SLT Institute of Pharmaceutical Sciences, Guru Ghasidas Vishwavidyalaya, Bilaspur (CG) 495009, IndiaChandigarh College of Pharmacy, Landran, Mohali (PB) 140307, India
r t i c l e i n f o
rticle history:eceived 26 April 2013eceived in revised form 16 June 2013ccepted 26 June 2013vailable online xxx
eywords:nkephalinrain deliveryrimethyl chitosan
a b s t r a c t
Leucine-enkephalin (Leu-Enk) is a neurotransmitter or neuromodulator in pain transmission. Due tonon-addictive opioid analgesic activity of this peptide, it might have great potential in pain manage-ment. Leu-Enk loaded N-trimethyl chitosan (TMC) nanoparticles were prepared and evaluated as a braindelivery vehicle via nasal route. TMC biopolymer was synthesized and analyzed by 1H NMR spectroscopy.TMC nanoparticles were prepared by ionic gelation method. Mean peptide encapsulation efficiency andloading capacity were 78.28 ± 3.8% and 14 ± 1.3%, respectively. Mean particle size, polydispersity indexand zeta potential were found to be 443 ± 23 nm, 0.317 ± 0.17 and +15 ± 2 mV respectively for optimizedformulations. Apparent permeability coefficient (Papp) of Leu-Enk released from nanoparticles across theporcine nasal mucosa was determined to be 7.45 ± 0.30 × 10−6 cm s−1. Permeability of Leu-Enk released
ntinociceptive from nanoparticles was 35 fold improved from the nasal mucosa as compared to Leu-Enk solution. Fluo-rescent microscopy of brain sections of mice showed higher accumulation of fluorescent marker NBD-Flabelled Leu-Enk, when administered nasally by TMC nanoparticles, while low brain uptake of markersolution was observed. Furthermore, enhancement in brain uptake resulted into significant improve-ment in the observed antinociceptive effect of Leu-Enk as evidenced by hot plate and acetic acid inducedwrithing assay.
Enkephalins are pentapeptides that are known to be neuro-ransmitters in pain transmission [1]. Because of non-addictivepioid analgesic activity, these neuropeptides might have greatotential in acute and chronic pain management. However, non-
nvasively administered Leucine-enkephalin (Leu-Enk) is rapidlyegraded and poorly absorbed from mucosal membranes, strongly
imiting the therapeutic application of this peptide [2,3]. Further,lood–brain barrier (BBB) restricts the passage of substances
ncluding enkephalins into the brain, when given systemically.ecently, the nasal route of administration has gained substantial
nterest for obtaining brain uptake of polar or hydrophilic drugs3,4]. The olfactory region connected to nasal cavity is the only sitef the body where the CNS is in direct contact with the external
nvironment. Consequently, a drug able to deposit on the olfactoryegions should have more chances to reach the cerebrospinal fluidCSF), upon diffusion across the mucosa. Afterwards, the drug
diffuses into the interstitial fluid from where it can penetrate thebrain parenchyma [5,6].
Nasal delivery of drugs has following general advantages: avoid-ance of degradation in the gut and first-pass metabolism, anextensive absorption surface in the nose, rapid diffusion in tosystemic circulation, and possibility of better patient compliancecompared to parenteral therapy [7]. However, polypeptides arepoorly absorbed from the nasal cavity due to their large size[8] and extensive degradation in the mucosal cavities [9]. Sev-eral specific enzymes have been suggested being responsible forthe metabolism of enkephalins such as Enkephalinase, Dipepti-dase, Aminopeptidase and Carboxypeptidases [10]. Effective brainuptake from nasal cavity requires formulations and devices capa-ble to provide drug deposition in the olfactory region, prolongedresidence time and high local drug concentration for diffusion.Trimethyl chitosan (TMC) is a permanently quaternized positivelycharged chitosan derivative which is water soluble, non-toxicand offer potential application as a mucoadheisve polymer and
targeted-carrier [11]. As a cationic ligand, TMC facilitate the activetransport of nanoparticles via absorption-mediated transcytosisthrough BBB and because of this; TMC nanoparticles could be usedas a drug carrier for brain delivery [12,13]. TMC conjugated PLGA
190 M. Kumar et al. / International Journal of Biolog
n[tb
nnas
2
2
daftumep
2
2
TwaEbb
2
t(idZismte
Fig. 1. 1H NMR spectrum of the N-trimethyl chitosan derivative dissolved in D2O.
anoparticles have been utilized for coenzyme Q10 delivery to brain14]. Recently, TMC nanoparticles with 36.1% degree of quaterniza-ion were also used for delivery of anti-neuroexcitation peptide torain following parenteral administration [15].
The present study was designed to explore the potential of TMCanoparticles as a brain-targeting delivery system for Leu-Enk viaasal route. Leu-Enk-loaded TMC nanoparticles were prepared byn ionic cross linking technique and extensively characterized byeveral bio-analytical methods.
. Materials and methods
.1. Materials
Leu-Enk, 4-fluoro-7-nitrobenzofurazan (NBD-F), chitosan (85%egree of deacetylation and molecular weight of 500 kDa), aceticcid, Sodium tripolyphosphate (TPP), Tween 80 were purchasedrom Sigma Chemical Co. (USA). TMC with a degree of quaterniza-ion (DQ) of 25% was synthesized by methylation of chitosan bysing CH3I in the presence of NaOH and analyzed by 1H nuclearagnetic resonance (NMR) spectroscopy as described by Sieval
t al. [16] (Fig. 1). All other chemicals were of analytical grade,urchased from local suppliers and used as received.
.2. Methods
.2.1. Preparation of TMC nanoparticlesThe TMC nanoparticles were prepared by the ionic gelation of
MC with TPP anions. TMC (10 mg, DQ = 25%) was dissolved in 5 mlater. Then, TPP solutions (0.6 mg/ml) were added drop wise to the
bove solution under magnetic stirring at room temperature. Leu-nk-loaded TMC nanoparticles were prepared as described abovey dissolving different amounts of Leu-Enk in 5 ml TMC solutionefore adding TPP.
.2.2. Characterization of TMC nanoparticlesMorphological examination of Leu-Enk loaded TMC nanopar-
icles were performed using a transmission electron microscopeJEM-1200EX, Japan). Particle size, zeta potential, polydispersityndex and water absorbing capacity of the TMC nanoparticles wereetermined by laser diffraction particles size analyser (Malverneta nano ZS, Malvern, UK). Size of nanoparticles was analyzedn paraffin oil (viscosity: 5mPa s) as a non-dissolving disper-
ion medium. Particles were suspended by sonication during theeasurements. For determination of swelling behaviour, nanopar-
icles were incubated in 100 mM phosphate buffer pH 6.8 prequilibrated to 37 ◦C. Swelling of nanoparticles was determined
ical Macromolecules 61 (2013) 189– 195
immediately after addition of the buffer and after 0.5, 1, 2, 3 and 4 hof incubation in the same buffer medium under continuous shakingat 100 rpm on water bath at 37 ◦C. Each experiment was performedin triplicate.
2.2.3. Encapsulation efficiency of TMC nanoparticlesThe peptide encapsulation efficiency and loading capacity of
TMC nanoparticles were determined by following method. FreeLeu-Enk was separated from the nanoparticles by centrifugationat 12,000 × g (Remi C-22, Mumbai, India) for 30 min. The amountof unentrapped peptide remaining in the supernatant was mea-sured by UV spectrophotmetry at 216 nm (Shimadzu 1800 UV-VisSpectrophotometer, Japan). The supernatant of non-loaded TMCnanoparticles suspension was used as a blank to correct for inter-ference by free TMC. Peptide encapsulation efficiency and loadingcapacity of the nanoparticles were calculated as follows:
Encapsulation efficiency
= Total amount of Leu Enk − Free Leu EnkTotal amount of Leu-Enk
× 100
Loading capacity = Total mass of Leu-Enk − Free Leu EnkTotal mass of nanoparticles
× 100
2.2.4. In vitro Leu-Enk release from nanoparticlesAliquots of 10 ml Leu-Enk loaded TMC nanoparticle dispersion
prepared with Tween 80 were centrifuged at 12,000 × g (Remi C-22,Mumbai, India) for 20 min. The supernatant was decanted and thepellet was re-suspended in 10 ml phosphate buffered saline. Dis-persion was distributed among 10 Eppendorf tubes (1.5 ml) eachcontaining 1 ml suspension. The tubes were incubated at 37 ◦C(under agitation 50 rpm) for 3 h At time 0 and at different timeintervals, a tube was taken and centrifuged at 15,000 × g for 20 min(Remi C-22, Mumbai, India). Released Leu-Enk in the supernatantwas determined by the UV spectroscopy at 216 nm (Shimadzu 1800UV-Vis Spectrophotometer, Japan). A sample consisting of onlynanoparticles re-suspended in PBS was used as background. Eachexperiment was performed in triplicate.
2.2.5. Ex vivo mucoadhesion studiesNasal mucosa (conchae nasals) from the porcine nose (obtained
fresh from local slaughter house) was excised and stored in Krebs-Ringer-Bicarbonate Buffer (HIMEDIA, Mumbai, India) at 37 ◦Cduring transport to the laboratory. Nasal mucosa was separatedfrom the underlying cartilage by blunt stripping using a pair oftweezers and cut into defined pieces of diameter 2.5 cm. 10 mgTMC nanoparticles were spread on to each pieces and stream of airwas blown over the nanoparticles for 15 s. Amount of nanoparticlessticking on the mucosa was determined indirectly by quantificationof Leu-Enk in the remaining nanoparticles. Nanoparticles stickingto the mucosa were recovered by flushing the mucosa with bufferand volume was adjusted to 10 ml. Quantitative determination ofLeu-Enk was performed by HPLC using 0.1% trifluoroacetic acid inwater/acetonitrile and 0.1% TFA (90:10) as mobile phase at a flowrate of 1 ml/min, by LC 10-AT vp pump (Shimadzu, Japan). 20 �lof injection volume was eluted in LUNA 54, C18, 4.6 mm × 150 mmcolumn (Phonomex, USA) at room temperature. The column eluantwas monitored at 220 nm using SPD-M10A vp UV detector (Shi-madzu, Japan) and were identified by comparison and co-migration
with peptide standards. Peak areas were directly proportional tomass of standards injected and peptide was quantified from inte-grated peak areas and molar absorbance values calculated fromstandards for Leu-Enk.
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.2.6. Ex vivo nasal permeation studiesEx vivo drug diffusion study was performed using Franz diffu-
ion cell with a diameter of 10 mm and mucosa thickness 0.20 mm17]. Excised nasal mucosa free from fat and adhered mucus was
ounted between the donor and receptor compartments with theucosal side facing the donor compartment. The tissue was sta-
ilized under phosphate buffer (pH 6.4) in both donor and thecceptor compartments with magnetically stirring at 200 rpm. Sub-equently, buffer from both the compartments were removed andhe receptor compartment was filled with fresh phosphate bufferpH 6.4). Ex vivo diffusion of pure drug was conducted by placing
ml Leu-Enk solution or peptide loaded nanoparticle dispersionequivalent amount of 2 mg/ml) onto the stabilized porcine nasal
ucosal membrane placed in the donor compartment and stirredlowly on a magnetic stirrer. Samples from the receptor phaseere withdrawn at periodic time intervals, filtered through 0.45icron filter and the amount of intact peptide drug permeatedas quantified via HPLC as described above. Each removed sampleas replaced by an equal volume of diffusion medium to maintain
ink conditions. The cumulative amount of peptide permeated pernit area was plotted as a function of time, the steady-state per-eation rate (Jss) and lag time (h) were calculated from the slope
nd X-intercept of the linear portion, respectively. The permeabil-ty coefficient Papp (×10−6 cm s−1) was calculated by the followingquations:
ss = Q
A · tand
app = Q
A · 60 · C0
here Q is the amount of Leu-Enk transported through the nasalucosa in time t, and A is the area of exposed membrane in cm2
nd C0 is the donor concentration of the Leu-Enk.Enhancement ratios (R) were calculated from Papp values by:
= Papp (sample)Papp (control)
Each experiment was performed three times.
.2.7. Location of NBD-F labelled Leu-Enk in mouse brainTo investigate whether TMC nanoparticles were able to deliver
eu-Enk in to brain, Leu-Enk was labelled with NBD-F (4-fluoro-7-itrobenzofurazan) by previously described method [18]. NBD-F
abelled Enk were encapsulated into TMC nanoparticles andnstilled into Balb/c mice similarly as described elsewhere inext. Nanoparticles were instilled into nostrils of mice and 45 minater, brains were removed and fixed in a PBS solution of 4%araformaldehyde overnight. After that, samples were placed in5% sucrose PBS solution for 12 h, which was then replaced with0% sucrose for 24 h. The samples were then processed for micro-omy. Frozen sections, 10 mm thick, were then observed under auorescence microscope (Axio Lab.A1, Carl Zeiss Micro ImagingmbH, Germany).
.2.8. Biological evaluation
.2.8.1. Nasal administration. Balb/c mice of either sex (weighing0–25 g) were used for in this study. The study protocol includingandling, care and treatment were approved by Institutional Ani-al Ethics Committee of SLT Institute of Pharmaceutical Sciences,uru Ghasidas Vishwavidyalaya, Bilaspur (CG), India. The studiesere carried out as per the guidelines of Council for the Purpose
f Control and Supervision of Experiments on Animals (CPCSEA),inistry of Social Justice and Empowerment, Government of India.Mice were divided into three groups and each group consists
f six animals. The mice were lightly exposed to ether vapour to
ical Macromolecules 61 (2013) 189– 195 191
induce sedation, avoiding the influence of ether on the writhingas far as possible before the experiments. Mice were intranasallyadministered with 5 �l per 20 g (body weight) of normal saline(Group 1), Leu-Enk solution (Group 2) and Leu-Enk loaded TMCnanoparticles formulations (equivalent to 0.1 mg/ml Leu-Enk inPBS pH 6.8) (Group 3) using a 50 �l Hamilton microsyringe.
2.2.8.2. In vivo antinociceptive efficacy. Antinociceptive efficacy ofLeu-Enk loaded nanoparticles was assessed in mice by the hot-plate (thermal stimulation) and acetic acid induced writhing tests(chemical stimulation).
2.2.8.3. Hot-plate test (thermal stimulation). In the hot-plate assay,mice were placed on a 54 ◦C surface (Stoelting, USA), and the timeto lick one of the paws or escape jump was recorded as the responselatency. Predosing latency was determined before administrationof the compounds and was 4.6 ± 1.6 s. The hot-plate latency wasdetermined at 0, 30, 60 and 90 min after intranasal administrationof free or Leu-Enk loaded nanoparticles. A maximal cut off time ofthe heat was 45 s to prevent tissue damage. Results are expressedas mean ± SD of the nociceptive behaviour latency [19].
2.2.8.4. Writhing test (chemical stimulation). Mice wereintranasally administered with Leu Enk solution and Leu Enkloaded nanoparticles 60 min before receiving a 0.6% acetic acidinjection (10 ml/kg, intraperitoneal). The number of contractionsor writhing, determined by abdominal muscle contractions andhind paw extension was recorded for 20 min, starting 10 min afterthe administration of acetic acid [20].
2.3. Statistical analysis
The one-way ANOVA was employed for simultaneous statisticalanalysis of three or more groups. For comparing between groups,Tukey’s post test was used. p-Values less than 0.05 were consideredstatistically significant.
3. Results and discussion
3.1. Preparation of Leu-Enk loaded TMC nanoparticles
Neuropeptide as a drug hold great promise for the treatmentof a wide variety of brain disorders such as ischaemia, inflamma-tory and non-inflammatory neurodegenerative disorders, as well asacute, chronic or neuropathic pain syndromes. However, it is widelyacknowledged that neuropeptides typically fail to reach their tar-get after systemic administration due to poor diffusion through theBBB. In the past few years, a number of different approaches havebeen developed for drugs to overcome this hurdle. Among these,non-invasive application of polymeric nanoparticles seems to beone of the most promising techniques for central nervous systemdrug delivery. Nanoparticles are increasingly demonstrating theiradvantage of effective transporting of various different drugs acrossthe BBB on the basis of their small size and appropriate surface func-tionalization [21]. It has been a widely held view that intranasaladministration provides a means of circumventing the BBB and thusmay allow increased CNS penetration of compounds that otherwisedisplay limited CNS exposure [22]. In this study, nasal absorptionstudies were carried out with TMC nanoparticles with a low DQbecause of practical advantage of higher peptide encapsulation effi-ciencies and it was also previously demonstrated that the DQ ofTMC plays only a minor role in its (nasal) permeation [23,24]. The
TMC nanoparticles were prepared by ionic gelation, a gentle tech-nique that involves the mixing of two aqueous solutions at ambienttemperature without any harsh chemicals or production stress suchas sonication or use of organic solvents [25]. Since proteins and
192 M. Kumar et al. / International Journal of Biological Macromolecules 61 (2013) 189– 195
pplwrtm5a
3
4TioLt21iaptocn4T
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Fig. 2. TEM image of Leu-Enk loaded TMC Nanoparticles (bar = 1 �m).
eptides are very sensitive molecules to several stress factors, thisreparation method is very appropriate for preparation of peptide
oaded nanoparticles. Several process and composition parametersere optimized those include size distribution; colloidal stability,
eproducibility of nanoparticles, encapsulation efficiency to selecthe best formulation parameters to prepare nanoparticles. The opti-
al TMC nanoparticles were formed when the TMC/TPP ratio was.5 (w/w) which is close to the optimal ratio for chitosan/TPP (w/w)s reported by several authors [11,15].
.2. Characterization of Leu-Enk loaded TMC nanoparticles
Leu-Enk loaded TMC nanoparticles had an average diameter of43 ± 23 nm with a poly-dispersity index of 0.317 ± 0.17 (n = 3).ransmission electron microscopy confirmed few spherical withrregular shaped particles of nanometric size (Fig. 2). Zeta-potentialf the peptide loaded nanoparticles was +15± 2 mV (n = 3) (Table 1).eu-Enk was efficiently associated with TMC nanoparticles, ashey showed mean encapsulation efficiency of 78.28 ± 3.8% (TMC:
mg/ml, Leu-Enk: 0.5 mg) and mean loading capacity of about4 ± 1.3% (w/w). Leu-Enk (pI = 5.2) had a negative charge before
t was added to TMC solution. Consequently, the ionic interactionnd ionic cross linking between negatively charged Leu-Enk andositively charged TMC were intense, leading to high encapsula-ion efficiency. Further, TMC nanoparticles demonstrated swellingf 137–151% of initial size at pH 6.8 over 4 h. There was no signifi-
ant difference observed in the percentage swelling between TMCanoparticles and Enk loaded TMC nanoparticles (Table 1). Within
h, the swelling had almost reached equilibrium in both samples.his may be accounted due to their small particle size. Similar
5. Loading capacity (%) NA 14 ± 1.36. Zeta potential (mV) 19 ± 3 15 ± 27. Ex vivo mucoadhesion
(%attached)62.7 61.3
ll values represent mean ± SEM, n = 3.
Fig. 3. In vitro Leu-Enk release from TMC nanoparticles (n = 3).
findings were observed with chitosan/TPP film with a drasticdecrease in the swelling ratio from pH 4 to 6.5 followed by a grad-ual decrease between pH 8 and 10 [26]. Higher swelling propertiesof TMC particles could be attributed to their ability to uncoil thepolymer to an extended structure and higher molecular weight[27].
3.3. In vitro release studies
Drug release from nanoparticles and microparticles occur byseveral mechanisms including surface erosion, disintegration, dif-fusion and desorption [28,29]. The release profile of Leu-Enk fromTMC nanoparticles into PBS (pH 7.4; 37 ◦C) are shown in Fig. 3. Leu-Enk loaded TMC nanoparticles showed an initial release of 2.6%which may be accounted for the Leu-Enk adsorbed to the surface.Over 2 h, 26% of Leu-Enk was released followed by slow release upto 4 h. The increased release in aqueous media from 2.6% to 26% dueto the swelling property of the polymer that was clearly observedin the swelling studies (Fig. 3). Similar results with an initial releaseof 10% encapsulated BSA were reported with chitosan nanoparti-cles and later followed slow release at a constant but different rate[30].
3.4. Mucoadhesive studies
Adhesion to mucous membrane in nasal cavity can bedescribed as mucoadhesion. Mucoadhesive polymers improveabsorption/bioavailability of drugs with poor absorption charac-teristics [31–33]. In our experiment, more than 60% of initialamount of nanoparticles were attached to nasal mucosa. This resultshows prominent mucoadhesive capacity of TMC nanoparticles.Moreover, Leu-Enk does not alter its mucoadhesive characteristics(Table 1). It is reported that mucoadhesion properties of TMC aredue to the electrostatic attraction between the positively chargedpolymer and negatively charged sialic acid group of mucin of thenasal mucosa [34]. This provides a prolonged contact time betweenthe polymeric system and mucous layer surface to enhance theabsorption of the drugs [35].
3.5. Ex vivo permeation studies
In vitro nasal permeation studies were carried out in isolatedporcine nasal mucosa since it closely resembles humans in itsanatomy and physiology as well as in histological and biochem-
ical aspects [36]. Permeability co-efficient of Leu-Enk from thenasal mucosa determined to be 0.20 ± 0.04 × 10−6 cm s−1. Althoughhigher permeability of Leu-Enk from nasal mucosa was observeddue to lower molecular weight of Leu-Enk as compared to nasal
M. Kumar et al. / International Journal of Biolog
Fig. 4. Transport of Leu-Enk through excised porcine nasal mucosa (n = 3).
Table 2Leu-Enk test formulations permeation across excised porcine nasal mucosa.
Formulation Flux Jss
(�g cm−2 h−1)Lag time(h)
Apparentpermeabilityco-efficientPapp
(×10−6 cm s−1)
Control (Leu-Enk in buffer) 1.49 ± 0.30 0.18 ± 0.02 0.20 ± 0.04
bsorption of peptide drugs of higher molecular weight, never-heless nasal Leu-Enk absorption is so far too low to reach theherapeutic level. Poor uptake of Leu-Enk from nasal cavity is
ainly based on the enzymatic degradation of this peptide [37].his poor membrane permeability of Leu-Enk, however, could betrongly improved by encapsulation in TMC nanoparticles. In Fig. 4,he permeation of Leu-Enk across the nasal mucosa is shown as aunction of time. Permeation of Leu-Enk across nasal tissues wasignificantly enhanced by (enhancement ratio = 35) when formu-ated in TMC nanoparticles as compared with Leu-Enk in buffer.he analysis of the permeation profiles reported in Fig. 4 showedag times of about 0.18 and 0.31 h for Leu-Enk in buffer and Leu-nk released from TMC nanoparticles. The permeation flux of theeu-Enk (1.49 ± 0.3 �g cm−2 h−1) was lower (p < 0.001) than that ofeu-Enk loaded into TMC nanoparticles (56.61 ± 1.41 �g cm−2 h−1)Table 2). Permeability Coefficient of Leu-Enk was significantly
−6 −1
ncreased from 0.20 to 7.45 × 10 cm s when loaded in to TMCanoparticles. The permeability of Leu-Enk from the nasal mucosaas even 35-fold improved. TMC nanoparticles not only provide
onger contact time with nasal mucosa due to mucoadhesive nature
ig. 5. Fluorescent micrographs of mouse brain sections after the intranasal administratiot 45 min.
ical Macromolecules 61 (2013) 189– 195 193
followed by improved nasal uptake but also prevent activity of nasalpeptidases responsible for degradation of freely available Leu-Enk.Moreover, TMC acts by opening the tight junctions between nasalepithelial cells to allow paracellular transport of large hydrophilicmolecules due to an interaction of a positively charged aminogroup of TMC with negatively charged sites on the cell membranes,which results in a structural reorganization of the tight junction-associated proteins [11].
3.6. Distribution of NBD-F labelled Leu-Enk in mouse brain
Microscopic observation of sections of mouse brain clearlyshowed presence of labelled Leu-Enk in brain tissues (Fig. 5A),while no brain uptake of Labelled Leu-Enk solution was detected(Fig. 5B). Although qualitative, the present results showed that TMCnanoparticles could exert enhanced delivery of Leu-Enk into thebrain after intranasal administration. Positive charge of TMC at thesurface of nanoparticles could cause an electrostatic interactionwith the anionic binding sites of the brain capillaries and transferthe labelled drug in to the brain. On the other hand, TMC formeda hydrophilic surface which also contributed to this enhancement,and avoided uptake by the mononuclear phagocytic system andincreased the possibility of entering the brain [15].
3.7. In vivo evaluation
Among the various analgesic tests using thermal nociceptivestimulation, the most popular are the tail-flick and the hot platetest. In the hot plate test, animals are exposed only once to theheat stimulus, resulting in minimal tissue injury. The assay maybe performed without any previous habituation and offers goodreliability and reproducibility. The hot-plate response has beenproposed to require the activation of supra spinal mechanisms toinhibit a behavioural response [38]. This test is commonly used toassess narcotic analgesics or other centrally acting drugs [39]. Thenociceptors seem to be sensitized by sensory nerves. The involve-ment of endogenous substances such as PGs may be minimized bythis model. The hot-plate test was performed for the assessmentof the central antinociceptive effect of Leu-Enk released from TMCnanoparticles (Fig. 6). Paired-samples post test results revealed thatLeu-Enk liberated from TMC nanoparticles significantly inhibitedthe reaction time to thermal stimuli at 60 and 90 min compared tocontrol (p < 0.001).
Furthermore, the acetic acid induced writhing reaction in
mice, described as a typical model for inflammatory pain, haslong been used as a screening tool for the assessment of anal-gesic or anti-inflammatory properties of new agents [40]. Inthe acetic acid-induced abdominal writhing test, the writhing
n of (A) NBD-F labelled Enk loaded TMC Nanoparticles and (B) NBD-F Enk in buffer
194 M. Kumar et al. / International Journal of Biolog
Fa
itiTibsTa[pnhbwtc
Ff
ig. 6. Effect of Leu-Enk formulations in mice offered to hot-plate test (n = 6). Valuesre shown as means ± SD.
nducer (acid) induces nociception when injected intraperi-oneally by stimulating nociceptive fibres. This process involvedon-sensitive peripheral polymodal nociceptors, such as ASIC,RPV1 and glutamate receptors, and the release of endogenousnflammatory mediators involved in pain modulation, includingradykinin, pro-inflammatory cytokines (TNF-�, IL1� and IL-8),erotonin, histamine and prostaglandins [41]. In the writhing test,MC nanoparticles significantly inhibited the acetic acid-inducedbdominal constrictions in mice (p < 0.001) (Fig. 7). Gwak et al.20] concluded from their experiments that Leu-Enk could showotent analgesic activity when administered nasally with a combi-ation of enzyme inhibitors and absorption enhancers. Further Itas been also hypothesized that high molecular weight drugs cane delivered directly into the brain by extracellular transport path-ays associated with components of the peripheral olfactory and
rigeminal systems [42]. In the present study, results from biologi-al evaluation confirmed that Leu-Enk can be successfully delivered
ig. 7. Inhibition (%) of writhing number following nasal administration of Leu-Enkormulations. Values are shown as means ± SD. p < 0.05 was found to be significant.
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ical Macromolecules 61 (2013) 189– 195
by TMC nanoparticles in active form to brain tissues through nasalcavity resulted into potent centrally acting analgesic activity.
TMC is an excellent polymer which not only protects Leu-Enkfrom the action of peptidase in nasal cavity but also enhances theabsorption of intact Leu-Enk by virtue its mucoadhesive naturewhich counteract mucociliary clearance and provide longer contacttime for absorption through mucosa. TMC also assist in paracel-lular transport of peptide/drugs by transiently opening the tightjunctions existing between the epithelial cells and suppressing theefflux transporters [8,11,23].
4. Conclusion
Management of pain and inflammation due to various clinicalconditions like arthritis, cancer, and vascular diseases are one ofmost challenging task so far. Leu-Enk is an endogenous opioid pep-tide neurotransmitter that is found naturally in the brains of manyanimals, including humans. It has potent analgesic action by bind-ing at both the �- and �-opioid receptors in brain. The developmentof this peptide as therapeutic agents has been hampered by its poorenzymatic stability and bioavailability due to poor absorption. Datafrom present study suggest that TMC nanoparticles could generatea significant improvement of bioactive Leu-Enk levels in the brainwhen intranasally administered.
Conflict of interest
The authors report no conflicts of interest. The authors alone areresponsible for the content and writing of the paper.
Acknowledgement
Authors are thankful to All India Institute of Medical Sciences(AIIMS, New Delhi, India) and for providing Electron Microscopyfacility.
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