Design and synthesis of potential polyfluorene carriers
Amino functionalized polyfluorenes have been made before24
but never exploited as protein delivery systems. Our early studies showed that only the quaternary ammonium system 1
was water soluble; corresponding primary amine, secondary N
-methylamine, and tertiary N
-dimethyl amines polymers were all surprisingly insoluble
in aqueous media, even at reduced pHs. However, the quaternary salt 1
, very similar to a known material,25
gave a clear solution in water even at neutral pH. Consequently we made a batch of this material via
a modification of the literature procedure25
that involved similar steps but in a different order (see ESI†
Material 1 is a polyamine derivative whereas the oligoarginines in HIV-TAT mimics contain multiple guanidine functionalities. Consequently, we also prepared the guanidine-functionalized polyfluorene 2. Synthesis of one monomer for the preparation of polymer 2 was achieved via modification of the protected diamine 3 into the masked guanidine 4 (). The complementary diboronate 7 was prepared via the azide intermediates 5 and 6. Finally, Suzuki coupling of monomers 4 and 7, then deprotection afforded the target material 2.
Polymers 1 and 2 had the weight average molecular weight (Mw) of 8200 (Poly Dispersity Index, PDI, 1.21) and 9400 (1.25), respectively, from gel permeation chromatography (GPC). Dynamic light scattering experiments (DLS) indicated polymer 1 in water aggregated to form particles that had average diameters of 140 nm. Polymer 2 was insoluble in water, but it appeared to dissolve in methanol while DLS indicated it had in fact aggregated to form particles with an average diameter of 45 nm. Both polymers exhibited a strong UV absorbance centered on 380 nm, and they fluoresced around 415 nm in water with quantum yields of 0.58 (1) and 0.42 (2).
We were particularly interested in the cytotoxicities of these nanoparticles, so they were tested using Clone 9 rat liver cells in an MTT assay. No significant cytotoxicity was observed for either polymer (IC50 100 μM for both; polymer 2 was delivered as a suspension in buffer). Based on these data it appeared that 1 and 2 were suitable for testing with regards to their abilities to act as carriers for protein import.
Delivery at 37 °C: uptake into punctate vesicular structures
Four proteins were selected for uptake experiments (). These were cationic (avidin) or anionic (GFP, β-galactosidase, streptavidin), with molecular masses between 26.9 and 540 kDa, and labeled with anionic dye (FITC, Alexa Fluor® 488), cationic dye (Texas Red® ), or no dye at all (GFP). Two cell lines, Clone 9 and Chinese hamster ovary (CHO), were used in the import studies.
Proteins for cellular uptake studies
Polymeric carrier-to-protein cargo ratios were optimized so that the signals observed in the cells were visible yet not saturated. A 1 : 1 ratio (mol : mol throughout) was thus found to be optimal for the two proteins of intermediate size. Conversely, less carrier was used for the smallest protein (GFP; 1 : 3 carrier : cargo), and more was necessary for the largest (β-galactosidase; 25 : 1.0 carrier : cargo). In each case, images for the uptake were recorded after 12 h of incubation, then checked after another 24 h. However, the signal for GFP was almost completely lost after 24 h indicating degradation, but the polymer fluorescence was still observed.
gives illustrative data for the cellular uptake experiments. This graphic features GFP, but similar images were obtained for all the proteins in both cell lines (see ESI†
). Without carrier, little or no visible GFP was imported into the cells (). A polyfluorene based polymer similar to 1
(different alkyl groups, and another monomer included) has been reported to be imported into cells.26
Consequently, we were not surprised that polymer 1
alone permeated into the cells (data not shown). Polymer 1
and GFP incubated with the cells for 12 h resulted in import of both
fluorescent molecules (b and c). shows the extent of fluorescence overlap from the polymer and protein; this may be expressed in terms of a “colocalization coefficient” deduced using laser scanning microscopy (LSM) software, with larger coefficients corresponding to greater overlap. In this particular case, i.e
. for GFP and polymer 1
, the coefficient was 0.48. After a further 24 h the signals were weaker, but no diffuse fluorescence was observed. Polymer 2
under the same conditions gave noticeably more uptake of GFP in experiments for which the polymer
signal inside the cells was qualitatively similar to the those with GFP/polymer 1
. However, more of the GFP colocalized with 2
(colocalization coefficient 0.68) than with polymer 1
. Images for import of the other proteins are shown in the ESI,†
and summarizes the colocalization coefficients. Overall, more protein was released from the quaternary amine polymer 1
than it was from the guanidine 2
Fig. 1 Delivery of GFP into Clone 9 cells at 37 °C mediated by polymers 1 and 2; a represents no carrier used, and e is a differential interference contrast (DIC) image. Data for polymer 1: b channel showing fluorescence by polymer 1; c channel showing (more ...)
Colocalization coefficients for polymers 1 (blue) and 2 (red) for the four featured proteins in Clone 9 cells.
Attempts to establish the location of the protein and polymer were made for each of the proteins imported. shows the data obtained to track the quaternary amine coated polymer 1, streptavidin-Texas Red®, and LysoTracker® Green. Three dyes are involved in these experiments and all of them show up distinctly (a shows channel to detect polyfluorene; b streptavidin-Texas Red® ; c LysoTracker® Green). Three colocalization coefficients are significant in these experiments: LysoTracker® Green/polymer 1, LysoTracker® Green/streptavidin-Texas Red®, and polymer 1/streptavidin-Texas Red®. These coefficients were 0.35, 0.56, and 0.46, respectively. This data indicates most of the polymer has escaped from the lysosomes, and less than half of the fluorescent protein escaped. Correlations between the polymer 1 and streptavidin-Texas Red® do not distinguish if they are together in the lysosomes, elsewhere, or both. However, show areas where all three fluorescent entities are colocalized in white. Quantitative analysis of this situation is difficult and somewhat unnecessary because it is evident that for the quaternary amine polymer 1 regions with distinct blue, red, and green signals predominate; conversely, white areas indicating colocalization of all three labels are prevalent for the guanidine-sheathed polymer 2.
Fig. 3 Delivery of streptavidin-Texas Red® into Clone 9 cells at 37 °C mediated by 1 and 2. Data for transport using polymer 1: (a) polymer 1 channel; (b) streptavidin-Texas Red® channel; (c) LysoTracker® Green; (d) colocalization (more ...)
Colocalization experiments that parallel those above were attempted with markers for other organelles. Specifically, streptavidin-Texas Red® was tested with compounds that are thought to localize in endosomes, and in the endoplasmic recticulum (ER). However, no significant colocalization was observed.
Similar series of experiments were attempted for the three other proteins. However, the labels involved (GFP, FITC, and Alexa Fluor® ) are all green, so LysoTracker® Green could not be used. LysoTracker® Red was tested, but crosstalk between the red and green channels precluded accurate analyses. Other labels could have been tested, but the chances of success were only moderate, and we made the decision that the scientific gains made by determining the degree of colocalization for these three proteins were not worth the costs and effort involved.
In previous studies, we had found that (Arg)8
mediated import of some of the same protein cargoes into lysosomes at 37 °C, but into the cytosol
at 4 °C.19
Experiments to test import with polymers 1
at 4 °C
showed both polymers impregnated into the cell wall membrane, and none of the four proteins were imported.
Futaki and co-workers have studied the influence of the lipophilic, fluorescent counter ion, pyrenebutyrate, in import mediated by oligoarginine-based cell penetrating peptides.22,23,27
Here import of bovine serum albumin-Texas Red®
(BSA, MW = 66 kDa, 538 amino acids, pI = 4.7) mediated by polymer 1
was briefly tested in the presence of pyrenebutyrate. This counterion dramatically increased the rate
of transport into the cells at 37 °C so that the protein was conspicuous after only 5 min incubation. However, most of the protein colocalized with LysoTracker®
Green and it was not liberated into the cytosol even after extended incubation. The same experiment but run at 4 °C showed only impregnation of the polymer into the membrane as in the absence of pyrenebutyrate (see ESI†