Bioconjugate Chem. 2007, 18, 2077–2084
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Degradable-Brushed pHEMA–pDMAEMA Synthesized via ATRP and Click Chemistry for Gene Delivery Xulin Jiang,†,‡ Martin C. Lok,† and Wim E. Hennink*,† Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS) Utrecht University, P.O. Box 80 082, 3508 TB Utrecht, The Netherlands, and Key Laboratory of Biomedical Polymers of Ministry of Education and Department of Chemistry, Wuhan University, Wuhan 430072, P.R. China. Received April 7, 2007; Revised Manuscript Received August 20, 2007
Brushed polymers composed of a backbone of poly(hydroxyethyl methacrylate) (pHEMA) onto which poly(2(dimethylamino)ethyl methacrylate)s (pDMAEMAs) was grafted via a hydrolyzable linker were synthesized and evaluated as nonviral gene delivery vectors. Both pDMAEMA and pHEMA polymers with controlled molecular weights and narrow distributions were synthesized by controlled atom transfer radical polymerization (ATRP). The azide initiator was used to ensure complete and monoazide functionalization of the pDMAEMA polymer chains. Click reaction between pHEMA with alkyne side groups and the azide end group in the pDMAEMA resulted in a high-molecular-weight polymer composed of low-molecular-weight constituents via an easily degradable carbonate ester linker. The length of the pDMAEMA grafts as well as the number of grafts of the brushed pHEMA–pDMAEMA can be easily varied. At physiological conditions (pH 7.4 and 37 °C), the brushed polymer degraded by hydrolysis of the carbonate ester with a half-life of 96 h. The molecular weights of the formed degradation products was very close to that of the starting pDMAEMA, which is likely below the renal excretion limit (75 kDa) were able to condense DNA into small particles (90–190 nm, at a polymer to plasmid mass ratio g6). This is in agreement with a previous report in which it was demonstrated that only high-molecular-weight pDMAEMA is able to condense the structure of plasmid DNA effectively (33). As can be seen in Figure 4, the size of the pHEMA– pDMAEMA-1 based polyplexes is smaller than that of the pHEMA–pDMAEMA-3-based polyplexes when the polymer/ DNA mass ratio is >3, although the weight-average molecular weight of pHEMA–pDMAEMA-1 is lower than that of pHEMA–pDMAEMA-3. This implies that the size of the polyplexes does not increase with the molecular weight of the polymer anymore when the molecular weight is high enough (Mw above 75 kDa). The zeta potentials of the brushed pHEMA–pDMAEMA-based polyplexes were independent of the molecular weight of pHEMA–pDMAEMA. The binding of polymers to plasmid DNA was also studied by ethidium bromide fluorescence measurement in HBS buffer (Table 4). This table shows that all tested polymers quenched the plasmid DNA/ethidium bromide fluorescence. The brushed pHEMA–pDMAEMAs showed more quenching than the starting pDMAEMA, indicating a weaker binding of this low-molecular-weight polymer to the plasmid DNA. However, the brushed pHEMA–pDMAEMAs
Table 3. SEC Results of Brushed pHEMA–pDMAEMA Copolymers (Alkyne/Azide Molar Ratio 4:1)a name
p(HEMA-co-HEMA-PPA) used
Mn, kDa
Mw, kDa
PDI
p(HEMA-co-HEMA-PPA)1 p(HEMA-co-HEMA-PPA)2 p(HEMA-co-HEMA-PPA)3
8.7 45.2 50.9 52.7
10.0 79.3 158 589
1.15 1.75 3.10 11.2
pDMAEMA-2 pHEMA-pDMAEMA-1 pHEMA-pDMAEMA-2 pHEMA-pDMAEMA-3 a
SEC method as described previously (48).
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Figure 4. Particle size and zeta potential of brushed pHEMA– pDMAEMA-based polyplexes as a function of polymer/DNA ratio. Square, size of pHEMA–pDMAEMA-1-based polyplexes; circle, size of pHEMA–pDMAEMA-2-based polyplexes; triangle, size of pHEMA– pDMAEMA-3-based polyplexes; open square, zeta potential of pHEMA–pDMAEMA-1-based polyplexes; open circle, zeta potential of pHEMA–pDMAEMA-2-based polyplexes; open triangle, zeta potential of pHEMA–pDMAEMA-3-based polyplexes.
Figure 6. β-Galactosidase expression after incubation of COS-7 cells with polyplexes based on the degradable brushed pHEMA–pDMAEMA polymers and the reference polymer linear PEI in the presence of INF-7 peptide.Square,pHEMA–pDMAEMA-1;circle,pHEMA–pDMAEMA-2; triangle, pHEMA–pDMAEMA-3; diamond, linear PEI.
Figure 7. Relative cell viability of polyplexes based on the degradable brushed pHEMA–pDMAEMA polymers and the reference polymers pDMAEMA and linear PEI in the presence of INF-7 peptide in COS-7 cells. Square, pHEMA–pDMAEMA-1; circle, pHEMA–pDMAEMA2; triangle, pHEMA–pDMAEMA-3; star, reference pDMAEMA; diamond, linear PEI.
Figure 5. Polyplex (polymer/DNA mass ratio 3:1) destabilization studied by DLS. Square, pHEMA–pDMAEMA-1-based polyplexes at pH 7.4 and 5 mM HEPES; triangle, pHEMA–pDMAEMA-3-based polyplexes at pH 7.4 and 5 mM HEPES; open square, pHEMA– pDMAEMA-1-based polyplexes at pH 5 and 10 mM NaAc; open triangle, pHEMA–pDMAEMA-3-based polyplexes at pH 5 and 10 mM NaAc; star, pHEMA–pDMAEMA-1-based polyplexes at pH 7.4 and HBS.
showed similar quenching as high-molecular-weight pDMAEMA. This is in agreement with the DLS results. The destabilization of pHEMA–pDMAEMA polyplexes was investigated by DLS. Figure 5 shows the particle size of pHEMA–pDMAEMA-1 and pHEMA–pDMAEMA-3 polyplexes (with the polymer to DNA mass ratio of 3) as a function of time at 37 °C and at pHs 5.0 and 7.4. The size of the polyplexes was constant at pH 5.0 in 10 mM NaAC over the investigated time period (7 days), while the size of the pHEMA–pDMAEMA-1-based polyplexes incubated at pH 7.4 increased substantially over time (Figure 5). The size of pHEMA–pDMAEMA-3 (relatively higher molecular weight) based polyplexes in 10 mM HEPES at pH 7.4 increased very
slowly, and the size of the polyplexes based on high-molecularweight pDMAEMA was very stable under the same conditions (data not shown). Likely, the higher-molecular-weight brushed pHEMA–pDMAEMA needs more time to degrade completely than the lower-molecular-weight ones. The observation that polyplexes based on high-molecular-weight pDMAEMA are stable over time after incubation at physiological conditions is in agreement with previous data that this polymer is very stable at pH 7.4 and 37 °C (51). The time period in which aggregation of the polyplexes occurred is on the same order of magnitude as pHEMA–pDMAEMA-1 polymer hydrolysis (compare Figure 5 (95 h) with Figure 3 (96 h)), which indicates that the degradation of brushed pHEMA–pDMAEMA polymers is hardly influenced by the complexation with DNA (45). The polyplexes in HBS buffer aggregated much more quickly than those in 5 mM HEPES buffer, especially for low polymer/DNA ratio or for polymers with low molecular weight (Figure 5). Obviously, chemical degradation results in a decrease in the colloidal stability of the polyplexes in HEPES-buffered saline. In Vitro Transfection Efficiency and Cytotoxicity Studies. The transfection activity and toxicity of the different polyplexes was studied in vitro in COS-7 cells (Figures 6 and 7). Figure 6 shows the expression levels of β-galactosidase after incubation of COS-7 cells for 1 h with polyplexes based on the
Table 4. Relative fluorescence of ethidium bromide/plasmid DNA incubated with different cationic polymers in HBS buffer (DNA concentration, 10 µg/mL; ethidium bromide/plasmid DNA, 1:10 (mol/mol)) polymer/DNA (w/w)
pDMAEMA-2
pHEMA–pDMAEMA-1
pHEMA–pDMAEMA-2
pHEMA–pDMAEMA-3
pDMAEMA ref
12 6 3
0.50 ( 0.01 0.50 ( 0.02 0.51 ( 0.01
0.33 ( 0.02 0.36 ( 0.01 0.39 ( 0.02
0.31 ( 0.02 0.33 ( 0.03 0.38 ( 0.02
0.34 ( 0.05 0.36 ( 0.05 0.45 ( 0.03
0.33 ( 0.07 0.36 ( 0.04 0.38 ( 0.05
Degradable-Brushed pHEMA–pDMAEMA for Gene Delivery
three degradable brushed pHEMA–pDMAEMA polymers and the reference polymer, linear PEI, as a function of the polymer to DNA ratio in the presence of INF-7 peptide. The transfection efficiency of the polyplexes based on high-molecular-weight pDMAEMA at a polymer/DNA ratio of 3/1 (w/w) was set at 1.0 (33). The highest transfection was observed for the polyplexes based on the highest-molecular-weight pHEMA– pDMAEMA-3 at a polymer/DNA mass ratio of 6/1, with a tranfection efficiency about 2.5 times higher than that of the polyplexes based on the reference pDMAEMA and about 3.1 times higher than that of the polyplexes based on linear PEI (Exgen 500). The polyplexes based on lower-molecular-weight brushed pHEMA–pDMAEMA showed lower transfection, which is in agreement with the results reported previously (33). The transfection of the pDMAEMA-2-based polyplexes was negligible (data not shown). We also studied the transfection activity of the polyplexes without addition of the INF peptide. In line with previous data (52), the transfection was lower (3–4 times), but the observed trends were the same as those observed for the polyplexes with INF. Importantly, the brushed pHEMA–pDMAEMAs showed lower cellular toxicity as compared to reference pDMAEMA (Figure 7). Of the investigated brushed polymers, pHEMA– pDMAEMA-3-based polyplexes showed the highest transfection efficiency and the lowest toxicity, making this polymer an interesting candidate for future studies.
CONCLUSION A new degradable brushed pHEMA–pDMAEMA was synthesized via ATRP and click chemistry for gene delivery. The click reaction and the degradation process were monitored clearly by aqueous SEC. The molecular weight of the final main degradation product pDMAEMA was very close to that of the starting pDMAEMA, which was coupled to p(HEMA-coHEMA-PPA) via the click reaction and is likely below the renal excretion range (
