Unique Triphenylphosphonium Derivatives for Enhanced

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Unique Triphenylphosphonium Derivatives for Enhanced Mitochondrial Uptake and Photodynamic Therapy Zhang Hu, Ying Sim, Oi Lian Kon, Wai Har Ng, António Ribeiro, Maria João Ramos, Pedro A. Fernandes, Rakesh Ganguly, Bengang Xing, Felipe Garcia, and Edwin K. L. Yeow Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00682 • Publication Date (Web): 03 Jan 2017 Downloaded from http://pubs.acs.org on January 5, 2017

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Bioconjugate Chemistry

Unique Triphenylphosphonium Derivatives for Enhanced Mitochondrial Uptake and Photodynamic Therapy

Zhang Hu,1 Ying Sim,1 Oi Lian Kon,2 Wai Har Ng,2 António J. M. Ribeiro,3 Maria J. Ramos,3 Pedro A. Fernandes,*,3 Rakesh Ganguly,1 Bengang Xing,1 Felipe García,*,§,1 and Edwin K. L. Yeow*,§,1

1

School of Physical and Mathematical Sciences, Division of Chemistry and Biological

Chemistry, Nanyang Technological University, 21 Nanyang Link, 637371, Singapore 2

Division of Medical Sciences, Laboratory of Applied Human Genetics, Humphrey Oei Institute

of Cancer Research, National Cancer Centre, 169610, Singapore 3

UCIBIO, REQUIMTE, Departamento de Química e Bioquímica,Faculdade de Ciências,

Universidade do Porto, Rua do Campo Alegre s/n, 4169-007, Portugal *Email: [email protected], [email protected], [email protected]

1 ACS Paragon Plus Environment


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Abstract In this study, unique methyl functionalized derivatives (T*PP+) of the drug carrier triphenylphosphonium (TPP+) that exhibit significant enhancement in the accumulation of both the cation and its conjugated cargo in cell mitochondria are designed. We show that the presence of methyl group(s) at key positions within the phenyl ring results in an increase in the hydrophobicity and solvent accessible surface area of T*PP+. In particular, when the paraposition of the phenyl ring in T*PP+ is functionalized with a methyl group, the cation is most exposed to the surrounding environment; leading to a high drop in water entropy, and an increase in Van der Waals interaction with and partition into a non-polar solvent. Therefore, a greater binding between the hydrophobic T*PP+ and mitochondrial membrane is formed. This is exemplified in a (hexachloro-fluorescein)-TPP+ conjugate system, where an ca. twelve times increase in mitochondrial uptake and two times increase in photodynamic therapy (PDT) efficacy against HeLa and FU97 cancer cells is achieved when TPP+ is replaced by T*PP+. Importantly, nearly all the FU97 cells treated with (hexachloro-fluorescein)-T*PP+ conjugate are killed as compared to only half the population of cells in the case of (hexachloro-fluorescein)-TPP+ conjugate at similar PDT light dosage. This study thus forms a platform for the healthcare community to explore alternative TPP+ derivatives that can act as optimal drug transporter for enhanced mitochondria-targeted therapies.


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INTRODUCTION Mitochondria are essential cell organelles responsible for cellular respiration and apoptosis. Several degenerative diseases including Parkinson’s disease, heart conditions, and cancer have been attributed to dysfunctional mitochondria.1-3 Therefore, many efforts have been focused on the design of optimal mitochondria-targeting drugs specifically delivered into the mitochondria.411

There are several strategies available to targeted mitochondrial delivery including the use of

Szeto-Schiller peptides, mitochondrial-targeting peptides and lipophilic cations (LC).12,13 LC are capable of effectively permeating mitochondrial membranes due to their large hydrophobic surface area and delocalized charge distribution.14,15 A large portion of LC, initially adsorbed on the outer surface of mitochondria, are transported through the outer and inner membranes and accumulated by several-hundredfold due to the large membrane potential across the inner mitochondrial membrane.16 One of the most commonly utilized LC for drug delivery is the triphenylphosphonium cation (TPP+) (1 in Figure 1a) that is conjugated to a therapeutic agent via a linker (e.g., alkyl chain).14,15 Previous studies have shown that modification of the hydrophobicity of the TPP+-drug conjugate system by lengthening the alkyl chain between TPP+ and drug results in a significant improvement on the accumulation of drugs in the mitochondria.17,18 However, the length and type of linkers have been known to affect the stability, toxicity and usage (e.g., drug release by cleaving the linker between drug and carrier) of drug conjugates.19,20 Moreover, there are no studies on the effect of phenyl substitution on the mitochondrial uptake properties of TPP+. Hence, it is imperative to investigate alternative strategies to control the hydrophobicity and mitochondrial uptake of TPP+-drug conjugates without involving changes to the pre-set linker.



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Photodynamic therapy (PDT) of cancer involves the light irradiation of photosensitizerstained tumour cells in the presence of molecular oxygen to produce reactive oxygen species (e.g., singlet oxygen 1O2) that are capable of damaging and killing cells.21,22 Since mitochondria are important regulators of cell death, PDT-induced cytotoxicity within the mitochondria is a promising cancer treatment with minimal damages to healthy tissues.21,23-26 Another advantage of mitochondria-targeted PDT is the avoidance of defective signalling routes often encountered in drug-induced apoptotic responses that give rise to resistance in conventional chemotherapy.21 So far, mitochondria-targeted PDT is still in its infancy, and the development of better mitochondria-targeting photosensitizers (e.g., fluorescein) is instrumental for its future progress.24-26 Fluorescein is capable of generating 1O2 when irradiated with light27 and is therefore commonly used as a photosensitizer (PS) in both antimicrobial photodynamic therapy28 and fluorophore-assisted light inactivation (FALI) of endogenous proteins.27,29,30 In FALI, a strategy employed to label proteins in vivo involves fusing FKBP12(F36V) to the targeted protein.29 A fluorescein-ligand conjugate, where the engineered ligand is not only cell permeable but also binds to FKBP12(F36V), results in efficient cellular protein labeling. The stained protein is subsequently inactivated by

1

O2 generated from the fluorescein conjugate during light

exposure.29 Therefore, fluorescein conjugated to suitable cell-permeable carriers can effectively function as a PDT agent. Our approach in this study is based on the facile methyl functionalization of TPP+ to enhance both delivery and mitochondrial uptake (see tri*phenylethylphoshonium cations T*PP+ 2-7 in Figure 1a). We have demonstrated that the accumulation of the modified T*PP+ species in the mitochondria of HeLa cells and FU97 gastric cancer cells increases significantly upon


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substitution of the phenyl rings. In addition, hexachloro-fluorescein conjugates 1a, 2a and 5a (Figure 1b) were prepared and their mitochondrial uptake and PDT efficacy in HeLa and FU97 cells determined. Unconjugated fluorescein does not accumulate significantly in the cell/mitochondria and therefore does not display any PDT effects. On the other hand, conjugates 1a, 2a and 5a permeate both plasma and mitochondrial membranes and are accumulated in the mitochondria; hence allowing efficient PDT to occur. When compared to TPP+, T*PP+ cations significantly increase the accumulation of photosensitizer in mitochondria which leads to a nearly two times improvement in the PDT of cancer cells. This study underscores the importance of the presence of the mitochondrial directing LC.

Figure 1. (a) Structures of triphenylethylphosphonium 1 and tri*phenylethylphosphonium (T*PP+; where *phenyl = mono- (2, 3 and 4), di- (5, and 6) and tri-methyl (7) functionalized phenyl); tri-p-tolylethylphosphonium (2), tri-m-tolylethylphosphonium (3), tri-o-tolyethylphos-phonium (4), tris-(3,5xylylethylphosphonium (5), tris-(2,4-xylylethylphospho-nium (6) and tris-mesitylethylphosphonium (7). (b) Structures of the (hexachloro-fluorescein)-TPP+/T*PP+ conjugates: (hexachloro-fluorescein)triphenylethylphosphonium

(1a),

(hexachloro-fluorescein)-tri-p-tolylethylphosphonium

(2a)

and

(hexachloro-fluorescein)-tris-(3,5-xylyl)ethylphosphonium (5a). 5 ACS Paragon Plus Environment


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RESULTS AND DISCUSSION Partition coefficient and mitochondrial uptake of cations 1-7. The octanol-water partition coefficients (P) for compounds 1-7 (i.e., ratio of the concentration of cation in 1-octanol to the concentration of cation in water) were determined to establish the relative hydrophobicity of the various cations (Figure 2a). T*PP+ cation 7, containing tri-methyl substituted phenyl rings, has the highest P and is the most hydrophobic followed by cations with bi-methyl substituted (5 and 6) and mono-methyl substituted (2, 3 and 4) phenyl rings. The least hydrophobic salt in the series is the commonly used TPP+ 1. As expected, a positive correlation exists between hydrophobicity and number of methyl groups present due to the additional non-polar substituents. Interestingly, not only the number but also the phenyl ring substitution pattern plays a significant role in the overall hydrophobicity of the cation. In the case of the mono-substituted phenyl, the hydrophobicity increases in the order 4 < 3 < 2; where the phosphonium salt displays greatest hydrophobicity when the paraposition is substituted and least hydrophobic when the orthoposition is substituted. In order to evaluate if the hydrophobic variation observed for compounds 1-7 would lead to different mitochondrial uptake, their accumulation in isolated mitochondria was determined. Basically, HeLa and FU97 mitochondria were isolated using a mitochondrial isolation kit (Miltenyi Biotec), and the protein quantity in the mitochondria was determined using the Bradford assay. Salt uptake was determined by first incubating freshly isolated mitochondria (~0.135 mg of proteins per L) with the various cations 1-7 (500 µM) for 5 min. The mitochondria were then lysed and the quantity of accumulated 1-7 (Cm) determined using HPLC. The results for HeLa and FU97 cells are presented in Figure 2b and S11 (Supporting Information), respectively. From Figure 2c, we note a positive correlation between Cm and Log P


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(i.e., hydrophobicity) where T*PP+ uptake by the mitochondria is enhanced for the more hydrophobic cations. In particular, there is a ca. 4 and 7 times enhancement in the uptake of 7 as compared to 1 for HeLa and FU97 mitochondria, respectively.

Figure 2. (a) Values of Log P and (b) amount of 1-7 accumulated (Cm) in isolated HeLa mitochondria. (c) Positive correlation between Log P and Cm, and (d) negative correlation between Cm and ∆Gaq→oct/vdW. The r2 values for the linear fits are provided.

Computational study. The free energy associated with the permeation of small charged molecules across a membrane from an aqueous environment can be decomposed into the Born free energy and hydrophobic (or neutral) free energy.14,31 The Born free energy WB = 339(Z2/r) is inversely proportional to the radius (r) of the cation,14,31,32 whereas the hydrophobic free energy was empirically found to be proportional to the accessible surface area of the cation ( ∝ r2).14,31



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To gain insight into the underlying cause of the trends observed in Section 2.1, molecular dynamics (MD) calculations were performed for molecules 1-7 in a cubic box containing either water or 1-octanol molecules. Thermodynamic integration was used to compute the free energy ∆Gaq→oct associated with moving a solute molecule from water (aq) to 1-octanol (oct). ∆Gaq→oct is defined to be the difference between the free energies for the annihilation of the solute in water (∆Gaq) and in 1-octanol (∆Goct) (i.e., ∆Gaq→oct = ∆Gaq - ∆Goct). The values of ∆Gaq→oct with the corresponding van der Waals (vdW) and electrostatic contributions to the free energy are provided in Table 1. Furthermore, the solvent accessible surface area (SASA) of the cations are almost equivalent in both water and 1-octanol (average unsigned difference of 0.5 Å2); showing that the structural conformations adopted by 1-7 are maintained in the two solvents (Table 1). Visualization of the trajectories confirms this observation.

Table 1. Free energy ∆Gaq→oct associated with moving a solute molecule from water (aq) to 1-octanol (oct), and corresponding van der Waals (vdW) and electrostatic interaction contributions in kcal mol-1. The solvent accessible surface area SASA for 1-7 are given.

cation

SASA/Å2

∆Gaq→oct

vdW

electrostatic

1

535.8±0.1

-17.7±0.2

-14.7±0.2

-3.0±0.1

2

626.1±0.1

-21.3±0.2

-17.5±0.2

-3.7±0.1

3

623.0±0.1

-20.7±0.2

-17.1±0.2

-3.5±0.1

4

566.3±0.1

-19.5±0.2

-16.1±0.2

-3.4±0.1

5

708.6±0.1

-23.6±0.3

-19.7±0.2

-3.9±0.1

6

653.3±0.1

-22.6±0.2

-19.3±0.2

-3.3±0.1

7

673.6±0.1

-23.6±0.3

-20.1±0.2

-3.5±0.2


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An almost negligible correlation exists between the electrostatic component of ∆Gaq→oct with the experimental partition coefficient P and T*PP+ uptake Cm (Table 1). This is because the calculated electrostatic solvation free energy is mostly dependent on the charge of the cation which remains invariant for the cations studied here. Furthermore, assuming a spherical model (calculated sphericity ~0.8 for 1 and 7), the calculated radius of 7 is only ~1.1 times larger than that of 1 which suggests that changes in the Born free energy between the various cations is not significant to fully explain the trends seen above.

Figure 3. Positive correlation between ∆Gaq→oct and vdW energy (a) and negative correlation between ∆Gaq→oct and SASA (b). The r2 values for the linear fits are provided and the corresponding cations for each point are given.

On the other hand, we note that the magnitudes of both the calculated ∆Gaq→oct and the vdW component of the free energy positively correlate with P and Cm (Figure 2d for HeLa cells). The vdW component describes the non-polar (hydrophobic) interaction and is thus dependent on the hydrophobicity of the cation. From Table 1, it is observed that a positive/negative correlation exists between ∆Gaq→oct and the vdW/SASA term (Figure 3). As the number of substituted 9 ACS Paragon Plus Environment


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methyl groups increases, a larger (hydrophobic) surface area of the cation is accessible to the solvent. This leads to a higher drop in water entropy as more water molecules surrounding the hydrophobic cation have less orientational freedom to form hydrogen bonds with other water molecules; resulting in a poorer solvation in water. On the other hand, when the larger surface area of the more hydrophobic cation is exposed to 1-octanol molecules, an increase in vdW interaction leads to a favorable partition into the organic solvent (i.e., more negative ∆Gaq→oct). A similar explanation is used to rationalize the trend observed within the monosubstituted cation group. In this case, the SASA increases in the order 4 < 3 < 2, indicating that when the paraposition is substituted, the methyl groups in 2 are better exposed to the solvent as compared to the ones in 3 and 4. In particular, methyl groups at the orthoposition in 4 are less accessible to solvent molecules due plausibly to steric hindrance arising from the adjacent phosphorous. Since more 1-octanol molecules are able to interact with 2, a stronger dispersion interaction gives rise to the observed increased partition in 1-octanol (Figure 2a). In water, the larger hydrophobic surface will give rise to a larger water entropy penalty. A similar driving force is responsible for facilitating the uptake of the more hydrophobic cation in the mitochondrial membrane where the greater binding of T*PP+ within the membrane may lead to the observed higher mitochondrial uptake.18

Mitochondrial uptake of dye conjugates increases with the hydrophobicity of the lipophilic cation. We further examine the ability to deliver a hexachloro-fluorescein dye (i.e., cargo sensitizer) into the mitochondria by T*PP+ of different hydrophobicity (i.e., carrier). In our study, HeLa and FU97 cells were utilized and first incubated with similar amounts (20 µM) of hexachloro-fluorescein, and conjugates 1a, 2a and 5a for 3 h. As control, the cells were also


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Figure 4. Confocal fluorescence microscopy imaging of FU97 cells treated with 5a, 2a and 1a. The fluorescence images upon 543 nm excitation of the dye-T*PP+ conjugates and 633 nm excitation of Mitotracker Deep Red are given in the first and second columns, respectively. Overlay images from the first and second columns are given in the third column. The scale bar represents 20 µm.

treated with Mitotracker Deep Red dye to monitor the progress of mitochondrial staining. After washing the cells to remove free dyes, fluorescence imaging experiments were conducted using laser light of 543 nm to excite hexachloro-fluorescein (first column in Figure 4) and 633 nm to excite Mitotracker Deep Red (second column in Figure 4). No obvious fluorescence was detected in the same cells after incubation with the unconjugated hexachloro-fluorescein; suggesting that dye alone under the conditions employed here is not effectively delivered into the cell mitochondria (Figure S12 and S13 in the Supporting Information for FU97 and HeLa cells, respectively). In contrast, similar incubation with conjugates 1a, 2a and 5a leads to distinct fluorescence images (Figure 4 and S13 in Supporting Information for FU97 and HeLa cells,



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respectively). Images from PS-TPP+ 1a and PS-T*PP+ 2a and 5a overlap with the ones with Mitotracker emission, suggesting that 1a, 2a and 5a are taken up by the cells and co-localized with Mitotracker Deep Red in the mitochondria. Moreover, the fluorescence intensity from the hexachloro-fluorescein moiety of the conjugates increases in the order: 1a < 2a < 5a (Figure S14 in the Supporting Information); suggesting that 5a is most readily taken up. We further quantify the uptake of PS-TPP+ and PS-T*PP+ by isolated HeLa and FU97 mitochondria using HPLC. After incubating freshly isolated

mitochondria (~0.135 mg of

proteins per L) with 1a, 2a and 5a (500 µM) for 5 min, the amounts of accumulated 1a, 2a and 5a in HeLa/FU97 cells were determined to be 1.1±0.1/1.6±0.2, 7.3±0.3/8.1±0.5 and 13.2±0.6/20.2±0.9 mmol g-1, respectively. These data positively correlate with the partition coefficients (i.e., Log P of 1a, 2a and 5a are 0.19 ± 0.02, 0.91 ± 0.01 and 3.66 ± 0.02, respectively), and increase in the order: 1a < 2a < 5a. In particular, we note that 5a is accumulated ~12 times more than 1a in the mitochondria. Amongst the 3 conjugate systems, the most hydrophobic tris(3,5-dimethylphenyl)phosphonium (T*PP+ 5) in 5a acts as the best cargo transporter, followed by tri-p-tolyphosphonium (T*PP+ 2) in 2a and triphenylphosphonium (TPP+ 1) in 1a. The fluorescence quantum yields and lifetimes of hexachloro-fluorescein, 1a and 5a in water did not vary significantly and were measured to be 0.94 and 3.9 ns, respectively; indicating that the photophysics of the sensitizer is not perturbed when linked to either TPP+ or T*PP+ (Figure S15 in Supporting Information). Therefore, it is most likely that the large accumulation of 5a in the mitochondria leads to the higher emission intensity observed in the fluorescence cell image (Figure 4) when compared to the other conjugates. When the mitochondrial membrane potential (∆ψm) was reduced by treating freshly isolated HeLa mitochondria with FCCP (carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone,


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500 nM) for 1 min before incubation with 5a, the latter was not detected in the mitochondria using HPLC. This indicates that 5a is not accumulated in the mitochondria when ∆ψm is abolished, and the accumulation of 5a is thus driven by the presence of the membrane potential.

PDT efficacy is enhanced when hydrophobicity of cation increases. We next examine the PDT efficacy of 3 compounds: hexachloro-fluorescein, 1a and 5a. Both HeLa and FU97 cells were first incubated with different concentrations (i.e., 10, 20, 40 and 60 µM) of hexachlorofluorescein, 1a and 5a for 3 h. The cells were then washed with PBS before exposure to white light. Resazurin assay was employed to evaluate the viability of the light-treated cells after incubation for 24 h. In the control experiment using free hexachloro-fluorescein, cell viability remains high (> 90 %) for both HeLa and FU97 cells after 15 min of light irradiation (light intensity of 14.3 J cm-2). PDT is not effective because of the small amounts of dye accumulated in the cells. In addition, the light dosages used here are insufficient to cause significant damages to the cells. In the absence of light irradiation, cells treated with 1a and 5a did not show obvious cytotoxicity and high cell viability is observed (i.e., close to 100 %) (Figure 5). Conjugate systems 1a and 5a are non-lethal at concentrations as high as 60 µM. The lack of dark cytotoxicity is consistent with the IC50 values determined for 1a (192 ± 8 µM for HeLa and 160 ± 12 µM for FU97) and 5a (200 ± 11 µM for HeLa and 207 ± 12 µM for FU97). After 15 min of light irradiation, cells treated with 5a clearly exhibited more cell deaths when compared to those treated with an equal amount of 1a (Figure 5). For example, the viabilities of HeLa cells treated with 60 µM of 5a and 1a are 32 % and 68 %, respectively, after 15 min of light exposure (Figure 5a). When FU97 cells were treated with 60 µM of 5a, nearly all of the cells were killed 13 ACS Paragon Plus Environment


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Figure 5. Phototoxicity of 5a at various concentrations (0, 10, 20, 40 and 60 µM) and light irradiation times (0, 5, 10 and 15 min) for (a) HeLa and (b) FU97 cells. The phototoxicity of 1a at different concentrations after 15 min light irradiation is also displayed.

as compared to only 55.3 % of cells treated with the same amount of 1a (Figure 5b). Furthermore, Figure 5 shows that elevated tumour cell damage occurs when the concentration of PS-T*PP+ conjugate used or light exposure time is increased; indicating that PDT is responsible for cell death. Upon light excitation, hexachloro-fluorescein, 1a and 5a in PBS react with the surrounding O2 to produce singlet oxygen 1O2 as detected by the ABDA (9, 10-anthracenediyl-bis (methylene) dimalonic acid) assay (Figure S16 in Supporting Information). Furthermore, the absorbance of ABDA decreases with light irradiation time as the amount of 1O2 produced increases. Previous studies have reported that late stages of apoptosis are triggered by 1O2 created during light irradiation of photosensitizers located in mitochondria.23 In this case, mitochondrial permeabilization and photodamage of antiapoptotic protein bcl-2 can induce the release of caspase activators such as cytochrome c or other pro-apoptotic molecules (e.g., apoptosis-inducing factor) into the cytosol.33-35 PDT-mediated apoptosis occurs when conjugates


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1a and 5a, localized in the mitochondria, are exposed to light as indicated by positive Annexin V staining. Annexin V emission from cells treated with 5a is, in general, brighter than the corresponding fluorescence from cells treated with 1a (Figure 6 for HeLa cells); demonstrating that at the same light dosage, the higher mitochondrial uptake of 5a leads to more cell deaths when compared to 1a.

Figure 6. Fluorescence imaging of HeLa cells treated with 1a and 5a (20 µM), irradiated with white light for 15 min, incubated for 1 day and then treated with Annexin V. Annexin V emission from cells treated with 5a is, in general, brighter than the corresponding fluorescence from cells treated with 1a (first column). Corresponding images from hexachloro-fluorescein conjugate fluorescence are given in the second column. Overlay images from the first and second columns are given in the third column. Scale bar represents 20 µm.



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CONCLUSIONS In summary, we have demonstrated a readily-accessible, enhanced alternative to the conventionally and widely used mitochondria-targeting triphenylphosphonium cation by simple modification using commercially-available phosphine reagents. Our experimental and computational studies have unambigiously demonstrated that increasing both the hydrophobicity of the cation and solvent accessible surface area by methyl substitution at various positions of the phenyl rings significantly improve the partition of T*PP+ cation in a hydrophobic environment, and enhances the accumulation of conjugated cargo within the mitochondria. In particular, the use of the more hydrophobic T*PP+ cationic carrier 5 facilitates a significantly better mitochondrial uptake of conjugated hexachloro-fluorescein in HeLa and FU97 cells which in turn leads to a two times improvement in PDT efficacy upon light irradiation. The LCs developed in our study can be easily conjugated to other therapeutic cargos such as anticancer prodrugs (e.g., doxorubicin) and antioxidants (e.g., ubiquinone).11 It is noteworthy to question, from our study, whether the inclusion of a broader range of organic substituents onto lipophilic cations may additionally enhance their cell uptake efficiency and hence further improve therapeutic applications. The rational design of optimal mitochondrial delivery systems is both an exciting challenge in the area of synthetic chemistry and is relavent to future mitochondrial therapies.

EXPERIMENTAL METHODS Chemicals and materials. Phosphines 97% were purchased from Strem Chemicals, 6-HEX dipivaloate (98%) was purchased from Chemodex, the other chemicals were purchased from Sigma Aldrich and all were used without further purification. Solvents were distilled over sodium/benzophenone, dried and stored under 4 Å molecular sieves. Reactions were carried out 16 ACS Paragon Plus Environment

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using standard inert-atmosphere, Schlenk techniques or performed under argon. Human epithelial carcinoma cell line (HeLa) was purchased from ATCC (ATCC no. CCL-2); Stomach carcinoma cell FU97 was obtained from Prof Oi Lian Kon’s research laboratory (National Cancer Centre). MitoTracker Deep Red FM and the apoptosis imaging reagent (Annexin V Alexa Fluor 488 conjugate) were purchased from Thermo Fisher Scientific. Mitochondria Isolation Kit was purchased from Miltenyi Biotec.

Instrumentation. 1H, 13C NMR (400/100 MHz) spectra were collected using a Bruker Avance DPX400 spectrometer with the 1H, 13C NMR chemical shifts internally referenced to the relevant residual solvent peaks. All NMR spectroscopic analysis were performed at room temperature (300 K). High-resolution mass spectra were obtained from Water Q-Tof Premier with ESI mode. Melting points were determined using a SRS-Optimelt MPA-100 apparatus using sealed glass capillaries under argon and were uncorrected. Infrared spectra were recorded as Nujol mulls using NaCl plates on a Shimadzu IR Prestige-21 FTIR spectrometer. Reverse-phase HPLC analysis was performed on a Shimadzu HPLC system using an Alltima C-18 (250 × 10 mm) column at a flow rate of 3.0 mL/min for preparation and a C-18 (250×4.6 mm) at a flow rate of 1.0 mL/min for analysis. Diffraction-quality crystals were obtained by slow evaporation of solvent from solutions in acetonitrile/water solvent mixtures at room temperature. The crystals were mounted onto quartz fibers, and the X-ray diffraction intensity data were measured at 103 K with a Bruker Kappa diffractometer equipped with a CCD detector, employing Mo K α radiation (λ = 0.71073 Å), with the SMART suite of programs.36 All data were processed and corrected for Lorentz and polarization effects with SAINT and for absorption effects with SADABS.37 Structural solution



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and refinement were carried out with the SHELXTL suite of programs.38 The structures were solved by direct methods or Patterson maps to locate the heavy atoms, followed by difference maps for the light, non-hydrogen atoms. All non-hydrogen atoms were refined with anisotropic thermal parameters. The crystals of 5 had acetonitrile and 6 has a molecule of water as solvent of crystallization. In 7 the bromide anion was modeled in two alternative sites (with 0.5 occupancy). Crystallographic data is available from the Cambridge Crystallographic Database Centre (CCDC 1478282-1478291).

Synthesis. TPP+ 1 and T*PP+ 2-7 (Figure 1a) were synthesized by dropwise addition of the respective tertiary phosphine to a solution of ethylbromide in acetonitrile and reacted under reflux or in a microwave. The (hexachloro-fluorescein)-phosphonium salt conjugates 1a, 2a and 5a (Figure 1b) were prepared by reacting the respective tertiary phosphine precursors with 2bromoethylamine hydrobromide in acetonitrile to obtain their tri*phenylethylaminephosphonium cation counterparts. The amino functionalized phosphonium cations were conjugated to a hexachloro-fluorescein through the coupling reaction between the amine group on the phosphonium salt and NHS group on the dye molecule. The products 1a, 2a and 5a were purified by HPLC and further characterized by NMR and MS analysis. Detailed information on the synthesis and characterization of the various compounds are provided in the Supporting Information.

Determination of the octanol-water partion coefficient. The extinction coefficients of 1-7 in water-saturated 1-octanol and 1-octanol-saturated water were determined using absorption spectroscopy (Cary 100 UV-Vis, Varian) and the Beer-Lambert law. An approximate amount of 1.00 – 2.50 mg of sample was weighed and dissolved in 50 – 70 ml of saturated 1-octanol. The 18 ACS Paragon Plus Environment

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mixture is allowed to stir to ensure the formation of a homogenous solution. Next, with the stock solution and 1-octanol-saturated water, 3 sets of different volume ratios of 1-octanol to water mixtures of each sample were prepared. In this study, the three volume ratios used were 1:5, 1:10 and 1:20 (1-octanol:water). Absorbance for each phase was recorded and used to calculate the partition coefficient P values: P = [LC]octanol/[LC]water where [LC]octanol and [LC]water are the concentrations of the lipophilic cation in octanol and water, respectively.

Determination of the half maximal inhibitory concentrations (IC50) of 1a, 2a and 5a. HeLa and FU97 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) solution with 10 % fetal bovine serum (FBS) and 1 % Penicillin/Streptomycin under humidified atmosphere of 5 % CO2 at 37 °C. The IC50 was measured using Resazurin assay. The HeLa and FU97 cells were seeded on a 96-wells containing 10000 cells per well in 100 µL DMEM media and incubated overnight before the addition of 1a, 2a and 5a. Upon incubation at 37 °C for an additional 72 h, the media were removed and the cells washed with PBS. Resazurin solution 100 µL (1:10 v/v ratio of resazurin solution:DMEM (no phenol red)) was added to each well before incubation for 2 h at 37 °C. The samples were excited using a 560 nm light and the fluorescence was recorded on a Tecan's Infinite M200 microplate reader using a 590 nm emission filter. Different concentrations of 1a, 2a and 5a were used and for each concentration, the experiment was repeated three times. The IC50 was determined from the plot of viability against concentration of conjugates.



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Isolated mitochondria preparation and mitochondrial quantification. Isolated mitochondria were prepared using a mitochondria isolation kit (Mitochondria Isolation Kit, human 130-094532, Miltenyi Biotec). First, Hela or FU97 cells were lysed and the mitochondria magnetically labeled with Anti-TOM22 microbeads. The monoclonal Anti-TOM22 antibody specifically binds to the translocase of outer mitochondrial membrane 22 (TOM22) of human mitochondria. The labeled cell lysate was loaded onto a LS column placed in a MidiMACS Separator. After washing, only magnetically labeled mitochondria remained on the column which were then removed from the separator. Functional human mitochondria were eluted from the column and stored in storage buffer. Western blot was used to ascertain that proteins isolated were from cell mitochondria. Bradford Assay was used to determine the concentration of proteins in mitochondria. MPER mammalian protein extraction reagent was used to extract all protein constituents of the mitochondria. Samples and diluted Bradford dye reagent (200 µL) were pipetted into separate microtiter plate wells and incubated at room temperature for 10 minutes. UV-absorbance was measured at 595 nm and the concentration of proteins was determined from a calibration curve obtained using a protein standard (i.e., BSA).

Mitochondrial uptake of salts. Freshly isolated mitochondria (0.135 mg protein) were incubated with the salts (500 µM) in 2 mL plastic tubes in a shaking water bath at 25 °C. After 5 min, the mitochondria were lysed by centrifugation (7500×g) for 10 min, and the pellets extracted by vortexing in 250 µL Buffer B (99.9 % acetonitrile (ACN), 0.1 % trifluoroacetic acid (TFA)) followed by centrifugation (7500×g) for 10 min. Supernatants (250 µL) were removed and stored at 4 °C in glass vials. All samples were diluted to 25 % ACN by addition of Buffer A (99.89 % H2O, 0.1 % TFA), filtered using a Milipore Millex syringe-driven filter unit (0.22 µm) 20 ACS Paragon Plus Environment

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and subsequently injected into a 2 mL sample loop and separated by RP-HPLC; a C18 column with a widepore C18 guard column were used. A gradient of buffer A and B was run at 1 mL min-1 (% B): 0–5 min, 5–15 %; 5–31 min, 15–100 %; 31–35 min, 100–5 %. Peaks were detected at 220 nm using a UV-Vis spectrophotometer. Known salt standards were used to determine elution times and calibration curves. To reduce the mitochondrial membrane potential, freshly isolated mitochondria (0.135 mg protein) was treated with 500 nM FCCP (carbonyl cyanide-4(trifluoromethoxy)phenylhydrazone) for 1 min. Fluorescence bio-imaging experiments. HeLa or FU97 cells were seeded in a µ-dish (iBidi, Germany) at a density of 100 000 cells (1 mL) per well one day prior to any measurements. Hexachloro-fluorescein, 1a, 2a and 5a (20 µM) were incubated with the cells at 37 oC for 3 h in DMEM. Mitotracker 633 (100 nM) was added 30 min before the end of the incubation period. Cells were then washed three times with PBS (1X) and imaged using a confocal microscope (Nikon Eclipse TE2000) equipped with 543 nm argon and 633 nm lasers.

PDT and cytotoxicity assay after light irradiation. HeLa/FU97 cells were seeded in 96-well at a density of 10 000 cells (100 µL) per well one day prior to experiments. The cells were treated with different concentrations of hexachloro-fluorescein, 1a and 5a (i.e., 10, 20, 40 and 60 µM) for 3 h. Cells were irradiated with white light (400-800 nm light) for different time durations (i.e., 5, 10 and 15 min), followed by 24 h incubation before performing the cell viability test (i.e., Resazurin assay). All experiments were repeated 3 times and the averaged taken.

Apoptosis imaging analysis. HeLa cells were seeded in a µ-dish (iBidi, Germany) at a density of 100 000 cells (1 mL) per well one day prior to experiments. The cells were incubated with 1a



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and 5a conjugates (20 µM) for 3h in DMEM. Subsequently, drug-containing medium was replaced with fresh culture medium and washed with PBS followed by irradiation with white light (400-800 nm, light intensity of 14.3 J cm-2) for 15 min. After an additional incubation time of 24 h, the cells were washed with PBS and redistributed in fresh medium. The apoptosis imaging reagent (Annexin V Alexa Fluor 488 Conjugate) was added and the cells further incubated for 15 min. Live cell imaging was carried out using a Nikon Eclipse TE2000 Confocal Microscope. Excitation filters were set as 488 nm and 543 nm for Annexin V and 1a/5a, respectively.

Fluorescence quantum yield, lifetime and singlet oxygen detection. Fluorescence quantum yields were performed using a fluorescence spectrophotometer (Varian Cary Eclipse) using an excitation wavelength of 500 nm. The fluorescence lifetime was recorded using a time-correlated single photon counting spectrofluorimeter (FluoroCube, Horiba Jobin Yvon) with a 466 nm-excitation laser. For the singlet oxygen detection, hexachloro-fluorescein, 1a and 5a (10 µM) were mixed with 9, 10-anthracenediyl-bis (methylene) dimalonic acid (ABDA) (200 µM) in PBS buffer (10 mM, pH 7.4) and placed in a 96-well. The ABDA absorbance at 378 nm was measured. The samples were then illuminated with white light (400 - 800 nm) for different time durations (i.e., 1, 5 and 10 min) and the absorbance of ABDA at 378 nm was re-measured. The blank solution in the UV-absorption experiment is the corresponding sample solution without ABDA. ABDA solution (200 µM) in the absence of any sensitizer was used as in the control experiment. The destruction of ABDA indicated the generation of singlet oxygen. Each assay was performed twice and the averaged taken.


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Molecular dynamics calculations. Parametrization of the cations and 1-octanol: The cations and 1-octanol structures were drawn in GaussView39 and firstly optimized at the B3LYP/6-31+G(d) level of theory40-42 in Gaussian09.43 We then followed the default antechamber44 protocol and settings to calculate all molecular mechanics parameters. Antechamber attributes GAFF45 parameters to atoms, bonds and angles, and calculates RESP point charges46 (at the HF/6-31G* level of theory)47 that are compatible with the amber force field.48 Preparation of the models: A cubic box of 300 1-octanol molecules with sides of 43 Å was built with the Packmol package.49 This software creates a packed box of the specified molecule that minimizes both steric hindrance and empty space.50 This box was further relaxed in a 1 ns molecular dynamics simulation with the isothermal-isobaric ensemble. The 300 1-octanol molecules optimized to a box of size of 43 Å x 43 Å x 43 Å and a density of 0.824 ± 0.003 (average over last half of the simulation; error is the standard deviation), which compares very well with the experimental density of 1-octanol at 20 ºC (0.827 g cm-3). We prepared the simulation boxes for every cation-solvent combination by adding 20 Å of either TIP3P51 water molecules or 1-octanol molecules (using the previous described box) in every direction from the cation. Minimization and molecular dynamics calculations: The protocol for the 14 molecular dynamics (MD) simulations (7 cations x 2 solvents) was the same: 1. Energy minimization of the solvent molecules; 2. Energy minimization of both solute and solvent; 3. Heating MD; 4. Equilibration MD; 5. Production MD. Both minimizations consisted of 2500 steps with the steepest descent algorithm followed by 2500 steps with



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the conjugate gradient algorithm. In the first minimization, the solute was restrained with a force constant of 10 kcal mol-1 Å-2. The heating MDs run for 1 ns in the canonical ensemble (NVT). The temperature was increased from 0 up to 300 K with the Langevin thermostat. We used the isothermal-isobaric ensemble (NTP) for the equilibration MDs. Temperature was kept constant at 300 K with the Berendsen thermostat, and the isotropic Berendsen barostat was used to keep the pressure constant at 1 bar.52 We ran the equilibration MDs for 1 ns for the simulation with water and 10 ns for the simulation with 1-octanol. These time lengths were enough to equilibrate the slowestconvergence property (solvent density) in the simulation boxes. Production MDs used the same parameters as equilibration MDs and run for 10 ns for all solute-solvent combinations. Explicit van der Waals interactions were truncated at 10 Å in all minimizations and molecular dynamics, and the Coulombic interactions were calculated with the particle mesh Ewald (PME) method,53 with the real part also truncated at 10 Å. We used the SHAKE algorithm in all MDs, which constrains the length of the bonds containing hydrogen atoms, to allow the use of a time step of 2 fs.54 All MD simulations were carried with the AMBER software. Calculation of the accessible volume and surface area: The solvent accessible surface area (SASA) and volume were calculated with the sasa command of Gromacs.55 A probe radius of 1.4 Å was used for both area and volume measurements. Values were calculated for 1000 equally space snapshots of the 10 ns production MDs.


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Thermodynamic

integration

calculations:

We

used

thermodynamic

integration

calculations to calculate the difference between hydration free energy and 1-octanol solvation free energy for every compound. We calculated the free energy associated with two alchemical transformations (Scheme 1): the annihilation of the solute in water and the annihilation of the solute in 1-octanol. The difference between these two energies is equivalent to the thermodynamic free energy of moving a solute molecule from water to 1-octanol. The MD sampling for each transformation was done in two steps: the first is the elimination of the atomic point charges, and the second the removal of the vdW components of the solute. The first step was divided in 4 windows, which correspond to lambdas 0.2, 0.4, 0.6, and 0.8. Since the effect of the neutralization of the atomic point charges in the potential energy is typically linear, four points are enough to calculate the TI integral. The second step was divided in 19 windows spaced by 0.05, from 0.05 up to 0.95. We used softcore potentials in the second step. For each window, 500 cycles of energy minimization and a 100 ps equilibration MD preceded the production dynamics. The production MD run for 1 ns in the water solvent systems, and 30 ns and 5 ns for the first step and second steps of the 1-octanol system. These larger simulation times were required to obtain well converged energies. TI calculations were done in AMBER 12. Besides the thermodynamic integration energy, we also calculated BAR and MBAR energies with the alchemical-analysis package.



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Scheme 1. Free energy associated with moving a solute molecule from water to 1-octanol.

ASSOCIATED CONTENT Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. Experimental details on synthesis and characterization are provided. AUTHOR INFORMATION Corresponding Author *Email: [email protected], [email protected], [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. §

F.G. and E.K.L.Y both jointly supervised the work.

ACKNOWLEDGMENT F.G. would like to thank NTU for a start-up grant (M4080552) and MOE Tier 1 grant (M4011441) for finantial support. P.A.F., A. J.M.R. and M.J.R. acknowledge financial support from the European Union (FEDER funds POCI/01/0145/FEDER/007728) and National Funds (FCT/MEC, Fundação para a Ciência e Tecnologia and Ministério da Educação e Ciência) under the Partnership Agreement PT2020 UID/MULTI/04378/2013.


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ABBREVIATIONS LC,

lipophilic

cations;

PDT,

photodynamic

therapy;

PS,

photosensitizer;

TPP+,

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Increasing the hydrophobicity and solvent accessible surface area of the widely-used triphenylphosphonium cation by methyl substitution at key position(s) of the phenyl rings substantially increases the mitochondrial uptake of conjugated fluorescent dyes which leads to enhanced photodynamic therapy against cancer cells.


Unique Triphenylphosphonium Derivatives for Enhanced

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