Figure 3.2: Site-selective delayed co-condensation approach for creating bifunctional MSNs. In a first step, a mixture of organosilane (green) and tetraethylorthosilicate (TEOS) in an aqueous solution containing template and base catalyst creates a functionalized nanoparticle core. Subsequently, the nanoparticle growth is completed by addition of pure TEOS (blue) resulting in an unfunctionalized silica shell around the core.
Finally, the addition of another organotriethoxysilane (RTES, R represents an organic moiety, red) and TEOS forms an external skin with different functionality.
reagents. [189, 218–220] In order to gain control over the location of the functional groups in silica nanoparticles, Bein and co-workers established a site-selective delayed co-condensation ap-proach. [37, 221] Here, bifunctional MSNs with a selective functionalization of the interior and an orthogonal functionality at the external particle surface in different onion-like shells can thus be prepared (Figure 3.2).This strategy opens new possibilities for the design of numerous highly functionalized porous nanoparticles with applications in controlled drug delivery.
3.2 Modification of silica cores
Surface modification with organic and inorganic species can introduce a large variety of function-alities for controlling diffusion and release of cargo molecules and cell surface recognition, among others. The potential to design biocompatible external surfaces of nanoparticles providing tunable interactions with the biological environment by attachment of molecular or macromolecular moi-eties for biomedical applications has been recently demonstrated. [40, 41, 86] The combination of the properties of such an external functional shell and the advantageous structural properties of the mesoporous silica core can create multifunctional drug carriers, making the delivery process highly controllable.
GatingOne of the important functionalities in this context is triggered release of the cargo through specially designed gating concepts. In general, gatekeepers can be classified into three different types, namely molecular/particle pore gating, surface coating, and internal pore modifications (cf. Figure 3.3). Pore gating systems can consist of either bulky molecular groups or nanoparti-cles, such as proteins, superparamagnetic iron oxide nanoparticles (SPIONs), or gold nanoparticles (Au-NPs) which block the pore entrances for efficient sealing of the interior mesoporous environ-ment. [40, 111, 166] These macromolecular structures are either degradable or attached to the silica particle surface via linkers that are cleavable upon exposure to certain stimuli. [125, 156] Very good pore sealing can also be achieved by a complete coating of the MSNs. For instance, poly-mers, oligonucleotides, or supported lipid bilayers (SLB) have been shown to prevent premature
3 Multifuncional mesoporous silica nanoparticles
Figure 3.3: Strategies for controlled release can be classified into three different types, molecular/particle pore gating (a,b), surface coating (c,d), and internal pore modification (e,f). (a) Mesoporous silica nanorods capped with superparamagnetic iron oxide nanoparticles (SPIONs) containing redox-responsive cleavable disulfide linkers; [111] (b) temperature-dependent programmable molecular valve system consisting of avidin caps being opened by melting the DNA linkers; [114] (c) temperature-dependent phase transition of PNIPAM-coating on MSNs; [118] (d) disulfide-linked polymeric network at the outlet of mesoporous silica allowing redox-responsive controlled release of the cargo [160] (e) schematic release mechanism for a pH-responsive system based on coordination bonding in mesopores; [144] and (f) light-activated cis/trans isomerization of azobenzene groups inside mesopores expels the cargo. [222]
cargo release. [15, 39, 51, 53, 95, 99, 108, 177, 223, 224] Often, phase transitions or competitive displacement reactions lead to opening of the pores and efficient cargo delivery. [117, 120] The third strategy for controlled cargo release involves attachment of the cargo molecules in the porous system of the silica nanocarriers. Coordinative or covalent bonds can be cleaved by certain stimuli such as competitively binding molecules or reducing agents to activate cargo release. [107, 109, 161] Zink and co-workers have presented different nanocarriers with on-demand controllable release mecha-nisms, including nanoimpellers consisting of azobenzene groups that have been described to trigger UV-light-activated release of a cell membrane-impermeable dye. [222]
Biocompatibility and StabilityFor applications of MSNs as nanocarriers, biocompatibility and
3.2 Modification of silica cores
low toxicity are required. A modification of the nanoparticle surface with functional shells, such as polymer coatings, charged groups, or a supported lipid bilayer was found to decrease particle aggregation and improve stability in biological media. For instance, functionalization of the particle surface with phosphonate groups was shown to improve the stability and dispersibility of MSNs in aqueous media. [128, 225] This modification helped to prevent interparticle aggregation, and redispersion after a drying process was highly improved. [16] In general, MSNs provide good bio-compatibility, but the high surface area and a low degree of condensation of the silica framework can promote a high rate of dissolution. [226, 227] Bare, nonfunctionalized MSNs featuring silanol groups at their surface dissolve fairly rapidly in simulated body fluid under physiological conditions and pro-duce soluble silicic acid species (which are found to be nontoxic). [228] The rate of silica dissolution is dependent on particle size, functionalization, degree of silica condensation, and pore morphology.
A surface functionalization can prevent fast degradation and provide prolonged stability of MSNs in biological media. For example, a hydrophilic polymer shell such as poly(ethylene glycol) (PEG) or an SLB on colloidal MSNs improves stability in water, maintains monodispersity, and can minimize nonspecific adsorption of proteins on the nanoparticle surface. [35, 53, 128] Such a polymer coating provides a protective shell for the silica surface, which is important when prolonged circulation time in an organism is required for effective drug delivery. PEGylation can hinder capture by organs of the reticuloendothelial system (RES) and consequently slow down biodegradation. [229] Hemo-compatibility is another important attribute of MSNs. Surface functionalization of bare MSNs can reduce or even completely prevent thrombogenic effects and nonspecific protein adsorption on MSN surfaces. [230] For example, heparin-coated core-shell MSNs have recently been described. [231] Hep-arin is a highly sulfated, anionic polysaccharide, known for its anticoagulant properties. This novel nanoscale system combines the efficiency of heparin in preventing blood-clotting with multifunc-tional core-shell MSNs featuring excellent structural properties and colloidal stability. In general, MSNs with organic shells offer multifunctionality and improved biocompatibility and hemocompat-ibility and are expected to have potential as blood-stream-injectable drug-delivery systems offering new options for cancer therapy.
4 Fluorescence live cell-imaging
A common technique to perform live-cell imaging is fluorescence microscopy because it offers the ability to monitor cell interactions after the cells were labeled, with fluorescent dyes. Nowadays, many site-specific fluorescent dyes to mark defined cell compartments are commercial available. [232]
Furthermore nanostructures such as mesoporous silica nanoparticles can be marked with fluorescent dyes and the interaction of particles with cells can then be monitored. [1, 14, 39, 86, 233] These experiments lead to a better understanding of the interaction between nanoparticles and cells, and are an important first step in developing new materials for medical applications.
In this thesis live-cell imaging was employed to observe the uptake and cellular fate of function-alized mesoporous silica nanoparticles, their release behavior as well as for the activation of the photoinduced endosomal escape.
The following section aims to give a general introduction to fluorescence principles on the basis of the Jablonsky-Diagram. Furthermore, requirements for dyes used in live cell imaging and related problems are discussed. Finally, the utilized spinning disc setup is described along with the principal for confocal microscopy.
4.1 Fluorescence principle
The basic physical principles behind fluorescent microscopy [234, 235] can be described by a look at the Jablonsky-Diagram (Figure 4.1). A fluorescent molecule is capable of gaining energy by absorption of photons if the energy of the photon is equal the energy difference of its energetic levels.
From the electronic ground state S0 the molecule gets excited to a higher (electronic) state (e.g.
S1, S2) and therein in vibrational and/or rotational excited states by absorbing a photon. Internal conversion, vibrational relaxation and intersystem crossing can then take place or relaxation by emission of a photon.
For live-cell fluorescent microscopy the energy of absorbed and emitted photons is normally in the visible light range. The energy E is inversely related to the wavelength (λ) of the absorbed photon’s:
E=h·c
λ (4.1.1)
Hereby, h is the Plank’s constant and c is the speed of light in vacuum.
To come from higher energy levels back to the ground state the molecule can emit a photon with the corresponding wavelength (fluorescence), which normally takes place in the nanosecond range.
Beside the singlet excited states also triplet excited state can be reached. This process is called intersystem crossing and is more likely if the triplet state vibrational energy levels overlap with
4 Fluorescence live cell-imaging
lowest energy levels in S1. In this case the electron undergoes a forbidden transition (spin transition) and owing to this, it can take microseconds for the electron to come back to the singlet ground state which involves another spin transition. Meanwhile it is also possible, that another photon excites a triplet-triplet transition and it would take even longer to come back to the ground state.
Figure 4.1: Jablonsky-Diagram displaying the energy states of a molecule and the times that the various steps in the fluorescence excitation and emission and phosphorescence take. The diagram is taken from ref. [234].
In addition to the light emitting processes also emission-free possibilities for returning to a favorable lower energy level. The vibrational energy is then transferred e.g. to neighboring molecules and lost for fluorescence. But it is unlikely for a fluorescent molecule to get back to the ground level only using vibrational relaxation. Beside vibrational relaxation internal conversion plays a role. In the case of internal conversion the system changes between electronic states without energetic loss, by going from a low vibrational state of a higher electronic state to a high vibrational state of a lower electronic state. Internal conversion can also be followed by vibrational relaxation. Within picoseconds a molecule is able to come to the lowest energy level of S1.
Because of the energy difference between the vibrational modes of the electronic ground state and the first singlet excited state, normally a total relaxation, without any photon emission, to the ground state is not preferred.
Due to the non-radiant relaxation behavior there is a shift to lower energy in the maximum of the emission wavelength in comparison to the maximum in the absorption wavelength, the so called
“Stokes shift”. Apart from that, the emission and absorption spectra exhibit a symmetry due to the similarity of transitions. Depending on the electronic, vibrational and rotational states of a fluorophore the excitation spectra a broader or smaller.