Tetraphenylporphyrin tethered chitosan based carriers for photochemical transfection

Article abstract of DOI:10.1021/jm301270r

Water-soluble amphiphilic chitosan nanocarriers tethered with the photosensitizer meso-tetraphenylporphyrin were synthesized in a seven-step procedure, starting from 3,6-di-O-tert-butyldimethylsilyl-chitosan and 5-(p-aminophenyl)-10,15,20-triphenylporphyr

Full text of DOI:10.1021/jm301270r

Article
pubs.acs.org/jmc
Tetraphenylporphyrin Tethered Chitosan Based Carriers for
Photochemical Transfection
Vivek S. Gaware,†,‡ Monika Hakerud,‡,§ Kristjan Leosson,∥ Sigrídur Jonsdottir,⊥ Anders Høgset,‡
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́
́
́
Kristian Berg,§ and Mar Masson*,†
́
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†Faculty of Pharmaceutical Sciences, School of Health Sciences, University of Iceland, Hofsvallagata 53, IS-107 Reykjavík, Iceland
‡PCI Biotech AS, N-1366 Lysaker, Norway
§Oslo University Hospital, The Norwegian Radium Hospital, Institute for Cancer Research, Department of Radiation Biology,
N-0310 Oslo, Norway
∥Department of Physics, Science Institute, University of Iceland, Dunhagi 3, IS-107 Reykjavik, Iceland
⊥Department of Chemistry, Science Institute, University of Iceland, Dunhagi 3, IS-107 Reykjavik, Iceland
S
* Supporting Information
ABSTRACT: Water-soluble amphiphilic chitosan nanocarriers
tethered with the photosensitizer meso-tetraphenylporphyrin
were synthesized in a seven-step procedure, starting from 3,6-di-
O-tert-butyldimethylsilyl-chitosan and 5-(p-aminophenyl)-
10,15,20-triphenylporphyrin. The lipophilic photosensitizer
could be introduced in a quantitative and reproducible reaction
to give either 0.1 or 0.25 degrees of substitution per
glucosamine monomer. Fluorescence and NMR investigations
revealed the dynamic structures of the carriers, which formed
nanoparticles in aqueous solution with a core of π-stacked
photosensitizers. These carriers can then unfold in the lipophilic environment, and the photosensitizer moiety can thus be
inserted into the cell membrane. The efficacy of the carriers for photochemical internalization (PCI) mediated gene delivery was
evaluated in vitro using the HCT116/LUC human colon carcinoma cell line. The efficacy of transfection was comparable to what
could be achieved by the reference compound and current clinical candidate TPCS2a.
accumulating in solid tumors, PCI can be highly specific; this
also contributes to the enhanced antitumor efficacy of the
drug.6
INTRODUCTION
■
Photochemical internalization (PCI) is a technology which
utilizes selected photosensitizers in combination with light to
induce release of trapped drugs and macromolecules from
endosomes into the cytosol.1 As in photodynamic therapy
(PDT), light activation of the photosensitizer (PS) after its
accumulation in the target tissue leads to the formation of
reactive oxygen species (ROS) which can kill cells directly by
apoptosis, necrosis, and autophagy or indirectly by vascular
shutdown and induced inflammatory responses.2 In PCI, light
excitation leads to ROS mediated damage to lysosomal and/or
endosomal membranes and the release of entrapped hydro-
philic drugs and macromolecules. Thereby, endocytosed
molecules can be released to reach their target of action before
being degraded in lysosomes.3 PCI has been shown to enhance
the biological activities of a variety of macromolecules that do
not readily penetrate through the plasma membrane. These
include type I ribosome-inactivating proteins, immunotoxins,
chemotherapeutic agents such as bleomycin and doxorubicin,
gene encoding plasmids, adenoviral-based gene delivery, and
oligonucleotides.4 It has been found to induce cytotoxicity in
deeper tissue layers than the corresponding PDT.5 Because of
the combination of targeted therapeutics along with the light-
activated cytosolic delivery induced by PS preferentially
The most hydrophilic PSs, such as aluminum phthalocyanine
tetrasulfonate (AlPcS4) and uroporphyrin (UP), do not insert
well into cell membranes and are therefore not suitable for PCI,
whereas amphiphilic PSs like meso-tetraphenylporphyrin
disulfonate (TPPS2a) and aluminum phthalocyanine disulfonate
(AlPcS2a) are most effective in PCI.7 They can be inserted into
plasma membranes but are unable to penetrate through this
barrier and will therefore be taken into cells by endocytosis.
The current clinical candidate for PCI is meso-tetraphenyl-
chlorin disulfonate (TPCS2a).8 A phase I/II study of TPCS2a
based PCI of bleomycin in patients with local recurrence or
advanced/metastatic cutaneous and subcutaneous malignancies
resulted in complete clinical regression of target tumors in
87.5% (14 of 16) of patients completing the first assessment
and in 72% (8 of 11) of patients completing the second
assessment.2,9
One of the common problems associated with PDT in
clinical application is skin photosensitivity and unfavorable
Received: September 4, 2012
Published: January 3, 2013
© 2013 American Chemical Society
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a
Scheme 1. Synthesis of Porphyrin Intermediate 5
a
Reagents and conditions: (a) propionic acid, reflux, 30 min (20%); (b) NaNO2 (1.8 equiv), TFA, 25 °C, 3 min (67%); (c) SnCl2·2H2O, concd
HCl, 60 °C, 1 h (88%); (d) bromoacetyl bromide, Et3N, CH2Cl2, 25 °C, 1 h (64%); (e) piperazine, CH2Cl2, 25 °C, 1 h (94%).
biodistribution of the PS.10 Nanocarriers such as dendrimers,
liposomes, and polymeric micelles have been introduced as an
approach to reduce side effects and improve pharmacokinetics
in PDT.11 One specific advantage of such systems is that they
have shown tumor selective accumulation due to the enhanced
permeation and retention (EPR) effect. PS linked cationic
polyamidomine (PAMAM) dendrimers and anionic dendrimer
photosensitizer-incorporated micelles have recently been
shown to be effective for PCI mediated gene delivery.12
Many such polymeric systems can be considered, but the
preferred delivery system has to be built from biocompatible,
nontoxic, and biodegradable entities.
of CS including N-(chloroacetyl)-chitosan, N-(2-(N,N,N-
trialkylammoniumyl)acetyl-chitosan,24 and N,N,N-trimethyl-
chitosan homopolymer.25 Silyl protected CS (DiTBDMS-CS)
is compatible with low polarity aprotic solvents such as
dichloromethane. This property is especially useful when
modification with lipophilic moieties is desired.
The aim of the current study was to develop novel
macromolecular nanocarriers based on PS functionalized
water-soluble CS derivatives. The highly lipophilic PS meso-
tetraphenylporphyrine (TPP) (log P ≥ 9) was covalently
attached to N-(bromoacetyl)-3,6-di-O-tert-butyldimethylsilyl-
chitosan (BrA-DiTBDMS-CS), and these materials were then
reacted with either trimethylamine (TMA) or 1-methylpiper-
azine (MP) in order to introduce a hydrophilic cationic moiety.
These reactions were carried out in organic solvent in order to
enable quantitative and reproducible attachment of functional
moieties to the polymer backbone. Finally, the TBDMS
protecting groups were removed and water-soluble carriers
were obtained. These carriers were designed to have properties
that should contribute to optimal endosomal uptake and PCI
efficacy. The structures of the macromolecular carriers, with
different degrees of TPP substitution, and all intermediates in
the synthesis were characterized by NMR and FT-IR. The
physicochemical properties of the carriers were investigated
with NMR, UV−vis, and fluorescence spectroscopy, scanning
electron microscopy (SEM), and dynamic light scattering
(DLS).
Chitosan (CS) (poly-β-1−4-N-glucosamine) is a nontoxic,13
biocompatible, and biodegradable polymer whose physiological
inertness contributes to the considerable interest in its use as a
pharmaceutical excipient.14 It is used as a neutraceutical and has
been investigated in mucoadhesive drug delivery systems and as
an absorption enhancer to stimulate opening of the tight-
junction barrier in epithelial membrane15 and to form
polyplexes with DNA plasmids for application in gene
delivery.16 However, low solubility at physiological pH is a
drawback for many applications of unmodified CS.17
Various CS derivatives have been designed and synthesized
in order to enhance solubility and further improve its physical,
chemical, and biological properties.18 For this purpose, some
protecting groups have been introduced to allow fully
chemoselective modification. Kurita and co-workers have
introduced procedures for N-phthaloyl protection of the
amino group and 6-O-trityl protection of the primary hydroxyl
group.19 Trityl protected CS have been used for the synthesis
of N-betaine,20 N-chloroacyl,21 and quaternary piperazine
derivatives of CS.22 We have recently reported 3,6-di-O-tert-
butyldimethylsilyl-chitosan (DiTBDMS-CS)23 as a useful
intermediate for N-selective modification of the polymer
backbone as well as the synthesis of more complex derivatives
The efficacy of the TPP tethered CS carriers for PCI
mediated gene delivery was evaluated in vitro using the
HCT116/LUC human colon carcinoma cell line.
RESULTS AND DISCUSSION
■
Synthesis and Characterization. Porphyrins can localize
in tumor tissue and have been investigated as sensitizing drugs
for application in PDT.26 The biological efficacy of a PS is
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a
Scheme 2. Synthesis of N-Modified CS Nanocarriers (16−19)
a
Reagents and conditions: (a) MeSO3H/H2O, 10−25 °C, 1 h (90%); (b) TBDMSCl, imidazole, DMSO, 25 °C, 24 h (96%); (c) bromoacetyl
bromide, Et3N, CH2Cl2, −20 °C, 1 h (92%); (d) compound 5 (0.1 or 0.25 equiv), Et3N, CHCl3, 25 °C, 12 h (90%); (e) NMe3 or 1-
methylpiperazine, CHCl3, 25 °C, 24 h; (f) concd HCl/MeOH, 25 °C, 24 h (batch A) or TBAF, NMP, 55 °C, 24 h (batch B).
dependent on efficient insertion into cellular membranes and
delivery into specific organelles within cancer cells. Amphiphilic
TPP derivatives bearing one, two, or three water solubilizing
did not give desired results in our initial experiments. Thus, in
order to convert this photosensitizer derivative into a more
potent nucleophile, aminoporphyrin 3 was first acylated using
bromoacetyl bromide to give 5-(4α-bromoacetylamidophenyl)-
10,15,20-triphenylporphyrin (4), followed by nucleophilic
substitution with excess piperazine to give nucleophilic
porphyrin intermediate 5-(4α-piperazineacetylamidophenyl)-
10,15,20-triphenylporphyrin (TPP-NH-Pip) (Scheme 1). The
piperazine motif has a positive charge under physiological
conditions (aqueous pH 7.4). The piperazine moiety is also
suited as a spacer due to its low toxicity and biotransformation
that involves several well-known metabolic reactions.31
+
groups like trimethylammonium (−N(CH3)3 ), sulfonate
−
(−SO3 ) etc. have demonstrated increased photodynamic
efficacy as compared to hydrophilic, symmetric macrocycles.27
TPP is one of the most commonly used porphyrinic system in
various fields such as chemistry, medicine, biochemistry,
physics, and materials sciences28 A variety of methodologies
for derivatization of TPP are well established, including
regioselective modifications such as mono-, di-, or tri-para-
phenyl nitration of TPP.29
Nucleophilic Porphyrin Intermediate. In the current
study, 1 (TPP) (see Experimental Section) was synthesized on
a large scale and used as the starting material. The mononitro
functionality was introduced regioselectively using 1.8 equiv of
NaNO2 in TFA to give 5-(p-nitrophenyl)-10,15,20-triphenyl-
porphyrin (2), followed by reduction with SnCl2·2H2O to
obtain the mono-aminoporphyrin, 5-(p-aminophenyl)-
10,15,20-triphenylporphyrin [TPP(p-NH2)1]. The previously
reported procedure29 was simplified by avoiding time-
consuming purification of the crude material 2 before the
reduction step. Aminoporphyrin 3 [TPP(p-NH2)1] was then
obtained after purification without compromising its overall
yield (Scheme 1).
Electrophilic CS Intermediate. CS was modified as
previously reported method23 to obtain TBDMS protected
chitosan 8. After protection of the hydroxyl groups, the
solubility of the biopolymer changes dramatically and it
becomes freely soluble in common organic solvents like
CH2Cl2, which facilitates its modification with lipophilic
moieties. Thus the electrophilic intermediate 9 was prepared
by reacting 8 with bromoacetyl bromide. This reaction requires
precise control of the reaction time, temperature, and
equivalent ratios of the different reagents in order to avoid
side reactions like deprotection and esterification. Reaction of 8
with bromoacetyl bromide at 0 °C for 1.5 h resulted in material
that was insoluble in CH2Cl2; FT-IR analysis revealed an ester
peak at 1760 cm−1 along with the amide peak at 1680 cm−1
(data not shown). Thus, it was necessary to precisely optimize
The aminoporphyrin 3 is known to be weakly nucleophilic,30
and attempts to link this compound to 9 (BrA-DiTBDMS-CS)
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the reaction in order to avoid side reactions and to obtain freely
soluble material. The optimized protocol for this reaction
required the use of exactly 4 equiv of bromoacetyl bromide and
5 equiv of Et3N and maintaining the reaction temperature at
−20 °C for 1 h. The obtained intermediate 9 was fully soluble
in CH2Cl2, and the correct structure could be confirmed by 1H
NMR and FT-IR. Compound 9 was then used as a key
electrophilic intermediate in the synthesis of the TPP modified
CS carriers (Scheme 2).
TPP Modified CS Nanocarriers (16−19). Four CS
carriers were synthesized as shown in Scheme 2. TPP was
covalently attached to the CS backbone by reaction of 9 with
the nucleophilic intermediate 5 (TPP-NH-Pip). The reaction
was quantitative, which facilitated good control of the degree of
substitution per glucosamine monomer (DS) in the resulting
material. Preliminary investigations had shown that carriers
with a high DS of porphyrin were insoluble in aqueous
solution; the modification was therefore limited to 0.1 and 0.25
DS.
Figure 1. Representative FT-IR spectra overlay of all intermediates
and the final carrier in the synthesis of 18: (A) 5, (B) 9, (C) 10, (D)
14, (E) 18 (material deprotected with HCl, batch A).
Thus, 5 was reacted in a controlled manner at either 0.1 or
0.25 equiv per glucosamine monomer unit of compound 9 to
afford the desired 0.1 or 0.25 DS products 10 or 11,
respectively. Progress of the reaction was monitored by TLC,
and the reaction was stopped when 5 was completely
consumed in order to avoid side reactions. H NMR analysis
of these materials was consistent with a quantitative reaction in
the covalent linkage of TPP to CS.
Cationic moieties were then introduced on the TPP
substituted CS backbone in order to enhance the aqueous
solubility of the carriers and provide affinity for the endocytic
membrane. Therefore, compounds 10 and 11 were reacted
with excess trimethylamine to yield trimethylammoniumyl
(TMA) derivative 12 and 13, respectively, with a fixed cationic
charge. Similarly, compounds 10 and 11 were reacted with
excess 1-methylpiperazine to yield 1-methylpiperazinyl (MP)
derivative 14 and 15, respectively. The crude materials were
used directly for the final deprotection steps.
bonds, and three strong peaks at 966, 799, and 700 cm−1 are
characteristic of the TPP ring system (shown by blue arrows).
The main characteristic peaks of the CS intermediate 9 were
observed at 2858−2957 cm−1, showing C−H stretching of Si−
CH3 as well as the amide peaks at 1682 and 1530 cm−1.
Additional peaks at 836 and 777 cm−1 represent Si−C
stretching and CH3 rocking (Figure 1B) (the last two peaks
are indicated by red arrows).
The appearance of characteristic TPP peaks in the spectrum
of intermediate 10 (Figure 1C) confirmed the covalent
attachment of porphyrin to CS. There was no marked change
observed in the spectrum when this was converted to crude 14.
However, the final TBDMS deprotection of the material to give
final carrier 18 was clearly indicated by the absence of the
characteristic Si−CH3 peaks and the appearance of the broad
O−H peak, whereas the characteristic TPP peaks remained
intact (Figure 1E).
1
1H NMR Analysis. A representative example of the overlay
of 1H NMR spectra of all intermediates and the final product in
the synthesis of final carrier 18 is shown in Figure 2. After
TBDMS protection, the material 8 became cleanly soluble in
CDCl3 and also showed well resolved peaks (Figure 2A) for
each proton in the CS backbone, with peaks at 0.90−0.89 (br s)
and 0.13−0.05 (br s) which can be assigned to (CH3)3C−Si
and (CH3)2Si, respectively.23b
Finally, the crude materials from the first batches of
compounds 12−15 were deprotected by concentrated HCl in
MeOH at room temperature for 12 h to give the corresponding
final products 16−19 (denoted as 16A−19A, for first batch
compounds), whereas crude materials from the second batches
were deprotected by TBAF/NMP at 60 °C for 24 h to give the
corresponding final products (denoted as 16B−19B, second
batch compounds), respectively. In both of the cases, the
deprotection step was repeated in order to remove trace
amounts of TBDMS (∼1.5−7%), which was still present after
the initial deprotection step. Deprotection with concentrated
HCl in MeOH was used in our earlier work,24 but recently the
milder TBAF/NMP deprotection of 8 derivatives has been
introduced.25 However, the disadvantage of the latter method is
that it requires a lengthy dialyzing process in order to remove
the NMP solvent. The final TMA derivatives of TPP-CS, 16
and 17, solubilized faster in water than the MP derivatives of
TPP-CS, 18 and 19. This may be due to the presence of the
fixed ionic charge in the former carrier compounds.
Bromoacetylation of 8 to give 9 was marked by a downfield
shift of the H-2 (GluN) peak. Also, all the CS backbone peaks
H-1 to H-6 along with the COCH2Br peaks came together at
4.40−3.26 ppm, while the TBDMS peaks shows no significant
change in position (Figure 2B).
1
The H NMR spectrum of the intermediate 10 (Figure 2C)
confirmed the characteristic peaks of the TPP moiety which can
be seen in the aromatic region. A peak at −2.7 ppm (not shown
in Figure 2C), typically associated with two pyrrolic inner
amine protons in the core of the free base porphyrin, could also
be identified. In addition, one distinct peak that can be assigned
to the half of the cyclic −CH2 group of the piperazine moiety
could be observed at 2.81−2.86 (m) ppm, and the remaining
cyclic −CH2 group of the piperazine fell under the broad region
(3.3−4.5 ppm) of the CS moiety.
FT-IR Analysis. A comparison of FT-IR spectra of various
intermediates in the synthesis of the representative final
compound 18 is shown in Figure 1. The characteristic peaks
for the porphyrin intermediate 5 (Figure 1A) are illustrated as
N−H stretching in the region 3310−3450 cm−1 and by
aromatic and aliphatic C−H stretching at 3022 and 2816 cm−1.
Peaks at 1691 and 1595 cm−1 are consistent with the amide
In the spectrum of 14, new peaks for the N-CH3 of the 1-
methylpiperazine moiety (Figure 2D) could be observed.
Purification was not done at this stage, so the spectrum thus
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Figure 2. Representative 1H NMR spectra overlay of all intermediates and the final carrier in the synthesis of carrier 18: (A) 8, (B) 9, (C) 10, (D)
14, (E) 18 (material deprotected with HCl, batch A).
shows excess 1-methylpiperazine peaks. After the final
deprotection step, a material with excellent aqueous solubility
1
was obtained. In the H NMR spectra of final carrier 18 in
D2O, all peaks of the CS backbone along with 1-
methylpiperazine peaks can be clearly identified; however,
there is no observable NMR signal for the TPP moiety (Figure
2E). The same results were observed for all deprotected final
carriers (16−19). In DMSO-d6, the TPP peaks could be
observed, but in this case the CS backbone peaks were
broadened and not clearly identifiable. The solubility of the
carriers were also poor in pure DMSO-d6, and it was discovered
that the best results were obtained using a mixture of these two
solvents (see later discussion). Compound 18 had a dark-red
Figure 3. Partitioning of compounds in two-phase system
(aqueous:CHCl3): (A) 3, (B) 5, (C) 17, (D) 19 (aqueous phase on
top).
color confirming the presence of the PS. A partitioning
experiment showed that the highly lipophilic PS moieties (3
and 4) did not partition into the aqueous phase in a two-phase
H2O/CHCl3 system (Figure 3), whereas the final nanocarriers
merged with the glucosamine signals in the 50−55 ppm region.
The solid-state 13C NMR were therefore consistent with the CS
carriers having covalently linked PS.
(16−19) were amphiphilic. They were fully solubilized in the
aqueous phase and did not partition into the organic phase.
Solid-State 13C NMR Analysis. The carbon peaks of the
final carriers could be observed by solid-state NMR (Figure 4).
The C2−C6 carbons of the glucosamine and the COCH2
carbon were observed in the 60−80 ppm range, and the C1
peak was clearly resolved at ∼102 ppm. The peaks between
115−155 ppm can be assigned as Cmeso (∼119), Cm,p (∼128),
Cβ (∼131), Co (∼138), Ci (∼140), and Cα (∼150) on the
TPP moiety; this assignment is fully comparable with the
solution state 13C NMR spectrum of intermediate 5 (see
Experimental Section). The carbonyl group (CS-NH-CO
and TPP-NH-CO) appears at 160−169 ppm. In the TMA
DS and Conversion Efficiency. DS for the final nano-
carriers are shown in Table 1. The aim in the current work was
to control the DS with the equivalent ratio between compound
5 and 9 in reaction step “d” (Scheme 2). The final DS matched
the target value based on equivalent in the reaction (0.1 or
0.25) within a 2% margin, indicating 100% efficiency in the
reaction. Covalent linking of various lipophilic moieties to CS
has been investigated previously.32 This includes drugs like
doxorubicin33 and paclitaxel;34 endogenous biomolecules like
5α-cholanic acid,35 5β-cholanic acid,36 deoxycholic acid,37 and
cholesterol38 and photosensitizers like chlorine e6 (Ce6)39 and
protoporphyrin IX (PpIX).40 In most cases, the DS is
significantly less than reported here for lipophilic TPP. Previous
investigators also observed that the conjugation efficiency
decreased significantly as the DS increased.39,40 The current
+
derivative 17, the quaternary methyl carbons (−N(CH3)3 ) can
be observed as an intense peak at 55.5 ppm. In the MP
derivative 19, the methyl carbon (N−CH3) can be observed at
45 ppm and the cyclic methylene (−CH2−N) carbons are
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peaks increases as the DMSO-d6 content increases in the
mixture; in almost pure DMSO-d6 (only 2% D2O), the
aromatic TPP peaks are clearly visible along with the H-1 and
+
remaining CS-backbone peaks and the −N(CH3)3 peak
(Figure 5I(A)). This also suggests that the carrier gets fully
unfolded under these conditions. This interpretation was
further supported by UV−vis and fluorescence studies.
The nanocarriers dissolved in water show a broad Soret band
for the π−π stacked PS with an absorption maximum at 417 nm
(Figure 5II). This peak becomes gradually sharper and slightly
shifted (to 420 nm in 100% DMSO) as the DMSO
concentration is increased and the structure unfolds. In pure
water, the fluorescence is almost fully quenched, which is also
consistent with aggregation of the photosensitizer moieties.
The fluorescence intensity increases as the DMSO content
increased to 50%, and there was a dramatic sharp increase in
the fluorescence intensity observed with the further increase in
DMSO content in the mixture (Figure 5III). The comparison
of fluorescence spectra for all carriers 16−19 in 100% DMSO
and 100% water (Figure S1, Supporting Information) clearly
confirms the high degree of aggregation in aqueous medium
which in turn causes fluorescence quenching.
Figure 4. Solid-state 13C NMR spectra of the representative final
carriers 17 and 19 (materials deprotected with TBAF, batch B).
Table 1. DS of CS Nanocarriers 16−19
An attempt was made to further characterize the nanocarrier
particles by SEM. Droplets of nanocarrier solution (aqueous)
were pipetted onto the surfaces of clean silicon wafers and
allowed to dry. The nanocarriers were subsequently imaged by
SEM (Figure 6). In some samples, nanoparticles with sizes in
the range 10−200 nm could be observed, either isolated
(Figure 6A), clustered, or crystallizing into dendrite structures
(Figure 6B), similar to those typically observed when drying
nanoparticle suspensions. Spin-coating of the samples was also
tried, but in this case featureless films with only a few visible
nanoparticles were observed. The film thickness after spin-
coating was estimated to be smaller than 10 nm using
spectroscopic ellipsometry. The maximum thickness of the
layers formed after drying of solutions on the surface, however,
was measured to be on the order of 100 nm. Water contact-
angle measurement revealed a relatively hydrophobic film
surface and that the contact angle was dependent on the
concentration of the applied carrier sample (Figure 6C).
These results are consistent with dynamic nanoparticles that
unfold when coated so the hydrophilic polymer backbone will
adhere to the polar silicon dioxide surface exposing the
hydrophobic photosensitizer moieties in the particle’s core.
Furthermore, DLS study confirmed the consistency of
nanoparticle formation by all nanocarriers (16−19), with a
mean average nanoparticle size always in the same range
between 150 and 200 nm (Table 2). The typical particle size
distribution (by intensity) of the TPP-CS nanocarrier is shown
in Figure 6D. The particles were positively charged and stable,
having a ζ potential in the range of 40−120 mV (Table 2).
Nanoparticle aggregation and physical instability leading to
formation of insoluble aggregates is commonly observed.
However, when the physical stability of the carrier 17 in 1
mg/mL aqueous solution was monitored (Supporting
Information, Figure S4) over a period of three months, no
precipitation of this compound was observed. This implies the
good stability of the carriers.
a
b
a
compd no.
TPP-NH-Pip used
DS (linked TPP moieties )
16
17
18
19
0.10
0.25
0.10
0.25
0.10
0.23
0.09
0.25
a
b
1
Equivalent per glucosamine monomer unit. DS determined by H
NMR (data for batch A compounds)
process has the advantage that it uses protected CS, which is
fully soluble in organic solvents and can therefore be efficiently
conjugate with the lipophilic PS moiety under mild conditions.
Excellent reproducibility of the reaction is also confirmed, as
the DS between the two batches (A and B) match within a 2%
margin.
Analysis of Carrier Self-Aggregation and Unfolding.
Aromatic porphyrins can form π−π stacking systems, which are
defined as J (red-shifted) or H (blue-shifted) type aggregates.
Peripheral substituent groups can contribute to aggregation
mechanisms.41 This aggregation can be observed by NMR,
UV−vis, or fluorescence spectroscopy. Thus, extreme broad-
ening of peaks and loss of peaks in 1H NMR has been reported
for carboxyphenylporphyrin (TCPP) aggregates,42 p-sulfonato-
phenyl and phenyl meso-substituted porphyrins,43 and three
kinds of cationic porphyrins.44 Similar observations, of loss of
signal due to immobilization, have been made in dispersion
copolymerization lipophilic n-butylmethancrylate with poly-
(ethylene oxide) macro monomer.45
In the current work, the lack of peaks in the aromatic region
in the H NMR of the final carriers 16−19 in D2O suggested
aggregation of the TPP moieties in the D2O due to π−π
stacking and hydrophobic interaction. NMR study of the
representative carrier 17 in the DMSO-d6:D2O cosolvents is
shown in Figure 5.
In pure D2O, the TPP moiety peaks are missing, and only
very weak peaks are observed in the aromatic region in the
DMSO-d6:D2O (25:75) mixture, while the CS backbone peaks
can be observed. When the DMSO-d6 content is further
increased to DMSO-d6:D2O (50:50), broad peaks can be
observed (Figure 5I(C). The relative intensities of the aromatic
1
The fluorescence quantum yields of intermediate 5 and CS
nanocarriers (16−19) were all similar (Table 2), but quantum
yield of aminoporphyrin 3 was less than that of its derivative 5
as well as the final CS naocarriers (16−19). This demonstrated
a high degree of excited state quenching of 3 compared to its
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Figure 5. (I) 1H NMR spectra of carrier 17 (batch B) in cosolvents: (A) DMSO-d6:D2O (98:2), (B) DMSO-d6:D2O (75:25), (C) DMSO-d6:D2O
(50:50), (D) DMSO-d6:D2O (25:75), (E) DMSO-d6:D2O (0:100). (II) UV−vis absorption spectra overlay of 17 at constant concentration (0.3 mg/
L) and (III) fluorescence emission spectra overlay of 17 (absorbance adjusted to 0.1 in UV−vis (data not shown) at λex = 420 nm, slit width (15−15
nm), and 1 cm path length) in cosolvents: (A) DMSO:H2O (100:0), (B) DMSO:H2O (75:25), (C) DMSO:H2O (50:50), (D) DMSO:H2O
(25:75), (E) DMSO:H2O (0:100).
derivatives. Also, absorption spectra of 3 indicate aggregation of
this compound, even at high dilution, whereas the absorption
data did not indicated aggregation of the other compounds
(data not shown).
Similar results of experiments for four different CS
nanocarriers are summarized in the Figure 8, and it can be
seen that all the nanocarriers tested were able to enhance
transfection in a light dose-dependent manner. It can be seen
that the carriers 17 and 19 seemed the more active, giving
maximal effect both at a lower photosensitizer dose and at a
lower light dose than the carriers 16 (from batches A and B).
However, it can also be seen that this enhanced efficiency
seems to coincide with a higher cell toxicity. Hence, all in all,
nanocarrier 16 (batch B) gave 100 times transfection
enhancement at a light dose killing only about 30% of the
cells, while with this degree of transfection enhancement, about
80% of the cells were killed with the nanocarriers 17 and 19
(batch A). In this experimental system, the effect of nanocarrier
16 (batch B) was also substantially better than what was
achieved with the monomeric photosensitizer TPCS2a (data not
shown), which is under clinical development as a PCI
photosensitizer.8
In Vitro Studies. The nanocarriers were investigated as
photosensitizing agents in PCI to enhance gene delivery. For
evaluation of the ability of the nanocarriers to induce PCI,
experiments were performed to investigate if the nanocarriers
could enhance gene transfection with a plasmid/poly lysine
complex.1,46 The plasmid employed encodes enhanced green
fluorescent protein (EGFP), and the expression of EGFP was
analyzed by flow cytometry as described (see Experimental
Section). Figure 7 shows the result of an experiment with the
nanocarrier 16 (batch B) and indicates that this carrier
compound induced at substantial enhancement of transfection
upon illumination (an increase in % EGFP-positive cells from
0.2% with no illumination to 20% with 120 s illumination).
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Figure 6. (A) SEM image of representative carrier 16 isolated nanoparticles. (B) SEM image of representative carrier 16 dendritic nanoaggregates.
(C) Graph of the contact angles of TPP-CS derivatives. (D) DLS measurements showing intensity particle size distribution of typical representative
nanocarrier 16 having mean diameter of 178.1 nm.
at a lower light dose than the 0.10 DS carriers. This shows that
close proximity of the photosensitizer on the polymer chain did
not lead to self-quenching when the nanocarriers were in
contact with the endocytic membrane. However, this enhanced
activity seems to coincide with a higher cell toxicity.
Table 2. Photophysicochemical Properties of CS
Nanocarriers (16−19)
relative
fluorescence
average
ζ
c
compd
no.
quantum
particle
potential
(mV)
a
b
CS derivatives
TPP(p-NH2)1
yield
size (nm)
Our hypothesis to explain these observations is illustrated in
Figure 9. In aqueous solution, the carriers assemble into
nanoparticle-like structures, with the cationic group and
polymer backbone forming an outer shell around a core
composed of the aggregated (π−π stacked) lipophilic TPP
moieties. The cationic groups and polymer backbone have
relatively free movement in the liquid and can therefore be
observed by NMR. In contrast, the TPP core is composed of
stacked PS with very limited movement and can therefore only
be detected in solid-state NMR. This structure exists in a
dynamic equilibrium with the unfolded structure and is
fluorescent TPP, becoming the dominant form in DMSO,
allowing detection of the TPP peaks by NMR. When the carrier
is in contact with a cell or endocytic membrane, the structure
unfolds and the lipophilic moieties are inserted into the
endocytic membrane, allowing for excitation and photo-
sensitization leading to strong PCI effects.
3
0.05
1.00
0.89
1.00
1.00
1.00
TPP-NH-Pip
5
TPPp0.1-CS-TMA0.9
TPPp0.25-CS-TMA0.75
TPPp0.1-CS-MP0.9
TPPp0.25-CS-MP0.75
16
17
18
19
158 64
191 77
149 77
136 69
136
96
6
7
3
5
110
128
a
Quantum yield measurements of all compounds were done in DMSO
and λex = 419 nm. The observed emission wavelength for all is λem
=
651 nm. The calculated ΦF of 5 (0.0036 when anthracene as standard)
was used as reference for the relative values (see Supporting
b
c
Information for details). Compounds from batch A. DLS and ζ
potential measurements of all nanocarriers were done in water.
CONCLUSION
■
In conclusion, we have shown that TBDMS protected CS can
be used for efficient synthesis of meso-tetraphenylporphyrin
tethered CS-based nanocarriers. The synthesis of these carriers
was fully reproducible, and the method allowed precise control
of the DS with a highly lipophilic photosensitizer. NMR,
fluorescence, and UV−vis studies were consistent with self-
association of the photosensitizer moieties. The carriers are
polar and show good aqueous solubility and physical stability.
In DMSO, the photosensitizer dissociates from its self-
association and therefore becomes fluorescent.
EXPERIMENTAL SECTION
■
General Experimental Methods. A parent CS polymer
GO30626−2 provided by Genis EHF, Iceland, was converted to CS-
mesylate (average MW = 10.5 kDa and polydispersity index = 1.04;
degree of deacetylation = 95% as determined by 1H NMR) by
previously reported procedure,23b and this was used as a starting
material for the synthesis. All solvents and reagents were purchased
commercially and used without further purification. NMR spectra were
recorded on a Bruker Avance 400 MHz NMR spectrometer at 298 K,
and chemical shifts were reported relative to the deuterated NMR
solvents. Solid-state 13C NMR of compounds 17 (batch B) and 19
The carriers were effective in photochemically enhanced
gene transfection. Nanocarriers with 0.25 DS are more active,
giving maximal effect both at a lower photosensitizer dose and
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Figure 7. Transfection with pEGFP-N1 (plasmid encoding enhanced green fluorescent protein) in HCT116/LUC cells employing nanocarrier 16
(from batch B, 16B). Cells were tranfected as described under In Vitro Biological Experiments using 16 in a concentration of 0.1 μg/mL (with
regard to TPP). Transfection was analyzed by flow cytometry 48 h after illumination. The gatings for EGFP negative (GFP−) and EGFP positive
(GFP+) cells, and the illumination times are indicated on the figure.
Figure 8. Transfection pEGFP-N1 in HCT116/LUC cells. Transfection was measured 48 h after illumination by flow cytometry. Cell survival was
measured by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. (a) 16 (batch A, 16A) (0.1 μg/mL TPP equivalent).
(b) 16 (batch B, 16B) (0.1 μg/mL TPP equivalent). (c) 17 (batch A) (0.05 μg/mL TPP equivalent). (d) 19 (batch A) (0.05 μg/mL TPP
equivalent).
(batch B) were obtained from the Department of Chemistry, Durham
University. HRMS spectra were recorded on a Bruker micrOTOF-Q
instrument with ESI. The purity of all synthesized compounds was
using Spectra/Por dialysis membrane (MWCO: 3500). UV−vis
measurements were recorded on a Perkin-Elmer Lambda 25 UV−vis
spectrometer. Fluorescence emission spectra for quantum yield were
obtained using a SPEX FluoroMax spectrometer (Spectrocell
Corporation, Oreland, PA, USA). Fluorescence spectra shown in
Figure 5III and Figure S1 (Supporting Information) were recorded on
a PerkinElmer LS50B fluorescence spectrometer (equipped with red
sensitive photomultiplier tube).
1
confirmed >95% by using HPLC, GPC, H NMR, and solid-state 13C
NMR. HPLC analysis of porphyrin intermediates were carried on a
Dionex system including a HPG-3x00 pump and PDA-3000 detector,
using reverse phase Phenomenex LUNA 5μ C18 (2) column with size
150 mm × 4.60 mm, 5μ micrometer. Gel permeation chromatography
(GPC) analysis of CS-mesylate and TPP-CS final carriers (16−19)
were performed using Polymer Standards Service (PSS) GmbH,
Germany, WinGPC Unichrom system equipped with a series of three
columns Novema 10 μ [guard (50 mm × 8 mm), 30A (150 mm × 8
mm), and 1000A (300 mm × 8 mm)] and PSS’s ETA-2010
viscometer/Shodex RI-101 detectors. FT-IR measurements were
performed with an AVATAR 370 FT-IR instrument (Thermo Nicolet
Corporation, Madison, WI, USA). Polymer samples were dialyzed
Stability Study. For the physical stability study, compound 17
(batch A) was dissolved in H2O (1 mg/mL), sonicated for 30 min,
centrifuged (on a HERMLE Z-320) at 4000 rpm for 10 min, decanted,
and wrapped with aluminum foil. UV−vis absorbance was measured at
λmax = 419 nm in H2O over a period of 0−90 days.
Determination of the DS by 1H NMR. For calculating DS from final
carriers 16−19, we used the integration values of TPP peaks (from
TPP-NH-Pip part) and that of the H-1 (from the CS part) peak. The
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1179, 1153, 1071, 1001, 965, 799, 728, 700 cm−1. 1H NMR (CDCl3):
δ = 9.51 (br s, 1H, TPP-NHCO), 8.89−8.93 (m, 8H, β-pyrrole H),
8.23−8.26 (m, 8H, tetraphenyl-HO), 8.01 (d, J = 8.0 Hz, 2H, Pip-NH-
phenyl-Hm), 7.75−7.81 (m, 9H, triphenyl-Hm,p), 3.31 (s, 2H,
COCH2−Pip), 3.11 (t, 4H), 2.77 (br t, 4H), 2.60 (br s, 1H, piperazine
NH), −2.71 (br s, α-pyrrole 2H) ppm. 13C NMR (CDCl3): δ =
168.75, 142.30, 138.26, 137.44, 135.33, 134.68, 131.26, 127.86, 126.83,
120.32, 119.66, 117.89, 62.87, 54.53, 46.24 ppm. HRMS (ESI): m/z
calcd for C50H42N7O ([M + H]+), 756.3445; found, 756.3418.
3,6-Di-O-tert-butyldimethylsilyl-chitosan (8). was synthesized
according to our previously published procedure.23b
N-Bromoacetyl-3,6-O-DiTBDMS-CS (9). Compound 8 (1 g, 2.60
mmol) was dissolved in dry CH2Cl2 (15 mL) in a round-bottom flask
under N2 atmosphere. Then the reaction mixture was cooled to −20
°C using an ice−salt mixture. Et3N (1.81 mL, 13 mmol) was added,
followed by slow dropwise addition of bromoacetylbromide (0.91 mL,
10 mmol). Stirring was continued for exactly 1 h. The reaction mixture
was diluted with CH2Cl2 and concentrated in vacuo. The crude
product was stirred in CH3CN, filtered, and washed with fresh
CH3CN. The dry material was dissolved and extracted in CH2Cl2,
washed with water and brine, and then dried over Na2SO4 and
concentrated in vacuo. A faint yellow powdered product 9 was
obtained, 1.2 g (92% yield). FT-IR (KBr): ν = 3402, 2957, 2931, 2886,
2858, 1682, 1530, 1473, 1391, 1362, 1311, 1259, 1101, 1005, 837, 777,
669 cm−1. 1H NMR (CDCl3) δ = 4.40 (br s, H-1), 4.02−3.26 (m, H-2
GlcN, H-3, H-4, H-5, H-6, H-6′ and 2H GluNH-COCH2Br), 0.90
and 0.88 (br s, (CH3)3C−Si), 0.13 and 0.07 (br s, (CH3)2−Si) ppm.
Compounds 10 and 11. Compounds 10 and 11 were synthesized
by reacting CS intermediate 9 and porphyrin intermediate 5 using
1:0.1 and 1:0.25 equiv per glucosamine monomer, respectively
(Supporting Information)
Figure 9. Schematic figure showing the proposed nanoparticle and its
unfolding in DMSO-d6. Possible insertion of the tethered photo-
sensitizer moieties into the cell/endocytic membrane is also shown.
integrals of TPP (four peaks from the aromatic region) were used,
while the integral of the CS backbone (H-1) group was used in order
to calculate DS using the following equation, DS = (∫ aromatic TPP
peaks/27)/(∫ H − 1 peak/1).
Although under this condition the polymer backbone is partially
overlapped by the HDO peak, the DS can be determined with good
accuracy from the relative ratio of the integrals of the H-1 peak and the
TPP peaks.
SEM and Ellipsometry. Solutions were spin-coated at 1000 rpm
onto pristine silicon ⟨100⟩ substrates (15 mm × 15 mm) using a
conventional spinner in a Class-100 clean room environment. The
silicon has a layer of native oxide approximately 15 Å in thickness.
Furthermore, 10 μL of the same solutions were pipetted directly onto
silicon substrates and allowed to dry in air at room temperature. The
coated substrates were imaged in a Zeiss LEO 1550 scanning electron
microscope at 10 keV acceleration voltage and 2 mm working distance
using an in-lens detector.
Water Contact Angle Measurements. Water contact angles were
determined using a KSV CAM 200 optical contact angle meter (KSV
Instruments). A 5 μL deionized water droplet was dropped on the
center of the silicon waver, and measurement was started 10 s after
drop down. Water contact angles were determined based on the
Laplace and Young equation. Measurements were done at room
temperature and ambient humidity.
DLS and ζ Potential. All DLS and ζ potential measurements were
performed at 25 0.5 °C on a Nanotrac wave-Zeta (ZS), (Microtrac,
USA) having scattering angle = 180°, laser wavelength = 780 nm, and
power = 3 mW. Samples were prepared by dissolving TPP-CS carriers
in deionized water, sonicating (40 min, 55 °C), and then filtering
through a 0.45 μL filter (Spartan 13/0.45 RC, Whatman) before
measurement.
Chemical Synthesis. Synthesis of Free Base Tetraarylporphyrins.
meso-Tetraphenylporphyrin (1), was synthesized according to Adler−
Longo method.47 5-(4-Nitrophenyl)-10,15,20-triphenylporphyrin (2)
and 5-(4-aminophenyl)-10,15,20-triphenylporphyrin (3) were pre-
pared using procedure reported by Luguya and co-workers.29
5-(4α-Bromoacetylamidophenyl)-10,15,20-triphenylporphyrin
(4). Bromoacetylation of aminoporphyrin 3 using 2-bromoacetyl
bromide in the presence of Et3N at room temperature in CH2Cl2
afforded 4 (Supporting Information).
5-(4α-Piperazineacetylamidophenyl)-10,15,20-triphenylporphyr-
in (5). Bromoacetylated porphyrin 4 (275 mg, 0.366 mmol) and excess
piperazine (158 mg, 1.83 mmol) were mixed together in CH2Cl2 (10
mL) and stirred at room temperature for 1 h under N2 atmosphere.
After completion of the reaction, the reaction mixture was diluted with
CH2Cl2 (85 mL) and washed with water (2 × 40 mL) and brine (35
mL). The organic layer was dried over Na2SO4 and concentrated in
vacuo. The crude product was purified by silica gel column
chromatography using MeOH:CH2Cl2, (1:12) as eluent to afford
title compound 5 (260 mg, 94%) as a purple solid. TLC (CH2Cl2/
MeOH, 9:1); Rf = 0.15. FT-IR (KBr): ν = 3442, 3312, 3052, 3022,
2903, 2816, 1691, 1596, 1557, 1517, 1471, 1439, 1400, 1349, 1309,
Compounds 12−15. Compounds 12 and 13 were prepared by
treating excess trimethylamine with 10 and 11, respectively, whereas
compounds 14 and 15 were prepared by treating excess 1-
methylpiperazine with 10 and 11, respectively (Supporting Informa-
tion)
General TBDMS Deprotection Procedure for Synthesis of
Carriers 16−19 (batch A). Compound 16 (TPPp0.1-CS-TMA0.9). The
material 12 (350 mg, 0.50 mmol) was dissolved in MeOH (5−10 mL),
followed by addition of concentrated HCl (2−3 mL). The reaction
mixture was stirred for 12 h at room temperature. Excess deionized
water was then added to the reaction mixture and stirred for 30 min
before dialysis against 8% NaCl for one day and against deionized
water for three days. During this time, the color of the solution
gradually changed from dark-green to red. The red-colored solution
was then freeze-dried to yield a brown sponge-like material. The
materials were again deprotected, ion exchanged, dialyzed, and freeze-
dried. The procedure was repeated using the same conditions in order
to remove trace amounts of TBDMS to obtain 16 (210 mg, 89%) as a
brown sponge material. FT-IR (KBr): ν = 3419, 3064, 1683, 1558,
1506, 1488, 1473, 1403, 1296, 1153, 1112, 1068, 1032, 966, 911, 800,
730, 701, 618 cm−1. 1H NMR (DMSO-d6:D2O, 96:4) δ = 8.81 (br m,
β-pyrrole H), 8.12−8.16 (br m, phenyl-Ho and Pip-NHTPP-phenyl-
Hm), 8.04 (br d, J = 8.0 Hz, RNTPP-phenyl-Ho), 7.75−7.84 (m,
triphenyl-Hm,p), 4.66 (br s, H-1), 4.21 (br s, BrCH2CO), 3.83−
3.54 (m, H-2 GlcNAc, H-3, H-4, H-5, H-6, H-6′), 3.32 (s,
+
−N(CH3)3 ) ppm.
Compound 17 (TPPp0.25-CS-TMA0.75). The general procedure was
followed using 13 (240 mg, 0.30 mmol) and concentrated HCl/
MeOH to give 17 as a purple solid (120 mg, 71%). FT-IR (KBr): ν =
3392, 3061, 2950, 1683, 1559, 1506, 1489, 1473, 1402, 1350, 1296,
1
1154, 1112, 1068, 1032, 966, 911, 800, 730, 701, 619 cm−1. H NMR
(DMSO-d6:D2O, 98:2) δ = 8.81 (br m, β-pyrrole H), 8.12−8.16 (br m,
phenyl-Ho and Pip-NHTPP-phenyl-Hm), 8.04 (br d, J = 8.0 Hz,
RNTPP-phenyl-Ho), 7.79−7.87(m, triphenyl-Hm,p), 4.50 (br s, H-1),
4.15 (br s, BrCH2CO), 3.27−3.65 (m, H-2 GlcNAc, H-3, H-4, H-5,
+
H-6, H-6′), 3.24 (s, −N(CH3)3 ) ppm.
Compound 18 (TPPp0.1-CS-MP0.9). The general procedure was
followed using 14 (350 mg, 0.52 mmol) and concentrated HCl/
MeOH to give 18 as a purple solid (200 mg, 87%). FT-IR (KBr): ν =
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3419 (br, O−H), 3057 (C−H), 1683, 1558, 1474, 1403, 1296, 1234,
(Becton Dickinson, CA, USA). EGFP was measured through a 425−
475 nm filter after excitation at 488 nm, and propidium iodide
(Calbiochem Corporation, CA, USA) was measured through a 600−
620 nm filter after excitation at 561 nm. Propidium iodide (1 μg/mL)
was used to discriminate dead cells from viable cells, and pulse-
processing was performed to discriminate cell doublets from single
cells. For each sample 10,000 events were collected and the data was
analyzed with BD FACSDiva Software (Becton Dickinson, CA, USA).
Measurement of Cell Survival. Cell survival was measured by a
method based on reduction of a water-soluble tetrazolium salt (MTT)
to a purple, insoluble formazan product by mitochondrial dehydro-
genases present in living, metabolically active cells. One-half mL of
medium containing 0.125 mg of MTT was added to the cells, followed
by a 2 h incubation at 37 °C, 5% CO2. The resulting formazan crystals
were dissolved by adding 500 μL of DMSO (Sigma-Aldrich, MO,
USA) per well. The plates were read by a PowerWave XS2 microplate
spectrophotometer (BioTek Instruments, VT, USA). Cell survival was
calculated as percent of controls (parallels with no light).
1
1154, 1112, 1068, 1032, 966, 930, 911, 799, 730, 701 cm−1. H NMR
(DMSO-d6:D2O, 96:4) δ = 8.83 (br m, β-pyrrole H), 8.13−8.19 (br m,
phenyl-Ho and Pip-NHTPP-phenyl-Hm), 8.08 (br d, J = 8.0 Hz,
RNTPP-phenyl-Ho), 7.79−7.87(m, triphenyl-Hm,p), 4.48 (br s, H-1),
3.24−3.78 (m, H-2 GlcNAc, H-3, H-4, H-5, H-6, H-6′), 3.92 (dd, J =
12 Hz, Pip-CH2CO), 2.30−2.67 (m, piperazine (CH2)4), 2.48 (br s,
piperazine, N−CH3) ppm.
Compound 19 (TPPp0.25-CS-MP0.75). The general procedure was
followed using 15 (200 mg, 0.26 mmol) and 1-methylpiperazine to
give 19 as a purple solid (80 mg, 58%). FT-IR (KBr): ν = 3392, 3056,
2947, 1683, 1558, 1520, 1489, 1472, 1458, 1400, 1349, 1309, 1248,
1
1154, 1068, 1031, 1001, 966, 911, 800, 729, 701, 658 cm−1. H NMR
(DMSO-d6:D2O, 96:4) δ = 8.83 (br m, β-pyrrole H), 8.14−8.20 (br m,
phenyl-Ho and Pip-NHTPP-phenyl-Hm), 8.08 (br d, J = 8.0 Hz,
RNTPP-phenyl-Ho), 7.78−7.86 (m, triphenyl-Hm,p), 4.50 (br s, H-1),
3.24−3.78 (m, H-2 GlcNAc, H-3, H-4, H-5, H-6, H-6′), 3.92 (dd, J
=12 Hz, Pip-CH2CO), 2.28−2.67 (m, piperazine (CH2)4), 2.48 (br
s, piperazine, N−CH3) ppm.
In Vitro Biological Experiments. Cell Lines and Culture
Conditions. The HCT116/LUC human colon carcinoma cell line
(permanently transfected with a gene encoding luciferase) was kindly
provided by Dr. Mohammed Amarzguioui, siRNasense, Oslo, Norway.
MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide)
was from Sigma-Aldrich (MO, USA; cat. no. M 2128) and dissolved in
PBS to a concentration of 5 mg/mL, sterile filtered, and stored at 4 °C.
The plasmid pEGFP-N1 was purchased from Clontech Laboratories
Inc. (CA, USA; cat. no. 6085−1), produced by ELIM Biopharma-
ceuticals, Inc. (CA, USA) (lot no.1002) and delivered at a
concentration of 2 mg/mL in sterile water. This stock solution was
aliquoted and kept at −20 °C. Poly-L-lysine HBr (MW 15000−30000)
from Sigma-Aldrich (MO, USA; cat. no. P 7890) was dissolved and
diluted in distilled water, sterilized by filtration, and stored at −20 °C.
Cell Cultivation. HCT116/LUC were cultured in DMEM
(Dulbecco’s Modified Eagle Medium; Lonza, Veviers, Belgium)
supplemented with 10% fetal calf serum (PAA Laboratories GmbH,
Pasching, Austria), 100 U/mL penicillin, and 100 μg/mL streptomycin
(Sigma-Aldrich, MO, USA) at 37 °C and 5% CO2 in a humid
environment.
ASSOCIATED CONTENT
■
S
* Supporting Information
General experimental methods and the copies of 1H, 13C NMR,
HRMS, and fluorescence spectra of key compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: +354-8228301. Fax: +354-5254071. E-mail:
mmasson@hi.is.
■
Notes
The authors declare the following competing financial
interest(s): Three of the co-authors, namely Vivek S. Gaware,
Monika Hakerud, and Anders Høgset, are employed by PCI
̊
Biotech. PCI Biotech is a Norwegian company that has
developed and patented photochemical drug delivery technol-
ogy for use in cancer therapy and other diseases. PCI Biotech
has obtained a grant for this work from the Scandinavian fund
Nordforsk Public Private Partnership (NPPP). Also, PCI
Biotech has signed collaborative agreements with University
of Iceland and Norwegian Radium Hospital. The synthesis
work was done by Vivek S. Gaware and the biological
Treatment of Cells. HCT116/LUC cells (1.5 × 105 cells per well
for transfection measurements, 3.75 × 105 cells per well for MTT
assay) were seeded into 6-well (transfection) and 24-well (MTT)
plates (Nunc, Roskilde, Denmark) and incubated for 24 h (5% CO2,
37 °C). The photosensitizer TPCS2a or the CS nanocarriers [16
(batch A), 16 (batch B), 17 and 19 (batch A)] were then added to the
cells, and the cells were incubated for 18 h (5% CO2, 37 °C). The cells
were then washed three times with cell culture medium and incubated
for 4 h (5% CO2, 37 °C) in medium containing the plasmid complex.
Then the cells were washed once. After addition of fresh medium, the
cells were illuminated with different light doses. After 48 h of
incubation, the expression of enhanced green fluorescent protein
(EGFP) was analyzed by flow cytometry. Cell survival was measured
by the MTT assay in parallel experiments.
The cells were exposed to light from a LumiSource (PCI Biotech,
Oslo, Norway). LumiSource is delivered with a bank of four light tubes
(4 × 18 W Osram L 18/67, Blue) emitting mainly blue light with a
peak wavelength in the region of 420−435 nm.
Preparation of Plasmid/Poly-L-lysine Complexes. Plasmid/poly-L-
lysine complexes with charge ratio 2.2 were formed by gentle mixing of
plasmid DNA and poly-L-lysine solutions. Then 2.5 μL of DNA (stock
solution 2 μg/ μL) was diluted with 47.5 μL of water, and 6.92 μL of
poly-L-lysine (1 μg/ μL) was diluted with 43.08 μL of water. After
mixing, the solution was incubated at room temperature for 30 min,
then diluted with culture medium to a final volume of 1 mL and added
to the cells (1 mL per well).
investigations by Monika Hakerud. Other authors have no
̊
conflicts of interest to declare..
ACKNOWLEDGMENTS
■
We thank Nordforsk Public Private Partnership and the
Technical Development fund for financial support and Genis
EHF and Dr. Jon M. Einarsson for the generous donation of
chitosan starting material and helpful advice. We thank Gissur
̈
́
Orlygsson and Marketa Foley for their skillful assistance in the
́
water contact angle measurements and Kristmann Gislason for
assisting with the quantum yield measurements. Also, we thank
Eydis Einarsdottir for her generous assistance in HPLC and
Victoria for her help with fluorescence measurements.
ABBREVIATIONS USED
■
AlPcS4, aluminum phthalocyanine tetrasulfonate; AlPcS2a,
aluminum phthalocyanine disulfonate; CS, chitosan; Ce6,
chlorine e6; DiTBDMS-CS, 3,6-di-O-tert-butyldimethylsilyl-
chitosan; DLS, dynamic light scattering; DMEM, Dulbecco’s
Modified Eagle Medium; DS, degree of substitution per
glucosamine monomer; EGFP, enhanced green fluorescent
protein; pEGFP-N1, plasmid encoding enhanced green
Measurement of Transfection. The cells were trypsinized in 100
μL of trypsin (Trypsin-EDTA, Sigma-Aldrich, MO, USA), resus-
pended in 500 μL of cell culture medium, and filtered through a 5 mL
polystyrene round-bottom tube with cell-strainer cap (BD Falcon) (50
μm mesh nylon filter) before analysis in a BD LSR flow cytometer
817
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fluorescent protein; EPR, enhanced permeation and retention;
GPC, gel permeation chromatography; kDa, kilo Dalton; MP,
1-methyl piperazine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide; PAMAM, polyamidomine; PCI,
photochemical internalization; PDT, photodynamic therapy;
PS, photosensitizer; PpIX, protoporphyrin IX; ROS, reactive
oxygen species; TBDMS, tert-butyldimethylsilyl; TCPP,
carboxyphenylporphyrin; TMA, trimethylamine; TPCS2a,
meso-tetraphenylchlorin disulfonate; TPP, meso-tetraphenylpor-
phyrin; TPPS2a, meso-tetraphenylporphyrin disulfonate; UP,
uroporphyrin
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