Chapter 3. Mixed-Charge Nanocarriers Allow for Selective Targeting of Mitochondria by Otherwise
3.3 Results
3.3.2 Supramolecular assembly of mixed-charge fluorescein nanoparticles
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experiments. This experiment confirmed that FL-C10-SS-C10-FL (compound 3b), but not FL-C10-SS- C10-COOH (compound 3a), undergoes partial FRET-based self-quenching due to a short distance between fluorophores – max 34 Å (shorter than Förster radius for fluorescein, 44 Å). 5- aminofluorescein (5-FAM, compound 2) was used as a control.
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concentrations of various ions (MUA-, TMA+, Au3+), separate calibration curves were established for nanoparticles with different ligand shell compositions (described in Methods section). We found that a larger number of fluorescein ligands were associated with mixed-charge than with singly-charged nanoparticles. On average 52, 94, 82, 47, and 12 ligands per nanoparticle were bound to [+]/FL, [+/-]9:3/FL, [+/-]6:6/FL, [+/-]3:9/FL and [-]/FL nanoparticles, respectively (Figure 3.3c, Table 3.1). The different fluorescence recovery values, f, (cf. above) for cationic, mixed-charge, and anionic particles hint at the complexity of the interactions, as well as fluorescence quenching mechanisms, the explaining of which certainly merits detailed theoretical study on its own. For example, the smaller f values for more anionic NPs (cf. above, especially compare f ~ 11 for [+]/FL versus f ~ 7 for [+/-]3:9/FL with a similar number of ligands bound to both particles) could be indicative of fewer covalently bound ligands onto particles with more MUAs, in agreement with the latter limiting the adsorption of fluorescent disulfides onto planar surfaces27.
To characterize the fluorescent ligand shells in more detail, the hydrodynamic diameters (DH) of the particles were calculated from the particle diffusion coefficients obtained by either dynamic light scattering (DLS) or fluorescence correlation spectroscopy (FCS)37. On one hand, DLS measurements confirmed that all particles were monodispersed with overall DH ~7.6 nm. On the other hand, the fluorescence-based measurements summarized in Figure 3.3f revealed that particles with more fluorescein ligands attached were larger than the ones with the least number of ligands attached (for example, mixed-charge particles with ~94 FL/NP were larger than anionic ones with ~12 FL/NP by
~1.85 nm; Table 3.1). One should keep in mind that low contrast between the refractive indices of the ligand shell and solvent typically precludes accurate measurements of very small changes in the particle diameters with DLS38. FCS, however, allows for the detection of particle size changes of ≥ 1 nm37. The fact that in our experiments fluorescein ligand shells are not “detectable” with DLS suggests that these shells are loosely-packed and permeated with solvent (water) (Table 3.1). Furthermore, the differences in ligand shell thickness observed with FCS were confirmed by TEM images of the nanoparticles counterstained with phosphotungstic acid to visualize the ligand layer – mixed-charge particles featured thicker shells than anionic particles or bare MCNPs (Figure 3.3f, Table 3.1).
Taken together, these results reflect the formation of a multi-layered shell of fluorescein ligands non-covalently bound to on-particle charged groups.
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Figure 3.3. Properties of the mixed-charge nanoparticles decorated with fluorescent disulfide assemblies. (a) Normalized UV-Vis spectra for various fluorescein nanoparticles prepared by the co- assembly method with FL-C10-SS-C10-COOH and standard mixed-charge 80:20 NPs without fluorescein (grey curve overlapping with colored curves). The subscripts indicate the ratios of [+] to [-] ligands present in solution during ligand exchange reaction (see Experimental Section). All nanoparticles were non-aggregated (see also NP sizes in Table 3.1). (b) The quenching effect of Au cores was assessed by comparing fluorescence intensities for nanoparticle solutions (2 nM in PBS, λex
= 495 nm) before (dashed lines) and after (solid lines) Au core digestion with potassium cyanide. (c) The total number of fluorescein ligands adsorbed onto various nanoparticles (see Experimental Section).
Data are mean ± s.d., Horizontal lines indicate median values and rectangular markers show the mean values. (d) Zeta potentials for various mixed-charge fluorescein nanoparticle assemblies, [+/-] FL NPs, are plotted as a function of the ligand shells' composition,
TMA:
MUA, at physi ological pH ~7.2-7.4. [+] = 100:0 / pure-TMA NPs; [+/-]9:3 = MCNPs with
TMA:
MUA = 80:20 (grey diamonds). The ratios of [+] to [-] ligands in solution are indicated by the subscripts, and the resulting surface ligand ratios,
TMA:
MUA, were established previously for mixed-charge NPs74
by core etching/NMR2524,39. Data are mean ± s.d. (e) TEM images of nanoparticles with the ligand layer stained by phosphotungstic acid show thicker ligand shells (indicated in red) for fluorescein nanoparticles [+/-]9:3/FL (ii) as compared to 80:20 MCNPs (i) (see Table 3.1). Scale bar, 20 nm. (f) Hydrodynamic diameters (DH) of co-assembled fluorescein/[+/-] nanoparticles were obtained from fluorescence correlation spectroscopy (FCS) measurements (see Table 3.1). The grey dashed line indicates the value of DH for bare 80:20 MCNPs from dynamic light scattering (DLS). Data ar e mean ± s.d. Note that FCS sizes are consistent with the number of FL ligands shown in c.
NPs
The molar ratio of ligands in
solution (TMA:MUA/
[+] : [-] )
aZeta potential
[mV]
pH 7.2-7.4
bHydrodynamic diameter, DH,
from DLS [nm]
cHydrodynami c diameter, DH, from FCS [nm]
dLigand shell thickness from TEM
[nm]
eFL molecul
es/ NP
[+] / FL 12:0 29.0± 2.1 7.7 ± 0.5 8.26 ±0.10 2.51 ± 0.75 52
[+/-]9:3 / FL 9:3 25.5 ±3.0 7.6 ± 0.4 9.55 ±0.14 3.80 ± 1.42 94
[+/-]6:6 / FL 6:6 16.2 ±2.5 7.6 ±0.1 8.33 ±0.20 2.55 ± 0.61 82 [+/-]3:9 / FL 3:9 -18.3 ± 5.1 7.3 ± 0.5 7.66 ±0.10 2.25 ± 0.41 47
[-] / FL 0:12 -34.1 ± 1.3 7.5 ± 0.7 7.60 ±0.14 2.18 ± 0.46 12
[+] = 100:0 12:0 27.0 ± 1.4 8.0 ± 0.1 NA 2.00 ± 0.12 0
[+/-]9:3 = 80:20 9:3 20.2 ± 1.4 7.6 ± 0.4 NA 1.94 ± 0.16 0
Table 3.1. Structure and surface characteristics of mixed-charge fluorescein nanoparticles.
Fluorescein nanoparticles were prepared with co-assembly method with FL-C10-SS-C10-COOH (compound 3a) as described in Figures 1.1 and 3.3. aParticle net surface charge shown as zeta potentials.
The hydrodynamic diameters (DH) as determined with bdynamic light scattering (DLS) or cfluorescence correlation spectroscopy (FCS). NA = not applicable. dLigand shell thickness from TEM images shown in Figure 3.3e. eThe average numbers of fluorescein (FL) ligand molecules per nanoparticle corresponding to the experiments shown in Figure 3.3c. The optimized molar ratio of thiol (MUA + TMA) ligands to asymmetric fluorescein ligands (compound 3a) was 60:1. For comparison, parameters for [+/-] NPs without fluorescein: [+] = 100:0 referring to cationic/pure-TMA NPs and [+/-]9:3 = 80:20 referring to NPs with surface composition of
TMA:
MUA = 80:20 – are also shown. All data are mean± s.d..
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Table 3.2. Non-covalent interactions in fluorescein nanoparticle assembly. Types of non-covalent interactions possible between on-particle TMA/MUA groups and asymmetric fluorescein disulfide (FL- C10-SS-C10-COOH, 3a). a −G binding free energies for interactions in aqueous solutions are listed. b Interactions upon transfer of the co-assembled particles to an aqueous solution (water, pH 8.0), or also during sequential assembly of particles in water. cπ-π, Lone pair-π interactions are predominantly between adjacent fluorescein ligands contributing to multi-layered ligand shell. Anion-π (in aqueous solutions), hydrogen bonds, and hydrophobic (in organic solvent) interactions are also possible between neighboring functional groups of fluorescein ligands. While π here refers to fluorescein’s two π systems, the interaction strength listed is from various π systems, mostly amino acids within proteins; FL2-, dianionic form of fluorescein.
Fluorescein nanoparticles systems prepared by sequential assembly. Motivated by results shown in Figure 3.3, we surveyed the probable non-covalent interactions – while cation-π interactions between fluorescein moieties and on-particle TMAs are present irrespective of solvent (organic versus aqueous), the types of interactions with on-particle MUAs change on transfer to an aqueous solution (Table 3.2). In organic solvent, protonated on-particle MUAs engage in hydrogen bonding and hydrophobic interactions, but upon transfer to an aqueous solution (pH 7.4 or pH 8), MUAs are de- protonated and engage in ion-pair and anion-π interactions (e.g., between TMA groups in the ligand shell and MUAs in FL disulfides).
Considering this, we probed if the electrostatic interactions alone were sufficient for mitochondrial-targeted particle assembly by performing so-called sequential assembly in which Fl- containing ligands were mixed with MCNPs with
TMA:
MUA = 80:20 for 18 h (equivalent time to theInteraction on-NP TMA/MUA ···
FL-C10-SS-C10-COOH
-D Ga (kJ/mol)
Co-assembly in organic solvent
Transfer to aqueous solutionb
Ref
[+/-]9:3/FL [+]/FL [+/-]9:3/FL [+]/FL
Cation-λ ̶ N+ (CH3)3 ··· π 1 – 5.5 + + ++ ++ 40,41
Hydrophobic ̶ COOH ··· π ~0-60
~0-60
+ - - - 41
Hydrophobic ̶ COOH ··· C10-alkyl chain + - - - 41
Hydrogen bond
̶ COOH ··· - OOCH 2-17 + - - - 41
cLone pair-π S: ··· π 2-8 + + + + 42
cπ-π π ··· π 7-17 + + + + 41
Salt bridge ̶ N+ (CH3)3 ··· ̶ COO- 5-8 - - + + 41
Ion pair ̶ N+ (CH3)3 ··· FL2- 0-20 - - + + 41
Anion-π ̶ COO- ··· π 1-2 - - + - 41
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ligand exchange), or as briefly as 20 min, in aqueous solution. For these experiments, we chose to use 80:20 NPs because the corresponding [+/-]9:3/FL NPs had the largest number of FL ligands bound in ex vivo experiments (shown in Figure 3.3c). As a result of this approach, assemblies with similar sizes to those created by co-assembly were formed. (Table 3.3). The nanoparticles formed with ligands FL- C10-S-S-C10-COOH have the thickest shells. Since, during the sequential assembly scheme, considerable place exchange of disulfide ligands with on-nanoparticle thiol ligands is not expected23,28, especially in the presence of MUAs27, these results provide further support for the role of non-covalent interactions in the assembly of disulfide-based fluorescein ligand shells, specifically highlighting the importance of the electrostatic interactions.
Hydrodynamic diameter, DH, from FCS [nm]
NPs Fluorescent compounds Fluorescein nanoparticles Free ligands
80:20
FITC 7.39 ± 0.28 nd
FL-C5 7.86 ± 0.26 nd
FL-C10 FL-C5-SS-C5-COOH
8.38 ± 0.41 9.36 ± 0.36 (90%)
nd 1.48 ± 0.20 (10%) FL-C10-SS-C10-COOH 9.90 ± 0.49 (70%) 2.18 ± 0.05 (30%)
FL-C10-SS-C10-FL NA NA
Table 3.3. Properties of sequentially-assembled mixed-charge fluorescein nanoparticles. F luorescein nanoparticles were prepared by mixing various fluorescent compounds with MCNPs with
TMA:
MUA = 80:20 for 18 h (equivalent time to the ligand exchange). The diameters of fluorescent particles from fluorescence correlation spectroscopy (FCS) generally present as one component, with an exception for FL-C5-SS-C5-COOH and FL-C10-SS-C10-COOH compounds consisting of two components: ligands bound to NPs and free ligands (ratio shown in percentages). nd = undetectable with FCS. NA = not applicable for compound FL-C10-SS-C10-FL due to the FRET-mediated fluorescein quenching. 80:20 refers to NPs with a surface composition of
TMA:
MUA = 80:20.77