Hyperbranched Polyglycerol Loaded with (Zinc ... - ACS Publications

Article Cite This: Biomacromolecules XXXX, XXX, XXX−XXX

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Hyperbranched Polyglycerol Loaded with (Zinc-)Porphyrins: Photosensitizer Release Under Reductive and Acidic Conditions for Improved Photodynamic Therapy Michael H. Staegemann,†,‡ Susanna Graf̈ e,‡ Burkhard Gitter,‡ Katharina Achazi,† Elisa Quaas,† Rainer Haag,*,† and Arno Wiehe*,†,‡ †

Institut für Chemie und Biochemie, Freie Universität Berlin, Takustr. 3, D-14195 Berlin, Germany Biolitec Research GmbH, Otto-Schott-Str. 15, D-07745 Jena, Germany

‡

S Supporting Information *

ABSTRACT: An adaptable approach toward cleavable nanoparticle carrier systems for photodynamic therapy (PDT) is presented, comprising a biocompatible carrier loaded with multiple photosensitizer (PS) molecules related to the clinically employed PS Temoporfin, two linkers cleavable under different triggers and glyco-targeting with mannose. A synthetic pathway to stimuli responsive hyperbranched polyglycerol (hPG) porphyrin conjugates via the copper(I)catalyzed 1,3-dipolar cycloaddition (CuAAC) or the strainpromoted alkyne−azide cycloaddition (SPAAC) has been developed. The PS 10,15,20-tris(3-hydroxyphenyl)-5(2,3,4,5,6-pentafluorophenyl)porphyrin was functionalized with disulfide containing cystamine and acid-labile benzacetal linkers. Conjugates with reductively and pH labile linkers were thus obtained. Cleavage of the active PS agents from the polymer carrier is shown in several different release studies. The uptake of the conjugates into the cells is demonstrated via confocal laser scanning microscopy (CLSM) and flow cytometry. Finally, the antitumor and antibacterial phototoxicity of selected conjugates has been assessed in four different tumor cell lines and in cultures of the bacterium Staphylococcus aureus. The conjugates exhibited phototoxicity in several tumor cell lines in which conjugates with reductively cleavable linkers were more efficient compared to conjugates with acid-cleavable linkers. For S. aureus, strong phototoxicity was observed for a combination of the reductively cleavable and the pH labile linker and likewise for the cleavable conjugate with mannose targeting groups. The results thus suggest that the conjugates have potential for antitumor as well as antibacterial PDT.

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INTRODUCTION Photodynamic therapy (PDT) is an established procedure for the treatment of tumors or in general of malignant tissues.1−3 A nontoxic dye compound, the photosensitizer (PS), in combination with a light source induces a phototoxic effect in the target tissue, for example, the tumor. When the tissue is irradiated with light of a specific wavelength, photochemical reaction cascades are initiated, and the cells die via necrosis or apoptosis.2,3 Another application is the treatment of infections known as antimicrobial photodynamic therapy (aPDT).4,5 aPDT is especially appealing for treating local infections caused by antibiotic-resistant bacteria.4,6 This method has found different medical applications including dental usage,7,8 burn wounds,9,10 and acne.11 Tetrapyrrolic systems are known to be essential in several biological processes. Because of that various applications have been studied in literature including PDT,2,12 light-harvesting13,14 or catalysis.15 Many tetrapyrrolic PS are known, for example, chlorins,2,16,17 porphyrins,18,19 phthalocyanines,20 and corroles.21,22 For our conjugate systems, we chose porphyrins © XXXX American Chemical Society

which are well-known PS; moreover, they can be transformed to chlorins which are even more potent PS.2,23−25 The structure of the PS chosen here, 10,15,20-tris(3-hydroxyphenyl)-5(2,3,4,5,6-pentafluorophenyl)porphyrin, is closely related to that of Temoporfin, an approved PS for PDT.26 In addition, such monopentafluorophenyl-substituted porphyrins have been shown to be particularly suitable for linker strategies related to bioconjugation.27 Currently, an intensively investigated approach in tumor therapy uses nanocarriers to increase the accumulation of active drug substances in the tumor tissue by exploiting the enhanced permeability and retention (EPR) effect.28−30 Hence, this concept has been applied to PDT by coupling or encapsulating PS into different carrier systems,31−33 for example, PLGA,31,33 albumin,31,33 gold,31,33 and chitosan31,33 nanoparticles; moreover, it has also been employed to aPDT.34,35 An important Received: October 15, 2017 Revised: November 18, 2017

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DOI: 10.1021/acs.biomac.7b01485 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules

For specific targeting, one conjugate decorated with additional mannose groups has been synthesized. Mannose units are known to interact with mannose receptors on bacterial membranes which makes porphyrin−mannose conjugates possible candidates for aPDT.66 Mannose-decorated PS systems have also been employed for targeting tumor cells.67,68 Recently, we could show that noncleavable hPG− PS conjugates with higher mannose loading (approximately >70 mannose units) benefit from a pronounced multivalency effect in their aPDT activity.40 With these systems, as for other nanoparticle systems, an interaction with proteins has been found to limit their PDT efficacy. These observations i.a. prompted us to develop systems with cleavable linkers. Thus, starting from hPG as a biocompatible carrier system loaded with porphyrin PS, and its combination with the two linkers, cleavable under different triggers, the targeting by mannose presents the third element in this approach for improved PDT, aiming at both antitumor as well as antibacterial PDT. The release of the porphyrin PS from hPG carrier has been intensively analyzed in a quenching experiment with fluorescence spectroscopy, release studies followed by sizeexclusion chromatography (SEC), dialysis, extraction, and thinlayer chromatography (TLC). The PDT activity of these porphyrin-hPG-conjugates with disulfide and pH cleavable linkers has been investigated in cellular assays with four different tumor cell lines as well as for aPDT with S. aureus as a typical Gram-positive germ. The cellular phototoxicity assays are complemented by cellular uptake experiments using confocal laser scanning microscopy (CLSM) and flow cytometry.

complementary element for such carrier systems are means by which the release of the active molecules (e.g., the PS) from the carrier is facilitated at the site of action (e.g., the tumor).32,36 Moreover, release systems avoid the problem of photophysical quenching which reduces the PDT activity. This quenching can occur with PS, which are densely packed on or within polymeric systems.37,38 In addition, the introduction of release systems may also help to overcome the problem of protein− nanoparticle interaction, which can adversely influence the activity of nanoparticle carrier systems.39−41 The release of PS from a polymeric carrier can be achieved by using degradable connection linkers. For this reason, linkers have to be chosen, which remain stable in the bloodstream and are rapidly cleaved in the target tumor tissue. Examples of possible linkers include: acid−cleavable hydrazine linkages,42,43 acetal,44,45 and cis-aconityl spacers;46 enzymatically cleavable oligopeptide linkers;47 and reductively cleavable disulfide linkers.48,49 Disulfide bonds have been used for the synthesis of several delivery systems, for example, antisense oligonucleotides,50 small interfering ribonucleic acid (RNA),51 plasmid deoxyribonucleic acid (DNA),52 and anticancer drugs.49,53 Acid-cleavable acetal linkers, on the other hand, have been employed in delivery systems with, for example, cells44 and enzymes.45 For our conjugate systems we chose reductively cleavable disulfide linkers and acid-cleavable acetal linkers. The cleavability of the disulfide bridge relies on the difference of the redox potentials between the cellular membrane and the bloodstream. It is known that the bloodstream has a global potential that is mildly oxidative.54,55 In contrast, the intracellular potential is reductive. The concentration of glutathione (GSH) within the cell is much higher compared to its counterpart, GSSG.56 For the release of drugs, it is thus described that disulfide bonds can be reduced intracellularly.55,57 The pH-triggered release on the other hand is based on pH differences between extracellular and intracellular compartments. The pH drops from 7.4 in the bloodstream to 5−6 in the endosomes and even to 4.5 in the lysosomes.54,58 These properties make acetal containing linkers stable in the blood.54 Once the conjugate is taken up by the cell via endocytosis, the linker can be cleaved in the endosomes or lysosomes, leading to the release of the active agent. Moreover, tumor tissue is known to be slightly acidic compared to healthy tissue, favoring the cleavage of such linkers there.59 As carrier system hyperbranched polyglycerol (hPG) was employed. hPG is an ideal candidate for carrier systems in biomedical applications. Its synthesis can easily be upscaled to kilogram scale,60 numerous hydroxyl groups are available for further functionalizations,45,60−62 it has a good biocompatibility in vitro and in vivo and is chemically stable.60,63−65 hPG is a highly polar carrier system. The linkage of the porphyrin PS with hPG combines the advantages of both systems for the benefit of PDT: The EPR effect,28−30 better cellular uptake,28 better water solubility (due to highly abundant hPG hydroxyl groups) of the conjugate, and the PS properties of the porphyrin. In this publication, the functionalization of hPG with multiple porphyrins using reductively and pH cleavable linkers is described and a convenient and easy way for the connection of the porphyrins to hPG is shown. Thus, based on the biocompatible carrier hPG a flexible linker approach is presented that allows different linkers to be employed and combined, eventually enabling release under two different triggers adjusted to the site of application.

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EXPERIMENTAL SECTION

Reagents. L-Ascorbic acid sodium salt (99%), dimethyl sulfoxide (DMSO; ≥99.7%) extra dry over molecular sieves, dimethylformamide (DMF; 99.8%) extra dry over molecular sieves, 1,3-dicyclohexylcarbodiimide (DCC; 99%), 1-hydroxybenzotriazole hydrate (HOBt hydrate), methanol (≥99.8%), triethylamine (≥99%), dichloromethane (DCM; ≥99%), sodium acetate × 3H2O for analysis (99.5%), zinc acetate × 2H2O for analysis (99.5%), ammonia solution (≥25%) pure, DMSO Rotidry (≤200 ppm of H2O; ≥99.5%), sodium sulfate (≥99%), and tetrahydrofuran (THF; ≥99.7%) for highperformance liquid chromatography (HPLC), acetone-d6 (99.8%), D2O (99.95%), and THF-d8 (99.5%) were purchased from commercial suppliers. All these chemicals were used without further purification. (1R,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-methyl (4-nitrophenyl) carbonate endo, (1R,8S,9r)-bicyclo[6.1.0]non-4-yn-9-methyl (4-nitrophenyl) carbonate exo, hPG19.5-acetal-azide (synthesized from an hPG with Mw = 19.5 kDa and Mn = 8.4 kDa) and hPG10.6-acetal-azide (synthesized from an hPG with Mw = 10.6 kDa and Mn = 7.9 kDa),44 hPG19.5mannose-azide (synthesized from an hPG with Mw = 19.5 kDa and Mn = 8.4 kDa),61 hPG19.5-azide (synthesized from an hPG with Mw = 19.5 kDa and Mn = 8.4 kDa),69 5,10,15-tris(3-hydroxyphenyl)-20-[4-((6(((((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)methoxy)carbonyl)amino)hexyl)amino)tetrafluorophenyl]porphyrin 4a,40 zinc-porphyrin 1127 and the noncleavable hPG19.5-porphyrin conjugate 1040 with approximately 13 porphyrin groups and approximately 110 mannose groups (synthesized from an hPG with Mw = 19.5 kDa and Mn = 8.4 kDa) were prepared according to the literature or with slight modifications. All solvents were dry or distilled prior to use. Quenching Experiment. Conjugate 5 (2.0 mg, 95.2 nmol, 150 nmol porphyrin and 352 nmol azide groups) was dissolved in degassed methanol/H2O = 1/1, v/v (20 mL) in the presence of dithiothreitol (DTT) (50 mM). The solution was heated to 37 °C. Fluorescence spectra (excitation wavelength = 395 nm, monitored wavelength = 604 B


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Biomacromolecules

Scheme 1. Substitution of the A3B Porphyrins 1a,b with Free Amine End Groups via Amide Coupling with Propiolic Acid or Bicyclo[6.1.0]non-4-yn-9-ylmethyl (4-nitrophenyl) Carbonate (endo or exo)a

a

Reagents and conditions: (a) propiolic acid, HOBt hydrate, DCC, THF, 130 min, RT; (b) bicyclo[6.1.0]non-4-yn-9-ylmethyl (4-nitrophenyl) carbonate (endo or exo), DMF, 15 to 20 min, RT;40 (c) Zn(OAc)2, NaOAc, MeOH, 30 min, RT (detailed conditions and yields are given in the Supporting Information). was stirred vigorously. The sample was stirred at 37 °C for 295 min. During this time UV/vis spectra of the aqueous phase were measured after 0, 35, 65, 90, 130, 155, 175, 205, 235, 265, and 295 min. Release Study of Conjugate 8 by UV/vis. Conjugate 8 (139 μg, 4.73 nmol, 39.9 nmol porphyrin, and 123 nmol azide groups) was dissolved in degassed PBS (pH 7.4)/DMSO = 162/1, v/v (2 mL). Ethyl acetate (4 mL) was added, and the solution was stirred vigorously. The sample was stirred at 37 °C for 195 min. During this time UV/vis spectra of the aqueous phase were measured after 0, 15, 30, 45, 60, 75, 105, 135, 165, and 195 min. Conjugate 8 (139 μg, 4.73 nmol, 39.9 nmol porphyrin and 123 nmol azide groups) was dissolved in degassed PBS (pH 4.0)/DMSO = 162/1, v/v (2 mL). Ethyl acetate (4 mL) was added, and the solution was stirred vigorously. The sample was stirred at 37 °C for 195 min. During this time, UV/vis spectra of the aqueous phase were measured after 0, 15, 30, 45, 60, 75, 105, 135, 165, and 195 min. Qualitative Release Study of Conjugate 6. Conjugate 6 (300 μg, 10.3 nmol, 86.4 nmol porphyrin, and 264 nmol azide groups) was dissolved in degassed PBS (pH 7.4)/DMSO = 81/1, v/v (700 μL). Ethyl acetate (300 μL) was added, and the solution was stirred vigorously. The sample was stirred at 40 °C for 30 min. Conjugate 6 (300 μg, 10.3 nmol, 86.4 nmol porphyrin, and 264 nmol azide groups) was dissolved in degassed PBS (pH 7.4)/DMSO = 81/1, v/v (700 μL) with 50 mM DTT. Ethyl acetate (300 μL) was added, and the solution was stirred vigorously. The sample was stirred at 40 °C for 30 min. Qualitative Release Study of Conjugate 8. Conjugate 8 (300 μg, 10.2 nmol, 86.1 nmol porphyrin, and 265 nmol azide groups) was dissolved in degassed PBS (pH 7.4)/DMSO = 81/1, v/v (700 μL). Ethyl acetate (300 μL) was added, and the solution was stirred vigorously. The sample was stirred at 40 °C for 30 min. Conjugate 8 (300 μg, 10.2 nmol, 86.1 nmol porphyrin and 265 nmol azide groups) was dissolved in degassed PBS (pH 2.0)/DMSO = 81/1, v/v (700 μL). Ethyl acetate (300 μL) was added, and the solution was stirred vigorously. The sample was stirred at 40 °C for 30 min. Cellular Uptake Experiments. The uptake of compounds in human epithelial carcinoma A431 cells was analyzed qualitatively by confocal laser scanning microscopy (CLSM) and quantitatively by flow cytometry. Cell lines were routinely propagated in DMEM medium supplemented with 2% glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% fetal calf serum at 37 °C with 5% CO2,

nm) were measured after 0, 204, 408, 816, 1020, 1224, 1428, 1632, 1837, 2041, 2245, 2449, 2653, 2857, 3061, 3265, and 3469 s. Release Study by Thin Layer Chromatography (TLC). Conjugate 6 (point of a spatula) was dissolved in degassed methanol (200 μL) as sample a. In a second sample b conjugate 6 (point of a spatula) was dissolved in degassed methanol with the addition of DTT (1 mg) and the solutions were heated to 40 °C for 10 min. A TLC of the two samples in DCM/methanol = 9/1, v/v was developed. Release Study by Size Exclusion Chromatography (SEC). Conjugate 6 (1.0 mg, 34.2 nmol, 288 nmol porphyrin, and 880 nmol azide groups) was dissolved in degassed DMSO (25.0 μL) as sample B and then diluted with H2O (475 μL). In a second sample A conjugate 6 (1.0 mg, 34.2 nmol, 288 nmol porphyrin, and 880 nmol azide groups) was dissolved in degassed DMSO (25.0 μL) and then diluted with H2O (475 μL). To this sample DTT (3.9 mg) was added. The solutions were kept at 40 °C for 10 min. An SEC in H2O was performed. Release Study of Conjugate 5 by Dialysis. Conjugate 5 (5.0 mg, 238 nmol, 375 nmol porphyrin, and 880 nmol azide groups) was dissolved in methanol/H2O = 1/1, v/v (5 mL). After the sample had been degassed with argon, it was filled into a dialysis tube. The sample was dialyzed in a beaker with DTT (50 mM) in methanol/H2O = 1/1, v/v (245 mL) for 6060 min. Conjugate 5 (5.0 mg, 238 nmol, 375 nmol porphyrin and 880 nmol azide groups) was dissolved in methanol/H2O = 1/1, v/v (5 mL). After the sample had been degassed with argon, it was filled into a dialysis tube. The sample was dialyzed in a beaker with methanol/H2O = 1/1, v/v (250 mL) for 6060 min. During the dialysis outside the tube, fluorescence spectra (excitation wavelength = 395 nm, monitored wavelength = 604 nm) were measured after 0, 140, 240, 1270, 1580, 3175, and 6060 min. Release Study of Conjugate 6 by UV/vis. Conjugate 6 (119 μg, 4.05 nmol, 34.2 nmol porphyrin, and 105 nmol azide groups) was dissolved in degassed PBS (pH 7.4)/DMSO = 162/1, v/v (2 mL). Ethyl acetate (4 mL) was added, and the solution was stirred vigorously. The sample was stirred at 37 °C for 295 min. During this time UV/vis spectra of the aqueous phase were measured after 0, 35, 65, 90, 130, 155, 175, 205, 235, 265, and 295 min. Conjugate 6 (119 μg, 4.05 nmol, 34.2 nmol porphyrin, and 105 nmol azide groups) was dissolved in degassed PBS (pH 7.4)/DMSO = 162/1, v/v (2 mL) with 1 mM DTT. Ethyl acetate (4 mL) was added, and the solution C


Article

Biomacromolecules Scheme 2. Conjugation of the A3B Porphyrins 3 and 4b to hPG19.5-azide or hPG19.5-mannose-azidea

a

Numbers in brackets give the approximate loading per polymer with porphyrin molecules, azide, and mannose groups, respectively. Reagents and conditions: (a) CuSO4•5H2O, L-ascorbic acid sodium salt, dimethyl sulfoxide (DMSO)/H2O, 24 h, RT; (b) DMSO, 1−24 h, RT (detailed conditions and yields are given in the Supporting Information).

Scheme 3. Conjugation of the A3B Porphyrin 4a to hPG10.6-acetal-azidea

a

The number in brackets gives the approximate loading of porphyrin molecules per polymer. Reagents and conditions: DMSO, 24 h, RT (detailed conditions and yields are given in the Supporting Information).

and subcultured twice a week. Samples were dissolved in water at a concentration of 2 mM and freshly prediluted 1:20 before adding them to the cells. For CLSM, 270 μL of cells were seeded in 8-well ibidi μ-slides (27000 cells/well) in cell culture medium. After adhesion of the cells, 30 μL of the compounds were added at a final test concentration of 10 μM and the cells were grown for another 4 or 24 h, respectively. Cell nuclei were stained with 1 μg/mL Hoechst 33342. Cells were washed and confocal images were taken with an inverted confocal laser

scanning microscope Leica DMI6000CSB SP8 with a 63x/1.4 HC PL APO CS2 oil immersion objective using the manufacturer given LAS X software. In the images, the porphyrin fluorescence is shown in red and the fluorescence of Hoechst 33342 in blue. Transmitted light images were also taken and are shown in gray scale. For flow cytometry, 450 μL of cells were seeded in 24-well plates (45000 cells per well) in cell culture medium. After adhesion of the cells, 50 μL of the compounds were added at a final test concentration of 10 μM and the cells were grown for another 4 or 24 h, respectively. D


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Biomacromolecules Scheme 4. Conjugation of the A3B Porphyrin 4b with hPG19.5-acetal-azidea

a

The number in brackets gives the approximate loading of porphyrin molecules per polymer. Reagents and conditions: DMSO, 64 h, RT (detailed conditions and yields are given in the Supporting Information).

Table 1. Overview on the Properties of the Synthesized Porphyrin-hPG Conjugates with Cleavable Linkers entry

approx. porphyrin loading: groups (loading)

1 2 3 4 5

2a (0.8%) 6 (2%) 16 (6%) 6 (4%) 15 (6%)

approx. mannose loading: groups (loading)

cleavable linker

size in nm (by DLS)

solubility

product

100 (38%)

disulfide disulfide disulfide acetal disulfide + actal

∼50b ∼200 ∼50 ∼200 ∼90

MeOH/H2O 1/1 MeOH acetone/H2O 9/1 acetone/H2O 9/1 DMSO

5 6 7 8 9

a

For entry 1, the PS is the corresponding zinc porphyrin; for entries 2−5, it is the free base porphyrin. bThe DLS for 5 shows a second peak of lower intensity. Phototoxicity Bacterial Testing. The bacterium S. aureus DSM11729 is a typcial Gram-positive member of the wound microflora. Cultured bacterial cells were suspended in sterile PBS, or sterile PBS supplemented with 10% sterile horse blood serum. The final OD (optical density) at 600 nm (1 cm cuvette path length) in all cases was 0.015. The bacterial suspensions were placed in sterile black well plates with clear bottoms. Concentrations of PS used in the study were 100 μM and 10 μM (for conjugate 9 and porphyrin 4b also 1 μM was tested). After an incubation time of 30 min at RT, the samples were irradiated at 652 nm at a dose rate of approximately 100 J cm−2. Control plates containing no PS were not exposed to light. The control samples for dark toxicity were only exposed to PS without any illumination.

Then, cells were washed, and detached by trypsin, transferred to an Eppendorf tube, centrifuged at 140g and 4 °C for 4 min, and resuspended in PBS. Fluorescence of at least 10,000 cells was measured (FL3: exc. 488 nm, em. > 670 nm) in a BD Accuri C6 and median fluorescence intensities were obtained by using the software FlowJo 10.2. Measurements were done in duplicate and repeated three times. Mean values with SD were plotted with the software Excel. Phototoxicity Cell Testing. Human epidermoid carcinoma A253, human epithelial carcinoma A431, human oral adenosquamous carcinoma CAL27, and colorectal adenocarcinoma HT29 cells were grown in Dulbecco’s modified eagle medium (DMEM) with 10% heat inactivated FCS, 1% penicillin (10000 IU) and streptomycin (10000 μg mL−1). A stock solution (2 mM) of the PS was prepared at 4 °C in DMSO and kept in the dark. DMEM (without phenol red) with 10% FCS was used for further dilution to reach concentration 2 or 10 μM of the PS, respectively. In microplates 2 × 104 cells per well were seeded in fresh medium (DMEM without phenol red) containing 10% FCS with 2 μM or 10 μM of the PS and incubated for 24 h. After exchange of medium (to remove any PS not taken up by the cells), the photosensitization was performed at RT with a white light source or with a laser at 652 nm at a dose rate of app. 50 J cm−2. The cell viability of the samples was assessed using the XTT assay70 and the absorbance was measured with a Tecan Infinite 200 microplate reader, at a wavelength of 490 nm. A wavelength of 690 nm was used to measure the reference absorbance.

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RESULTS AND DISCUSSION Synthesis. It is possible to functionalize hPG with different methods,45,61,62,69 starting from the large number of available hydroxyl groups. To obtain the azide functionality, one approach is mesylation of the hydroxyl groups followed by nucleophilic substitution with sodium azide leading to the azido-substituted hPG. To control the degree of functionalization, the stoichiometry of the reactants can be varied.69 Another approach is the formation of hPG-acetal using 4-(3azidopropoxy)-2-methoxybenzaldehyde dimethyl acetal or 4(3-azidopropoxy)benzaldehyde dimethyl acetal by using an E


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Figure 1. (Left) Release study of conjugate 6 (disulfide linker) followed by TLC in DCM/methanol = 9/1, v/v (a and b). (Right) Release study of conjugate 6 followed by SEC in methanol (A and B; detailed conditions are given in the Experimental Section).

Figure 4. Time-dependent release of porphyrin dye from conjugate 8 (acetal linker) in PBS (pH 4.0)/DMSO = 162/1, v/v or PBS (pH 7.4)/DMSO = 162/1, v/v. UV/vis spectra were taken after 0, 15, 30, 45, 60, 75, 105, 135, 165, and 195 min. For all UV/vis measurements the peak maximum of the Soret band was used (421 nm; detailed conditions are given in the Experimental Section).

acid-catalyzed transacetalization with hPG.44 With this method the acid-cleavable benzacetal group is incorporated already in the hPG. In the next step the azido-functionalized hPG can easily be functionalized with active groups, namely, PS molecules or mannose units, using the well-known copper(I)-catalyzed 1,3dipolar cycloaddition (CuAAC), known as click-reaction,71,72 and the alternative metal-free strain-promoted alkyne−azide cycloaddition (SPAAC),44,73,74 by reacting it with the respective alkyne-substituted molecular building blocks. Thereby, porphyrin-hPG conjugates with cleavable linkers are obtained where the porphyrin can be released from the substrate, which is of interest for biological and medical applications.54 For this work we chose hPG with a core size of 19.5 kDa, which leads to functionalized conjugates with sizes between 21 and 64 kDa. A higher cellular uptake is observed with hPG with a size larger than 20 kDa.75 Additionally, we chose one example with an hPG core size of 10.6 kDa. The reaction sequences started with the amino-substituted porphyrins 1a,b27 (Scheme 1). To obtain the alkyne functionality for the following CuAAC reaction porphyrin 1b was reacted with propiolic acid in the presence of dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole hydrate (HOBt hydrate). This method is routinely used in peptide synthesis and known to prevent the formation of Nacyl urea.76 The porphyrin 1b, propiolic acid, HOBt hydrate, and DCC in tetrahydrofuran (THF) were stirred for 130 min at room temperature (RT). The crude product was purified by column chromatography to afford porphyrin 2 with 77% yield.27 In the second sequence, an active ester was used to obtain the cyclooctyne functionality for the following SPAAC. We used bicyclo[6.1.0]non-4-yn-9-ylmethyl (4-nitrophenyl) carbonate (endo or exo) in a direct reaction with the porphyrins 1a,b (Scheme 1). The porphyrins 1a,b, triethylamine, and bicyclo[6.1.0]non-4-yn-9-ylmethyl (4-nitrophenyl) carbonate (endo or exo) in dimethylformamide (DMF) were stirred for 15−20 min at RT. The crude products were purified by column chromatography to afford the porphyrins 4a−c with yields of

Figure 2. Time-dependent release of porphyrin dye from conjugate 5 (disulfide linker) in 50 mM DTT in methanol/H2O = 1/1, v/v or methanol/H2O = 1/1, v/v without DTT. Fluorescence spectra were taken after 0, 140, 240, 1270, 1580, 3175, and 6060 min. For all fluorescence measurements, an excitation wavelength of 395 nm and a monitoring wavelength of 604 nm was used (detailed conditions are given in the Experimental Section).

Figure 3. Time-dependent release of porphyrin dye from conjugate 6 (disulfide linker) in 1 mM DTT in PBS (pH 7.4)/DMSO = 162/1, v/ v or PBS (pH 7.4)/DMSO = 162/1, v/v without DTT. UV/vis spectra were taken after 0, 35, 65, 90, 130, 155, 175, 205, 235, 265, and 295 min. For all UV/vis measurements the peak maximum of the Soret band was used (421 nm; detailed conditions are given in the Experimental Section). F


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Figure 5. Schematic representation of the porphyrin PS release under reductive conditions. The blue sphericals represent the hPG and the red ones the porphyrins. In the test tubes, the upper phase is ethyl acetate, whereas the lower is PBS.

cyclooctyne functionality is used for the transition metal-free conjugation via SPAAC. The SPAAC avoids the use of toxic substances and does not require any precomplexation of the porphyrin with a metal (as protection for the inner core of the porphyrin) for further functionalization. For a later release of the porphyrin from the carrier, it was necessary to introduce the cleavable linkers. One example is the disulfide linker contained in porphyrins 2 and 4b,c (Scheme 1). Using these porphyrins, we were able to obtain cleavable systems with our conjugates in the next step (Schemes 2 and 3). CuAAC is a fast, easy, and versatile reaction. It is often used in organic, polymer, materials, and medicinal chemistry.71,72 LAscorbic acid sodium salt, copper(II) sulfate, the porphyrin 3, carrying an alkyne-substituted linker, and the corresponding hPG19.5-azide69 were dissolved in a DMSO/H2O mixture under argon (Scheme 2). The solution was stirred for 24 h. After this time, TLC of the reaction mixture showed that almost all of the starting material had been consumed and that a new redcolored substance had formed which stayed at the baseline. The crude product was purified by dialysis to obtain the hPG19.5 conjugate 5 with disulfide linkers and approximately two zinc porphyrin groups. The amount of porphyrin covalently bound to the polymer was determined via NMR as described in the literature.27,40,77−79 SPAAC is a newer reaction type which improves the conditions of the CuAAC and does not need additional catalysts or transition metals.44,73,74 The porphyrin 4b with the cyclooctyne moiety and the corresponding hPG19.5-azide69 or hPG19.5-mannose-azide61 were dissolved in DMSO under argon (Scheme 2). For the synthesis of conjugate 6, the solution was stirred for 1 h, followed by purification by dialysis, to obtain the hPG19.5 conjugate with disulfide linkers and approximately eight porphyrin groups. Targeting groups are of great interest for use in PDT, as they can make the PS conjugates more specific.2,80,81 Therefore, we used as well hPG19.5-azide prefunctionalized with mannose units as targeting groups.61 Again, the cyclooctynelinked porphyrin 4b and the corresponding hPG19.5-mannoseazide were dissolved in DMSO under argon (Scheme 2). The solution was stirred for 24 h. After this time, TLC of the reaction mixture showed that almost all of the starting material had been consumed and that a new red-colored substance had formed that stayed at the baseline. The crude product was purified by dialysis to obtain the hPG19.5 conjugate 7 with

Figure 6. Release study of conjugates 6 (disuifide linker) and 8 (acetal linker), followed by extraction. The upper phase is the organic one (ethyl acetate), whereas the aqueous is the lower one. (A) Conjugate 6 in PBS (pH 7.4)/DMSO = 81/1; (B) Conjugate 6 in PBS (pH 7.4)/ DMSO = 81/1with 50 mM DTT; (C) Conjugate 8 in PBS (pH 7.4)/ DMSO = 81/1; (D) Conjugate 8 in PBS (pH 2.0)/DMSO = 81/1 (detailed conditions are given in the Experimental Section).

Figure 7. Time-dependent fluorescence measurements of the conjugate 5 (disulfide linker) in 50 mM DTT methanol/H2O = 1/ 1, v/v. Fluorescence spectra were taken after 0, 204, 408, 816, 1020, 1224, 1428, 1632, 1837, 2041, 2245, 2449, 2653, 2857, 3061, 3265, and 3469 s. For all fluorescence measurements an excitation wavelength of 395 nm and a monitoring wavelength of 604 nm was used (detailed conditions are given in the Experimental Section).

88 and 98%.40 Both isomers of the cyclooctyne active ester can be employed to obtain the corresponding porphyrin. The G


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Figure 8. Time-dependent fluorescence measurements of the conjugate 5 (disulfide linker) in 50 mM DTT methanol/H2O = 1/1, v/v. Fluorescence spectra were taken after 0, 204, 408, 816, 1020, 1224, 1428, 1632, 1837, 2041, 2245, 2449, 2653, 2857, 3061, 3265, and 3469 s. For all fluorescence measurements an excitation wavelength of 395 nm was used (detailed conditions are given in the Experimental Section).

approximately 16 porphyrin groups (attached via disulfidecontaining linkers) and approximately 100 mannose units. The amount of porphyrin covalently bound to the polymer was again determined via NMR.27,40,77−79 As mentioned above, noncleavable hPG−PS conjugates with higher mannose loading (approximately >70 mannose units) benefit from a multivalency effect in their aPDT activity,40 hence, the mannose loading of conjugate 7 (approx. 100 mannose units). With this large number of mannose groups, conjugate 7 is prone to act via the multivalency effect.27,61,82 As acid-cleavable systems, we used hPG-acetal-azides with and without an additional methoxy group at the aromatic ring (Schemes 3 and 4).44 These two acetals show different degradation kinetics, with the methoxy derivative degrading slightly faster.44 First, the cyclooctyne porphyrin 4a and the corresponding hPG10.6-acetal-azide44 (with methoxy group) were dissolved in DMSO under argon (Scheme 3). The solution was stirred for 24 h, followed by purification with dialysis in the presence of triethylamine to avoid unintentional cleavage, to obtain the hPG10.6 conjugate 8 with acetal linkers and approximately six porphyrins. For the hPG-porphyrin conjugate 9, the cyclooctyne porphyrin 4c and the corresponding hPG19.5-acetal-azide44 (without methoxy group) were dissolved in DMSO under argon (Scheme 4). The solution was stirred for 64 h followed by purification with dialysis (again basified with aqueous ammonia to avoid unintentional cleavage) to obtain the hPG19.5-porphyrin conjugate 9 with acetal and disulfide linkers, exemplifying that with this method it is even possible to obtain conjugates with two different cleavable linkers. The loading of the polymers with porphyrins (conjugates 5− 9) and mannose (for conjugate 7) was determined by NMR, following the literature.27,40,77−79 In addition, all conjugates were characterized by DLS (data see Supporting Information). Table 1 gives an overview on the synthesized conjugates with cleavable linkers. Altogether five conjugates were synthesized, three (conjugates 5−7) with disulfide linkers, one (conjugate 8) with acetal linkers, and one conjugate 9 with both disulfide and

acetal linkers. Among the three conjugates with disulfide linkers, 5−7, conjugate 7 carries additionally mannose substitution for improved targeting, thus, combining PS release with nanoparticle targeting.40,66−68 It is noteworthy that the parent PS molecules 1a,b as well as their derivatives 3, and 4a−c are non-water-soluble; however, after linkage to hPG, the resulting PS-hPG conjugates show an increased solubility in polar solvents, and conjugates 5, 7, and 8 become partly watersoluble. This increased polarity is advantageous for a desired medical application. After linker-cleavage and release of the individual PS molecules these are again non-water-soluble, amphiphilic, and highly membrane affine. This membrane affinity and amphiphilicity is an important prerequisite for effective antitumor PS.83 Similarly, for aPDT it has been shown that an uptake by the bacteria is not always necessary to obtain an aPDT effect, a release in the close proximity of the bacterial membrane maybe sufficient.84 In order to assess the cleavability of the PS from the nanocarrier, first, the release of the porphyrin PS was investigated in a series of experiments. Porphyrin Release Studies. Porphyrins exhibit a strong fluorescence and characteristic UV/vis spectra. These properties can be used to monitor the release of porphyrin from the conjugate in the following release studies. Release Study Followed by TLC. The different TLC retention behavior of porphyrin bound to hPG and of the cleaved free porphyrin can be used to qualitatively show the release of the active substance (Figure 1 (left)). Conjugate 6 (disulfide linker) was dissolved in methanol as sample a. A second sample b was prepared in methanol, and DTT as a reducing agent was added. The solutions were heated to 40 °C for 10 min. After that, a TLC of the samples was performed. The obtained TLC is shown in Figure 1 (left). Only the sample b is moving on the TLC plate, which indicates the release of the porphyrin. The sample a as control instead stayed at the baseline, which shows that conjugate stayed intact. a+b in the middle shows the TLC of both samples a and b combined. Release Study Followed by SEC. Another experiment that relies on the different sizes of the intact porphyrin-hPG conjugate and the cleaved free porphyrin is the SEC (Figure 1 (right)). On SEC column larger molecules have a higher flow H


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than that of the cleaved porphyrin dye (873 g/mol). Conjugate 6 was dissolved in H2O/DMSO = 20/1, v/v as sample A. A second sample B was prepared in H2O/DMSO = 20/1, v/v, and DTT as reducing agent was added. The solutions were kept at 40 °C for 10 min. After that, an SEC of the samples in H2O was performed. In Figure 1 (right) the SEC columns are shown. Column A is the control experiment where no porphyrin was cleaved, and only the conjugate moves on the column (ii). Column B indicates that most of the porphyrin is cleaved which stayed at the start (i). The column in the middle shows the SEC of both samples A and B combined. Release Study Followed by Dialysis. With a dialysis assay the different sizes of the bound porphyrin-hPG conjugate and the free cleaved porphyrin can also be separated. Conjugate 5 (disulfide linker) was dissolved in methanol/H2O = 1/1, v/v, and filled in a dialysis tube (molecular weight cutoff = 12000). Dialysis was performed against 50 mM DTT solution in methanol/H2O = 1/1, v/v, over 101 h. The release of the porphyrin dye was measured by the increasing fluorescence signal of aliquots taken from the outside of the dialysis tube. Figure 2 shows how the fluorescence outside the tube increases. After 4 d, the equilibrium had nearly been reached, and the fluorescence did not increase significantly anymore. In a control experiment where conjugate 5 was dialyzed against methanol/ H2O = 1/1, v/v, a slower increase of the fluorescence was observed. It is possible that part of the intact conjugate diffused slowly from the dialysis tube because of the nature of polymers, which do not have a monomodal size distribution. Therefore, a smaller part of the porphyrin-hPG conjugate is released from the dialysis tube. Release Study by Extraction Followed by Measuring the UV/Vis Absorption. With an assay by extraction and measuring the UV/vis absorption the different solubilities of the bound porphyrin-hPG conjugate and the free cleaved porphyrin can be used. Conjugate 6 (disulfide linker) was dissolved in PBS (pH 7.4)/DMSO = 162/1, v/v with 1 mM DTT. Ethyl acetate was added to obtain two separated phases. The sample was stirred at 37 °C over 295 min. The release of the porphyrin dye was monitored by the decreasing fluorescence signal of aliquots taken from the aqueous phase. Figure 3 shows how the fluorescence in the aqueous phase decreases. In a control experiment where conjugate 6 was stirred without the addition of DTT, a slower decrease of the fluorescence was observed. Conjugate 8 (acetal linker) was dissolved in PBS (pH 4.0)/ DMSO = 162/1, v/v. Ethyl acetate was added to obtain two separated phases. The sample was stirred at 37 °C over 195 min. The release of the porphyrin dye was monitored by the decreasing fluorescence signal of aliquots taken from the aqueous phase. Figure 4 shows how the fluorescence in the aqueous phase decreases. In a control experiment where conjugate 8 was stirred in PBS (pH 7.4)/DMSO = 162/1, v/v a slower decrease of the fluorescence was observed. This phenomenon could also be observed visually with the naked eye when the conjugates 6 (disulfide linker) and 8 (acetal linker) were treated in the same way as in the extraction experiment. Figure 5 shows the extraction experiment where the solution has stirred for 10 min in the presence of 50 mM DTT or pH 2.0 with conjugates 6 and 8, respectively. The higher concentration of DTT and the lower pH 2.0 was chosen to increase release speed of the porphyrin dye and show the qualitative results in Figure 6. In PBS (pH 7.4) both conjugates 6 and 8 stayed in the aqueous phase. The aqueous phase (lower

Figure 9. CLSM images of conjugates 6, 7, 9, and the comparator compounds 4b and 10 after 4 and 24 h. In the images, the porphyrin fluorescence is shown in red and the fluorescence of Hoechst 33342 in blue (nuclei staining). The transmitted light images are shown in gray scale.

rate compared to smaller molecules. The molecular weight (MW) of the conjugate 6 (29300 g/mol) is significantly larger I


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Figure 10. Overview on flow cytometry investigations for conjugates/compounds 4b, 6, 7, 9, and 10. Bars represent the mean intensity with SEM of three independent measurements. Two times 10000 cells were counted for each measurement.

one) is slightly red in A and C, whereas the organic phase is colorless (upper one). With addition of DTT to the solution of conjugate 6 (sample B) or lowering the pH of the solution of conjugate 8 (sample D) the porphyrin is cleaved leading to a better solubility in the organic phase. In this case, the previously colorless organic phase turned red for sample B and D. Quenching Experiment. Porphyrins that are in close vicinity to each other can transfer energy to one another upon irradiation. This effect leads to a quenching of their intrinsic fluorescence known as self-quenching.85 Porphyrins bound to hPG have a short distance to each other which can lead to a quenching effect. If the porphyrin is cleaved, the fluorescence should increase over time. Conjugate 5 (disulfide linker) was dissolved in degassed methanol/H2O = 1/1, v/v in the presence of DTT (50 mM). The solution was heated to 37 °C, and fluorescence spectra were measured over 58 min. In Figures 7 and 8, the time-dependent fluorescence measurement is shown. These diagrams indicate that the porphyrin has been set free and the fluorescence increased significantly. On the hPG, the porphyrin dyes are densely packed and show a selfquenching behavior. After they are cleaved, the porphyrin dye is set free, which nullifies this effect and leads to increasing fluorescence. Cellular Uptake of Compounds. The uptake of the conjugates was evaluated by confocal laser scanning microscopy (CLSM) and flow cytometry in the human epithelial carcinoma A431 cell line. Uptake was exemplarily evaluated with the conjugates carrying the disulfide linkers (6 and 7) and the compound carrying both, disulfide and acetal linkers (9). The cells were incubated with solutions of conjugates 6, 7, and 9 at a final test concentration of 10 μM PS for 4 or 24 h (detailed conditions are given in the methods section, additional data in the Supporting Information). As comparators for the cleavable conjugates two compounds were chosen: The precursor porphyrin 4b carrying the disulfide linker and a noncleavable hPG-porphyrin conjugate 10 (for structure, see Supporting Information, Figure S27) carrying additionally mannose units (analogously to 7), whose synthesis and photobiological properties we described recently.40

In Figure 9 CLSM pictures of conjugates 6, 7, 9, and the comparator compounds 4b and 10 are shown after 4 and 24 h. In the CLSM images (Figure 9) it can be seen, that conjugates 7 (with disulfide linkers and mannose substitution) and 9 (with disulfide and acetal linkers) show the highest uptake, as their fluorescence is already visible after 4 h (Figure 9, left column). Especially, conjugate 7 showed a pronounced uptake after 24 h as the whole cellular interior exhibits intense fluorescence; additionally, the conjugate already seems to have a negative effect on the cells (nuclei appearance, Figure 9, right column). In comparison, conjugate 6 with disulfide linkers but no mannose substitution showed a slower uptake and fluorescence from the conjugates is visible only after 24 h. Interestingly, for the noncleavable hPG-porphyrin conjugate 10 with mannose substitution, nearly no porphyrin fluorescence could be detected from within the cells, even after 24 h of incubation (Figure 9, bottom). Though the fluorescence intensity for 10 would be somewhat lower due to the close interaction of the porphyrins within the conjugate, the release experiments above show that even the noncleaved conjugates still show fluorescence which would easily allow their intracellular detection if they were taken up to a larger amount. Also, for the comparator porphyrin 4b, no considerable uptake was observed. Cellular uptake was additionally investigated by flow cytometry (detailed conditions are given in the Experimental Section); the results are shown in Figure 10. The flow cytometry results confirmed the findings from CLSM. A high fluorescence (corresponding to a high cellular uptake) was observed for conjugate 7 with disulfide linkers and mannose substitution and conjugate 9 with the combination of disulfide and acetal linkers. Also, conjugate 6 with disulfide linkers and no mannose substitution is taken up at a medium level by the cells, whereas the precursor porphyrin with the disulfide linker (4b) as well as the noncleavable conjugate 10 only show a very low uptake. The results suggest that the introduction of cleavable linkers especially in combination with mannose targeting is beneficial regarding cellular uptake. With these data on cellular uptake the photocytotoxicity of the J


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Figure 11. Phototoxicity of porphyrin-hPG conjugate 5 (acetal linkers) and comparator zinc porphyrin 11 in cellular assays with human epidermoid carcinoma A253, human epithelial carcinoma A431, human oral adenosquamous carcinoma CAL27, and colorectal adenocarcinoma HT29 cells, irradiated with a white light source (see methods section for details). DT: dark toxicity.

conjugates was finally evaluated in four different tumor cell lines and the Gram-positive bacterium S. aureus. Photocytotoxicity in Cellular Assays. The photocytotoxicity of the conjugates 5−9 was evaluated in cellular assays in human epidermoid carcinoma A253, human epithelial carcinoma A431, human oral adenosquamous carcinoma

CAL27, and colorectal adenocarcinoma HT29 cells (Figures 11 and 12; see Experimental Section for details). The assays were carried out after incubation for 24 h, with the PS in medium containing 10% fetal calf serum (FCS). After the 24 h incubation, the medium was exchanged to ensure that only PS that has been taken up by the cells contributes to the observed K


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Figure 12. Phototoxicity of porphyrin-hPG conjugates 6, 7 (disulfide linkers), 8 (acetal linkers), 9 (acetal and disulfide linkers), and the comparator porphyrin 4b in cellular assays with human epidermoid carcinoma A253, human epithelial carcinoma A431, human oral adenosquamous carcinoma CAL27, and colorectal adenocarcinoma HT29 cells, irradiated with a laser at 652 nm (see Experimental Section for details). DT: dark toxicity.

the HT29 cell line where conjugate 5 reduces the cell viability to about 60% whereas zinc porphyrin 11 seems to induce sublethal stress in the cells, leading to a slightly increased cell growth. Neither conjugate 5 nor zinc porphyrin 11 exhibit any remarkable dark toxicity (only for 11 a small dark toxic effect is observed at 10 μM in the A253 cell line). Additionally, the noncleavable hPG-zinc-porphyrin conjugate 10 with mannosesubstitution was tested in the A431 cell line, which had also been taken for the cell uptake experiments. In this case, nearly no dark or phototoxicity was observed corresponding to the low uptake of the conjugate in the cell uptake experiments. In Figure 12 the linker-substituted porphyrin 4b (incorporating a disulfide linker) is compared to the hPG19.5-porphyrin conjugates having disulfide linkers (6), disulfide linkers and mannose targeting groups (7), acetal linkers (8), and the conjugate having acetal as well as disulfide linkers (9). None of

effect. Both, the dark and the phototoxicity were determined at two different sensitizer concentrations (2 and 10 μM). A white light source at a dose rate of app. 50 J/cm2 or a laser with a wavelength of 652 nm at a dose rate of 50 J/cm2 was used for irradiation. As comparators, we used the metal-free porphyrin 4b and the zinc porphyrin complex 11,27 respectively (for structure of 11 see Supporting Information, Figure S28). Conjugate 5 with the zinc porphyrins attached shows a pronounced phototoxic effect at 10 μM in the A431, A253, and CAL27 cell lines with the cell viability being reduced to less than 50%, for CAL27 even below 10% (Figure 11). However, the simple comparator zinc porphyrin 11 shows similar effects in the A253 and CAL27 cell line. On the other hand, the hPG19.5-porphyrin conjugate (at 10 μM) exhibits a higher phototoxicity in the A431 and HT29 cell line compared to the zinc porphyrin complex 11. This is especially pronounced for L


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horse serum the conjugates 6−9 were added in amounts equal to 10 and 100 μM PS concentration, respectively. The samples were incubated for 30 min in the absence of light and afterward irradiated with a laser at 652 nm. In control experiments, the effect of incubation with the photosensitizing agent alone (100 μM concentration) without subsequent illumination was assessed (dark toxicity) (see Experimental Section for details). For comparison with the conjugates 6−9 again the free porphyrin dye 4b was tested. As can be seen from Figure 13, neither the conjugates nor the comparator porphyrin 4b exhibit any dark toxic effect on S. aureus. Conjugate 6 does not show any antibacterial activity in PBS; conjugate 7 on the other hand, having additional mannose units, eradicates all bacteria at both concentrations in PBS. The mannose functionalities on this conjugate lead to a multivalent targeting effect,40,66 resulting in a better attachment of the conjugate to the bacteria and increasing the antibacterial photodynamic activity. This is in line with our previous results on noncleavable conjugates.40 Conjugate 8 shows a low toxicity against bacteria, whereby 37% and 64% of them died at a concentration of 10 and 100 μM, respectively. Conjugate 9 combines the disulfide and acetal linker. In this case, again a complete eradication of the bacteria at both concentrations is observed. Thus, conjugate 9, with both linkers performs significantly better than the conjugates with either acetal (8) or disulfide (6) linkers alone. Remarkably, conjugate 9 exhibits this high phototoxicity despite the fact that it lacks additional targeting groups like conjugate 7. The comparator porphyrin 4b also exhibits a significant phototoxic effect on S. aureus though with a lower activity than conjugate 7 or 9. Also, 4b is completely water-insoluble (and, hence, was given in DMSO to the bacterial suspension), which is problematic for the use under physiological conditions. The experimental setting also allows a direct comparison of the PDT activity of the conjugates in tumor cells and in S. aureus. For example, conjugates 8 and especially 9, possessing acid cleavable acetal linkers, which were less active in the tumor cell cultures (see above) show a higher phototoxic activity in S. aureus. Studies have shown that bacteria, like Staphylococcus epidermis or aureus, can create an acidic environment when they grow.88,89 This is i.a. attributed to acidic substances (e.g., lactic acid), which are formed as metabolic products.88,89 Therefore, the observed effect may be explained by the acidic environment of the bacteria, which cleaves the acetal-bound porphyrin and increases the antibacterial activity. As stated above, the high PDT activity of the mannose decorated conjugate 7 in tumor cells as well as in S. aureus may be rationalized by unspecific interactions of the mannose and an increased solubility of the conjugate for the former and multivalent interactions with bacterial mannose receptors for the latter. For a medical application activity in complex biological media is required.9,10,90,91 Therefore, the antibacterial tests of the conjugates were additionally performed in PBS supplemented with 10% horse serum (see Supporting Information, Figure S26). The presence of horse serum had a pronounced effect on the antibacterial activity. For all conjugates, the PDTactivity vanished completely. Previously it has been described that tetrapyrrolic systems interact with proteins, which are a main component of serum.92−94 Around nanoparticles, a protein corona can be formed which changes the biological behavior.41,95,96 As mentioned above, this protein interaction has been investigated in previous publications.40,92−96

Figure 13. Antibacterial toxicity of the synthesized hPG19.5-porphyrin conjugates 6−8, 9, and the comparator porphyrin 4b against S. aureus after 30 min of incubation. The antibacterial toxicity is expressed as the logarithm of the number of colony-forming units [lg (CFU mL−1)]; that is, the reduction from 6 to 5 corresponds to an eradication of about 90% of the bacteria. “↓” indicates complete eradication of the bacteria.

the compounds exhibits any remarkable dark toxic effect. Small dark toxic effects were observed for conjugate 9 at 2 and 10 μM PS concentration in the A253 and HT29 cell line. The comparator porphyrin 4b does not have any strong phototoxic effect whereas all conjugates exhibit stronger phototoxic effects at least in some cell lines. The conjugate with the lowest phototoxic activity is 8 (with the acetal linker). A possible explanation for this could be the smaller conjugate size, which may lead to a decreased uptake.75 All other conjugates reduce the cell viability in the investigated tumor cell lines to at least 60%. It should also be considered that the cell lines typically show differences in their sensitivity to cellular stress and PDT treatment,86 for example, the HT29 cell line in nearly all cases shows a higher resistance to the PDT treatment than the other cell lines. Interestingly, the strongest phototoxic effect is observed for conjugate 7 carrying disulfide linkers and mannose targeting groups. This is also the only conjugate which exhibits a pronounced phototoxicity already at 2 μM PS concentration, reducing, for example, the cell viability of the A253 and CAL27 cells to below 20%. The cell testing results for the A431 cell line correspond very nicely to the cell uptake experiments discussed above (cf. Figure 10), with conjugates 7 and 9 exhibiting the strongest phototoxicity in this cell line. Though the investigated cell lines do not have specific mannose receptors, unspecific interactions of the mannose and an increased solubility of the conjugate may account for the observed higher phototoxicity of 7. These preliminary results show that the conjugates with the cleavable linkers exhibit phototoxicity in several tumor cell lines. In these tests, the conjugates with the reductively cleavable linkers were more efficient than the one with the acidcleavable linker. Moreover, it seems that the decoration of the conjugates with mannose units appears to have a beneficial effect on the PDT activity. Phototoxicity against S. aureus. The bacteria S. aureus and methicillin-resistant S. aureus (MRSA) are common standards in the investigation of antibacterial activity.87 To suspensions of S. aureus bacterial cells in PBS or PBS + 10% M


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CONCLUSION A new approach to functionalize hPG with porphyrins has been developed providing hPG-porphyrin nanoparticle conjugates containing cleavable linkers. We could obtain reductively cleavable hPG-porphyrin conjugates with disulfide bridges and pH sensitive hPG-porphyrin conjugates with acetal linkers. Apart from CuAAC, the linkage to hPG could also be performed with SPAAC, thereby allowing the connection of porphyrins to hPG without any addition of catalysts or transition metals. Even one conjugate having a combination of disulfide and acetal linkers could be synthesized, allowing a release under two different triggers. In addition, the hPGporphyrin conjugates could also be loaded with mannose units enabling a multivalent targeting. The cleavability of the active PS’s from the conjugates could be shown in different release studies. Cell uptake experiments in the human epithelial carcinoma A431 cell line showed that the introduction of cleavable linkers especially in combination with mannose targeting was beneficial regarding cellular uptake of the PS. Finally, these conjugates showed a promising phototoxicity against tumor cells as well as against S. aureus. Especially, the conjugate with a combination of disulfide linker and mannose targeting proved to be effective against tumor cells as well as against the bacterium S. aureus, suggesting such systems may be promising candidates for applications in antitumor as well as antibacterial PDT. Future work will be directed toward the detailed investigation of the in vitro release of the porphyrins from the conjugates in cells.

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REFERENCES

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.7b01485. 1. Materials and Methods; 2. NMR spectra of porphyrin 4b and the conjugates 5−9; 3. ESI-MS of porphyrin 4b; 4. Antibacterial photodynamic activity of the conjugates/ porphyrins 6−8, 9, and 4b; 5. Antibacterial toxicity of 6− 8, 9, and 4b in the presence of 10% serum; 6. Structural formula of conjugate 10; 7. Structural formula of porphyrin 11; 8. DLS data of compounds; 9. CLSM images (PDF).

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Rainer Haag: 0000-0003-3840-162X Arno Wiehe: 0000-0002-6289-7672 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the DFG (SFB 765), the core facility “Biosupramol” (supported by the DFG) and the German Bundesministerium für Bildung und Forschung (BMBF; Project “CryNaPhot”, FKZ 031A405A, biolitec research GmbH) for financial support, as well as Dr. Florian Paulus and Dr. Dirk Steinhilber for the scientific support. N


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