Los Puentes: Descripción de la comunidad y la escuela rural

To overcome the endosomal membrane barrier separating CMS nanoparticles from the cytosol and to gain mechanistic insight into the release at a single-cell level in real-time, photoinduced endosomal release is a promising strategy. In this method, the photosensitizer TPPS2a is incubated with the

cells and incorporates into membranes via the endocytic pathway. By 405 nm laser light, TPPS2a

is excited to its singlet state, followed by intersystem crossing to its triplet state. This excited state is then quenched by triplet oxygen producing singlet oxygen. Singlet oxygen is highly reactive and leads to a collapse of the endosomal membrane followed by release of the endosomal content into the cytosol.

To ensure that 405 nm light-activated TPPS2ain solution has no influence on the fluorescence inten-

sity of CMS-CysATTO633core-NH2shell nanoparticles, control measurements have been performed

in vitroon glass. For this, redox-cleavable CMS-CysATTO633_core-NH2shellnanoparticles in TPPS2a

solution have been sedimented on glass coverslips on the microscope. After activation of TPPS2aby

405 nm laser light for 1 min, a movie was recorded under 642 nm illumination. The CysATTO633 flu- orescence intensities of single particles and background have been extracted, normalized and plotted versus time (see Figure 8.5). The fluorescence intensities of both the background (Figure 8.5 gray curve) and the particles (black curves) remain constant. This indicates that activated TPPS2a in

solution has no influence on the fluorescence intensity of CMS-CysATTO633core-NH2shell particles,

which is a prerequisite for further experiments in living cells.

To evaluate photochemically induced endosomal escape of CMS nanoparticles in living cells, a marker for endosomal opening was needed. For this purpose, we incubated the cells with the fluid phase marker Alexa Fluor 488 dextran (AFD). AFD is internalized by fluid-phase endocytosis

8.4 Photochemically-induced endosomal release

Figure 8.6: Photoinduced endosomal release of CMS-CysATTO633_core-NH2shell and fluid

phase marker AFD inside living HuH7 cells monitored by wide-field fluorescence microscopy. The cells were exposed to the nanoparticles overnight. (a) Intensity plot of three exemplary tracked endo- somes (highlighted by small circles in b and c) over time. The fluorescence intensity of CysATTO633 (upper three curves) showed a sudden increase concomitant to the decrease in AFD fluorescence intensity (corre- sponding lower three curves) due to endosomal rupture and AFD dye release. (b) Fluorescence microscopy image overlays of the CysATTO633 (magenta) and fluid phase marker AFD (green) signal at activation of the photosensitizer and (c) 4 min later. The cell nucleus is indicated with the large white circle. Scale bar: 10 µm.

and is used as a label for endosomes [45]. Along with AFD, we incubated the cells with CMS- CysATTO633core-NH2shell nanoparticles and the photosensitizer. After 18-24 h of incubation, the

cells were examined by wide-field fluorescence microscopy.

As a result, we found all CMS nanoparticles that exhibit typical intracellular motion to be co- localized with AFD. This indicates successful internalization of the CMS nanoparticles into en- dosomes. The AFD fluorescence intensity of the endosomes varied depending on the amount of internalized AFD. Excitation of the photosensitizer was achieved by illumination of the sample with 405 nm laser for 1 min. This resulted in deceleration of endosomal motion until arrest, as reported previously [30]. Depending on the amount of photosensitizer incorporated in the endosomal mem- brane, the endosomes were ruptured within 1-4 min after 405 nm illumination. Endosomal rupture lead to a spontaneous release of AFD into the cytoplasm as indicated by a sudden drop in endosomal AFD fluorescence.

Concomitant with endosomal rupture and AFD release, the fluorescence intensity of the CMS- CysATTO633core-NH2shellnanoparticles increased due to the dequenching effect, as described above

and presented in Figure 8.3a. Directly after photosensitizer activation the endosomes still show predominantly AFD fluorescence (see Figure 8.6b depicted in green). Strikingly, only 4 min later the same endosomes show only CysATTO633 fluorescence (Figure 8.6c depicted in magenta). Please note that the diffuse magenta fluorescence within the nucleus area (big, white circle) is due to out of focus particle fluorescence. With a size of 10 kDa, AFD can diffuse almost freely after endosomal release, and is dispersed within the cytosol [252]. In contrast, due to their large size and impaired

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 2 4 6 8 10

relative fluorescence intensity [a.u.]

time [min]

a b c

Figure 8.7: Photoinduced endosomal release of CMS-ATTO633core-NH2shell and fluid phase

marker AFD inside living HuH7 cells monitored by wide-field fluorescence microscopy. (a) Intensity plot of three exemplary tracked endosomes (highlighted by small circles in b and c) over time. The fluorescence intensity of ATTO633 (upper three curves) showed no or only slight increase concomitant to the decrease in AFD fluorescence intensity (corresponding lower three curves) due to endosomal rupture and AFD dye release. (b) Fluorescence microscopy image overlays of the ATTO633 (magenta) and fluid phase marker AFD (green) signal at activation of the photosensitizer and (c) 4 min later. The cell nucleus is indicated with the large white circle. Scale bar: 10 µm.

motion in the crowded cell interior, the nanoparticles remain at their location.

As a control measurement, noncleavable CMS-ATTO633core-NH2shell nanoparticles without cysteine

linker were incubated with AFD and the photosensitizer for 12-24 hours. The internalized nanopar- ticles showed co-localization with AFD until endosomal rupture. At endosomal rupture, the relative fluorescence intensity of AFD showed a sudden drop, whereas the ATTO633 fluorescence remained largely constant with a slight intensity increase by a factor of 1.25 (Figure 8.7a). This increase might be due to a small amount of unreacted dye inside the mesopores, which is released from the endosome after disruption of the endosomal membrane. The disruption of the endosomal mem- brane and release of AFD occurred within 4 min, and the CMS-ATTO633core-NH2shell nanoparticle

fluorescence remained at the former endosomal regions (Figure 8.7c).

8.5 Summary

In this chapter, we presented mechanistic insights into disulfide-based drug delivery from CMS nanoparticles. After particle synthesis,in vitrosingle-particle measurements of the nanoparticles on

cover glass showed that disulfide-bound CysATTO633 is released from CMS nanoparticles in 10 mM GSH. Evaluation of the particle’s fluorescence intensity revealed that the dye release is accompanied by a dequenching of the ATTO633 fluorescence. We therefore show, for the first time, that linkage of ATTO633 at high concentration in the pores of silica nanoparticles results in quenching of the ATTO633 fluorescence. This unexpected effect permits a sensitive readout for successful dye release. Although the particles were shown to be functional and to release their cargo upon addition of a

8.5 Summary

reducing agent, disulfide-based CysATTO633 release into HuH7 cells was not observed even after up to 49 h incubation of CMS-CysATTO633core-ATTO488shell nanoparticles with the cells. Inefficient

endosomal escape of the nanoparticles is a bottleneck for molecular delivery into the cytoplasm. To overcome the endosomal barrier, we used the photosensitizer TPPS2a. Upon activation of

the photosensitizer, CMS-CysATTO633corenanoparticles successfully released disulfide-bound Cys-

ATTO633 into the cytoplasm, however the resolution of our measurement setup does not allow conclusions on the locus of reductive cleavage (endosome or cytoplasm). Our measurements confirm that endosomal escape is the main bottleneck for disulfide-based drug delivery. In case of noncleav- able CMS-ATTO633corenanoparticles without cysteine linker, release of ATTO633 was not observed

after endosomal rupture.

The proposed drug delivery system allows us to control the time of the endosomal rupture by use of the 405 nm laser. However, TPPS2ashows a high toxicity to the cells, as highly reactive oxygen

species are produced where the photosensitizer is present. For further studies, we want to achieve spatial control on the endosomal escape by covalently particle-bound photosensitizer. Using this particle, we plan to limit singlet oxygen production and endosomal rupture only to the endosomes containing photosensitizer-equipped nanoparticles (see Chapter 9).

9 Cascaded photoinduced drug delivery from

mutifunctional PpIX-mesoporous silica

In the previous chapter, we reported on an approach using reductive cleavage of disulfide-bridged compounds bound to the pore walls of mesoporous silica nanoparticles. We showed that endosomal escape represents a bottleneck for this approach. Photochemical internalization (PCI) of a pho- tosensitizer (PS) that induces endosomal escape by creating singlet oxygen has been proven to be powerful to overcome this barrier (see Chapter 8). The PS, the porphyrin derivative TPPS2a, is

incubated with the cells. It inserts into the plasma membrane and, after endocytosis, locates in the membrane of all endocytic vesicles. Therefore, all PS containing vesicles are subject to photoactiva- tion upon irradiation and the spatial control on endosomal escape and the induced phototoxicity is limited. To obtain spatial control on the photoactivity, association of the PS with the nanoparticles is desirable.

In current literature, different groups have applied PS associated with mesoporous silica for other purposes than PCI, such as photodynamic therapy or two-photon imaging [166, 253–255]. In addi- tion, there are initial approaches to combine PS with mesoporous silica as drug carrier. However, in these studies, the PS is not or not completely bound to the particles [256, 257]. This can lead to uncontrolled leaching of the compounds and highly toxic effects on the cells [30]. To achieve a more precise, “surgical” function of PS-equipped nanodevices and to minimize their toxicity on cells, it is desirable to bind the PS directly to the surface of the mesoporous particles.

Therefore, we report how multifunctional core-shell colloidal mesoporous silica nanoparticles com- bined with covalently surface-linked protoporphyrin IX (PpIX) as an on-board photosensitizer show localized photoactivity along with controlled drug release. PpIX is a photosensitizer naturally oc- curring in many cell types. The iron(II) complex of PpIX, called heme, is needed for binding to different proteins to form hemoproteins such as hemoglobin [258]. For photochemical internaliza- tion, PpIX in solution is not useful due to its low water solubility (around 1 µg/mL) [259]. The

low solubility can be overcome by delivery of PpIX into cells by a nanoparticle drug carrier. Once inside the cell, PpIX can be effectively excited in its Soret band at around 410 nm. This excitation initiates a cascade in which energy is transferred to molecular oxygen that oxidizes amino acids, unsaturated fatty acids, cholesterol and various cellular compartments enabling endosomal escape [80].

In this chapter, the synthesis of the CMS with covalently surface-linked PpIX and the applied particle designs will be presented briefly. Afterwards live-cell imaging studies of CMS-PpIX for disulfide-based drug delivery similar to the studies presented in Chapter 8 will be shown. To improve the readout signal for successful drug delivery, we exchanged disulfide-based drug loading

a

b

Figure 9.1: PpIX attachment to CMS nanoparticles. Attachment of amino-terminated PpIX (PpIX- NH2) (a) to the PEGylated surface of CMS, leading to the sample CMS-PpIXshell(b).

towards adsorption of “model drugs”. To seal the pores after drug loading, the particles were further equipped with a SLB. The activity of these SLB-coated CMS-PpIX nanodevices for photochemical release was examined with regard to the release efficiency by wide-field microscopy on a single cell level. In addition, successful drug release of “model drugs” of varying size was shown by spinning disk confocal microscopy. The results give insight into the characteristics of this newly developed drug delivery system. The project was performed in collaboration with Dr. Axel Schlossbauer, Dr. Valentina Cauda and Alexandra Schmidt from the group of Prof. Thomas Bein; Dr. Hanna Engelke from the group of Prof. Joachim Rädler and with Dr. Kourosh Zolghadr and Dr. Ulrich Rothbauer from the group of Prof. Heinrich Leonhardt (all LMU Munich). Main parts of this chapter are derived from a manuscript submitted for publication [14].

9.1 Synthesis of CMS-NH

-PpIX

CMS nanoparticles with covalently surface-linked PpIX have been developed by Dr. Axel Schloss- bauer and have been synthesized for our experiments by him, Dr. Valentina Cauda and Alexandra Schmidt from the group of Prof. Thomas Bein (LMU Munich).

In brief, the recently developed sequential co-condensation process [9, 260] was used to generate core- shell bifunctionalized colloidal mesoporous silica, exhibiting aminopropyl-groups on the inner pore walls and 3-mercaptopropyl on the particles’ outer surface (sample CMS-NH2core-SHshell). For the

synthesis, a mixture of tetraethylorthosilicate (TEOS), 3-aminopropyl triethoxysilane (APTES), phenyltriethoxysilane (PhTES), cetyltrimethylammonium chloride (CTAC) and triethanolamine (TEA) was stirred for hydrolysis of the silanes. PhTES was used as an additive to improve the struc- tured co-condensation with APTES in the synthesis. 30 minutes after seed generation, a thin shell of silica was generated on the surface by adding four equal portions of TEOS to the synthesis (each with 2.5% of the total Si content of the synthesis). The outer shell of thiols was grown after an additional aging time of 30 minutes. To this end, a mixture of TEOS and 3-mercaptopropyltriethoxysilane