Letter pubs.acs.org/macroletters
MAG versus PEG: Incorporating a Poly(MAG) Layer to Promote Colloidal Stability of Nucleic Acid/“Click Cluster” Complexes Daniel J. Buckwalter,† Antons Sizovs,† Nilesh P. Ingle,‡ and Theresa M. Reineke*,‡ †
Macromolecules and Interfaces Institute, Department of Chemistry, Virginia Polytechnic Institute, Blacksburg, Virginia 24061, United States ‡ Department of Chemistry, University of Minnesota, 207 Pleasant Street SE, Minneapolis, Minnesota 55455, United States S Supporting Information *
ABSTRACT: Herein, we demonstrate the reversible addition− fragmentation chain transfer (RAFT) synthesis of an adamantaneconjugated glycopolymer, poly(2-methacrylamido-2-deoxy glucopyranose) (Ad-pMAG), as a hydrophilic coating to promote colloidal stability of click cluster-pDNA complexes in biological media. The AdpMAG is assembled via noncovalent interactions through inclusion complex formation between adamantane (Ad) and the β-cyclodextrin (βCD) core of the click cluster/pDNA and then further assembled with plasmid DNA to form polyplexes. Ad-pMAG incorporation was favorable over Ad-poly(ethylene glycol) (Ad-PEG) due to the enhanced colloidal stability of the click cluster/pDNA polyplex under physiological salt conditions at high N/P ratios. Interestingly, the uptake and reporter gene expression with polyplexes coated with the Ad-pMAG was much lower in HeLa cells than that observed with two glioma cell lines (U87 and U251 cells) in vitro, possibly indicating some delivery specificity.
P
oly(ethylene glycol) (PEG) has become a widely used polymer for incorporation of hydrophilic nanoparticle layers and, in many cases, for increased colloidal stability, reduced toxicity, and imparting passive nanoparticle targeting.1−4 PEG conjugation has provided delivery systems with these desired properties; for example, Davis et al. have shown clinical success with a cyclodextrin-based polycation siRNA delivery vehicle conjugated with adamantane PEG (AdPEG).5,6 However, this is not true in all cases. Recent studies have also revealed concerns of the accelerated blood clearance (ABC) phenomenon when using PEGylated systems, which could be detrimental for therapies that involve repetitive administration of drugs.7−9 PEG, while being well suited for easy conjugation to many delivery systems, does have drawbacks that warrant investigations into alterative polymers for use as stealth coatings for nanosystems and biologic drugs. The aim of this initial investigation is to study the difference in physical and biological properties of polyplex formulations that include adamantane conjugated to either PEG or poly(2methacrylamido-2-deoxy glucopyranose) (p(MAG)), a potentially new stealth coating for stabilizing nanosystems. The novelty of this study lies in the synthesis and application of an adamantane-terminated p(MAG) (Ad-p(MAG)) as an alternative to commonly used PEG. The click cluster delivery vehicles, developed by Srinivasachari et al.,10 are utilized in this study to form noncovalent inclusion complexes with the Ad-X polymers (Figure 1), which are investigated as pDNA delivery vehicles. Previous studies with HeLa (human adenocarcinoma) cells have demonstrated high transfection efficiency and low © 2012 American Chemical Society
Figure 1. Structures of (a) click clusters, (b) Ad-PEG, and (c) Adp(MAG).
toxicity of polyplexes created from pDNA complexed with click cluster macromolecules. Although these click cluster/pDNA polyplexes are colloidally stable in water, there is a high tendency for them to aggregate in physiological conditions, such as salt- and serum-containing media, which can be detrimental for in vivo delivery applications. Therefore, these polyplexes provide a suitable system for comparison of the Adp(MAG) and Ad-PEG effects on the polyplex properties. Received: February 18, 2012 Accepted: April 4, 2012 Published: April 19, 2012 609
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Scheme 1. Synthesis of Ad-pMAG
pDNA movement at an N/P = 1. When Ad-p(MAG)52 is introduced into the polyplex formulation at a 1:1 molar ratio (mol Ad/mol βCD) via inclusion complex formation, the pDNA complexation was not completed until N/P = 5, at which pDNA migration was fully retarded (Supporting Information, SI Figure 7). This decrease in pDNA binding ability suggests that p(MAG) could sterically hinder the click cluster−pDNA binding to a larger extent than PEG-modified click clusters. If Ad-p(MAG)52 inclusion complex formation with click clusters affects their binding to pDNA, this interaction could presumably impact the size of the polyplex nanoparticles. Dynamic light scattering (DLS) was used to determine the size of polyplexes for each different formulation in pure water (Supporting Information, SI Figure 8). There was no difference in size between unmodified and Ad-PEG-containing polyplexes, both of which ranged from 40 to 60 nm in hydrodynamic diameter at N/P ratios of 5−20. Interestingly, a formulation including Ad-p(MAG)52 showed larger polyplexes, ranging from 90 to 110 nm in size. This result agrees with the gel electrophoresis data in that it indicates weaker interactions between click cluster and pDNA in the presence of p(MAG), which could lead to a more relaxed polyplex core and, thus increased polyplex size. Zeta potential measurements also revealed a difference in the surface charge of the nanoparticles upon modification (Supporting Information, SI Figure 9). The zeta potential of unmodified polyplexes increased with increasing N/P ratio, reaching a value of 52 mV at N/P = 20. Ad-PEG inclusion reduced zeta potential values, which was especially noticeable at higher N/P ratios (at N/Ps of 10 and 20, zeta potentials reached 30 and 35 mV, respectively). The influence of AdpMAG52 was more profound: in its presence, the zeta potential remained between 22 and 30 mV at all tested N/Ps. One of the primary functions of the Ad-PEG and Adp(MAG) coating is to provide colloidal stability of polyplexes in physiologically relevant conditions. We used DLS to measure the polyplex size over time after dilution with salt- (reducedserum Opti-MEM) or serum-containing media (DMEM + 10% fetal bovine serum, FBS). Upon dilution of unmodified polyplexes (N/P = 5 or 20) with reduced-serum medium (Opti-MEM), there is a dramatic increase in polyplex size from around 50 nm to over 1 μm after 4 h incubation, as seen in Figure 2. The inclusion of Ad-PEG into the formulations did not impart evident colloidal stability to the delivery system as
Ad-p(MAG) was synthesized through reversible addition− fragmentation chain transfer (RAFT) polymerization using an adamantyl-functionalized chain transfer agent (Ad-CTA), 2 (Scheme 1). The consumption rate of the MAG monomer, compound 3, was established in a water/DMSO solvent mixture at a MAG/CTA/initiator molar ratio of 100:1:0.1 (Supporting Information, SI Figures 2 and 3). We targeted the degree of polymerization (DP) of 50, and this choice was based on the previous successful use of p(MAG) having DP = 46 in previous nucleic acid delivery vehicles developed in our lab.11 The polymer was purified by dialyzing the reaction mixture against ultrapure water. A degree of polymerization of 52 (Adp(MAG)52) was established by 1H NMR (Supporting Information, SI Figure 4). Ad-PEG was synthesized using a commercially available, mono-NHS-activated PEG5000 and adamantane methylamine according to a previously published procedure.12 It is essential for these Ad-X polymers to form strong inclusion complexes with the hydrophobic cyclodextrin (CD) core of the click clusters to impart a stable stealth effect on the polyplexes. To quantify this interaction, NMR titration experiments were utilized to verify efficient binding of the adamantane-containing macromolecule. The analysis of three independent titrations resulted in a binding constant (K) of approximately 36000 M−1 for the Ad-PEG inclusion with click clusters (Supporting Information, SI Figure 6). While there was potential that the seven cationic arms of the click cluster could distort the CD cup in a way that would hinder the binding of adamantane, the high K value indicated that this was not the case and correlated well with published values for a similar polymeric system.13 This method was not suitable for measurement of the Ad-p(MAG)52 polymer, however, due to overlapping NMR resonance peaks. Because of the structural similarity in the adamantane−polymer linkages, it was assumed that the Ad-p(MAG)52 would exhibit a similar binding constant to the click clusters. Future studies utilizing Ad-p(MAG) host− guest chemistry will include measurements of the binding constant between Ad-p(MAG) and the click clusters. Gel electrophoresis assays were performed for polyplexes that were unmodified, Ad-PEG-modified, or Ad-p(MAG)modified to measure the minimum N/P ratio (click cluster amines/phosphate units in pDNA backbone) that is necessary to fully complex the pDNA. Polyplexes that were formulated with pDNA and click cluster only (unmodified), as well as a formulation that included Ad-PEG, appeared to fully retard the 610
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To investigate the effect that PEG and pMAG might have on polyplex endocytosis, polyplexes were prepared with Cy5labeled pDNA and incubated with each of these three cell lines. Flow cytometry was utilized to quantify the number of cells positive for the presence of Cy5 (Cyanine5: excitation/ emission: 649 nm/670 nm)-labeled pDNA. Figure 3 shows
Figure 2. Hydrodynamic diameters of polyplexes after dilution with 700 μL of Opti-MEM of each different formulation.
similar rapid increases in size were observed upon dilution into Opti-MEM. When Ad-p(MAG)52 was added instead, at N/P = 20, there was a dramatic increase in the colloidal stability for up to 2 h (Supporting Information, SI Figure 11). This is likely due to the increased bulk of p(MAG) versus PEG due to the sugar units along its backbone. The size of the Ad-p(MAG)52modified polyplexes at N/P = 20 remains steady, around 100 nm, for 2 h until the size gradually increases. The size reaches a maximum between 5 and 8 h. At an N/P = 5, the colloidal stability of Ad-pMAG-modified polyplexes actually decreases compared to unmodified and Ad-PEG-modified analogs, exhibiting rapid aggregation. At this formulation N/P ratio, the polyplex is weakly held together, as observed via gel electrophoresis. Thus, we suspect that a small amount of charge screening by the salt can cause the rapid aggregation and even polyplex disassembly, leading to rapid aggregate formation. Also, it is likely at an N/P = 5 there is not a sufficient number of CD moieties present on the particle surface for Adp(MAG)52 complexation to provide enough colloidal stability. In the case of N/P = 20 there are likely more CD cups present on the surface and thus more Ad-p(MAG)52 can coat the polyplex leading to an increase in colloidal stability. When the polyplexes were diluted into medium containing 10% FBS, a slight difference in stabilizing effect was noticed when comparing the Ad-p(MAG) polyplex coating to Ad-PEG (Supporting Information, SI Figure 10). Ad-PEG performed better at lower N/P ratio of 5 than the Ad-p(MAG)52 coating, which could be related to the steric hindrance in the binding and likely incomplete polyplex formation. However, at a higher N/P ratio (20), the Ad-p(MAG)52 exhibited similar colloidal stability to the Ad-PEG polyplex coating. Further studies are currently ongoing to provide a more complete understanding of the dynamics of this aggregation in salt and serum conditions as well as to test the effects of pMAG molecular weight on the colloidal stability of click cluster polyplexes. The data shown thus far indicate that inclusion of Adp(MAG)52 in polyplex formulations causes a significant impact on the physical properties of the pDNA/click cluster polyplexes. To probe how these effects may influence the nucleic acid delivery properties of the click cluster, several biological assays were conducted using three human cell lines: HeLa cells (human cervical cancer) and two brain tumor cell lines: U-251 and U-87 MG [human glioblastoma multiforme (GBM) cells].
Figure 3. Cellular uptake (percent Cy5 positive cells) of each polyplex formulation at N/P = 10 in HeLa (blue), U-87 MG (red), and U-251 (green) cell lines.
that both of the GBM cell lines have around 90% cellular uptake for all polyplex formulations. HeLa cells, on the other hand, largely reject the polyplexes formulated with Adp(MAG)52. Only a little more than 20% of the HeLa cells treated with Ad-pMAG-modified polyplexes were positive for Cy5. These results could be related to the high amount of glucose transporter-1 present on the cell surface of the GBM cells.14 This effect, with respect to HeLa cells, should be further investigated with other cell lines to determine whether Adp(MAG)52 can be used for reducing nonspecific cellular uptake while potentially providing enhanced delivery to cell types that have increased level of glucose transporters. Delivery of gWiz luc-pDNA, which encodes for the luciferase protein, was carried out on all three cell lines to study the effect that Ad-X inclusion had on gene expression efficiency. After 4 h of cellular incubation in reduced-serum medium (Opti-MEM), followed by 42 h in DMEM containing 10% fetal bovine serum, expression was assayed. It was found that incorporation of AdPEG in polyplex formulations did not effect gene expression as compared to the click cluster only in U-251 and HeLa cells (SI Figures 12 and 13) and was slightly lower with U-87 cells (Figure 4). Ad-p(MAG)52 incorporation, on the other hand, does impact gene expression with the different cell lines. With U-251 cells (SI Figure 12), there appears to be minimal transgene expression at N/P 5 and 10, whereas luciferase levels increase for N/P 20 and 50, reaching gene expression levels similar to those of unmodified polyplexes. When comparing all of the polyplex formulations for transfection efficiency with U-87 MG cells (Figure 4), little to no difference in gene expression levels was observed. Interestingly, the results for HeLa cells reveal a much lower level of gene expression when using Ad-p(MAG)52, compared to Ad-PEG and unmodified polyplexes (Supporting Information, SI Figure 13). These data correlated to the cellular uptake data that showed minimal cellular internalization of Adp(MAG)52 containing polyplexes by HeLa cells. These studies 611
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methylamine and HBTU coupling reagent to yield the Ad-CTA derivative. Ad-CTA was subsequently used in the synthesis of AdpMAG in a deoxygenated mixture of water, acetate buffer, and DMSO using a V-501 initiator at 70 °C. The monomer consumption and kinetics were studied to target a particular DP (Supporting Information, SI Figures 2 and 3). Ad-PEG polymers were produced in high yields and purity using a NHS carbonate-functionalized PEG with an average molecular weight of 5000 g/mol. Click clusters were synthesized via the Cu(I) catalyzed azide−alkyne click reaction in high purity, as evident by the MALDI-TOF, 1H NMR (Supporting Information, SI Figure 1a), and 13C NMR analyses, performed according to a published procedure with minor modifications.10 NMR titration experiments were carried out on a Varian MR400 NMR. In short, 10 samples were prepared in 5 mm NMR tubes with 1.2 mM AD-PEG in D2O and increasing amounts of click cluster from 0 to 6.6 mM. The shift in the resonance peak of the tertiary proton of adamantane was plotted against click cluster concentration. Conner’s equation17 was then used to fit the data and obtain the K of adamantane/CD complexation (Supporting Information, SI Figures 5 and 6). All polyplexes were formed in DNase/RNase-free distilled water, at the designated N/P value, and then diluted with water, DMEM with 10% FBS, or Opti-MEM before use. All polyplexes were incubated for 1 h at room temperature before use. Polyplex formulations were prepared at a concentration of 0.02 μg/μL pDNA, with an exception of those used in the gel binding assays, which were formulated using 0.1 μg/μL pDNA. Gel electrophoresis assays were run using agarose gel in TAE (Tris-Acetate-Ethylenediaminetetraacetic acid) buffer, with ethidium bromide for visualization of the pDNA. Gels were visualized and photographed using a UV (ultraviolet) gel box. A Malvern Zetasizer DLS was used to measure polyplex sizes and zeta potentials in the three media described above. Colloidal stability was assessed by measuring hydrodynamic diameters at various time points. Cellular uptake studies were carried out using Cy5-labeled pDNA for polyplex formation and subsequently used in transfections of each cell line at N/P 10. Cells were incubated with the labeled polyplexes for 4 h before washing with PBS buffer and CellScrub to remove any cell surface-bound polyplexes. The number of cells positive for Cy5 fluorescence was measured on a BD FACS Canto II using BD FACSDiva software. Gene expression was evaluated by transfecting cells with polyplexes formed with gWiz luciferase pDNA for 4 h in reduced-serum medium then in DMEM containing 10% FBS for 42 h. A total of 46 h after transfection, luciferin was added to cellular lysate and fluorescence was measured with a TECAN Genios Pro plate reader. Gene expression was evaluated based on the fluorescence normalized to a total amount of protein present. Toxicity was measured using an MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay after 48 h incubation of cells with treatments. Transfection and incubation was carried out identical to that of the luciferase transfection experiments except the transfected cells were incubated with MTT for 1 h then lysed with DMSO (dimethyl sulfoxide). The absorbance was measured at 570 nm, using the same plate reader, to determine the relative cell viability of transfected cells.
Figure 4. Transfection efficiency of the three polyplex formulations using gWiz-luciferase plasmid DNA in U-87 MG cells.
suggest there may be a cell type dependence on transfection when using Ad-p(MAG)52 in the polyplex formulations, which, as previously mentioned, could be due to differences in the cell surface chemistry and glucose transporters among cell types. MTT toxicity assays confirmed the previously reported10 trend of increased cellular toxicity with the increase in N/P for polyplexes created with unmodified click cluster and pDNA (Supporting Information, SI Figure 14). However, this was not the case when incorporating Ad-p(MAG)52 into the formulations. Remarkably, AD-pMAG-modified polyplexes at all N/Ps (up to 50) showed little to no toxicity in all three cell lines. A similar result was obtained for polyplexes formulated with pDNA and click clusters modified with Ad-PEG. In this study, we have shown the successful polymerization of Ad-p(MAG)52 and that incorporation of Ad-p(MAG)52 into click cluster/pDNA polyplexes provides improved colloidal stabilization under physiological conditions. The length of p(MAG) used in this initial study was a DP=52 and additional studies need to be conducted to probe the effect of Adp(MAG) molecular weight on the colloidal stability similar or better than Ad-PEG at higher N/P ratios of polyplex formulation. The toxicity properties of the click clusters appeared to be significantly improved by p(MAG) as no change in cell viability was observed even at a high N/P ratio of 50. The decrease in toxicity, along with a trithiocarbonate “handle” useful for chemical modifications, make p(MAG) a good candidate for incorporation of other targeting groups. There was a significant effect of Ad-p(MAG) on the biological properties of the polyplexes, which appears to be dependent on the cell type studied. Ad-p(MAG)52-containing polyplexes were poorly taken up by HeLa cells. Cellular internalization in both GBM cell lines, however, was very efficient. Cell-type dependence is currently being further investigated to reveal the full potential of p(MAG) as a coating layer for click clusters. Overall, the use of p(MAG), instead of PEG, could be advantageous in some applications to impart colloidal stability, hydrophilicity, and a terminal thiol for facile inclusion of targeting moieties or other biologically active molecules.
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ASSOCIATED CONTENT
S Supporting Information *
Synthetic methods, material characterization, polyplex formation and characterization, biological evaluation, and binding constant determination procedures. This material is available free of charge via the Internet at http://pubs.acs.org.
EXPERIMENTAL METHODS
The Ad-pMAG polymer used in the polyplex formulations was synthesized through a RAFT polymerization technique using an adamantane derivative of 4-cyano-4-(propylsulfanylthiocarbonyl) sulfanylpentanoic acid (CPP). The chain transfer agent (CTA) and 2-deoxy-2-methacrylamido glucopyranose monomers were synthesized according to previously published procedures.15 The CPP, synthesized according to a published procedure,16 was reacted with adamantane
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. 612
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Notes
The authors declare the following competing financial interest(s): T.M.R. is a consultant to and has stock options in Techulon, Inc.
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ACKNOWLEDGMENTS Special thanks go out to Techulon, Inc., for donation of the Glycofect transfection reagent. Major funding was provided by the Virginia Tech Department of Chemistry as well as National Institutes of Health (NIH; #DP2OD006669-01), Alfred P. Sloan Foundation, and the Dreyfus Foundation. Sincere thanks to Keith Ray for all the MALDI analysis as well as Dr. Mehdi Ashraf-Khorassani for all the LC-MS and HPLC analysis.
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REFERENCES
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