Nanotechnology and Light against Pathogens

Targeting, Labeling and Photoinactivate Antibiotic Resistant Bacteria

  • Fig. 1: Pictorial view of the multifunctional nanomaterial used to target, label and photoinactivate antibiotic resistant bacteria. The zeolite L nanocrystal is loaded with the DXP emitter (green ellipsoid), and its surface is functionalized with a phthalocyanine derivative (red ellipsoid), as well as with amino groups (blue circles). On the right side, a schematic view showing the connection of the functional groups to the zeolite L framework is depicted.Fig. 1: Pictorial view of the multifunctional nanomaterial used to target, label and photoinactivate antibiotic resistant bacteria. The zeolite L nanocrystal is loaded with the DXP emitter (green ellipsoid), and its surface is functionalized with a phthalocyanine derivative (red ellipsoid), as well as with amino groups (blue circles). On the right side, a schematic view showing the connection of the functional groups to the zeolite L framework is depicted.
  • Fig. 1: Pictorial view of the multifunctional nanomaterial used to target, label and photoinactivate antibiotic resistant bacteria. The zeolite L nanocrystal is loaded with the DXP emitter (green ellipsoid), and its surface is functionalized with a phthalocyanine derivative (red ellipsoid), as well as with amino groups (blue circles). On the right side, a schematic view showing the connection of the functional groups to the zeolite L framework is depicted.
  • Fig. 2: Spectra and fluorescence microscopy images of the hybrid nanomaterial. (a) Excitation (dotted line, λem = 620 nm) and emission (solid line, λexc = 480 nm) spectra of DXP in a suspension of the nanomaterial in CH2Cl2. (b) Excitation (dotted line, λem = 750 nm) and emission (solid line, λexc  = 630 nm) spectra of PC in a suspension of the nanomaterial in CH2Cl2. (c) Excitation (dotted line, λem = 1275 nm) and emission (solid line, λexc  = 690 nm) spectra of PC and 1O2, respectively, in a suspension of the nanomaterial in CD2Cl2. Fluorescence microscopy pictures were acquired by exciting at 420 nm – 490 nm (a) or at 575 nm – 630 nm (b).
  • Fig. 3: Interaction of the hybrid nanomaterials with E. coli cells. (a) Bright field microscopy, (b) fluorescence microscopy (λexc = 470 nm – 490 nm) and (c) SEM images of bacteria treated with the amino-functionalized nanomaterial. Inset in (c) shows a magnification of a portion of the bacterium showing the adhesion of the nanomaterial to the cell surface. (d) Bright field microscopy, (e) fluorescence microscopy (λexc = 470 nm – 490 nm) and (f) SEM images of bacteria treated with the nanomaterial without amino functionalization.
  • Fig. 4: Time-lapse fluorescence microscopy view and percentage of inactivated cells during the photodynamic treatment in PBS. (a) Images of the chloramphenicol-resistant E. coli cells recorded with excitation in the range 470 nm – 490 nm. (b) Percentage of inactivated E. coli and (c) N. gonorrhoeae cells. Green bars correspond to irradiated hybrid nanomaterial (with DXP, PC and amino groups, Sample 1), red bars to the dark control (with DXP, PC and amino groups, Sample 2). Gray and black bars show control experiments with the nanomaterial without PC functionalization, with and without irradiation (with DXP and amino groups, Samples 3 and 4, respectively).

We designed a multifunctional nanomaterial, able to target, label and photoinactivate antibiotic resistant bacteria. Highly luminescent dyes were inserted into the channels of zeolite L nanocrystals for imaging and labeling purposes. The outer surface was functionalized with a photosensitizer that forms singlet oxygen upon illumination, and finally coated with targeting moieties. The nanomaterial shows intense fluorescence, efficient 1O2 photoproduction, and consequently targets, labels and photoinactivates antibiotic resistant bacteria.

In the fight against cancers and infections deseases, phototherapeutic agents constitute a powerful armory [1-3], and recent advances in nanotechnology made multifunctional arrays with targeted cytotoxicity and labeling capabilities possible [4-6]. Materials employed must not only be robust, but also well characterized and producible at industrial scale. In photodynamic therapy (PDT), a photosensitizer is used in order to generate cytotoxic 1O2 upon irradiation with light. A single structure possessing targeted 1O2 photoproduction and imaging capacities can be regarded as the ultimate goal [1-3].

Zeolite L is an aluminosilicate possessing channels able to host a variety of dyes [7], constituting a non-toxic nanocarrier that can be functionalized on its surface and orthogonally modified on the channel entrances [8]. Phthalocyanines are excellent for the development of phototherapeutic agents due to their low toxicity, high stability, efficient 1O2 generation and intense light absorption in the therapeutic window [1-2, 9].

We started the funcionalisation of zeolite L nanocrystals, 50 nm both in length and diameter, by loading with DXP, (N,N'-bis(2,6-dimethylphenyl)perylene-3,4,9,10-tetracarboxylic diimide). The dye-loaded nanocrystals were further functionalized with tetra-tert-butyl Si(IV) phthalocyanine dihydroxide (PC) by axial attachment to the zeolite L surfaces (fig. 1). Finally, the nanomaterial was coated with amino groups to promote the adhesion to bacteria [10].

The photophysical properties are shown in figure 2. It represents microscope fluorescence pictures of the hybrid system, as well as excitation and emission spectra.

Figure 2a depicts the characteristic emission and excitation spectra of DXP. Figure 2b shows the distinctive excitation and emission spectra of PC. The phosphorescence of 1O2 with its characteristic maximunm at 1275 nm can be seen in figure 2c.

In in vitro experiments, the nanomaterial labeled the E. coli cells resistant against chloramphenicol, as can be seen on the microscopy images (fig. 3a and 3b). In order to confirm the adhesion, we recorded SEM pictures (fig. 3c and inset): the amino groups provide the effective anchorage to the bacterial surface due to their hydrogen and electrostatic bonding ability. When using zeolites without amino groups, no cell adhesion could be seen (fig. 3d-3f).

The photodynamic treatment was carried out by suspending E. coli cells with the hybrid nanomaterial, and irradiating the sample for 2.5 hrs between 570 nm and 900 nm (irradiance of 3 mW cm-2). Samples were stained with propidium iodide (PI): the living cells exhibit green emission from DXP, and the inactivated ones show the red fluorescence from the PI (fig. 4). The photodynamic inactivation was complete after 2 hrs, corresponding to a light dose of 27 J cm-2. Similar results were obtained with a pathogenic strain of tetracycline resistant N. gonorrhoeae (fig. 4c).

In conclusion, we developed an innovative potential phototherapeutic tool by using a multifunctional nanoarchitecture constructed on the zeolite L platform, easy to synthesize by combining industry standard chromophores and a well-defined solid substrate. We proved that the hybrid nanomaterial efficiently produces singlet oxygen and adheres to bacterial surfaces, leading to targeting, labeling and photoinactivation capabilities against antibiotic resistant bacteria. These results open fascinating possibilities for the photodynamic treatment of infectious and neoplastic diseases, shining new light onto the design of third generation photosensitizers for PDT [11].

References
[1] Dougherty T.J. et al.: J. Natl. Cancer Inst. 90, 889-905 (1998)
[2] Juzeniene A.et al.: Photochem. Photobiol. Sci. 6, 1234-1245 (2007)
[3] Hamblin M.R. and Hasan T.: Photochem. Photobiol. Sci. 3, 436-450 (2004)
[4] Choi M.-R. et al.: Nano Lett. 7, 3759-3765 (2007)
[5] Ferrari M.: Nature Rev. Cancer 5, 161-171 (2005)
[6] Whitesides G.M.: Small 1, 172-179 (2005)
[7] Calzaferri G. et al.: Angew. Chem. Int. Ed. 42, 3732-3758 (2003)
[8] Busby M. et al.: Adv. Mat. 20, 1614-1618 (2008)
[9] Strassert C.A. et al.: Photochem. Photobiol. Sci. 7, 738-747 (2008)
[10] Popovic Z. et al.: Angew. Chem. 119, 6301-6304 (2007); Angew. Chem. Int. Ed 42, 3732-3758 (2003)
[11] Strassert C. A. et al.: Angew. Chem. Int. Ed. 48, 7928-7931 (2009)

Figures 1 to 4:
Strassert CA, Otter M, Albuquerque RQ, Höne A, Vida Y, Maier B, De Cola L. Photoactive hybrid nanomaterial for targeting, labeling, and killing antibiotic-resistant bacteria. Angew. Chem. Int. Ed. 2009, 48 (42), 7928-7931. Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission.

 

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