Glucose-Responsive Peptide Coacervates with High Encapsulation

Jun 26, 2018 - A new glucose-responsive insulin delivery system is fabricated using biomimetic peptide coacervates derived from the Humboldt squid ...
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Communication Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Glucose-Responsive Peptide Coacervates with High Encapsulation Efficiency for Controlled Release of Insulin Zhi Wei Lim,† Yuan Ping,† and Ali Miserez*,†,‡ †

Centre for Biomimetic Sensor Science, School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang Drive, 637553 Singapore ‡ School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, 637551 Singapore

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ABSTRACT: A new glucose-responsive insulin delivery system is fabricated using biomimetic peptide coacervates derived from the Humboldt squid (Dosidicus Gigas) beak. Both insulin and glucose oxidase are coencapsulated within coacervate microdroplets. The glucose oxidase quickly responds to increasing glucose levels to generate a local acidic environment, thereby rapidly triggering the dissociation of pH-sensitive coacervates to release the insulin cargo. The rate of insulin release is dependent on the glucose level, increases under hyperglycemic conditions, and decreases under normoglycemic conditions. This glucose responsiveness mimics pancreatic β-cell function by releasing insulin according to glucose levels.

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concentrated protein microdroplets. DgHBP coacervates have been hypothesized to infiltrate the chitin-DgCBPs scaffold28 followed by interchain covalent cross-linking during maturation, with the very high cross-link density imparting the beak with its impressive mechanical properties.29,31 All DgHBPs have been sequenced and exhibit a two-domain organization. The N-terminal domains contains nonrepetitive, long stretches of Alanine (Ala) and Histidine (His)-rich regions, whereas the C-terminal domains consist of tandem His- and Gly-rich pentarepeats (GAGFA, GHGXX′/X″, or GHGXY, where X represents a hydrophobic residue, X′ usually represents tyrosine, and X″ represents either glycine or alanine32) that were found to be responsible for DgHBPs self-coacervation properties.32 Because coacervate droplets are known to be able to sequester both low- and high-molecular-weight therapeutics33,34 and because coacervation of DgHBPs C-termini is pH-sensitive,32 we hypothesized that a consensus peptide from the C-termini of DgHBPs could be used to create a GRIDS. More specifically, we reasoned that coacervates of DgHBP-2 peptide could coencapsulate insulin and glucose oxidase (GOx). In this design, the coacervate droplets would act as an insulin reservoir whereas GOx would trigger the release of insulin upon exposure to glucose, based on the conversion of glucose to gluconic acid that would dissociate the pH-sensitive

iabetes is a chronic metabolic disease that is characterized by abnormally high levels of fasting blood glucose. As a general practice, insulin is often administrated to control the blood glucose level in both type I and II diabetic patients.1 To obtain ideal therapeutic results, a strict insulin administration program is needed for diabetic patients. Hence, many investigations have been dedicated to develop environmentally responsive delivery systems.2 Commonly, a glucoseresponsive insulin delivery system (GRIDS) is able to sense the increased glucose concentration and subsequently release the required amount of insulin according to the glucose level.3−11 GRIDS may not only replace the recurrent insulin injections, but can also reduce the patient’s direct involvement in glucose control and prevent insulin from excessive or insufficient dosage.1,3,9−14 One approach used for sensing glucose is to incorporate a glucose-responsive element such as glucose oxidase (GOx) into the delivery system, whereby GOx catalyzes the conversion of D-glucose into gluconic acid to reduce the local pH.14−23 The acidic pH subsequently results in the conformational or structural changes of the carrier and ultimately releases the insulin.14,20,23,24 Such a strategy has been notably employed in pH-responsive hydrogels.22,25−27 Recently, we have isolated and sequenced the proteins of the Humboldt squid beak,28 a hard biomolecular composite made of chitin and proteins.29,30 Two families of proteins, chitin binding beak proteins (DgCBPs) and histidine-rich beak proteins (DgHBPs), were discovered within the beak. DgCBPs likely bind to chitin to form a chitin-DgCBPs scaffold, while DgHBPs exhibit self-coacervation ability, a liquid−liquid phase separation process resulting in the formation of highly © XXXX American Chemical Society

Received: May 26, 2018 Revised: June 17, 2018

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DOI: 10.1021/acs.bioconjchem.8b00369 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Communication

Bioconjugate Chemistry

(Figure 1C). Even though ionic strength is usually a key factor governing coacervation,35−37 ionic strength in the range 0.1 to 1 M NaCl did not greatly affect DgHBP-2 peptide coacervation, suggesting that charge shielding of protonated His residues below pH 6 is already efficient at 0.1 M ionic strength. Since DgHBP-2 peptide coacervation did not occur in acidic pH, we reasoned that the coacervates may serve as a pH-responsive carrier whose release of therapeutic cargo could be triggered by local changes of pH. Through dynamic light scattering (DLS) measurements (Figure S1A), the average size of DgHBP-2 peptide coacervates was ca. 1 μm and increased by 6% to ca. 1.06 μm after insulin encapsulation (Figure S1B). We next elected to encapsulate insulin within the coacervate droplets by adding insulin to the phosphate buffer solution. To verify encapsulation, insulin was labeled with fluorescein isothiocyanate (FITC) to obtain FITC-labeled insulin (FITC-insulin). Following coacervation, FITC-insulin-loaded coacervates were imaged under an inverted fluorescence microscope. As shown in Figure S2, the coacervates and green fluorescence completely overlapped with each other, demonstrating successful encapsulation of insulin by coacervates, with nearly 100% efficiency. We also verified the cytotoxicity of DgHBP-2 peptide in solution and in the coacervate state on mouse fibroblasts using a live/dead cell assay. As shown in Figure S3, the cytotoxicity of DgHBP-2 peptide and insulin-loaded coacervates was negligible at the tested concentrations. The encapsulation efficiency (EE) was measured at different insulin/peptide ratios. At 1 mg mL−1 of peptide, we found a very high EE, reaching nearly 100% regardless of FITC-insulin concentration (from 0.01 to 0.4 mg mL−1) (Table S1). The EE slightly dropped only when DgHBP-2 peptide concentration was below 0.3 mg mL−1 (Table S2). These results demonstrate the very high loading capacity of DgHBP-2 peptide coacervates, which corroborates previous studies conducted with other types of coacervates.38 This high EE was further confirmed for labeled-free insulin using reverse phase high performance liquid chromatography, where native insulin was detected only in the coacervates pellet but not in supernatant (Figure S4), thus verifying that insulin encapsulation occurs regardless of FITC functionalization. The underlying mechanism behind the very high EE of insulin within the coacervate microdroplets is still unclear. Nonspecific interactions between DgHBP-2 peptide and insulin are likely involved, notably hydrophobic and π−π interactions given the high content of hydrophobic and Tyr residues in DgHBP-2 peptide, but this remains to be validated. Next, we tested whether GOx could be coencapsulated with insulin inside the coacervate droplets. As shown in Figure 2, the green fluorescence and the red fluorescence representing FITCinsulin and rhodamine B isothiocyanate-labeled GOx (RITCGOx), respectively, fully overlapped and yielded yellow fluorescence. These results established that both insulin and GOx could be coencapsulated by DgHBP-2 peptide coacervates with a high efficiency (Figure 2D). To investigate whether insulin release could be triggered from the coacervates by glucose-sensing, we prepared GOX +insulin coloaded coacervates (1 mg mL−1 of DgHBp-2 peptide, 0.01 mg mL−1 of GOx, and 0.1 mg mL−1 of insulin) as well as insulin-loaded (1 mg mL−1 of DgHBp-2 peptide, 0.1 mg mL−1 of insulin) coacervates (free of GOx) in phosphate buffer and exposed the coacervate droplets to glucose. As shown in Figure 3A, the insulin release rate was much faster in the presence of both GOx and glucose, indicating that glucose

coacervate droplets and thereby release the insulin cargo (Scheme 1). Scheme 1. Schematic Representation of the GlucoseResponsive Insulin Delivery System (GRIDS) Based on DgHBP-2 Peptide Coacervatesa

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(A) Illustration of the coacervation and encapsulation process. (B) When the coacervate droplets are exposed to glucose, the latter diffuses into the droplets and is converted into gluconic acid. This results in a local decrease of pH and in turn to the dissociation of coacervate droplets with the concomitant release of insulin.

We used a 26-amino-acid-long consensus peptide derived from DgHBP-2 (DgHBP-2 peptide) that consists of 5 GHGXY repeats with a C-terminus tryptophan residue (Figure 1A). To

Figure 1. Coacervation of DgHBP-2 peptide. (A) Sequence of DgHBP-2 peptide. (B) Relative turbidity of DgHBP-2 peptide at different pH and ionic strength (n = 3, mean values ± S.D.). (C) Microscopy image of DgHBP-2 peptide coacervates at pH 7.5, 0.1 M ionic strength.

prepare DgHBP-2 peptide coacervates, we tested buffers of different pH and ionic strength to determine the optimal conditions for coacervation, which was inferred by turbidimetry. Optimal coacervation occurred between neutral (pH 7.4) to slightly alkaline pH (pH 9.5) (Figure 1B) and the formation of coacervates was confirmed by the presence of liquid-like droplets that could be clearly observed by optical microscopy B

DOI: 10.1021/acs.bioconjchem.8b00369 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Communication

Bioconjugate Chemistry

We next examined the ability of GOx+insulin coacervates to respond to changes in glucose concentration. The coacervates displayed a pulsatile release of insulin when the glucose concentration was altered between normal (1 mg mL−1) and hyperglycemic levels (4 mg mL−1) every 1.5 h (Figure 3B). The coacervates could reversibly vary the rate of insulin release between higher and slower rates in response to hyperglycemic and normal levels. Such a glucose-triggered insulin release behavior could be repeated for at least 3 cycles, indicating that only a fraction of insulin was released during each cycle, in agreement with the sustained release obtained in Figure 3A. The maximum number of cycles over which insulin can be released is yet unknown and will be tested in follow-up work. An important parameter to ensure that our GRIDS system could be used for glucose management is to verify retention of insulin activity following entrapment within the coacervate droplets33 and subsequent release from the local acidic microenvironment. We first used circular dichroism (CD) measurements to assess possible changes in secondary structure (Figure 4A). CD spectra of the native insulin

Figure 2. Microscopy images of DgHBP-2 peptide coacervates loaded with FITC-insulin and RITC-GOx. (A) Green fluorescence micrograph is FITC-insulin (0.1 mg mL−1). (B) Red fluorescence micrograph is RITC-GOx (0.1 mg mL−1). (C) Light microscopy micrograph is DgHBP-2 peptide coacervates (1 mg mL−1). (D) Merged (green, red, and light) micrograph image. Yellow fluorescence of the droplets indicate coencapsulation of FITC-insulin and RITCGOx.

Figure 4. Comparison of released insulin from dissociated coacervates and native insulin. (A) CD spectra of native insulin, denatured insulin, insulin released from dissociated coacervates, and DgHBP_2 peptide. (B) Enzyme-linked immunosorbent assay (ELISA) of native insulin and insulin released from dissociated coacervates.

solution displayed the classical α-helical signature (two minima at 208 and 222 nm). After coacervation and release, the CD signature was a mix of insulin and DgHBP-2 peptide, with the slight decrease in intensity attributed to the weaker ellipticity of the peptide. However, the characteristic minima of α-helices at 208 and 222 nm were still visible. In addition, CD spectra of native insulin at pH 5.5 to 7. (Figure S5) indicated that the secondary structure was not disrupted at mild acidic conditions, strongly suggesting that released insulin retained its bioactivity. We also measured the CD spectrum of thermally denatured insulin (Figure 4A), which exhibited important differences with native/released insulin, specifically the loss of the 195 nm maximum and of the 222 nm minimum, as well as a peak shift of the 208 nm minimum toward lower wavelength. To further verify insulin activity, we subjected the insulinDgHBP-2 peptide solution after release to an ELISA assay. The released insulin was detected to similar levels compared to native insulin (Figure 4B), suggesting that the conformational epitope was not disrupted since antibodies could equally recognize the coacervate-released insulin. Together, these data are in agreement with recent studies showing that therapeutic biomacromolecules retain or even exhibit enhanced bioactivity inside the coacervate phase,33,39−41 and that folded proteins are preferentially sequestrated within coacervate microdroplets when compared to their unfolded state.40

Figure 3. In vitro release assay of insulin from DgHBP-2 peptide coacervates (0.1 M NaCl). (A) GOx+insulin with 4 mg mL−1 of glucose (square, black); GOx+insulin (triangle, blue); insulin (inverted triangle, pink); and insulin with 4 mg mL−1 of glucose (circle, red). (B) Alternate high glucose (4 mg mL−1, blue) and low glucose (1 mg mL−1, green) solution. (n = 3, mean values ± S.D.)

readily diffused into the coacervates and converted to gluconic acid by GOx, resulting in local acidification that dissociated the coacervates. Although some leakage of insulin was observed in the absence of glucose, the rate and the degree of insulin release over 48 h were much higher in the presence of glucose. C

DOI: 10.1021/acs.bioconjchem.8b00369 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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In summary, we have synthesized a simple and efficient GRIDS based on biomimetic peptide coacervates loaded with GOx and insulin. The GRIDS can be easily prepared by simply mixing biomimetic DgHBP-2 peptide into aqueous buffer solutions containing insulin and GOx. Coacervation ensues and acts as an insulin reservoir with very high encapsulation efficiency. Under hyperglycemic conditions, glucose molecules diffuse into the coacervates where GOx readily converts glucose into gluconic acid, resulting in a local acidic environment that triggers the dissociation of coacervates and eventually leads to glucose-triggered release of insulin, with a release kinetics that can be altered in response to glucose levels. Furthermore, the secondary structure and the activity of insulin are retained following exposure to acidic microenvironment, which can primarily be attributed to the use of aqueous solvents. Our results indicate that this new GRIDS combines the advantages of both glucose responsiveness and high loading capacity, representing a promising potential for diabetes management. Further optimization to enhance coacervate stability is currently underway, notably to prevent premature leakage in the absence of glucose exposure, which may be achieved by introducing simple mutations in the peptide sequence or by varying the number of tandem pentapeptides.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.8b00369.

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Communication

Detailed experimental methods; DLS of coacervates before and after encapsulation; additional optical and fluorescence microscopy of insulin-loaded DgHBP-2 coacervates; cell (NIH3T3 mouse fibroblasts) viability against DgHBP-2 peptides and coacervates; RP-HPLC of coacervation process with labeled-free insulin; CD spectra of insulin at different pHs; EE of FITC-insulin (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Ali Miserez: 0000-0003-0864-8170 Author Contributions

Z.W.L. conducted all experiments. Y.P. and A.M. supervised and designed the research. All authors contributed to writing of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported the Singapore Ministry of Education (MOE) through an Academic Research Fund (AcRF) Tier 2 grant (# MOE2015-T2-1-062). We also acknowledge funding support from a joint Singapore National Research Foundation (NRF)/Agence Nationale de la Recherche, France (ANR) Joint Grant (NRF2015-NRF-ANR000). D

DOI: 10.1021/acs.bioconjchem.8b00369 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Communication

Bioconjugate Chemistry (23) Zhao, L., Xiao, C., Wang, L., Gai, G., and Ding, J. (2016) Glucose-sensitive polymer nanoparticles for self-regulated drug delivery. Chem. Commun. (Cambridge, U. K.) 52 (49), 7633−52. (24) Zhao, W., Zhang, H., He, Q., Li, Y., Gu, J., Li, L., Li, H., and Shi, J. (2011) A glucose-responsive controlled release of insulin system based on enzyme multilayers-coated mesoporous silica particles. Chem. Commun. (Cambridge, U. K.) 47 (33), 9459−61. (25) Qi, X., Wei, W., Li, J., Zuo, G., Pan, X., Su, T., Zhang, J., and Dong, W. (2017) Salecan-Based pH-Sensitive Hydrogels for Insulin Delivery. Mol. Pharmaceutics 14 (2), 431−440. (26) Kang, S. I., and Bae, Y. H. (2003) A sulfonamide based glucoseresponsive hydrogel with covalently immobilized glucose oxidase and catalase. J. Controlled Release 86 (1), 115−21. (27) Traitel, T., Cohen, Y., and Kost, J. (2000) Characterization of glucose-sensitive insulin release systems in simulated in vivo conditions. Biomaterials 21 (16), 1679−87. (28) Tan, Y., Hoon, S., Guerette, P. A., Wei, W., Ghadban, A., Hao, C., Miserez, A., and Waite, J. H. (2015) Infiltration of chitin by protein coacervates defines the squid beak mechanical gradient. Nat. Chem. Biol. 11 (7), 488−95. (29) Miserez, A., Li, Y., Waite, J. H., and Zok, F. (2007) Jumbo squid beaks: inspiration for design of robust organic composites. Acta Biomater. 3 (1), 139−49. (30) Miserez, A., Schneberk, T., Sun, C., Zok, F. W., and Waite, J. H. (2008) The transition from stiff to compliant materials in squid beaks. Science 319 (5871), 1816−9. (31) Miserez, A., Rubin, D., and Waite, J. H. (2010) Cross-linking chemistry of squid beak. J. Biol. Chem. 285 (49), 38115−24. (32) Cai, H., Gabryelczyk, B., Manimekalai, M. S. S., Gruber, G., Salentinig, S., and Miserez, A. (2017) Self-coacervation of modular squid beak proteins - a comparative study. Soft Matter 13 (42), 7740− 7752. (33) Blocher, W. C., and Perry, S. L. (2017) Complex coacervatebased materials for biomedicine. Wiley Interdiscip Rev. Nanomed Nanobiotechnol 9 (4), e1442. (34) Chu, H., Gao, J., Chen, C. W., Huard, J., and Wang, Y. (2011) Injectable fibroblast growth factor-2 coacervate for persistent angiogenesis. Proc. Natl. Acad. Sci. U. S. A. 108 (33), 13444−9. (35) Perry, L. S., Li, Y., Priftis, D., Leon, L., and Tirrell, M. (2014) The Effect of Salt on the Complex Coacervation of Vinyl Polyelectrolytes. Polymers 6 (6), 1756. (36) Wang, Y., Kimura, K., Huang, Q., Dubin, P. L., and Jaeger, W. (1999) Effects of Salt on Polyelectrolyte−Micelle Coacervation. Macromolecules 32 (21), 7128−7134. (37) Joshi, N., Rawat, K., and Bohidar, H. B. (2018) pH and ionic strength induced complex coacervation of Pectin and Gelatin A. Food Hydrocolloids 74 (Supplement C), 132−138. (38) Johnson, N. R., and Wang, Y. (2014) Coacervate delivery systems for proteins and small molecule drugs. Expert Opin. Drug Delivery 11 (12), 1829−32. (39) Black, K. A., Priftis, D., Perry, S. L., Yip, J., Byun, W. Y., and Tirrell, M. (2014) Protein Encapsulation via Polypeptide Complex Coacervation. ACS Macro Lett. 3 (10), 1088−1091. (40) Martin, N., Li, M., and Mann, S. (2016) Selective Uptake and Refolding of Globular Proteins in Coacervate Microdroplets. Langmuir 32 (23), 5881−9. (41) Pippa, N., Karayianni, M., Pispas, S., and Demetzos, C. (2015) Complexation of cationic-neutral block polyelectrolyte with insulin and in vitro release studies. Int. J. Pharm. 491 (1−2), 136−43.

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DOI: 10.1021/acs.bioconjchem.8b00369 Bioconjugate Chem. XXXX, XXX, XXX−XXX