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. 2020 Mar 12;12(3):259.
doi: 10.3390/pharmaceutics12030259.

An Enteric-Coated Polyelectrolyte Nanocomplex Delivers Insulin in Rat Intestinal Instillations when Combined with a Permeation Enhancer

Svenja Sladek  1 , Fiona McCartney  1 , Mena Eskander  2 , David J Dunne  2 , Maria Jose Santos-Martinez  2   3 , Federico Benetti  4 , Lidia Tajber  2 , David J Brayden  1
Affiliations

An Enteric-Coated Polyelectrolyte Nanocomplex Delivers Insulin in Rat Intestinal Instillations when Combined with a Permeation Enhancer

Svenja Sladek et al. Pharmaceutics. .

Abstract

The use of nanocarriers is being researched to achieve oral peptide delivery. Insulin-associated anionic polyelectrolyte nanoparticle complexes (PECs) were formed that comprised hyaluronic acid and chitosan in an optimum mass mixing ratio of 5:1 (MR 5), followed by coating with a pH-dependent polymer. Free insulin was separated from PECs by size exclusion chromatography and then measured by HPLC. The association efficiency of insulin in PECs was >95% and the loading was ~83 µg/mg particles. Dynamic light scattering and nanoparticle tracking analysis of PECs revealed low polydispersity, a negative zeta potential range of -40 to -50 mV, and a diameter range of 95-200 nm. Dissolution studies in simulated small intestinal fluid (FaSSIF-V2) revealed that the PECs were colloidally stable. PECs that were coated with Eudragit® L-100 delayed insulin release in FaSSIF-V2 and protected insulin against pancreatin attack more than uncoated PECs. Uncoated anionic PECs interacted weakly with mucin in vitro and were non-cytotoxic to Caco-2 cells. The coated and uncoated PECs, both concentrated further by ultrafiltration, permitted dosing of 50 IU/kg in rat jejunal instillations, but they failed to reduce plasma glucose or deliver insulin to the blood. When ad-mixed with the permeation enhancer (PE), sucrose laurate (100 mM), the physicochemical parameters of coated PECs were relatively unchanged, however blood glucose was reduced by 70%. In conclusion, the use of a PE allowed for the PEC-released bioactive insulin to permeate the jejunum. This has implications for the design of orally delivered particles that can release the payload when formulated with enhancers.

Keywords: chitosan; hyaluronic acid; insulin; intestinal permeation enhancers; nanomedicine; oral peptide delivery.

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Conflict of interest statement

D.J.B. consults for pharmaceutical companies working in oral peptide delivery.

Figures

Figure A1
Figure A1
ATP and NR Uptakes assays on Caco-2 cells exposed to different concentrations of PECs. ATP content in unloaded and insulin-loaded PECs (100 µg/mL) was determined after (A) 2 h, and (B) 24 h. NR Uptake was carried out only with unloaded PECs after (C) 2 h, and (D) 24 h. Symbols: ■ PEC; ● Insulin-loaded PEC.
Figure A2
Figure A2
Photographs of samples containing EL100 added to either water, HA, or CS solution in 25 mL glass beakers. Note the clear solutions in water and HA, and the opalescence in CS.
Figure A3
Figure A3
(A) Insulin released from uncoated and coated PECs loaded with insulin FaSSIF-V2 over 6 h. Groups: uncoated PEC, 100 µg/mL (o); uncoated PEC, 500 µg/mL (☐); coated PEC, 100 µg/mL (●); coated PEC, 500 µg/mL (■). Data expressed in mean ± SD (n = 3–4). (B) Model parameter estimates and correlation coefficient for insulin release data fitted to a first order model, where W∞ is the amount of insulin released at infinity and k is the release rate constant. (n = 3–4).
Figure A4
Figure A4
Particle diameter (A), PdI, (B) and (C) ZP of PECs loaded with 100 µg/mL insulin in water at t = 0 and after 6 h incubation in FaSSIF-V2 at 37 °C. ■ Uncoated; ■ Coated. The uncoated PECs gave PdI values ~1 and aggregated in FaSSIF after 6 h. Mean ± SD (n = 3–7). * p < 0.05, paired t-test. Data was similar for PECs loaded with 500 µg/mL insulin (data not shown).
Figure 1
Figure 1
Particle diameter (open rectangles) and zeta potential (ZP) (open triangles) of polyelectrolyte complexes (PECs) in relation to mass mixing ratio (MR) and charge mixing ratio (CMR) of the ratio of hyaluronic acid (HA): chitosan (CS). Data expressed as mean ± SD (n = 3).
Figure 2
Figure 2
(A) MTS assay of PEC prototype MR 5 exposure to Caco-2 cells after incubation for 2 h (black bars) and 24 h (white bars). Data is expressed as mean ± SEM (n = 3–5). 0.01% (w/v) Triton™ X-100 was the positive control. One-way ANOVA with Dunnett’s post-test (* p < 0.05, ** p < 0.01 and *** p < 0.001 versus untreated control). (B) Light microscopy images of Caco-2 cells after 24 h incubation with PEC prototype, MR 5 versus native Caco-2 cells (negative control) and staurosporine (1 µM; positive control). Magnification: ×200. Scale bar = 100 µm.
Figure 3
Figure 3
Mucoadhesion of PECs using Quartz Crystal Microbalance with Dissipation (QCM-D). (A,E) Representative traces from the 5th overtone recorded by the device showing the effects of uncoated, unloaded PECs (MR 1.3) and (MR 5) on frequency (blue line, left axis) and dissipation (red line, right axis). (B,F) Representative micrographs of the surface of mucin-coated quartz crystals as viewed by phase contrast microscopy (×20). Quantitative analysis of the effects of (C,D) PECs (MR 1.3) and (G,H) PECs (MR 5) on frequency and energy dissipation. The data is expressed as mean ± SEM (n = 4). One-way ANOVA with Dunnett’s post-test (ns—not significant, * p < 0.05, ** p < 0.01 versus control (mucin)).
Figure 4
Figure 4
Nanoparticle tracking analysis (NTA) analysis of PECs that were loaded with 100 µg/mL insulin. (A) number-based size distributions of uncoated (grey) and coated (black) sample, (B) representative video frame of uncoated sample, representative 3D graph (particle diameter vs. intensity vs. concentration of (C) uncoated and (D) coated PECs, scattergram of intensity (A.U.) vs. particle diameter of (E) uncoated and (F) coated PECs. Data of number-based size distribution is expressed as mean (n = 2 for both samples, three measurements each).
Figure 5
Figure 5
Stability of EL100-coated PECs (100 µg insulin concentration) in (A) Simulated Intestinal Fluid (SIF) and (B) SIF supplemented with pancreatin. Readout was the % change in particle diameter (solid line) and derived count rate (DCR) as measured by DLS (dashed line). Data expressed as mean ± SD (n = 3–5).
Figure 6
Figure 6
(A) Cumulative release of insulin from uncoated PEC (open circle), EL100-coated PECs (open square) and coated PECs + SL (100 mM) (closed square), all loaded with 500 µg/mL insulin, in FaSSIF-V2 at 37 °C. Two-way ANOVA with Bonferroni’s post-test (* p < 0.05 uncoated vs. coated PEC, # p < 0.05 coated PECs vs. coated PECs + SL). (B) Particle diameter, PdI, ZP of coated PECs loaded with 500 µg insulin with and without the addition of SL in FaSSIF-V2. Data expressed as mean ± SD (n = 3–4). Paired Student’s t-test (NS, not significant; * p < 0.05 for coated PECs + SL versus coated PECs). Data expressed as mean ± SD (n = 3).
Figure 7
Figure 7
(A) Degradation profile of insulin solution as compared to insulin entrapped in coated and uncoated PECs and incubated in pancreatin-supplemented SIF. Prototypes were loaded with 500 µg/mL of insulin. Insulin solution (●), Insulin-loaded uncoated PECs (o), Insulin-loaded coated PECs (□), Data expressed as mean ± SD (n = 3–4). (B) Plasma glucose levels following jejunal instillation of insulin (50 IU/kg). Symbols as in A, but with an additional group, insulin-loaded coated PECs co-administered with 100 mM SL (■). S.C. insulin (1 IU/kg) was used as control (▲). The blood glucose levels were standardised to t = 0 min (100%). (C) Insulin serum levels following instillations. Symbols as in B, except uncoated PECs were not tested. (D) Haemotoxylin and eosin staining of jejunal loops following 120 min instillations. Bars = 250 µm. Two-way ANOVA followed by Bonferroni’s post-test. Mean ± SEM (n = 4–6). * p < 0.05, vs. insulin solution in A, B, and C.
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References

    1. Hu Q., Luo Y. Recent advances of polysaccharide-based nanoparticles for oral insulin delivery. Int. J. Biol. Macromol. 2018;120:775–782. doi: 10.1016/j.ijbiomac.2018.08.152. - DOI - PubMed
    1. Batista P., Castro P.M., Madureira A.R., Sarmento B., Pintado M. Recent insights in the use of nanocarriers for the oral delivery of bioactive proteins and peptides. Peptides. 2018;101:112–123. doi: 10.1016/j.peptides.2018.01.002. - DOI - PubMed
    1. Sonia T.A., Sharma C.P. An overview of natural polymers for oral insulin delivery. Drug Discov. Today. 2012;17:784–792. doi: 10.1016/j.drudis.2012.03.019. - DOI - PubMed
    1. Nur M., Vasiljevic T. Can natural polymers assist in delivering insulin orally? Int. J. Biol. Macromol. 2017;103:889–901. doi: 10.1016/j.ijbiomac.2017.05.138. - DOI - PubMed
    1. Wong C.Y., Al-Salami H., Dass C.R. Potential of insulin nanoparticle formulations for oral delivery and diabetes treatment. J. Control. Release. 2017;264:247–275. doi: 10.1016/j.jconrel.2017.09.003. - DOI - PubMed

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