Spectroscopic Investigations on the Binding of the Photosensitizer Chlorin p 6 with Amine-modiﬁed Silica Nanoparticles in Aqueous Media Beena Jain, Abha Uppal, Pradeep K. Gupta and Kaustuv Das* Laser Bio-Medical Applications & Instrumentation Division, Raja Ramanna Center for Advanced Technology, Indore, Madhya Pradesh, India Received 18 November 2008, accepted 9 January 2009, DOI: 10.1111 ⁄ j.1751-1097.2009.00545.x ABSTRACT Absorption and emission properties of the amphiphilic photo- sensitizer Chlorin p 6 were investigated in aqueous medium in the presence of silica nanoparticles (SiNPs) having positively charged amino groups. The results of these studies reveal that the acid–base ionization equilibrium of Chlorin p 6 in aqueous medium is signiﬁcantly affected as a result of strong electrostatic binding between the negatively charged drug and SiNP. The spectroscopic signature of the drug bound to SiNPs suggests that the tri-anionic form of the drug remains bound to the positively charged SiNPs at pH 8.0. As the pH is progressively decreased the formation of hydrophobic aggregates is disrupted signiﬁ- cantly due to the presence of electrostatic binding force, which competes with intermolecular hydrophobic forces. The interplay of hydrophobic and electrostatic forces in the drug–nanoparticle binding process might affect the relative uptake and photo- dynamic efﬁcacy of the free drug and the drug–nanoparticle complex in cancer cells. INTRODUCTION Chlorin p 6 is an interesting photosensitizer as it has a strong absorption band at 660 nm which lies in the so-called therapeutic window (650–900 nm), a fairly good triplet yield and good tumor selectivity (1–4). The photophysical and photochemical parameters of different porphyrin and chlorin type photosensitizers have been studied earlier (5). Chlorin p 6 has three ionizable carboxylic acid groups and the different ionic structures involving protonation–deprotonation of the three carboxylic acid groups of the molecule are shown in Fig. 1. It is pertinent to note that the sequence of the structures shown in Fig. 1 is only notional. The pH-depen- dent acid–base equilibrium of Chlorin p 6 has been studied by absorption and ﬂuorescence spectroscopy (6). It has been observed that at physiological pH the drug exists predomi- nantly as negatively charged (i.e. closer to structure D in Fig. 1) and as the pH is progressively lowered, successive protonation of the carboxyl groups (forming structures C to A in Fig. 1) results in aggregation of the drug in aqueous medium. The hydrophobic aggregates were observed to be nonﬂuorescent (6). It has been observed that by controlling the relative populations of the species A–D, pH plays an important role in the relative binding of the drug with various systems like micelles, liposomes, proteins and bio- polymers (6–11). Indeed it was observed that with a decrease in the pH of the incubation medium (from 7.4 to 6.0) the uptake of the drug increased in human colon adenocarci- noma (Colo-205) cells (4). Further, it has also been observed that the electrostatic binding of the drug with charged surfactants can affect the ionization equilibrium of the drug in aqueous medium (7,8). Thus it is to be expected that electrostatic binding with charged silica nanoparticles (SiNPs) would affect its acid–base ionization equilibrium and thus cellular uptake. To investigate this aspect we investigated the acid–base ionization equilibrium of the negatively charged drug and positively charged SiNP by monitoring the spec- troscopic (absorption and ﬂuorescence) properties of the drug. MATERIALS AND METHODS Vinyl-triethoxysilane (VTES), 3-aminopropyl-triethoxysilane (APTS) and Aerosol OT (AOT) were purchased from Sigma and used as received. Chlorin p 6 was synthesized in-house following the procedure described in Datta et al. (6). All the solvents used were of spectroscopic grade. Amine-modiﬁed SiNPs were synthesized in the nonpolar core of AOT ⁄ 1-Butanol ⁄ water micellar system using VTES and APTS as described in the literature (12). The SiNPs were puriﬁed from AOT and butanol by dialyzing (using a membrane having a molecular cutoff of 10 kDa) against neutral water for a period of 96 h. The average diameter of these particles was 25 nm as determined by transmission electron microscopy. At neutral water (pH = 7.0) the amine groups present at the surface are expected to be positively charged as the pK a value of the 3-aminopropyl group of APTS is 9 (13). The concentration of Chlorin p 6 was ﬁxed at 1 lM for all experiments. The pH of the aqueous medium was varied between 3.0 and 8.0 by adding microliter amounts of 1(N) HCl or NaOH. Experiments were not conducted below pH 3.0 as it resulted in nanoparticle aggregation and not above pH 8.0 to ensure that all the 3-amino propyl groups on the surface of the SiNP remain positively charged to bind the drug. Absorption and emission spectra of Chlorin p 6 were recorded with 1 and 4 nm resolution, respectively. Emission lifetimes of the drug were recorded by excitation at 405 nm using a picosecond diode laser and using the time-correlated single photon counting module Lifespec-RED from Edinburgh Instruments, the details of which have been described earlier (9). The instrument response function of this setup is 200 ps. The lifetime values of the drug were calculated by using the software supplied by the manufacturer. The goodness of the ﬁt was judged by the chi-squared values and by visual inspection of the residuals. The residuals for some of the decays (described in Figs. 3–5) are provided in the Supporting Information. *Corresponding author email: kaustuv@rrcat.gov.in (Kaustuv Das) Ó 2009 The Authors. Journal Compilation. The American Society of Photobiology 0031-8655/09 Photochemistry and Photobiology, 2009, 85: 927–933 927