Endosome Targeting meso-Tetraphenylchlorin-Chitosan Nanoconjugates for Photochemical Internalization

Article abstract of DOI:10.1021/acs.biomac.6b01670

Four amphiphilic covalently linked meso-tetraphenylchlorin-chitosan nanoconjugates were synthesized and evaluated for use in photochemical internalization (PCI) in vitro and in vivo. The synthetic protocol for the preparation of two different hydrophobic chlorin photosensitizers, 5-(4-aminophenyl)-10,15,20-triphenylchlorin and 5-(4-carboxyphenyl)-10,15,20-triphenylchlorin, was optimized. These monofunctional photosensitizers were covalently attached to carrier chitosan via silyl-protected 3,6-di-O-tert-butyldimethylsilyl-chitosan (Di-TBDMS-chitosan) with 0.10 degree of substitution per glucosamine (DS). Hydrophilic moieties such as trimethylamine and/or 1-methylpiperazine were incorporated with 0.9 DS to give fully water-soluble conjugates after removal of the TBDMS groups. A dynamic light scattering (DLS) study confirmed the formation of nanoparticles with a 140-200 nm diameter. These nanoconjugates could be activated at 650 nm (red region) light, with a fluorescence quantum yield (Φ_F) of 0.43-0.45, and are thus suitable candidates for use in PCI. These nanoconjugates were taken up and localized in the endocytic vesicles of HCT116/LUC human colon carcinoma cells, and upon illumination they substantially enhanced plasmid DNA transfection. The nanoconjugates were also evaluated in preliminary in vivo experiments in tumor-bearing mice, showing that the nanoconjugates could induce a strong photodynamic therapy (PDT) and also PCI effects in treatment with bleomycin.

Full text of DOI:10.1021/acs.biomac.6b01670

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Article
Endosome Targeting meso-Tetraphenylchlorin-Chitosan
Nano-Conjugates for Photochemical Internalization
VIVEK SAMBHAJI GAWARE, Monika Hakerud, Asta
Juzeniene, Anders Høgset, Kristian Berg, and Már Másson
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01670 • Publication Date (Web): 28 Feb 2017
Downloaded from http://pubs.acs.org on March 1, 2017
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Endosome Targeting meso-Tetraphenylchlorin-Chitosan
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Nano-Conjugates for Photochemical Internalization
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Vivek S. Gaware1, 2, Monika Håkerud2, 3, Asta Juzeniene3, Anders Høgset2, Kristian Berg3, Már
Másson1*
1 Faculty of Pharmaceutical Sciences, School of Health Sciences, University of Iceland,
Hofsvallagata 53, ISꢀ107 Reykjavik, Iceland
2 PCI Biotech AS, Strandveien 55, Nꢀ1366 Lysaker, Norway
3 Oslo University Hospital, The Norwegian Radium Hospital, Institute for Cancer Research,
Department of Radiation Biology, Montebello, Nꢀ0310 Oslo, Norway
ABSTRACT
Four amphiphilic covalently linked mesoꢀtetraphenylchlorinꢀchitosan nanoꢀconjugates were
synthesized and evaluated for use in photochemical internalization (PCI) in vitro and in vivo. The
synthetic protocol for the preparation of two different hydrophobic chlorin photosensitizers, 5ꢀ(4ꢀ
aminophenyl)ꢀ10,15,20ꢀtriphenylchlorin and 5ꢀ(4ꢀcarboxyphenyl)ꢀ10,15,20ꢀtriphenylchlorin, was
optimized. These monoꢀfunctional photosensitizers were covalently attached to carrier chitosan
via silylꢀprotected 3,6ꢀdiꢀOꢀtertꢀbutyldimethylsilylꢀchitosan (DiꢀTBDMSꢀchitosan) with 0.10
degree of substitution per glucosamine (DS). Hydrophilic moieties such as trimethylamine and/or
1ꢀmethylpiperazine were incorporated with 0.9 DS to give fully waterꢀsoluble conjugates after
removal of the TBDMS groups. A dynamic light scattering (DLS) study confirmed the formation
of nanoparticles with a 140–200 nm diameter. These nanoꢀconjugates could be activated at 650
nm (red region) light, with a fluorescence quantum yield (ΦF) of 0.43–0.45, and are thus suitable
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candidates for use in PCI. These nanoꢀconjugates were taken up and localized in the endocytic
vesicles of HCT116/LUC human colon carcinoma cells and upon illumination they substantially
enhanced plasmid DNA transfection. The nanoꢀconjugates were also evaluated in preliminary in
vivo experiments in tumorꢀbearing mice showing that the nanoꢀconjugates could induce a strong
photodynamic therapy (PDT) and also PCI effects in treatment with bleomycin.
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KEYWORDS: chitosan, nanoparticles, DiꢀTBDMSꢀchitosan, PCI, photosensitizer, Endosome,
transfection.
INTRODUCTION
Photochemical Internalization (PCI) is a technology that utilizes amphiphilic photosensitizer (PS)
molecules and light for a siteꢀspecific release of endocytosed macromolecules or
chemotherapeutics into the cell cytosol, thereby addressing one of the significant challenges in
macromolecular anticancer therapy.1ꢀ4
Like photodynamic therapy (PDT), the PCI technology has three possible mechanisms for
tumor therapy: (i) direct cytotoxic effects on the tumor cells, (ii) vascular shutdown, and (iii)
possible activation of the immune system. In addition, PCI also has a fourth and very important
effect: siteꢀspecific photochemicallyꢀinduced release of drugs from endocytic vesicles into the
cytosol. Unlike in the case of PDT, in PCI the photosensitizers need to be located in the
endocytic vesicles of the targeted cells for the release of endocytosed therapeutic agents by
reactive oxygen species (ROS)ꢀmediated rupture of the endocytic vesicles.
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PCI can be used with biologically active molecules that have not been exploited in the
clinic due to their inability to penetrate the plasma membrane to reach their intracellular targets.
PCI has been demonstrated in vitro and in vivo with type I ribosomeꢀinactivating proteins5,
immunotoxins6, adenovirus7, nucleic acids8, (plasmids, siRNA, mRNA) and chemotherapeutic
drugs like bleomycin9, 10 and doxorubicin11. Therefore, in principle PCI can be used for the
treatment of many types of cancers1 as well as for nonꢀmalignant diseases.2ꢀ4, 12, 13 The main
clinical advantage of PCI is the possibility for siteꢀspecific drug delivery leading to better
utilization of drugs, thereby potentially reducing some of the severe side effects seen with most
current cancer therapies.4, 14, 15
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With PCI, the structure of the PS employed is essential for the therapeutic outcome. Over
the past decade, substantial efforts have been put into developing various classes of PSs for better
light absorption, greater tumor specificity, and less cutaneous photosensitivity compared to the
firstꢀgeneration photosensitizer Photofrin for PDT. In most cases, second generation PSs are
based on porphyrin and chlorin structures as they often accumulate preferentially in neoplastic
lesions as compared to the surrounding normal tissue. Chlorins are preferred over porphyrins for
PDT and PCI as they offer the use of light with a deeper tissue penetration. This is due to their
increased absorption on the red end of the spectrum and because they often have a very good
singlet oxygen quantum yield.2, 16 For effective PCI, however, the PS must also localize in
endocytic vesicles. Amphiphilic PSs have been found to be the most efficient for PCI as their
hydrophilic part prevents them from passing through the plasma membrane, allowing
accumulation of the PS in the inner leaflet of the endocytic vesicles.8 Therefore, PSs like mesoꢀ
tetraphenylporphyrin disulfonate (TPPS2a) and aluminum phthalocyanine disulfonate (AlPcS2a)
are very effective in PCI. MesoꢀTetraphenylchlorin disulfonate (TPCS2a, fimaporfin) is a similar
PS that like TPPS2a and AlPcS2a has sulfonate groups in adjacent positions; and that is currently
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being tested in clinical trials. A clinical Phase I fimaporfin dose escalating study in patients with
cutaneous or subꢀcutaneous head and neck and skin neoplasms has recently been completed. In
this study fimaporfin was used in combination with the cytotoxic drug bleomycin, and the results
indicated a strong tumor response in most treated tumors, with good safety. 17 A Phase I/II safety
and efficacy study of fimaporfin in combination with gemcitabine for bile duct
(cholangiocarcinoma) cancer has recently been initiated.18, 19
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In order to further optimize the efficacy and selectivity of PDT, different strategies have
been conceived, in which PSs are covalently attached to targeting vehicles. Thus, monoclonal
antibodies (Mabs),20ꢀ22 other proteins,23, 24 polymers25ꢀ30 and sugars,31ꢀ35 have been designed to
improve cell typeꢀspecific targeting. With this approach, a wide range of selective targets on the
molecular, cellular, and tissue levels could be exploited. However, the use of large proteins, such
as antibodies, often has limitations due to difficulties both in escaping from blood vessels and in
moving through the interstitial space of the tumors. Still, macromolecules are very well suited for
PCI since they usually get endocytosed through receptorꢀmediated endocytosis, adsorptive
endocytosis, or pinocytosis. PolymerꢀPS conjugates could be a very good targeting system for PS
in PCI since such structures could take advantage of the leaky vasculature and poor lymphatic
drainage in tumors by the soꢀcalled enhanced permeability and retention (EPR) effect.
Chitosan is a very good polymer candidate as a carrier for PS attachment due to its nonꢀ
toxic, biocompatible and biodegradable nature, as well as the fact that it can form nanoparticles
spontaneously in aqueous conditions. In an earlier study, conjugation of free base mesoꢀ
tetraphenylporphyrin (TPP) to cationic chitosan carriers has shown promising in vitro results for
PCI mediated photochemical transfection.36 We have developed a quantitative, reproducible and
efficient synthetic method for the preparation of amphiphilic TPPꢀchitosan nanoꢀconjugates with
the aid of organoꢀsoluble tertꢀbutyldimethylsilyl protected chitosan (DiꢀTBDMSꢀchitosan). Using
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this method, highly lipophilic PS monoꢀamino functionalized TPP (log P ≥ 9) was covalently
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linked in a controlled manner (with a degree of substitution per glucosamine monomer (DS) of
0.10 or 0.25), followed by the incorporation of a hydrophilic moiety such as trimethylamine or 1ꢀ
methylpiperazine onto the chitosan backbone with a higher DS to enhance aqueous solubility.
With these structures, nanoꢀconjugates with a DS of 0.10 of the photosensitizer (TPP in this case)
showed better properties in PCIꢀmediated photochemical transfection than nanoꢀconjugates with
a DS of 0.25. While these porphyrin based TPPꢀchitosan nanoꢀconjugates have shown promising
results in vitro, they are not optimal for use in vivo. This is mainly because porphyrinꢀbased
molecules do not absorb enough light in the red region of the visible spectrum and thus would not
be activated in the deeper layers of the tumors.
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Having established proofꢀofꢀconcept with TPP, the logical next step was to synthesize
structurally similar chitosan nanoꢀconjugates with a PS that would have a better absorbance in the
red spectral region. This could be achieved by converting porphyrin into chlorin by reducing one
of the pyrrolic double bonds of the tetrapyrrolic macrocycle. The synthesis of pure chlorin
derivatives is however considerably more challenging compared to that of porphyrins. In the
current study, we established a method for the synthesis of four different free base mesoꢀ
tetraphenylchlorin (TPC)ꢀbased TPCꢀchitosan nanoꢀconjugates (with a DS of 0.10). To address
the challenge of synthesizing TPC conjugate two synthesis strategies were tried, starting from
either the carboxyl (ꢀCOOH) or the amino (ꢀNH2) derivative of the PS. In the former case the
“spacer” group, linking the PS to the biopolymer, will be slightly shorter and more rigid. Both
strategies were successful and the effect of the spacer group could therefore also be investigated.
These amphiphilic nanoꢀconjugates are completely soluble in water and have been thoroughly
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scattering (DLS). The molecular weight was determined by gel permeation chromatography
(GPC) techniques. The efficacy of the TPCꢀchitosan nanoꢀconjugates for PCI mediated gene
delivery was evaluated in vitro using the HCT116/LUC human colon carcinoma cell line and in
vivo using a xenograft model in nude mice.
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EXPERIMENTAL SECTION
Materials
The chitosan (CS) polymer GO30626ꢀ2 (provided by Genis EHF, Iceland) was first converted to
chitosan mesylate [weightꢀaverage molecular weight (Mw) = 10.5 kDa] and polydispersity index
(PDI) = 1.04; degree of deacetylation (DD) = 95% (as determined by 1H NMR)] by a previously
reported procedure.37 This was used as a starting material for the synthesis. Porphyrin and chlorin
intermediate compounds were purified by flash column chromatography using silica gel 60 Å
(0.040ꢀ0.063 mm) (230–400 mesh ASTM) which was purchased from Merck Millipore,
Germany. The Rf values of compounds were determined by using thin layer chromatography
(TLC) silica gel 60 F254 aluminum sheets, and compounds were visualized by ultraviolet (UV)
and visible light. MesoꢀTetraphenylporphyrin was purchased from SigmaꢀAldrich, Germany and
used as a reference compound for fluorescence quantum yield measurements. All other reagents
and solvents were purchased commercially and used without further purification.
Equipment
Nuclear Magnetic Resonance (NMR) spectra were recorded on a DRX 400 MHz Bruker NMR
spectrometer at 298 K and the chemical shifts were reported in parts per million (ppm) relative to
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the residual proton signal (for H NMR) and the carbon signal for (13C NMR) of the deuterated
solvent used [1H NMR: CDCl3 (7.26 ppm), DMSOꢀd6 (2.50 ppm); 13C NMR: CDCl3 (77.16
ppm), DMSOꢀd6 (39.52 ppm)]. All coupling constants were reported in Hertz. The acetone peak
(2.22 ppm) was used as the internal reference for D2O as solvents. The protons (ortho, meta,
para) on the phenyl rings of the porphyrin/chlorin system are identified with respect to their
positions relative to the porphyrins/chlorins ring system and not with the respective substituent
on the phenyl ring. Mass spectra were recorded on Bruker Autoflex III or a Bruker micro TOFꢀ
Q11. The molecular mass of organic compounds was determined by highꢀresolution mass spectra
(HRMS) recorded on a Bruker micrOTOFꢀQ instrument with ESI. Fourier transform infrared
(FTꢀIR) spectra were recorded on an AVATAR 370 FTꢀIR instrument (Thermo Nicolet
Corporation, Madison, U.S.) by preparing sample pellets with KBr using a Specac compressor
(Specac Inc., Smyrna, U.S.). Melting Points (mp) were recorded on Buchi Melting Point Bꢀ540.
Polymer samples were dialyzed using Spectra/Por Dialysis Membrane (MWCO: 3500) and were
freeze dried on a Snijders Scientific freeze dryer. Water from MilliQ® (Millipore, Billerica, MA,
U.S.) with a resistivity above 18.2 Mꢁ·cm was used for all aqueous mobile phase and sample
preparation. PerkinElmer Lambda 35 UVꢀvis Spectrophotometer and LS 55 Fluorescence
Spectrometer were used for acquiring absorption and fluorescence emission spectra, respectively.
High performance liquid chromatography (HPLC) measurements were performed on Dionex
Softron GmbH (Germany) Ultimate 3000 series system. Gel permeation chromatography (GPC)
measurements were carried out on Dionex Softron GmbH (Germany) Ultimate 3000 series
system equipped with PSS´s ETAꢀ2010 viscometer/Shodex RIꢀ101 detectors. Dynamic Light
Scattering (DLS) and zeta potential measurements were performed on a Nanotrac waveꢀZeta
(ZS), (Microtrac, U.S.) instrument having a scattering angle = 180°, a laser wavelength = 780 nm
and a power of 3 mW.
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Methods
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HPLC of Intermediate Porphyrin, and Chlorin Compounds
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The purity (>95%) of porphyrin and chlorin intermediates was confirmed by HPLC, and for the
final TPCꢀchitosan nanoꢀconjugates with GPC analysis and 1H NMR analysis. HPLC
measurements were performed on a Dionex Softron GmbH (Germany) Ultimate 3000 series
consisting of a DGPꢀ3600A pump with a builtꢀin degasser, a WPSꢀ3000 auto sampler, a TCCꢀ
3100 column compartment and a Photodiode Array Detector (PDAꢀ3000). A reverse phase
Phenomenex LUNA 5 µm C18 (2) column with size 150 × 4.60 mm (Phenomenex, UK), was
used for this purpose. All samples were prepared by first dissolving ~1 mg of compound in 100
µL DMSO, and then diluting it with CH3CN in more than 10 fold this volume. Solutions were
filtered through a 0.45 µm (Spartan 13/0.45 RC, Whatman) filter before measurements. The flow
rate was 1.0 mL/min, the injection volume 20 µL and the temperature of the column
compartment was 25 °C. Four different wavelengths (254 nm, 366 nm, 420 nm and 650 nm) were
used for detection and recording. The isocratic mode was used for all measurements and the
mobile phase consisted of a three solvent system out of solvent A = 0.2% (v/v) TFA in CH3CN,
solvent B = water, and solvent C = 10% (v/v) MeOH in water. In the mobile phase, retention time
and ratio of isomers (in case of chlorin derivatives) for individual compounds are shown in Table
S2, in the section HPLC Results.
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Gel Permeation Chromatography of Chitosan Derivatives
Gel permeation chromatography (GPC) measurements of chitosan derivatives were performed
using WinGPC Unichrom software [Polymer Standards Service (PSS) GmbH (Germany)], on
Dionex HPLC system (auto sampler WPSꢀ3000, Pump LPGꢀ3400 A, Column compartment
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TCCꢀ3000), equipped with a series of three columns [Novema 10µ guard (50 × 8 mm), Novema
10µ 30A (150 × 8 mm) and Novema 10µ 1000A (300 × 8 mm)] and PSS´s ETAꢀ2010
viscometer/Shodex RIꢀ101 detectors. For GPC measurement of the parent chitosan material:
eluent 0.1% (v/v) TFA in 0.1 M (aqueous) NaCl, and Poly(2ꢀvinylpyridine) standards with
varying average molecular weights (provided by PSSꢀkit), were used for generating a universal
calibration curve. For GPC measurements of the chitosan mesylate and the final TPCꢀchitosan
nanoꢀconjugates (18, 19, 23, 24): the eluent was 0.1 M (aqueous) NaCl, and Dextran standards
with varyingaverage molecular weights (Amersham Bioscience AB, Sweden) were used for
obtaining a universal calibration curve. All samples were dissolved in respective eluents and
filtered through a 0.45 ꢂm filter (Spartan 13/0.45 RC, Whatman) prior to measurements. GPC
results consisting of viscosity and refractive index chromatograms were acquired at ambient
temperature using the flow rate of 1 mL/min and with a 100µL sample volume.
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Absorption and Fluorescence Properties
Absorption spectra were recorded on a PerkinElmer Lambda 35 UVꢀvis Spectrophotometer.
Steady state fluorescence emission spectra were acquired on a PerkinElmer LS55 Fluorescence
Spectrometer equipped with a red sensitive photomultiplier tube (Rꢀ928 PMT). The excitation
source was a Xenon discharge lamp, equivalent to 20 kW for 8 µs duration and a MonkꢀGillieson
type monochromator was employed. For fluorescence quantum yield (ΦF) measurements the
conditions were as follows: (i) for small porphyrin and chlorin intermediate compounds, stock
solutions were prepared in DMSO (0.01 mg/mL); the stock solution of the standard mesoꢀ
tetraphenylporphyrin (TPP) (0.1 mg/mL) was prepared in toluene. (ii) For the final TPCꢀchitosan
nanoꢀconjugates (18, 19, 23 and 24) the following solutions were created: (a) for ΦF
measurements in DMSO, samples were dissolved by first add ing 100 µL of water to 1 mg of the
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sample, it was then vortexed and finally 900 µL of DMSO was added and this solution was then
further diluted ten times with DMSO and used as a stock solution; (b) for ΦF measurements in
H2O, stock solutions were prepared by dissolving samples (0.1 mg/mL) in deionized H2O. All the
samples were wrapped in aluminum foil prior to analysis and used within 3 h of initial
preparation.
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The UVꢀvis absorption and fluorescence emission spectra were recorded at ambient
temperature using a quartz cuvette with a 10 mm path length. For this purpose, diluted solutions
with a final concentration below 10–6 M resulting in an absorbance of <0.1 for porphyrin
derivatives and <0.04 for chlorin derivatives at the excitation wavelength were used to avoid an
innerꢀfilter effect. Fluorescence emission spectra of all the samples and reference compounds
were measured under identical instrumental parameters: with the a constant slit width of 2.5 nm
for both excitation and emission, auto PMT voltage, and spectra were averaged over three scans
automatically by the acquisition software (FL WinLab software V4.00.03). For ΦF calculations,
fluorescence emission spectra were corrected for spectral sensitivity of the instrument (LS55
spectrometer) for PMT Rꢀ928, by calibration using a standard tungsten lamp (LSꢀ1ꢀCAL,
LSC172 36251, Ocean optics, Dunedin, U.S.). The ΦF values of all compounds (either in DMSO
or H2O) were determined relative to a standard TPP (ΦF = 0.11, in toluene λex = 514 nm),38 taking
into account the refractive index of the solvents, using the following eq (1):39
ꢃ
GradX
ηX
(ΦF)X = ΦST ꢀ
ꢁ ꢂ ꢃꢄ
(1)
GradST ηST
Where, ΦF = fluorescence quantum yield; Grad = gradient from the plot of integrated fluorescence intensity versus
absorbance; η = the refractive index of the solvent; and the subscripts ST and X refer to the standard and the
unknown test sample, respectively.
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Dynamic Light Scattering (DLS)
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For DLS and zeta potential measurements, TPCꢀchitosan (18, 19 and 23, 24) samples were
dissolved in deionized water, sonicated (40 min, 55 °C), and then filtered through a 0.45 ꢂm filter
(Spartan 13/0.45 RC, Whatman) prior to measurements. All acquisitions were carried out at 25 ±
0.5 °C, and the results were the average of five runs of 30 s each, for each sample. The
reproducibility within the same sample was checked by performing measurements at two
different times and concentrations (0.1 mg/mL, and 1 mg/mL). The surface charge of the nanoꢀ
conjugates was determined by the estimation of zeta potential.
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Degree of Substitution (DS)
1
The DS of chlorin PSs in nanoꢀconjugates (18, 19, 23 and 24) is determined by H NMR of the
key intermediate compounds 15 and 20, by using the following eq (2):
(
)
Aromatic TPC peaks + α pyrrole NH peak
30
ꢆ
( )
2
DS = ꢅ
ꢇ . ꢅ
ꢇ
28
(
)
TBDMS peaks
ꢆ
Where DS = degree of substitution of the photosensitizer per glucosamine monomer unit; Aromatic TPC peaks =
sum of the integration of peaks of linked photosensitizer TPCN1P or TPCC1P moieties in the aromatic region; α
pyrrole NH peak = sum of the integration of inner NH proton peaks of linked TPCN1P/TPCC1P moieties in the
aliphatic region; TBDMS peak = sum of the integration of peaks of TBDMS groups of backbone chitosan polymer in
the aliphatic region.
Chemical Synthesis
mesoꢀTetraphenylporphyrin (1) (TPP). Porphyrin 1 was prepared by the procedure as described
in our previous article following the literature procedure.36, 40 TLC (Hexane/CH2Cl2, 1:1): Rf =
0.66. FTꢀIR (KBr): ν = 3309 (N–H), 3051, 3022 (aryl C–H), 1595, 1556 (phenyl ring), 1469,
1
1440, 1347, 1176, 1001, 966, 793, 750, 726, 696, 657 cm−1. H NMR (CDCl3): δ = 8.90 (s, 8H,
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βꢀpyrroleꢀCH), 8.27 (d, J = 8 Hz, 8H, tetraphenylꢀHo), 7.75–7.83 (m, 12H, tetraphenylꢀHm,p),
−2.70 (s, 2H, αꢀpyrroleꢀNH). 13C NMR (CDCl3): δ = 142.34 (q, ipsoꢀC), 134.72 (t, orthoꢀC),
131.24 (br, βꢀpyrroleꢀC), 127.86 (t, paraꢀC), 126.83 (t, metaꢀC), 120.31 (q, mesoꢀC). HRMS
(ESI): m/z calcd for C44H31N4 ([M+H]+), 615.2543; found, 615.2545.
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5ꢀ(4ꢀAminophenyl)ꢀ10,15,20ꢀtriphenylporphyrin (2) (TPPN1). Aminoꢀporphyrin 2 was prepared
by the procedure as described in our previous article following the literature procedure.36 TLC
(Hexane/CH2Cl2, 3:7): Rf = 0.23. FTꢀIR (KBr): ν = 3470, 3381 (NH2), 3314 (N–H), 3051, 3023
(aryl C–H), 1618, 1594, 1557 (aryl C–C), 1472, 1441, 1349, 1178, 1154, 1071, 1001, 965, 798,
746, 723, 699 cm−1. 1H NMR (CDCl3): δ = 9.01 (d, J = 4.0 Hz, 2H, βꢀpyrroleꢀCH), 8.92–8.93 (m,
6H, βꢀpyrroleꢀCH), 8.29–8.31 (d, J = 8 Hz, 6H, triphenylꢀHo), 8.03 (d, J = 8.0 Hz, 2H, NH2ꢀ
phenylꢀHo), 7.77–7.84 (m, 9H, triphenylꢀHm,p), 6.99 (d, J = 8.0 Hz, 2H, NH2ꢀphenylꢀHm), 3.87
(s, 2H, NH2), −2.62 (br s, 2H, αꢀpyrroleꢀNH) ppm. 13C NMR (CDCl3): δ = 146.12, 142.44,
142.39, 135.81, 134.71, 132.45, 131.21, 127.80, 126.81, 121.06, 120.15, 119.92, 113.52 ppm.
HRMS (ESI): m/z calcd for C44H32N5 ([M+H]+), 630.2652; found 630.2660.
5ꢀ(4ꢀAminophenyl)ꢀ10,15,20ꢀtriphenylchlorin (3) (TPCN1). Aminoꢀporphyrin 2 (1.5 g, 2.38
mmol) was dissolved in pyridine under N2 while protected from light. To this solution, K2CO3
(2.96 g, 21.5 mmol) and pꢀtoluenesulfonyl hydrazide (0.887 g, 4.77 mmol) were added and the
resulting reaction mixture was heated under reflux. Additional portions of pꢀtoluenesulfonyl
hydrazide (0.887 g, 4.77 mmol) were added after the interval of 2, 4, 6 and 8 h. The stirring was
continued under reflux for 24 h. The reaction mixture was then poured into EtOAc/H2O (2:1, 900
mL) and heated under reflux for 1 h. After cooling to a 25 °C, the organic phase was separated
and washed with 2 N HCl (3 × 200 mL) followed by a washing with H2O (2 × 100 mL) and
saturated aqueous NaHCO3 (2 × 150 mL). The organic phase was then dried over Na2SO4 and
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concentrated in vacuo to afford a 1.3 g crude product. Analysis of the visible spectrum of the
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crude product showed that it was a mixture of chlorin and bacteriochlorin (band at 651 and 737
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nm, respectively). Furthermore, the analysis by H NMR spectra confirmed that there was no
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trace amount of starting porphyrin material left unreacted.
This crude product (1.3 g) (chlorin and bacteriochlorin mixture) was dissolved in CH2Cl2
(100 mL) and then tetrachloroꢀoꢀbenzoquinone (oꢀchloranil) (420 mg, 2.7 mmol) was added in
one portion while stirring at 25 °C. The progress of the reaction was monitored simultaneously by
UVꢀvis spectroscopy. As soon as the peak of bacteriochlorin (738 nm) had completely
disappeared, the reaction mixture was quenched with solid sodium bisulfite (NaHSO3) and
washed with a 5% aqueous NaHSO3 solution (2 × 125 mL), followed by a washing with H2O
(100 mL), 5% aqueous NaOH (2 × 150 mL), and finally with H2O (150 mL). The organic phase
was collected, dried over Na2SO4, and concentrated in vacuo to afford exclusively a chlorin
compound, i.e. aminoꢀchlorin 3 (1.2 g, 80%) as a brown colored solid. Compound 3 and its
derivatives exist in two isomeric forms (confirmed by HPLC). TLC (Hexane/CH2Cl2, 3:7): Rf =
0.23. 1H NMR (CDCl3): δ = 7.86–8.66 (m, 14H, βꢀpyrroleꢀCH & tetraphenylꢀHo), 7.63–7.73 (m,
9H, triphenylꢀHm,p), 7.00 (d, J = 8 Hz, 2H, NH2ꢀphenylꢀHm), 4.14–4.23 (m, 4H, chlorin βꢀ
pyrroleꢀCH2), 3.95 (br s, 2H, NH2), −1.38 and −1.46 (br s, 2H, αꢀpyrroleꢀNH) ppm. HRMS (ESI):
m/z calcd for C44H34N5 ([M+H]+), 632.2809; found 632.2808.
5ꢀ(4ꢀ(2ꢀ(1ꢀpiperazinyl)acetyl)aminophenyl)ꢀ10,15,20ꢀtriphenylchlorin (4) (TPCN1P). Compound 3
(600 mg, 0.95 mmol) was dissolved in CH2Cl2 (15 mL) and stirred under N2 while protected from
light. To this solution, Et3N (0.32 mL, 2.27 mmol) was added followed by a dropwise addition of
chloroacetyl chloride (0.092 mL, 1.15 mmol) at 25 °C for 2 h. An excess quantity of piperazine
(0.328 g, 3.8 mmol) was then added and the stirring was continued overnight. The reaction
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mixture was diluted with CH2Cl2 (85 mL), washed with H2O (3 × 35 mL) and brine (35 mL),
dried over Na2SO4, and concentrated in vacuo. The crude product was purified by silica gel
column chromatography using MeOH/CH2Cl2 (8:92) as eluent, to afford the desired compound 4
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(440 mg, 61%) as a brown solid. TLC (CH2Cl2/MeOH, 9:1): Rf = 0.15. H NMR (CDCl3): δ =
9.34, 9.39 (s, 1H, TPCꢀNH), 7.86–8.65 (m, 16H, βꢀpyrroleꢀCH, triphenylꢀHo & RꢀNHTPCꢀ
phenylꢀHo,m ), 7.66–7.73 (m, 9H, triphenylꢀHm,p), 4.18–4.19 (br s, 4H, chlorin βꢀpyrroleꢀCH2),
3.29 (s, 2H, ArNHCOCH2ꢀpip), 3.17 (br m, 4H, piperazine ringꢀCH2), 2.81 (br m, 4H, piperazine
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ringꢀCH2), −1.37 (br s, 2H, αꢀpyrroleꢀNH) ppm. C NMR (CDCl3): δ = 168.37, 167.48, 152.61,
143.14, 142.22, 140.86, 139.20, 138.32, 137.19, 136.99, 135.33, 134.64, 133.98, 133.01, 132.37,
132.12, 131.96, 128.17, 127.69, 126.81, 123.56, 123.38, 122.79, 122.08, 119.22, 117.94, 112.41,
111.65, 62.63, 53.50, 45.59, 35.90 ppm. HRMS (ESI): m/z calcd for C50H44N7O ([M+H]+),
758.3602; found 758.3613.
Methylꢀ4ꢀformylbenzoate (5). 4ꢀCarboxybenzaldehyde (4 g, 26.6 mmol) was suspended in
anhydrous MeOH (60 mL) and stirred under N2. The reaction mixture was cooled to 0 °C and
then acetyl chloride (9.5 mL, 133 mmol) was added dropwise. The resulting mixture was allowed
to warm up and then stirred for 12 h at 25 °C before it was concentrated in vacuo to remove the
MeOH completely. The crude residue was then diluted with EtOAc (120 mL), washed with 1 N
aqueous NaOH (5 × 30 mL) and brine (2 × 25 mL), dried over Na2SO4, and concentrated in
vacuo. The crude solid obtained was finally recrystallized with EtOAc and petroleum ether, to
afford the pure ester compound 5 (3.8 g, 87%) as a white solid. TLC (Hexane/CH2Cl2, 3:7): Rf =
0.36. mp: 61–63 °C (lit. mp 59–64 °C). 1H NMR (CDCl3): δ = 10.06 (s, 1H, CHO), 8.15 (d, J = 8
Hz, 2H), 7.91 (d, J = 8 Hz, 2H), 3.92 (s, 3H) ppm. 13C NMR (CDCl3): δ = 191.66, 166.07,
139.21, 135.13, 130.23, 129.55, 52.62 ppm.
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5ꢀ(4ꢀMethoxycarbonylphenyl)ꢀ10,15,20ꢀtriphenylporphyrin
(6)
(TPPMC1).
Methylꢀ4ꢀ
5
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7
8
formylbenzoate 5 (2 g, 12.2 mmol) and benzaldehyde (3.7 mL, 36.6 mmol) was dissolved in
CHCl3 (800 mL) and stirred under N2. To the reaction mixture, pyrrole (3.39 mL, 48.8 mmol)
was added followed by the dropwise addition of BF3.Et2O (0.2 mL, 1.6 mmol). The reaction
mixture was stirred overnight at 25 °C and then pꢀchloranil (5.1 g, 20.7 mmol) was added and
stirring continued at 25 °C for 24 h. The reaction mixture was concentrated in vacuo and the
crude residue obtained was purified by a silica gel column chromatography using
CH2Cl2/petroleum ether (3:7 to 2:3 as eluent) to afford the compound 6 (1.2 g, 14.6%) as a purple
solid. TLC (CH2Cl2/Hexane, 1:1): Rf = 0.28. 1H NMR (CDCl3): δ = 8.92–8.94 (m, 6H, βꢀpyrroleꢀ
CH), 8.85 (d, J = 4 Hz, 2H, βꢀpyrroleꢀCH), 8.49 (d, J = 8 Hz, 2H, RꢀCOTPCꢀphenylꢀHm), 8.36
(d, J = 8 Hz, 2H, RꢀNHTPCꢀphenylꢀHo), 8.26–8.28 (m, 6H, triphenylꢀHo), 7.76–7.83 (m, 9H,
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triphenylꢀHm,p), 4.15 (s, 3H, OCH3), −2.68 (br s, 2H, αꢀpyrroleꢀNH) ppm. C NMR (CDCl3): δ
= 167.49, 147.22, 142.20, 134.69, 131.38, 129.72, 129.65, 128.06, 127.93, 126.86, 120.74,
120.55, 120.31, 118.69, 52.56 ppm. HRMS (ESI): m/z calcd for C46H33N4O2 ([M+H]+),
673.2598; found 673.2581.
5ꢀ(4ꢀCarboxyphenyl)ꢀ10,15,20ꢀtriphenylporphyrin (7) (TPPC1). Compound 6 (1.2 g, 1.78 mmol)
was dissolved in a mixture of THF/pyridine (10:1, 100 mL). Then 2N methanolic KOH (120 mL)
was added and the reaction mixture was refluxed for 24 h. The reaction mixture was allowed to
cool to 25 °C and was then neutralized with a saturated aqueous citric acid solution. Subsequently
the reaction mixture was concentrated in vacuo to remove the MeOH and THF. The crude residue
was then diluted with CH2Cl2 (150 mL) and H2O (120 mL) and the aqueous phase was extracted
with CH2Cl2 (3 × 50 mL). The combined organic phase was washed with water (2 × 40 mL) and
brine (35 mL), dried over Na2SO4, and concentrated in vacuo. The crude product was purified by
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a silica gel column chromatography using MeOH/CH2Cl2 (0:100 to 4:96 as eluent) to afford the
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acid compound 7 (0.83 g, 71%) as a purple solid. TLC (CH2Cl2/MeOH, 95:5): Rf = 0.54. H
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NMR (DMSOꢀd6): δ = 8.84 (br s, 8H, βꢀpyrroleꢀCH), 8.33–8.39 (m, 4H, RꢀCOTPPꢀphenylꢀ
Ho,m), 8.21–8.23 (m, 6H, triphenylꢀHo), 7.81–7.88 (m, 9H, triphenylꢀHm,p), −2.92 (s, 2H, αꢀ
pyrroleꢀNH). HRMS (ESI): m/z calcd for C45H31N4O2 ([M+H]+), 659.2442; found 659.2446.
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5ꢀ(4ꢀCarboxyphenyl)ꢀ10,15,20ꢀtriphenylchlorin (8) (TPCC1). Compound 7 (600 mg, 0.9 mmol)
and anhydrous K2CO3 (1.13 g, 8.2 mmol) were dissolved in pyridine (42 mL) under N2 and
protected from light. pꢀtoluenesulfonyl hydrazide (340 mg, 1.8 mmol) was then added to the
reaction mixture and stirred with heating under reflux. Additional quantities of pꢀtoluenesulfonyl
hydrazide (340 mg, 1.8 mmol) in 3mL of pyridine were added after the periods of 2, 4, 6, 8 and
10 h, and stirring continued under reflux for 24 h. The reaction mixture was then allowed to cool
down to 25 °C before it was poured into EtOAc/H2O (2:1, 750 mL) and again stirred under reflux
for 1 hour. After that it was cooled to 25 °C, the organic phase was separated and washed with 2
N HCl (2 × 150 mL), followed by washing with H2O (2 × 150 mL), dried over Na2SO4 and
concentrated in vacuo to give 565 mg of the crude compound. Analysis of the UVꢀvis spectrum
of the crude revealed that it was the mixture of chlorin and bacteriochlorin (band at 651 nm and
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738 nm respectively). Furthermore, analysis by H NMR spectra confirmed that there was no
trace amount of starting porphyrin material left unreacted.
The above crude material (chlorin/bacteriochlorin mixture, 565 mg) was completely
dissolved in a 40 ml mixture of CH2Cl2/MeOH (3:1) under N2. To this solution, oꢀchloranil (180
mg, 0.7 mmol) was added in one portion at 25 °C, and the progress of the reaction monitored
simultaneously by UVꢀvis. As soon as the absorption peak of bacteriochlorin (738 nm)
diminished, the reaction mixture was quenched with a solid NaHSO3, followed by a washing of
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the organic phase with 5% aqueous NaHSO3 (2 × 150 mL), H2O (100 mL) and 5% aqueous
NaOH (2 × 150 mL) and finally, again with H2O (120 mL). The emulsion that formed during the
extraction process was removed by washing the organic phase with the saturated aqueous citric
acid solution. The organic phase was dried over Na2SO4, filtered and concentrated in vacuo to
give exclusively the chlorin compound 8 (420 mg, 70%) as a brown solid. Compound 8 and its
derivatives exist in two isomeric forms (confirmed by HPLC). TLC (CH2Cl2/MeOH, 95:5): Rf =
0.54. 1H NMR (DMSOꢀd6): δ = 7.91–8.58 (m, 16H, βꢀpyrroleꢀCH, triphenylꢀHo & RꢀCOTPCꢀ
phenylꢀHo,m), 7.68–7.77 (m, 9H, triphenylꢀHm,p), 4.12–4.13 (m, 4H, chlorin βꢀpyrroleꢀCH2),
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−1.53 and −1.60 (br s, 2H, αꢀpyrroleꢀNH) ppm. H NMR (CDCl3): δ = 7.87–8.60 (m, 16H, βꢀ
pyrroleꢀCH, triphenylꢀHo & RꢀCOTPCꢀphenylꢀHo,m), 7.64–7.74 (m, 9H, triphenylꢀHm,p), 4.16–
4.18 (m, 4H, chlorin βꢀpyrroleꢀCH2), −1.39 and −1.49 (2 br s, 2H, αꢀpyrroleꢀNH) ppm. HRMS
(ESI) calcd for C45H33N4O2 ([M+H]+), 661.2598; found 661.2612.
Nꢀ(tertꢀButoxycarbonyl)ꢀpiperazine (9) (1ꢀBocꢀpiperazine). Piperazine (6 g, 69.6 mmol) was
dissolved in CH2Cl2 (120 mL) and the solution was cooled to 0 °C. To the reaction mixture,
solution of diꢀtertꢀbutyl dicarbonate (Boc2O) (7.6 g, 34.8 mmol) in CH2Cl2 (80 mL) was added
dropwise and the stirring was continued for 24 h. The precipitate formed was filtered off and
washed with CH2Cl2 (2 × 20 mL), and the combined filtrate was separated and washed with H2O
(3 × 40 mL), brine (30 mL), dried over Na2SO4 and concentrated in vacuo to afford the
compound 5 (6.5 g, 50%) as a white solid. mp: 44–46 °C (lit. mp 46–47 °C); 1H NMR (CDCl3): δ
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= 3.32 (t, J = 4 Hz, 4H), 2.74 (t, J = 4 Hz, 4H), 1.60 (s, 1H, NH), 1.39 (s, 9H) ppm. C NMR
(CDCl3): δ = 154.85, 80.00, 79.52, 45.96, 44.45, 28.45 ppm. MS (ESI): m/z calcd for C9H19N2O2
([M+H]+), 187.1441; found 187.1434.
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5ꢀ(4ꢀ(4ꢀtertꢀButoxycarbonylpiperazinꢀ1ꢀyl)carbonylphenyl)ꢀ10,15,20ꢀtriphenylchlorin (10). The
chlorin compound 8 (500 g, 0.76 mmol) and tertꢀbutyl piperazineꢀ1ꢀcarboxylate 9 (155 mg, 0.83
mmol) were dissolved in DMF (4 mL) under N2 and protected from light. Nꢀ(3ꢀ
dimethylaminopropyl)ꢀN′ꢀethylcarbodiimide hydrochloride (EDCI·HCl) (174 mg, 0.91 mmol)
and 1ꢀhydroxybenzotriazole hydrate (HOBt) (123 mg, 0.91 mmol) were added to the reaction
mixture, followed by an addition of Et3N (0.26 mL, 1.82 mmol). The reaction mixture was stirred
overnight at 25 °C before it was slowly poured into stirring H2O (100 mL). The solid obtained
was filtered off, washed with plenty of H2O, dried, and then the crude product was purified by a
silica gel column chromatography (CH2Cl2/MeOH, 100:0 to 99:1 as eluent) to afford the
compound 10 (340 mg, 54%) as a brown solid. TLC (CH2Cl2/MeOH, 99:1): Rf = 0.74. 1H NMR
(CDCl3): δ = 7.74–8.59 (m, 16H, βꢀpyrroleꢀCH, triphenylꢀHo & RꢀCOTPCꢀphenylꢀHo,m), 7.65–
7.72 (m, 9H, triphenylꢀHm,p), 4.16–4.17 (m, 4H, chlorin βꢀpyrroleꢀCH2), 3.78–3.86 (br m, 4H,
piperazine ringꢀCH2), 3.63 (br m, 4H, piperazine ringꢀCH2), 1.53 (s, 9H, Boc (CH3)3), −1.39 and
−1.47 (br s, 2H, αꢀpyrroleꢀNH) ppm.
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5ꢀ(4ꢀ(1ꢀpiperazinyl)carbonylphenyl)ꢀ10,15,20ꢀtriphenylchlorin (11) (TPCC1P). The compound 10
(320 mg, 0.39 mmol) was dissolved in CH2Cl2 (8 mL) under N2 and with protection from light.
To this solution TFA/CH2Cl2 (1:1, 4 mL) was added and the reaction mixture was stirred at 25 °C
for 1 h, before it was diluted with CH2Cl2 (40 mL), and the organic phase was washed with H2O
(2 × 15 mL), saturated aqueous NaHCO3 (2 × 15 mL), and brine (15 mL). The organic phase was
then dried over Na2SO4 and concentrated in vacuo. The crude residue was then purified by a
silica gel column chromatography (CH2Cl2/MeOH, 100:0 to 92:8 as eluent) to afford the key
intermediate compound 11 (250 mg, 89%) as a brown solid. TLC (CH2Cl2/MeOH, 9:1): Rf =
1
0.35. H NMR (CDCl3): δ = 7.74–8.59 (m, 16H, βꢀpyrroleꢀCH, triphenylꢀHo & RꢀCOTPCꢀ
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phenylꢀHo,m), 7.64–7.72 (m, 9H, triphenylꢀHm,p), 4.16–4.17 (m, 4H, chlorin βꢀpyrroleꢀCH2),
3.73–3.90 (br m, 4H, piperazine ringꢀCH2), 3.04 (br m, 4H, piperazine ringꢀCH2), −1.40 and
−1.47 (br s, 2H, αꢀpyrroleꢀNH) ppm. HRMS (ESI): calcd for C49H41N6O ([M+H]+), 729.3336;
found 729.3332.
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Chitosan mesylate (12). This compound was synthesized according to our previously published
procedure.37 FTꢀIR (KBr): ν = 3342 (br, O–H), 3312, 3052, 3022, 2816 (m, C–H), 1691 (vs, C=O
amide I), 1596 (vs, C=O amide II), 1557, 1471, 1439, 1400, 13408, 1178, 965, 799, 700 cm−1. 1H
NMR (D2O): δ = 4.86 (Hꢀ1, partly overlapped with HDO peak), 3.73–3.92 (m, Hꢀ2 GlcNAc, Hꢀ3,
1
Hꢀ4, Hꢀ5, Hꢀ6, Hꢀ6´), 3.18 (m, Hꢀ2 GlcN), 2.81 (s, CH3S), 2.06 (s, CH3CO) ppm. H NMR
(DMSOꢀd6): δ = 8.26 (br s, 2H), 5.38–4.80 (m, 3H), 3.50–3.71 (m, 4H), 2.87 (br s, 1H), 2.42 (s,
13
3H) ppm. 13C NMR (D2O): δ = 100.09, 78.84, 77.28, 72.62, 62.49, 58.32, 41.05, 13.80 ppm. C
NMR (DMSOꢀd6): δ = 97.73, 77.80, 74.90, 70.22, 60.39, 55.83 ppm.
3,6ꢀDiꢀOꢀtertꢀbutyldimethylsilylꢀchitosan (13) (DiꢀTBDMSꢀchitosan). This compound was
synthesized according to our previously published procedure.37 FTꢀIR (KBr): ν = 2957, 2931,
2886, 2858 (s, C–H, TBDMS), 1474, 1390, 1362, 1258, 108, 1005, 836, 777 (Si–C), 670 cm−1.
1H NMR (CDCl3) δ: 4.30 (br s, Hꢀ1), 3.89 and 3.85 (br s, Hꢀ6, Hꢀ6’), 3.68 (br s, Hꢀ4), 3.50 (br s,
Hꢀ3), 3.33 (br s, Hꢀ5), 2.72 (br s, Hꢀ2 GlcN), 0.90 and 0.89 (br s, (CH3)3CꢀSi), 0.13, 0.10, 0.06,
0.05 (br s, (CH3)2Si) ppm.
Nꢀbromoacetylꢀ3,6ꢀdiꢀOꢀtertꢀbutyldimethylsilylꢀchitosan (14) (BrAꢀDiꢀTBDMSꢀchitosan). Silyl
compound 13 (1 g, 2.60 mmol) was dissolved in dry CH2Cl2 (15 mL) under N2 atmosphere. This
solution was cooled to a −20 °C by using an ice/salt mixture and then Et3N (1.81 mL, 13 mmol)
was added, followed by a slow dropwise addition of bromoacetyl bromide (0.91 mL, 10 mmol).
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The stirring was continued for exactly 1 h at −20 °C, before the reaction mixture was diluted with
CH2Cl2 (40 mL) and concentrated in vacuo. The obtained crude residue was triturated and stirred
with CH3CN (30 mL), filtered and washed with fresh CH3CN (3 × 15 mL) and dried. The dry
material was dissolved in CH2Cl2 and the organic phase was washed with H2O (3 × 30 mL) and
brine (25 mL), dried over Na2SO4, and concentrated in vacuo to afford the bromoacetyl
compound 14 (1.2 g, 92%) as a faint yellow powder. FTꢀIR (KBr): ν = 3402 (br , NH), 2957,
2931, 2886, 2858 (s, C–H, TBDMS), 1682 (vs, C=O amide I), 1530 (vs, C=O amide II), 1473,
1391, 1362, 1311, 1259, 1101, 1005, 837, 777 (Si–C), 669 cm−1; 1H NMR (CDCl3) δ = 4.40 (br s,
Hꢀ1), 4.02–3.26 (m, Hꢀ2 GlcN, Hꢀ3, Hꢀ4, Hꢀ5, Hꢀ6, Hꢀ6’ and GlcNHCOCH2Br), 0.90 and 0.88
(br s, (CH3)3CꢀSi), 0.13 and 0.07 (br s, (CH3)2Si) ppm.
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[Nꢀ(2ꢀ(4ꢀ(Nꢀ(4ꢀ(10,15,20ꢀtriphenylchlorinꢀ5ꢀyl)phenylamino)carbonylmethyl)piperazinꢀ1ꢀ
yl)acetyl)]0.1[Nꢀ(2ꢀbromoacetyl)]0.9ꢀ3,6ꢀdiꢀOꢀtertꢀbutyldimethylsilylꢀchitosan
(15) (TPCN1P)0.1(Br)0.9ꢀAꢀDiꢀTBDMSꢀchitosan). The chitosan intermediate 14 (800 mg, 1.58
mmol) and the aminoꢀchlorin intermediate 4 (TPCN1P) (120 mg, 0.158 mmol) were dissolved in
CH2Cl2 (25 mL) under N2 and with protecion from light. An exact equimolar quantity of Et3N (22
ꢂL, 0.158 mmol) with respect to 4 was added and the reaction mixture was stirred at 25 °C for 24
h. The full consumption of the starting material was confirmed by TLC. The reaction mixture
was diluted with CH2Cl2 (55 mL) and washed with H2O (2 × 25 mL) and brine (25 mL). The
organic phase was dried over Na2SO4, and concentrated in vacuo to afford compound 15 (700
1
mg, 78%) as a brown solid. H NMR (CDCl3): δ = 9.21, 9.25 (s, TPCꢀNHCO), 7.86–8.60 (m, βꢀ
pyrroleꢀCH, triphenylꢀHo & RꢀNHTPCꢀphenylꢀHo,m), 7.65–7.73 (m, triphenylꢀHm,p), 3.35–4.50
[br m, chitosan (Hꢀ1, Hꢀ2 GlcN, Hꢀ3, Hꢀ4, Hꢀ5, Hꢀ6, Hꢀ6’ and Hꢀ2 GlcNAc, CH2CONHGlc),
TPCꢀNHCOCH2ꢀpip, piperazine ringꢀCH2 and chlorin βꢀpyrroleꢀCH2], 2.77–2.83 (m, piperazine
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ringꢀCH2), 0.88–0.89 [br s, (CH3)3CꢀSi], 0.02–0.13 [(br m, (CH3)2Si], −1.44 (br s, 2H, αꢀpyrroleꢀ
NH).
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[Nꢀ(2ꢀ(4ꢀ(4ꢀ(10,15,20ꢀtriphenylchlorinꢀ5ꢀyl)phenylcarbonyl)piperazinꢀ1ꢀyl)acetyl)]0.1[Nꢀ(2ꢀ
bromoacetyl)]0.9ꢀ3,6ꢀdiꢀOꢀtertꢀbutyldimethylsilylꢀchitosan (20) ((TPCC1P)0.1(Br)0.9ꢀAꢀDiꢀTBDMSꢀ
chitosan). The chitosan intermediate 14 (800 mg, 1.58 mmol) was dissolved in NꢀMethylꢀ2ꢀ
pyrrolidone (NMP) (15 mL) under N2 and with protection from light. The carboxylꢀchlorin
intermediate 11 (TPCC1P) (125 mg, 0.173 mmol), and NaHCO3 (0.29 g, 3.45 mmol) were added
and the reaction mixture was heated to 75 °C and stirred overnight, before it was cooled to 25 °C,
and poured into stirring H2O. The obtained crude solid was filtered off, washed with plenty of
H2O and dried. The crude material was dissolved in CH2Cl2, washed with H2O (2 × 25 mL and
brine (25 mL), filtered, dried over Na2SO4, and concentrated in vacuo to obtain the compound 20
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(810 mg, 89%) as a brown solid. H NMR (CDCl3): δ = 7.75–8.60 (m, βꢀpyrroleꢀCH, triphenylꢀ
Ho and RꢀCOTPCꢀphenylꢀHo,m), 7.64–7.71 (m, 9H, triphenylꢀHm,p), 3.38–4.5 [br m, chitosan
(Hꢀ1, Hꢀ2 GlcN, Hꢀ3, Hꢀ4, Hꢀ5, Hꢀ6, Hꢀ6’ and Hꢀ2 GlcNAc, CH2CONGlc), piperazin ringꢀCH2
and chlorin βꢀpyrroleꢀCH2], 2.76–2.84 (m, piperazin ringꢀCH2), 0.89–0.92 [br s, (CH3)3CꢀSi],
0.02–0.10 [ (br m, (CH3)2Si) ], −1.40 and −1.48 (br s, 2H, αꢀpyrroleꢀNH) ppm.
General Procedure A for the Synthesis of Compounds 16 and 21.
[Nꢀ(2ꢀ(4ꢀ(Nꢀ(4ꢀ(10,15,20ꢀtriphenylchlorinꢀ5ꢀyl)phenylamino)carbonylmethyl)piperazinꢀ1ꢀ
yl)acetyl)]0.1[Nꢀ(2ꢀ(N,N,Nꢀtrimethylammoniumyl)acetyl)]0.9ꢀ3,6ꢀdiꢀOꢀtertꢀbutyldimethylsilylꢀ
chitosan bromide (16) (TPCN1P)0.1ꢀDiꢀTBDMSꢀchitosanꢀTMA. Compound 15 (350 mg, 0.61
mmol) was dissolved in CH2Cl2 (15 mL) under N2 and with protection from light. Excess Me3N
(31–35 wt % in EtOH, 4.2 M) (15 mL) solution was added to the reaction mixture and it was
stirred at 25 °C for 24 h. The reaction mixture was concentrated in vacuo and the crude material
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was completely dried under high vacuum, yielding the crude compound 16 (355 mg) as a brown
solid. The material 16 was used directly for the next step without further purification.
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[Nꢀ(2ꢀ(4ꢀ(4ꢀ(10,15,20ꢀtriphenylchlorinꢀ5ꢀyl)phenylcarbonyl)piperazinꢀ1ꢀyl)acetyl)]0.1[Nꢀ(2ꢀ
(N,N,Nꢀtrimethylammoniumyl)acetyl)]0.9ꢀ3,6ꢀdiꢀOꢀtertꢀbutyldimethylsilylꢀchitosan bromide (21)
(TPCC1P)0.1ꢀDiꢀTBSꢀchitosanꢀTMA. The general procedure A was followed using 20 (350 mg,
0.61 mol) and trimethylamine solution to give 21 as crude solid (360 mg), which was used
without further purification for the next step.
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General Procedure B for the Synthesis of Compounds 17 and 22.
[Nꢀ(2ꢀ(4ꢀ(Nꢀ(4ꢀ(10,15,20ꢀtriphenylchlorinꢀ5ꢀyl)phenylamino)carbonylmethyl)piperazinꢀ1ꢀ
yl)acetyl)]0.1ꢀ[Nꢀ(2ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)acetyl)]0.9ꢀ3,6ꢀdiꢀOꢀtertꢀbutyldimethylsilylꢀchitosan
(17) (TPCN1P)0.1ꢀDiꢀTBDMSꢀchitosanꢀMP. Compound 15 (350 mg, 0.61 mmol) was dissolved in
CH2Cl2 (15 mL) under N2 and with protection from light. Excess of 1ꢀmethylpiperazine (10 mL)
was added to the reaction mixture that was stirred at 25 °C for 24 h. The reaction mixture was
concentrated in vacuo and the crude product was completely dried under high vacuum yielding
the corresponding crude product 17 (330 mg), which was used without further purification for the
next step.
[Nꢀ(2ꢀ(4ꢀ(4ꢀ(10,15,20ꢀtriphenylchlorinꢀ5ꢀyl)phenylcarbonyl)piperazinꢀ1ꢀyl)acetyl)]0.1[Nꢀ(2ꢀ(4ꢀ
methylpiperazinꢀ1ꢀyl)acetyl)]0.9ꢀ3,6ꢀdiꢀOꢀtertꢀbutyldimethylsilylꢀchitosan (22) (TPCC1P)0.1ꢀDiꢀ
TBDMSꢀchitosanꢀMP. The general procedure B was followed using the compound 20 (250 mg,
0.38 mol) and 1ꢀmethylpiperazine to yield the compound 22 (265 mg) as a crude solid, which
was used as it is for the next step.
Final TBDMSꢀdeprotection was carried out by following general procedure C.
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Synthesis of the TPCꢀchitosan nanoꢀconjugates (18, 19, 23 and 24).
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The compound (16, 17, 21, 22) was dissolved in MeOH (20 mL) under N2 and the reaction
mixture was protected from light. The reaction mixture was degassed by purging it with N2 for 5
minutes and subsequently cooled to 0 °C, before the addition of concd HCl (4 mL). The reaction
mixture was allowed to warm up to 25 °C and stirred for 12 h. The reaction mixture was then
diluted and ionꢀexchanged by the addition of 5% NaCl (aqueous) (40 mL) to the solution. It was
then stirred for 1 h before it was dialyzed against 8% NaCl (aqueous) for 24 h, and then again
against deionized water for two days. The clean brown solution was subsequently freeze dried to
afford the corresponding final nanoꢀconjugates (18, 19, 23, 24) as a brown fluffy material.
Sometimes the reaction needed to be repeated in order to get rid of a trace amount of TBDMS
that was left unꢀdeprotected from the chitosan backbone.
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[Nꢀ(2ꢀ(4ꢀ(Nꢀ(4ꢀ(10,15,20ꢀtriphenylchlorinꢀ5ꢀyl)phenylamino)carbonylmethyl)piperazinꢀ1ꢀ
yl)acetyl)]0.1[Nꢀ(2ꢀ(N,N,Nꢀtrimethylammoniumyl)acetyl)]ꢀchitosan chloride (18) [(TPCN1P)0.1ꢀCSꢀ
TMA]. The general procedure C was followed by using compound 16 (325 mg, 0.52 mmol) and
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concd HCl/MeOH to afford the nanoꢀconjugate 18 (175 mg, 85%) as a brown solid. H NMR
(DMSOꢀd6/D2O, 96:4): δ = 7.83–8.62 (m, βꢀpyrroleꢀCH, triphenylꢀHo & RꢀNHTPCꢀphenylꢀ
Ho,m), 7.69–7.77 (m, triphenylꢀHm,p), 4.52 (br s, Hꢀ1), 4.11–4.14 (m, CH2CONGlc and chlorin
βꢀpyrroleꢀCH2), 3.26–3.67 (br m, partially overlapped with HDO peak, Hꢀ2 GlcNAc, Hꢀ3, Hꢀ4,
+
Hꢀ5, Hꢀ6, Hꢀ6´, Hꢀ2 GlcNHCO, TPCNHCOCH2ꢀpip, piperazine ringꢀCH2), 3.24 (s, N(CH3)3))
ppm. UVꢀvis (DMSO): λmax = 421, 520, 549, 599, 651 nm.
[Nꢀ(2ꢀ(4ꢀ(Nꢀ(4ꢀ(10,15,20ꢀtriphenylchlorinꢀ5ꢀyl)phenylamino)carbonylmethyl)piperazinꢀ1ꢀ
yl)acetyl)]0.1ꢀ[Nꢀ(2ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)acetyl)]ꢀchitosan (19) [(TPCN1P)0.1ꢀCSꢀMP]. The
general procedure C was followed by using compound 17 (300 mg, 0.45 mmol) and concd
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HCl/MeOH to afford the nanoꢀconjugate 19 (165 mg, 84%) as a brown solid. 1H NMR (DMSOꢀ
d6/D2O, 96:4): δ = 7.83–8.62 (m, βꢀpyrroleꢀCH, triphenylꢀHo & RꢀNHTPCꢀphenylꢀHo,m), 7.66–
7.75 (m, triphenylꢀHm,p), 4.50 (br s, Hꢀ1), 4.10–4.14 (m, chlorin βꢀpyrroleꢀCH2), 2.92–3.55 (m,
partially overlapped with HDO peak , Hꢀ2 GlcNAc, Hꢀ3, Hꢀ4, Hꢀ5, Hꢀ6, Hꢀ6´, Hꢀ2 GlcNHCO,
CH2CONGlc, TPCNHCOCH2ꢀpip), 2.33–2.63 (m, partially overlapped with DMSOꢀd6 peak,
piperazine ringꢀCH2, piperazineꢀNꢀCH3) ppm. UVꢀvis (H2O): λmax = 412, 430, 531, 560, 611, 664
nm. UVꢀvis (DMSO): λmax = 421, 521, 548, 596, 651 nm.
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[Nꢀ(2ꢀ(4ꢀ(4ꢀ(10,15,20ꢀtriphenylchlorinꢀ5ꢀyl)phenylcarbonyl)piperazinꢀ1ꢀyl)acetyl)]0.1[Nꢀ(2ꢀ
(N,N,Nꢀtrimethylammoniumyl)acetyl)]ꢀchitosan chloride (23) [(TPCC1P)0.1ꢀCSꢀTMA]. The
general procedure C was followed using the compound 21 (300 mg, 0.48 mmol) and concd
HCl/MeOH to afford the nanoꢀconjugate 23 (170 mg, 89%) as a brown solid. FTꢀIR (KBr): ν =
3353, 3061, 2950, 1683, 1580, 1473, 1440, 1376, 1291, 1154, 1112, 1067, 1032, 970, 911, 794,
1
703 cm−1. H NMR (DMSOꢀd6/D2O, 96:4): δ = 7.89–8.62 (m, βꢀpyrroleꢀCH, triphenylꢀHo & Rꢀ
COTPCꢀphenylꢀHo,m), 7.67–7.76 (m, triphenylꢀHm,p), 4.50 (br s, Hꢀ1), 4.06–4.16 (m,
CH2CONGlc and chlorin βꢀpyrroleꢀCH2), 3.26–3.75 (m, partially overlapped with HDO peak, Hꢀ
+
2 GlcNAc, Hꢀ3, Hꢀ4, Hꢀ5, Hꢀ6, Hꢀ6´, Hꢀ2 GlcNHCO, piperazine ringꢀCH2), 3.24 (s, N(CH3)3))
ppm. UVꢀvis (DMSO): λmax = 420, 520, 547, 599, 651 nm.
[Nꢀ(2ꢀ(4ꢀ(4ꢀ(10,15,20ꢀtriphenylchlorinꢀ5ꢀyl)phenylcarbonyl)piperazinꢀ1ꢀyl)acetyl)]0.1[Nꢀ(2ꢀ(4ꢀ
methylpiperazinꢀ1ꢀyl)acetyl)]ꢀchitosan (24) [(TPCC1P)0.1ꢀCSꢀMP]. The general procedure C was
followed using the compound 22 (240 mg, 0.38 mmol) and concd HCl/MeOH to afford the
compound 24 (85 mg, 52%) as a brown solid. FTꢀIR (KBr): ν = 3349, 2927, 1644, 1580, 1461,
1440, 1374, 1285, 1070, 1043, 985, 945, 794, 719, 703 cm−1. 1H NMR (DMSOꢀd6/D2O, 96:4): δ
= 7.86–8.63 (m, βꢀpyrroleꢀCH, triphenylꢀHo & RꢀCOTPCꢀphenylꢀHo,m), 7.67–7.76 (m,
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triphenylꢀHm,p), 7.69–7.80 (m, triphenylꢀHm,p), 4.50 (br s, Hꢀ1), 4.08–4.14 (m, chlorin βꢀ
pyrroleꢀCH2), 2.92–3.55 (m, partially overlapped with HDO peak, Hꢀ2 GlcNAc, Hꢀ3, Hꢀ4, Hꢀ5,
Hꢀ6, Hꢀ6´, Hꢀ2 GlcNHCO, CH2CONGlc), 2.27–2.63 (m, partially overlapped with DMSOꢀd6
peak, piperazine ringꢀCH2, piperazine NꢀCH3) ppm. UVꢀvis (DMSO): λmax = 421, 520, 547, 599,
651 nm.
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Porphyrin (TPP) analogues of 18, 19, 23 and 24.
Unexpected results (backꢀoxidation of TPCꢀchitosan nanoꢀconjugates to their TPP analogue
nanoꢀconjugates by TBAF/NMP) were observed when the following general TBDMSꢀ
deprotection procedure D was followed:
Example: TPP analogue of nanoꢀconjugate 18.
[Nꢀ(2ꢀ(4ꢀ(Nꢀ(4ꢀ(10,15,20ꢀtriphenylporphyrinꢀ5ꢀyl)phenylamino)carbonylmethyl)piperazinꢀ1ꢀ
yl)acetyl)]0.1[Nꢀ(2ꢀ(N,N,Nꢀtrimethylammoniumyl)acetyl)]ꢀchitosan chloride [(TPPN1P)0.1ꢀCSꢀ
TMA]. The compound 16 (600 mg, 0.86 mmol) was dissolved in NMP (5–10 mL) at 55 °C under
N2 atmosphere and protected from light. Excess amount of tetraꢀnꢀbutyl ammonium fluoride
(TBAF) was added to the reaction mixture and the stirring continued for 24 h at 55 °C. The
reaction mixture was cooled to 25 °C, diluted and ion exchanged with the 5% NaCl (aqueous, 40
mL) for 1 hour. The solution was then dialyzed against 8% NaCl (aqueous) for 24 h, and then
again against deionized water for two days. The red colored solution was then freezeꢀdried to
yield purple colored spongeꢀlike material. Surprisingly, after analysis, it was established that due
to backꢀoxidation the chlorin compounds were converted back to their TPP analogues that were
confirmed by 1H NMR and UVꢀvis (as the characteristic peak at 650 vanished) (Data not shown).
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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 [3ꢀ(4,5ꢀDimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide] was from Sigmaꢀ
Aldrich (MO, U.S.; cat. no. M 2128), and was dissolved in phosphateꢀbuffered saline (PBS) to a
concentration of 5 mg/mL, sterile filtered and stored at 4 °C. A plasmid encoding enhanced green
fluorescent protein (pEGFPꢀN1) was purchased from Clontech Laboratories Inc. (CA, U.S.; Cat.
No. 6085ꢀ1), produced by ELIM Biopharmaceuticals, Inc. (CA, U.S.) (lot# 1002) and delivered
at a concentration of 2 mg/mL in sterile water. This stock solution was aliquoted and kept at −20
°C. PolyꢀLꢀLysine hydrobromide (Mw 15–30 kDa) was purchased from SigmaꢀAldrich (MO,
U.S.; cat. no. P 7890). PolyꢀLꢀLysine hydrobromide was dissolved and diluted in distilled water,
sterilized by filtration and stored at –20 °C.
In Vitro Cell Culture Studies
Cell Cultivation. HCT116/LUC were cultured in Dulbecco's Modified Eagle's Medium
(DMEM) (Lonza, Veviers, Belgium), supplemented with 10% fetal calf serum (PAA
Laboratories GmbH, Pasching, Austria) 100 U/mL penicillin and 100 µg/mL streptomycin
(SigmaꢀAldrich, MO, U.S.) at 37 °C and 5% CO2 in a humid environment.
Treatment of the Cells. HCT116/LUC cells (1.5 × 105 cells per well for the transfection
expriments, 3.75 × 105 cells per well for the MTT assay) were seeded into 6ꢀwell (transfection)
and 24ꢀwell (MTT) plates (Nunc, Roskilde, Denmark) and incubated for 24 h (5% CO2, 37 °C).
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The photosensitizer TPCS2a or the TPCꢀchitosan nanoꢀconjugates (18, 19, 23, and 24) 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 the cell culture medium and incubated for 4 h (5% CO2, 37 °C) in a
medium containing the plasmid complex. The cells were washed once and after the addition of
fresh medium, the cells were illuminated with different light doses. After 48 h of incubation, the
expression of EGFP (Enhanced Green Fluorescent Protein) was analyzed by flow cytometry. Cell
survival was measured by the MTT assay in parallel experiments.
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The cells were exposed to light from LumiSource® (PCI Biotech, Oslo, Norway).
LumiSource® is delivered with a bank of 4 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 and an irradiance of 13
mW/cm2.
Preparation of Plasmid/poly-L-lysine Complexes. Plasmid/polyꢀLꢀlysine complexes with a
charge ratio of 2.2:1 (charge ratio is: (number of positive charges (primary amines) in polyꢀLꢀ
lysine)/number of negative charges in the phosphate groups of the DNA) were formed by the
gentle mixing of plasmid DNA and polyꢀLꢀlysine solutions. 2.5 µl of DNA (stock solution 2
µg/µL) was diluted with 47.5 µL water, and 6.92 µl polyꢀLꢀlysine (1 µg/µL) was diluted with
43.08 µL water. After mixing, the solution was incubated at 25 °C for 30 min, then diluted with
the culture medium to a final volume of 1 mL and added to the cells (1 mL per well).
Measurement of Transfection. The cells were trypsinized in 100 µL trypsin (Trypsinꢀ EDTA,
SigmaꢀAldrich, MO, U.S.), reꢀsuspended in a 500 µL cell culture medium and filtered through a
5 mL Polystyrene RoundꢀBottom Tube with a CellꢀStrainer Cap (BD Falcon) (50 µm mesh nylon
filter) before analysis in a BD LSR flow cytometer (Becton Dickinson, CA, U.S.). EGFP was
measured through a 500ꢀ550 nm filter after excitation at 450ꢀ490 nm, and the propidium iodide
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(Calbiochem Corporation, CA, U.S.) 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
pulseꢀprocessing was performed to discriminate cell doublets from single cells. 10,000 events
were collected for each sample and the data was analyzed with BD FACSDiva Software (Becton
Dickinson, CA, U.S.).
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Measurement of Cell Survival. Cell survival was measured by a method based on the reduction
of a waterꢀsoluble tetrazolium salt (MTT) to a purple, insoluble formazan product by the
mitochondrial dehydrogenases present in living, metabolically active cells. 0.5 mL medium
containing 0.125 mg 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 DMSO (SigmaꢀAldrich,
MO, U.S.) per well. The plates were read by a PowerWave XS2 Microplate Spectrophotometer
(BioꢀTek Instruments, VT, U.S.). Cell survival was calculated as the percent of controls (parallels
with no light).
In Vivo Studies
Animals. Hsd:Athymic nudeꢀFoxn1nu female mice were bred at the animal department at the
Norwegian Radium Hospital. The mice were kept under specific pathogenꢀfree conditions. Water
and food was given ad libitum. All procedures involving mice were carried out in agreement with
protocols approved by the animal care committee at the Norwegian Radium Hospital, following
the National Ethical Committee’s guidelines on animal welfare.
The six mice were 22–25 g (5–8 weeks old) when included in the experiment. The
HCT116/LUC cells were cultured at 37 °C and 5% CO2 in a humid environment before
transplantation. 1.5 × 106 cells were injected subcutaneously on the right hip of each mouse.
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