University of Groningen DNA-based drug carriers and dynamic proteoids with tunable properties Liu, Yun

DNA-based drug carriers and dynamic proteoids with tunable properties Liu, Yun

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Chapter 3

Lipid-Modified DNAs as Solubilizers of

Foscan

In this chapter, we designed and synthesized lipid-mofidified DNAs by using solid-phase DNA synthesis. Owing to their self-assembly properties, they were capable of solubilizing the photosensitizer Foscan for photodynamic therapy at high loading capacity without influencing its activity.

Part of this chapter will be submitted for publication:

Y. Liu,‡ _{J. W. de Vries,}‡ _{Q. Liu, S. Wieczorek, H. G. Börner, E. Buhler, M. C. A. Stuart, W. R.}

3.1 Introduction

With the advent and fast development of high-throughput (HTS) and ultra- high-throughput screening (uHTS) technologies for drug discovery over the past two decades,[1] compound libraries have yielded an increasing number of potential candidates that exhibit a high affinity for their targets. However, a substantial number of these pharmaceutically active compounds suffer from lower water solubility, which hinders their development and delays market entry. Even for already marketed drugs, more than 40% are poorly water soluble.[2]

To enable the use of these active compounds and reduce their side effects, numerous strategies have been proposed, one of them being the use of drug-delivery vehicles. These include employing dendrimers,[3] liposomes,[4] cyclodextrins,[5] chitosan,[6] nanoporous silica particles,[7] liquid crystals[8] and micelles.[9] Among them, micelles are most widely used due to attractive properties such as high solubilizing efficiency, good reproducibility, simple preparation procedures and the possibility to make them stimuli-responsive.[10]

Despite various amphiphilic materials being used, it is still a challenge to construct a biocompatible, effective and targeted micellar drug-delivery system. In the meantime, previous studies showed that amphiphilic DNA-based copolymers self-assemble into uniform micelles above their critical micelle concentration (CMC) and are able to accommodate drugs of interest in the hydrophobic core.[11] These constructs have several advantages over those formed from synthetic polymers. First, being formed from biomaterials, DNA-based micelles are more biocompatible and biodegradable and have shown no observable toxicity and little immunogenicity.[12] Secondly, they can be easily synthesized by automated solid-phase synthesis.[13] Most importantly, DNA-based micelles can be modified in a straight forward fashion by employing highly specific hybridization, which conveniently endows the system with targeting or imaging properties.[14] All these beneficial properties give them great potential to be used as targeted drug-delivery vehicles.

Photodynamic therapy (PDT) is a non-invasive therapeutic treatment used to battle many tumors.[15] It involves the administration of a natural or synthetic photosensitizer (PS), followed by the activation of the PS with light at a specific wavelength. The PS transfers energy from the light to the biological substrates or supplies molecular oxygen to generate reactive oxygen species (ROS), which includes free radicals and singlet oxygen.[16] These ROSs then induce cellular damage and cause the death of tumor cells. In PDT, the localization of the PS in the tumor and the controllable activation of the PS through exposure of only the tumor site to light lead

41 to dual selectivity. As a result, treatment of tumors without affecting normal tissues is possible. The reduced porphyrin meta-tetra-hydroxyphenyl-chlorin (mTHPC, Scheme 1a), also known as Foscan and Temoporfin,[17] is a poorly water-soluble second-generation PS that has been widely used in PDT for decades. Because of its high quantum yield (ΦΔ) and good biological activity it is a great candidate for

clinical applications, and it has been approved in Europe for the treatment of head and neck cancer.[18] However, due to the poor water solubility and tumor-targeting properties for conventional formulations, it remains a challenge to develop novel formulations for mTHPC that omit these problems and allow easy functionalization.

Based on the considerations outlined above, nanocarriers made of lipid-modified DNA amphiphiles (Scheme 1c) are excellent candidates to be used as solubilizers for poorly soluble medication. Here, we report the successful use of lipid-modified DNAs to render mTHPC water-soluble with high drug loading capacities (LCs) that do not compromise the biological activity of the active pharmaceutical ingredient.

N HN O O O O C10H21 O O P N O CN N NH N HN OH OH HO HO 5' _3' 3' 5' 5' 3' 1 2 a 11mer 5'-TTTGGCGTCTT-3' 12mer 5'-TTTTTTGGATTC-3' c11mer 5'-AAGACGCCAAA-3' b c ds11mer d

Scheme 1. Representation of a) meta-tetra-hydroxyphenyl-chlorin (mTHPC); b) 5-(dodec-1-ynyl)uracil deoxyribophosphoramidite used in solid-phase synthesis of lipid-modified DNAs, this nucleotide building block is abbreviated as U in the corresponding sequences; c) lipid-modified DNAs (UU11mer, double-stranded UU11mer (dsUU11mer) and UUUUUU12mer) used; d) pristine control DNAs (11mer, complementary 11mer (c11mer), double-stranded 11mer (ds11mer) and 12mer) used.

3.2 Results and discussion

3.2.1 Synthesis and determination of critical micelle concentration (CMC) of the lipid-modified DNAs

Figure 1. Characterization of amphiphilic oligonucleotides by MALDI-TOF mass spectrometry. Spectrum of (A) UU11mer (calc. 3629 g/mol, found 3622 g/mol) and (B) UUUUUU12mer (calc. 4534 g/mol, found 4527 g/mol).

Lipid-modified DNAs with different hydrophobicity were synthesized by using the alkyl modified 5-(dodec-1-ynyl)uracil phosphoramidite 2 (U, Scheme 1b) using standard solid-phase synthesis. It was reported that the alkyl chains did not influence the hybridization of the DNA.[11b] Two sequences were designed and synthesized by an automated DNA synthesizer. The first sequence, an 11-mer (UU11mer, 5’-UUTGGCGTCTT-3’) contained two modified uracil bases and the second oligonucleotide, a 12-mer (UUUUUU12mer, 5’-UUUUUUGGATTC-3’) (Scheme 1c) was comprised of six modified uracil bases. The crude products were purified by reversed phase (RP) chromatography and characterized by MALDI-TOF mass spectrometry for molecular weights (Figure 1). Furthermore, RP high-performance liquid chromatography (RP-HPLC) spectra illustrate the high purity of the products (Figure 2). Before the experiments, the aqueous DNA solutions were heated to 90 °C followed by slow cooling to room temperature to allow the formation of micelles with a narrow size distribution.

The critical micelle concentration (CMC) is an essential parameter to evaluate the thermodynamic stability of micellar systems upon dilution. The CMC values of UU11mer and UUUUUU12mer were determined by measurement of changes in the fluorescence intensity upon solubilization of the hydrophobic probe 1,6-diphenyl-1,3,5-hexatriene (DPH).[19] Due to the encapsulation of DPH into the

43 hydrophobic interior offered by the formed DNA micelles, an abrupt increment in fluorescence intensity can be observed at the CMC. To determine the CMC, a constant amount of solid DPH (10 pmol) was taken, and various concentrations of the lipid-modified DNAs ranging from 0.0025 to 1 g/L were added to the fluorescent probe. The fluorescence spectra (375–500 nm) were recorded, and the intensity at 425 nm (maximum) was plotted against the logarithm of the lipid-modified DNA concentration (Figure 3). Accordingly, the CMCs were determined to be 29 and 24 µM for UU11mer and UUUUUU12mer, respectively.

Figure 2. Purity analysis of a) UU11mer and b) UUUUUU12mer characterized with RP-HPLC, while monitoring at 260 nm.

oligonucleotides. Fluorescence spectra of micelle-incorporated 1,6-diphenyl-1,3,5-hexatriene (DPH) at different concentrations (g/L) (left) and intensity at 425 nm (maximum) plotted against the logarithm of the concentration (right) for a) UU11mer and b) UUUUUU12mer.

3.2.2 Screening of solubilizers for mTHPC

To identify the specific solubilizers for mTHPC, three different types of lipid-modified DNAs were used for screening. The micelles investigated were single stranded (ss) UU11mer, double-stranded (ds) UU11mer (dsUU11mer) and ss UUUUUU12mer (Scheme 1c). Meanwhile, the pristine DNA counterparts with the same nucleic acid sequences, but in which the modified uracils were replaced by thymines, were used as controls. This includes the ss 11mer (5’-TTTGGCGTCTT-3’), ss complementary 11mer (c11mer, 5’-AAGACGCCAAA-3’), ds 11mer (ds11mer) and the ss 12mer (5’-TTTTTTGGATTC-3’) (Scheme 1d). The samples were prepared at a concentration of 50 µM both for lipid-modified DNAs and controls during the screening for solubilizers. At this concentration the formation of micelles is ensured as it is higher than the CMC of UU11mer and UUUUUU12mer. The samples were prepared by incubating the aqueous solutions of DNA together with the solid mTHPC to incorporate mTHPC into the micelles. After centrifugation, mTHPC-loaded samples (supernatant) were carefully obtained for further characterization, without touching the unsolubilized mTHPC (pellet).

The unloaded lipid-modified DNA micelles were visualized and characterized by cryo-transmission-electron microscopy (cryo-TEM) to characterize their size and morphology. As expected, the cryo-TEM images show the formation of micelles with a narrow size distribution and regular shape for UU11mer, dsUU11mer and UUUUUU12mer micelles. Also, no obvious aggregation is visible for UU11mer and dsUU11mer micelles, while bigger aggregates form for UUUUUU12mer (Figure 4), which might be ascribed to the hydrophobic interactions of the six alkyl chains. On the other hand, the diameter of UUUUUU12mer containing six alkyl chains (8.2 ± 1.8 nm), is smaller than micelles of UU11mer (9.8 ± 1.0 nm) and dsUU11mer (9.9 ± 2.0 nm) bearing two alkyl chains (Table 1). This observation indicates that the degree of hydrophobicity is the key factor in the micelle size, and six hydrophobic alkyl chains make UUUUUU12mer micelles more stable and compact than UU11mer and dsUU11mer micelles. Meanwhile, a slight size increment of dsUU11mer is observed compared with that of UU11mer, which is in line with what was reported earlier for lipid-modified DNAs[11b] and can be explained by the increased hydrophilic segment resulting from hybridization.

45 Figure 4. Cryo-transmission-electron microscopy (cryo-TEM) images of a) UU11mer micelles; b) dsUU11mer micelles; c) UUUUUU12mer micelles; d) mTHPC-loaded UU11mer micelles; e) mTHPC-loaded dsUU11mer micelles; f) mTHPC-loaded UUUUUU12mer micelles. No stain was used and image acquisition was achieved at a 2 μm defocus. Scale bar = 50 nm.

Table 1. Diameters of lipid-modified DNA micelles before and after maximum mTHPC loading obtained through cryo-TEM.

Sample UU11mer (–) UU11mer (+) dsUU11mer (–) dsUU11mer (+) UUUUUU12mer (–) UUUUUU12mer (+) Diameter (nm) 9.8 ± 1.0 11.1 ± 1.7 9.9 ± 2.0 11.4 ± 1.6 8.2 ± 1.8 9.0 ± 2.0

Figure 5. Absorption spectra of mTHPC-solubilized supernatants for solubilizer-screening experiments. The inset shows the region where mTHPC exhibits an absorption maximum (417 nm).

Solubilizers for mTHPC were screened by using UV/vis spectroscopy. Based on the absorption spectra, all mTHPC-loaded lipid-modified DNA (UU11mer, dsUU11mer and UUUUUU12mer) supernantants show typical absorption of mTHPC at 417 nm, which proves the incorporation of mTHPC into the aqueous solutions (Figure 5). In contrast, the pristine DNAs do not show any mTHPC absorption, indicating no solubilization of the drug. The observed differences in mTHPC absorbance values can be attributed to the varied abilities to solubilize mTHPC. In this regard the dsUU11mer micelles are most efficient in incorporating the compound, followed by UU11mer and finally UUUUUU12mer. This observation suggests that the lipid-modified DNA micelles with a less hydrophobic core or more hydrophilic corona feature bigger particles and core spaces, enabling more efficient solubilization of mTHPC. To conclude, the hydrophobicity-hydrophilicity balance is a key factor in determining micelle size and drug-solubilizing ability of lipid-modified DNA micelles.

3.2.3 Determination of maximum LC and characterization of mTHPC-loaded micelles

47 To determine the maximum LCs of the different lipid-modified DNAs, they were firstly incubated with a suspension of mTHPC in a mixture of H2O and EtOH,

followed by lyophilization and redissolution in H2O. The nonsolubilized mTHPC was

removed by centrifugation to obtain maximum mTHPC-loaded lipid-modified DNA micelles. During the experiments, H2O and an aqueous solution of MgCl2 (Mg2+ was

used to stabilize the double-helix of the ds DNA) were used as controls.

The loading concentrations and LCs were determined by RP-HPLC (Figure 6) with a method of lyophilization-redissolution and calculated using the calibration curve (Figure 7). Each measurement was performed in triplicate and LCs were calculated by using equation 1:

%LC = Weight of mTHPC loaded

Weight of DNA × 100% (1)

To further confirm the found high loading concentrations, fluorescence-emission spectra of the maximum mTHPC-loaded micelles and their dilutions with equivalent volumes of EtOH were recorded (Figure 8). Abrupt increments are observed after dilution with EtOH, which illustrates the intermolecular quenching of mTHPC caused by the high concentrations inside the lipid-modified DNA micelles.

Cryo-TEM images of mTHPC-loaded micelles (Figure 4) show the formation of micelles with a narrow size distribution and regular shape after mTHPC maximum loading. By analogy to unloaded micelles, no obvious aggregation is visible for UU11mer and dsUU11mer micelles, while big aggregates are formed for UUUUUU12mer. Also similar, the diameter of the loaded UUUUUU12mer (9.0 ± 2.0 nm) is smaller than that of loaded UU11mer (11.1 ± 1.7 nm) and dsUU11mer (11.4 ± 1.6 nm) (Table 1). As expected, the diameters of all the lipid-modified DNA micelles increase slightly after mTHPC loading.

The loading concentrations and LCs of lipid-modified DNAs are presented in Table 2. As is visible, mTHPC is efficiently loaded into UU11mer, dsUU11mer and UUUUUU12mer micelles and can be found at high concentrations and corresponding LCs, while the controls show minimal solubilization of mTHPC. Additionally, the maximum mTHPC loading concentration of dsUU11mer (39.0 μM) is markedly higher than that of UU11mer (30.4 μM) and UUUUUU12mer (16.5 μM), which is in good agreement with the results found in the solubilizer-screening experiment. In contrast, the mTHPC LC of UU11mer (11.4%) is higher than that of dsUU11mer (7.6%), which is due to the higher molecular weight of the hybridized DNA.

Figure 6. RP-HPLC spectra of a) H2O; b) MgCl2 (10 mM) aqueous solution; c)

UU11mer (50 µM); d) dsUU11mer (50 µM); e) UUUUUU12mer (50 µM) mTHPC-loaded samples for maximum loading capacity (LC) determination and f) mTHPC-loaded UU11mer (50 µM) and dsUU11mer (50 µM) micelles in D2O for 1

49 Figure 7. Calibration curve of mTHPC obtained through RP-HPLC in ethanol, while monitoring at 417 nm.

Table 2. Concentrations and loading capacities (LC) of mTHPC-loaded samples determined by RP-HPLC.

Samples Integral Concentration (μM) Average concentration (μM) LC (%) H2O-1 0 0 0 0 H2O-1 0 0 H2O-1 0 0 MgCl2-1 15454 0.6 0.7 - MgCl2-2 34958 0.8 MgCl2-3 16779 0.6 UU11mer -1 4235026 30.5 30.4 11.4 UU11mer -2 4438431 31.9 UU11mer -3 4015920 28.9 dsUU11mer -1 5162632 37.0 39.0 7.6 dsUU11mer -2 5543864 39.7 dsUU11mer -3 5639095 40.4 UUUUUU12mer -1 2316226 16.9 16.5 5.0 UUUUUU12mer -2 2183281 16.0 UUUUUU12mer -3 2286009 16.7

Figure 8. Fluorescence-emission spectra of mTHPC-loaded lipid-modified DNA aqueous solutions (blue line) and dilutions with 1 equivalent volume of EtOH (red line) at an excitation wavelength of 417 nm. a) UU11mer (50 µM); b) dsUU11mer (50 µM); c) UUUUUU12mer (50 µM).

3.2.4 Activities of mTHPC-loaded lipid-modified DNA micelles

Figure 9. Absorption spectra of the samples for 1O2-generation experiment measured

51 Table 3. Concentrations of the samples used for 1O2-generation experiments measured

in D2O determined by RP-HPLC.

Samples Integral Concentration (μM)

UU11mer 319170 2.8

dsUU11mer 366328 3.1

Figure 10. Singlet oxygen luminescence spectra of mTHPC-loaded UU11mer and dsUU11mer micelles compared to reference compound (Ru(bpy)3Cl2) in D2O.

Given that 1O2 plays a key role in killing tumor cells during PDT, the activities of

mTHPC-loaded lipid-modified DNA micelles were evaluated by measuring the 1O2

generation, which was monitored by near-infrared (NIR) emission spectroscopy of produced 1O2 at around 1270 nm. To facilitate the detection of 1O2, all measurements

were performed in D2O, which elongates the 1O2 lifetime compared to H2O.[20]

For this purpose, mTHPC-loaded micelles prepared by incubating the pretreated UU11mer and dsUU11mer micelles with solid mTHPC in D2O were characterized by

UV/vis spectroscopy and RP-HPLC before 1O2 generation experiments. The measured

absorption spectra (Figure 9) illustrate that dsUU11mer encapsulates a higher amount of mTHPC than UU11mer. The loading concentration was determined by RP-HPLC (Figure 6f) by lyophilization of the loaded sample and dissolving the drug in ethanol. It is found that 12% more mTHPC is solubilized in dsUU11mer (3.1 μM) than in UU11mer micelles (2.8 μM) (Table 3), which is in line with the results found from the absorption spectra.

The 1O2 phosphorescence-emission spectra by sensitization of mTHPC-loaded

UU11mer or dsUU11mer micelles in D2O demonstrate that mTHPC retains its

activity in both micelles after loading (Figure 10). The observed difference in phosphorescence intensity are expected and correspond to the differences in the absorbance of the mTHPC loaded in dsUU11mer micelles compared to UU11mer micelles, i.e. a higher concentration is achieved with the dsUU11mer micelles.

3.3 Conclusions

We have reported herein two types of lipid-modified DNA polymers (UU11mer and UUUUUU12mer) containing differing numbers of hydrophobic alkyl chains as solubilizers of the poorly water-soluble drug, mTHPC used in PDT. The sequences were designed and synthesized using standard solid-phase synthesis using an automated DNA synthesizer. Additionally, we determined their CMC values. Consequently, we successfully used UU11mer, dsUU11mer and UUUUUU12mer micelles to solubilize mTHPC with high loading concentrations and LCs. The dsUU11mer micelles solubilize the most mTHPC, while UU11mer has the highest LC. We conclude that lipid-modified DNAs micelles with a less hydrophobic core or more hydrophilic corona result in increased particles and core spaces, which leads to enhanced solubilization of mTHPC. Finally, we evaluated the activity of mTHPC-loaded UU11mer and dsUU11mer micelles in D2O by 1O2 generation, which

was monitored by NIR emission spectroscopy at around 1270 nm. The generated phosphorescence demonstrates that mTHPC remains active in D2O. Based on the

calculable deuterium-isotope solvent effect and oxygen generating activity in D2O, we

believe that mTHPC loaded into UU11mer and dsUU11mer micelles also maintains its activity in H2O. In conclusion, this work illustrates the successful use of

lipid-modified DNA micelles to solubilize mTHPC with high LCs while keeping the biological activity of the active pharmaceutical ingredient. Interestingly, their size and morphology are related to the hydrophobicity of lipid-modified DNAs, which can be adjusted by hybridization with the complementary strand or altering the number of incorporated modified uracil nucleotides. Thus, the present results offer a basis for the rational design of a novel class of drug-delivery vehicle based on lipid-modified DNA vehicles. Notably, hybridization offers a facile route for further functionalization of the drug-delivery system, as complements bearing different moieties can be used for hybridization. This method offers the possibility to design more convenient, rational, flexible and controllable drug carriers compared to using other methods for surface

53 modification. Therefore, our lipid-modified DNA micellar drug-delivery system holds great potential for further development and application in biomedical fields.

3.4 Experimental section

3.4.1 Materials and methods

All chemicals and reagents were purchased from commercial suppliers and were used without further purification, unless otherwise noted. In all experiments, MilliQ standard water (Millipore Inc., USA) with a typical resistivity of 18.2 MΩ/cm was used. The 1-dodecyne, copper(I) iodide, tetrakis(triphenylphosiphine)palladium(0) and diisopropylamine were purchased from Sigma-Aldrich and used as received. mTHPC was provided kindly by Professor Mathias O. Senge (School of Chemistry, SFI Tetrapyrrole Laboratory, Trinity College Dublin, Dublin 2, Ireland). Other special chemicals acquired from different chemical sources were 5’-DMT-5-iodo deoxy uridine (Chemgenes). All lipid-modified DNAs (ODNs) were synthesized using standard automated solid-phase phosphoramidite coupling methods on an ÄKTA oligopilot plus (GE Healthcare) DNA synthesizer. All solvents and reagents for DNA synthesis were purchased from Novabiochem (Merck, UK) and SAFC (Sigma-Aldrich, Netherlands). Solid supports (Primer SupportTM, 200 μM/g) from GE Healthcare were used for the synthesis of DNA. Lipid-modified DNAs were purified by reversed-phase high performance liquid chromatography (RP-HPLC) using a C15 RESOURCE RPCTM 1 ml reverse phase column (GE Healthcare) through custom gradients using elution buffers (A: 100 mM triethylammonium acetate (TEAAc) and 2.5% ACN, B: 100 mM TEAAc and 65% ACN). Afterwards the lipid-modified DNAs were characterized by MALDI-TOF mass spectrometry using a 3-hydroxypicolinic acid matrix. Spectra were recorded on an ABI Voyager DE-PRO MALDI-TOF (delayed extraction reflector) Biospectrometry Workstation mass spectrometer. The concentrations of the DNA were determined on a Jasco V-630 spectrophotometer (Jasco Benelux B.V., Netherlands) using 1 cm light-path quartz cuvette. Pristine DNAs were purchased from Biomers.net at HPLC purification grade. Absorption spectra were recorded on a Specord S 600 (Analytic Jena) spectrometer. A 0.1 x 1 cm cuvette was used for solubilizer-screening experiments and 1 x 1 cm cuvette for other measurements. The loading concentrations of mTHPC were determined by RP-HPLC using Xterra Prep MS C18, 10 µm, 7.8 x 150 mm column (Waters) through custom gradients using elution buffers (A: H2O (0.1% TFA), B:

ACN (0.1% TFA)) at a wavelength of 417 nm. Fluorescence spectra were recorded on a Varian Cary Eclipse fluorometer (Varian Nederland B.V.) at room temperature, an

excitation wavelength of 417 nm was used for mTHPC-loaded samples.

3.4.2 Synthesis and characterization of lipid-modified DNAs

N HN O O OH O O O N HN O O OH O I O O N HN O O O O C10H21 O O P N O CN a b 2 3 4

Scheme 2. Synthetic scheme of 5-(dodec-1-ynyl) uracil phosphoramidite. a) 1-dodecyne, Pd(PPh3)4, CuI, DMF/(iPr)2N (v/v = 1/1), room temperature 18 h, yield =

70 %; b) N-diisopropyl-2-cyanoethyl-chlorophosphoramidte, (iPr)2EtN, CH2Cl2, room

temperature 3 h, yield = 80 %.

The modified 5-(dodec-1-ynyl)uracil phosphoramidite 2 was synthesized in two steps as previously reported starting from 1 (Scheme 2).[22] Subsequently, the modified uracil phosphoramidite was dissolved in ACN to adjust the concentration to 0.15 M and directly connected to the DNA synthesizer. All oligonucleotides were synthesized on a 50 μmol scale on an ÄKTA oligopilot plus (GE Healthcare) DNA synthesizer using standard β-cyanoethylphosphoramidite coupling chemistry. Deprotection and cleavage from the PS support was carried out by incubation in concentrated aqueous ammonium hydroxide solution at 55 °C for 5 h. Following this step, the oligonucleotides were purified by using RP-HPLC, using a C15 RESOURCE RPCTM 1 mL reversed phase column (GE Healthcare) through custom gradient elution (A: 100 mM TEAAc and 2.5% ACN, B: 100 mM TEAAc and 65% ACN). Fractions were desalted using centrifugal dialysis membranes (MWCO 3000, Sartorius Stedim) or a HiTrap Desalting column (GE Healthcare). DNA concentrations were determined by UV absorbance using their respective extinction coefficients. The purity was characterized by RP-HPLC (Figure 2) on a Shimadzu VP series HPLC system with PDA detector using a C4 Jupiter 5 µm 300 Å 1 mL reversed phase column (Phenomenex) through custom gradient elution (A: 100 mM TEAAc and 5% ACN, B: Methanol). Finally, the identity of the oligonucleotides was confirmed by MALDI-TOF mass spectrometry (Figure 1).

3.4.3 Characterization of lipid-modified DNAs micelles Pretreatment of DNA aqueous solutions

55 Single-stranded DNAs (11mer, c11mer, 12mer, UU11mer, UUUUUU12mer, 50 µM), dsUU11mer (UU11mer 50 µM, c11mer 50 µM, MgCl2 10 mM) and ds11mer

(11mer 50 µM, c11mer 50 µM, MgCl2 10 mM) were thermally cycled (90 °C, 30 min;

–1 °C/2 min until room temperature) by using a polymerase chain reaction (PCR) thermocycler (Biorad, USA) before use.

CMC determination

For CMC determination, firstly 10 pmol of 1,6-diphenyl-1,3,5-hexatriene (DPH) was loaded in Eppendorf DNA low-binding tubes using a solution in acetone (1 µM). The solvent was allowed to evaporate at room temperature for 5 h after which DNA amphiphile solution (100 µL) was added. The oligonucleotides were prepared at concentrations ranging from 0.0025 to 1 g/L in 1x TAE buffer (10 mM Tris Acetate, 0.2 mM EDTA, 20 mM NaCl, 12 mM MgCl2, pH 8.0) and thermally cycled before

use. After addition to the DPH-containing tubes, the solutions were shaken at 37 °C overnight. Subsequently, fluorescence spectra (375–500 nm) were recorded on a Varian Cary Eclipse fluorometer (Varian Nederland B.V.) at room temperature using an excitation wavelength of 350 nm. The CMCs were determined to be 29 and 24 µM for UU11mer and UUUUUU12mer, respectively.

Cryo-TEM

Cyro-TEM was performed according to a standard procedure. A sample suspension (3 µL) was placed on a glow-discharged holy carbon coated grid (Quantifiol 3.5/1) blotted and vitrified in a Vitrobot (FEI). Samples were observed in a Gatan 626 cryo-stage in a Philips CM 12 or CM120 operating at 120 keV or in a FEI Tecnai T20 operating at 200 keV. Images were recorded under low-dose conditions on a slow-scan CCD camera.

Dynamic Light Scattering (DLS)

The measurements used a 3D DLS spectrometer (LS Instruments, Fribourg, Switzerland) equipped with a 25mW HeNe laser (JDS uniphase) operating at =632.8 nm, a two channel multiple tau correlator (1088 channels in autocorrelation), a variable-angle detection system, and a temperature-controlled index matching vat (LS Instruments). The scattering spectrum was measured using two single mode fibre detections and two high sensitivity APD detectors (Perkin Elmer, model SPCM-AQR-13-FC).

Fluctuations in the scattered intensity with time I(q,t) (also called count rate), measured at a given scattering angle  or equivalently at a given scattering wave

vector q=(4n/)sin(/2), are directly reflecting the so-called Brownian motion of the scattering particles (refractive index n=1.33 at 20 °C). In dynamic light scattering (DLS), the fluctuation pattern is translated into the normalized time autocorrelation function of the scattered intensity,

For a diffusive process, with a characteristic time inversely proportioned to q², g(2)(q,t)~exp(-2Dq2t), with D the mutual diffusion coefficient. The Stokes-Einstein relation allows one to determine the hydrodynamic radius Rh of the scattered objects;

Rh=kT/6πηD, if the temperature T and solvent viscosity  are known (here =1.002 cP

at 20 °C for water). The size distribution was determined using the CONTIN algorithm based on the inverse Laplace transform of the correlation function.

3.4.4 Samples preparation and determination of LCs

Preparation of samples for solubilizer-screening experiment

First, mTHPC (340 µg, 0.5 µmol) in ethanol (1 mg/mL) was loaded into a vial and the solvent was removed under vacumn at 30 °C for 3 h. The pretreated DNA aqueous solution (500 µL) was added directly to mTHPC, and the mixture was stirred (1000 r/min) at 37 °C for 12 h. After centrifugation (10000 r/min) for 15 min, the supernatant loaded with mTHPC was obtained without touching the pellet. Aluminum foil was used to cover the samples to avoid photochemical degradation of mTHPC throughout the experiments.

Preparation of maximum mTHPC-loaded lipid-modified DNA micelles

mTHPC (680 µg, 1 µmol) in ethanol (1 mg/mL) was mixed with pretreated aqueous DNA solution (1000 µL) and stirred (1000 r/min) at room temperature for 1 h. After that, the mixture was lyophilized, and H2O (1000 µL) was added. The mixture was

stirred (1000 r/min) at room temperature for 1 h. After centrifugation (10000 r/min) for 15 min, the supernatant loaded with mTHPC was obtained without touching the pellet. Aluminum foil was used to cover the samples to avoid photochemical degradation of mTHPC throughout the experiments.

Determination of mTHPC loading concentrations and LCs

To determine the LCs, the isolated supernatant (400 µL) was lyophilized, followed by the addition of cold ethanol (400 µL) to extract mTHPC. After centrifugation (10000 r/min) for 15 min, the supernatant was removed to determine the drug loading

2 ) 2 ( ) 0 , ( ) , ( ) 0 , ( ) , ( q I t q I q I t q g 

57 concentration and LC through RP-HPLC measurement.

3.4.5 Singlet oxygen generation experiments

Preparation of samples for 1O2-generation experiment

mTHPC 680 µg (1 µmol) in ethanol (1 mg/mL) was loaded into a vial, the solvent was removed under vacuum at 30 °C for 5 h. In the meantime, the aqueous solution of UU11mer (50 µM in D2O, 4000 µL) and dsUU11mer (UU11mer 50 µM, c11mer 50

µM, MgCl2 10 mM in D2O, 4000 µL) was prepared and thermally cycled as described

above. After that, the solutions obtained were added directly to mTHPC, and the mixtures were stirred (1000 r/min) at room temperature for 6 h. After centrifugation (10000 r/min) for 15 min, the supernatants were obtained without touching the pellet. Aluminum foil was used to cover the sample to avoid photochemical degradation of mTHPC throughout the experiments.

Setup for 1O2 generation experiments

A 1 x 1 cm cuvette was used for the measurements. Near–infrared emission spectra were recorded using an Andor iDus InGaAs detector coupled with a Shamrock 163 spectrograph with excitation using a 4 mW 405 nm diode laser (Thorlabs LDM 405). Tris(bipyridine)ruthenium(II) chloride (Ru(bpy)3Cl2) was used as reference sensitizer.

3.5 Contributions from co-authors

The design and synthesis of the lipid-modified DNAs was performed by Dr. J. W. de Vries from the group of Prof. Dr. A. Herrmann. Lipid-modified DNA used for cell-based testing was synthesized by Q. Liu from the group of Prof. Dr. A. Herrmann. Cyro-TEM measurements were performed by Dr. M. C. A. Stuart. Singlet oxygen generation experiments were performed by Prof. Dr. W. R. Browne. Dr. S. Wieczorek and Prof. Dr. H. Börner are acknowledged for providing mTHPC and sharing their experience on mTHPC solubilization.

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