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Nanoparticles of cationic chimeric peptide and sodium polyacrylate exhibit striking antinociception activity at lower dose
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Journal of Controlled Release j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j c o n r e l
Nanoparticles of cationic chimeric peptide and sodium polyacrylate exhibit striking antinociception activity at lower dose Kshitij Gupta a,c, Vijay P. Singh a, Raj K. Kurupati a, Anita Mann a, Munia Ganguli a, Yogendra K. Gupta b, Yogendra Singh a, Kishwar Saleem c, Santosh Pasha a,⁎, Souvik Maiti a,⁎ a b c
Institute of Genomics and Integrative Biology, Mall Road, Delhi-110007, India Department of Pharmacology, All India Institute of Medical Sciences, New Delhi-110029, India Department of Chemistry, Faculty of Natural Sciences, Jamia Millia Islamia, New Delhi-110025, India
a r t i c l e
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Article history: Received 20 March 2008 Accepted 10 October 2008 Available online 1 November 2008 Keywords: Nanoparticles Chimeric peptide Cytotoxicity Hematological parameters Antinociception
a b s t r a c t The current study investigates the performance of polyelectrolyte complexes based nanoparticles in improving the antinociceptive activity of cationic chimeric peptide-YFa at lower dose. Size, Zeta potential and morphology of the nanoparticles were determined. Size of the nanoparticles decreases and zeta potential increases with concomitant increase in charge ratio (Z+/−). The nanoparticles at Z+/−12 are spherical with 70 ± 7 nm diameter in AFM and displayed positive surface charge and similar sizes (83 ± 8 nm) by Zetasizer. The nanoparticles of Z+/− 12 are used in this study. Cytotoxicity by MTT assay on three different mammalian cell lines (liver, neuronal and kidney) revealed lower toxicity of nanoparticles. Hematological parameters were also not affected by nanoparticles compared to normal counts of water treated control group. Nanoparticles containing 10 mg/kg YFa produced increased antinociception, ~ 36%, in tail-flick latency test in mice, whereas the neat peptide at the same concentration did not show any antinociception activity. This enhancement in activity is attributed to the nanoparticle associated protection of peptide from proteolytic degradation. In vitro peptide release study in plasma also supported the antinociception profile of nanoparticles. Thus, our results suggest of a potential nanoparticle delivery system for cationic peptide drug candidates for improving their stability and bioavailability. © 2008 Elsevier B.V. All rights reserved.
1. Introduction In recent years, significant efforts have been put in to develop nanotechnology for drug delivery. Nanotechnology offers better prospects over conventional procedures [1–4] for delivering small molecular weight drugs, as well as macromolecules (proteins, peptides or genes) by either localized or targeted delivery to the tissue of interest [5–11]. It focuses on formulating therapeutic agents in biocompatible nanocomposites such as nanoparticles, nanocapsules and micellar systems. Therapeutic molecules within the nanocomposites possess a higher stability in biological fluids and are resistant towards enzymatic metabolism [12]. As a successful drug delivery vehicle, polymeric nanoparticles have the ability to escape from the reticuloendothelial system, resulting in longer stay in circulation [5]. These nanoparticles can penetrate deep into tissues through capillaries, cross the fenestration present in the polyepithelial lining, and are generally taken up efficiently by the cells [13]. A wide variety of techniques are available for producing nanoparticles including solvent evaporation [14] interfacial polymerization ⁎ Corresponding authors. Fax: +91 11 27667471. E-mail addresses: [email protected] (S. Pasha), [email protected] (S. Maiti). 0168-3659/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2008.10.008
[15] and emulsion polymerization methods [16]. However, most of these approaches involve the use of organic solvents, heat or vigorous agitation, which are potentially harmful to sensitive biomolecules. In recent years, self assembly of proteins/peptides/drug candidates with natural or synthetic polyelectrolyte to form Polyelectrolyte Complexes (PECs) has drawn increased interest of chemists and pharmacologists [17]. PEC formation leads to particles with dimensions on a colloidal level generating optically homogeneous and stable nano-dispersions. Additionally, such methods are advantageous as they do not require sonication and organic solvents during preparation, thereby minimizing possible damage to drug candidates during PEC formation. We have also demonstrated that such type of PECs of nanometer size can be prepared through electrostatic interaction of cationic peptides containing varying number of lysine residues with polyanionic polymer of sodium salt of polyacrylic acid (PAA) [18]. The polymer PAA has been used as an excellent protectant additive to preserve the bioactivity of L-lactate dehydrogenase (LDH) as it interacts with alginate microparticles [19]. Furthermore, PAA has been widely employed for buccal [20] and ophthalmic drug delivery systems [21]. In our previous study, it has been shown that cationic chimeric peptide (YGGFMKKKFMRFa-YFa) of Met-enkephalin (YGGFM) and
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FMRFa, joined by three lysine residues, induces antinociceptive action at doses of 40 mg/kg and above [22]. However, at lower dose of 10 mg/kg of YFa no antinociception effect was observed. This may be due to the peptidic nature of YFa which renders it susceptible to protease degradation and hence resulting in lower bioavailability. In the current study, to increase the delivery efficiency of the cationic chimeric peptide-YFa, we have adopted a PECs formation strategy to obtain nanoparticles from polyanionic polymer PAA and YFa to achieve significant antinociception effect at lower dose of peptide. Size and zeta potential of nanoparticles were characterized by Zetasizer and morphology of nanoparticles was determined by Atomic Force Microscopy. Cytotoxicity assay on mammalian cell lines and evaluation of hematological parameters were done to understand the toxicity behavior of nanoparticles. In vitro peptide release in blood plasma was done to understand the release profile of peptide from nanoparticles.
to 1.0 mL for measurements, which were carried out in an automatic mode. The values of size and zeta potential are the average of 30 runs. The Smoluchowski approximation was used to calculate zeta potential from the electrophoretic mobility.
2. Experimental section
Two millilitre of PAA-YFa nanoparticles containing 10 mg/kg YFa, 10 mg/kg neat YFa and neat PAA of same concentration that was used for the formation of nanoparticles at Z+/−12 were ultracentrifuged (Beckman Coulter, USA) at 21,7000 ×g for 60 min to obtain supernatants. YFa in the supernatant was quantitated by RP-C18 column (µBondapak™ 5 µm, 3.9 × 300 mm, Waters, USA) at 220 nm with a photodiode array detector (Waters 996, USA) on a reverse phase HPLC (Waters Delta 600, USA). The mobile phase consisted of acetonitrile/water (10:90 linear gradient for 40 min) and the flow rate was 0.8 mL/min. The peptide entrapment efficiency was expressed as the percentage of YFa difference between the total amount of the YFa added and in the supernatant relative to the total amount of YFa added to form nanoparticles. All samples were measured in triplicate.
2.1. Chemicals Sodium salt of polyacrylic acid (Mw = 10,000), N,N′diisopropyl carbodiimide (DIPCI) and trifluoroacetic acid were obtained from Sigma-Aldrich. Naloxone hydrochloride was purchased from Sigma (St. Louis, MO). All Fmoc-amino acids, 1-hydroxybenzotriazole (HOBt), and Rink amide resin were purchased from Nova Biochem (Switzerland). Acetonitrile was obtained from Merck Ltd. (India). 2, 5dihydroxybenzoic acid was supplied by Bruker Daltonics (Germany). Cell lines Hep G2 (liver, human), N2a (neuronal, mouse), and HEK 293 (kidney, human) were obtained from National Centre for Cell Sciences (NCCS), Pune, India. Dulbecco's modified eagle's medium (DMEM), fetal bovine serum, sodium bicarbonate, antibiotic–antimycotic solution (100X), Trypsin-EDTA, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and Dimetyl sulfoxide (DMSO) were procured from Sigma. 2.2. Peptide synthesis Peptide-YFa was synthesized by the solid phase method on automated peptide synthesizer (Advanced chemtech, USA), using the standard chemistry of fluorenylmethoxy carbonyl (Fmoc) amino acids and 1-hydroxybenzotriazole (HOBt)/N,N′-diisopropyl carbodiimide (DIPCI) activation method on a rink amide resin. The peptide was purified by RP-C18 column (µBondapak™ 10 µm, 7.8 × 300 mm, Waters, USA) on semi-preparative reverse phase HPLC (Waters 600, USA) with a 40 min linear gradient from 10% to 90% acetonitrile containing 0.05% trifluoroacetic acid in water. The mass analysis of the peptides was done in linear positive ion mode by MALDI-Tof-Tof (Bruker Daltonics Flex Analysis, Germany) with 2, 5 dihydroxybenzoic acid as the matrix. The peptide sequence was confirmed by automated peptide sequencing (Procise 491 Applied Biosystems, USA). 2.3. Preparation of polyelectrolyte complexes The complexes of PAA and chimeric peptide were prepared by electrostatic interaction as described in our previous study [18]. In brief, different dilutions of stock 1.0 mg/mL peptide-YFa were added to 1 × 10− 3 M (in acrylate unit of PAA) solution of PAA to obtain complexes of different Z+/− i.e. 2–12. YFa and PAA solutions were prepared in MilliQ-water, pH = 6.8–7.0. 2.4. Size and zeta potential measurements Zetasizer Nano ZS (Malvern Instruments, UK) determined size and zeta potential of peptide–polymer complexes of different Z+/− i.e. 2–12. One hundred microlitre of complex samples of each Z+/− were diluted
2.5. Atomic force microscopy (AFM) measurements All AFM images were procured on PicoSPM equipment (Molecular Imaging, Tempe, AZ, USA) using AAC mode. Two microlitre of complex solution was deposited on a fresh piece of mica (1 × 1 cm) and allowed to adsorb for 2 min at room temperature. Imaging was performed under dried condition in air with 250 µm long cantilevers with a resonance frequency of about 41 kHz. Minimal image processing (flattening only) was carried out in the presented image. 2.6. Evaluation of entrapment efficiency of peptide
2.7. Cytotoxicity measurements The toxicity of the PAA-YFa nanoparticles was studied by MTT colorimetric assay [23]. HepG2 (liver, human), N2a (neuronal, mouse), and HEK293 (kidney, human) cells were grown in Dulbecco's modified eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 3.7 g/L sodium bicarbonate and antibiotic–antimycotic solution (10,000 units/mL penicillin G, 10 mg/mL streptomycin sulfate and 25 µg/mL amphotericin B, 100×) at 37 °C in a humidified incubator with 5% CO2. Cells were detached using trypsin-EDTA from the culture flask upon attaining 70–80% confluency. The HepG2/ N2a/ HEK293 cells were seeded on 96-well plate at a density of approximately 10,000 cells/well. After 16 h of incubation, cells were treated with PAA-YFa nanoparticles of different concentrations at Z+/−12 and with similar concentrations of neat peptide, neat polymer and water as used in the preparation of nanoparticles at Z+/−12 as controls for 48 h. One hundred microlitre of 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) 0.5 mg/mL of DMEM, was added in each well and incubated for 2 h. Accumulated formazan crystals were solubilized in DMSO and absorbance (A) was measured in an ELISA plate reader (Spectra Max 384 spectrophotometer, Molecular devices, USA) at 560 nm and 630 nm. The relative cell viability (%) compared to control cells was calculated by the formula (Asample / Acontrol) × 100. The assay was performed in triplicate in all the cell lines. 2.8. Animal studies 2.8.1. Animals For all the experiments, male Swiss albino mice weighing 25–30 g were used. The animals were acclimatized at 22–25 °C for 5 days in the animal house prior to experiments. Food and water was allowed ad libitum. The animals were handled according to the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India. CPCSEA and Institute Animal Ethical
Committee, (Institute of Genomics and Integrative Biology) approved the study. For all the studies described below, PAA-YFa nanoparticles of Z+/− = 12 (containing 10 mg/kg YFa), 10 mg/kg neat YFa, PAA polymer (same concentration as used in the preparation of nanoparticles at Z+/− = 12) were used. 2.8.2. Analysis of hematological parameters Test samples of PAA-YFa nanoparticles, neat YFa, PAA polymer and water were injected intraperitoneally (IP) to each group of seven mice. All the test samples were prepared in MilliQ-water at 10 mL/kg. The mice were sacrificed by an overdose of thiopentone sodium (100 mg/mL in 0.9% saline, IP) and 1.0 mL of blood was collected by cardiac puncture in tubes containing heparin as anticoagulant. The total red blood cells (RBC), white blood cells (WBC) and platelet counts for each group were determined on an automated haematology analyzer (Sismex KR 21, Japan). 2.8.3. In vitro peptide release study Blood was collected from three mice per group by the method as described in Section 2.8.2. to obtain plasma. Aliquots of 180 µL of mouse plasma in triplicates were placed in 1.5 mL centrifuge tubes for the time points 5, 15, 30, 45 and 60 min. Twenty microlitres of PAA-YFa nanoparticles, neat YFa and PAA was added to each tube, vortexed briefly and incubated immediately at 37 °C for proteolytic reaction which was terminated at the end of each incubation period by adding 200 µL of CH3CN and placing the tube on ice. The tubes were centrifuged at 3000 ×g [22]. All the supernatant samples were ultracentrifuged (Beckman Coulter, USA) at 21,7000 ×g for 60 min and YFa in the supernatant was quantitated on reverse phase HPLC as described in Section 2.6. 2.8.4. Measurement of the antinociceptive effect The antinociceptive effect was measured by the tail-flick method using local analgesiometer [24] in seven mice per group. The tail flick response was elicited by applying radiant heat to the dorsal surface of the tail. The intensity of heat stimulus in the tail-flick apparatus was adjusted to elicit a response in control or untreated animals within 3–5 s and to minimize tail skin tissue damage cut-off time was set at 10 s. The mice were given three base line trials, each separated by 10 min and were then injected with the test samples of PAA-YFa nanoparticles, neat YFa, neat PAA, water and naloxone hydrochloride (5 mg/kg). The samples were tested for tail-flick response at 5, 15, 30,
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45 and 60 min. All the test samples were prepared in MilliQ- water and administered IP in mice at 10 mL/kg. The inhibition of the tailflick response was expressed as percentage maximum possible effect (%MPE); calculated as [(T1 − T0) / (T2 − T0)] × 100, where T0 and T1 are the tail-flick latency before and after the injection of each test sample and T2 is the cut-off time. The %MPE is reported as Mean ± SEM. Statistical significance between the test samples was determined by a one-way ANOVA and subsequent post hoc Tukey comparison. Significance level was set at P b 0.05. 3. Results and discussion 3.1. Size, zeta potential and morphology of the complexes The chemical structures of peptide and polymer are shown in Fig. 1. The cationic peptide-YFa mediates electrostatic interactions with PAA to form complexes whose sizes are in the nanometric range at different charge ratios (Z+/−), where the charge ratio is defined as the ratio of concentration of cationic units in peptide to that of anionic units in the polymer present in the system. The size, zeta potential and morphology of nanoparticles are mainly governed by the charge ratio at which they were prepared. There was an inverse relationship between size and charge ratio of the nanoparticles and direct relationship between zeta potential and charge ratio of the nanoparticles (Fig. 2a). At charge ratio 2 and 4, complexes of size 778 ± 77 nm and 423 ± 42 nm were formed, respectively, whereas smaller complexes of 83 ± 8 nm size were formed at Z+/−12. Zeta potential gradually increased from negative to positive with corresponding increment in the value of Z+/−, as can be seen in Fig. 2b. The changes in size and zeta potential indicate interaction between the cationic chimeric peptide and oppositely charged polymer to form complexes at different charge ratios. The three dimensional morphology of the complexes was determined by AFM. For size determination, all nanoparticles seen within a representative scan area (4.0 × 4.0 µm) were considered. Elongated structures were formed at lower Z+/− having an average length in the range of 320–1000 nm and width in the range of 55–90 nm while spherical particles of size 70 ± 7 nm were formed at Z+/−12, as illustrated in Fig. 3a and b. The height of the complexes at all Z+/− was generally in the range of 10–20 nm in positive Z direction. A histogram representing the percentage size distribution of nanoparticles at Z+/−12, has been shown in Fig. 3c.
Fig. 1. Chemical structures: a) Chimeric peptide, b) Sodium salt of Polyacrylic acid.
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are observed in the chromatogram of the PAA-YFa nanoparticles after 30 min and similar peaks are also observed in the chromatogram of the polymer after 30 min. The appearance of the peaks at this time interval in the chromatograms of polymer and nanoparticles may be due to the presence of low molecular weight PAA (oligomers) present in the system.
Fig. 2. (a) Size and, (b) Zeta potential of complexes at Z+/−2–12, are measured through Zetasizer Nano ZS. Decrease in size and increase in zeta potential is observed on increment of Z+/−.
The reduction in size observed in Zetasizer and AFM is due to the charge neutralization of the polymer by the increasing concentration of cationic peptides at increasing charge ratios, which means that as the hydrophobicity of the peptide–polymer complexes increases the size of the complexes decreases. The extent of complexation increased at high charge ratios and thus smallest particle size was detected at Z+/−12, signifying complete particle formation. Although, when two oppositely charged polyelectrolytes interact, the largest size particles are generally formed when Zeta potential approaches zero. However, we observed that the sizes were reduced from 800 nm to 360 nm when the Zeta potential approached zero. Such deviations are, however, often observed when polyelectrolytes differ significantly in charge density, dimensions in solution or one of the components is highly branched [25–27]. Some more factors like molecular weight of the polyelectrolytes, ionic strength of the polyelectrolytes in solution and molar ratio of the mixing polyelectrolytes, excess of one of the polyelectrolyte may be the other contributing factors for the observed deviation that can contribute to particle stability. In the present case two counter interacting partners differ in dimensions and one of the components (peptide) is branched. Thus, added contributions of these factors can be responsible for the deviation in the present study. 3.2. Entrapment efficiency of peptide Entrapment efficiency of the peptide in the nanoparticles was calculated by analyzing the absorbance in the chromatograms in Fig. 4 and it was found to be approximately 90 ± 3%. Some additional peaks
Fig. 3. AFM images of complexes of polyanionic polymer and cationic peptide-YFa (YGGFMKKKFMRFa) at Z+/−2 and 12 in the scan range of 4.0 × 4.0 µm and a histogram of complexes at Z+/−12. (a) Elongated particles having length in the range of 320–1000 nm and width in the range of 55–90 nm are observed at Z+/−2. (b) Spherical particles with an average size of 70 ± 7 nm are seen at Z+/−12. Height of nanoparticles is observed in the range of 10–20 nm (c) A histogram showing the nanoparticles of PAA and YFa from AFM image at Z+/− 12. Histogram is generated by measuring sizes of over 60 particles.
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Fig. 4. Entrapment of the peptide-YFa in a nanoparticle. Chromatograms of neat peptide-YFa 10 mg/kg (without polymer) with absorbance 0.3AU (trace a), PAA-YFa nanoparticle solution containing YFa 10 mg/kg with absorbance 0.03AU (trace b), and neat Polymer PAA (without peptide) after ultracentrifugation (trace c).
3.6. Antinociceptive activity 3.3. Cytotoxicity measurements The MTT assay performed on three cell lines revealed almost similar trend of cytotoxicity for the PAA-YFa nanoparticles, neat YFa and neat PAA (Fig. 5). Nanoparticles were found less toxic in comparison to neat peptide at as high a concentration as of 0.5 mM (in acrylate unit of PAA in nanoparticles). This shows that the complex formation of peptide with PAA in nanoparticles reduces the toxicity of YFa.
3.4. Hematological parameters RBC, WBC and platelets counts for all the four samples viz; PAAYFa nanoparticles, neat YFa, neat PAA and water at different time points in blood are provided in Table 1. RBC, WBC and platelets counts for PAA-YFa nanoparticles were found to be in the normal range [28] when compared with the remaining groups. The data further confirm that PAA-YFa nanoparticles do not have any adverse effect at the dose of 10 mg/kg of YFa in nanoparticles.
3.5. In vitro peptide release study Fig. 6 shows the detectable amount of the peptide in plasma at 5, 15, 30, 45 and 60 min after adding free YFa and PAA-YFa nanoparticles. For the sample added with free YFa, the peptide concentration dropped to ~85% of original in 5 min and to 0% in 45 min, suggesting a rapid proteolysis of YFa in plasma that can lead YFa blood concentration below effective level, whereas for the nanoparticle formulation the peptide concentration dropped to ~ 50% of original in 5 min, thence onwards the drop in concentration was gradual and it dropped to ~ 10% over 60 min. This suggested the effect of initial burst release followed by sustained release [29–31] of the peptide from nanoparticles. This result also suggests that complexation of YFa with PAA into nanoparticles may extend effective blood concentration of YFa in vivo.
In our previous work, IP administration of neat YFa did not show any significant antinociceptive activity in tail-flick test in male Swiss albino mice at a dose of 10 mg/kg but at higher dose i.e. 40 mg/ kg and above [22]. A dose response curve of peptide administered alone has been shown in Fig. 7a. At 40 mg/kg, YFa showed a maximum %MPE of about 29% at 30 min and thereafter the activity dropped to ~18% in 60 min. Peptides are prone to degradation by enzymes like peptidases and proteases and hence higher concentration of peptides would be required to sustain the effect at the target site. Hence, in an attempt to enhance the stability and bioavailability of the peptides at the target site at low dose, we designed a nanoparticle delivery system. The IP administration of PAA-YFa nanoparticles at Z+/−12 containing 10 mg/kg YFa elicited significant antinociception ~20% just after 5 min. The activity increased to a maximum of ~ 36% in 30 min and subsided to ~ 11% by 60 min (Fig. 7b). PAA-YFa nanoparticles illustrated similar antinociception activity profile as elicited by 40 mg/kg dose of neat YFa (Fig. 7a) substantiating that nanoparticle system containing 10 mg/kg of peptide is as equipotent as 40 mg/kg of neat peptide administered in mice. Lower dose of PAA-YFa nanoparticles (containing 5 mg/kg of peptide) at Z+/−12 did not show considerable antinociception effect, only a maximum of ~12% antinociception at 30 min was observed (Fig. 7b). Water, neat PAA and neat YFa used for PAA-YFa nanoparticles formation did not show any significant antinociceptive action (Fig. 7c), suggesting that the observed antinociception activity was due to the PAA-YFa nanoparticles system only. These results suggest that PAA-YFa nanoparticles protect the peptide from proteolysis on complexation with PAA and ensure sustained release of the peptide at the site of action i.e. opioid receptors to cause the antinociception. The in vitro peptide release from nanoparticles in mouse plasma also supports our antinociception profile. Previously, poly butylcyanoacrylate (PBCA) nanoparticles have been employed to increase the antinociception efficacy of peptides at lower doses [32–34]. For example, systemic administration of peptide dalargin at higher doses did not contribute any antinociceptive effect. However, the bioavailability of dalargin was
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K. Gupta et al. / Journal of Controlled Release 134 (2009) 47–54 Table 1 Hematological parameters Blood samples
Time (min) 5 min
15 min
30 min
Red blood cells count ( × 106/µL) PAA-YFa nanoparticles 9.46 ± 0.06 YFa 9.45 ± 0.60 PAA 9.48 ± 0.50 Water 9.48 ± 0.30
9.45 ± 0.19 9.39 ± 0.34 9.44 ± 0.58 9.46 ± 0.04
9.52 ± 0.05 9.50 ± 0.08 9.53 ± 0.40 9.55 ± 0.05
9.50 ± 0.46 9.48 ± 0.25 9.56 ± 0.28 9.53 ± 0.36
9.40 ± 0.15 9.39 ± 0.20 9.45 ± 0.10 9.43 ± 0.30
White blood cells count ( × 103/µL) PAA-YFa nanoparticles 6.21 ± 0.30 YFa 6.19 ± 0.35 PAA 6.26 ± 0.45 Water 6.22 ± 0.41
6.22 ± 0.60 6.18 ± 0.64 6.22 ± 0.54 6.18 ± 0.14
6.23 ± 0.25 6.18 ± 0.52 6.22 ± 0.42 6.20 ± 0.62
6.120 ± 0.38 6.119 ± 0.45 6.122 ± 0.29 6.125 ± 0.43
6.20 ± 0.33 6.18 ± 0.28 6.25 ± 0.32 6.23 ± 0.09
Platelets count ( × 103/µL) PAA-YFa nanoparticles YFa PAA Water
683 ± 63 676 ± 74 680 ± 37 682 ± 58
658 ± 92 654 ± 86 660 ± 34 656 ± 66
657 ± 63 659 ± 84 663 ± 45 660 ± 68
675 ± 78 673 ± 43 676 ± 92 678 ± 80
668 ± 90 666 ± 73 670 ± 81 670 ± 93
45 min
60 min
of 3.41% after 5 min (Fig. 7b). This inhibition by naloxone suggested that the antinociception effect of nanoparticles was mediated by central opioid receptors. A separate group of mice was also injected with 5 mg/kg naloxone (IP) as control which did not show any antinociceptive activity up to 60 min, as shown in Fig. 7c.
4. Conclusions
Fig. 5. Cytotoxicity measurements at different concentrations of PAA-YFa nanoparticles (●), neat peptide-YFa (■) and neat polymer PAA (▲) at Z+/−12 on various mammalian cell lines, (a) HepG2 (liver, human), (b) N2a (Neuronal, mouse), (c) HEK293 (Human embryonic kidney). The data is expressed as concentration (mM) of PAA (in acrylate unit) at Z+/−12 in nanoparticles at the x-axis.
improved by PBCA nanoparticles which showed remarkable enhancement in antinociception at low doses [32–34] suggesting that dalargin was protected from proteolysis with entrapment in the nanoparticles, thereby showing improved bioavailability and consequently enhanced antinociception. Further, YFa resembles endogenously occurring opioid peptide the dynorphins [35–37] in containing positively charged hydrophilic sequence and adopting alpha helical structure [38], therefore it may also show similar pharmacological action like dynorphins through peripheral mechanisms [39]. In fact, the early onset of antinociceptive effect in just 5 min by administration of nanoparticles favors the peripheral opioid receptors stimulation by YFa and then subsequent action through central mechanisms. The antinociceptive action of PAA-YFa nanoparticles mediated by central opioid receptors was assessed by a central opioid receptor antagonist naloxone [32,40]. Pretreatment of animals with 5 mg/kg (IP) of naloxone 5 min before the administration of PAA-YFa nanoparticles did not show any pronounced antinociception up to 60 min. The antinociception effect observed was almost at the base line level showing maximum %MPE
Nanoparticles of cationic chimeric peptide, YFa and oppositely charged polymer, PAA were formed via electrostatic interactions. Size of the particles decreased and zeta potential increased on increment of Z+/−. Nanoparticles at Z+/−12 were spherical with cationic surface charge. These nanoparticles displayed less toxicity on different mammalian cell lines, did not affect the hematological parameters and showed enhanced antinociception in tail-flick test in mice. Our results suggest of a potential nanoparticle delivery system for cationic peptide drug candidates in providing better stability and bioavailability.
Fig. 6. In vitro peptide release of PAA-YFa nanoparticles (■) and peptide-YFa (●) at Z+/− = 12 in mouse plasma.
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References
Fig. 7. Time course of the antinociceptive response of IP administered (a) Neat chimeric peptide-YFa at 10 mg/kg, 40 mg/kg and 60 mg/kg, (b) PAA-YFa nanoparticles containing 5 mg/kg and 10 mg/kg YFa at Z+/−12, 10 mg/kg neat YFa and 5 mg/kg naloxone 5 min before the nanoparticles (c) Neat PAA, neat 10 mg/kg YFa, 5 mg/kg naloxone and water as controls, by tail-flick test in mice. The %MPE values of the test samples are depicted as Mean±SEM. The %MPE values of nanoparticles containing 10 mg/kg YFa are found to be statistically significant compared to the %MPE values of the controls and naloxone pretreated mice (Pb 0.05). (⁎) Indicates significant differences from control groups and naloxone pretreated group.
Acknowledgements CSIR supported this work under the project: Nanomaterials and Nanodevices for applications in Health and Disease, a network project. K. Gupta is a CSIR-SRF.
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Journal of Controlled Release j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j c o n r e l
Nanoparticles of cationic chimeric peptide and sodium polyacrylate exhibit striking antinociception activity at lower dose Kshitij Gupta a,c, Vijay P. Singh a, Raj K. Kurupati a, Anita Mann a, Munia Ganguli a, Yogendra K. Gupta b, Yogendra Singh a, Kishwar Saleem c, Santosh Pasha a,⁎, Souvik Maiti a,⁎ a b c
Institute of Genomics and Integrative Biology, Mall Road, Delhi-110007, India Department of Pharmacology, All India Institute of Medical Sciences, New Delhi-110029, India Department of Chemistry, Faculty of Natural Sciences, Jamia Millia Islamia, New Delhi-110025, India
a r t i c l e
i n f o
Article history: Received 20 March 2008 Accepted 10 October 2008 Available online 1 November 2008 Keywords: Nanoparticles Chimeric peptide Cytotoxicity Hematological parameters Antinociception
a b s t r a c t The current study investigates the performance of polyelectrolyte complexes based nanoparticles in improving the antinociceptive activity of cationic chimeric peptide-YFa at lower dose. Size, Zeta potential and morphology of the nanoparticles were determined. Size of the nanoparticles decreases and zeta potential increases with concomitant increase in charge ratio (Z+/−). The nanoparticles at Z+/−12 are spherical with 70 ± 7 nm diameter in AFM and displayed positive surface charge and similar sizes (83 ± 8 nm) by Zetasizer. The nanoparticles of Z+/− 12 are used in this study. Cytotoxicity by MTT assay on three different mammalian cell lines (liver, neuronal and kidney) revealed lower toxicity of nanoparticles. Hematological parameters were also not affected by nanoparticles compared to normal counts of water treated control group. Nanoparticles containing 10 mg/kg YFa produced increased antinociception, ~ 36%, in tail-flick latency test in mice, whereas the neat peptide at the same concentration did not show any antinociception activity. This enhancement in activity is attributed to the nanoparticle associated protection of peptide from proteolytic degradation. In vitro peptide release study in plasma also supported the antinociception profile of nanoparticles. Thus, our results suggest of a potential nanoparticle delivery system for cationic peptide drug candidates for improving their stability and bioavailability. © 2008 Elsevier B.V. All rights reserved.
1. Introduction In recent years, significant efforts have been put in to develop nanotechnology for drug delivery. Nanotechnology offers better prospects over conventional procedures [1–4] for delivering small molecular weight drugs, as well as macromolecules (proteins, peptides or genes) by either localized or targeted delivery to the tissue of interest [5–11]. It focuses on formulating therapeutic agents in biocompatible nanocomposites such as nanoparticles, nanocapsules and micellar systems. Therapeutic molecules within the nanocomposites possess a higher stability in biological fluids and are resistant towards enzymatic metabolism [12]. As a successful drug delivery vehicle, polymeric nanoparticles have the ability to escape from the reticuloendothelial system, resulting in longer stay in circulation [5]. These nanoparticles can penetrate deep into tissues through capillaries, cross the fenestration present in the polyepithelial lining, and are generally taken up efficiently by the cells [13]. A wide variety of techniques are available for producing nanoparticles including solvent evaporation [14] interfacial polymerization ⁎ Corresponding authors. Fax: +91 11 27667471. E-mail addresses: [email protected] (S. Pasha), [email protected] (S. Maiti). 0168-3659/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2008.10.008
[15] and emulsion polymerization methods [16]. However, most of these approaches involve the use of organic solvents, heat or vigorous agitation, which are potentially harmful to sensitive biomolecules. In recent years, self assembly of proteins/peptides/drug candidates with natural or synthetic polyelectrolyte to form Polyelectrolyte Complexes (PECs) has drawn increased interest of chemists and pharmacologists [17]. PEC formation leads to particles with dimensions on a colloidal level generating optically homogeneous and stable nano-dispersions. Additionally, such methods are advantageous as they do not require sonication and organic solvents during preparation, thereby minimizing possible damage to drug candidates during PEC formation. We have also demonstrated that such type of PECs of nanometer size can be prepared through electrostatic interaction of cationic peptides containing varying number of lysine residues with polyanionic polymer of sodium salt of polyacrylic acid (PAA) [18]. The polymer PAA has been used as an excellent protectant additive to preserve the bioactivity of L-lactate dehydrogenase (LDH) as it interacts with alginate microparticles [19]. Furthermore, PAA has been widely employed for buccal [20] and ophthalmic drug delivery systems [21]. In our previous study, it has been shown that cationic chimeric peptide (YGGFMKKKFMRFa-YFa) of Met-enkephalin (YGGFM) and
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FMRFa, joined by three lysine residues, induces antinociceptive action at doses of 40 mg/kg and above [22]. However, at lower dose of 10 mg/kg of YFa no antinociception effect was observed. This may be due to the peptidic nature of YFa which renders it susceptible to protease degradation and hence resulting in lower bioavailability. In the current study, to increase the delivery efficiency of the cationic chimeric peptide-YFa, we have adopted a PECs formation strategy to obtain nanoparticles from polyanionic polymer PAA and YFa to achieve significant antinociception effect at lower dose of peptide. Size and zeta potential of nanoparticles were characterized by Zetasizer and morphology of nanoparticles was determined by Atomic Force Microscopy. Cytotoxicity assay on mammalian cell lines and evaluation of hematological parameters were done to understand the toxicity behavior of nanoparticles. In vitro peptide release in blood plasma was done to understand the release profile of peptide from nanoparticles.
to 1.0 mL for measurements, which were carried out in an automatic mode. The values of size and zeta potential are the average of 30 runs. The Smoluchowski approximation was used to calculate zeta potential from the electrophoretic mobility.
2. Experimental section
Two millilitre of PAA-YFa nanoparticles containing 10 mg/kg YFa, 10 mg/kg neat YFa and neat PAA of same concentration that was used for the formation of nanoparticles at Z+/−12 were ultracentrifuged (Beckman Coulter, USA) at 21,7000 ×g for 60 min to obtain supernatants. YFa in the supernatant was quantitated by RP-C18 column (µBondapak™ 5 µm, 3.9 × 300 mm, Waters, USA) at 220 nm with a photodiode array detector (Waters 996, USA) on a reverse phase HPLC (Waters Delta 600, USA). The mobile phase consisted of acetonitrile/water (10:90 linear gradient for 40 min) and the flow rate was 0.8 mL/min. The peptide entrapment efficiency was expressed as the percentage of YFa difference between the total amount of the YFa added and in the supernatant relative to the total amount of YFa added to form nanoparticles. All samples were measured in triplicate.
2.1. Chemicals Sodium salt of polyacrylic acid (Mw = 10,000), N,N′diisopropyl carbodiimide (DIPCI) and trifluoroacetic acid were obtained from Sigma-Aldrich. Naloxone hydrochloride was purchased from Sigma (St. Louis, MO). All Fmoc-amino acids, 1-hydroxybenzotriazole (HOBt), and Rink amide resin were purchased from Nova Biochem (Switzerland). Acetonitrile was obtained from Merck Ltd. (India). 2, 5dihydroxybenzoic acid was supplied by Bruker Daltonics (Germany). Cell lines Hep G2 (liver, human), N2a (neuronal, mouse), and HEK 293 (kidney, human) were obtained from National Centre for Cell Sciences (NCCS), Pune, India. Dulbecco's modified eagle's medium (DMEM), fetal bovine serum, sodium bicarbonate, antibiotic–antimycotic solution (100X), Trypsin-EDTA, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and Dimetyl sulfoxide (DMSO) were procured from Sigma. 2.2. Peptide synthesis Peptide-YFa was synthesized by the solid phase method on automated peptide synthesizer (Advanced chemtech, USA), using the standard chemistry of fluorenylmethoxy carbonyl (Fmoc) amino acids and 1-hydroxybenzotriazole (HOBt)/N,N′-diisopropyl carbodiimide (DIPCI) activation method on a rink amide resin. The peptide was purified by RP-C18 column (µBondapak™ 10 µm, 7.8 × 300 mm, Waters, USA) on semi-preparative reverse phase HPLC (Waters 600, USA) with a 40 min linear gradient from 10% to 90% acetonitrile containing 0.05% trifluoroacetic acid in water. The mass analysis of the peptides was done in linear positive ion mode by MALDI-Tof-Tof (Bruker Daltonics Flex Analysis, Germany) with 2, 5 dihydroxybenzoic acid as the matrix. The peptide sequence was confirmed by automated peptide sequencing (Procise 491 Applied Biosystems, USA). 2.3. Preparation of polyelectrolyte complexes The complexes of PAA and chimeric peptide were prepared by electrostatic interaction as described in our previous study [18]. In brief, different dilutions of stock 1.0 mg/mL peptide-YFa were added to 1 × 10− 3 M (in acrylate unit of PAA) solution of PAA to obtain complexes of different Z+/− i.e. 2–12. YFa and PAA solutions were prepared in MilliQ-water, pH = 6.8–7.0. 2.4. Size and zeta potential measurements Zetasizer Nano ZS (Malvern Instruments, UK) determined size and zeta potential of peptide–polymer complexes of different Z+/− i.e. 2–12. One hundred microlitre of complex samples of each Z+/− were diluted
2.5. Atomic force microscopy (AFM) measurements All AFM images were procured on PicoSPM equipment (Molecular Imaging, Tempe, AZ, USA) using AAC mode. Two microlitre of complex solution was deposited on a fresh piece of mica (1 × 1 cm) and allowed to adsorb for 2 min at room temperature. Imaging was performed under dried condition in air with 250 µm long cantilevers with a resonance frequency of about 41 kHz. Minimal image processing (flattening only) was carried out in the presented image. 2.6. Evaluation of entrapment efficiency of peptide
2.7. Cytotoxicity measurements The toxicity of the PAA-YFa nanoparticles was studied by MTT colorimetric assay [23]. HepG2 (liver, human), N2a (neuronal, mouse), and HEK293 (kidney, human) cells were grown in Dulbecco's modified eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 3.7 g/L sodium bicarbonate and antibiotic–antimycotic solution (10,000 units/mL penicillin G, 10 mg/mL streptomycin sulfate and 25 µg/mL amphotericin B, 100×) at 37 °C in a humidified incubator with 5% CO2. Cells were detached using trypsin-EDTA from the culture flask upon attaining 70–80% confluency. The HepG2/ N2a/ HEK293 cells were seeded on 96-well plate at a density of approximately 10,000 cells/well. After 16 h of incubation, cells were treated with PAA-YFa nanoparticles of different concentrations at Z+/−12 and with similar concentrations of neat peptide, neat polymer and water as used in the preparation of nanoparticles at Z+/−12 as controls for 48 h. One hundred microlitre of 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) 0.5 mg/mL of DMEM, was added in each well and incubated for 2 h. Accumulated formazan crystals were solubilized in DMSO and absorbance (A) was measured in an ELISA plate reader (Spectra Max 384 spectrophotometer, Molecular devices, USA) at 560 nm and 630 nm. The relative cell viability (%) compared to control cells was calculated by the formula (Asample / Acontrol) × 100. The assay was performed in triplicate in all the cell lines. 2.8. Animal studies 2.8.1. Animals For all the experiments, male Swiss albino mice weighing 25–30 g were used. The animals were acclimatized at 22–25 °C for 5 days in the animal house prior to experiments. Food and water was allowed ad libitum. The animals were handled according to the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India. CPCSEA and Institute Animal Ethical
Committee, (Institute of Genomics and Integrative Biology) approved the study. For all the studies described below, PAA-YFa nanoparticles of Z+/− = 12 (containing 10 mg/kg YFa), 10 mg/kg neat YFa, PAA polymer (same concentration as used in the preparation of nanoparticles at Z+/− = 12) were used. 2.8.2. Analysis of hematological parameters Test samples of PAA-YFa nanoparticles, neat YFa, PAA polymer and water were injected intraperitoneally (IP) to each group of seven mice. All the test samples were prepared in MilliQ-water at 10 mL/kg. The mice were sacrificed by an overdose of thiopentone sodium (100 mg/mL in 0.9% saline, IP) and 1.0 mL of blood was collected by cardiac puncture in tubes containing heparin as anticoagulant. The total red blood cells (RBC), white blood cells (WBC) and platelet counts for each group were determined on an automated haematology analyzer (Sismex KR 21, Japan). 2.8.3. In vitro peptide release study Blood was collected from three mice per group by the method as described in Section 2.8.2. to obtain plasma. Aliquots of 180 µL of mouse plasma in triplicates were placed in 1.5 mL centrifuge tubes for the time points 5, 15, 30, 45 and 60 min. Twenty microlitres of PAA-YFa nanoparticles, neat YFa and PAA was added to each tube, vortexed briefly and incubated immediately at 37 °C for proteolytic reaction which was terminated at the end of each incubation period by adding 200 µL of CH3CN and placing the tube on ice. The tubes were centrifuged at 3000 ×g [22]. All the supernatant samples were ultracentrifuged (Beckman Coulter, USA) at 21,7000 ×g for 60 min and YFa in the supernatant was quantitated on reverse phase HPLC as described in Section 2.6. 2.8.4. Measurement of the antinociceptive effect The antinociceptive effect was measured by the tail-flick method using local analgesiometer [24] in seven mice per group. The tail flick response was elicited by applying radiant heat to the dorsal surface of the tail. The intensity of heat stimulus in the tail-flick apparatus was adjusted to elicit a response in control or untreated animals within 3–5 s and to minimize tail skin tissue damage cut-off time was set at 10 s. The mice were given three base line trials, each separated by 10 min and were then injected with the test samples of PAA-YFa nanoparticles, neat YFa, neat PAA, water and naloxone hydrochloride (5 mg/kg). The samples were tested for tail-flick response at 5, 15, 30,
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45 and 60 min. All the test samples were prepared in MilliQ- water and administered IP in mice at 10 mL/kg. The inhibition of the tailflick response was expressed as percentage maximum possible effect (%MPE); calculated as [(T1 − T0) / (T2 − T0)] × 100, where T0 and T1 are the tail-flick latency before and after the injection of each test sample and T2 is the cut-off time. The %MPE is reported as Mean ± SEM. Statistical significance between the test samples was determined by a one-way ANOVA and subsequent post hoc Tukey comparison. Significance level was set at P b 0.05. 3. Results and discussion 3.1. Size, zeta potential and morphology of the complexes The chemical structures of peptide and polymer are shown in Fig. 1. The cationic peptide-YFa mediates electrostatic interactions with PAA to form complexes whose sizes are in the nanometric range at different charge ratios (Z+/−), where the charge ratio is defined as the ratio of concentration of cationic units in peptide to that of anionic units in the polymer present in the system. The size, zeta potential and morphology of nanoparticles are mainly governed by the charge ratio at which they were prepared. There was an inverse relationship between size and charge ratio of the nanoparticles and direct relationship between zeta potential and charge ratio of the nanoparticles (Fig. 2a). At charge ratio 2 and 4, complexes of size 778 ± 77 nm and 423 ± 42 nm were formed, respectively, whereas smaller complexes of 83 ± 8 nm size were formed at Z+/−12. Zeta potential gradually increased from negative to positive with corresponding increment in the value of Z+/−, as can be seen in Fig. 2b. The changes in size and zeta potential indicate interaction between the cationic chimeric peptide and oppositely charged polymer to form complexes at different charge ratios. The three dimensional morphology of the complexes was determined by AFM. For size determination, all nanoparticles seen within a representative scan area (4.0 × 4.0 µm) were considered. Elongated structures were formed at lower Z+/− having an average length in the range of 320–1000 nm and width in the range of 55–90 nm while spherical particles of size 70 ± 7 nm were formed at Z+/−12, as illustrated in Fig. 3a and b. The height of the complexes at all Z+/− was generally in the range of 10–20 nm in positive Z direction. A histogram representing the percentage size distribution of nanoparticles at Z+/−12, has been shown in Fig. 3c.
Fig. 1. Chemical structures: a) Chimeric peptide, b) Sodium salt of Polyacrylic acid.
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are observed in the chromatogram of the PAA-YFa nanoparticles after 30 min and similar peaks are also observed in the chromatogram of the polymer after 30 min. The appearance of the peaks at this time interval in the chromatograms of polymer and nanoparticles may be due to the presence of low molecular weight PAA (oligomers) present in the system.
Fig. 2. (a) Size and, (b) Zeta potential of complexes at Z+/−2–12, are measured through Zetasizer Nano ZS. Decrease in size and increase in zeta potential is observed on increment of Z+/−.
The reduction in size observed in Zetasizer and AFM is due to the charge neutralization of the polymer by the increasing concentration of cationic peptides at increasing charge ratios, which means that as the hydrophobicity of the peptide–polymer complexes increases the size of the complexes decreases. The extent of complexation increased at high charge ratios and thus smallest particle size was detected at Z+/−12, signifying complete particle formation. Although, when two oppositely charged polyelectrolytes interact, the largest size particles are generally formed when Zeta potential approaches zero. However, we observed that the sizes were reduced from 800 nm to 360 nm when the Zeta potential approached zero. Such deviations are, however, often observed when polyelectrolytes differ significantly in charge density, dimensions in solution or one of the components is highly branched [25–27]. Some more factors like molecular weight of the polyelectrolytes, ionic strength of the polyelectrolytes in solution and molar ratio of the mixing polyelectrolytes, excess of one of the polyelectrolyte may be the other contributing factors for the observed deviation that can contribute to particle stability. In the present case two counter interacting partners differ in dimensions and one of the components (peptide) is branched. Thus, added contributions of these factors can be responsible for the deviation in the present study. 3.2. Entrapment efficiency of peptide Entrapment efficiency of the peptide in the nanoparticles was calculated by analyzing the absorbance in the chromatograms in Fig. 4 and it was found to be approximately 90 ± 3%. Some additional peaks
Fig. 3. AFM images of complexes of polyanionic polymer and cationic peptide-YFa (YGGFMKKKFMRFa) at Z+/−2 and 12 in the scan range of 4.0 × 4.0 µm and a histogram of complexes at Z+/−12. (a) Elongated particles having length in the range of 320–1000 nm and width in the range of 55–90 nm are observed at Z+/−2. (b) Spherical particles with an average size of 70 ± 7 nm are seen at Z+/−12. Height of nanoparticles is observed in the range of 10–20 nm (c) A histogram showing the nanoparticles of PAA and YFa from AFM image at Z+/− 12. Histogram is generated by measuring sizes of over 60 particles.
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Fig. 4. Entrapment of the peptide-YFa in a nanoparticle. Chromatograms of neat peptide-YFa 10 mg/kg (without polymer) with absorbance 0.3AU (trace a), PAA-YFa nanoparticle solution containing YFa 10 mg/kg with absorbance 0.03AU (trace b), and neat Polymer PAA (without peptide) after ultracentrifugation (trace c).
3.6. Antinociceptive activity 3.3. Cytotoxicity measurements The MTT assay performed on three cell lines revealed almost similar trend of cytotoxicity for the PAA-YFa nanoparticles, neat YFa and neat PAA (Fig. 5). Nanoparticles were found less toxic in comparison to neat peptide at as high a concentration as of 0.5 mM (in acrylate unit of PAA in nanoparticles). This shows that the complex formation of peptide with PAA in nanoparticles reduces the toxicity of YFa.
3.4. Hematological parameters RBC, WBC and platelets counts for all the four samples viz; PAAYFa nanoparticles, neat YFa, neat PAA and water at different time points in blood are provided in Table 1. RBC, WBC and platelets counts for PAA-YFa nanoparticles were found to be in the normal range [28] when compared with the remaining groups. The data further confirm that PAA-YFa nanoparticles do not have any adverse effect at the dose of 10 mg/kg of YFa in nanoparticles.
3.5. In vitro peptide release study Fig. 6 shows the detectable amount of the peptide in plasma at 5, 15, 30, 45 and 60 min after adding free YFa and PAA-YFa nanoparticles. For the sample added with free YFa, the peptide concentration dropped to ~85% of original in 5 min and to 0% in 45 min, suggesting a rapid proteolysis of YFa in plasma that can lead YFa blood concentration below effective level, whereas for the nanoparticle formulation the peptide concentration dropped to ~ 50% of original in 5 min, thence onwards the drop in concentration was gradual and it dropped to ~ 10% over 60 min. This suggested the effect of initial burst release followed by sustained release [29–31] of the peptide from nanoparticles. This result also suggests that complexation of YFa with PAA into nanoparticles may extend effective blood concentration of YFa in vivo.
In our previous work, IP administration of neat YFa did not show any significant antinociceptive activity in tail-flick test in male Swiss albino mice at a dose of 10 mg/kg but at higher dose i.e. 40 mg/ kg and above [22]. A dose response curve of peptide administered alone has been shown in Fig. 7a. At 40 mg/kg, YFa showed a maximum %MPE of about 29% at 30 min and thereafter the activity dropped to ~18% in 60 min. Peptides are prone to degradation by enzymes like peptidases and proteases and hence higher concentration of peptides would be required to sustain the effect at the target site. Hence, in an attempt to enhance the stability and bioavailability of the peptides at the target site at low dose, we designed a nanoparticle delivery system. The IP administration of PAA-YFa nanoparticles at Z+/−12 containing 10 mg/kg YFa elicited significant antinociception ~20% just after 5 min. The activity increased to a maximum of ~ 36% in 30 min and subsided to ~ 11% by 60 min (Fig. 7b). PAA-YFa nanoparticles illustrated similar antinociception activity profile as elicited by 40 mg/kg dose of neat YFa (Fig. 7a) substantiating that nanoparticle system containing 10 mg/kg of peptide is as equipotent as 40 mg/kg of neat peptide administered in mice. Lower dose of PAA-YFa nanoparticles (containing 5 mg/kg of peptide) at Z+/−12 did not show considerable antinociception effect, only a maximum of ~12% antinociception at 30 min was observed (Fig. 7b). Water, neat PAA and neat YFa used for PAA-YFa nanoparticles formation did not show any significant antinociceptive action (Fig. 7c), suggesting that the observed antinociception activity was due to the PAA-YFa nanoparticles system only. These results suggest that PAA-YFa nanoparticles protect the peptide from proteolysis on complexation with PAA and ensure sustained release of the peptide at the site of action i.e. opioid receptors to cause the antinociception. The in vitro peptide release from nanoparticles in mouse plasma also supports our antinociception profile. Previously, poly butylcyanoacrylate (PBCA) nanoparticles have been employed to increase the antinociception efficacy of peptides at lower doses [32–34]. For example, systemic administration of peptide dalargin at higher doses did not contribute any antinociceptive effect. However, the bioavailability of dalargin was
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K. Gupta et al. / Journal of Controlled Release 134 (2009) 47–54 Table 1 Hematological parameters Blood samples
Time (min) 5 min
15 min
30 min
Red blood cells count ( × 106/µL) PAA-YFa nanoparticles 9.46 ± 0.06 YFa 9.45 ± 0.60 PAA 9.48 ± 0.50 Water 9.48 ± 0.30
9.45 ± 0.19 9.39 ± 0.34 9.44 ± 0.58 9.46 ± 0.04
9.52 ± 0.05 9.50 ± 0.08 9.53 ± 0.40 9.55 ± 0.05
9.50 ± 0.46 9.48 ± 0.25 9.56 ± 0.28 9.53 ± 0.36
9.40 ± 0.15 9.39 ± 0.20 9.45 ± 0.10 9.43 ± 0.30
White blood cells count ( × 103/µL) PAA-YFa nanoparticles 6.21 ± 0.30 YFa 6.19 ± 0.35 PAA 6.26 ± 0.45 Water 6.22 ± 0.41
6.22 ± 0.60 6.18 ± 0.64 6.22 ± 0.54 6.18 ± 0.14
6.23 ± 0.25 6.18 ± 0.52 6.22 ± 0.42 6.20 ± 0.62
6.120 ± 0.38 6.119 ± 0.45 6.122 ± 0.29 6.125 ± 0.43
6.20 ± 0.33 6.18 ± 0.28 6.25 ± 0.32 6.23 ± 0.09
Platelets count ( × 103/µL) PAA-YFa nanoparticles YFa PAA Water
683 ± 63 676 ± 74 680 ± 37 682 ± 58
658 ± 92 654 ± 86 660 ± 34 656 ± 66
657 ± 63 659 ± 84 663 ± 45 660 ± 68
675 ± 78 673 ± 43 676 ± 92 678 ± 80
668 ± 90 666 ± 73 670 ± 81 670 ± 93
45 min
60 min
of 3.41% after 5 min (Fig. 7b). This inhibition by naloxone suggested that the antinociception effect of nanoparticles was mediated by central opioid receptors. A separate group of mice was also injected with 5 mg/kg naloxone (IP) as control which did not show any antinociceptive activity up to 60 min, as shown in Fig. 7c.
4. Conclusions
Fig. 5. Cytotoxicity measurements at different concentrations of PAA-YFa nanoparticles (●), neat peptide-YFa (■) and neat polymer PAA (▲) at Z+/−12 on various mammalian cell lines, (a) HepG2 (liver, human), (b) N2a (Neuronal, mouse), (c) HEK293 (Human embryonic kidney). The data is expressed as concentration (mM) of PAA (in acrylate unit) at Z+/−12 in nanoparticles at the x-axis.
improved by PBCA nanoparticles which showed remarkable enhancement in antinociception at low doses [32–34] suggesting that dalargin was protected from proteolysis with entrapment in the nanoparticles, thereby showing improved bioavailability and consequently enhanced antinociception. Further, YFa resembles endogenously occurring opioid peptide the dynorphins [35–37] in containing positively charged hydrophilic sequence and adopting alpha helical structure [38], therefore it may also show similar pharmacological action like dynorphins through peripheral mechanisms [39]. In fact, the early onset of antinociceptive effect in just 5 min by administration of nanoparticles favors the peripheral opioid receptors stimulation by YFa and then subsequent action through central mechanisms. The antinociceptive action of PAA-YFa nanoparticles mediated by central opioid receptors was assessed by a central opioid receptor antagonist naloxone [32,40]. Pretreatment of animals with 5 mg/kg (IP) of naloxone 5 min before the administration of PAA-YFa nanoparticles did not show any pronounced antinociception up to 60 min. The antinociception effect observed was almost at the base line level showing maximum %MPE
Nanoparticles of cationic chimeric peptide, YFa and oppositely charged polymer, PAA were formed via electrostatic interactions. Size of the particles decreased and zeta potential increased on increment of Z+/−. Nanoparticles at Z+/−12 were spherical with cationic surface charge. These nanoparticles displayed less toxicity on different mammalian cell lines, did not affect the hematological parameters and showed enhanced antinociception in tail-flick test in mice. Our results suggest of a potential nanoparticle delivery system for cationic peptide drug candidates in providing better stability and bioavailability.
Fig. 6. In vitro peptide release of PAA-YFa nanoparticles (■) and peptide-YFa (●) at Z+/− = 12 in mouse plasma.
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References
Fig. 7. Time course of the antinociceptive response of IP administered (a) Neat chimeric peptide-YFa at 10 mg/kg, 40 mg/kg and 60 mg/kg, (b) PAA-YFa nanoparticles containing 5 mg/kg and 10 mg/kg YFa at Z+/−12, 10 mg/kg neat YFa and 5 mg/kg naloxone 5 min before the nanoparticles (c) Neat PAA, neat 10 mg/kg YFa, 5 mg/kg naloxone and water as controls, by tail-flick test in mice. The %MPE values of the test samples are depicted as Mean±SEM. The %MPE values of nanoparticles containing 10 mg/kg YFa are found to be statistically significant compared to the %MPE values of the controls and naloxone pretreated mice (Pb 0.05). (⁎) Indicates significant differences from control groups and naloxone pretreated group.
Acknowledgements CSIR supported this work under the project: Nanomaterials and Nanodevices for applications in Health and Disease, a network project. K. Gupta is a CSIR-SRF.
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