, the absorption spectrum of XF73 in pure ${\mathrm{H}}_2\mathrm{O}$ clearly depended on XF73 concentration. The absorption cross-section decreased with increasing XF73 concentration from ${10}^{-5}$ to $2·{10}^{-3}\mathrm{M}$ and the absorption maximum (Soret band) shifted to shorter wavelengths ($\sim 7\mathrm{nm}$) [Fig. 3(a)]. Both effects indicate aggregation of XF73 molecules.

Fig. 3

(a) Absorption spectrum of XF73 with increasing PS concentration. A blue-shift of the absorption maxima of 7 nm was detected when increasing the concentration from ${10}^{-5}$ to ${10}^{-3}\mathrm{M}$. (b) Comparison of the influence of the single components of PBS on the absorption spectrum of XF73. A PS concentration of $2\times {10}^{-5}\mathrm{M}$ has been used and NaCl, KCl, ${\mathrm{KH}}_2{\mathrm{PO}}_4$, and ${\mathrm{Na}}_2{\mathrm{HPO}}_4$ had each a concentration of 0.1 M. The table shows the wavelengths ${\lambda }_{\max }$ of the absorption maximum and its value ${\sigma }_{\max }$ for each component of PBS, for PBS and ${\mathrm{H}}_2\mathrm{O}$.

3.2.

Absorption Spectroscopy in Aqueous XF73 Solution with PBS or PBS Constituents

The PBS and cytosol of living cells contain various ions like $K^{+}$, ${\mathrm{Na}}^{+}$, ${\mathrm{Cl}}^{-}$, ${{\mathrm{HCO}}_3}^{-}$, ${{\mathrm{Mg}}_2}^{+}$, ${{\mathrm{Ca}}_2}^{+}$, and ${{\mathrm{HPO}}_4}^{2-}$. As a first approximation to cellular environment, XF73 was dissolved in PBS solution. As XF73 was not easily soluble in PBS, the maximum concentration of PBS was 50% in ${\mathrm{H}}_2\mathrm{O}$. Absorption spectra of XF73 ($2\times {10}^{-5}\mathrm{M}$) were recorded in pure ${\mathrm{H}}_2\mathrm{O}$, in 50% ${\mathrm{H}}_2\mathrm{O}$ plus 50% PBS, and in 100% ${\mathrm{H}}_2\mathrm{O}$ adding single constituents of PBS such as KCl, NaCl, ${\mathrm{NaH}}_2{\mathrm{PO}}_4$, or ${\mathrm{KH}}_2{\mathrm{PO}}_4$, 0.1 M each [Fig. 3(b)].

In the presence of ${\mathrm{Na}}_2{\mathrm{HPO}}_4$ or ${\mathrm{KH}}_2{\mathrm{PO}}_4$, the absorption cross-section showed no wavelength shift or new absorption maxima within given experimental accuracy ($\pm 2\mathrm{nm}$) when compared with pure ${\mathrm{H}}_2\mathrm{O}$. The maximum value of absorption cross-section at ($402\pm 2$) nm decreased from ${\sigma }_{\max }=0.71\times {10}^{-15}{\mathrm{cm}}^2$ (pure ${\mathrm{H}}_2\mathrm{O}$) to $\sigma =0.41\times {10}^{-15}{\mathrm{cm}}^2$ or $\sigma =0.48\times {10}^{-15}{\mathrm{cm}}^2$ when ${\mathrm{Na}}_2{\mathrm{HPO}}_4$ or ${\mathrm{KH}}_2{\mathrm{PO}}_4$ was added, respectively.

When adding PBS, ${\sigma }_{\max }$ decreased from $0.71\times {10}^{-15}{\mathrm{cm}}^2$ to $0.25\times {10}^{-15}{\mathrm{cm}}^2$ and shifted to longer wavelengths (red shift) of $24\pm 2\mathrm{nm}$.

When adding NaCl or KCl to XF73 solution, ${\sigma }_{\max }$ decreased to $0.25\times {10}^{-15}{\mathrm{cm}}^2$ for each. In addition, ${\sigma }_{\max }$ shifted to the red by about $25\pm 3\mathrm{nm}$. At the same time, the absorption spectrum showed new absorption maxima within the spectral range of the Soret band. Addition of ${\mathrm{Cl}}^{-}$ leads to a fundamental change of the absorption spectrum including a red shift. It is suggested that ${\mathrm{Cl}}^{-}$ affects the tetrapyrrol ring system and enhances the aggregation, which was already reported for other porphyrin structures.³²

A visible precipitation of the solute started when using $>10\%$ $\mathrm{PBS}+{\mathrm{H}}_2\mathrm{O}$. This effect was shown to be reversible by diluting the solution with pure ${\mathrm{H}}_2\mathrm{O}$. As a consequence of this dilution, the absorption spectrum of XF73 in PBS changed back to the absorption spectrum in pure ${\mathrm{H}}_2\mathrm{O}$ (data not shown). The precipitation does not affect the absorption measurements because the probes are directly used after being diluted and the precipitation effect needs several hours to develop. No light scattering effect in solutions was detectable by checking the absorption spectrum at shorter wavelengths.

3.3.

Photostability

Also, the photostability and hence the change of absorption spectrum during irradiation may affect ${}^1{\mathrm{O}}_2$ luminescence. Therefore, the photostability of XF73 in solution containing PBS was investigated when illuminating the samples up to $54.8\mathrm{J}{\mathrm{cm}}^{-2}$.

No changes in the absorption spectrum of TMPyP were noticed within irradiation time of upto 60 min (data not shown). The XF73 in ${\mathrm{H}}_2\mathrm{O}$ and in 50% $\mathrm{PBS}+{\mathrm{H}}_2\mathrm{O}$ showed a decrease in absorption that was mainly detected in the spectral range of the Soret band (Fig. 4). Obviously, the presence of PBS, i.e., its ions, can additionally reduce radiation absorption of XF73. These effects may also affect the use of XF73 when applied for photodynamic inactivation of microorganisms.

Fig. 4

Photostability measurements with XF73 show a decrease of the absorption cross section with the time of illumination and therefore the applied energy. The light source was the Waldmann-UV236 lamp with an applied energy dose of 13.7 or $54.7\mathrm{J}{\mathrm{cm}}^{-2}$, respectively. XF73 with a concentration of ${10}^{-5}\mathrm{M}$ has been investigated in pure ${\mathrm{H}}_2\mathrm{O}$ and in PBS (50% in ${\mathrm{H}}_2\mathrm{O}$).

In case of ${}^1{\mathrm{O}}_2$ experiments (see below), XF73 solutions were irradiated with 1 J of laser energy (532 nm). It is expected that $\sigma$ values do not significantly change under these experimental conditions.

3.4.

${}^1{\mathrm{O}}_2$ Luminescence Experiments without PBS

Incubation of bacteria or human cells with XF73 and subsequent irradiation yielded effective cell killing by means of ${}^1{\mathrm{O}}_2$ generation, which was confirmed by adding ${}^1{\mathrm{O}}_2$ quencher ${\mathrm{NaN}}_3$ that significantly reduced the cell toxicity.¹⁶ Since detailed studies on ${}^1{\mathrm{O}}_2$ generation of the novel porphyrin molecule XF73 were missing, we investigated XF73 in pure aqueous solution according to previous studies on other photosensitizers.²⁷

After dissolving $[\mathrm{XF}73]=5\times {10}^{-5}\mathrm{M}$ in air saturated ($[{}^3{\mathrm{O}}_2]=2.7\times {10}^{-4}\mathrm{M}$), pure ${\mathrm{H}}_2\mathrm{O}$, the rise and decay part of the time resolved signals could be assigned to the decay time ${\tau }_{\mathrm{\Delta }}$ of ${}^1{\mathrm{O}}_2$ and the decay time ${\tau }_{T1}$ of PS, respectively. Experiments yielded ${\tau }_{T1}=1.6\pm 0.2\mu \text{s}$ and decay time ${\tau }_{\mathrm{\Delta }}=3.5\pm 0.3\mu \text{s}$ [Fig. 5(a)]. The decay time is in good correlation with the lifetime of ${}^1{\mathrm{O}}_2$ in pure water.^33–35 The spectrally resolved ${}^1{\mathrm{O}}_2$ luminescence revealed a peak at 1270 nm, which clearly confirmed the generation of ${}^1{\mathrm{O}}_2$ [Fig. 5(d)]. The ${}^1{\mathrm{O}}_2$ quantum yield ${\mathrm{\Phi }}_{\mathrm{\Delta }}$ of XF73 was determined in air saturated, pure ${\mathrm{H}}_2\mathrm{O}$, using TMPyP as reference. The ${\mathrm{\Phi }}_{\mathrm{\Delta }}$ values of TMPyP are 0.74²¹ and $0.77\pm 0.04$.¹³ Using the previously reported technique,²¹ XF73 showed a value of ${\mathrm{\Phi }}_{\mathrm{\Delta }}=0.57\pm 0.06$.

Fig. 5

(a)–(c) ${}^1{\mathrm{O}}_2$ luminescence signals of $[\mathrm{XF}73]=5\times {10}^{-5}\mathrm{M}$ with different PBS concentrations in ${\mathrm{H}}_2\mathrm{O}$ with an oxygen concentration of $[{}^3{\mathrm{O}}_2]=2.7\times {10}^{-4}\mathrm{M}$. (d) Spectroscopically resolved ${{}^1\mathrm{O}}_2$ luminescence signal, generated by XF73 in ${\mathrm{H}}_2\mathrm{O}$ with an oxygen concentration of $[{\mathrm{O}}_2]=2.7\times {10}^{-4}\mathrm{M}$. A Lorentz-shaped curve has been fitted through the measurement points. (e) ${{}^1\mathrm{O}}_2$ luminescence generated by XF73 in ${\mathrm{H}}_2\mathrm{O}+20\%\mathrm{PBS}$ at 1270 nm with $2\times {10}^{-3}\mathrm{M}$ NaCl in solution. (f) Spectroscopically resolved ${{}^1\mathrm{O}}_2$ luminescence signal, generated by XF73 in 30% $\mathrm{PBS}+{\mathrm{H}}_2\mathrm{O}$ with an oxygen concentration of $[{\mathrm{O}}_2]=2.7\times {10}^{-4}\mathrm{M}$. A Lorentz-shaped curve has been fitted through the measurement points.

When changing the concentration of ${\mathrm{O}}_2$ in the solution at a constant concentration of $[\mathrm{XF}73]=5\times {10}^{-5}\mathrm{M}$, the meaning of the rates $K_{\mathrm{\Delta }}$ and $K_{T1}$ at $[{}^3{\mathrm{O}}_2]=1.1\times {10}^{-4}\mathrm{M}$ changed according to the decay paths of ${}^1{\mathrm{O}}_2$ and $T_1$ [Fig. 6(a)].²⁰ This change occurs at a crossing point of ${t_1}^{-1}$ and ${t_2}^{-1}$, which was about $[{}^3{\mathrm{O}}_2]=(0.11\pm 0.02)$ ${10}^{-3}\mathrm{M}$ for XF73. By extrapolating ${t_2}^{-1}$, $K_{T1}$ ($([{\mathrm{O}}_2]=0\mathrm{M})=0.03{\mu \text{s}}^{-1}$ was determined yielding a lifetime of the triplet $T_1$-state of $(33\pm 5)\mu \text{s}$ in aqueous solution without oxygen quenching. The quenching rate constant $k_q$ for quenching of the excited triplet state of XF73 by oxygen is therefore $k_q=2.3\times {10}^9{\mathrm{s}}^{-1}{\mathrm{M}}^{-1}$ resulting from the Stern-Volmer-plot in Fig. 6(a), where the oxygen concentration was varied and the triplet decay of XF73 was determined.

Fig. 6

(a) Rates ${t_1}^{-1}$ and ${t_2}^{-1}$ of the time resolved ${}^1{\mathrm{O}}_2$ signal depending on the concentration of ${\mathrm{O}}_2$. The meaning of the two rates changes at the crossing point of the curves. (b) The rate ${t_2}^{-1}$ characterizes the decay time of the triplet-${\mathrm{T}}_1$-state and changes with the XF73 concentration; here the oxygen concentration is kept constant at $[{}^3{\mathrm{O}}_2]=5.4\times {10}^{-5}\mathrm{M}$.

As a next step, XF73 concentration was varied from $[\mathrm{XF}73]={10}^{-6}$ to $5\times {10}^{-3}\mathrm{M}$ at [$[{{}^3\mathrm{O}}_2]=5.6\times {10}^{-5}\mathrm{M}$ [Fig. 6(b)]. The value of ${t_2}^{-1}$ increased with increasing concentration that indicated a clear self-quenching effect of the excited triplet-$T_1$-state for [XF73] up to about $2\times {10}^{-4}\mathrm{M}$. Above this concentration, the quenching effect decreased and reached a plateau at ${t_2}^{-1}=0.205{\mu \text{s}}^{-1}$, which is equivalent to a decay time of the triplet-$T_1$-state of $t_{T1}=4.9\mu \text{s}$ [Fig. 6(b)]. According to the absorption spectroscopy data, a stacking of XF73 molecules occurred, which is easily detectable for XF73 concentration higher than $1\times {10}^{-4}\mathrm{M}$ [Fig. 3(a)]. Obviously, the stacking process had already led to the formation of dimers or oligomers of XF73 molecules at this concentration. Besides a different absorption cross-section, these aggregates also show different deactivation of triplet $T_1$-state as compared with XF73 monomers [Fig. 6(b)].

3.5.

${{}^1\mathrm{O}}_2$ Luminescence Experiments with PBS

In light of the results above, ${{}^1\mathrm{O}}_2$ luminescence signals should be affected by molecule stacking, in particular when the photosensitizer in located in C. albicans cells [Fig. 2(b)]. Therefore, we investigated the PBS effect on time-resolved ${{}^1\mathrm{O}}_2$ luminescence generated by XF73 in air saturated solution at a concentration of $5\times {10}^{-5}\mathrm{M}$, for which stacking due to PS concentration should be still minimal [Fig. 3(a)]. The results clearly show that ${{}^1\mathrm{O}}_2$ luminescence substantially changed with increasing PBS concentration [Fig. 5(a)–5(c)]. From 0% to 50% PBS in ${\mathrm{H}}_2\mathrm{O}$, the rising part of ${{}^1\mathrm{O}}_2$ luminescence signal disappeared, whereas the decaying part shortened. Now, the luminescence signals at high PBS concentrations [Fig. 5(c)] were similar to those recorded for XF73 in C. albicans cells [Fig. 2(b)] yielding again a multiexponential decay.

When adding ${{}^1\mathrm{O}}_2$ quencher ${\mathrm{NaN}}_3$^36,37 to the 20% PBS solution up to a high concentration of $2\times {10}^{-3}\mathrm{M}$ ${\mathrm{NaN}}_3$, the ${{}^1\mathrm{O}}_2$ luminescence signal almost disappeared. The residual signal should not originate from ${{}^1\mathrm{O}}_2$ luminescence [see Fig. 5(e)]. The same residual signal was detected in solutions without ${\mathrm{NaN}}_3$ and without oxygen (data not shown).

${{}^1\mathrm{O}}_2$ luminescence was also spectrally resolved for PBS 0% and 50% in ${\mathrm{H}}_2\mathrm{O}$ [Fig. 5(d) and 5(f)]. A Lorentz-shaped curve has been fitted through the measurement points and the values were normalized to the maximal value. Without PBS, the fit shows a clear maximum at 1270 nm that confirms the generated ${{}^1\mathrm{O}}_2$.³⁸ At 50% PBS, the maximum at 1270 nm almost disappeared, the baseline moved for wavelengths $<1270\mathrm{nm}$, and the signal-to-noise ratio decreased, which indicates a substantial decrease of ${{}^1\mathrm{O}}_2$ generation.

Comparable to absorption spectroscopy, the changes of time- and spectral resolved ${{}^1\mathrm{O}}_2$ luminescence signals, induced by PBS, could be simply reversed by diluting the used solutions with ${\mathrm{H}}_2\mathrm{O}$ and hence reducing the PBS concentration. A high degree of dilution of PBS concentration yielded time- and spectrally resolved ${{}^1\mathrm{O}}_2$ luminescence signals comparable with Fig. 5(a) and 5(d).

Scattering of photons within solution might also cause a ${{}^1\mathrm{O}}_2$ luminescence signal equal to the one in Fig. 5(e), and might be originating from precipitation due to the stacking of the porphyrins. To exclude any scattering effects, the scattering agent ${\mathrm{SiO}}_2$ was added to aqueous solutions containing $5\times {10}^{-5}\mathrm{M}$ XF73 or TMPyP. No effect on the shape of the ${{}^1\mathrm{O}}_2$ luminescence signal and no change of the rise and decay times were detected for both photosensitizers. Additionally there was no scattering effect visible in the absorption spectrum of XF73 in ${\mathrm{H}}_2\mathrm{O}+50\%$ PBS.