Adsorption of tetrakis(4-sulfophenyl)porphyrin onto liposomal surfaces composed of neutral diacylphosphatidylcholine and release by cyclodextrin

Anionic tetrakis(4-sulfophenyl)porphyrin (TPPS) interacts with liposomal surfaces composed of neutral diacylphosphatidylcholine at high lipid concentrations. TPPS interacted with liposomal surfaces through four contact points. The association constant was obtained to be 9.0 × 105 M−4. TPPS was peeled off the liposomal surfaces by the addition of cyclodextrin.


Results and discussion
Interaction between anionic porphyrin and neutral lipid The interactions between anionic porphyrin 3 and the neutral lipid 1 [1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), Fig. 1] were investigated. Concentration dependent UV-vis absorption spectra of 3 were measured by the addition of liposomes composed of 1 (liposomes-1) (Fig. 2) and the red shi in the Soret band was observed at 20 C (413 / 415.5 nm, DAbs ¼ 2.5 nm). The red shi was not because of the formation of onedimensional self-assembled structures because: (i) 3 cannot form self-aggregates under neutral conditions because protonation of 3 is essential for self-association, and (ii) the shi value is too small for self-aggregates because the Soret band of the porphyrin J-aggregates displayed a signicant red shi to 491 nm. [15][16][17][18] If sulfo groups of 3 interact with ammonio groups of 1 on the liposomal surface, the addition of cationic lipids in the liposome should facilitate the formation of strong interactions between anionic 3 and the liposomal surface via electrostatic interactions. [19][20][21] Therefore, a cationic lipid (2) was mixed with lipid 1 in the liposomes {[1] : [2]  Therefore, the red shi suggests an interaction between 3 and lipid 1. The shis of Dl max in the Soret band of 3 were plotted against the concentration of 1 in Fig. 2B. We were not able to determine the association constant between 3 and lipid 1 from a curve in Fig. 2B. The association constant determined from 1 H NMR spectrum is described below. In contrast, we investigated interactions between cationic porphyrin 4 ( Existence of porphyrin on the liposomal surface C 60 is known to act as a quencher in liposomes. 22 To conrm that 3 exists on the liposomal surface, we measured uorescence quenching of 3 by C 60 in lipid-membrane-incorporated C 60 (LMIC 60 , Fig. 3). The presence of C 60 led to a uorescence quenching of 48%, indicating that 3 exists in the neighborhood of C 60 .

Form of porphyrin on the liposomal surface
In the 1 H NMR spectra, 3 gives rise to two peaks arising from the phenyl protons in the ortho and meta positions because of the D 4h symmetry of 3 ( Fig. 4A and S2A †). Although the peak assigned to the phenyl protons in the meta position (8.2 ppm) appeared as a sharp doublet, the corresponding peak for the protons in the ortho position (7.7 ppm) was broader and this line-broadening was ascribed to self-aggregation of 3. 23,24 In Fig. 4B, the phenyl protons in the ortho positions of 3 were observed as a pronounced broaden peak (grey circle). 23 In contrast, when 3 interacted with liposome-1, two pairs of phenyl protons in the ortho and meta positions appeared in a 1 : 1 ratio [ Fig. 4B (red circles) and S2B †]. The result suggests the following three models for the interaction between 1 and 3: C 4v , C 2v and C 2v symmetries, depending on the interaction on the liposomal  surface (Scheme 2). In Scheme 2A, 3 interacts with liposomal surfaces by four point interactions. The porphyrin face of 3 is shaped asymmetrical at the upper and lower sides. If the rotation of phenyl units decreases and is slower than the NMR time scale because of steric hindrance by the liposome surface, the two ortho and two meta protons in one phenyl unit are in different chemical environments (see Scheme 2A) and give rise to the four signals in Fig. 4B (red circles). For the other possible models, 3 interacts with the liposomal surface by only two SO 3 À in the cis-position with C 2v (Scheme 2B) or the trans-position with C 2v (Scheme 2C), in which 3 interacts with the N + of P À -N + in liposome-1. Because 3 has different symmetries in the three models, the b-pyrrole protons should appear as one, four or two sets of peaks in the NMR spectrum for models in Schemes 2A, B and C, respectively. As shown in Fig. 4B (red circle), the bpyrrole protons appeared as a single broad peak, suggesting that the model in Scheme 2A is correct.

Association constant between anionic porphyrin and neutral lipid
As shown in Scheme 2A, porphyrin 3 interacted with the liposome surface consisted of lipids 1 through four point interactions. Therefore, the equilibrium is dened as (1): The concentrations of free 3 and 3 on the liposomal surface were determined by the peak intensities in Fig. 4B. When these values were substituted into eqn (2), we obtained the association constant (K a ) ¼ 9.0 Â 10 5 M À4 .
Porphyrin derivatives can form a 1 : 2 complex with TMe-b-CDx. [24][25][26] Furthermore, all of the tetraphenylporphyrin (5) were released from the TMe-b-CDx cavities and transferred to the lipid membrane aer mixing the 5$TMe-b-CDx complex with liposome-1 at 30 C for 1 h. 25 Therefore, we attempted the exchange reaction of 3 from TMe-b-CDx cavities to lipid membranes or the formation of direct interactions between liposome surfaces and the 3$TMe-b-CDx complex. Aer mixing the 3$TMe-b-CDx complex with liposome-1 under the same conditions reported previously, 25 all peaks assignable to TMe-b-CDx in the 3$TMe-b-CDx complex remained in the 1 H NMR spectrum (blue circles in Fig. 4D, E, S2D and E †). The result shows that porphyrin 3 did not transfer from the TMe-b-CDx cavities to the liposomes and the porphyrin remained in the TMe-b-CDx cavities (Scheme 1C). This observation suggests that 3 is too hydrophilic to incorporate into the hydrophobic lipid membrane. Furthermore, no chemical shi changes of these peaks indicate that the 3$TMe-b-CDx complex did not interact with the liposome surface by only a single point interaction because of steric hindrance by the two TMe-b-CDxs. Because K a for the four point interactions was 9.0 Â 10 5 M À4 , K a ' per a single point interaction was estimated to be approximately 30 M À1 at most even if entropy and enthalpy gains by multi point interactions were excluded. Therefore, the 3$TMe-b-CDx complex cannot interact with the liposome surface by only a single point interaction. Consequently, the interactions between 3 and 1 need at least four point interactions.

Exfoliation of porphyrin from the liposome surface by cyclodextrin
To control the association-dissociation of 3 onto the liposomal surface, TMe-b-CDx was added to the mixture of 3 and liposome-1. Aer adding TMe-b-CDx, although the l max of the Soret band of 3 is barely shied (i.e., 415 to 416 nm), the l max in the Q-band of 3 is shied from 517 to 511 nm. These l max were the same as that observed for the 3$TMe-b-CDx complex (416 and 511 nm) ( Fig. 2A, green and orange). Furthermore, peaks were observed in the 1 H NMR spectrum for the 3$TMe-b-CDx complex ( Fig. 4C and S2C †). The results show that most of 3 peeled off the liposomal surface and had formed a complex with two TMe-b-CDxs (Scheme 1B).

Conclusions
In summary, anionic 3 was adsorbed onto liposomal surfaces composed of neutral lipid 1 at high lipid concentrations. The interaction of 3 with 1 was via four contact points. In contrast, the 3$TMe-b-CDx complex cannot be adsorbed onto liposomal surfaces because 3 interacts with 1 by only a single point and is encapsulated by two TMe-b-CDxs. Addition of TMe-b-CDx caused the release of 3 from the liposomal surface and complex formation with two TMe-b-CDxs. Consequently, the association-dissociation of 3 onto the liposomal surfaces can be controlled by the addition of TMe-b-CDx.

Preparation of liposome-1 and liposome-1-2
An appropriate amount of 1 or a mixture of 1 and 2 ([1] : [2] ¼ 7 : 3 mol mol À1 ) was dissolved in chloroform. The solvent was evaporated under a gentle stream of nitrogen, followed by a period under vacuum to remove any traces of solvent. The resulting thin lipid lms were hydrated on the wall of the vial above the phase transition temperature with an appropriate amount of phosphate buffer. The hydrated materials were subjected to eight freeze-thaw cycles (À195 and 50 C) to give unilamellar vesicles, which were extruded 11 times through 0.05 mm pores using a LiposoFast miniextruder from Avestin (Ottawa, Canada) above the phase transition temperature. The resulting liposomes were uniform in size with a diameter of approximately 80 nm. The nal lipid concentration was 3.0 mM.

Preparation of the 3$TMe-b-CDx complex
Compound 3 (5.00 mg, 5.26 Â 10 À6 mol) and TMe-b-CDx (15.0 mg, 1.05 Â 10 À5 mol) were placed in an agate capsule with two agate-mixing balls. The resulting mixture was agitated vigorously at 30 Hz for 20 min using a high-speed vibration mill (MM 200; Retsch Co., Ltd., Haan, Germany). The solid mixture was suspended in either phosphate buffer or D 2 O-phosphate buffer (1.5 mL) to produce a dark purple emulsion. Subsequent centrifugation (18 000 Â g, 25 C, 20 min) removed nondispersed 3 from the solution. The concentration of 3 in the 3$TMe-b-CDx complex was determined to be 0.03 mM by measuring the absorbance of the solution at l max in water. The molar absorption coefficient for the water-soluble 3$TMe-b-CDx complex is 3 416 ¼ 3.79 Â 10 5 dm 3 mol À1 cm À1 .

Conflicts of interest
There are no conicts to declare.