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Synthesis, Characterization and In Vitro Evaluation of a Novel Glycol Chitosan-EDTA Conjugate to Inhibit Aminopeptidase-Mediated Degradation of Thymopoietin Oligopeptides Jiao Feng 1 , Yan Chen 1,2 , Feng Li 1 , Lili Cui 3 , Nianqiu Shi 2,4 , Wei Kong 1,2 and Yong Zhang 1,2, * ID 1

2 3 4

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National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun 130012, China; [email protected] (J.F.); [email protected] (Y.C.); [email protected] (F.L.); [email protected] (W.K.) Key Laboratory for Molecular Enzymology and Engineering the Ministry of Education, School of Life Sciences, Jilin University, Changchun 130012, China; [email protected] Institute of Pharmaceutical Science, King’s college London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, UK; [email protected] Department of Pharmaceutics, School of Pharmacy, Jilin Medical University, Jilin 132013, China Correspondence: [email protected]; Tel.: +86-431-8516-7674

Received: 21 June 2017; Accepted: 24 July 2017; Published: 26 July 2017

Abstract: In this study, a novel conjugate consisting of glycol chitosan (GCS) and ethylene diamine tetraacetic acid (EDTA) was synthesized and characterized in terms of conjugation and heavy metal ion chelating capacity. Moreover, its potential application as a metalloenzyme inhibitor was evaluated with three thymopoietin oligopeptides in the presence of leucine aminopeptidase. The results from FTIR and NMR spectra revealed that the covalent attachment of EDTA to GCS was achieved by the formation of amide bonds between the carboxylic acid group of EDTA and amino groups of GCS. The conjugated EDTA lost part of its chelating capacity to cobalt ions compared with free EDTA as evidenced by the results of cobalt ion chelation-mediated fluorescence recovery of calcein. However, further investigation confirmed that GCS-EDTA at low concentrations significantly inhibited leucine aminopeptidase-mediated degradation of all thymopoietin oligopeptides. Keywords: chitosan; peptide degradation; chelation; conjugation

1. Introduction Glycol chitosan is one of the polysaccharides that are soluble at pH values from 0 to 14, while conventional chitosan cannot disperse at a monomolecular level at pH values higher than 6.5 [1,2]. The significant difference originates from the replacement of hydrogen by ethylene glycol residues at the hydroxyl group bonded to the 6th C of the glucosamine ring of chitosan (Figure 1). The solubility property gives glycol chitosan great advantages over chitosan in conjugation modifications, especially for the reaction conditions required at neutral or alkali pH values. Furthermore, the resultant conjugates are usually soluble at all pH values due to the intrinsic solubility property of glycol chitosan [3]. In addition, similar to chitosan, glycol chitosan is biodegradable and biocompatible. Recently, glycol chitosan has been proved to have the ability to stabilize lipid rafts in the intestinal brush border and to inhibit the P-glycoprotein efflux pump through membrane binding [4,5]. Covalent attachment of functional molecules, such as enzyme inhibitors and thiol bearing ligands, to chitosan has been widely reported [6]. The main purpose is to endow chitosan with other functions, such as enzyme inhibition and stronger mucoadhesive property. However, few studies involve glycol Molecules 2017, 22, 1253; doi:10.3390/molecules22081253

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chitosan and its modification mainly focuses on hydrophobic ligand conjugation to yield amphiphilic polymers capable of self-assembling in aqueous solutions [7–9].and In our previous reported studies, we reported a novel glycol chitosan-bestatin conjugate, evaluated its studies, potentialwe application aasnovel glycol chitosan-bestatin and evaluatedoligopeptides its potential application as an aminopeptidase an aminopeptidase inhibitor conjugate, to protect thymopoietin from enzymatic degradation [3]. inhibitor to protect thymopoietin oligopeptides from enzymatic degradation [3]. Aminopeptidase, Aminopeptidase, as zinc ion-dependent metalloenzyme, is widely distributed in nasal mucosa and as ion-dependent metalloenzyme, is widely distributed mucosa andtothe brush-border thezinc brush-border membrane of the gastrointestinal tract and in is nasal the key enzyme rapidly degrade membrane of the gastrointestinal tract and is the key enzyme to rapidly degrade thymopoietin thymopoietin oligopeptides via nonparenteral administration routes [10–12]. Inhibition of oligopeptides viacan nonparenteral routes [10–12]. of aminopeptidase can be aminopeptidase be achievedadministration through competitive bindingInhibition of the active site of the enzyme or achieved through competitiveremoval bindingof of zinc the active site of enzyme through chelation-mediated through chelation-mediated ions from itsthe active site. or Glycol chitosan–bestatin is an removal zinc ions frombestatin its active site. Glycol chitosan-bestatin is interaction. an effective However, inhibitor as free effective of inhibitor as free through enzyme–substrate binding highly bestatin through enzyme-substrate binding interaction. However, highly efficient inhibition can only efficient inhibition can only be achieved in the presence of a high concentration of conjugates [3]. be achieved in the presence of a high concentration of conjugates [3]. Specific inhibition may be less Specific inhibition may be less advantageous in protecting peptides and proteins from extensive advantageous in protecting peptides and proteins from extensive degradation by other enzymes via degradation by other enzymes via the nonparenteral administration routes, such as nasal mucosal the nonparenteral administration routes, such as nasal mucosal and oral delivery. and oral delivery.

1. Structures of EDTA EDTA (A) (A) and and glycol glycol chitosan chitosan (GCS) (GCS) (B). The The protons protons along along the the backbones backbones of Figure 1. EDTA EDTA and and GCS GCS are are numbered numberedfor forsubsequent subsequentpeak peakassignments assignmentsin inNMR NMRspectra. spectra.

EDTA is is an an efficient efficient broad-spectrum broad-spectrum inhibitor inhibitor of of metalloproteases metalloproteases via via the the metal metal ion-chelating ion-chelating EDTA mechanism, and EDTA with with free free mechanism, and usually usually requires requires low low concentration concentration such such as as 1–10 1–10 µM. µM. However, However, EDTA carboxyl groups is barely soluble and often requires higher pH than 7 to achieve monomolecular carboxyl groups is barely soluble and often requires higher pH than 7 to achieve monomolecular dispersion, where addition, when used as an dispersion, where two two of of four four carboxyl carboxylgroups groupswill willbe beionized ionized[13]. [13].In In addition, when used as enzyme inhibitor for oral delivery of proteins and peptides, high dose is generally needed because of an enzyme inhibitor for oral delivery of proteins and peptides, high dose is generally needed extensive dilution and clearance during passage. In this case, the safety problem may arise especially for because of extensive dilution and clearance during passage. In this case, the safety problem may the long-term usage duelong-term to the possible metal ion-dependent biological processes [14]. arise especially for the usageinterference due to thetopossible interference to metal ion-dependent Conjugation of EDTA to chitosan can of partly solve the above through mucoadhesion. biological processes [14]. Conjugation EDTA to chitosan canproblems partly solve the above problems However, the synthesis reaction requires complicated pH adjustment, and the resultant conjugate is through mucoadhesion. However, the synthesis reaction requires complicated pH adjustment, and insoluble in acid medium due to the of due EDTA to primary amino groups chitosan [15]. the resultant conjugate is insoluble in attachment acid medium to the attachment of EDTA toof primary amino In addition, the conjugate usually forms transparent gel, but not real solution, in neutral and alkali groups of chitosan [15]. In addition, the conjugate usually forms transparent gel, but not real solution, solutions [15,16]. Considering the intrinsic solubility property of glycol chitosan, using glycol in neutral and alkali solutions [15,16]. Considering the intrinsic solubility property of glycol chitosan, chitosan as conjugation willmatrices probablywill have more advantages, as yielding solution at using glycol chitosan as matrices conjugation probably have more such advantages, suchaas yielding all pH values. In this study, glycol chitosan-EDTA was synthesized by forming amide bonds between a solution at all pH values. In this study, glycol chitosan-EDTA was synthesized by forming amide the amino group ofamino the latter andofthe group of the former. influence conjugation bonds between the group thecarboxylic latter andacid the carboxylic acid groupThe of the former.ofThe influence onconjugation the chelation of EDTA to cobalt ionstowas evaluated. potential its application of oncapacity the chelation capacity of EDTA cobalt ions wasMoreover, evaluated.itsMoreover, potential as a metalloenzyme inhibitor in solution was investigated using three thymopoietin oligopeptides as model peptide drugs in the presence of leucine aminopeptidase.

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application as a metalloenzyme inhibitor in solution was investigated using three thymopoietin 3 of 11 oligopeptides as model peptide drugs in the presence of leucine aminopeptidase.

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2. 2. Results Results and and Discussion Discussion 2.1. 2.1. FTIR FTIR Measurement Measurement The The FTIR FTIR spectra spectra of of EDTA-Na EDTA-Na22,, glycol glycol chitosan chitosan(GCS) (GCS)and andGCS-EDTA GCS-EDTAare are shown shownin in Figure Figure2.2. Among Among the spectra, EDTA-Na22 gave gave three three typical typical absorptions absorptions of of carboxylic carboxylic groups groups at at 1673.9, 1673.9, 1627.6 1627.6 −1 − 1 and 1396.2 cm , respectively. The results agreed very well with previous reports [17,18]. The and 1396.2 , respectively. The results agreed very well with previous reports [17,18]. The peak peak −1 comes assignment broad peak at 3374.8 cm−1cm comes from from amineamine N-H assignment of of glycol glycolchitosan chitosanisisasasfollows. follows.TheThe broad peak at 3374.8 −1 stretch, whichwhich is overlapped with O-H vibration [19]. Peaks at 2915.8 and 2871.5 cm−1 are N-H stretch, is overlapped withstretch O-H stretch vibration [19]. Peaks at 2915.8 and 2871.5 cmthe typical stretch Two peaks centered 1664.3 and 1600.6and cm−11600.6 represented amide I and are the C-H typical C-Hvibration. stretch vibration. Two peaks at centered at 1664.3 cm−1 represented −1 −1 II respectively In the GCS-EDTA spectrum, the peaks at 1673.9 and 1664.3 cm disappeared and amide I and II[16]. respectively [16]. In the GCS-EDTA spectrum, the peaks at 1673.9 and 1664.3 cm −1 −1 , indicating adisappeared new band appeared at 1635.3 cm , indicating thecm formation of amide although the groups, typical and a new band appeared at 1635.3 thegroups, formation of amide −1 might overlap with amide − 1 absorption of carboxylic groups at 1627.6 cm I. Another new strong bandI. although the typical absorption of carboxylic groups at 1627.6 cm might overlap with amide −1 − 1 centered 1403.9 cmband , which is characteristic of carboxylic groups of EDTA,offurther confirmed Another at new strong centered at 1403.9 cm , which is characteristic carboxylic groupsthe of successful attachment of EDTA to glycol chitosan. It should be noted that, before FTIR measurement, EDTA, further confirmed the successful attachment of EDTA to glycol chitosan. It should be noted GCS and GCS-EDTA experienced extensive dialysis experienced treatment, and therefore the peaks in FTIR that, before FTIR measurement, GCS and GCS-EDTA extensive dialysis treatment, and spectra of the GCS-EDTA originate from the conjugate but not from EDTA.but In addition, therefore peaks in can FTIRonly spectra of GCS-EDTA can only originate fromthe thefree conjugate not from considering theInexcessive used the in the conjugation reaction, mole freereaction, amino one group is the free EDTA. addition, EDTA considering excessive EDTA used in theone conjugation mole expected togroup conjugate with one EDTAwith group. free amino is expected tomole conjugate one mole EDTA group.

Figure Figure 2. 2. The TheFTIR FTIRspectra spectraof ofNa2-EDTA Na2 -EDTA (A); (A); GCS GCS (B); (B);GCS-EDTA GCS-EDTA (C) (C) synthesized synthesized with withaaGCS:EDTA GCS:EDTA mass EDTA. mass ratio ratio of 1:30 and followed by complete removal of free EDTA.

2.2. 2.2. NMR NMR Measurement Measurement To further confirm confirm the the successful successful conjugation, conjugation, NMR To further NMR was was used used to to characterize characterize the the resultant resultant 11H-NMR spectra of EDTA-Na2, unmodified and EDTA-modified conjugates in an alkali solution. The conjugates in an alkali solution. The H-NMR spectra of EDTA-Na2 , unmodified and EDTA-modified GCS 3. In GCS are are shown shown in in Figure Figure 3. In the the case case of of EDTA-Na EDTA-Na22,, peaks peaks at at 3.97 3.97 and and 3.53 3.53 ppm ppm resulted resulted from from protons protons H-a H-a and and H-b H-b on on the the methylene methylene groups groups of of EDTA, EDTA, respectively, respectively, which which agreed agreed well well with with the the ratio of relative integrals. For GCS, the peaks assigned to the protons along the backbones of GCS are ratio of relative integrals. For GCS, the peaks assigned to the protons along the backbones of GCS numbered in Figure 1B. The assignments of GCS 2.69 (3H, CH3 of CH3 the acetyl are numbered in Figure 1B. proton The proton assignments ofwere: GCS δwere: δ 2.69 (3H, of thegroup), acetyl δ 3.34 (1H, H-2 (D)), δ 4.00–4.60 (5H, H-3, 4, 5, and 6 of glucosamine ring), δ 5.09 (1H, H-1 (D)) and δ 5.28 (1H, H-1 (A)) [20,21]. The degree of acetylation (DD) calculated by the equation of DD = (ICH3/3)/(IH-1total) was 26.08% [16]. In contrast with GCS, GCS-EDTA showed characteristic proton signals in the range 3.0–4.0 ppm. The peak centered at 3.75 ppm was the proton signal of H-a on the

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group), δ 3.34 (1H, H-2 (D)), δ 4.00–4.60 (5H, H-3, 4, 5, and 6 of glucosamine ring), δ 5.09 (1H, H-1 (D)) and δ 5.28 (1H, H-1 (A)) [20,21]. The degree of acetylation (DD) calculated by the equation of DD = (ICH3 /3)/(IH-1total ) was 26.08% [16]. In contrast with GCS, GCS-EDTA showed characteristic proton signals Molecules 2017,in 22,the 1253range 3.0–4.0 ppm. The peak centered at 3.75 ppm was the proton signal 4 ofof 11H-a on the methylene groups of EDTA, while the peak at around 3.26 ppm was the overlap of those of groups of the EDTA, while the peak atofaround was the overlap thoseand of H-2 H-2 methylene of GCS and H-b on methylene groups EDTA.3.26 Theppm peaks arising at 3.75ofppm 3.26ofppm GCS and H-b on theconjugation methylene groups EDTA. Theaspeaks arising experienced at 3.75 ppm extensive and 3.26 ppm indicated the successful of GCSofwith EDTA GCS-EDTA dialysis indicated the successful conjugation of GCS with EDTA as GCS-EDTA experienced extensive dialysis treatment before NMR measurement. In addition, the peak resulted from H-1 (D) of GCS disappeared treatment before NMR measurement. In addition, the peak resulted from H-1 (D) of GCS disappeared in the spectra of GCS-EDTA, suggesting that there was no free amino group present in this molecule. in the spectra of GCS-EDTA, suggesting that there was no free amino group present in this molecule. The calculated degree of acetylation was 26.71%, in agreement with that of free GCS, suggesting The calculated degree of acetylation was 26.71%, in agreement with that of free GCS, suggesting that that conjugation reaction did not have a remarkable effect on DD. Together with the results of FTIR, conjugation reaction did not have a remarkable effect on DD. Together with the results of FTIR, it is it is reasonable toconclude concludethat that conjugation of EDTA to glycol chitosan is successfully achieved. reasonable to thethe conjugation of EDTA to glycol chitosan is successfully achieved.

Figure 3. 1H-NMR spectra of Na2-EDTA, GCS and GCS-EDTA synthesized with a GCS:EDTA mass

Figure 3. 1 H-NMR spectra of Na2 -EDTA, GCS and GCS-EDTA synthesized with a GCS:EDTA mass ratio of 1:30 in pOD 10 solutions at 70 °C. The numbered protons along the backbones of EDTA and ratio of 1:30 in pOD 10 solutions at 70 ◦ C. The numbered protons along the backbones of EDTA GCS are shown in Figure 1. The (D) refers to deacetylated residues, while the (A) refers to acetylated and residues. GCS are shown in Figure 1. The (D) refers to deacetylated residues, while the (A) refers to acetylated residues.

2.3. Evaluation of Chelating Ability

2.3. Evaluation of Chelating Ability

Whether the successful conjugation will influence the chelation capacity of EDTA is a key point to its future application. Figure 4conjugation shows the effect EDTA and differentofconcentrations onpoint the to Whether the successful willofinfluence theGCS-EDTA chelation at capacity EDTA is a key normalized fluorescence intensity of calcein in the presence of cobalt ions. It should be noted that its future application. Figure 4 shows the effect of EDTA and GCS-EDTA at different concentrations molar concentrations of the EDTA groupofwere used EDTAof and GCS-EDTA. the latter, on the normalized fluorescence intensity calcein infor theboth presence cobalt ions. It For should be noted the EDTA molar concentration was calculated according to the speculation that all free amino groups that molar concentrations of the EDTA group were used for both EDTA and GCS-EDTA. For the latter, of glycol chitosan were conjugated with EDTA at 1:1 molar ratio. The reasonable speculation is the EDTA molar concentration was calculated according to the speculation that all free amino supported by the results of FTIR and NMR assays and other studies [15]. As shown in Figure 4, with groups of glycol chitosan were conjugated with EDTA at 1:1 molar ratio. The reasonable speculation the increasing concentration of EDTA, the fluorescence intensity of calcein gradually increased, is supported the results of FTIR and NMR assays andtoother [15]. EDTA As shown in Figure 4, suggestingby concentration-dependent chelation of EDTA cobaltstudies ions. When concentration withwas the higher increasing concentration of EDTA, the fluorescence intensity of calcein gradually than 0.67 µM, a plateau was reached, indicating the almost complete removal of increased, cobalt suggesting concentration-dependent chelation of EDTA to cobalt ions.was When ions from the calcein/cobalt ion complex. In contrast, similar tendency alsoEDTA foundconcentration in the presencewas higher than 0.67 µM, a plateau was reached, indicating the almost removal of cobalt of GCS-EDTA. However, compared with free EDTA, GCS-EDTA in complete all molar concentration rangesions slightly reduced capacity to cobaltsimilar ions, which is probably due to the loss one of four of fromshowed the calcein/cobalt ionchelation complex. In contrast, tendency was also found inof the presence carboxyl groups that resulted from the conjugation. The reasonable speculation is supported by the fact GCS-EDTA. However, compared with free EDTA, GCS-EDTA in all molar concentration ranges showed that the chelation of EDTA to metal ions usually involves its two amines and four carboxyl groups. slightly reduced chelation capacity to cobalt ions, which is probably due to the loss of one of four

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carboxyl groups that resulted from the conjugation. The reasonable speculation is supported by the fact that the chelation of EDTA to metal ions usually involves its two amines and four carboxyl groups.

Figure 4. The effect of cobalt ion chelation resulted from EDTA and GCS-EDTA with different The effect of cobalt resulted from EDTA and GCS-EDTA withintensity different EDTA EDTAFigure group4.concentrations on ion the chelation normalized maximum fluorescence emission of calcein group concentrations on the normalized maximum fluorescence emission intensity of calcein (0.0625 (0.0625 µg/mL). The molar ratio of calcein to cobalt dichloride was 1:1. Data were fitted with µ g/mL). The molar ratioModel of calcein to cobalt dichloride 1:1. Data8.5 were fitted withNorthampton, the AsymptoticMA, the Asymptotic Regression in software OriginLabwas of version (OriginLab, Regression Model2 in software OriginLab of version 8.5 (OriginLab, Northampton, MA, USA), and USA), and adjusted R EDTA and GCS-EDTA were 0.97 and 0.98, respectively. adjusted R2 EDTA and GCS-EDTA were 0.97 and 0.98, respectively.

2.4. Inhibition of Aminopeptidase-Mediated Peptide Degradation 2.4. Inhibition of Aminopeptidase-Mediated Peptide Degradation

Here,Here, we investigated the potential application of GCS-EDTA to inhibit aminopeptidase-mediated we investigated the potential application of GCS-EDTA to inhibit aminopeptidasepeptide degradation. The results were shown in Figure 5A–C for TP5, TP4 respectively. mediated peptide degradation. The results were shown in Figure 5A–C for and TP5, TP3, TP4 and TP3, In therespectively. case of TP5, aminopeptidase at 0.01 at U/mL caused significant In theleucine case of TP5, leucine aminopeptidase 0.01 U/mL caused significantdegradation degradation as as evidenced the decreased concentration TP5over overtime. time. In In addition, of GCS evidenced by the by decreased concentration of of TP5 addition,the theaddition addition of GCS slightly accelerated TP5 degradation, which might be due to the residual metal ions-mediated slightly accelerated TP5 degradation, which might be due to the residual metal ions-mediated enzyme activation from GCS. Similar phenomena were were also our previous studies [3]. [3]. enzyme activation from GCS. Similar phenomena also observed observedinin our previous studies However, in the presence of GCS-EDTA, TP5 degradation that resulted from leucine aminopeptidase However, in the presence of GCS-EDTA, TP5 degradation that resulted from leucine aminopeptidase was significantly inhibited and, at almost all sampling points, the remaining TP5 possessed higher was significantly inhibited and, at almost all sampling points, the remaining TP5 possessed higher concentrations than those without GCS-EDTA. For TP4 and TP3, similar phenomena were also concentrations than those For TP4 and TP3,inhibit similar were also observed. These resultswithout indicatedGCS-EDTA. that GCS-EDTA can markedly thephenomena activity of leucine observed. These results indicated that GCS-EDTA can markedly inhibit the activity of aminopeptidase. However, unexpectedly, 100-time improvement in GCS-EDTA concentration leucine did aminopeptidase. However, unexpectedly, 100-timethymopoietin improvement in GCS-EDTA concentration not completely inhibit aminopeptidase-mediated oligopeptide degradation. Leucine did not completely inhibit oligopeptide Leucine aminopeptidase hasaminopeptidase-mediated been reported to include two thymopoietin zinc ions at its active site in the degradation. C-terminal domain [22,23]. One of them is responsible for forming a stable complex with the substrate and another is to aminopeptidase has been reported to include two zinc ions at its active site in the C-terminal activate the catalytic water located in the active site. Removal of one of them may markedly influence domain [22,23]. One of them is responsible for forming a stable complex with the substrate and another its activity removing all of them will leadactive to complete inactivation incomplete GCSis to activate theand catalytic water located in the site. Removal of [22]. one The of them may markedly EDTA inhibition of leucine aminopeptidase may be due to the dynamic equilibrium of zinc ions influence its activity and removing all of them will lead to complete inactivation [22]. The incomplete between the GCS-EDTA/zinc ion complex and enzyme/zinc ion complex. To further reveal GCSGCS-EDTA inhibition of leucine aminopeptidase may be due to the dynamic equilibrium of zinc EDTA inhibition of leucine aminopeptidase, the degradation clearance of three peptides in the ions between the GCS-EDTA/zinc ion complex enzyme/zinc complex. absence and presence of the conjugate were and calculated, and the ion result is shownToinfurther Figure reveal 6. GCS-EDTA inhibition of leucine aminopeptidase, the clearance of three peptides in Degradation clearance data agreed well with those of degradation degradation kinetics discussed previously. the absence and presence of the conjugate were calculated, the result in Figure Interestingly, at the same conditions, three peptides showedand different levels is of shown susceptibility to 6. Degradation clearance dataand agreed withofthose degradation kinetics previously. leucine aminopeptidase gave well the order TP4 >of TP5 > TP3. The results are discussed in agreement with several previously published reports [3,24], and can be explained by their different binding energies Interestingly, at the same conditions, three peptides showed different levels of susceptibility to leucine to aminopeptidase [3]. The that>combining peptides (such as TP4 aminopeptidase and gave the findings order ofsuggested TP4 > TP5 TP3. TheGCS-EDTA results areand in agreement with several and TP3) with robust resistance against enzymatic degradation might achieve an efficient delivery previously published reports [3,24], and can be explained by their different binding energies to via mucosal routes. aminopeptidase [3]. The findings suggested that combining GCS-EDTA and peptides (such as TP4 and TP3) with robust resistance against enzymatic degradation might achieve an efficient delivery via mucosal routes.

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Figure 5. 5.Degradation absence and and presence presenceof ofGCS GCS Figure Degradationkinetics kineticsof ofTP5 TP5(A), (A), TP4 TP4 (B) (B) and and TP3 TP3 (C) (C) in in the the absence Figure 5. Degradation kineticseach of TP5 (A), TP4 (B) andwas TP3incubated (C) in thewith absence and presence ofatGCS ◦ and GCS-EDTA. In all groups, peptide (0.1 mM) LAP (0.01 U/mL) 37 and GCS-EDTA. In all groups, each peptide (0.1 mM) was incubated with U/mL) at 37 °C.C. and GCS-EDTA. In all groups, each peptide (0.1 mM) was incubated with LAP (0.01 U/mL) at 37 °C. GCS, 0.50% (w/v); with aa GCS:EDTA GCS:EDTAmass massratio ratioofof1:30 1:30 GCS, 0.50% (w/v);the theconcentrations concentrations of of GCS-EDTA GCS-EDTA synthesized with GCS, 0.50% (w/v); the concentrations of GCS-EDTA synthesized with a GCS:EDTA mass ratio of 1:30 inin group GCS-E respectively. group GCS-E1 1and and2 2were were0.0001 0.0001mg/mL mg/mL and and 0.01 mg/mL, mg/mL, respectively. in group GCS-E 1 and 2 were 0.0001 mg/mL and 0.01 mg/mL, respectively.

Figure clearance of of TP3, TP3, TP4 TP4and andTP5 TP5in inthe theabsence absenceand andpresence presenceofofGCS GCSand andGCSGCSFigure 6. Degradation clearance Figure 6. Degradation clearance of TP3, TP4 and TP5 in the absence and presence of GCS and GCS-EDTA. EDTA. thymopoietinoligopeptide oligopeptide(0.1 (0.1mM) mM)was wasincubated incubatedwith withLAP LAP(0.01 (0.01U/mL) U/mL) EDTA. In all groups, each thymopoietin ◦ Inat all37 groups, each thymopoietin oligopeptide of (0.1 mM) was synthesized incubated with LAP (0.01 U/mL) atratio 37 °C. GCS, 0.50% the concentrations concentrations ofGCS-EDTA GCS-EDTA synthesized with GCS:EDTA mass ratioC. at 37 (w/v); the with a aGCS:EDTA mass GCS, 0.50% (w/v);GCS-E the concentrations of GCS-EDTA synthesized with a GCS:EDTA mass ratio of 1:30 of in group and 22 were were 0.0001 0.0001 mg/mLand and 0.01mg/mL, mg/mL, respectively. of 1:30 1:30 1 and mg/mL 0.01 respectively. in group GCS-E 1 and 2 were 0.0001 mg/mL and 0.01 mg/mL, respectively.

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at 37 °C. GCS, 0.50% (w/v); the concentrations of GCS-EDTA synthesized with a GCS:EDTA mass ratio of 1:30 in group GCS-E 1 and 2 were 0.0001mg/mL and 0.01mg/mL, respectively. Molecules 2017, 22, 1253of 2.5. Cytotoxicity

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The cytotoxicity of GCS-EDTA and GSC was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay after 24-h incubation with Madin-Darby Canine Kidney 2.5. Cytotoxicity of GCS-EDTA (MDCK) cells. The results were shown in Figure 7. Both GCS and GCS-EDTA showed doseThe cytotoxicity of GCS-EDTA and GSC was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2, dependent negative effects on MDCK cell viability, with the latter resulting in a slightly lower level 5-diphenyltetrazolium bromide (MTT) assay after 24-h incubation with Madin-Darby Canine Kidney of cell viability. However, even at all concentrations used in this study, cell viabilities of more than (MDCK) cells. The results were shown in Figure 7. Both GCS and GCS-EDTA showed dose-dependent 93% were obtained with all tested samples of GCS-EDTA, suggesting an acceptable level of negative effects on MDCK cell viability, with the latter resulting in a slightly lower level of cell viability. biocompatibility. However, even at all concentrations used in this study, cell viabilities of more than 93% were obtained with all tested samples of GCS-EDTA, suggesting an acceptable level of biocompatibility.

Figure 7. Effect of GCS and GCS-EDTA on the viability of Madin-Darby Canine Kidney (MDCK) cells. Figure 7. Effect of GCS and GCS-EDTA on the viability of Madin-Darby Canine Kidney (MDCK) cells. 3. Materials and Methods

3. Materials Materials and Methods 3.1.

(Arg-Lys-Asp, TP3), thymocartin (Arg-Lys-Asp-Val, TP4), thymopentin 3.1.Thymotrinan Materials (Arg-Lys-Asp-Val-Tyr, TP5) and their metabolites were provided by GL Biochem Ltd. Thymotrinan TP3), thymocartin (Arg-Lys(Shanghai, China).(Arg-Lys-Asp, All peptide purities were(Arg-Lys-Asp-Val, higher than TP4), 98% thymopentin as evidenced by Asp-Val-Tyr, TP5) and their metabolites were provided by GL Biochem Ltd (Shanghai, China). All RP-HPLC assay (Agilent Technologies, Santa Clara, CA, USA). Calcein, cobalt hydrodichloride, peptide purities were higher than 98% as evidenced by RP-HPLC assay (Agilent Technologies, Santa N-hydroxysuccinimide (NHS), leucine aminopeptidase (EC 3.4.11.2, microsomal from porcine kidney), Clara, California, USA). Calcein, cobalt hydrodichloride, (NHS), leucine 0 -ethylcarbodiimide N-(3-dimethylaminopropyl)-N hydrochlorideN-hydroxysuccinimide (EDAC), ethylene diamine tetraacetic aminopeptidase (EC 3.4.11.2, porcine kidney), N-(3-dimethylaminopropyl)-N′acid (EDTA), and glycol chitosan microsomal (GCS, degreefrom of deacetylation ca. 75.0%, 250 KDa) were purchased ethylcarbodiimide hydrochloride (EDAC), ethylene diamine tetraacetic acidIsotope (EDTA), and glycol from Sigma (St. Louis, MO, USA). Deuterium oxide was provided by Cambridge Laboratories, chitosan (GCS, degree of Laboratories, deacetylation Tewksbury, ca. 75.0%, 250 KDa) wereThese purchased fromand Sigma (St. Louis, Inc. (Cambridge Isotope MA, USA). peptides compounds MO, USA). Deuterium oxide was provided by Cambridge Isotope Laboratories, Inc (Cambridge were used as received without further purification. Isotope Laboratories, Tewksbury, MA, USA). These peptides and compounds were used as received without further purification. 3.2. Synthesis of Glycol Chitosan-EDTA Conjugate Glycol chitosan-EDTA was synthesized in a slightly modified way as described by 3.2. Synthesis of Glycol Chitosan-EDTA Conjugate Bernkop-Schnurch ˝ et al. [3,15]. An amount of 500 mg of glycol chitosan was dissolved in 50 mL of deionized double-distilled water. Furthermore, 15 g of EDTA was added to 20 mL deionized double-distilled water and the pH value was kept constant at pH 8 by continuously adding 5 M sodium hydroxide until EDTA was completely dissolved. Deionized double-distilled water was added to make a final volume of 50 mL. Thereafter, the abovementioned solutions were mixed evenly under

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stirring and the pH value was adjusted to 6.0 with 5 M sodium hydroxide. In order to catalyze the formation of amide bonds between the amino groups of glycol chitosan and the carboxyl groups of EDTA, EDAC was added at a final concentration of 0.1 M. The reaction mixture was incubated at room temperature under continuous stirring for 14 h. The resulting conjugate was isolated by exhaustive dialyzing against 0.05 M sodium hydroxide and then exhaustively against deionized double-distilled water. The purified product was lyophilized and stored at −20 ◦ C until use. Further solubility characterization revealed that glycol chitosan-EDTA was soluble at 10 mg/mL in both pH 6.5 phosphate buffered saline and Hank’s balanced salt solution without Ca2+ /Mg2+ at 25 ◦ C. The resultant solutions are transparent without any precipitate. Moreover, even at pH lower than 2.0, 20 mg/mL glycol chitosan-EDTA is still soluble and the resultant solution is transparent but with increasing viscosity. These results suggest that conjugation of EDTA to glycol chitosan significantly improved the solubility of EDTA. 3.3. FTIR and NMR Characterization Fourier transform infrared (FTIR) spectra measurements were performed using a DTGS detector-equipped Bruker Vertex 80V instrument (Billerica, MA, USA). Data were collected on the transmittance mode over a frequency region of 4000–400 cm−1 for 32 interferograms with a resolution of 4 cm−1 at 25 ◦ C. Samples were prepared by mixing 200 mg KBr with 1 mg sample. Finally, the spectra were presented in absorption mode after the baseline correction. The 1 H-NMR spectra measurements were conducted with a Bruker Avance-400 Ultrashield spectrometer (Billerica, MA, USA) equipped with a 5 mm probe at 70 ◦ C [3]. Samples were prepared at 20 mg/mL in deuterium oxide, and pH was adjusted to 10 with 5 M sodium hydroxide (6 µL) prior to running [21]. All chemical shifts were referenced to the HOD peak as a primary reference, and spectral data were collected and analyzed using Bruker’s Topspin software (Version 2.1, Billerica, MA, USA). 3.4. Evaluation of Chelating Ability of GCS-EDTA to Calcein It is well known that calcein fluorescence can be strongly quenched by cobalt ions at physiological pH, while removal of cobalt ion from the complex by EDTA-mediated chelation will lead to a significant increase in fluorescence intensity. Thus, the chelating capacity of the resultant conjugate can be determined by measuring the cobalt ion-chelating-mediated fluorescence recovery of calcein. That is, the fluorescence spectra of calcein coupled with cobalt ions at 1:1 molar ratio were measured with a RF-5301 fluorospectrophotometer (Shimadzu, Tokyo, Japan) in the presence of GCS-EDTA with different concentrations. The excitation wavelength of calcein was set at 490 and its emission was monitored in the range of 500–650 nm. The slits of excitation and emission were set at 5 and 3 nm, respectively. All background effects were subtracted. In addition, the same experiment was done in the presence of EDTA. Finally, the normalized maximum fluorescence emission intensity (calculated as F/F0) of the calcein at each time point was used, where F is the maximum fluorescence emission at each point and F0 is the maximum emission intensity of calcein in the absence of GCS-EDTA or EDTA. 3.5. In Vitro Evaluation of Enzyme Inhibition Efficiency of GCS-EDTA An amount of 0.01 U/mL Leucine aminopeptidase was suspended in 5 mL Krebs phosphate buffer (pH 7.0, 37 ◦ C) which contains 1.3 mM calcium chloride, 1.2 mM magnesium sulfate, 16.5 dibasic sodium phosphate, 120.8 mM sodium chloride, and 4.8 mM potassium chloride. After a 30-min enzymatic activation, the stock solutions of three thymopoietin oligopeptides were added into the above solution and made the final concentration of peptides at 0.1 mM. At specified time intervals after adding peptide, 100 µL aliquots were sampled and diluted with an equal volume of 1 M perchloric acid incubated in an ice bath to stop reaction. Meanwhile, a 100 mL aliquot of the abovementioned buffer was added to the incubation medium. To evaluate the enzyme inhibition efficiency of GCS-EDTA, further investigations were performed with a mixture of the pure enzyme with the conjugates at the same conditions as the degradation experiment. After centrifugation, the concentrations of intact

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peptide and metabolite in aliquots were analyzed by RP-HPLC as described in our previous studies [3]. All experiments were replicated at least three times. 3.6. Calculation of Degradation Clearances The degradation clearances of three peptides with/without GCS and GCS-EDTA were calculated from the total metabolized peptide amount (ΣM) in the incubation medium at each time point and the area under the peptide concentration-time curve (AUC) according to the equation: ΣM = Cldeg × AUC. Cldeg was determined by linear regression analysis from plots of ΣM versus the AUC at different time points [3,11]. 3.7. Cytotoxicity Studies MDCK cells were transferred to 96-well plates (Corning Inc, New York, NY, USA) at a density of 5 × 103 cells/well and cultured for 24 h in a humidified incubator, at 37 ◦ C with 5% CO2 . The culture medium was then replaced with Dulbecco minimum essential medium containing GCS and GCS-EDTA of different concentrations, respectively. After 24 h coincubation at 37 ◦ C, the medium was replaced with 20 µL of MTT (5 mg/mL in phosphate buffered saline) and 100 µL of incubation medium and incubated for a further 4 h at 37 ◦ C. After incubation, the medium was removed, and 100 µL of dimethyl sulfoxide (DMSO) was added to the residual precipitates. The absorbance of formazan was determined at 490 nm, using an iMark™ microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). Cell viability was expressed as a percentage of the absorbance relative to that of the control. Control cells were not exposed to any materials. The experiments were performed with three replicate wells for each sample and control. 3.8. Statistical Analysis All results are expressed as mean ± standard deviation. Statistical analysis was performed using a two-tailed unpaired Student’s t-test in software OriginLab of version 8.5 (OriginLab, Northampton, MA, USA). Difference was considered statistically significant at p-values < 0.05. 4. Conclusions In summary, a novel glycol chitosan conjugate was synthesized by forming an amide bond between the amino group of glycol chitosan and carboxyl group of EDTA. The resultant conjugate is soluble in both PBS (pH 6.5) and Hank’s balanced salt solution without Ca2+ /Mg2+ and is compatible with MDCK cells at high concentrations. Moreover, it still keeps the chelation capacity of EDTA to cobalt ions but shows slightly reduced efficiency compared with the free EDTA. Further investigation reveals that GCS-EDTA at low concentrations can significantly protect thymopoietin oligopeptides from leucine aminopeptidase-mediated degradation. This study suggests that GCS-EDTA might be used as an efficient metalloenzyme inhibitor to protect peptides and proteins from enzymatic degradation. Its potential application as a bioadhesive material and permeation enhancer is to be further investigated. Acknowledgments: This work was supported by China Postdoctoral Science Foundation (No. 20110491321), Hebei Provincial Natural Science Foundation of China—Shijiazhuang Pharmaceutical Group (CSPC) Foundation (No. C2011319001). We thank the anonymous reviewers and the editor for their constructive comments and suggestions. Author Contributions: Y.Z. and L.C. conceived and designed the experiments; Y.Z. and J.F. performed the experiments; J.F., L.C., F.L. and N.S. analyzed the data; W.K. and Y.C. contributed reagents/materials/analysis tools; Y.Z. and L.C wrote the paper. Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

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Sample Availability: No sample of the compounds is available from the authors. © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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