Recently we described the development of a new class of diblock copolymers that are able to deliver siRNA from the endosomal pathway into the cellular cytoplasm.29
These polymers contain a poly(DMAEMA) cationic block for siRNA binding and a hydrophobic ampholyte block capable of mediating endosomal escape in response to endosomal acidification (). This membrane interactive block contains PAA and DMAEMA, which are designed to change from an inactive conformation at physiological pH to a more hydrophobic membrane-interactive conformation in response to the endosomal pH drop. The addition of hydrophobic BMA residues increases the hydrophobicity of the copolymer, increasing the pKa of the PAA carboxylate residues, thus raising the pH at which protonation occurs. The optimal incorporation of hydrophobic BMA residues was found to be between 40 and 50 mol %. In all cases the molecular weight of both blocks was held constant at approximately 10 kDa. At polymer concentrations of between 0.1 and 10 mg/ml at pH=7.4, these materials did not form micelles or any higher order structures according to light scattering data. However, upon the addition of siRNA, particles with sizes between 85 and 236 nm were observed depending on the +/− charge ratio. Here we prepared a diblock copolymer with a hydrophobic endosomolytic second block approximately 2.5 times larger than the hydrophilic first block () in order to investigate the structural and biological impact of such a modification. The polyDMAEMA macroCTA was first prepared by polymerizing DMAEMA in the presence of the RAFT CTA and a radical initiator. The resultant polyDMAEMA block (Mn
= 9,100 g/mol; PDI =1.19) was then used to prepare the poly[DMAEMA-b-(BMA-co-DMAEMA-co-PAA)] diblock copolymer (Mn
= 31,000 g/mol; PDI = 1.57). Copolymer composition of the second block was determined to be 52 % BMA, 26 % DMAEMA, and 22 % PAA via a combination of 1
H NMR and size exclusion chromatography.
Synthetic structure, molecular weight, and chemical composition of the diblock copolymer siRNA carrier employed in these studies.
3.1 Aqueous particle sizes and solution morphology of the diblock copolymer
Particle sizes for the diblock copolymer alone and in the presence of siRNA were investigated at various charge ratios using DLS. Lyophilized polymer was first dissolved in ethanol at a concentration of 10–50 mg/mL. The ethanolic solutions were then diluted 10-fold into phosphate buffer at pH of 7.4. Following dissolution, the diblock copolymers at a final aqueous concentration of 1 mg/mL spontaneously self assembled to form particles with average particle sizes of 45 nm. For comparison, no particles were observed for any of our previous diblock copolymer systems even at similar comonomer compositions. This result suggests that increasing the second block size and the ratio of the hydrophilic DMAEMA block to the endosome pH responsive block is sufficient to induce the formation of micelles. The addition of free siRNA to the micelle solutions did not significantly change the particle sizes over a range of +/− charge ratios. For example, at a +/− charge ratio of 4:1 the complexed particles showed hydrodynamic diameters of 47 nm as compared to 45 nm for the free polymer. It should be noted that complete siRNA binding to the polymer was observed at theoretical +/− charge ratios of 4:1 and higher. For both the free diblock copolymer as well as the polymer/siRNA complexes, near uniform distributions (PDI < 0.1) were observed. This result strongly suggests that the addition of the negatively charged siRNA molecules is not causing bridging between the positively charged micelles.
Particle morphology of micelles prepared from aqueous solutions of the diblock copolymer was also evaluated by electron microscopy (). From the electron micrograph the average diameter of the particles, which appear spherical, was determined to be 27.5 nm ± 3.9 nm (n =45). The surface-exposed shell of the particles appears to be collapsed on the core as indicated by an electron dense region surrounding the particle cores. It is likely that the hydrophilic poly(DMAEMA) block, which stabilizes the particles under aqueous conditions, is collapsed under the anhydrous conditions of the experiment.
Figure 2 Transmission electron microscopy image of micelles formed from an aqueous solution of the diblock copolymer. A 0.5 mg/ml solution of the diblock copolymer in PBS was applied to a carbon coated copper grid for 30 minutes. The grid was fixed in Karnovsky’s (more ...)
Additional evidence that the diblock copolymers exist as particles stabilized by a hydrophilic poly(DMAEMA) block was provided by 1H NMR spectroscopy. Shown in are 1H NMR spectra for diblock copolymer in CDCl3 and D2O respectively. The 1H NMR spectrum of the diblock copolymer in CDCl3 shows resonances associated with both the poly(DMAEMA) block (e.g. (CH3)2N at 2.38 ppm and OCH2CH2N at 4.1 ppm) as well as those associated with the pH responsive block (e.g., BMA OCH2CH2C at 3.95 ppm). Under these conditions both blocks are solvated and show free segmental motion, which is consistent with a molecularly dissolved unimeric polymer. In contrast the 1H NMR spectrum of diblock copolymer in D2O () shows significant peak suppression and broadening of the resonances associated with the comonomers present in the hydrophobic block (i.e., BMA, PAA, and DMAEMA). Resonances associated with the DMAEMA residues remain visible in D2O, however, the signal is significantly attenuated. For example, the signal associated with the dimethyl groups (δ = 2.28 ppm) is reduced from 5600 in CDCl3 to 2800 in D2O.
Figure 3 1H NMR spectra of the diblock copolymer in deuterated chloroform (CDCl3) and deuterated water (D2O) at 25ºC. In CDCl3 resonances associated with both the poly(DMAEMA) block as well as the endosomolytic block (poly(BMA-co-DMAEMA-co-PAA) are visible. (more ...)
These findings provide direct spectroscopic evidence for the formation of a core-shell structure under aqueous conditions with a hydrated poly(DMAEMA) corona stabilizing a hydrophobic core composed of hydrophobic BMA units and electrostatically stabilizing units of opposite charge PAA and DMAEMA).
3.2 Effect of pH on polymer structure and CMC
CMC values of particles formed from the diblock copolymer were also measured by a DLS-based dilution method. Hydrodynamic diameters of the particles in pH 7.4 PBS buffer at a concentration of 1 mg/mL were measured by dynamic light scattering over a 5-fold range of serial dilutions from 1 mg/mL to 1.6 μg/mL. Using this method the particles were observed to remain stable at 45 nm with a low PDI down to a polymer concentration of about 10 μg/mL (> 100-fold dilution). CMC values at pH 7.4 were determined to be 2 ug/ml (pyrene assay) and 4 ug/ml (DLS dilution assay). As the diblock copolymer concentration was further reduced below approximately 5 μg/mL the particles become increasingly unstable suggesting a CMC value in this concentration range. This result is in good agreement with the pyrene assay, where individual polymer chains appear to dissociate and form non-specific aggregates (data not shown). A significant pH dependence was observed for CMC values as determined by DLS. Under physiological pH conditions (i.e. pH 7.4) the diblock copolymer micelles remain stable at concentrations as low as 5 μg/ml. In contrast, CMC values determined at pH 4.7 show a large increase in the concentration at which a significant population shows sizes of 1–8 nm, which is consistent with unimeric polymer chains. The CMC value at pH 4.7 was estimated to be approximately100 ug/ml (DLS dilution assay). This finding is likely a result of increased ionization of DMAEMA residues from both the corona and core forming segments. The increase in net positive charge would serve to increase the overall hydrophilicity of the diblock copolymer enhancing the micellar exchange rate. These conditions of low pH reflect the natural pH gradient found within endosomal compartments and could provide the basis by which the polymer mediates cytoplasmic delivery of siRNA.
3.3 pH responsive membrane destabilizing activity of polymeric micelles and their siRNA complexes
The pH-responsive membrane destabilizing activity of diblock copolymer was assayed using a red blood cell hemolysis assay. Three different pH conditions were used to mimic endosomal pH environments: extracellular pH = 7.4, early endosome pH = 6.6, late endosome pH = 5.8 (). The dependence of hemolytic properties on the morphology of the diblock copolymer was evaluated by incubating red blood cells with concentrations above and below the polymer CMC. Intact red blood cells were then removed by centrifugation and the amount of hemoglobin in solution was determined by measuring the absorbance of the supernatant at 540 nm. In order to establish that complexation of the diblock copolymer micelle to a hydrophilic nucleic acid does not interfere with the intrinsic membrane disruptive properties of the polymer, hemolysis experiments were conducted on both the polymer alone as well as the polymer/siRNA complexes. Polymer/siRNA complexes at theoretical +/− charge ratios of 8:1, 4:1, 2:1 and 1:1 were prepared by adding increasing quantities of 25 nM siRNA to a fixed concentration of the diblock copolymer. The concentrated polymer and polymer/siRNA stocks were then added to the red blood cell suspensions to prepare solutions with a final polymer concentration between 2.2 and 18 μg/mL. No significant hemolytic activity was observed at pH 7.4 for polymer and polymer/siRNA complexes at any of the concentrations evaluated. Significant increases in red blood cell lysis were observed as the pH was reduced to 6.6. Under these conditions very similar hemolytic properties were observed between the polymer and polymer/siRNA complexes at a given pH and polymer concentration. For example, at a diblock copolymer concentration of 18 μg/mL the polymer alone showed 52 % red blood cell lysis at pH 6.6 which increased to 102 % at pH 5.8. (). In comparison, the polymer/siRNA complexes showed 42 % red blood cell lysis at pH 6.6 with a similar increase to 98 % at pH 5.8 (). Taken together, there is less than a 23 % and 4 % difference between the polymer and the polymer/siRNA at pH 6.6 and 5.8 respectively. Given these trends it was concluded that the complexation of siRNA to the diblock copolymer micelles does not significantly compromise the hemolytic activity.
Figure 4 Hemolysis of the (a) diblock copolymer as a function of pH at concentrations of 2.2, 4.5, 9.0, and 18 μg/mL and (b) diblock copolymer/siRNA complexes at theoretical charge ratios of 1:1, 2:1, 4:1 and 8:1 (25 nM siRNA). Hemolytic activity is normalized (more ...)
3.4 Knockdown activity and toxicity of siRNA-polymer complexes in cultured mammalian cells
The ability of the polymeric micelles to deliver siRNA to the cellular cytoplasm in an active form was evaluated by conducting mRNA knockdown experiments in HeLa cells (). Solutions of the diblock copolymer were mixed with siRNA targeting GAPDH as well as a scrambled sequence to serve as a negative control. These solutions were then allowed to incubate for 30 minutes after which time they were added directly to HeLa cells in media containing 10 % FBS. Final siRNA concentrations were evaluated at 12.5, 25, 50, and 100 nM at polymer concentrations such that the theoretical +/− charge ratios were 8:1, 4:1, 2:1, and 1:1 to determine what conditions result in highest knockdown activity. It should be noted that at these polymer concentrations LDH cytotoxicity experiments showed complete HeLa cell viability (data not shown). Specific gene knockdown activity was then measured 24 hours post transfection via real-time quantitative PCR. At a theoretical +/− charge ratio of 4/1, high mRNA knockdown was observed at all four siRNA concentrations. The observed knockdown at this charge ratio varies between greater than 90% at 25, 50, and 100 nM to around 75 % at 12.5 nM. In contrast to the 4/1 charge ratio, a precipitous decline in mRNA knockdown as a function of siRNA concentration was observed at +/− charge ratios of 2/1 and 1/1. Under these conditions high mRNA knockdown efficiency was retained at 100 nM for both charge ratios, however, a dramatic loss in mRNA knockdown was seen as the siRNA concentration is reduced to 25 and 50 nM for the 2/1 and 1/1 charge ratios respectively. These trends are also associated with differences in polymer concentration which are necessary in order to prepare different charge ratios at a given siRNA concentration. Under these conditions (i.e., [polymer] < 5 μg/mL) the particles are below the CMC of the polymer. In this concentration region destabilization of the micelle core results in almost complete loss of knockdown activity for all but the highest siRNA concentrations where siRNA binding may stabilize the micelle. These results suggest self-assembly of the diblock copolymer into stable micelles results in a significant enhancement of siRNA mediated mRNA knockdown. This knockdown is greater at higher +/− ratios and siRNA concentrations but significant mRNA knockdown levels remain high (~90 %) even at siRNA concentrations as low as 12.5 nM.
Figure 5 GAPDH knockdown in HeLa cells was measured using real time RT-PCR following a 24 hour incubation period with the diblock copolymer/siRNA complexes. Final siRNA concentrations were evaluated at 12.5, 25, 50, and 100 nM at polymer concentrations such that (more ...)
3.5 Cell uptake properties and cellular distribution of diblock copolymer/siRNA complexes
The cellular internalization of the diblock copolymer-siRNA complexes at a 4:1 charge ratio was examined using flow cytometry with untreated cells and the commercial transfection reagent Lipofectamine (L2k) serving as the negative and positive controls respectively (). Measurements were taken with and without the addition of the fluorescence quenching agent Trypan blue in order to control for surface bound noninternalized fluorescence. As expected, untreated cells showed negligible fluorescence while the L2k control showed moderate fluorescence with approximately 39 % cellular uptake of the FAM-labeled siRNA (). By comparison, very high cellular uptake of the labeled siRNA was observed for cells treated with the diblock copolymer complexes. Indeed, near quantitative siRNA delivery (91 %) was observed for the diblock copolymer at a charge ratio of 4:1 following a 24 hour incubation period. The mean fluorescent intensity for the diblock copolymer and positive controls was also evaluated via flow cytometry (). From these experiments a dramatic increase in the fluorescent intensity is observed for cells treated with the diblock copolymer/siRNA complexes as compared to L2k/siRNA samples.
Figure 6 (a) HeLa cell internalization of 25 nM FAM-labeled siRNA, Lipofecatmine/siRNA, and polymer/siRNA complexes formed with the polymers at a theoretical charge ratios of 4:1 (after 4 h). Trypan blue was utilized for quenching of extracellular fluorescence (more ...)
In order to better understand the cellular distribution of the polymer/siRNA complexes fluorescent microscopy experiments were performed (). Shown in is a fluorescent microscopy image of L2K mediated delivery of FAM labeled siRNA to HeLa cells. DAPI staining, shown in blue, was employed in order to visualize the nucleus. While some diffuse fluorescence (green) is observed for the L2k based system, the majority of the fluorescence is located in discrete punctuate spots. This observation suggests that most of the FAM-labeled siRNA is confined to endosomal compartments. Because siRNA must reach the cytoplasm in order to become active this is clearly not optimal. In comparison, the diblock copolymer micelle mediated delivery of siRNA shows diffuse fluorescence throughout the cellular cytoplasm (). Taken with the high mRNA knockdown levels these results suggest that the diblock copolymer is able to transition to a membrane disruptive conformation in response to endosomal pH changes, resulting in endosomal release of the siRNA.
Figure 7 Polymer enhanced intracellular delivery of FAM labeled siRNA. Representative images illustrating (a) punctate staining (green) in the samples treated with lipofectamine/siRNA complexes alone and (b) dispersed fluorescence within the cytosol following (more ...)