B. Jain et al. / Journal of Molecular Structure 1060 (2014) 24–29
25
hydrophobic photosensitizers has attracted tremendous interest
2. Materials and methods
for their use in PDT of cancer. Different types of nanoparticles
are being currently explored for the delivery of such drugs [5–
10]. For drug delivery it is required that the NPs are biocompatible,
stable in physiological conditions and able to carry the drug
without any chemical alteration [11]. Among these, organically
modified silica nanoparticles (SiNPs) prepared using triethoxyvi-
nylsilane and/or 3-aminopropyl triethoxysilane have shown to sat-
isfy these features [12–15]. Due to the presence of a hydrophobic
core, these NPs can be loaded with hydrophobic drugs and since
the surface is charged these can be easily solublized in aqueous
medium [11–15,16]. Such NPs have been used for gene delivery,
entrapping dyes/probes for imaging, and hydrophobic photosensi-
tizers for PDT, etc. [17].
All the reagents used in the experiment were of spectroscopic
grade. These were mainly surfactant dioctyl sulfosuccinate (AOT,
99%), poly-lactic-co-glycolic acid (PLGA), phosphatidylcholine and
polyvinyl alcohol (PVA) from Sigma, co-surfactant 1-butanol, ace-
tone from Merck, triethoxyvinylsilane (VTES, 97%) and 3-amino-
propyl triethoxysilane (APTS 99%) from Acros chemicals and Fetal
bovine serum (serum, used as 10% by volume) from Himedia,
Mumbai, India. All the reagents were used as received. SiNPs were
synthesized in the non polar core of AOT/1-Butanol/water micellar
system using VTES and APTS silica precursors as described in liter-
ature [12]. In short SiNPs were synthesized in the non polar core of
AOT/1-Butanol/water micellar system. The micelles were prepared
Earlier, it has been reported that purpurin 18 (Pp18), a hydro-
phobic chlorophyll derivative, can be incorporated in liposomes
and the formulation was found to deliver the photosensitizer suc-
cessfully in human colon cancer cells [18]. Pp18 shows promising
optical properties for use in PDT such as an absorption band at
ꢁ 700 nm with extinction coefficient ꢁ41,800 MÀ1 cmÀ1. However,
since Pp18 contains an anhydride ring in the molecule, it under-
goes rapid hydrolysis at physiological pH (shown in Scheme 1)
which results in the formation of chlorin p6 (Cp6), a water soluble
by dissolving 0.44 g AOT and 800
by vigorous magnetic stirring. Neat VTES (200
micellar system and the resulting solution was stirred until clear.
After this20 l of either ammonia solution (to make SiNP-V) or
l
l of 1-butanol in 20 ml of water
ll) was added to the
l
APTS (to make SiNP-VA) was added and stirred for about 20 h.
The entire reaction was carried out at room temperature. At the
end of the process, a bluish white translucency indicating the for-
mation of nanoparticles was observed. The SiNPs were purified
from AOT and butanol by dialyzing (using a membrane having a
molecular cut-off 10 kDa) against neutral Millipore water for a
period of ꢁ96 h. Nano-PLGA formulations were prepared by
nano-precipitation technique with minor modifications [22]. In
brief, 90 mg of PLGA was dissolved in 10 ml of HPLC grade acetone
over a period of 3 h to get a uniform PLGA solution. This solution
was added drop wise to 20 ml of aqueous solution containing
1.5% of PVA over a period of 10 min on a magnetic stir plate oper-
ated at 800 rpm. Within a few minutes, precipitation of nanoparti-
cles was observed. This suspension was stirred at room
temperature for 24 h to evaporate the acetone completely. Larger
aggregates and free PLGA/PVA polymers were removed by centri-
fugation at 5000 rpm for 10 min. Then centrifuged at 18,000 rpm
for 1 h and washed the sediment with water. Small unilamellar lip-
osomes were prepared by ethanolic injection method. In short
photosensitizer, having
Q band absorption blue shifted to
ꢁ656 nm with decreased extinction coefficient (ꢁ24,800 MÀ1
-
cmÀ1). Although it was possible to stabilize Pp18 in liposomes pre-
pared at lower pH (6.0) and thus deliver Pp18 in cells [18], during
incubation for time periods beyond 1 h significant conversion of
Pp18 into Cp6 was observed [18]. Other carriers such as Cremophor
EL and Poly L lactic acid nanofibers have also been used to carry
Pp18 [19,20]. Recently, Droget et al. reported synthesis of a stable
and water soluble Pp18 in which the anhydride ring is chemically
modified into cycloimide and the carboxylic group at ‘17’ carbon
position (IUPAC system) is attached to polyethylenimine, a water
soluble polycationic molecule [21]. However, the singlet oxygen
yield of this derivative was substantially compromised (ꢁ 4 times
lower) as compared to Pp18.
100 l )
l ethanolic solution of phosphatidylcholine (20 mg mlÀ1
In this paper we investigated spectroscopically the suitability of
SiNPs as carriers of Pp18 in comparison with liposomes and poly-
meric NPs, which are the conventionally used drug carriers. This
was studied by monitoring the stability of Pp18 against its conver-
sion to its hydrolytic product Cp6 in aqueous buffer (at physiolog-
ical pH). To mimic biological environment, the same study was
done in the presence of 10% serum (as it forms the major part of
a biological system). The conversion was monitored by measuring
the absorption and fluorescence of the drug in the four NP systems
at various time intervals, over the period of 24 h.
was injected into 1.0 ml solution of 10 mM phosphate buffer at
pH 7.4. Purpurin 18 and Cp6 were prepared from dry spinach leaves
following the reported procedure [23]. The stock was prepared in
acetone.
The size and zeta potential of these nanoparticles was measured
using a 90 Plus particle size analyzer from Brookhaven Instruments
Corporation, USA. The average diameter of both SiNP particles was
measured to be ꢁ 32 nm using dynamic light scattering and the
zeta potential of the SiNP-VA and SiNP-V was ꢁ À 36.0 3.0 mV
Scheme 1. Chemical structure of Pp18 and Cp6 and the conversion of Pp18 to Cp6, due to its hydrolysis in the presence of water.
Jain, Beena
