Development of Poly(β-amino ester)-Based Biodegradable Nanoparticles for Nonviral Delivery of Minicircle DNA

Gene therapy provides a powerful tool for regulating cellular processes and tissue repair. Minicircle (MC) DNA are supercoiled DNA molecules free of bacterial plasmid backbone elements and have been reported to enhance prolonged gene expression compared to conventional plasmids. Despite the great promise of MC DNA for gene therapy, methods for safe and efficient MC DNA delivery remain lacking. To overcome this bottleneck, here we report the development of a poly(β-amino ester) (PBAE)-based, biodegradable nanoparticulate platform for efficient delivery of MC DNA driven by a Ubc promoter <i>in vitro</i> and <i>in vivo.</i> By synthesizing and screening a small library of 18 PBAE polymers with different backbone and end-group chemistry, we identified lead cationic PBAE structures that can complex with minicircle DNA to form nanoparticles, and delivery efficiency can be further modulated by tuning PBAE chemistry. Using human embryonic kidney 293 cells and mouse embryonic fibroblasts as model cell types, we identified a few PBAE polymers that allow efficient MC delivery at levels that are comparable or even surpassing Lipofectamine 2000. The biodegradable nature of PBAE-based nanoparticles facilitates <i>in vivo</i> applications and clinical translation. When injected <i>via</i> intraperitoneal route <i>in vivo</i>, MC alone resulted in high transgene expression, and a lead PBAE/MC nanoparticle formulation achieved a further 2-fold increase in protein expression compared to MC alone. Together, our results highlight the promise of PBAE-based nanoparticles as promising nonviral gene carriers for MC delivery, which may provide a valuable tool for broad applications of MC DNA-based gene therapy

Additional file 3: Figure S3. of The effects of immunomodulation by macrophage subsets on osteogenesis in vitro

Showing that polarized macrophages retain phenotypes in coculture. Supernatant from MC3T3 monocultures and macrophage-MC3T3 co-cultures were collected at 72 hours and weeks 1, 2, and 3 and analyzed for TNF-Îą a and IL-1ra b protein levels by ELISA. IL-1ra/TNF-Îą ratio was calculated c. Cells were also labeled with CD11b, iNOS, and CD206 anti-mouse antibodies and analyzed by flow cytometry. After gating for CD11b+ cells, iNOS and CD206 expression was analyzed d. *p <0.05, **p <0.01, ***p <0.001, and ****p <0.0001. IL interleukin, iNOS inducible nitric oxide synthase (TIF 930 kb

Additional file 4: Figure S4. of The effects of immunomodulation by macrophage subsets on osteogenesis in vitro

Showing that IL-4 treatment of M1-MC3T3 cocultures modulates M1 macrophages to the M2 phenotype. Supernatant from untreated and IL-4 treated M1-MC3T3 co-cultures and untreated M2-MC3T3 co-cultures were collected at 72 hours and week 1 and analyzed for TNF-Îą a and IL-1ra b protein levels by ELISA. IL-1ra/TNF-Îą ratio was calculated c. **p <0.01, ***p <0.001, and ****p <0.0001. IL interleukin (TIF 2669 kb

Detection of reporter macrophages from tissue sections.

<p>Lentivirus and cyclosporine transduced reporter macrophages where injected into the tail vein of mouse model of biomaterial induced local inflammation and osteolysis. Reporter cell accumulation to the right distal femur was confirmed by bioluminescence imaging. After 21 days the right and left femur were collected, processed into frozen sections and the reporter macrophages detected by FLUC immunostaining. <b>a)</b> Low power micrographs showing the transverse sections of distal metaphyseal region of left and right femur. On the right side the channel left behind by the titanium rod is evident (asterisk). <b>b)</b> High power micrographs showing several aggregations of FLUC positive cells (arrow heads) in the bone marrow surrounding the rod channel on the right side while only few FLUC positive cells are detected in the bone marrow of the left femur. <b>c)</b> GFP-FLUC transduced and non-transduced RAW 264.7 cells stained for FLUC served as additional positive and negative immunostaining controls. Scale bars 200μm.</p

Additional file 5: Figure S5. of The effects of immunomodulation by macrophage subsets on osteogenesis in vitro

Showing that IL-4 treatment of MC3T3 cells at 72 hours does not affect their osteogenic ability. Untreated and IL-4 treated MC3T3 monocultures cultured in MC3T3 growth medium or mixed medium were stained with Alizarin Red, destained, and quantified by absorbance at 562 nm. IL interleukin, n.s. not significant, OD optical density (TIF 43 kb

Reporter macrophages in in vivo bioluminescence imaging.

<p>Lentivirus and cyclosporine transduced reporter macrophages where injected into the tail vein of mouse model of biomaterial induced local inflammation and osteolysis. The cell trafficking was followed by bioluminescence imaging. Luminescence emanating from the local area of inflammation in the right distal femur as well as left distal femur serving as a control was determined from images obtained every other day up to 20 days post injection. <b>a)</b> Bioluminescence images showing the accumulation of reporter cells to the right distal femur starting on the day 2 after the systemic injection of the reporter cells and persisting over the 20 day imaging period. <b>b)</b> XY-blot showing the total flux from regions of interest over the right and left distal femurs. * = p > 0.05; ** = p > 0.01, *** = p > 0.001.</p

GFP expression in reporter macrophages.

<p>GFP and FLUC expressing reporter macrophages were produced by transducing mouse bone marrow macrophages with lentivirus vector in the presence of cyclosporine, DEAE-dextran or polybrene. The 24-hour virus infection was done either once or repeated after a 24-hour rest period (single vs. double infection). <b>a)</b> Fluorescence microscopy images of the GFP positive macrophages in the indicated treatment groups three days after the second infection. <b>b)</b> Representative scatter blots of GFP positive reporter macrophage in flow cytometry. <b>c)</b> Percentage of GFP positive macrophages in the indicated treatment groups as determined by flow cytometry. Cy–Cyclosporine; De–DEAE-dextran; Po–Polybrene; D–Double infection; S–Single infection; *** = p > 0.001 cyclosporine double infection vs. dextran and polybrene single and double infection; * = p > 0.05 cyclosporine double vs. single infection; + = p > 0.05 cyclosporine single infection vs. dextran and polybrene single and double infection.</p

Reporter macrophage viability.

<p>GFP and FLUC expressing reporter macrophages were produced by transducing mouse bone marrow macrophages with lentivirus vector in the presence of cyclosporine, DEAE-dextran or polybrene. The 24-hour virus infection was done either once or repeated after a 24-hour rest period (single vs. double infection). The percentage of viable cells was assessed three days after the second infection by flow cytometry and propidium iodide staining.</p

Additional file 1: Figure S1. of Preconditioning of murine mesenchymal stem cells synergistically enhanced immunomodulation and osteogenesis

Showing immunophenotypes of murine bone marrow-derived MSCs. Surface marker expression in murine MSCs at passage 4 analyzed by flow cytometry. Figure S2. showing titration of LPS dose for MSC preconditioning with TNF-α to modulate murine macrophage polarization. Murine MSCs were preconditioned with TNF-α (20 ng/ml) plus LPS (1–20 μg/ml) for 3 days, and cocultured with M1 macrophages for 24 hours. M2 (Arg1 and CD206) and M1 (TNF-α/IL-1Ra) macrophage marker expression in macrophages measured by quantitative PCR. Data presented as fold-change compared to macrophages cocultured with unstimulated control MSCs. *p < 0.05, **p < 0.01, ***p < 0.005. Figure S3. showing increased osteogenic differentiation in the MSCs with temporal inhibition of COX2 signaling. MSCs were treated with 25 μM Celecoxib in osteogenic media at indicated time points. Osteogenic differentiation ability examined by bone mineralization (Alizarin Red staining) at week 3. *p < 0.05, ***p < 0.005. Figure S4. showing increased iNOS expression in MSCs preconditioned with TNF-α and/or LPS. Murine MSCs were preconditioned with TNF-α (20 ng/ml) plus LPS (1–20 μg/ml) for 3 days, and washed out for 24 hours. Expression of iNOS determined by quantitative PCR. Data presented as fold-change compared to macrophages cocultured with unstimulated control MSCs. ***p < 0.005. (PDF 3973 kb

GFP and FLUC expression in polarized reporter macrophages.

<p>Lentivirus and cyclosporine transduced reporter macrophages were polarized into M1 and M2 phenotypes or left untreated (M0 macrophages) and the expression of GFP and FLUC determined 48 hours after the end of the polarization treatment. <b>a)</b> Fluorescence microscopy images of the GFP positive macrophages in the indicated treatment groups. <b>b)</b> Ratio of GFP positive macrophages in the indicated treatment groups compared to non-polarized M0 reporter macrophages as determined by flow cytometry <b>c)</b> FLUC activity in indicated treatments groups compared to the non-polarized M0 reporter macrophages as determined by Luciferase Assay System kit and luminometer.</p