Fullerene Polyglycerol Amphiphiles as Unimolecular Transporters

Article pubs.acs.org/Langmuir

Fullerene Polyglycerol Amphiphiles as Unimolecular Transporters Ievgen S. Donskyi,† Katharina Achazi,† Virginia Wycisk,† Kai Licha,† Mohsen Adeli,*,†,‡ and Rainer Haag*,† †

Institut für Chemie und Biochemie, Freie Universität Berlin, Takustr. 3, 14195 Berlin, Germany Department of Chemistry, Faculty of Science, Lorestan University, Khorram Abad, Iran

‡

S Supporting Information *

ABSTRACT: Due to their unique structure and properties, water-soluble fullerene derivatives are of great interest for various biomedical purposes. In this work, solution behavior, encapsulation and release properties, biocompatibility, and cellular uptake pathways of fullerene-polyglycerol amphiphiles (FPAs) with defined structures are investigated. The number of polyglycerol branches attached to the surface of fullerene affects the physicochemical properties of FPAs dramatically but not their cellular uptake. Release of encapsulated hydrophobic dyes from FPAs strongly depends on the number of their branches. Conjugation of a pH-sensitive dye to the FPAs as a probe showed that their self-assemblies are taken up through endocytotic pathways. It was observed that FPAs are able to transfer small molecules into cells both above and below their critical aggregation concentration. Taking advantage of the water solubility, biocompatibility, and transfer-ability of FPAs, they might find use as unimolecular carriers for future biomedical applications.

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great interest for biomedical applications.2 For example, it is possible to use them as nanocarriers to encapsulate and deliver various diagnostic and therapeutic agents ranging from MRI agents to HIV inhibitors.3,4 Polyglycerol, as a water-soluble, highly functional and biocompatible polyol of dendritic structure, has been used to prepare a wide range of nanostructures for biomedical applications.22,23 On the basis of these envisioned outcomes, combining polyglycerol and fullerenes results in amphiphiles with the ability of load, transfer, and release of therapeutic agents as well as interesting biological behaviors such as high cellular uptake and low toxicity. In this work, fullerene-polyglycerol amphiphiles (FPAs) consisting of different number of polyglycerol branches and a fullerene core have been synthesized by a facile and gram-scale method. Self-assembly, cellular compatibility, and uptake of the synthesized FPAs as well as their ability to load, transfer, and release of small molecules have been investigated. It was found that self-assembly, loading, release, and transport properties of FPAs are controlled by the number of polyglycerol branches. Conjugation of the pH-sensitive dye to FPAs shows that the compounds are taken up by acidic compartments of the cells endosomes and lysosomes. FPAs were able to transfer hydrophobic dye inside the cells as the unimolecular transporter or in the aggregated forms.

INTRODUCTION Fullerenes are carbon-based nanomaterials with a defined structure.1 They have been proposed for a wide range of biomedical applications.2−6 For example, fullerenes are found to be powerful antioxidants that react readily and quickly with the free radicals.5 The formation of singlet oxygen species is observed for water-soluble fullerene derivatives.7 Therefore, they could be considered as active agents for the photodynamic therapy. Cationic fullerenes are able to enhance the destruction of cancer cells and pathogenic microorganisms.8,9 It has also been shown that fullerene derivatives can inhibit enzyme activity and therefore decrease the speed of the development of Alzheimer’s disease.10 Use of fullerenes in many bioapplications is limited because of the intrinsic hydrophobicity and poor water solubility of fullerenes. Conjugation of hydrophilic (macro)-molecules onto the surface of hydrophobic fullerenes is one of the main approaches to improve their water solubility and to form fullerene-based amphiphiles.11,12 Various hydrophilic (macro)-molecules such as cyclodextrins and polymers are conjugated to the surface of fullerenes.13−15 Accordingly, highly water-soluble fullerene amphiphiles that form structurally persistent micelles are synthesized by conjugation of dendritic structures to their surfaces.16−18 The hydrophobic nature of fullerenes plays a key role in the self-assembly of fullerene-based amphiphiles.19,20 On the contrary, the structure of (macro)-molecules attached to the surface of fullerenes affects not only the solubility and selfassembly of fullerene-based amphiphiles in aqueous media but also their behavior inside the body.11,21 Due to the versatility in their solution properties, fullerene-based amphiphiles are of © 2017 American Chemical Society

Received: January 18, 2017 Revised: March 17, 2017 Published: April 7, 2017 6595

DOI: 10.1021/acs.langmuir.7b00183 Langmuir 2017, 33, 6595−6600

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EXPERIMENTAL SECTION

number of polyglycerol branches was conducted according to our recently reported method.24 The number of polyglycerol (PG) branches conjugated to the surface of fullerene and therefore the properties of FPAs can be tuned using different ratios of t-BuO−K+ and fullerene. To form fullerenes with different numbers of carbanionic centers, the fullerene and glycidol feed ratio were kept constant, while the amount of tBuO−K+ was changed. This resulted in FPAs with different numbers of branches (FPA1, FPA2, FPA3) and therefore physicochemical properties (Figure 1). Content of fullerene in FPAs was determined using thermogravimetric analysis (Figure S3).

Materials. Fullerene (Sigma Aldrich, 98%), potassium tert-butoxide in THF (1M) (Sigma Aldrich), mesyl chloride (MsCl, Acros Organics), sodium azide (NaN3, Alfa Aesar), triphenylphosphine (PPh3, Merck Chemicals), and triethylamine (TEA, Sigma Aldrich) were used without further purification. IDCC dyes were purchased from IC Discovery company. IDCC−CH3 was synthesized according to the reported procedure in literature.24 Glycidol (SigmaAldrich) was dried over CaH2, distilled before use, and stored at 4 °C. All other reagents and solvents were purchased from different commercial suppliers and used as received. Anhydrous solvents were either obtained from MBraun MB SPS-800 solvent purification system or purchased as ultradry solvents from Acros Organics company. Water was used from Milli-Q Advantage A10 water purification system in all experiments. Phosphate-buffered saline (PBS) (10×) pH 7.4 (ThermoFisher Scientific) was diluted 10 times with Millipore quality water. Sephadex G-25 Superfine (GE Healthcare Life Sciences) was mixed with PBS solution for 3 h before preparation of the SEC columns. Benzoylated cellulose dialysis tubes (width: 32 mm, MWCO > 1000 g/mol) were purchased from Sigma-Aldrich, and they were used for purification of the synthesized compounds. Synthesis. Synthesis of Fullerene-Polyglycerol Amphiphiles. FPAs were synthesized according to the previously reported procedure.24 In brief, fullerene (0.3 g) was mixed with potassium tert-butoxide (t-BuO−K+); then, the mixture was heated to 80 °C and glycidol (5 mL) was added dropwise. After the addition of glycidol, the mixture was stirred for 6 h, then cooled to room temperature, and the product was dissolved in methanol and precipitated in acetone. The precipitate was redissolved in water and dialyzed against this solvent for 5 days. The purified compounds were lyophilized. The yield was 75−80%. Synthesis of Amino-Functionalized FPAs. FPAs were mesylated, azidated, and reduced according to the reported procedure previously for polyglycerol (Figure S1).25 Ratios of the used compounds are shown in Table S1. For each step of the reaction, the amount of the added compounds, yield, and assignments of the peaks in NMR spectra are explained in the Supporting Information. Conjugation of IDCC Dyes to FPA-Amine. FPA-amine (10 mg) was dissolved in 1 mL of PBS. Then, IDCC/(IDCCpH)-NHS dye (0.5 mg) was added to each vial and stirred overnight. The separation of unreacted dye from conjugated products was performed by SEC columns using PBS as eluent. The ratio of volume of reaction mixture to the volume of the column was 1:6. Collected products were lyophilized. Encapsulation Studies. IDCC−CH3 (0.5 mg) was dissolved in methanol; then, solvent was evaporated. A solution of FPA in PBS was added to the vials with an evenly distributed thin layer of dye and stirred at 1200 rpm for 72 h. Encapsulated dye was separated from nonencapsulated by purification through a Sephadex column using PBS as eluent. The amount of encapsulated dye was studied by a UVvis spectrophotometer in the range between 600 and 750 nm (absorption maximum (Amax) of the dyes was 650 nm) using a calibration curve. Extinction coefficients of the hydrophobic dye were 202 000 (methanol) and 242 000 (PBS). The loading capacity and loading efficacy were measured according to the mol ratio of the encapsulated dye per mol of carrier and to the mol ratio of the encapsulated dye per mol of dye, respectively. Release Profiles of the Encapsulated Dyes. FPAs (1 μM concentration) loaded with dye were dissolved in PBS, and the amount of the loaded dye was determined by UV spectra. A part of PBS solution was then removed at specified time intervals and purified with a Sephadex column to separate the released dye. Then, encapsulated dye was determined by UV, and it was subtracted from the initial loading capacity to calculate the amount of released dye.

Figure 1. Schematic representation of the structure of various FPAs. Number of branches (n) can be tuned by manipulating the amount of t-BuO−K+ used in the synthesis.

The synthesized FPAs self-assemble in the form of nanoparticles with different sizes in aqueous solution. The behavior of FPAs in the aqueous solutions was investigated by DLS. Self-assembly of FPAs depends strongly on the number of polyglycerol branches. It was found that the size of the FPAs decreased with an increasing number of PG branches conjugated to the surface of fullerene. Consistent with the DLS data (Figure S4a), SEM images showed a correlation between the size and number of branches of FPAs. The size of FPAs with the lowest number of branches was 19 nm, while those with more branches showed sizes of 8− 10 nm (Figure 2). The molecular weight of the FPAs measured by GPC decreased with an increase in the number of branches (Table 1). Variation in the resulting molecular weight of FPAs is due to the difference in their hydrodynamic radius. As the amount of t-BuO−K+ in the synthetic protocol increases, the number of branches, and therefore hydrodynamic volume in a constant fullerene/glycidol feed ratio, decreases (Table 1). A higher number of PG branches around the fullerenes hinders their interactions and prevents their self-assembly in aqueous solutions. Critical aggregation concentration (CAC) measurements also supported results obtained by DLS and GPC measurements. CAC of FPAs was measured using DLS and pendant drop methods.26,27 In the first method, a series of FPA aqueous solutions with different concentrations were prepared, and using DLS software, count rates were derived for each sample. The intersection of the approximated lines gave values of CAC for each FPA (Figure S4b). FPAs with the lower number of PG branches showed CAC in the range of 14−17 μM. CAC measurements were also performed using the pendant drop method (Figure S5). Similar to DLS measurements, FPA3 did

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RESULTS AND DISCUSSION Gram-scale and direct synthesis of FPAs is a significant advantage over existing strategies for the preparation of fullerene-based amphiphiles. Synthesis of FPAs having different 6596

DOI: 10.1021/acs.langmuir.7b00183 Langmuir 2017, 33, 6595−6600

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Figure 2. SEM images and corresponded size profiles of FPAs.

CAC appeared at lower concentrations (Figures S4b and S5). Due to steric hindrance of the shell surrounding the fullerene, the FPAs with the highest number of branches did not show CAC even at high concentrations (Figures S4b and S5). In the case of FPA1 and FPA2, fullerene is not fully covered by PG branches, and hydrophobic interactions between fullerene segments result in the formation of nanoparticles in aqueous solutions (Figure 3). Therefore, they behave as micellar systems. To investigate the interactions between cells and synthesized fullerene derivatives and to evaluate the effect of FPAs on cell viability, real-time cell analysis (RTCA) was performed. A549 cells (adenocarcinoma human alveolar epithelial cells) were treated with FPAs having different number of branches for 48 h (Figure S6). None of the synthesized compounds showed considerable toxicity, and cell viability was similar to that of PG and untreated control cells. On the basis of these results, the synthesized compounds are promising candidates for biomedical applications such as drug delivery.

Table 1. Characterization of the Synthesized FPAs Obtained by Different Analytical Methods compound

FPA1

FPA2

FPA3

fullerene content molecular weight (Mn) PDI average number of branches24 degree of branchinga average size of particlesb critical aggregation concentrationc loading capacityIDCC−CH3 (mmol/mol) loading efficacyIDCC−CH3 (mmol/mol)

15% 8400 Da 1.4 2 54% 19 14 μM 23 14.8

14% 4900 Da 1.4 3 54% 10 17 μM 15 9.9

12% 3000 Da 1.3 5 49% 8 n.d.d 14 9.5

a

For calculations, see the Supporting Information. bBased on SEM data. cBased on DLS method measurements. dNot detectable.

not show CAC, while FPA1 and FPA2 showed CAC values in the range of 6−12 μM. In both methods, CAC of FPAs depended on the number of PG branches conjugated to the surface of fullerenes. As the number of branches decreased,

Figure 3. Schematic representation of the aggregation of various FPAs in aqueous solutions. FPA1 forms aggregates with the biggest size due to the less hindered fullerene core and hydrophobic interactions. However, with an increase in number of branches (FPA2, FPA3), interactions between fullerene cores are hindered and size of the aggregates decreases. The number of PG branches is schematic and is not based on quantitative calculations. 6597

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Figure 4. Schematic representation of the synthesis of FPAs-IDCCpH. First, a few hydroxyl groups were converted to amino groups; then, pHsensitive dye was attached to the FPAs.

To study cellular uptake of FPAs, indodicarbocyanine (IDCC) dye, a Cy5 analogue operating in the visible spectral range, was covalently conjugated via an amine bond to the FPAs, forming FPAs-IDCC. Successful attachment of the dye to FPAs was proven by UV spectroscopy (Figure S7). Confocal laser scanning microscopy (CLSM) showed that FPAs-IDCC are quickly taken up by A549 cells (Figures S8 and S9). The localization of FPAs inside the cells was studied by CLSM to identify their uptake pathway and to track their fate. Compartments of A549 cells were labeled with different CellLight reagents before incubating with FPAs-IDCC (1 mg/ mL). The nucleus was also labeled with Hoechst 33342. Merged colors in the overlay images indicate colocalization of stained structures and the IDCC dye covalently bound to the FPAs. Figure S9 shows that FPAs-IDCC are not localized in mitochondria, Golgi apparatus, and endoplasmic reticulum. However, after 4 h of incubation they could be recognized inside late endosomes and lysosomes. Precise localization of the conjugates was proven by covalent conjugation of a pH-sensitive IDCC dye (IDCCpH)28 to the FPAs (Figure 4). IDCCpH dye is slightly fluorescent in cell medium and becomes highly fluorescent upon protonation in acidic compartments due to a pKa value of 7.1. The uptake experiments further proved that synthesized FPAs are localized and accumulate over time in acidic compartments (endosomes and lysosomes), therefore indicating that the compounds are taken up by an endocytotic pathway. Concentration of the FPAs-dye conjugates was calibrated using fluorescence spectroscopy to comprise a similar intensity of all conjugates. Cells were incubated with FPAs-IDCCpH for 24 h; then, confocal images were recorded (Figure 5). The yellow color, which is due to the merged red (FPAs-IDCCpH) and green (CellLight Lysosomes-GFP) colors, proves penetration of FPAs-IDCCpH inside the lysosomes. Flow cytometry also showed that the fluorescence intensity of the conjugated IDCCpH dye increases with an increase in the incubation time, proving penetration of all FPAs-IDCCpH in acidic compartments of the cells (Figure S10). These quantitative results are in full accordance with the confocal microscopy studies, showing higher cellular uptake at longer incubation times. All synthesized FPAs showed high ability to encapsulate hydrophobic dyes and therefore a high potential as nanocarriers for biomedical applications (Table 1). Hydrophobic cyanine

Figure 5. Confocal microscopy images of A549 cells stained with Hoechst 33342 (blue), labeled with CellLight Lysosomes-GFP reagent (green), and treated for 22 h hours with FPA-IDCCpH conjugates (red) (120 μM): (a) nontreated control, (b) FPA1−IDCCpH, (c) FPA2−IDCCpH, and (d) FPA3−IDCCpH. Merged yellow color implies colocalization of the FPAs−IDCCpH conjugates with the LysosomeGFP reagent.

dye (IDCC−CH3) bearing methyl side chains was used as a model drug for encapsulation by FPAs. There was a strong correlation between the loading capacity (LC), loading efficacy (LE), and number of branches of FPAs (Table 1). With an increasing number of branches, LC and LE decrease. A reason for such an observation is the hampered aggregation of fullerenes covered by PG branches. Hence, with an increasing number of branches, the interactions between fullerenes and hydrophobic dyes decrease, leading to lower LC and LE. The release of IDCC−CH3 from FPAs was also investigated. The release profiles are shown in Figure 6. The rate of release of dye from FPAs was inversely correlated to their number of PG branches. Because of the fact that FPA3 did not show CAC values by surface tension and DLS methods, release of dye from this amphiphile mostly depends on the strength of interactions between dye and fullerene core. However, FPA1 and FPA2 form aggregations and dye is encapsulated inside these aggregations. Therefore, release of dye is faster than FPA3. As a result, kinetic 6598

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Figure 6. Release profiles of encapsulated IDCC−CH3 from FPAs. Amount of released dye was calculated based on the Amax of the dye encapsulated by FPAs.

of release of encapsulated dye from FPAs could be changed by manipulation of the number of their branches. To investigate the ability of FPAs as nanocarriers in the unimolecular form, they were loaded with almost the same amount of dye and then diluted below their CAC. Subsequently, they were purified through a Sephadex column to remove the released dyes. A control experiment showed that IDCC−CH3 stayed on the Sephadex column due to its hydrophobicity, while the loaded dye with FPAs passed through the column. To perform a comparative confocal microscopy study, the amount of dye was calibrated using fluorescence spectroscopy. Loaded and free IDCC−CH3 dye was incubated with A549 cells for 4 h. The cellular uptake of FPAs with encapsulated molecules shows the ability of these nanocarriers to transfer IDCC−CH3 dye into the cells. FPAs with different number of branches transfer a similar amount of the dye inside the cells, according to fluorescence intensity of confocal images (Figure 7b−d). Because of this fact, it is proposed that at low concentrations FPAs penetrate into the cells as unimolecular transporters. Hydrophobic interactions between the fullerene surface of FPAs and IDCC−CH3 dye are the main driving force to retain the dye near the core of the amphiphiles at concentrations lower than CAC. This result proves that FPAs are able to load and transfer small guest molecules in to the cells.

Figure 7. Confocal microscopy images of A549 cells treated for 4 h with (a) IDCC−CH3 dye. (b) FPA1 (1 μM), (c) FPA2 (1 μM), and (d) FPA3 (1 μM). FPAs are loaded with the same amount of IDCC− CH3 (2 × 10−5 μM). Blue and red colors correspond to the nucleus (stained using Hoechst 33342) and IDCC−CH3 dye, respectively. High intensity of the red color clearly proves cellular uptake of the IDCC−CH3 dye carried by FPAs inside the cells without penetration into the nucleus.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.7b00183. Details of the methods, synthesis and characterization of the properties, and behavior of the materials. (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*M.A.: Tel: +49-30-838-58083. E-mail: [email protected]. *R.H.: Tel: +49-30-838-52633. E-mail: [email protected]. de. ORCID

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Ievgen S. Donskyi: 0000-0001-6181-4030 Rainer Haag: 0000-0003-3840-162X

CONCLUSIONS FPAs and their dye-labeled derivatives were successfully synthesized on the gram scale. The number of PG branches attached to the surface of fullerene dramatically affects their physicochemical properties such as size of their aggregates and their loading capacity and efficacy. Moreover, it also provides control over the release profiles of encapsulated hydrophobic dye. However, it does not dominate their biological behavior. Synthesized FPAs showed low toxicity against A549 cells up to 1.2 mM. Cellular uptake studies by confocal microscopy indicate an endocytotic uptake pathway for FPAs. Because of their ability to transfer small guest molecules through the cell membrane even at concentrations lower than their CAC and low toxicity, FPAs are promising candidates for future biomedical applications.

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank our colleagues Cathleen Schlesener for performing GPC experiments and Paul Hillmann for performing flow cytometry measurements. Also, the authors thank Guy Guday for language polishing of the manuscript. We acknowledge the Helmholtz Association for partial funding of this work through Helmholtz-Portfolio Topic “Technology and Medicine”. We acknowledge support by CRC 765 of the DFG. 6599

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DOI: 10.1021/acs.langmuir.7b00183 Langmuir 2017, 33, 6595−6600