An in vitro and in vivo study on the properties of hollow polycaprolactone cell-delivery particles

Abstract

The field of dermal fillers is evolving rapidly and numerous products are currently on the market. Biodegradable polymers such as polycaprolactone (PCL) have been found to be compatible with several body tissues, and this makes them an ideal material for dermal filling purposes. Hollow PCL spheres were developed by the Council for Scientific and Industrial Research (CSIR) to serve both as an anchor point and a "tissue harbour" for cells. Particles were tested for cytotoxicity and cell adherence using mouse embryo fibroblasts (MEF). MEFs adhered to the particles and no significant toxic effects were observed based on morphology, cell growth, cell viability and cell cycle analysis, suggesting that the particles are suitable candidates for cell delivery systems in an in vivo setting. The objective of providing a "tissue harbour" was however not realized, as cells did not preferentially migrate into the ported particles. In vivo studies were conducted in BALB/c mice into whom particles were introduced at the level of the hypodermis. Mice injected with PCL particles (ported and non-ported; with or without MEFs) showed evidence of local inflammation and increased adipogenesis at the site of injection, as well as a systemic inflammatory response. These effects were also observed in mice that received apparently inert (polystyrene) particles. Ported PCL particles can therefore act as a cell delivery system and through their ability to induce adipogenesis, may also serve as a dermal bulking agent.

Fig 1

A) Optical image of ported PCL ported particles; B) SEM image of a ported PCL particle. A: scale bar = 100 μm and B: scale bar = 10 μm.

Fig 2. ASF (dark grey) cell numbers are expressed as a percentage of cells relative to the number of MEFs (light grey—100%) counted 24 h after seeding.

No significant differences in cell growth were observed after 72 between the two cell types.

Fig 3

Viability of MEFs cultured in Kimble glass tubes in growth medium (A) and growth medium together with ported PCL particles (B), 24 h after seeding. No statistically significant difference was observed (P-value > 0.05).

Fig 4

Cell cycle histograms (FL3 Lin) of MEFs cultured in Kimble glass tubes in growth medium (A) and growth medium together with ported PCL particles (B), 24 h after seeding. No significant differences were observed.

Fig 5. Acridine orange-stained MEFs attached to ported PCL particles.

Cells were propagated in glass Kimble tubes together with particles (A and B) and stained 24 h after seeding. MEFs attached to particles as clumps.

Fig 6

Scanning electron micrographs of MEFs attached to ported PCL particles 24 h after seeding (A and B). MEFs grew between particles causing the particles to clump together (A). A: scale bar = 100 μm and B: scale bar = 10 μm.

Fig 7

(A-D) Fibrin network formation as assessed by scanning electron microscopy in BALB/c control (A) MEF-injected (B) ported PCL particle-injected (C) and ported PCL particle+MEFs injected animals (D). (E-H) Light microscopy of control (E), cell injected (F), PCL particle injected (G), and PCL particle+MEFs injected (H) hypodermal loose connective tissue. Thick, white arrow = major, thick fibers; thin, white arrow = minor, thin fibers. Areas of typical fibrin morphology are present in the control and MEF-injected animals (A and B). No areas of typical fibrin morphology are present in ported PCL particle-injected (C) and ported PCL particle+MEFs injected animals (D). Large adipocytes (black arrow), blood vessels (white arrow) and fibroblasts (red arrow) are observed in control and cell injected animals (E and F). PCL particle injected specimens presented with large adipocytes (black arrows) surrounded by smaller adipocytes (green arrows) (G). PCL particle+MEFs injected specimens presented with smaller, but more numerous adipocytes (green arrows) and an infiltration of fibroblasts and/or white blood cells (H). Scale bar in A-D = 200nm; magnification in E, G and H = 40x; magnification in F = 100x.

Fig 8

Light microscopy images (A-D), scanning electron micrographs of fibrin networks (E-H) and red blood cells (I-L) for controls, non-ported PCL particles, ported PCL particles and PS particles in BALB/c mice after three weeks exposure. Fibrous thickening, leukocyte infiltration and adipocyte formation occurred in the hypodermis in all animals that received particles when compared to controls (A-D). By SEM, a fine fibrin fibre network (E) and healthy red blood cells (I) were observed in controls. However, a thickened and matted fibrin network and changes in red blood cell morphology indicative of inflammation were observed in mice injected with non-ported PCL particles (F and J), ported PCL particles (G and K) and PS particles (H and L). Scale bar in A-D = 50 μm, and in E, J and L = 2 μm, F-H = 200 μm; and in I and K = 1 μm.

Fig 9

Monocyte (A), neutrophil (B), eosinophil (C) and lymphocyte (D) profiles at 1, 2, 4 and 8 weeks in mice injected with non-ported PCL, ported PCL and PS particles. No significant differences were observed between the test groups and the controls at 1, 2, 4 and 8 weeks. Increases in monocyte, neutrophil and eosinophil counts and decreases in lymphocyte counts were seen between week 1 and subsequent time points in all particle injected mice. Connecting lines indicate a P-value <0.05.