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. 2011 Jan;6(1):25-42.
doi: 10.2217/nnm.10.129.

Polyelectrolyte Complex Optimization for Macrophage Delivery of Redox Enzyme Nanoparticles

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Free PMC article

Polyelectrolyte Complex Optimization for Macrophage Delivery of Redox Enzyme Nanoparticles

Yuling Zhao et al. Nanomedicine (Lond). .
Free PMC article

Abstract

Background: We posit that cell-mediated drug delivery can improve transport of therapeutic enzymes to the brain and decrease inflammation and neurodegeneration seen during Parkinson's disease. Our prior works demonstrated that macrophages loaded with nanoformulated catalase ('nanozyme') then parenterally injected protect the nigrostriatum in a murine model of Parkinson's disease. Packaging of catalase into block ionomer complex with a synthetic polyelectrolyte block copolymer precludes enzyme degradation in macrophages.

Methods: We examined relationships between the composition and structure of block ionomer complexes with a range of block copolymers, their physicochemical characteristics, and loading, release and catalase enzymatic activity in bone marrow-derived macrophages.

Results: Formation of block ionomer complexes resulted in improved aggregation stability. Block ionomer complexes with ε-polylysine and poly(L-glutamic acid)-poly(ethylene glycol) demonstrated the least cytotoxicity and high loading and release rates. However, these formulations did not efficiently protect catalase inside macrophages.

Conclusion: Nanozymes with polyethyleneimine- and poly(L-lysine)(10)-poly(ethylene glycol) provided the best protection of enzymatic activity for cell-mediated drug delivery.

Figures

Figure 1
Figure 1
A pictorial scheme of different block copolymers used in this study. EPL: ε-polylisine; PEG: Polyethylene glycol; PEI: Polyethyleneimine; PGLU: Poly(L-glutalic) acid; PL: poly(L-lysine).
Figure 2
Figure 2
AFM images of naked catalase (A) and different catalase nanozymes (Z=1): PEI50-PEG/catalase (B); PL10-PEG/catalase (C); PEI50/catalase (D); PL50-PEG/catalase (E); and PGLU50-PEG/catalase (F). Nanozymes were prepared at an enzyme concentration of 1 mg/mL, and then diluted 100 times in PBS prior to the application on positively charged mica (APS) for 2 minutes, washed with deionized water and dried under an argon flow. Images revealed sharp differences between large aggregates of naked catalase and spherical particles with size corresponding to a single catalase globule in case of nanozymes.
Figure 3
Figure 3
Effect of block copolymer structure on catalase nanozyme uptake in BMM. A) Kinetics of representative catalase nanozymes (Z = 1) accumulation in BMM: PL50-PEG/catalase (filled squares); PL10-PEG/catalase (filled diamonds); PGLU50-PEG/catalase (filled triangles); EPL/catalase (empty squares); catalase alone (empty circles). B) Accumulation of representative catalase nanozymes (Z = 1) in BMM. Cells were treated with (A) the RITC-labeled enzyme (1 mg/ml) in assay buffer alone or different nanozymes for various time points or (B) 125I-labeled enzyme or nanozymes for one hour. Following incubation, (A) the cells were washed three times with ice-cold PBS, solubilized in Triton X100 (1%), and the amount of fluorescence was measured by fluorescent spectrophotometer (λex=488 nm, λem=510 nm), or (B) the cellular content was precipitated by TCA, and the amount of radioactivity in the precipitate was measured by radioactivity counter. Results from N=8 wells (± SEM) demonstrating significant increase in accumulation of nanozyme without PEG corona; decrease of nanozyme obtained with PEG and positively-charged block copolymer, and no effect on nanozyme comprised of PEG and negatively-charged block copolymer, compared to “naked” catalase. Statistical significance is shown by asterisk: p<0.05 (*), and p<0.005 (**).
Figure 4
Figure 4
Release profile of different nanozymes from BMM: PL50-PEG/catalase (filled triangles); PL10-PEG/catalase (filled squares); PEI50-PEG (filled diamonds); PGLU50-PEG/catalase (crosses); catalase alone (empty diamonds). Cells were loaded with RITC-labeled catalase/block copolymer complex (1 mg/ml, Z = 1) for two hours, washed with PBS, and incubated with catalase-free media for various time intervals. Then, the media was collected and fluorescence in the each sample was accounted by fluorescent spectrophotometry as described in Figure 2 legend. The amount of the released RITC-labeled nanozyme was normalized for protein content and expressed in μg enzyme per ml media. Results from N=8 wells (± SEM) demonstrating significant increase in release of nanozyme without PEG corona; decrease of nanozyme obtained with PEG and positively-charged block copolymer, and no effect on nanozyme comprised of PEG and negatively-charged block copolymer, compared to “naked” catalase. Statistical significance is shown by asterisk: p<0.05 (*), and p<0.005 (**).
Figure 5
Figure 5
Effect of the amount of block copolymer in BIC on enzymatic activity of catalase in different BIC determined by hydrogen peroxide decomposition. The catalase activities were measured by spectrophotometry. In particular, 1-4 μL H2O2 (7.5 – 30 % v/w), and 2 μL catalase (0.06 - 0.5 mg/ml) or various nanozymes with different Z ratios were added into a cuvette with 1 mL PBS, and the changes in absorbance at 240 nm were monitored. Incorporation of catalase into polyelectrolyte complex resulted in a significant increase of catalase activity in PEG-containing nanozymes, and decrease in non-PEG nanozymes. Values are means ± SEM (N = 8), P<0.05 (*) and P<0.005 (**) compared with naked catalase.
Figure 6
Figure 6
Preservation of catalase enzymatic activity in selected nanozymes. The stability of catalase in BIC was examined upon incubation of different nanozymes (0.5 mg/ml catalase) with trypsin (10-5 M), or pronase (2×10-1 mg/ml) for 3 hours at 37°C. Following incubation, the aliquots were subjected for catalytic activity assessment as described in Figure 4 legend. A residual activity of catalase is expressed as a ratio of enzyme activity after 3h of incubation in the presence of pronase at 37°C to the initial one (at time point 0). Results from N=4 experiments (± SEM) demonstrating that incorporation of catalase into BIC with all studied polymer (with and without PEG) drastically increased stability of catalase against a mixture of proteinases (pronase). Statistical significance is shown by asterisk: P<0.005 (**) compared to “naked” catalase.
Figure 7
Figure 7
Modulation of BMM-derived ROS by catalase nanozymes (Z = 1). BMM were stimulated with LPS (20 ng/ml) and γ-INF 2 (μg/ml) for 24 hours, and then the media was supplemented with: catalase alone (empty circles); PL50-PEG/catalase (filled squares); PL10-PEG/catalase (filled diamonds); PEI50-PEG (crosses); PGLU50-PEG/catalase (filled triangles); fresh media (empty triangles). Control non-activated BMM were incubated with fresh media (empty squares). Then cell media was supplemented with Amplex Red Dye stock solution (10 U/mL HRP, 10 mM Ampex Red) for 30 minute, and the amount of H2O2 produced by BMM and decomposed by catalase nanozymes was measured by fluorescence at λex=563 nm, λem=587 nm. The maximal activity of nanozymes was observed for BIC obtained with positively-charged block copolymers. Data represent means ± SEM (N = 8). Statistical significance of the amount of H2O2 decomposed by nanozyme or catalase, compared to activated microglia is shown by asterisks: (*) p<0.05, (**) p<0.005.
Figure 8
Figure 8
Preservation of enzymatic activity of catalase against degradation in BMM. “Naked” catalase (first bar) or different nanozymes (Z = 1, 2, or 5) were loaded into BMM, cells were washed and incubated with catalase-free media for two hours. Then, the media was collected, and the activity of catalase released from BMM was accounted by spectrophotometry as described in Figure 4 legend. All nanozymes with PEG corona comprising of positively-charged block copolymers showed efficient protection of catalase inside macrophages compared to catalase loaded alone. Increasing of the amount of the block copolymer in the BIC leaded to better protection of the enzyme. In contrast, nanozymes based on positively-charged monopolymers without PEG corona, or negatively-charged block copolymer did not protect catalase inside the host cells. Data represent means ± SEM (N = 4). Statistical significance of nanozyme activity compared to catalase alone is shown by asterisks: (*) p<0.05, (**) p<0.005.
Figure 9
Figure 9
Effect of catalase nanozymes on endosomal acidification in BMM. Mature mouse BMM were loaded with RITC-labeled catalase alone (A) or nanozymes (1 mg/ml, Z = 5); PEI50-PEG/catalase (B); PEI50/catalase (C), PGLU50-PEG/catalase (D), EPL/catalase (E), or PL10-PEG/catalase (F) for two hours, washed with PBS, and stained with 1 μM LysoSensor dye. Confocal images indicate that nanozymes comprising of catalase and positively-charged polymers, especially, with PEI block, substantially increased the pH in the compartments containing catalase. Oppositely, loading of BMM with negatively-charged containing nanozyme resulted in further decreases in endosomal pH compared to “naked” catalase.
Figure 10
Figure 10
A pictoral scheme for different nanozyme structures evaluated for cell-mediated drug delivery. Three types of catalase nanozymes: Nanozyme I: contaning a negatively cherged block copolymer (with PEG) showed low toxicity, high loading capacity and sustained release from BMMs, but limited enzyme protection inside cell carriers; Nanozyme II: containing positively charged block copolymers (with PEG) showed increased cytotoxicity and low loading and release rates, but high level of nanozyme protection ; Nanozymes III: containing monopolymers (without PEG corona) showed higher loading and release rates, but low enzyme protection inside BMMs along with decreased catalase activity in cell-free system. The optimal nanozyme formulation selected from nanozyme II group is based on positively-charged block copolymers (PEI48-PEG/catalase and PL10-PEG/catalase) that demonstrate efficient protection of catalase enzymatic activity along with relatively high loading and release rates and limited cytotoxicity.

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