A naonoporous cell-therapy device with controllable biodegradation for long-term drug release

Abstract

Herein we describe the development and implementation of a nanoporous cell-therapy device with controllable biodegradation. Dopamine-secreting PC12 cells were housed within newly formulated alginate-glutamine degradable polylysine (A-GD-PLL) microcapsules. The A-GD-PLL microcapsules provided a 3-D microenvironment for good spatial cell growth, viability and proliferation. The microcapsules were subsequently placed within a poly(ethylene glycol) (PEG)-coated poly(ε-caprolactone) (PCL) chamber covered with a PEG-grafted PCL nanoporous membrane formed by phase inversion. To enhance PC12 cell growth and to assist in controlled degradation of both the PC12 cells and the device construct, small PCL capsules containing neural growth factor (PCL-NGF) and a poly(lactic-co-glycolic acid) pellet containing glutamine (PLGA-GLN) were also placed within the PCL chamber. Release of NGF from the PCL-NGF capsules facilitated cell proliferation and viability, while the controlled release of GLN from the PLGA-GLN pellet resulted in A-GD-PLL microcapsule degradation and eventual PC12 cell death following a pre-specified period of time (4 weeks in this study). In vivo, our device was found to be well tolerated and we successfully demonstrated the controlled release of dopamine over a period of four weeks. This integrated biodegradable device holds great promise for the future treatment of a variety of diseases.

Fig. 1

Design of a multifunctional and biodegradable cell-based device. A-GD-PLL microcapsules containing PC12 cells were enclosed within a large nanoporous PCL chamber and cultured with a small PCL-NGF chamber. Therapeutic levels of dopamine were produced and released from the integrated device. A-GD-PLL microcapsule degradation was initiated following exposure to GLN. After a designated time (varies based on PCL outer chamber degradation rate), the entire device degraded and PC12 cells were cleared by the host immune system.

Fig. 2

Schematic diagram of DTBP-PLL synthesis and GLN cleavage. A-GD-PLL microcapsules were generated by cross-linking dimethyl dithiobispropionimidate (DTBP) with PLL at a 2:1 molar ratio of DTBP to primary amine. The addition of GLN results in DTBP-PLL cleavage.

Fig. 3

A-GD-PLL microcapsules containing PC12 cells in a 24-well plate on Day 14: Phase contrast images of (A) PC12 cells grow to small clusters without NGF, (B) PC12 cells grow to large spheroids with NGF supplementation, and fluorescence images of (C) PC12 cells growth without NGF, and (D) PC12 cells growth with NGF. In images (C) & (D), the living cells showed the green color and the dead cells stained the red color. Phase contrast images of (E) PC12 cell growth within PCL devices without PCL-NGF capsules, (F) PC12 cell growth in the presence of PCL-NGF capsules. Scale bar: 100 μm.

Fig. 4

A-GD-PLL microcapsule degradation following GLN exposure. A-GD-PLL microcapsules were incubated in PC12 culture medium (A) without GLN, (B) with GLN for 30 min, (C) with GLN for 24 h, and (D) with GLN/Alkali. In C, the pH of the culture medium increased to 8.5 because of the presence of GLN. The pH value in D was manually adjusted by adding sodium hydroxide solution. Scale bar: 200 μm.

Fig. 5

Microencapsulated PC12 cell viability in the presence of GLN. Comparison of microencapsulated PC12 cell viability following incubation with or without a GLN solution for 30 min which was subsequently replaced with fresh containing 1 mg/ml IgG for 24 and 48 h. Data were derived from n=3 samples. *p<0.005.

Fig. 6

(A) Comparison of dopamine release profiles in vitro. PC12-derived dopamine was measured following culture within alginate-PLL microcapsules or implantation in a fully integrated PCL device loaded with A-GD-PLL microcapsules. (B) Comparison of serum dopamine concentrations in mice implanted with PCL devices loaded with PBS, alginate-PLL microcapsules, or A-GD-PLL microcapsules. Data were derived from n=3 devices for each condition. *Statistically significant increase in serum dopamine concentration compared to PBS control device (p<0.005).

Fig. 7

Comparison of CRP levels in the serum of mice implanted with various PCL devices. Control conditions consisted of untreated mice and mice implanted with alginate-PLL microcapsule alone. Data were derived from n=3 devices for each condition. * Statistically significant decrease in serum CRP concentration as compared to controls (p<0.005).

Fig. 8

Microscopic images of the inflammatory reaction produced by (A) PBS loaded capsules, (B) alginate-PLL microcapsules, (C) capsules loaded with alginate-PLL microcapsules, and (D) capsules loaded with A-GD-PLL microcapsules in immunocompetent mice on Day 30. All images taken at 200×. A: Occasional mast cells and lymphocytes. B: Prominent chronic inflammation with a predominance of lymphocytes. C: Mixed inflammatory infiltrate with a predominance of neutrophils, occasional eosinophils and lymphocytes. D: Rare lymphocytes present (near normal histology).