RESULTS AND DISCUSSION
3.2. Glycosylated Cell-Penetrating Poly(disulfide)s
3.2.2. Mechanistic Investigations
All glycoCPDs showed excellent uptake, localizing in cytosol, nucleus and nucleoli as shown by the confocal microscopy images (section 3.1.3.3.). In particular, aggregation could be avoided, as shown in Figure 91, where colocalization experiments were performed using Glu-CPD 205 and reference
O NH
Figure 91. CLSM images of HeLa cells after 15 min incubation with 2.5 µM concentration of CPDs a) 177 and b) Glu-CPD 205 together with Hoechst 33342 at 37 °C in Leibovitz’s medium. Left: Hoechst, LP = 6.5%; centre: CPDs, LP = 14.5%; right: merged. Scale bar = 10 µm. Arrows indicate CPD precipitates.
With such efficient delivery, we decided to analyse the cellular uptake behaviour of the polymers also quantitatively using FACS (fluorescence-activated cell sorting). This technique measures cellular uptake by analysing the fluorescence of cell-by-cell (total of 10000 cells at least) and therefore enables a fast and objective quantification. The cellular uptake of fluorescent CPDs in HeLa cells was quantified after 15 min incubation at 37 °C using a concentration of 2.5 µM, the optimal concentration established during CLSM experiments (Figure 92).
a)
b)
Figure 92. Flow cytometry analysis counting fluorescent HeLa cells after 15 min incubation without (blank, B) and with 2.5 µM of CPDs 177 (grey), 165 (purple), Tre-CPDs 207 (blue), Gal-CPDs 206 (red) and Glu-CPDs 205 (green) at 37 °C in Leibovitz’s medium (normalized to fluorescence intensity I with 205).
Flow cytometry analysis revealed a more efficient cellular uptake for all glycoCPDs 205-207 compared to reference 177 and azide 165. Among the carbohydrate-containing polymers, Glu-CPD 205 was the most active, closely followed by Gal-CPD 206 and then Tre-CPD 207. Overall these results suggested that the uptake of unmodified CPDs could be enhanced by the addition of carbohydrates to the sidechain. Due to the presence of receptors on the cell membrane that regulate the binding and entry of carbohydrates,[104-108, 125] we decided to investigate the mechanism of entry of the new glycoCPDs, in particular, Glu-CPD 205 which showed the best uptake by both confocal microscopy and flow cytometry. In particular, we were eager to see whether glucose receptors, like GLUT-1 which can be abundantly found in cancer cell lines,[146] could be involved.
Before starting any mechanistic investigations, we measured the cytotoxicity of the CPDs at the concentration used for all the experiments and after 24 h incubation in HeLa cells. We decided to use the MTT assay, a colorimetric
read-0 I (rel)
1
0.5
B 177 165 207 206 205
intensity of absorption measured can therefore be correlated to an active metabolism inside the cell and consequently to the cell viability. The results of the cytotoxicity assay are shown in Figure 93 where all CPDs tested revealed the same absorbance as the blank, unlike positive control polyarginine which is known to be toxic to the cells at high concentration.
Figure 93. Cell viability measured with the MTT assay without (blank, B) and with 2.5 µM of CPDs 177 (grey), 165 (purple), Tre-CPDs 207 (blue), Gal-CPDs 206 (red), Glu-CPDs 205 (green) and polyarginine 10 µM (polyR, light blue, positive control) after 24 h incubation at 37 °C in MEM (normalized to fluorescence intensity I with B).
In order to investigate on the mechanism of entry, we decided to start with Ellman’s inhibition to assess if thiol-mediated uptake was still contributing to the efficiency of glycoCPDs. We decided to use flow cytometry to analyse the results of the inhibition studies, as shown in Figure 94.
0 I (rel)
1
0.5
B 177 165 207 206 205 polyR
Figure 94. Flow cytometry analysis counting fluorescent HeLa cells after 15 min incubation without (blank, B) and with 2.5 µM of CPDs 177, 165, Tre-CPDs 207, Gal-CPDs 206 and Glu-CPDs 205, after 30 min pre-incubation with 0 mM (filled), 1.2 mM (striped) and 5.0 mM (empty) DTNB, at 37 °C in Leibovitz’s medium (normalized to fluorescence intensity I with 205 and 0 mM DTNB).
The cellular uptake of all CPDs was significantly reduced of a least half after treatment with Ellman’s reagent, with very little dependence on the concentration of DTNB used (1.2 mM or 5.0 mM). The decrease in uptake for Tre-CPD 207 could be observed only when 5.0 mM DTNB were used for the oxidation of the exofacial thiols. The inhibition of cellular entry mediated by DTNB proved that disulfide exchange with exofacial thiols was necessary for the delivery of the fluorophore attached to the CPDs.
Another experiment that proves the occurrence of thiol-mediated uptake is serum inhibition.[226] Indeed, when extra serum is added to the medium, uptake mediated by the interaction with thiols of proteins embedded on the surface of the cell membrane is slowed down.[227-228] Serum serves as a nutrient source for the cells and contains proteins, carbohydrates, fats that can enter the cells through different mechanisms (diffusion or active transport like endocytosis),[226] and
0 I (rel)
1
0.5
B 177 165 207 206 205
of uptake of reference CPD 177 and Glu-CPD 205 by confocal laser scanning microscopy. The images shown in Figure 95 revealed that the uptake of both polymers did not change with time.
Figure 95. CLSM images of HeLa cells after incubation with 2.5 µM of CPDs a) 177 and b) Glu-CPD 205 after, from left to right,15 min, 1 h, 2 h, 4 h and 6 h at 37 °C in Leibovitz’s medium. LP = 2%; scale bar = 10 µm.
In order to appreciate even small differences in the decrease of uptake, inhibition with 10% FBS (fetal bovine serum) was analysed by flow cytometry.
As shown in Figure 96, a slowdown in the uptake of reference CPD 177 could be observed, while, on the contrary, Glu-CPD 205 did not seem affected by the presence of serum with only a slight inhibition in the first 2 hours of incubation.
A possible explanation for this insensitivity towards the presence of serum could come from the fact that thiol-mediated uptake did not account for the major mechanism of entry of the glycopolymers, as well as the fact that the presence of the carbohydrate could increase the stability towards serum.[216]
a)
b)
15 min 1 h 2 h 4 h 6 h
15 min 1 h 2 h 4 h 6 h
Figure 96. Flow cytometry analysis counting fluorescent HeLa cells after incubation with 2.5 µM of CPDs 177 (top) and Glu-CPD 205 (bottom) after 1 h, 2 h, 4 h and 6 h at 37 °C in the absence (full) and in the presence (striped) of 10%
FBS. Data are normalized against I of CPDs 177 (top) and 205 (bottom) after 1 h, 2 h, 4 h and 6 h at 37 °C.
We therefore decided to investigate the dependence of the uptake on the presence of D-glucose, a competitive inhibitor for a glucose-mediated entry.
Cellular uptake mediated by glucose-receptors, such as the abundant GLUT-1, is indeed proven by inhibition experiments with different concentrations of D-glucose,[105, 114] as shown in Figure 97.
0 I (rel)
1
0.5
1 h 2 h 4 h 6 h
0 I (rel)
1
0.5
1 h 2 h 4 h 6 h
Figure 97. Flow cytometry data for the uptake of HeLa cells cultured in medium containing 0 mM (filled), 25 mM (striped), 50 mM (dotted), 100 mM (empty) D-glucose, after incubation without (blank, B) and with 2.5 µM of CPDs 177, 165, Tre-CPDs 207, Gal-CPDs 206 and Glu-CPDs 205, at 37 °C in Leibovitz’s medium (normalized to fluorescence intensity I with 205 and 0 mM D-glucose in the medium).
By culturing cells with an increasing amount of D-glucose, we were delighted to see complete insensitivity of reference 177, even at the highest concentration of inhibitor used. On the other hand, the cellular uptake of Glu-CPD 205 decreased with increasing concentration of D-glucose. The trend of inhibition for Tre-CPD 207 was quite surprising, with a maximum of decrease observed for the lowest concentration of D-glucose used. No explanation could be found for this reverse trend compared to Glu-CPD 205, even though, trehalose being a dimer of glucose, an entry mediated by glucose-receptors is plausible.
Interestingly, Gal-CPD 206 was not inhibited when using 50 mM of D-glucose, suggesting that its uptake was not glucose-mediated. These key inhibition experiments suggested overall that the cellular uptake of Glu-CPD 205 is glucose-dependent. In order to prove this hypothesis, we tested the uptake of the CPDs in the presence of the non-natural enantiomer, L-glucose.
Inhibition with L-glucose is a key experiment to prove glucose-mediated uptake.[105, 114] Since L-glucose is not recognized by the receptors and
0 I (rel)
1
0.5
B 177 207 206 205
transporters on the cell membrane, it does not act as an inhibitor of the uptake.
As shown in Figure 98, Glu-CPD 205 was not inhibited by L-glucose at neither lower or higher concentration, proving that its cellular entry was indeed mediated by specific receptors that recognize the naturally occurring monosaccharide. On the other hand, reference CPD 177, which was not inhibited by D-glucose, was also, as expected, not affected by L-glucose. As for Tre-CPD 207, the uptake could be partially inhibited by D-glucose, but a concentration-independent inhibition was also observed with L-glucose, suggesting that glucose-receptors were not involved in the mechanism of entry.
Figure 98. Flow cytometry data for the uptake of HeLa cells cultured in medium containing 0 mM (filled), 25 mM (striped), 50 mM (empty), L-glucose, after incubation without (blank, B) and with 2.5 µM of CPDs 177, Tre-CPDs 207 and Glu-CPDs 205, at 37 °C in Leibovitz’s medium (normalized to fluorescence intensity I with 205 and in the absence of L-glucose in the medium).
At this point, we decided to also investigate the influence of D-trehalose on the cellular uptake. Once again, HeLa cells were cultured in the presence of increasing concentrations of D-trehalose which by itself cannot enter mammalian cells and is not known to be recognized by any specific receptors.[229] Figure 99 shows the results of FACS analysis for reference CPD 177 and glycoCPDs 205
0 I (rel)
1
0.5
B 177 207 205
trehalose, while Glu-CPD 205 showed a concentration-dependent inhibition.
This observation might suggest that the glucose-receptors could also partially recognize the D-glucose unit present in D-trehalose which therefore acted as a competitive inhibitor of the uptake of CPD 205. On the other hand, the cellular entry of Tre-CPD 207 was not affected by the presence of the disaccharide at lower concentrations, while at higher concentrations, the fluorescence intensity decreased by half. It is to be noted that higher concentrations of D-trehalose could not be used for inhibition since they proved to be significantly cytotoxic.
Overall, no clear trend could be found to explain the cellular uptake of CPD 205, strongly suggesting though that its uptake was not glucose-mediated.
Figure 99. Flow cytometry data for the uptake of HeLa cells cultured in medium containing 0 mM (filled), 25 mM (striped), 50 mM (empty), D-trehalose, after incubation without (blank, B) and with 2.5 µM of CPDs 177, Tre-CPDs 207 and Glu-CPDs 205, at 37 °C in Leibovitz’s medium (normalized to fluorescence intensity I with 205 and 0 mM D-trehalose in the medium).
Another key experiment was to evaluate the cellular uptake at 4 °C. CPD 177, which enters through a dual mechanism of counterion-mediated and thiol-mediated uptake, has been shown to be inhibited when incubated at 4 °C where all energy-dependent pathways are shutdown, suggesting endocytosis. However, the uptake of 177 and other systems that operate through thiol-mediated uptake
0 I (rel)
1
0.5
B 177 207 205
was found to be insensitive to common endocytosis inhibitors as discussed in the introduction. As shown in Figure 100, when CPDs were incubated at 4 °C, their uptake decreased. The scaffold similarity of the glycoCPDs 205-207 to reference 177 would suggest that the uptake decrease could stem from changes in membrane fluidity and thiol-reactivity that could occur by lowering the temperature. However, no further inhibitors were tested and therefore the occurrence of endocytosis could not be completely ruled out.
Figure 100. Flow cytometry data for the uptake of HeLa cells after incubation without (blank, B) and with 2.5 µM of CPDs 177, Tre-CPDs 207, Gal-CPDs 206 and Glu-CPDs 205, at 37 °C (filled) and 4 °C (striped) in Leibovitz’s medium (normalized to fluorescence intensity I with 205 at 37 °C).
We then decided to have a closer look at Gal-CPD 206 since, similarly to Glu-CPD 205, it showed excellent cellular uptake by confocal microscopy reaching the cytosol, nucleus and nucleoli. Moreover, quantitatively as well, the fluorescence count was in the same range as the other glycoCPDs 205 and 207.
In the inhibition studies, uptake efficiency decreased only at 4 °C and in the presence of DTNB. Insensitivity towards glucose, both D and L isomers, supported the evidence that binding to glucose-receptors was not involved,
0 I (rel)
1
0.5
B 177 207 206 205
CPD 177 and Glu-CPD 205 did not vary with the addition of 50 mM of D-galactose to the medium, while on the other hand, a promising decrease in the uptake of Gal-CPD 206 could be observed as well as for the unpredictable Tre-CPD 207.
Figure 101. Flow cytometry data for the uptake of HeLa cells cultured in medium containing 0 mM (filled), 50 mM (striped), D-galactose, after incubation without (blank, B) and with 2.5 µM of CPDs 177, Tre-CPDs 207, Gal-CPDs 206 and Glu-CPDs 205, at 37 °C in Leibovitz’s medium (normalized to fluorescence intensity I with 205 and 0 mM D-galactose in the medium).
The inhibition of uptake with D-galactose for Gal- but not Glu-CPDs, and viceversa, the inhibition with D-glucose for Glu- but not Gal-CPDs, would suggest the binding of the two glycoCPDs to two different types of receptors or transporters, glucose- and galactose- specific.[105] We could also observe that the loss of activity for Gal-CPD 206 was proportionate to the increase in concentration of D-galactose. By plotting the CPD activity against the concentration of inhibitor, we obtained IC50 values for 205-206.
As shown in Figure 102, an IC50 value of around 20 mM D-glucose was obtained for Glu-CPD 205. On the other hand, a higher IC50 value of around 110
0 I (rel)
1
0.5
B 177 207 206 205
mM D-galactose was found for Gal-CPD 206. This less efficient inhibition with D-galactose would suggest that galactose-specific receptors were less powerful compared to thiol-mediated uptake in promoting the entry of Gal-CPDs, consistent as well with the fact that these receptors are poorly characterized in the literature.[104-108]
Figure 102. Cellular uptake of Glu-CPD 205 (¡) and Gal-CPD 206 (¨) into HeLa cells as a function of the concentration of D-glucose and D-galactose, respectively, in the medium.
Figure 103 shows in graphical form the comparison between reference unmodified CPD 177 and Glu-CPD 205. By studying the influence of the uptake in the presence of different inhibitors, we were able to prove that in addition to the now well established mechanism of uptake of traditional CPDs combining disulfide exchange with exofacial thiols and interaction with exofacial anions, a new type of interaction on the cell surface could be exploited to further enhance cell entry at high concentrations. The interaction of Glu-CPDs with glucose-receptors was proven by concentration-dependent inhibition with D-glucose and
0 I (rel)
1
0.5
0 20 40 60 80 100
c (mM)
exchange contributed to the efficient cell entry of Glu-CPDs. In sharp contrast to the decrease in cellular uptake of 205 with different inhibitors, the cellular uptake of CPD 177 was only reduced at 4 °C and in the presence of Ellman’s reagent.
Figure 103. Summary of flow cytometry data for Glu-CPD 205 (top) and CPD 177 (bottom).
In conclusion, sidechain modification was applied to improve solubility by introducing different carbohydrates on the sidechain of the CPDs. Once again, thanks to the method developed previously, glycosylation could be achieved in high yield and reproducibility. Cellular uptake at different concentrations was evaluated next, revealing high solubility for all glycoCPDs compared to unmodified ones that tended to aggregate and precipitate. Confocal laser scanning microscopy revealed localization of all glycoCPDs in the cytosol, nucleus and nucleoli of HeLa cells, while flow cytometry enabled to quantify the efficiency of uptake and draw comparisons with unmodified CPDs.
GlycoCPDs, therefore, proved to not only solve the solubility issue of common CPDs, but also revealed a superior uptake. Mechanistic investigations were conducted next using flow cytometry to evaluate the effect of different inhibitors and led to the conclusion that, thanks to glycosylation, a multifunctional efficient uptake could be obtained. In particular, thiol-mediated uptake was involved in the cell entry of all CPDs, while CPDs bearing glucose and galactose units on their sidechain were proven to bind to glucose and galactose receptors with different affinity.
Finally, with the introduction of carbohydrates we were able to integrate a new cell entry pathway to the already cooperative effect of thiol- and counterion- mediated uptake. The efficient cellular uptake achieved in particular with Glu-CPDs made them intriguing to be evaluated in protein delivery.