Use of Light-Degradable Aliphatic Polycarbonate Nanoparticles As Drug Carrier for Photosensitizer

Article abstract of DOI:10.1021/acs.biomac.8b01446

Aliphatic poly(carbonate)s (APCs) with rapid and controlled degradation upon specific stimulation have great advantages for a variety of biomedical and pharmaceutical applications. In the present work, we reported a new poly(trimethylene carbonate) (PTMC)

Full text of DOI:10.1021/acs.biomac.8b01446

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Article
Use of light-degradable aliphatic polycarbonate
nanoparticles as drug carrier for photosensitizer
Jingjiang Sun, Wolfgang Birnbaum, Juliane Anderski, Marie-
Theres Picker, Dennis Mulac, Klaus Langer, and Dirk Kuckling
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01446 • Publication Date (Web): 15 Nov 2018
Downloaded from http://pubs.acs.org on November 15, 2018
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Use of light-degradable aliphatic polycarbonate
nanoparticles as drug carrier for photosensitizer
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Jingjiang Sun†∥, Wolfgang Birnbaum†, Juliane Anderski‡, Marie-Theres Picker†, Dennis Mulac‡,
Klaus Langer‡, Dirk Kuckling†*
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†Paderborn University, Department of Chemistry, Warburger Str. 100, D-33098 Paderborn,
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Germany
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‡University of Münster, Institute of Pharmaceutical Technology and Biopharmacy, Corrensstr. 48,
D-48149 Münster, Germany
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∥Qingdao University of Science and Technology, School of Polymer Science and Engineering,
Zhengzhou Rd. 53, CN-266042 Qingdao, China
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ABSTRACT:
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Aliphatic poly(carbonate)s (APCs) with rapid and controlled degradation upon specific stimulation
have great advantages for a variety of biomedical and pharmaceutical applications. In the present
work, we reported a new poly(trimethylene carbonate) (PTMC)-based copolymer containing
multiple 4,5-dimethoxy-2-nitrobenzyl photo cleavable groups as pendent chains. The six-
membered light-responsive cyclic carbonate monomer (LrM) was first prepared from 2-
(hydroxymethyl)-2-methylpropane-1,3-diol and 4,5-dimethoxy-2-nitrobenzyl alcohol, and then
copolymerized with trimethylene carbonate (TMC) by 1,8-diazabicyclo(5.4.0)undec-7-ene (DBU)
catalyzed ring-opening polymerization (ROP) to afford the light-responsive polycarbonate
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(LrPC). The light-triggered decomposition of LrM and LrPC was studied by NMR, UV/VIS
spectroscopy, size exclusion chromatography (SEC), as well as ESI-ToF mass spectrometry (ESI-
ToF-MS). Stable monodisperse nanoparticles with hydrodynamic diameter of 100 nm could be
formulated from 25% LrPC and 75% poly(lactic-co-glycolic acid) (PLGA) and applied for the
encapsulation of temoporfin. Upon irradiation with UV light these particles displayed a significant
decrease of the particle countrate and increased the release rate of temoporfin in comparison to
standard PLGA nanoparticles. This work demonstrated that combination of encapsulation of
photosensitizer and light degradation using light-responsive polymers is suitable to enhance
photodynamic therapy (PDT).
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INTRODUCTION
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In recent years, aliphatic polycarbonates (APCs) have been intensively studied and used as
materials in the biomedical and pharmaceutical fields due to their low toxicity, good
biocompatibility and biodegradability.1-3 In contrast to other approaches to obtain APCs, such as
polycondensation of diols with dialkyl carbonates4-6 and copolymerization of CO2 and epoxides7,8,
the ring-opening polymerization (ROP) is the most important method to synthesize functionalized
APCs with controlled molar masses and polymer structures9-12. Although the degradation rates of
APCs could be accelerated under acid, basic or enzymatic environment, the controlled degradation
of APCs upon internal or external stimuli, such as pH13-15, temperature16,17, oxidation18-20
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enzyme11 and light21-23, is of great interest for their biomedical applications, such as drug or gene
delivery systems. Among various stimuli, the degradation rate of light-responsive polymers can
be controlled by adjusting light wavelength, intensity or duration of the irradiation, while other
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stimuli (e.g. pH, oxidation) only provide limited control and acceleration of the decomposition
process over the time.24,25
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Figure 1. Illustration of different light-responsive degradation systems: a) light-responsive groups
end-capped polymers; b) polymers with light-responsive linkers along the polymer backbone; c)
polymers with light-responsive pendent groups.
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The light-responsive self-immolative polymers can be divided into three classes (Figure 1): a)
Polymer backbones are end-capped with light-responsive groups, which can lead to a fast
degradation via a cascade of self-immolative reactions upon triggering with light.26-28 b)
Photocleavable linkers as monomers are directly incorporated into polymer backbones e.g. by
polycondensation. The degradation of polymers is achieved by cleaving self-immolative
linkers.29,30 c) Light-responsive self-immolative groups are constructed as pendent chains along
polymer backbones, whose degradation can be accelerated by intramolecular cyclization.17,31,32
From the view of biomedical applications, most polymers used in these degradation systems are
polyesters, polycarbonates and poly(ester-amide)s. Recently, Almutairi et al. reported a series of
novel functional poly(ԑ-caprolactone)31, poly(lactide-co-glycolide) (PLGA)32 and quinone-
methide26,30 based polyesters and polyurethanes with light-responsive pendent groups or end
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groups. Upon irradiation, these polymers degraded into small molecules completely via an
intramolecular cyclization or a quinone-methide self-immolative reaction. Li and Du have
prepared oxidation-promoted degradable APCs attached with phenylboronic ester pendent chain
via a carbamate linker. The H2O2-initiated oxidation led to a consecutive degradation process of
cleavage of phenylboronic ester, CO2-release, formation of free amino pendent group and
decomposition of the polymer backbone by intramolecular cyclization.18,19
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Figure 2. Illustration of the formulation of nanoparticles from LrPC, PLGA and temoporfin, and
nanoparticle decomposition upon irradiation with UV light.
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Photodynamic therapy (PDT) is a novel and efficient option for cancer treatment involving the use
of photosensitizers to generate reactive singlet oxygen species upon photoirradiation to destroy
tumor cells.33-35 Efficient anti-tumor photosensitizers are usually highly lipophilic macrocyclic
tetrapyrroles, such as porphyrins and chlorins. Due to the high lipophilicity and low water
solubility of anti-tumor photosensitizers, there is a need to develop new carriers for efficient load
and controlled release of the photosensitizer molecules upon internal or external stimuli.36-38 To
expand the family of light-responsive polymers and their applications, we report a new type of
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light-responsive APC which could be used as photosensitizer carrier to combine drug release and
PDT together upon irradiation. Toward this aim, a novel six-membered cyclic carbonate monomer
(LrM) with a light-responsive 4,5-dimethoxy-2-nitrobenzyl pendent group attached via a
carbamate linkage was synthesized (Scheme 1) and copolymerized with TMC to afford light-
responsive copolycarbonate (LrPC). By exposure to 300 – 365 nm UV light the photolabile o-
nitrobenzyl group can be removed according to the redox photoisomerization leading to the release
of functional amine group and forming an o-nitrosobenzylaldehyde side product.30 The LrPC was
stable to air and moisture but decomposed rapidly into small molecules via intramolecular
cyclization when the light-responsive pendent groups were cleaved. Furthermore, nanoparticles
prepared from 25% of this APC mixed with 75% PLGA are able to significantly increase the
release rate of the photosensitizer payload upon irradiation compared to standard PLGA
nanoparticles (Figure 2).
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EXPERIMENTAL SECTION
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Materials. Benzyl alcohol (BnOH) (99%, Acros Organics) was distilled over calcium hydride and
stored under Ar. Dichloromethane (DCM) (98%, Stockmeier Chemie) was dried over CaCl2 then
distilled over calcium hydride. Tetrahydrofuran (THF) (98%, Stockmeier Chemie) was dried over
KOH then distilled over calcium hydride. 1,3-dioxan-2-one (TMC) (99%, Shanghai Worldyang
Chemical Co.) was purified by column chromatography with ethyl acetate/n-hexane (3: 1). The
photosensitizer 5,10,15,20-tetrakis(m-hydroxyphenyl) chlorin (mTHPC, temoprofin) was kindly
provided by biolitec research GmbH (Jena, Germany). The polymer poly(DL-lactide-co-glycolide)
(PLGA), characterized by a copolymer ratio of 50: 50 (Resomer® RG 502H), was purchased from
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Evonik Industries (Darmstadt, Germany). Acetone (98%, Stockmeier Chemie), acetonitrile
(MeCN) (>99.9%, Carl Roth), ammonium formate (99%, Acros Organics), benzoic acid (>99%,
Merck), Celite (Fluka), 1,8-diazabicyclo(5.4.0)undec-7-ene (DBU) (99%, Alfa Aesar),
diisopropylethylamine (DIPEA) (99%, Alfa Aesar), dimethylformamide (DMF) (99.9%, Alfa
Aesar), 4,5-dimethoxy-2-nitrobenzyl alcohol (DMNA) (98%, Alfa Aesar), 1,4-dioxane (99.8%,
Acros Organics), Dulbecco’s Modified Eagle Medium (DMEM) (Biochrom AG, Berlin,
Germany), ethyl chloroformate (>98%, Fluka), 2-(hydroxymethyl)-2-methylpropane-1,3-diol
(97%, Alfa Aesar), fetal bovine serum (FBS) (Biochrom AG, Berlin, Germany), methanol (99.5%,
Grüssing), 4-nitrophenyl chloroformate (97%, abcr), Pd/C (10% Pd, Acros Organics), poly(vinyl
alcohol) (PVA; average molar mass 30-70 kDa) (Sigma-Aldrich), pyridine (>99%, Alfa Aesar),
sodium azide (99%, Acros Organics), p-toluenesulfonic acid monohydrate (TsOH) (99%, Merck),
4-toluenesulfonyl chloride (TsCl) (>98%, Merck), triethylamine (TEA) (99%, Acros Organics)
were used as received.
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Measurements. H and 13C nuclear magnetic resonance (NMR) spectra were recorded using
Bruker AV 500 spectrometer at 500 MHz and 125 MHz, respectively. Chloroform-d (CDCl3-d,
99.8 D%) or dimethylsulfoxide-d6 (DMSO-d6, 99.8 D%) were used as solvent for NMR
measurements. Electrospray ionization time-of-flight (ESI-ToF)-mass spectra were measured on
a SYNAPT G2 HDMS TM from Waters. Data were obtained with Mass Lynx 4.1. Fourier transform
infrared (FTIR) spectra were recorded on Vertex 70 spectrometer (Bruker Optik, Ettlingen,
Germany) with a RT_DLaTGS Detector. All samples were measured in the wavenumber region
of 4,000 cm-1 to 400 cm-1. UV/VIS spectra were recorded on Specord 50 PLUS UV/VIS
spectrophotometer from Analytik Jena using Aspect UV 1.1 software. The molar masses and
dispersities (ĐM) were analyzed employing a size exclusion chromatography (SEC) system
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(advanced polymer chromatography from Waters) equipped with two consecutive columns
(Acquity APC XT columns filled with polyethoxysilane with a defined porosity of 125 Å and
45 Å, respectively) and an Acquity APC RI-detector. The system was operated at a flow rate of
0.7 mL/min with THF/DMF (v/v = 80/20) as solvents. Poly(methyl methacrylate) (PMMA)
standards were used for calibration. Differential scanning calorimetry (DSC) was performed with
Netzsch DSC 204 F1 Phönix at a heating rate of 10 K min-1 under a nitrogen atmosphere. The
glass transition temperature (Tg), melting point (Tm) and decomposition temperature (Tdec.) were
determined with NETZSCH Proteus 6.1.0B. Melting point was measured by employing Büchi
Melting Point B-545. Light degradation experiments were performed using an OmniCure S1500
Curing System from Lumen Dynamics with a power of 0.607 W/cm2 (320 to 480 nm).
Nanoparticle sizes and polydispersities (PDI) were measured by photon correlation spectroscopy
(PCS) using a Malvern Zetasizer Nano ZS system. Drug load of the nanoparticles as well as drug
release were analyzed via high performance liquid chromatography (HPLC-DAD system, Agilent
Technologies 1200 series). Freeze-drying was performed in an Epsilon 2-4 single chamber-system
(Martin Christ, Osterode am Harz, Germany).
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Synthesis of light-responsive cyclic carbonate monomer (LrM) (Scheme 1).
Synthesis of (2,2,5-trimethyl-1,3-dioxan-5-yl)methanol (1)
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1 was synthesized according to the literature.39 2-(Hydroxymethyl)-2-methylpropane-1,3-diol
(80 g, 0.67 mol), acetone (320 mL), and TsOH (100 mg, 0.53 mmol) were stirred overnight at
room temperature. The reaction was quenched by addition of TEA (150 µL, 1.1 mmol). The
organic solvent was removed under reduced pressure and the residue was dissolved in DCM
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(200 mL). The precipitate was filtered off, the DCM was removed under reduced pressure, and the
residue was distilled at 0.2 mbar to achieve 86 g of a slightly viscous liquid (yield = 81%).
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(Figure S1)
1H-NMR (500 MHz, CDCl3): δ (ppm) = 0.82 (s, 3 H, CH3), 1.39 (s, 3 H, CH3), 1.43 (s, 3 H,
1CH3), 1.92 (m, 1 H, OH), 3.60 (m, 2 H, 3CH2), 3.67 (m, 2 H, 3CH2), 3.68 (s, 2 H, 6CH2). 13C-NMR
(125 MHz, CDCl3): δ (ppm) = 17.70 (1 C, 5CH3), 20.33 (1 C, 1CH3), 27.33 (1 C, 1CH3), 34.87 (1
C, 4Cq), 66.01 (1 C, 6CH2), 66.46 (2 C, 3CH2), 98.10 (1 C, 2Cq).
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Synthesis of (2,2,5-trimethyl-1,3-dioxan-5-yl)methyl 4-methylbenzenesulfonate (2)
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2 was synthesized according to the literature.40 A solution of TsCl (108 g, 0.57 mol) in pyridine
(140 mL) was dropped over 30 min to a solution of 1 (86 g, 0.54 mol) in pyridine (270 mL). The
resulting solution was stirred for 40 min at 100 °C, cooled and poured in an excess of ice water.
The precipitate was collected, washed with water and dried in vacuo to yield 140 g of an off-white
solid (yield = 83%). The crude product was used without further purification in the next step.
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(Figure S2)
1H-NMR (500 MHz, CDCl3): δ (ppm) = 0.81 (s, 3 H, CH3), 1.22 (s, 3 H, CH3), 1.36 (s, 3 H,
1CH3), 2.43 (s, 3 H, 13CH3), 3.54 (s, 4 H, 3CH2), 4.07 (s, 2 H, 6CH2), 7.34 (d, 3JHH = 8.3 Hz, 2 H,
9,11CH), 7.79 (d, 3JHH = 8.3 Hz, 2 H, 8,10CH). 13C-NMR (125 MHz, CDCl3): δ (ppm) = 17.34 (1 C,
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5CH3), 19.50 (1 C, CH3), 21.72 (1 C, CH3), 27.71 (1 C, CH3), 34.06 (1 C, Cq), 65.65 (2 C,
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3CH2), 72.61 (1 C, 6CH2), 98.18 (1 C, 2Cq), 128.18 (2 C, 8,10CH), 129.94 (2 C, 9,11CH), 132.85(1
C, 7Cq), 144.87 (1 C, 12Cq).
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Synthesis of 5-(azidomethyl)-2,2,5-trimethyl-1,3-dioxane (3)
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3 was synthesized according to the literature.41 2 (37.72 g, 120 mmol), NaN3 (23.41 g, 360 mmol),
water (20 mL) and DMF (200 mL) were stirred at 100 °C for 68 h. The mixture was poured in
water and extracted four times with Et2O (130 mL). The organic phase was dried over anhydrous
MgSO4 and the solvent was removed under reduced pressure. The residue was purified by column
chromatography with silica gel (100 g) and ethyl acetate/n-hexane (1/4) to give 20.9 g of a
colorless liquid (yield = 94%).
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1H-NMR (500 MHz, CDCl3): δ (ppm) = 0.81 (s, 3 H, CH3), 1.38 (s, 3 H, CH3), 1.41 (s, 3 H,
1CH3), 3.50 (s, 2 H, 6CH2), 3.57 (m, 4 H, 3CH2). 13C-NMR (125 MHz, CDCl3): δ (ppm) = 18.13 (1
C, 5CH3), 19.81 (1 C, 1CH3), 27.73 (1 C, 1CH3), 34.55 (1 C, 4Cq), 56.14 (1 C, 6CH2), 66.78 (2 C,
3CH2), 98.22 (1 C, 2Cq).
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Synthesis of (2,2,5-trimethyl-1,3-dioxan-5-yl)methanamine (4)
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4 was synthesized similar to the literature.42 3 (9.26 g, 50 mmol) and ammonium formate (12.6 g,
200 mmol) were dissolved in dry MeOH (130 mL). The solution was purged with argon for
20 min. The freshly degassed mixture was held under Ar atmosphere while Pd/C (10%) (0.9 g)
was added. The reaction mixture was stirred at room temperature in the opened reaction vessel.
After a few min the solution became slightly warm and a large gas formation was noticed. From
now the reaction mixture was stirred at room temperature for further 4.5 h. Pd/C was removed by
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filtration through a pad of Celite and the solvent was removed under reduced pressure. The residue
was dissolved in water (25 mL) and the resulting solution was extracted three times with Et2O
(3 x 50 mL). The aqueous phase was basified with KOH (25 g) and extracted five times with
MeCN (5 x 25 mL). The organic phase was dried over anhydrous MgSO4 and the solvent was
removed under reduced pressure to yield 5.91 g of a colorless liquid (yield = 74%). The crude
product was used without further purification in the next step.
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(Figure S4)
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1H-NMR (500 MHz, CDCl3): δ (ppm) = 0.78 (s, 3 H, CH3), 1.21 (b, 2 H, NH2), 1.36 (s, 3 H,
1CH3), 1.40 (s, 3 H, 1CH3), 2.76 (s, 2 H, 6CH2), 3.58 (m, 4 H, 3CH2). 13C-NMR (125 MHz, CDCl3):
δ (ppm) = 17.80 (1 C, 5CH3), 20.97 (1 C, 1CH3), 26.31 (1 C, 1CH3), 34.10 (1 C, 4Cq), 46.15 (1 C,
6CH2), 67.06 (2 C, 3CH2), 97.64 (1 C, 2Cq).
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Synthesis of 4,5-dimethoxy-2-nitrobenzyl ((2,2,5-trimethyl-1,3-dioxan-5-yl)methyl)carbamate (5)
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A mixture of 4 (2.954 g, 18.55 mmol), 7 (5.965 g, 15.77 mmol), TEA (4.9 mL, 35.16 mmol), and
dry MeCN (120 mL) was stirred at room temperature overnight. After adding DCM (100 mL) the
organic phase was washed twice with 0.1 M Na2CO3 solution (2 x 100 mL) and twice with 0.3 M
NaCl solution (2 x 100 mL). The organic phase was dried over anhydrous MgSO4 and the solvent
was removed under reduced pressure to yield 7.68 g of a viscous yellow liquid (yield = 84%). The
crude product was used without further purification in the next step.
O
O2N
10 11
O
7
3
6
14 O
9
O
2
1
1
NH
8
4
16
12
13 O
5
O
15
3
19
(Figure S5)
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1H-NMR (500 MHz, CDCl3): δ (ppm) = 0.81 (s, 3 H, CH3), 1.40 (s, 3 H, CH3), 1.43 (s, 3 H,
1CH3), 3.40 (d, 3JHH = 6.1 Hz, 2 H, 6CH2), 3.60 (m, 4 H, 3CH2), 3.95 (s, 3 H, O15CH3), 3.97 (s, 3
H, O16CH3), 5.22 (b, 1 H, NH), 5.52 (s, 2 H, 8CH2), 7.01 (s, 1 H, 12CH), 7.71 (s, 1 H, 11CH).
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Synthesis
of
4,5-dimethoxy-2-nitrobenzyl
(3-hydroxy-2-(hydroxymethyl)-2-
methylpropyl)carbamate (6)
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1 N HCl (25 mL) was added to a mixture of 5 (5.465 g, 13.70 mmol) and THF (25 mL). The
suspension was stirred at room temperature overnight. After addition of DCM (100 mL) the
mixture was washed three times with 0.1 M Na2CO3 solution (3 x 100 mL) and twice with 0.3 M
NaCl solution (2 x 100 mL). The organic phase was dried over anhydrous MgSO4 and the solvent
was evaporated under reduced pressure.
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Purification method 1: Et2O was added to the crude product and stirred/ triturated until a fine
precipitate appears. A large excess of Et2O was reduced before the precipitate was collected by
filtration and dried under reduced pressure to achieve 4.326 g of a yellow solid (yield = 88%).
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Purification method 2: The crude product was purified by column chromatography with silica gel
and ethyl acetate to give 4.410 g of a yellow solid (yield = 90%).
O
O2N
8
9
5
O
1
12 O
7
4
HO
HO
NH
6
2
14
10
11 O
3
13
1
16
17
18
19
20
(Figure S6)
1H-NMR (500 MHz, DMSO-d6): δ (ppm) = 0.73 (s, 3 H, 3CH3), 2.98 (d, 3JHH = 6.3 Hz, 2 H, 4CH2),
3.21 (d, 3JHH = 5.5 Hz, 4 H, 1CH2), 3.87 (s, 3 H, O13CH3), 3.91 (s, 3 H, O14CH3), 4.32 (t, 3JHH
5.5 Hz, 2 H, OH), 5.34 (s, 2 H, 6CH2), 7.19 (s, 1 H, 10CH), 7.22 (t, 3JHH = 6.2 Hz, 1 H, NH), 7.70
=
9
13
3
2
(s, 1 H, CH). C-NMR (125 MHz, DMSO-d6): δ (ppm) = 17.08 (1 C, CH3), 41.36 (1 C, Cq),
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44.29 (1 C, CH2), 56.22 (1 C, O13CH3), 56.32 (1 C, O14CH3), 62.44 (1 C, CH2), 64.55 (2 C,
1CH2), 108.32 (1 C, 9CH), 110.64 (1 C, 10CH), 128.11 (1 C, 7Cq), 139.43 (1 C, 8Cq), 147.85 (1 C,
11Cq), 153.48 (1 C, 12Cq), 156.55 (1 C, 5Cq). FTIR (cm-1): 3291 (ν-O-H), 1690 (ν-C=O, carbamate),
1581 (ν-C=C, aromatic), 1519 (νas-NO2), 1319 (νs-NO2), 1270 (ν-C-O, carbamate) and 1223 (ν-C-
O, ether). ESI-ToF-MS (m/z): [M + Na]+ calculated for C15H22N2O8Na+, 381.1274; found,
381.1271. Melting point (Tm) = 99.4 °C.
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Synthesis of 4,5-dimethoxy-2-nitrobenzyl ((5-methyl-2-oxo-1,3-dioxan-5-yl)methyl)carbamate
(LrM)
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6 (4.132 g, 11.53 mmol) was suspended in anhydrous THF (30 mL) under Ar atmosphere and
cooled to 0 °C. Subsequently, TEA (4.22 mL, 30.45 mmol) and ethyl chloroformate (2.55 mL,
26.65 mmol) were added and the suspension was stirred at room temperature for 24 h. The
precipitate was collected, washed with THF (100 mL), stirred in water (100 mL) for 30 min,
filtered off, washed twice with water (2 x 100 mL) and three times with Et2O (3 x 100 mL), and
was dried in vacuo to afford 4.011 g of an off-white solid (yield = 73%).
O
O2N
9
10
6
O
2
13 O
8
5
O
1
NH
7
3
O
15
11
12 O
O
4
14
2
15
16
17
18
19
20
(Figure S7)
1H-NMR (500 MHz, DMSO-d6): δ (ppm) = 0.93 (s, 3 H, 4CH3), 3.13 (d, 3JHH = 6.4 Hz, 2 H, 5CH2),
3.87 (s, 3 H, O14CH3), 3.91 (s, 3 H, O15CH3), 4.12 (d, 2JHH = 10.6 Hz, 2 H, 2CH2), 4.22 (d, 2JHH
=
10.6 Hz, 2 H, 2CH2), 5.35 (s, 2 H, 7CH2), 7.20 (s, 1 H, 11CH), 7.67 (t, 3JHH = 6.3 Hz, 1 H, NH), 7.70
(s, 1 H, 10CH). 13C-NMR (125 MHz, DMSO-d6): δ (ppm) = 16.70 (1 C, 4CH3), 32.70 (1 C, 3Cq),
5
7
43.11 (1 C, CH2), 56.23 (1 C, O14CH3), 56.35 (1 C, O15CH3), 62.74 (1 C, CH2), 73.90 (2 C,
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2CH2), 108.35 (1 C, 10CH), 111.01 (1 C, 11CH), 127.60 (1 C, 8Cq), 139.60 (1 C, 9Cq), 147.65 (1 C,
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13
6
2
1Cq), 147.95 (1 C, Cq), 153.39 (1 C, Cq), 156.53 (1 C, Cq). ESI-ToF-MS (m/z) (Figure S8):
[M + Na]+ calculated for C16H20N2O9Na+, 407.1066; found, 407.1066. FTIR (cm-1): 3083 (ν-C-H,
aromatic), 1738 (ν-C=O, carbonate), 1714 (ν-C=O, carbamate), 1584 (ν-C=C, aromatic), 1523 (νas-
NO2), 1322 (νs-NO2), 1274 (ν-C-O, carbamate), 1245 (ν-C-O, carbonate), 1221 (ν-C-O, ether).
Melting point (Tm) = 192.5 °C.
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Synthesis of 4,5-dimethoxy-2-nitrobenzyl(4-nitrophenyl)carbonate (7)
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7 was synthesized according to the literature.30 DIPEA (15 mL, 86.11 mmol) was added dropwise
to a mixture of DMNA (8.80 g, 41.28 mmol), 4-nitrophenyl chloroformate (15.55 g, 77.15 mmol)
and DCM (90 mL) under Ar atmosphere. The solution was stirred at room temperature for 1.5 h
and was then diluted with DCM (90 mL). After stirring at room temperature overnight the solvent
was removed under reduced pressure. The resulting product was suspended in ethanol (290 mL)
and heated to 85 °C for 45 min until a fine precipitate was formed. The precipitate was collected
and dried in vacuo to afford 14.16 g of a yellowish solid (yield = 91%).
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13
14
O
O
O2N
2
2
3
3
8
9
5
O
1
O2N
10 O
4
7
6
14
11
12 O
13
15
16
17
18
19
(Figure S9)
1H-NMR (500 MHz, DMSO-d6): δ (ppm) = 3.89 (s, 3 H, O13CH3), 3.92 (s, 3 H, O14CH3), 5.61 (s,
2 H, 6CH2), 7.26 (s, 1 H, 11CH), 7.59 (d, 3JHH = 9.1 Hz, 2 H, 3CH), 7.73 (s, 1 H, 9CH), 8.33 (d, 3JHH
= 9.1 Hz, 2 H, 2CH). 13C-NMR (125 MHz, DMSO-d6): δ (ppm) = 56.14 (1 C, O13CH3), 56.32 (1
C, O14CH3), 67.22 (1 C, 6CH2), 108.30 (1 C, 9CH), 112.00 (1 C, 11CH), 122.57 (2 C, 3CH), 124.37
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(1 C, 7Cq), 125.45 (2 C, 2CH), 139.04 (1 C, 8Cq), 145.25 (1 C, 1Cq), 148.40 (1 C, 12Cq), 151.62 (1
C, 5Cq), 153.17 (1 C, 10Cq), 155.17 (1 C, 4Cq).
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General procedure for the synthesis of light-responsive (co)polycarbonate (LrPC) by ROP.
Take the preparation of LrPC-2 (Table 1) as an example. In a Schlenk tube, LrM (0.461 g,
1.20 mmol), TMC (0.123 g, 1.20 mmol) and BnOH (4,16 µL, 0.04 mmol) were suspended in dry
1,4-dioxane (2 mL) under Ar atmosphere. After adding DBU (9.06 µL, 0.06 mmol) the reaction
mixture was stirred at 80 °C for 22 h and was then quenched by adding benzoic acid (10 mg,
0.07 mmol). The polymer was purified by precipitation into Et2O and dried in vacuo to give a
yellow solid (yield = 73%).
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O
O
a
a
b
1
1
3
3
H
a
a
O
O
O
O
O
2
m
n
4
5
a
x
O
NH
O
6
NO2
7
9
8
O
O
10
10
11
12
13
14
15
(Figure S10)
1H-NMR (500 MHz, DMSO-d6): δ (ppm) = 0.70 – 1.00 (m, 3 H, 4CH3), 1.80 – 2.00 (m, 2 H, 2CH2),
2.90 – 3.10 (m, 2 H, 5CH2), 3.75 – 4.00 (m, 3 H, O9CH3; 3 H, O10CH3; 4 H, 3CH2), 4.00 – 4.25 (m,
4 H, 1CH2), 5.11 (s, 2 H, bCH2), 5.25 – 5.40 (m, 2 H, 6CH2), 7.00 – 7.85 (m, 1 H, NH; 2 H, 7,8CH;
5 H, aCH). FTIR (cm-1): 1749 (ν-C=O, carbonate and carbamate), 1584 (ν-C=C, aromatic), 1525
(νas-NO2), 1330 (νs-NO2), 1281 (ν-C-O, carbamate), 1241 (ν-C-O, carbonate), 1221 (ν-C-O, ether).
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Kinetic of ring-opening (co)polymerization. For kinetic investigation, the (co)polymerization
was conducted by the same procedure aforementioned. At specific times, 0.1 mL of the reaction
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mixture was taken out, quenched by adding benzoic acid and studied by 1H NMR spectroscopy.
Conversions of LrM and TMC were calculated from the relative integrals of the monomer
(2.05 ppm for TMC and 3.25 ppm for LrM) and copolymer (1.95 ppm for PTMC and 3.11 ppm
for LrPC) peaks in 1H NMR spectra (Figure S11).
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Light decomposition of LrM followed by ESI-ToF-MS. DCM (3 mL) containing LrM (5 mg)
(1.67 mg/mL) was charged into a vial, and then irradiated with UV light (0.607 W/cm2, 320 –
480 nm) for 15 min. After removing the DCM under reduced pressure, the degradation products
were studied by ESI-ToF-MS.
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Light responsiveness of LrPC-2 monitored by UV/VIS spectrophotometer. A polymer
solution of LrPC-2 was prepared in DCM (0.075 mg/mL). The LrPC-2 solution was irradiated in
a quartz cuvette with UV light (0.607 W/cm2, 320 – 480 nm) for the specified periods of time (up
to 180 s). UV/VIS absorbance spectra were recorded after each irradiation.
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Light degradation of LrPC-2 followed by SEC. Five polymer solutions of LrPC-2 were
prepared in SEC vials in THF (2 mg/mL). These samples were irradiated with UV light
(0.607 W/cm2, 320 – 480 nm) for the specified times of 0, 1, 5, 15, and 30 min, and then analyzed
by SEC.
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Light degradation of LrPC-2 followed by H NMR spectroscopy. LrPC-2 (7.5 mg) was
dissolved in DMSO-d6 (0.5 mL) (15 mg/mL). The LrPC solution was irradiated directly in vial
1
with UV light (0.607 W/cm2, 320 – 480 nm) for 15 min and H NMR spectra before and after
irradiation were taken.
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Nanoparticle preparation, lyophilization, and characterization. Nanoparticles (NP) with a
hydrodynamic diameter of about 100 nm were prepared using a solvent displacement preparation
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technique. In brief, LrPC-2 (7.5 mg) and PLGA (22.5 mg) were dissolved in acetone (1 mL),
respectively. After combining both organic solutions, the photosensitizer temoporfin (mTHPC)
(3 mg) was added and dissolved. The organic solution was injected into an aqueous PVA solution
(2% (w/v)) (4 mL). The nanoparticle suspension was stirred overnight, whereby evaporation of the
organic solvent led to the final particle formation. Afterwards, purification of the nanoparticle
suspension was conducted three times via centrifugation (30,000 g, 1.5 h) and redispersion in
water. The NP were referred to as mTHPC-LrPC-PLGA-NP. To obtain unloaded NP (LrPC-
PLGA-NP) the preparation process was performed in the absence of the photosensitizer.
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Temoporfin loaded nanoparticle suspensions were freeze-dried in the presence of 3% trehalose
using the following parameters: Freezing at -40 °C for 7 h, a primary drying step at -34 °C and a
vacuum of 0.05 mbar for 40 h, followed by a secondary drying phase at 20 °C and 0.025 mbar for
11 h.
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The amount of the particle incorporated mTHPC was determined by dissolving nanoparticles
(1 mg) in acetone (1 mL). The released photosensitizer was quantified via HPLC with the aid of a
calibration curve of pure mTHPC (concentration range from 10 to 100 µg/mL). A reversed phase
column (Gemini RP 18, 250 x 4.6 mm, particle diameter 5 µm, Phenomenex Inc., Aschaffenburg,
Germany) was used. An isocratic elution was performed at a flow rate of 1.0 mL/min. The mobile
phase consisted of 42.5% water with 0.1% (w/v) trifluoroacetic acid and 57.5% acetonitrile. The
photosensitizer was detected at a wavelength of 415 nm.
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Light-induced nanoparticle decomposition. Light-depending particle degradation was
investigated after illumination of a diluted nanoparticle suspension (0.1 mg NP/mL suspension)
with light of a wavelength of 365 nm (Thorlabs´ Mounted LED M365LP1, 1,150 mW, Ø57.0 mm,
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1.76 mW/cm2) for 5 min. Variations in the count rate of the nanoparticle suspensions were
observed over 24 h via PCS measurements at a temperature of 22 °C and a backscatter angle of
173°. In between the measurements, samples were stored in the dark at room temperature. All
measurements were performed with unloaded nanoparticles.
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Photosensitizer temoporfin release. The temoporfin release kinetics were determined by first
dispersing lyophilized nanoparticles in Dulbecco’s Modified Eagle Medium (DMEM) containing
10% (v/v) fetal bovine serum (FBS) at a concentration of 0.4 mg NP/mL. Afterwards, aliquots of
1 mL were irradiated for 5 min with light of a wavelength of 365 nm and afterwards incubated at
37 °C for defined times (15 min, 30 min, 60 min, 2 h, 4 h, 6 h, 24 h). The collected samples were
centrifuged (30,000 g, 15 min) and the supernatants were collected. 150 µL of each supernatant
was mixed with acetone (450 µL) to precipitate remaining serum protein and to dissolve the drug.
In a further centrifugation step (20,000 g, 10 min), the precipitated proteins were separated and the
photosensitizer content of the supernatants was determined via HPLC-FLD analysis. The HPLC-
parameters were the same as described above, except that released mTHPC was detected with a
fluorescence detector at 421 nm excitation and 653 nm emission wavelength.
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Atomic force microscopy (AFM) measurements. The visualization of the NP was performed by
AFM employing a Bruker Dimension 3100 atomic force microscope, equipped with a Nanoscope
IIIa controller (Bruker, Karlsruhe, Germany) before and after illumination of the NP suspensions.
Samples were prepared as follows: NP suspensions were diluted to a concentration of 0.16 mg
NP/mL. An aliquot (3 µL) of the NP suspension was pipetted on a specimen slide and dried under
a darkened fume cupboard. The remaining NP suspension was illuminated for 5 min with light of
a wavelength of 365 nm and stored for 24 h in the dark at room temperature. Afterwards, 3 µL of
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the treated NP suspension was pipetted on a second specimen slide and also dried under a darkened
fume cupboard until AFM measurements were conducted.
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AFM measurements were performed in intermittent contact mode (Tapping Mode®) with n-type
silicon cantilevers (HQ:NSC14/Al BS, nominal tip radius < 10 nm, typical resonant frequency of
about 160 kHz and a nominal spring constant of 5 N/m; manufactured by µmash, Sofia, Bulgaria),
2% below resonance frequency at a RMS amplitude of around 2. Further data analysis was carried
out using Nanoscope analysis software version 1.5.
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RESULTS AND DISCUSSION
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Synthesis and characterization of LrM. The six-membered cyclic carbonate monomer LrM was
prepared from 2-(hydroxymethyl)-2-methylpropane-1,3-diol following the synthetic route as
shown in Scheme 1. In a four-step reaction one hydroxy group (1) was converted into an amino
group (4).39-42 Amine 4 was then protected with the light-cleavable 4,5-dimethoxy-2-nitrobenzyl
group via a carbamate linkage to give compound 5. The acetal 5 was hydrolyzed with HCl to reveal
the diol 6, which was cyclized with ethyl chloroformate to form the cyclic LrM. The overall yield
for LrM was 29% over seven steps. The structure of LrM was confirmed by NMR spectroscopy
(Figure S7) and ESI-ToF-MS (Figure S8). The LrM possess poor solubility (only soluble in
DMSO and DMF at room temperature) and very high melting point, which were probably caused
by strong intermolecular hydrogen bonding (Figure S12) between carbamate and carbonate groups
due to the presence of the electron withdrawing o-nitrobenzyl group. In addition, based on the
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DSC measurement (Figure S13) the melting point (Tm) and decomposition temperature (Tdec.) of
LrM were found to be 193 °C and 202 °C, respectively.
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OH OH
O
O
O
O
O
O
i)
ii)
iii)
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OH
N3
OH
OTs
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2
3
iv)
O
OH OH
HN
O
O
O
O
O
O
O
HN
O
HN
O
vii)
vi)
v)
O
O
O
O
O
NO2
NO2
NH2
NO2
O2N
O
NO2
4
O
O
O
O
O
O
7
O
O
LrM
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Scheme 1. Synthetic route for LrM. Reagents and conditions: i) acetone, TsOH, rt, 20 h; ii) TsCl,
pyridine, rt, 30 min → 100 °C, 40 min; iii) NaN3, DMF/H2O (10/1), 68 h, 100 °C; iv) ammonium
formate, Pd/C, MeOH, rt, 4 h; v) 7, TEA, MeCN, rt, overnight; vi) THF/1 N HCl (1/1), rt,
overnight; vii) ethyl chloroformate, TEA, THF, 0 °C → rt, overnight.
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O
a)
O
O
O
H
n
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O
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O
O
NH
HN
O
homopolymerization
O
DBU, BnOH
1,4-dioxane, 80 °C, 46 h
O
NO2
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NO2
O
O
O
LrM
LrPC-1
O
b)
O
O
O
O
O
H
O
O
O
O
n
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x
O
O
O
NH
HN
O
copolymerization
+
O
O
O
O
DBU, BnOH
NO2
1,4-dioxane, 80 °C, 22 h
NO2
O
O
O
TMC
LrM
LrPC-2
1
2
Scheme 2. Preparation of the light-responsive homopolymer (a) and copolymer (b) by ROP in 1,4-
3
4
dioxane using DBU as catalyst and BnOH as initiator.
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Synthesis of light-responsive (co)polycarbonate by ROP. ROP of cyclic carbonate monomers
catalyzed by organo-catalysts, such as DBU, 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) and (-)-
sparteine, have been intensively investigated and proved to be efficient method for the APC
preparation.43-45 We attempted to synthesize light-responsive homo- and copolycarbonates by
employing DBU as organo-catalyst and BnOH as initiator for the ROP of LrM (Scheme 2).
Attempts of ROP at room temperature in various aprotic organic solvents were unsuccessful due
to the poor solubility of LrM. Through optimizing the polymerization conditions, we found that
LrM could be polymerized in 1,4-dioxane at 80 °C. First, the kinetic of homopolymerization of
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LrM was investigated and followed by proton NMR spectroscopy. As shown in Figure 3a and b,
LrM was polymerized very slowly with polymerization rate constants of 0.0138 h-1 (0 – 22 h) and
0.0256 h-1 (22 – 46 h), respectively (Figure S14 a and b). The monomer conversion reached only
64% after 46 h. During the polymerization, the mixture remained in the suspension state, strongly
limiting the polymerization rate and conversion. The final number averaged molar mass (Mn) of
the obtained homopolycarbonate (LrPC-1) was 2,200 g/mol with dispersity (ĐM) of 1.23
(Table 1).
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Figure 3. Kinetics of homopolymerization of LrM (a and b) and copolymerization of LrM with
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Copolymerization is a facile method to provide the opportunity to modify polymer properties. To
increase the molar masses of obtained polymers, we further attempted the copolymerization of
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LrM and commercially available TMC with a molar feed ratio of 1: 1 (Scheme 2b). Fortunately,
the solubility of the monomer mixture containing 50% TMC was significantly enhanced, because
TMC could form new single hydrogen bonding with the carbamate group instead of double
hydrogen bonding between LrMs, thereby changing the hydrogen bonding system and leading to
a faster polymerization rate. Initially, we thought that for the copolymerization of LrM and TMC
a pseudo-block copolymer, PTMC-b-LrPC, could be obtained, since TMC has a very high ROP
rate catalyzed by DBU, as previously reported46. However, the kinetic analysis (Figure 3c and d)
suggested that TMC and LrM were polymerized at similar rates leading to a statistic copolymer.
In the first 8.5 h, due to the poor monomer solubility the ROP of both LrM and TMC showed
slower rate constants of 0.0460 h-1 and 0.0417 h-1 for LrM and TMC, respectively. As the
polymerization proceeded, the polymerization rate increased to 0.1045 h-1 and 0.0949 h-1 for LrM
and TMC (Figure S14c and d), respectively, with the increase of the monomer solubility in
polycarbonate solution. The suspension cleared after 20 h, and maximal conversion of 80% for
both monomers was reached after 22 h, thereafter, their conversions remained unchanged.
According to the previous reports by Endo et al.47,48 the ROP equilibrium of some 5,5-disubstituted
TMC could be strongly affected by polymerization temperature. The higher the polymerization
temperature, the lower the maximal monomer conversion that could be reached. The final
copolycarbonate had Mn of 4,400 g/mol and ĐM of 1.47. The thermal properties of LrPC-2 were
investigated by DSC (Figure S13). The glass transition temperature (Tg), Tm and Tdec. were
detected at 41 °C, 64 °C and 166 °C, respectively. Moreover, the thermal and hydrolytic stability
of LrPC-2 was studied in DMSO-d6/D2O (v/v = 5/1, 16.7 mg/mL) solution at 37 °C by 1H NMR
spectroscopy. As shown in Figure S15, both chemical shifts and relative intensities of polymer
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signals in 1H NMR spectra remained unchanged up to 21 days incubation at 37 °C pointing out the
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excellent stability of LrPC-2.
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Table 1. Results of the synthesis of homo- and copolycarbonates by ROP of LrM.
b
e
e
[LrM]:[TMC]:[I]a
Mn,th
t
(h)
46
22
conv.c (%)
DPd Mn,SEC ĐM
(g/mol)
7,400
LrM TMC (LrM + TMC) (g/mol)
LrPC-1
LrPC-2
30: 0: 1
64
81
-
5 + 0
2,200 1.23
4,400 1.47
30: 30: 1 11,500
79
10 + 10
aBoth polymerizations were conducted in 1,4-dioxane at 80 °C using DBU as the organo-catalyst
b
and BnOH as the initiator. Mn,th = ([LrM]/[I]) x M(LrM) x conv.(LrM) + ([TMC]/[I]) x
c
1
M(TMC) x conv.(TMC) + M(BnOH). Determined by H NMR spectra of polymer solution
d
1
(Figure S11). Determined by H NMR spectra of purified polymers; DP = degree of
polymerization. eDetermined by SEC in THF/DMF (8/2) with PMMA standards.
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The copolymerization of LrM and TMC appeared to suffer from side reactions, such as
transcarbamation, leading to lower Mn than that (Mn,th) predicted from the molar feed ratio [LrM]:
[TMC]: [I] and conversion as well as higher ĐM. The possible mechanism of the transcarbamation
is shown in Figure 4a. Since 4,5-dimethoxy-2-nitrobenzyl group is a strong electron withdrawing
group, the carbamate group could be deprotonated by DBU and attacked the carbonate group in
polymer backbone via intramolecular cyclization at evaluated temperature leading to polymer
fragmentation and loss of expected benzyl alcohol end groups. As a result of the transcarbamation
a small signal at 5.42 ppm corresponding to the carbamate end group (e’) could be observed after
the ROP, while the CH2 (l) for the benzyl alcohol end group at 5.12 ppm almost disappeared
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(Figure 4b). Moreover, the ratio of the relative intensities for the NH (d, 7.54 ppm) and CH2 (e +
e’, 5.33 ppm and 5.42 ppm, respectively) groups was found to be 1: 3.02 (Figure S10), which was
smaller than the ratio in the LrM spectrum of 1: 2.27 (Figure S7), providing circumstantial
evidence for this mechanism. The structure of LrPC-2 was confirmed by 1H NMR spectroscopy
(Figure 4b). Comparing the relative intensity at 1.93 ppm for PTMC and 3.07 ppm for LrPC, we
determined that the final copolycarbonate contained 52% light-responsive part, which was in
agreement with the kinetic results. DPs were calculated from the relative integrals of the polymer
(1.93 ppm for PTMC and 3.07 ppm for LrPC) and the end group peaks (5.12 ppm (i) for BnOH
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and 5.42 ppm (e’) for o-nitrobenzyl carbamate end groups, respectively) in H NMR spectra. In
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addition, differing from LrM the obtained light-responsive homo- and copolycarbonates are
soluble in common organic solvents, such as acetone, DCM, DMSO, DMF, THF and MeCN.
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Figure 4. a) Transcarbamation side reaction catalyzed by DBU during the ROP; b) H NMR
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Figure 5. a) Degradation mechanism of LrM upon irradiation. b) ESI-ToF mass spectrum for
degradation products of LrM after irradiation (320 – 480 nm, 0.607 W/cm2) for 15 min. [III+H]+,
calcd: 146.0812, found: 146.0803; [III+Na]+, calcd: 168.0631, found: 168.0645; [II+H]+, calcd:
196.0604, found: 196.0622; [V+H]+, calcd: 323.1238, found: 323.1209; [LrM+H]+, calcd:
385.1242, found: 385.1222; [LrM+Na]+, calcd: 407.1061, found: 407.1066; [IV+H]+, calcd:
530.1980, found: 530.1999.
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Light degradation study of LrM followed by ESI-ToF-MS and NMR. To clearly determine the
degradation products, LrM was irradiated with UV light (320 – 480 nm, 0.607 W/cm2) for 15 min
and studied by ESI-ToF-MS. As shown in Figure 5, upon irradiation LrM was decomposed as
expected into an intermediate 5-(aminomethyl)-5-methyl-1,3-dioxan-2-one (I) and 4,5-
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dimethoxy-2-nitrosobenzaldehyde (II) via a radical reduction mechanism, which has been
discussed elsewhere49,50. The reactive intermediate I transformed then into the thermally stable
six-membered cyclic carbamate III via intramolecular transcarbamation of the functional amine
group with the carbonate group. Since LrM was irradiated in the suspension state, differing from
other previously published works18, we observed the intermolecular degradation product IV as
well. In the presence of LrM, IV was formed by the intermolecular ring-opening reaction of I with
LrM. In addition, it was well known that the condensation reaction between primary amine and
aldehyde leads to the formation of imine functionalities. Imine V could be detected as a minor
degradation product generated from amine I and aldehyde II. The proton NMR analysis (Figure
S16) showed that, after irradiation for 15 min 91% 4,5-dimethoxy-2-nitrobenzyl group has been
cleaved based on the significant signal reduction of the benzylic CH2 group at 5.35 ppm. The
remaining signals indicated the emergence of aldehyde, alcohol and cyclic carbamate.
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Change in absorbance of LrPC-2 upon irradiation. As shown in Figure 6a, the
photoisomerization of 4,5-dimethoxy-2-nitrobenzyl group was carried out upon irradiation with
UV light into an o-nitrosobenzylaldehyde leading to release of a free amine pendent group. To
follow the deprotection rate, the absorbance changes of LrPC-2 in DCM (0.075 mg/mL) were
investigated by employing a UV/VIS spectrophotometer. Upon irradiation the absorbance at
346 nm decreased due to the cleavage of 4,5-dimethoxy-2-nitrobenzyl light-sensitive protecting
group, while a new absorbance appeared at 430 nm, which could be assigned to the degradation
product of 4,5-dimethoxy-2-nitrosobenzaldehyde. The UV/VIS spectrum remained unchanged
after irradiation for 180 s indicating complete cleavage of protecting group from polymer side
chains.
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irradiation with UV light. b) Change of the UV/VIS absorbance of LrPC-2 in DCM (0.075
mg/mL) upon irradiation with UV light (320 – 480 nm, 0.607 W/cm2) for various irradiation times
up to 180 s.
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Light degradation study of LrPC-2 followed by SEC. The light degradation of LrPC-2 in
dependence on the irradiation time (0.607 W/cm2, 320 – 480 nm for 0, 1, 5, 15, 30 min) was
analysed by SEC. As designed the residual amino function formed upon irradiation initiated the
polymer degradation by a subsequent intramolecular cyclization. In Figure 7 is shown that the
degradation strongly depends on the time of irradiation. In this case a shift of the high molar mass
peak maximum can be observed, which indicates the cleavage of the light-responsive side groups.
At the same time, the peak area decreased, which is proportional to the number of polymer chains.
This means that the degradation of the chain took place immediately after the side chain is split
off, which suggests a rapid intramolecular reaction. The longer the sample was exposed to the UV
light, the more the molecular weight decreased and the portion of the degradation products (elution
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time 2.3 to 2.6 min in Figure 7) increased. Irradiation times longer than 15 min did not show much
differences in the molecular weight and elugramm. Hence, it can be assumed that the degradation
was finished within 15 min. The degradation products are oligomers with Mn less than
1,000 g/mol. All samples were measured immediately after irradiation, it is therefore clear that the
polymer degradation occurred only during the irradiation time, and was not propagated beyond.
The degree of degradation can thus be controlled well by the duration of the exposure.
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Figure 7. SEC traces of LrPC-2 after exposure to UV light (320 – 480 nm, 0.607 W/cm2) for 0,
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