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2013
http://informahealthcare.com/ddiISSN: 0363-9045 (print), 1520-5762 (electronic)
Drug Dev Ind Pharm, Early Online: 1–9! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/03639045.2013.809533
ORIGINAL ARTICLE
Novel application of polioviral capsid: development of a potentand prolonged oral calcitonin using polioviral binding ligand andTat peptide
Jiradej Manosroi1,2, Warangkana Lohcharoenkal1, Friedrich Gotz3, Rolf G. Werner4, Worapaka Manosroi5, andAranya Manosroi1,2
1Faculty of Pharmacy, Chiang Mai University, Chiang Mai, Thailand, 2Natural Product Research and Development Center (NPRDC), Science and
Technology Research Institute (STRI), Chiang Mai University, Chiang Mai, Thailand, 3Department of Microbial Genetics, Faculty of Biology, University
of Tuebingen, Germany, 4Boehringer Ingelheim Company, Ingelheim am Rhein, Germany, and 5Department of Medicine, Faculty of Medicine,
Chiang Mai University, Chiang Mai, Thailand
Abstract
Context: Poor absorption and proteolytic degradation are major obstacles of orally administeredpeptide drugs including calcitonin. Cell penetrating peptides (CPPs) and receptor bindingligands are interesting tools for the application in the delivery of these drugs.Objective: To investigate the enhancements of in vitro and in vivo salmon calcitonin (sCT)activity by Tat, a trans-activating transcriptional peptide and VP1 peptide (V) from polioviralcapsid.Materials and methods: Tat/sCT, V/sCT and V/Tat/sCT mixtures at various molar ratios wereprepared and investigated for in vitro and in vivo activities of sCT.Results: Tat could increase in vitro sCT activity both in colon adenocarcinoma (HT-29) andmouth epidermal carcinoma (KB) cells. V/sCT (6:1) showed significant increase of intracellularcalcium in HT-29 cells. V/Tat/sCT (6:1:1) gave highest increase of intracellular calcium in bothcells. Oral administered Tat/sCT (1:1) showed comparable hypocalcemic effect to sCT injectionwith prolonged action. V/Tat/sCT (6:1:1) demonstrated hypocalcemic effect at 12 h afteradministration but no hypocalcemic effect was observed from V/sCT.Discussion: Positive charge from Tat might facilitate sCT uptake and absorption. Increasingof intracellular calcium in HT-29 cells by V but lacking of hypocalcemic effect from V/sCTin mice indicated the ligand-receptor mediated delivery of sCT by the interaction betweenV and PVR.Conclusion: Potential application of V and Tat in oral calcitonin delivery system wasdemonstrated. Further study in a proper PVR bearing host is still needed to provide moreuseful information for the application of V in the development of drug delivery systems.
Keywords
Calcitonin, cell penetrating peptide,oral administration, peptide delivery,viral ligand
History
Received 25 December 2012Revised 20 May 2013Accepted 21 May 2013Published online 26 June 2013
Introduction
Calcitonin (CT), an endogenous 32 amino acids peptide hormoneplays a crucial role both in calcium homeostasis and boneremodeling1,2. Four forms of CT including synthetic humanCT (hCT), synthetic salmon CT (sCT), natural porcine CT anda synthetic analogue of eel CT are used clinically. In order toeffectively inhibit the manifestations of metabolic bone disorderssuch as Paget’s disease and osteoporosis, a frequent and relativelyhigh dose of CT administration is needed3. Nowadays, CT canbe administered only parenterally or nasally4,5. Generally, theoral administration of drug is preferred to the parenteral routeespecially for the long-term treatment of chronic diseasesdue to ease of administration and non-invasion. However, the
development of oral peptide and protein drugs has been impedednot only from the poor absorption across intestinal membranesbut also from rapid proteolytic degradation, resulting in lessthan 1–2% bioavailability6–8. Extensive proteolytic degradation ofsCT in the gastro-intestinal lumen and low intrinsic intestinalmembrane permeability have been reported9,10. Insufficient oralbioavailability of CT necessitate high doses (4000–6000 IU/mg)of sCT, 20–30 times more potent than hCT has been reported.To solve these problems, several carrier-mediated deliverysystems have been developed. Recently, much attention hasbeen focused on the application of cell penetrating peptide (CPP)-based delivery systems. One of the most well-studied and efficientCPP is trans-activating transcriptional (Tat), the peptide fromhuman immunodeficiency virus type 1 (HIV-1)11. The basicregion of Tat at the amino acid position of 47–5712 is crucial formany key functions of the protein including interaction withthe transactivation-responsive region in viral mRNA13, nuclearlocalization14 and most importantly, cellular uptake15,16. Tat hasbeen shown to direct the internalization of an extensive list of
Address for correspondence: Jiradej Manosroi, Faculty of Pharmacy,Chiang Mai University, Chiang Mai 50200, Thailand. Tel: +66-53-894806. Fax: +66-53-894169. E-mail: [email protected],[email protected]
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cargoes ranging from small peptides to proteins17, polymers11,18,19,liposomes20, plasmid DNAs20 and even nanoparticles21. It has alsobeen used in vivo to deliver biologically active b-galactosidase intoall tissues of the mouse, even the brain22. Most of these studies usethe cross-linked conjugates of CPPs and cargoes. This type ofconjugates require chemical coupling and purifying, which mayaffect the physico-chemical properties and biological efficacy ofthe conjugates. The cargoes in these conjugates may not be releasedfrom the conjugate due to lacking of the enzyme which cleaves thecovalent bond between the cargoes and the CPPs. Recently, severalin vitro and in vivo studies of delivery systems using co-incubationmixture of various CPPs and peptides, proteins and antibodies havebeen reported23,24.
During the last decade, ligand-receptor mediated cellularevents in designing site-specific and target-oriented deliverysystems have received major attention. Most bio-relevant ligandssuch as antibodies, polypeptides, oligosaccharides, viral proteins,fusogenic residues and molecules of endogenous origin can beused for the site-specific delivery of various bioactives. Theseligands bind to their specific receptor for examples; bloodcarbohydrate (lectin) receptors, Fc receptors, complementreceptors, interleukin receptors, lipoprotein receptors, transferrinreceptors, scavenger receptors, receptors/epitopes expressed ontumor cells and cell adhesion receptors25.
Poliovirus (PV) is a small non-enveloped faecal-oral routetransmitted virus in Enteroviruses genera, Picornaviridae family.The structure of PV, like other picornaviruses, consists of anicosahedral protein shell that encapsidates a positive sense RNAgenome. The capsid consists of 60 copies each of three surfaceviral proteins: VP1, VP2 and VP3 (32, 29 and 26 kDa, respect-ively) and 60 copies of the internally located VP4 (7 kDa)26.PV infection is initiated by the attachment of capsid proteinto specific receptor molecules at the gastrointestinal epithelium.A narrow surface depression called ‘‘canyon’’ which is arrangedfrom VP1, VP2 and VP3 is a binding ligand to poliovirus receptor(PVR, CD155). VP1 BC loop (V) and VP1 bE to aB loop are theimportant parts involved in PVR binding27. Hence, the bindingability of these loops to the PVR in the upper gastrointestinal tractwhich result in the avoidance of the enzymatic degradationby the gastric juice, might be beneficial for the developmentof orally drug delivery system. In this study, Tat/sCT, V/sCT andTat/V/sCT mixtures were prepared and tested both in vitro andin vivo. The binding and translocation enhancement of sCT by Vand Tat to the gastrointestinal tract and the increase of sCTactivity were anticipated.
Materials and methods
Materials
sCT and VP1 BC loop (V) peptides [Thr-Val-Asp-Asn-Pro-Ala-Ser-Thr-Thr-Asn-Lys-Asp-Lys-Leu-Phe-Ala-Val-Arg-Lys (MW¼2105.3)] were purchased from Chengdu Biopharm, Chengdu,China. Tat [Gly-Arg-Lys-Lys-Arg-Arg-Glu-Arg-Arg-Arg-Pro-Pro-Glu-Arg-Lys-Cys (MW¼ 2106.5)] was from EMCCompany, Tubingen, Germany. All peptides purity was �90%.The Calcium OCPC kit was obtained from Coral ClinicalSystems, Verna, India. One-step RT-PCR kit was purchasedfrom Invitrogen, Carlsbad, CA. All other reagents were analyticalgrade. The male Sprague Dawley rats, weighing between 150 and200 g were from National Laboratory Animal Center, MahidolUniversity, Salaya, Nakhon Pathom, Thailand.
Cell culture
KB (human mouth epidermal carcinoma) and HT-29 (colonadenocarcinoma) cell lines were cultured in Dulbecco’s Modified
Eagle’s Medium (DMEM) with 10% fetal bovine serum, 100 U/mL penicillin and 100mg/mL streptomycin. All cell lines weregrown at 37 �C in a humidified 5% CO2 atmosphere incubator.
Preparation of the V/Tat/sCT mixture
Mixtures of various molar ratio of Tat/sCT (1/3:1–6:1), V/sCT(1/3:1–6:1) and V/Tat/sCT (1/3:1:1–6:1:1) were prepared inMilliQ water and incubated at room temperature (25� 2 �C) for1 h. In all systems, the final concentration of sCT was fixedat 100 pg/ml. All samples were freshly prepared before use.
Characteristics of V/Tat/sCT mixture
Particle size and � (zeta) potential
The mean particle size of sCT, Tat, V and the mixtures at variousmolar ratios were measured at 25 �C by a dynamic light scattering(DLS) (Zetasizer Nano Series Nano-S, Malvern Instrument Ltd.,Malvern, UK) using the non-negative constrained least squares(NNLS) algorithm mode particle size distribution (PSD) analysis.The measurements were performed in triplicate at 30 s with 10 spulse between cycles with three cycles in each measurement. The�-potential values of all samples were obtained from the phaseanalysis light scattering (PALS) software. The time-dependentcorrelation function on the scattered light intensity was measuredat a scattering angle of 90 �. The measurements were performedin triplicate with five cycles in each measurement.
Differential scanning calorimetry (DSC)
Thermograms of sCT, Tat, V, Tat/sCT (1:1 molar ratio), V/sCT(1:1 molar ratio) and V/Tat/sCT (1:1:1 molar ratio) wereinvestigated by a differential scanning calorimeter, DSC (Model4000, Perkin Elmer, Waltham, MA). Briefly, 3 mg of eachlyophilized sample was placed in the standard aluminum pans andsealed. An empty pan was used as a reference. DSC scans wereperformed at a heating rate of 10 �C/min under nitrogenatmosphere. To calibrate the temperature and energy scale ofthe DSC instrument, aluminum oxide was used as a standardreference material.
Fourier transform infrared (FT-IR) spectroscopy
The FT-IR spectrum of sCT, Tat, V, Tat/sCT (1:1 molar ratio),V/sCT (1:1 molar ratio) and V/Tat/sCT (1:1:1 molar ratio) wereperformed to observe the interaction among sCT, Tat and V.Samples were lyophilized and mixed with KBr powder. The KBrdisks were prepared by pressing at 7.54 kgf/cm2. The IRabsorbance was scanned at the range of 400–4000 cm�1 inFT-IR spectrophotometer (470 FT-IR, Nicolet, MA).
Determination of intracellular calcium concentrationof KB and HT-29 cells
The calcitonin activity of Tat/sCT, V/sCT and V/Tat/sCT inHT-29 and KB cells as determined by the increase of intracellularcalcium concentration was compared with free sCT. An amountof 1� 105 cells were seeded into each well of the 24-well platesand grown to 90% confluence. The culture medium was removedand replaced with 1 ml of sCT, Tat or V at the final concentrationof 100 pg/ml (29.15 pM), 61.22 pg/ml (29.15 pM) and 61.22 pg/ml(29.15 pM), respectively, or the mixtures at various molar ratio.Final concentration of sCT in all systems was 100 pg/ml. Allsamples were prepared in DMEM medium containing 0.2 mg/mlcalcium chloride. After 1 h incubation at 37 �C, the supernatantwas removed and the cells were washed twice with PBS. DI water(500 ml) was added into each well and the cells were lysed bysonication for 1 h in an ultrasonic bath (Model 690D, Crest, NJ).
2 J. Manosroi et al. Drug Dev Ind Pharm, Early Online: 1–9
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After removal of the cell debris by centrifugation at 10 000� g,the supernatant was collected for intracellular calcium determin-ation using calcium OCPC kit. Calcium concentration wascalculated from the standard curve of the plot between standardcalcium concentration and absorbance at 570 nm. All experimentswere performed in triplicate and the data was present asmean�SD. The results were presented as relative intracellularcalcium content of control as follows:
Relative intracellular calcium ð%Þ
¼ Intracellular calcium after treated with sample ðmg=mlÞIntracellular calcium of control ðmg=mlÞ
� 100
Determination of PVR in KB and HT-29 cells
PVR expression was determined to demonstrate and confirm thepresence of PVR in the cells. Total cellular RNA was extractedfrom cultured cells using Nucleospin RNA II (Macherey-nagel,Duren, Germany) according to the manufacturer’s instructions. Thereverse transcription PCR was performed in iCycler thermal reactor(Biorad, Milan, Italy). Primers were designed from the sequence ofthe human CD155 gene28. The primers were used to locate theexpression of the CD155 gene in a region corresponding to theextracellular domain of PVR, which is conserved in all splicevariants29. The sequences of these primers were50-TATCTGGCTCCGAGTGCTT GCC-30 for forward primer and50-ATCATAGCCA GAGATGGATACC-30 for reverse primer.Amplified fragments were identified by electrophoresis in a 1%agarose gel and stained with ethidium bromide. For quantification,the bands densities were determined by the Quantity One ProgramAnalysis using Gel documentation (BioRad, Milan, Italy).
Hypocalcemic activity
Animals
After 3 days of acclimatization, the rats were divided randomlyinto three groups. The experiment was performed conforming tothe guidelines of the Animal Welfare Commission of Chiang MaiUniversity, Thailand.
Subcutaneous administration of sCT
The sCT in 0.9% normal saline solution (NSS) pH 6.5 at the doseof 10, 50, 100, 250 and 500mg/kg were subcutaneously injected toeach group of the rats at the dorsal skin using a 27-gauge needle.At 0, 1, 2, 4, 8, 12 and 24 h after injection, the blood samples(100ml) were collected from the tail vein and immediatelycentrifuged at 5000� g (Universal 32 R, Hettich Zentrifugen,Kirchkengern, Germany), 15 �C for 10 min. The supernatant wascollected and assayed for the serum calcium level using calciumOCPC kit. The data was presented as relative serum calcium levelcalculated as follows:
Relative serum calcium level
¼ Serum calcium level at each sampling time
Serum calcium level at initial ðtime ¼ 0Þ � 100
Oral administration of the samples
The samples were orally administered to each group of the ratsusing a 4 inches long feeding tube. The dose of sCT, Tat and Vwere 50, 32 and 32 mg/kg, respectively. For Tat/sCT, V/sCT andV/Tat/sCT, the mixtures at molar ratios which showed thehighest in vitro activity in KB and HT-29 cells were used inthe experiment. The dose of sCT in all mixtures was fixedat 50mg/kg. At 0, 1, 2, 4, 8, 12 and 24 h after administration, the
blood samples (100 ml) were collected and assayed for the serumcalcium as described above.
Results
Characteristics of sCT, Tat, V and the mixtures
Particle size and � (zeta) potential
Particle sizes and zeta potential of sCT, Tat, V and the mixturesat various molar ratios were shown in Table 1. At 29.15 pM,the particle sizes of sCT, Tat and V were 235.90� 77.36,1106.00� 191.90 and 502.60� 104.10 nm, respectively, whereasthe peptide mixtures were in the range of 222.90� 71.28 to504.90� 24.53 nm. Zeta potential values of sCT, Tat and V inMQ water were (�) 5.92� 1.36, (�) 3.03� 2.53 and (�)17.80� 2.73 mV, respectively. Tat/sCT mixtures at all molarratios gave positive zeta potential values (11.4� 0.79 to13.9� 0.32 mV), while V/sCT and V/Tat/sCT mixtures showednegative zeta potential value.
Differential Scanning Calorimetry (DSC) and FourierTransform Infrared (FT-IR) spectroscopy
Physico-chemical characteristics of the mixtures were determinedby DSC and FT-IR spectroscopy. Figure 1 showed the DSCthermograms of sCT, Tat, V and Tat/sCT, V/sCT and V/Tat/sCTmixtures. sCT and V showed a single exothermic peak at 155.84and 166.98 �C, respectively, whereas Tat demonstrated somealterations at over 200 �C. Tat/sCT, V/sCT and V/Tat/sCTmixtures showed no exothermic peak at the temperature under-studied. For FT-IR, sCT showed one sharp peak of the stretchingvibration of hydroxyl group (–OH) at 3307.99 cm�1 and 2 peaksof N-H bending at 1657.95 and 1538.25 cm�1, respectively(Figure 2A). Two sharp peaks at 2350.38 and 661.89 cm�1 werefrom asymmetrical stretching and symmetrical bending of C¼O,respectively. C-N stretching peak of amide was detected at1415.53 cm�1, while the vibration peaks of C–O from phenolgroup of tyrosine were recorded at 1254.20 and 1118.94 cm�1.For Tat, besides the O-H stretching, N–H bending and C–Nstretching peak of amide as observed in sCT, C–N stretchingpeak of amine and C-H rocking peaks were also detected at1195.58 cm�1 and 599.96–805.19 cm�1, respectively (Figure 2B).For V, C–N stretching peak of both amide and amine were
Table 1. Particle size (nm) and zeta potential (mV) of sCT, Tat, V andV/Tat/sCT mixtures at various molar ratios.
Samples Particle size (nm) Zeta potential (mV)
Water – (�)17.60� 7.46sCT 100 pg/ml (29.15 pM) 235.90� 77.36 (�)5.92� 1.36Tat 61.22 pg/ml (29.15 pM) 1,106.00� 191.90 (�)3.03� 2.53V 61.22 pg/ml (29.15 pM) 502.60� 104.10 (�)17.80� 2.73Tat/sCT (1/3:1) 347.80� 38.07 11.40� 0.79Tat/sCT (1:1) 222.90� 71.28 12.40� 6.40Tat/sCT (3:1) 290.80� 54.22 13.00� 2.06Tat/sCT (6:1) 337.00� 92.34 13.90� 0.32V/sCT (1/3:1) 338.80� 66.06 (�)2.53� 1.02V/sCT (1:1) 340.00� 40.30 (�)7.72� 3.10V/sCT (3:1) 326.90� 23.70 (�)6.24� 1.63V/sCT (6:1) 301.20� 29.90 (�)7.54� 2.00V/Tat/sCT (1/3:1:1) 335.80� 30.22 (�)8.09� 3.36V/Tat/sCT (1:1/3:1) 380.10� 67.95 (�)12.20� 3.33V/Tat/sCT (1:1:1) 330.90� 68.06 (�)6.40� 5.39V/Tat/sCT (1:3:1) 504.90� 24.53 (�)6.20� 1.17V/Tat/sCT (1:6:1) 398.70� 17.46 (�)2.43� 0.28V/Tat/sCT (3:1:1) 226.80� 4.27 (�)12.60� 4.21V/Tat/sCT (6:1:1) 274.20� 130.20 (�)13.90� 0.32
In mixture form, the concentration of sCT was fixed at 100 pg/ml.
DOI: 10.3109/03639045.2013.809533 Polioviral ligand and Tat as oral calcitonin carrier 3
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detected at 1440.41 and 1193.12 cm�1, respectively (Figure 2C).The peaks of N–H bending and O–H stretching werealso observed with C–H stretching peak at 2966.20 cm�1.In Figure 2(D), the increase in peak intensity of C–H stretchingat 1100–1200 cm�1 and the decrease of C¼O stretching peakat 2349.42 cm�1 of sCT were observed in Tat/sCT mixture.A slight decrease of C–N stretching peak and the disappearanceof C–H bending of Tat at 1438.16 and 719.73 cm�1, respectively,were also detected in Tat/sCT mixture. For V/sCT and V/Tat/sCTmixtures, sharp peak of C¼O stretching at 2350.38 cm�1 of sCTwas disappeared and the FT-IR spectrum showed the similarpattern with V peptide (Figure 2E and F).
Enhancement of intracellular calcium in KB andHT-29 cells
Most of intracellular calcium is sequestered in mitochondriaand endoplasmic reticulum with fluctuate concentration rangingfrom 100 nM to greater than 1mM, due to the release fromcellular stores or influx from extracellular fluid controlledby parathyroid hormone (PTH) and calcitonin. The extent ofintracellular calcium accumulation indicated the extent of cellular
level calcitonin activity which is directly related to the concen-tration of calcium in medium30. In this study, concentration ofsCT at 100 pg/ml which was the serum concentration was used.The in vitro sCT activity was determined from the increasein intracellular calcium concentration after incubation with thesamples. The relative intracellular calcium (%) in HT-29 and KBcells after incubation with samples at various molar ratios werepresented in Figure 3. In both cell types, sCT, Tat or V aloneshowed no effect on relative intracellular calcium. When mixedwith Tat, the activity of sCT was increased in both cell lines withthe maximum relative intracellular calcium of 116.46� 0.57%(3:1) and 172.14� 4.12% (1:1) in HT-29 and KB cells, respect-ively (Figure 3A). V/sCT at 6:1 molar ratio showed significant anincrease in intracellular calcium only in HT-29 cells of 152.07%of control but not in KB cells (Figure 3B). This might be due tothe lack of PVR (CD155) in KB cells (Figure 4). In V/Tat/sCT,the similar sCT activity as either Tat/sCT or V/sCT in both celllines was observed. For V/Tat/sCT at 6:1:1 molar ratio, thehighest relative intracellular calcium level in HT-29 cells whichwas similar to V/sCT (6:1). Also, the V/Tat/sCT (6:1:1) andTat/sCT (1:1) showed higher sCT activity in KB than HT-29 cells(Figure 3C).
Figure 1. Differential scanning calorimetry (DSC) thermograms of sCT (A), Tat (B), V (C), Tat/sCT mixture at 1:1 molar ratio (D), V/sCT mixtureat 1:1 molar ratio (E) and V/Tat/sCT mixture at 1:1:1 molar ratio (F).
4 J. Manosroi et al. Drug Dev Ind Pharm, Early Online: 1–9
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Figure 2. FT-IR spectra of sCT (A), Tat (B), V (C), Tat/sCT mixture (D), V/sCT mixture (E) and V/Tat/sCT mixture (F).
Figure 3. Relative intracellular calcium (%) in HT-29 and KB cells after incubation with Tat/sCT (A), V/sCT (B) and V/Tat/sCT (C) at various molarratios. sCT concentration in all samples was fixed at 100 pg/ml (29.15 pM). Tat and V at 61.22 pg/ml (29.15 pM) were included as single protein control* ¼ significant different from control with 0.05 level of probability (p50.05).
DOI: 10.3109/03639045.2013.809533 Polioviral ligand and Tat as oral calcitonin carrier 5
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Hypocalcemic effect of Tat/V-sCT mixtures
The oral hypocalcemic activity of sCT and mixtures whichshowed the highest sCT activity in HT-29 and KB cells whichwere Tat/sCT mixtures at 3:1 and 1:1; V/sCT mixtures at 6:1 and3:1; and V/Tat/sCT mixture at 6:1:1 molar ratio were investigatedin rats (Figure 5). For sCT, the positive control, a significantlowering of serum calcium similar to the previous report wasobserved in all doses after 1–2 h of subcutaneous injection andremained for over 12 h (Figure 5A)31. The dose of 50 mg/kg wasselected for oral administration study due to the lowest in dosewith effective serum calcium lowering with the lowest fluctu-ation. Oral administration of sCT at 50 mg/kg showed the decreasein serum calcium with the minimum relative serum calciumof 64.13� 2.93% at 1 h (Figure 5B). Administration of Tat or Vdid not affect serum calcium level (Figure 5B). Interestingly, Tat/sCT mixture at 1:1 molar ratio at the dose of sCT at 50mg/kgwhich was equivalent to 300 IU/kg (6000 IU¼ 1 mg) demon-strated the reduction in the serum calcium level of about 18–31%of the control (distilled water) with the prolonged activity of over24 h (Figure 5C)32. The hypocalcemic effect was not observed inthe V/sCT mixture fed rats. For V/Tat/sCT mixture, hypocalcemicactivity could be observed at 4 h with the maximum at 12 h afteradministration (Figure 5C).
Discussion
In vitro and in vivo enhancements of sCT activity byco-administration with Tat or V peptide were described.Tat/sCT, V/sCT and V/Tat/sCT mixtures at various molar ratioswere prepared and characterized by DLS, DSC and FT-IRtechniques. All mixtures showed smaller particle size than theirparent peptides, indicating the dense and homogeneous arrange-ment between peptide molecules in the mixture (Table 1)33. Theincrease in zeta potential value of sCT (�5.92� 1.36 mV) and Tat(�3.03� 2.53 mV) comparing with MQ water (�17.6� 7.46 mV)indicating their higher charge positivity, while V showed negativezeta potential value of �17.80� 2.73 mV. Tat/sCT mixtures atall molar ratios gave positive zeta potential values (11.4� 0.79to 13.9� 0.32 mV) resulting from the net charge of Tat.
Tat/sCT mixture at the higher Tat molar ratio gave higherpositive charge. V/sCT and V/Tat/sCT mixtures demonstratednegative zeta potential values resulting from the charge negativityof V molecule. DSC is one of major methods for the thermalanalysis of proteins. DSC can detect and quantitate changes in thetransition temperature and enthalpy due to formulation variablessuch as pH, ionic strength and stabilizers in both solutions andlyophilized forms34. FT-IR spectroscopy (FT-IR) is widely usedto study protein secondary structure in lyophilized solids35.Both DSC and FT-IR have been used together in several studiesfor complex formation characterization of proteins and peptidesincluding calcitonin33,36. As known, the DSC and FT-IR resultsof lyophilized samples have been widely used to demonstratephysico-chemical interaction of several peptide complexes33,36,37,the lyophilized mixtures as well as the lyophilized of each nativecomponent were used for DSC and FT-IR experiments in thisstudy. The DSC thermograms demonstrated the disappearanceof sharp exothermic peak of single peptides in the mixture formsindicated the physico-chemical interaction among sCT, V andTat (Figure 1)38,39. Due to the thermal analysis data is just thepreliminary study of peptide-peptide interaction and the inter-pretation of DSC results for freeze-dried protein formulationscan be difficult as a result of the more complex thermal eventsoccurring with this technique40, FT-IR technique was also used inthis study for the better interpretation. Three different methodswere used in this study in order to characterize the complexformation between peptides. The DLS results represented thecomplex formation as indicated by the decrease of the particlesizes. The relationship between the increase of the complexationefficiency and the decrease of particle sizes of several proteinsand peptides has been reported33,36. DSC and FT-IR experimentswere performed to investigate interaction between peptides inthe mixtures. FT-IR spectra of Tat/sCT, V/sCT and V/Tat/sCTmixtures indicated some interactions with complex formationamong sCT and Tat or V (Figure 2)33,41. H-bond formation mightbe involved in Tat/sCT complex since the increase in C-Hstretching of sCT and the disappearance of C-H bending of Tatwere observed42. The similar FT-IR spectra pattern of V/sCTand V/Tat/sCT mixtures in comparing with V peptide mightbe resulting from the cage formation around Tat and sCT byhydrophobic residues in V peptide43.
The in vitro sCT activity was enhanced by co-incubation withTat or V. Without Tat or V, sCT activity could not be detected.This might be due to the lack of CT receptor (CTR) on themembrane of these cells. CTR is a member of a new family ofrelated G-protein coupled receptors which includes the parathy-roid hormone (PTH), secretin and glucagon receptors44,45. It isabundant in the bone, hypothalamus and kidney46. CT activity innon-osteoclast cell transfected with cDNA encoding CTR hasbeen reported47. This indicated the significance of CTR for sCTactivity. Hence, for the translocation of sCT into non-CTRbearing cell, carrier is needed. The increase of sCT activity inboth cell lines by Tat peptide might be due to the enhancement ofsCT uptake and absorption by Tat, the highly positive chargearginine rich peptide which could facilitate the translocation byelectrostatic interaction with cellular surface48. The intracellularcalcium was remarkably increased by Tat/sCT mixture in KBcells in comparing with HT-29 cells. This might be resulting fromthe variation of cytoplasmic and nucleic distribution of Tat indifferent cell lines49. Tat/sCT mixture at 1:1 molar ratio which hasthe smallest particle size (222.90� 71.28 nm) demonstrated thehighest increase in sCT activity. Thus, not only the translocationby Tat, but also the particle size might be also involved in theinternalization. The comparison of internalized extent of particleswith the size range between 50 and 500 nm has been reported.The amount and rate of internalization will be decreased when the
Figure 4. Agarose gel electrophoresis of poliovirus receptor (PVR,CD155) transcripts amplified by RT-PCR from HT-29 cells (lane 2) andKB cells (lane 3). Lane 1 was 100 kb DNA ladder.
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particle size was increased50. After incubation with V/sCTmixture, the significant increasing of intracellular calcium inHT-29 cells was observed indicating the ligand-receptor mediatedcellular delivery of sCT by the interaction between V and PVR.Cytotoxicity of the carrier peptides used in this study has beenassessed. Our previous study reported lower cytotoxicity of Vthan Tat in HT-29 and KB cells. Mixtures of these carrier peptidesand green fluorescent protein (GFP) as a model protein were
prepared and evaluated for cellular uptake and cell toxicity.GFP uptake enhancement with higher cell viability than singlepeptide after V/Tat/GFP treatment has been demonstrated51.
In in vivo studies, Tat/sCT mixture at 1:1 molar ratiodemonstrated hypocalcemic effect with prolonged activity ofover 24 h. This might be due to the slow release of sCT fromsCT/Tat complex33. The therapeutic dose of sCT is in the rangeof 50–400 IU/day. After subcutaneous injection of calcitonin,
Figure 5. Hypocalcemic activity as relative serum calcium level of: (A) Subcutaneous administration of sCT at various doses of 10, 50, 100, 250and 500 mg/kg, (B) Oral administration of sCT at the dose 50mg/kg (300 IU/kg) or 3.64 mM, Tat at 32mg/kg (3.64 mM) and V at 32mg/kg (3.64 mM)and (C) Oral administration of Tat/sCT, V/sCT and Tat/V/sCT mixtures containing sCT equivalent to 50mg/kg BW.
DOI: 10.3109/03639045.2013.809533 Polioviral ligand and Tat as oral calcitonin carrier 7
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serum calcium level decreased about 20–30%52. When adminis-tered orally, Tat/sCT mixture at 1:1 molar ratio could reducethe serum calcium level of about 18–31% of the control whichwas comparable to subcutaneous injection. These results indicatedthe potential of this mixture as an oral sCT administration.The maximum serum calcium level reduction at 1 h after oraladministration of sCT has been previously reported31. Maximumpercentage reduction of serum calcium after orally Tat/sCTmixture administration was in the same range as oral sCTadministration. This result was similar to other previous studieswhich showed the comparable change in blood calcium concen-tration but longer duration of sCT in nanoparticle carriers53.In rat, V/sCT mixtures showed no hypocalcemic effect. Thismight be due to the lack of PVR in rat resulting from the rapidchange of the PVR gene during evolution, whereas in Tatcontaining mixtures, hypocalcemic effects were still observed.The rodent species were not susceptible to PV due to the lowamino acid sequence similarity of rodent PVR to human PVR54.The long-acting pattern of V/Tat/sCT mixture might be resultedfrom the slow release of Tat/sCT from the cage forming byhydrophobic residue of V.
In conclusion, this study demonstrated the innovative findingsof in vitro and in vivo enhancement of sCT activity by mixingwith Tat or V peptide. The physico-chemical interactions betweenthe single peptides and also the cage formation with V peptidemight play role in the complexes formation of Tat/sCT, V/sCTand V/Tat/sCT mixtures. Tat/sCT (1:1) demonstrated theenhancement of in vitro and in vivo sCT activity comparingwith sCT as indicated by the increasing of intracellular calciumlevel and the prolonged hypocalcemic effect, respectively.Ligand-receptor mediated cellular delivery of V/sCT by theinteraction between V and PVR was demonstrated by significantincrease in intracellular calcium only in PVR-bearing cells and nohypocalcemic effect in PVR-lacking animal. Also, cage formationby hydrophobic residue of V might responsible for the slow-actingof Tat/sCT from V/Tat/sCT. The results of this study can beapplied for further development of oral calcitonin and otherpeptide drug delivery systems. However, further in vivo investi-gation in appropriate PVR bearing animal model should beperformed.
Declaration of interest
The authors report there is no known conflict of interests.This work was supported by the Thailand Research Fund (TRF) under
the RGJ-PhD program, Nanoscience and Nanotechnology ResearchCenter Project, Faculty of Sciences, Chiang Mai University, Thailandand Natural Product Research and Development Center (NPRDC),Science and Technology Research Institute (STRI), Chiang MaiUniversity, Thailand.
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