Intramuscular delivery of DNA releasing microspheres: microsphere properties and transgene expression

Abstract

Plasmid-loaded microspheres can provide localized and sustained release into the target tissue, and thus have the potential to enhance the efficiency of naked DNA at promoting transgene expression. In this report, microsphere design parameters are investigated by correlating the extent and duration of transgene expression intramuscularly to the polymer molecular weight and the mass of DNA delivered. Plasmid DNA was incorporated into poly (lactide-co-glycolide) microspheres using a cryogenic double emulsion process, and microspheres were injected intramuscularly. Bolus injection of naked plasmid was used for control, which exhibited transfection of muscle cells with transgene expression that gradually decreased over time. Microspheres fabricated from low molecular weight polymer had expression levels that increased from day 1 to day 92, which subsequently decreased through day 174. Decreasing the microsphere mass delivered resulted in steady expression during the same time. However, microspheres fabricated with high molecular weight polymer had expression for only 14 days. Intramuscular injection resulted in a foreign body response to the microspheres, and these infiltrating cells adjacent were primarily transfected. This understanding of microsphere properties that determine transgene expression and the distribution of transfected cells may facilitate their application to fields such as tissue engineering or DNA vaccines.

Fig. 1

Scanning electron photomicrograph of DNA-loaded microspheres fabricated with high molecular weight (A) and low molecular weight (B) polymer. Size bar represents 10μm. (C) Photomicrograph of agarose gel with plasmid extracted from microspheres fabricated with high MW. Lane 1: molecular weight marker, lane 2: incorporated DNA in microspheres, and lane 3: unincorporated DNA.

Fig. 2

Bioluminescence imaging of intramuscular DNA delivery. pLuc (50 μg) was delivered as a bolus (A) or encapsulated within low MW PLG microspheres (B, 5 μg/mg microsphere). (A, B) Images showing a single mouse treated with bolus or microspheres at several time points. (C) CCD signal intensity (photons/s) for bolus delivery of pLuc (■), or microspheres with encapsulated pLuc. (●). Control conditions included microspheres without pLuc (x) and background light emission (○) (i.e., imaging before D-luciferin injection) (n=4).

Fig. 3

In vivo bioluminescence signal intensities for varying doses of plasmid-loaded microsphere (low MW). PLG microspheres with similar DNA loading (approximately, 5 μg of DNA/mg of microspheres) were injected intramuscularly. (A) The mass of microspheres delivered was 10.9 mg (50 μg initial loading) (●) or 4.4 mg (20 μg initial loading) (▼). The signal intensities shown in Fig. 2 (10.9mg) were replotted for convenient comparison. Control conditions included microspheres without pLuc (x) and background light emission (○). (B) Average level of transgene expression between days 1–14 (□) and days 56–92 (■) for each condition. The symbols *, ** indicate significant differences between the average levels for 50 μg-loading (P<0.001) and between average levels at later time points for each DNA loading (P=0.003), respectively.

Fig. 4

In vivo bioluminescence signal intensities for microspheres of varying molecular weight PLG. Two populations of microspheres with the same DNA loading (approximately, 5 μg of DNA/mg of microspheres) were fabricated with high (◆) and low (●) molecular weight PLG. Signal levels for (●) are those shown in Fig. 2 through 35 days, and are replotted for convenient comparison. Control conditions included microspheres without pLuc (x) and background light emission (○).

Fig. 5

Muscle tissue containing injected microspheres at low magnification. Tissue samples were retrieved and sectioned at day 2 (A, B) and day 50 (C, D). Multiple images were assembled to represent the entire region containing microspheres. The polymer used for microsphere fabrication was high MW (A, C) and low MW (B, D). Labels indicate polymer (P) and muscle tissue (M). Scale bar represents 1.7 mm.

Fig. 6

Inflammatory cell infiltration into muscle tissue. Tissues with injected DNA (bolus and microspheres) were retrieved and sectioned at day 2 (A, C, E) or day 50 (B, D, F). DNA was delivered by bolus injection (A, B), high molecular weight PLG microspheres (C, D), or low molecular weight PLG microspheres (E, F). Labels indicate polymer (P), muscle tissue (M), and inflammatory cells (arrows). The scale bar equals 100 μm.

Fig. 7

Immunohistochemical staining for luciferase expression at day 2 and day 50. Tissues were retrieved and sectioned at day 2 (A, C, E) or day 50 (B, D, F). The sections were stained following injection of plasmid as a bolus (A), and encapsulated within high MW (C, D) or low MW PLG microspheres (E, F). An example of control staining (no primary antibody) is shown (B). Magnification is 200× (A, C, D, E, F) and 400× (B). The label indicates polymers (P). Scale bar represents 100 μm (A, C, D, E, F) and 50 μm (B).