Chitosan nanofibers for transbuccal insulin delivery

Abstract

In this work, they aimed at producing chitosan based nanofiber mats capable of delivering insulin via the buccal mucosa. Chitosan was electrospun into nanofibers using poly(ethylene oxide) (PEO) as a carrier molecule in various feed ratios. The mechanical properties and degradation kinetics of the fibers were measured. Insulin release rates were determined in vitro using an ELISA assay. The bioactivity of released insulin was measured in terms of Akt activation in pre-adipocytes. Insulin permeation across the buccal mucosa was measured in an ex-vivo porcine transbuccal model. Fiber morphology, mechanical properties, and in vitro stability were dependent on PEO feed ratio. Lower PEO content blends produced smaller diameter fibers with significantly faster insulin release kinetics. Insulin showed no reduction in bioactivity due to electrospinning. Buccal permeation of insulin facilitated by high chitosan content blends was significantly higher than that of free insulin. Taken together, the work demonstrates that chitosan-based nanofibers have the potential to serve as a transbuccal insulin delivery vehicle. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1252-1259, 2017.

Keywords: chitosan; electrospun fiber; insulin; transbuccal delivery.

Figure 1. At least 20% high molecular weight polymer is necessary for stable fiber formation

Poly (ethylene oxide) (Mw 900kDa) was added to dissolved chitosan electrospinning solution in decreasing ratios. At 20% PEO solution content intermittent beading can be observed. Scale bar 10µm.

Figure 2. CS:PEO fibers dissolve slowly under physiologic conditions

10mg samples of CS:PEO fiber blends (n=3) were immersed in 5ml of PBS at 37°C. At predetermined time points PBS was removed. Samples were then washed once with diH2O and freeze dried overnight. Mass loss approximated PEO content (A). SEM micrographs showed no major morphological changes in fibers over the degradation period (B, scale bar represents 50 µm). The average fiber diameter measured in these micrographs also showed no significant changes within individual fiber blends after hydration (C). (* denotes statistically significant difference between all groups p < 0.05)

Figure 3. Polysaccharide content controls insulin release kinetics

Fiber blends of different chitosan:PEO ratios were immersed in PBS at 37°C. Insulin release was quantified at 15 minutes, 6 hours, and 1 day by ELISA assay. Various models were applied to fit the data. Curves corresponding to the Ritger-Peppas equation are shown. CS:PEO20 fibers show the fastest release profile, but there was no difference observed between blends at 24 hours.

Figure 4. Insulin remains bioactive after electrospun fiber fabrication

3T3-L1 preadipocytes incubated for 10 minutes in either fresh growth medium (control), 6 hour CS:PEO20 fiber release medium, or insulin containing media (7.98 µg/mL). Western blots of cell lysates were run for p-Akt expression then stripped and re-probed for total Akt1 as loading controls. Cells exposed to fiber release medium showed 3.5 fold higher p-Akt/Akt expression ratio than control cells. (* denotes statistically significant difference from all other groups p < 0.05)

Figure 5. Chitosan enhances insulin transbuccal permeability

Insulin transport across the buccal membrane was determined using a Franz diffusion cell. Measurements of the insulin concentration of the acceptor chamber showed that membranes were largely impermeable to both dissolved insulin and the lower chitosan blend ratios, with those groups delivering less than 1% of their total protein over 6 hours. CS:PEO20 fibers however showed significantly higher insulin delivery over the other groups at all time points over 2 hours. The permeability coefficient of the buccal mucosa to each of these insulin compounds was calculated from the steady state flux region of the tests and CS:PEO20 fibers showed around a 500 fold increase in permeability over the other fiber blends and a 16 fold increase over naked insulin.