Silicon(IV) Phthalocyanine-Decorated Cyclodextrin Vesicles as a Self

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Silicon(IV)Phthalocyanine-Decorated Cyclodextrin Vesicles as a Self-Assembled Phototherapeutic Agent against MRSA Anzhela Galstyan, Ulrike Kauscher, Desiree Block, Bart Jan Ravoo, and Cristian Alejandro Strassert ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b02132 • Publication Date (Web): 21 Apr 2016 Downloaded from http://pubs.acs.org on April 26, 2016

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ACS Applied Materials & Interfaces

Silicon(IV)Phthalocyanine-Decorated Cyclodextrin Vesicles as a Self-Assembled Phototherapeutic Agent against MRSA Anzhela Galstyan,a,b Ulrike Kauscher,c Desiree Block,d Bart Jan Ravooc* and Cristian A. Strasserta* a

Physikalisches Institut and CeNTech, Westfälische Wilhelms-Universität Münster,

Heisenbergstrasse 11, 48149 Münster (Germany). E-mail: [email protected] b

c

European Institute for Molecular Imaging, Waldeyerstrasse 15, 48149 Münster (Germany)

Organic Chemistry Institute, Westfälische Wilhelms-Universität Münster, Corrensstrasse 40,

48149 Münster (Germany). E mail: [email protected] d

Institute for Medical Microbiology, University Hospital Münster, Domagkstrasse 10, 48149

Münster (Germany) ABSTRACT The host-guest complexation of a tailored Si(IV)phthalocyanine with supramolecular β– cyclodextrin vesicles (CDV) was studied, revealing a reduced aggregation of the photoactive center upon binding to the CDV, even in aqueous environments. For this purpose, a photosensitizing unit axially decorated with one adamantyl group and one pyridinium moiety on the other side was obtained by two successive click reactions on a bis-azido-functionalized derivative of Si(IV)phthalocyanine. To evaluate its potential as a photosensitizer against antibiotic-resistant bacteria , comparative studies of the photophysical properties including 1 ACS Paragon Plus Environment


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absorption and emission spectroscopy, lifetimes as well as fluorescence and singlet oxygen quantum yields were determined for the Si(IV) phthalocyanine alone and upon self-assembly on the CDV surface. In vitro phototoxicity against the methicillin-resistant Staphylococcus aureus (MRSA) USA300 was evaluated, showing an almost complete inactivation.

KEYWORDS

Cyclodextrin vesicle - Self-assembly – Phthalocyanine – Singlet Oxygen – MRSA

INTRODUCTION The increasing occurrence of infectious diseases caused by antibiotic resistant bacteria has become a global concern to modern medicine.1 Because of the ineffectiveness of conventional antimicrobial strategies, the search for new therapeutic strategies against infections caused by multiresistant bacteria has become an important research area.2 Photodynamic inactivation (PDI) of microorganisms provides an alternative mode of action and was suggested as a promising treatment modality.3,4 PDI is based on a non-toxic light-activated dye called photosensitizer (PS), which is excited upon irradiation with visible electromagnetic radiation. Both photoinduced electron transfer from or to biomolecules as well as Dexter energy transfer to ground-state triplet oxygen ultimately generate reactive oxygen species (ROS) and concomitant cellular damage. Therefore, these highly cytotoxic species induce photoinactivation of pathogenic microorganisms.5 One major drawback regarding PDI is the nonspecific binding of the PS to both healthy and affected tissues, which results in a nonspecific phototoxic effect.6 Additionally, most of the used PS are macrocyclic molecules with extended conjugated π-systems that usually result in poor water solubility and π-stackingmediated H-aggregate formation. Such aggregation processes severely limit the production of 2 ACS Paragon Plus Environment

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ROS by radiationless deactivation pathways. To overcome both drawbacks, significant research efforts have been directed to develop biocompatible and biodegradable delivery platforms that would suppress undesired aggregation phenomena.7,8 An optimal approach would include a platform that allows the targeting and labelling of the multifunctional array, leading to a specific uptake by diseased cells and also the visualization of affected tissues or pathogens. The delivery platform should be biocompatible to avoid a nonspecific toxicity as well as an unspecific immune response, which would result in the degradation of the system.9

Scheme 1. Schematic representation of cyclodextrin vesicles (CDV) decorated with a tailored phthalocyanine photosensitizer.

A well-established nanovector is represented by functionalized vesicles.10,11 Vesicles are supramolecular assemblies of amphiphiles which self-organize into a bilayer membrane to enclose an inner aqueous compartment. In 2000, Darcy and Ravoo implemented a new class of multivalent vesicles, which consist of amphiphilic cyclodextrins (CD).12 The amphiphiles are obtained via a modification of βcyclodextrin (β-CD) with alkyl chains (C12 or C16) on the primary side and oligoethyleneglycol units on the secondary side in a straightforward 3-step synthesis. 3 ACS Paragon Plus Environment


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These amphiphiles spontaneously form cyclodextrin vesicles (CDV) in aqueous solution with bilayer membranes. CDs are a well-known class of host molecules with appropriate characteristics for drug delivery.13,14 These molecules exhibit a hydrophilic exterior and a hydrophobic inner cavity that allows the CD to complex hydrophobic molecules with moderate binding constants that depend on their size and fitting. Ravoo and coworkers showed that even the cavities of amphiphilic CDs embedded in bilayer membranes are available for host guest inclusion with hydrophobic molecules.15 Once formed in aqueous solution, the CDV are stable colloids that can be easily decorated with numerous functions by simple addition of a guest to the aqueous solution. One exemplary guest with a moderate binding constant is adamantane. Any molecule that possess an adamantane function can therefore be brought to the surface of the vesicles and immobilized by host-guest interaction.16-18 Phthalocyanines are macrocyclic molecules with a planar conjugated core possessing 18 π-electrons. Besides their unique advantages as catalysts and as optoelectronic materials, they show outstanding properties as phototherapeutic agents. They exhibit high molar absorption coefficients in the phototherapeutic window (650-750 nm), high intersystem crossing quantum yields, long excited triplet state lifetimes and high singlet oxygen quantum yields (between 30% and 100%, depending on the central metal ion and the degree of aggregation).19-22 In the near infrared region of the electromagnetic spectrum, light is less absorbed by the tissue and can therefore penetrate deeper. The chemical structure of phthalocyanines can be modified with substituents on the aromatic ring or by changing the coordinating central metal or pseudo metal. This allows for a tailored modification depending on the intended application, and particularly to enhance their solubility, biocompatibility and processability in general. Soluble Zn(II) phthalocyaninates can be obtained by introduction of adequate hydrophilic substituents23-26 but we have shown that even 4 ACS Paragon Plus Environment

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eight ammonium groups do not suffice to avoid aggregation of such planar systems in aqueous media27. Thus, incorporation into diverse amphiphiles has been explored to partially overcome this trend.28-30 In contrast, Si(IV) phthalocyanines with axial substituents exhibit particularly striking photophysical properties, including high fluorescence and singlet oxygen quantum yields (typically 50% for both) and long fluorescence lifetimes (roughly 5 ns) as a consequence of reduced aggregation.31,32 In fact, Pc4, a Si(IV) phthalocyanine derivative, is currently in clinical use for the phototherapy of diverse cancer affections. The asymmetric substitution pattern including a hydrophilic amino group on one side and the hydrophobic macrocycle on the other side favors the uptake by cellular membranes presumably in a flip-flop-like mechanism in which amphiphilicity plays a crucial role.33 We have recently also shown that Si(IV) phthalocyanines can be axially linked as monomeric species onto zeolite L,34 or adsorbed onto layered nanoclays35,36 yielding nanostructured hybrid materials able to photoinactivate both Gram negative and Gram positive pathogens, despite their antibiotic resistance and without major synthetic efforts. Despite their enhanced dispersability in water,37 such architectures are not fully biodegradable and could hardly be employed for parentheral administration of phototherapeutic drug. We have also demonstrated that Zn(II) phthalocyanines can be laterally immobilized and aggregation can be partially suppressed with the aid of CDV, even though the strong H-aggregation still prevented an optimal photosensitizing performance.38 In this article we present the tailored synthesis of an asymmetrically decorated Si(IV)phthalocyanine (7), which was employed to decorate CDV by axial immobilization (Scheme 1). This array combines the high performance of Si(IV)phthalocyanines with the multivalency of CDV as a self-assembled nanocarrier. Since the PS carries one axially bound adamantane moiety, it can dock on the CDV via host-guest inclusion. An axial pyridinium moiety provides the required solubility, and 5 ACS Paragon Plus Environment


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both axial substituents hinder undesired H-aggregation phenomena in water. The photophysical characterization revealed enhanced fluorescence and singlet oxygen quantum yields upon surface binding, as required the photoinactivation of microorganisms.

RESULTS AND DISCUSSION

SYNTHESIS

Scheme 2 shows the synthetic strategy towards the asymmetrically substituted phthalocyanine bearing both an adamantyl group for host-guest complexation and a water-soluble pyridinium moiety. The positive charge of the pyridinium ion and the bulky nature of the adamantyl group aid the suppression of aggregation. Moreover, cationic groups are known to favour the interaction of PS with bacterial membranes, whereas the amphiphilic nature facilitates the internalization across lipidic bilayers in living cells.39 It should be pointed out that only a monovalent PS (i.e., with only one axial adamantyl unit) was intended, since a planar chromophore bearing two guest moieties could lead to clustering of the vesicles.16,18 A “stepwise click” approach was implemented for the ambipolar decoration of the phthalocyanine, which is based on our previously described modular building block containing two axial azide functionalities (3).40 Briefly, the photosensitizing module possesses a Pc4-inspired hydrophobic C3-SiO-Si-O-Si-C3 central backbone that avoids hydrolysis of the axial substituents and Haggregation of the photoactive macrocycle. The PS unit was obtained by axial functionalization of Si(IV) phthalocyanine dihydroxide with (3-aminopropyl) dimethylethoxy-silane under basic conditions, and the azide function was inserted via amide formation with azidoacetic acid. In analogy to a previously reported alkenesubstituted polyethyleneglycol,41 we now used an alkyne-functionalized spacer for the 6 ACS Paragon Plus Environment

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adamantyl group, in view of its biocompatibility and hydrophilicity. Additionally, a positively charged custom-made pyridinium moiety was introduced to enhance the water solubility. Thus, 1-(((3s,5s,7s)-adamantan-1-yl)oxy)-3,6,9,12-tetraoxapentadec14-yne (2) was prepared by reaction of corresponding alcohol with propargyl bromide, and 1-(methyl)-4-(prop-2-ynyloxy) pyridinium iodide (6) was obtained by methylation of 4-(prop-2-ynyloxy)pyridine using an excess of methyl iodide.

Scheme 2. Synthetic pathway towards asymmetric functionalized Si(IV) phthalocyanine 7. (I) tBuOK, propargylbromide, THF, 18h, (II) CuSO4 5H2O, sodium ascorbate, CH2Cl2/H2O 1:1, rt, 18h, (III) K2CO3, propargylbromide, rt → 50°C, 2h, (IV) MeI, rt, 45°C, (V) CuSO4 5H2O, sodium ascorbate, CH2Cl2/H2O 1:1, rt, 18h. 7 ACS Paragon Plus Environment


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The intermediate product 4 was obtained in excellent yield via a copper-catalyzed Huisgen-cycloaddition in a two phase system (water and chloroform) using 2 and an excess of 3. A second reaction was then performed to introduce the pyridinium moiety. Column chromatography on neutral alumina was employed to obtain the pure product 7, which is soluble in organic solvents such as dichloromethane and was fully characterized by UV-Vis, NMR and MALDI-TOF-MS analysis (see ESI). The spectral features are in good agreement with the predicted structure shown in Scheme 2. All syntheses proceeded in good yields, and a detailed description of the experimental procedure can be found in the supporting information. Amphiphilic β-CD with C12 alkyl substituents was synthesized as described.15 Vesicles were prepared from amphiphilic β-CD via hydration of a thin film cast by rotary evaporation, as previously described.12,15,16,17 The aqueous dispersion was extruded through a polycarbonate membrane with a pore size of 100 nm to form unilamellar CDV. The formation was confirmed via dynamic light scattering (DLS) and the size distribution of the CDV can be found in the supporting information.

PHOTOPHYSICAL CHARACTERIZATION The photophysical behaviour of compound 7 was studied in dichloromethane and in water (pre-dissolving the sample in dimethylsulfoxide, which never exceeded 1% of the total volume of aqueous solution and can therefore be neglected). The data are summarized in Table 1 along with Figure 1 and Figures S2-S6 of the ESI.

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Table 1. Photophysical parameters of 7 in DCM and in double distilled H2O, and of 7+CDV in double distilled H2O. “sh” denotes a shoulder.

7+CDV[a]

7 in DCM

H2 O

352 (52),

λabs/nm -3

357 (46),

608,

616 (27),

642,

698 (57)

671

604 (24), -1

-1

(ε* 10 / [M cm ])

639 (22), 669 (158)

675,

λem /nm

672

680 709sh

[a]

ΦF (±0.02)

0.15[b]

0.01[b]

0.12[b]

Φ∆ (±0.02)

0.47[c]

0.17[d]

0.21[d]

τ / ns (±0.05)

5.15

4.44

5.03

[CDV] = 0.2M, [7] = 5 µM. [b] Quantum yield was measured in an integrating sphere

system. [c] Quantum yield was measured using the relative method. [d] Quantum yield was measured using photochemical monitor bleaching rates (for details see supporting information).

In dichloromethane, 7 showed a clear monomeric behavior as evidenced by a single Q band at 669 nm together with two vibrational bands at 639 and 604 nm, and a B-band at 352 nm. The spectra of 7 in aqueous media (Figure 1a) show a band that is redshifted by nearly 30 nm, which can be attributed to the formation of J-aggregates on the basis of the exciton theory of Kasha et al.,42 and points towards the formation of 9 ACS Paragon Plus Environment


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self-assembled clusters with slipped co-facial architecture. In contrast, incorporation of 7 (5 µM) into water with increasing amounts of CDV (from 0.02 mM to 0.2 mM)

causes the progressive reduction of the absorption shoulder at 695 nm and a sharpening of the absorption signal at 672 nm (Figure 1b), which is clearly indicative of the suppression of aggregation upon host-guest interaction with the CDV.

Figure 1. (a) Normalized absorption spectra of 7 in dichloromethane and water; (b) normalized absorption spectra of 7 immobilized on the surface of CDV ([β-CD] = 0.0 - 0.2 mM). (c) Emission spectra of 7 immobilized on the surface of CDV ([β-CD] = 0.0 - 0.2 mM; [7] = 5 µM) and Langmuir regression of the fluorescence titration (d).


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Since the vesicles consist of a bilayer of β-CD with only about half the molecules pointing to the outer surface, approximately half of the cavities are available for the host-guest complexation. For the binding studies, we therefore spanned a host (amphiphilic β-CD) concentration ranging from a twofold of the guest (7) concentration up to a 20-fold excess. The stability of the vesicles in the presence of the photosensitizer was verified by DLS, showing an average particle size of around 100 nm, even after 1 day of incubation, thus confirming the stability of the Si(IV)phthalocyanine-decorated CDV (Figure S1, ESI). We measured fluorescence spectra of increasingly concentrated solutions of CDV (0.02M to 0.2M) at constant concentration of 7. As can be seen from Figure 1c, we did not obtain any significant fluorescence signal from the aqueous solution of 7, and the absolute fluorescence quantum yield determined in an integrating sphere system was found to be 0.01. This

ΦF reduction in comparison to the monomeric species (0.15, in dichloromethane) can be attributed to the formation of J-type aggregates. Notably, a gradual increase of the fluorescence signal at 675 nm for increasing concentrations of CDV was observed, reaching a maximum of 0.12 at a CDV concentration of 0.2M (Figure S2, ESI). This behavior can be explained by the deaggregation of 7 upon binding of the adamantane moiety to the cyclodextrin cavities. The immobilization of Si(IV)phthalocyanine at the surface of the vesicles prevents the aggregation and the associated radiationless deactivation at dark energy traps.43 The occurrence of J-type aggregates was also suggested by fluorescence lifetime measurements of 7. In comparison to the monomeric species, it was shortened from 5.15 ns (in dichloromethane) to 4.44 ns (in water). In contrast, the τF of 7+CDV (5.03 ns) is very close to the monomeric species, indicating suppression of aggregation of 7 upon binding to CDV.



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Based on the fluorescence intensity, it was also possible to estimate a binding constant. To this end, the emission intensity of 7 at 675 nm was plotted against the concentration of β-CD at the CDV surface available for the complexation. Figure 1d shows the resulting Langmuir regression curve, which gives a binding constant of 33x103 M-1.44 This value is consistent with host-guest inclusion of CD and adamantane, possibly enhanced by secondary interactions of the positively charged pyridinium group at the negatively charged CDV surface15 as well as hydrophobic interaction of 7 with the CD bilayer membrane.

Figure 2. Emission spectra of ADMADM at different irradiation times (0 s, 60 s and 100 s) in double distilled water with: A) MB; B) 7 and C) 7+CDV. D) Decay of ADMADM for MB (black line), for 7 (red line) and 7+CDV (blue line).


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The singlet oxygen quantum yield (Φ∆) of 7 was determined by comparison of the singlet oxygen phosphorescence intensity with a reference in dichloromethane, and by the rate of photochemical bleaching of 9,10-anthracenediyl-bis(methylene)dimalonic acid ADMADM) in water (Figure 2). The Φ∆ in pure water (where the J-aggregates are predominant) was 0.17, compared to 0.47 for the monomeric species in dichloromethane. This observation indicates that these aggregated species are still useful as photosensitizers, as opposed to their counterparts, namely the rather inactive H-aggregates. When 7 is immobilized at CDV, it can also induce photodegradation of ADMADM with comparable efficiencies (Φ∆ = 0.21), which indicates that the suppressed aggregation upon binding also leads to a recovery of the Φ∆ in water. A significant photobleaching of the PS itself was not observed, both in the presence and in the absence of the CDV.

PHOTOBIOLOGICAL EVALUATION

10

10

light control dark control CDV + light CDV - light 7 + light 7 - light CDV + 7 + light CDV + 7 - light

9

10

8

10

7

10 CFU /ml

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6

10

5

10

4

10

3

10

2

10

1

10

0

1

2

3

Time [h]

Figure 3. Bacterial survival expressed as log CFUmL-1.



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A methicillin-resistant Staphylococcus aureus strain (MRSA, USA300) was selected for the studies. This strain was firstly reported in the USA and has spread to Europe, South America and Australia in the last decade. USA300 is known to cause skin and soft tissue infections, even though isolates have also been recovered from cases of invasive diseases such as bacteremia, endocarditis, severe necrotizing pneumonia and osteomyelitis.45 The samples were prepared by incubation of 7 or 7+CDV with a bacterial suspension (optical density OD600 = 0.5) for 1h at 37°C. Irradiation proceeded with polychromatic light of a projector lamp passing through a 610 nm cut-off filter for up to 3h (10 mW/cm2; total radiant exposure 108 J/cm2). Aliquots of samples and controls were taken in 60 min intervals and colony forming units (CFU) were determined by serial dilutions, plating on agar plates and counting afterwards. As can be observed in Figure 3, both 7 alone and 7+CDV are non-toxic in the absence of light, but they become cytotoxic upon irradiation. The results demonstrate that in fact both induce significant bacterial inactivation (7 log10 units) measured as the number of CFU per mL. Interestingly, the antimicrobial activity was found to be dependent on the light dosis, but not significantly affected by the binding of the PS to the CDV, which therefore does not restrict the uptake of the PS by the bacterial surface. Indeed, already after a 1h exposure (36 J/cm2), the inactivation of S. aureus USA300 with 7+CDV was comparable to 7 alone. The results also clearly indicate that an effective monomerization of 7 occurs upon binding to the bacteria, just as measured upon binding to CDV. The fact that the complete bacterial inactivation is observed for both the free (yet aggregated in water) and the bound (yet monomeric at the CDV) phthalocyaninate suggests that suppression of the aggregation of 7 compensates its binding to the CDV when it comes to the delivery of the drug to the microorganism. Future studies will elucidate the role of CDV Gram negative bacteria and eukaryotic cells with active uptake mechanisms, particularly in vivo. 14 ACS Paragon Plus Environment

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CONCLUSIONS

In summary, we have prepared and characterized a novel asymmetric Si(IV)phthalocyanine with amphiphilic properties. The formation of J-aggregates in aqueous environments significantly diminishes the fluorescence and singlet oxygen quantum yields. The axial adamantyl moiety enables the immobilization on βcyclodextrin vesicles as multivalent biocompatible nanocarriers, a process that is driven by host-guest interactions. The efficient singlet oxygen photoproduction of the new asymmetrically decorated, amphiphilic phthalocyaninate makes it an interesting photosensitizer for the therapy of cancer and infectious diseases, despite the formation of J-aggregates in pure water. However, a significant enhancement of the fluorescence and singlet oxygen quantum yields can be observed upon complexation with CDV, indicating monomerization of the photoactive center. The photobiological evaluation against Staphylococcus aureus USA300 (MRSA) demonstrates that almost complete inactivation can be reached. The multivalent nature of the CDV provides the opportunity for further decoration with antibiotic or chemotherapeutic drugs, with luminescent tags, with NMR-active or radioactive markers, as well as with tailored targeting units to yield a multimodal theranostic nanoarray against bacterial and fungal infections or neoplastic affections. Future studies will elucidate the role of CDV for the the in vivo delivery of our new photosensitizer against Gram negative bacteria, fungal and eukaryotic cells with active uptake mechanisms such as pino- and phagocytosis. Moreover, the realization of solution-processable light-harvesting units in aqueous photocatalysis as well as in water-based inks and dyes can be envisaged also for optoelectronic applications.



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Supporting information for this article, including the synthesis, characterization of all compounds, the photophysical characterization of all photoactive compounds is available.

ACKNOWLEDGEMENTS

Financial support from by the DFG EXC 1003 Cells in Motion–Cluster of Excellence, Münster, Germany is gratefully acknowledged.

NOTES AND REFERENCES

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Silicon(IV) Phthalocyanine-Decorated Cyclodextrin Vesicles as a Self

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