http://informahealthcare.com/drd ISSN: 1071-7544 (print), 1521-0464 (electronic) Drug Deliv, Early Online: 1–8 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10717544.2014.916769
RESEARCH ARTICLE
Insulin-loaded alginic acid nanoparticles for sublingual delivery Nilam H. Patil and Padma V. Devarajan
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Department of Pharmaceutical Science and Technology, Institute of Chemical Technology, Mumbai, Maharashtra, India
Abstract
Keywords
Alginic acid nanoparticles (NPs) containing insulin, with nicotinamide as permeation enhancer were developed for sublingual delivery. The lower concentration of proteolytic enzymes, lower thickness and enhanced retention due to bioadhesive property, were relied on for enhanced insulin absorption. Insulin-loaded NPs were prepared by mild and aqueous based nanoprecipitation process. NPs were negatively charged and had a mean size of 200 nm with low dispersity index. Insulin loading capacities of 495% suggested a high association of insulin with alginic acid. Fourier Transform Infra-Red Spectroscopy (FTIR) spectra and DSC (Differential Scanning Calorimetry) thermogram of insulin-loaded NPs revealed the association of insulin with alginic acid. Circular dichroism (CD) spectra confirmed conformational stability, while HPLC analysis confirmed chemical stability of insulin in the NPs. Sublingually delivered NPs with nicotinamide exhibited high pharmacological availability (4100%) and bioavailability (480%) at a dose of 5 IU/kg. The high absolute pharmacological availability of 20.2% and bioavailability of 24.1% in comparison with subcutaneous injection at 1 IU/kg, in the streptozotocin-induced diabetic rat model, suggest the insulin-loaded alginic acid NPs as a promising sublingual delivery system of insulin.
Circular dichroism, diabetic rats, FTIR, insulin, nanoparticles, permeation enhancer, stability, sublingual delivery
Introduction Insulin is currently the only drug for the treatment of insulindependent diabetes, which when genetic, afflicts even infants. Current therapy, namely the subcutaneous injection presents clear disadvantages ranging from severe patient discomfort and pain, significant variations in absorption and the possibility of hyperinsulinemia. Mucosal routes of administration have been extensively researched as alternatives and among the various mucosal routes the oral route, although biomimetic presented serious challenges, due to high concentration of proteolytic enzymes (Aungst et al., 1988). The nasal route appeared most promising, and triggered significant interest (Hinchcliffe & Illum, 1999; Sintov et al., 2010). Nasally delivered bioadhesive nanoparticles (NPs), enabled enhanced residence and hence increased bioavailability (Illum et al., 2001; Varshosaz et al., 2006; Jain et al., 2008). Polymeric NPs and microparticles exhibited enhanced insulin absorption, which was further increased by the inclusion of permeation enhancers (Krauland et al., 2006; Bhumkar et al., 2007; Wang et al., 2009). Nevertheless, mucociliary clearance, nasal irritation, inconsistent absorption and enzymatic degradation by proteases in the nasal mucosa, proved to be practical limitations of nasal delivery (Hinchcliffe & Illum, 1999; Sintov et al., 2010).
Address for correspondence: Padma V. Devarajan, Head, Department of Pharmaceutical Science and Technology, Institute of Chemical Technology, Mumbai 400019, Maharashtra, India. Tel: +91 22 33612201. Fax: +91 22 3361 1020. Email: [email protected]
History Received 18 March 2014 Revised 12 April 2014 Accepted 16 April 2014
The lower concentration of proteolytic enzymes compared to the nasal mucosa presents the buccal and sublingual route as a more viable option (Aungst et al., 1988; Aungst & Rogers, 1989; Oh & Ritschel, 1990). A novel insulin buccal spray containing soy lecithin and propranediol showed good hypoglycaemic effect in rabbits (Xu et al., 2002). Enhanced insulin bioavailability using permeation enhancers was achieved following buccal administration in rats and rabbits (Aungst & Rogers, 1989; Oh & Ritschel, 1990). Insulinloaded pelleted bioadhesive NPs exhibited significant glucose lowering following buccal administration (Venugopalan et al., 2001). A micellar insulin formulation Oral-Lyn has been commercialized. Although the buccal mucosa (500–600 mm) in thickness, presents a greater expanse, the lower thickness of the sublingual mucosa (100–200 mm) could provide improved permeation of insulin. Nevertheless, inclusion of permeation enhancers is essential to maximize insulin absorption. Among the enhancers reported to increase mucosal permeation, most were surfactants, known to disrupt the mucosa although temporarily (Cui et al., 2005). However, for administration of insulin which would be long term, safer permeation enhancers are imperative. Nicotinamide, a vitamin, is reported to enhance bioavailability of drug by increasing blood flow at the site of action, provides a promising alternative (Gupta et al., 2006). The highly vascularised sublingual mucosa prompted us to evaluate nicotinamide as a promising and safe permeation enhancer for effective sublingual delivery. A major requirement for sublingual delivery of insulin is retention of the system at the absorption site coupled with enhanced permeation. Insulin-loaded NPs using bioadhesive
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polymer alginic acid with nicotinamide as enhancer was designed to simultaneously address both issues. The aim of the study was the design stable insulin-loaded alginic acid NPs, with the objectives of achieving good decrease in serum glucose level and increase in serum insulin level in diabetic rats.
Materials and methods
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Materials Recombinant human insulin (28.7 IU/mg) was a gift sample from USV Ltd, Mumbai, India. Streptozotocin (STZ, MW 265.2) was purchased from Sigma-Aldrich, Mumbai, India. Alginic acid was a gift from Signet Co. and Pluronic F 68 from BASF Ltd., Mumbai, India. Nicotinamide was purchased from SD Fine Chemicals Ltd, Mumbai, India. GOD-POD Autopac kit was purchased from Accurex Biomedical, Mumbai, India. ELISA Rat Insulin Kit (Mercodia) was purchased from R. K. Diagnostics, Mumbai, India. All other chemicals were of analytical grade and commercially available. Methods Nanoparticles preparation Recombinant human insulin NPs (Rh-INS-NPs) were prepared by nanoprecipitation. Alginic acid (10 mg), pluronic F 68 (2.5 mg) and insulin (3 mg) were dissolved in sodium hydroxide 0.025 N (3 mL). Hydrochloric acid 0.025 N (2.5 mL) was subsequently added to the above solution under stirring using a mechanical stirrer and stirring was continued for 15 min followed by probe sonication for 5 min. The nanoparticulate dispersions were centrifuged at 15 000 rpm for 30 min at 25 C. Insulin in the supernatant was analyzed by HPLC at lmax of 214 nm to monitor entrapment efficiency. Nicotinamide (2 mg) was dissolved in double-distilled water (5 mL) using a vortex mixture. The pellet was re-dispersed in 5 mL solution of the nicotinamide. Albumin was added as cryoprotectant such that the ratio of solid nanoparticulate content: albumin was 1:2. Samples were frozen at 80 C in amber glass vials, and dried in a chamber at 0 C for 48 h at 0.133 mbar, corresponding to a condenser temperature of 50 C using a Lyph-lock 6 apparatus (Labconco, Boston, MA). Particle size determination Particle size measurements were performed by dynamic light scattering (photon correlation spectroscopy (PCS)) using an N4 Particle Analyser (Beckman Coulter Inc., Mumbai, India). Measurements were carried out in triplicate at a detection angle of 90 . Each sample was analyzed in triplicate and average particle size and polydispersity index (PI) measured. Zeta potential analysis Zeta potential measurements were performed by laser Doppler electrophoresis using a Zetasizer Nano ZS (Malvern Instruments Ltd., UK). Measurements were carried out in a folded capillary electrophoresis cell (Malvern Aimil Instruments, Mumbai, India) using ultrapure water as diluent. Data are presented as mean and standard deviation of triplicate runs.
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Drug loading Accurately weighed freeze-dried NPs (10 mg) were suspended in 2 mL of 0.1 N HCl and sonicated for 5 min to dissolve the loaded insulin. Aliquot of 1 mL was collected and further diluted with phosphate buffer saline (pH 7.4) to 5 mL and centrifuged for 15 min at 5000 rpm. The supernatant containing insulin was separated and analyzed by a developed HPLC method. HPLC (Shimadzu Co., Japan) equipped with a quaternary pump, a UV detector set at max of 214 nm, a reversed-phase X-Terra RP 18 column 5 mm, 4.6 250 mm (Merck, Darmstadt, Germany). The mobile phase comprised phosphate buffer pH 2.5 and acetonitrile (72:28), degassed and isocratically run at a flow rate of 1 mL/min. The injection volume was 100 mL. Percent Drug loading (DL %) was calculated using the equation: DL ð%Þ ¼ WDL =WNP 100
ð1Þ
where, WDL ¼ weight of drug in Np and WNP ¼ weight of Np. In-vitro release study of insulin nanoparticles Insulin NPs (10 mg) were suspended in PBS (pH 6.8, 1 mL), sealed in dialysis bags (M.W. 12 000 cut-off, Sigma) and immersed in 25 mL of PBS (pH 6.8) at 37 C under magnetic stirring. Samples were withdrawn at scheduled intervals of 1, 2, 4, 6, 8 and 12 h replaced with equivalent amount of dissolution medium. Insulin content was estimated by reverse phase HPLC method. The release study was performed in triplicate. Scanning electron microscopy The morphology/shape of NPs was determined by scanning electron microscopy (SEM) (JSM-6380-LA, JEOL, Tokyo, Japan). A drop of colloidal dispersion was deposited onto a carbon tape and dried under vacuum. The samples were sputtered with platinum using an auto fine coater prior to analysis (JFC-1600, JEOL, Tokyo, Japan). Ex-vivo bioadhesion studies Mucoadhesive properties of insulin-loaded NPs were evaluated by Texture analyzer (CT3 TA) using porcine sublingual mucosa. Sections of sublingual mucosa from healthy porcine were obtained from slaughter house. The underlying connective tissue was removed carefully using a surgical scissor, making sure that the basal membrane was not damaged. Membrane of a 1.6 cm diameter (2 cm2 area) were cut using the surgical scissor and equilibrated in phosphate buffer (pH 6.8) for 30 min before mounting to ensure removal of the soluble components present. The membranes were then attached on the base of texture analyzer in a holder and to the upper probe using two sided adhesive tape. The probe was fixed to the mobile arm of the texture analyzer. Freeze dried NPs (10 mg) were transferred onto the centre of the membrane placed in the holder, and moistened with 1 mL of phosphate buffer (pH 6.8). The mobile arm was lowered at a rate of 0.5 mm s1 until contact with the formulation was made. A contact force of 10 g was maintained for 500 s, after which the probe was withdrawn from the membrane.
Insulin-loaded alginic acid NPs
DOI: 10.3109/10717544.2014.916769
The force (N) required to separate the adhesive bond was recorded as mucoadhesive strength. Insulin solution prepared using phosphate buffer saline (pH 7.4) was used as control (Thirawong et al., 2007). Fourier transform infra-red spectroscopy FTIR studies was performed on Perkin Elmer RX1 (Mumbai, India) using a KBr pellet to determine drug excipient interaction. Briefly insulin, alginic acid and lyophilized insulin NPs were crushed to a fine powder, mulled with KBr and compressed to form a thin transparent pellet at a force of 10–11 KN for 2 min using a KBr press model no. H-15 (Technosearch Instruments, Mumbai, India). The IR spectra were recorded over wavenumber range of 4000 to 500 cm1
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Differential scanning calorimetry Thermal behavior of insulin NPs was determined by DSC. Samples were accurately weighed (5 mg) in aluminium pans, crimped and subjected to DSC under constant purging of nitrogen at 20 mL/min using a Perkin Elmer Pyris 6 (Mumbai, India) DSC thermal analysis instrument. Thermograms were recorded by heating samples from 40 C to 350 C at a heating rate of 10 C min1 with empty aluminium pan as the reference. Circular dichroism spectroscopy Circular dichroism (CD) spectra were obtained with a Jasco J-810 (Easton, MD) spectropolarimeter equipped with a temperature controller to examine the secondary structure of insulin in particles. Spectra were collected at 25 C using a 0.1 cm cell over the wavelength range of 200–250 nm. A resolution of 0.2 nm and scanning speed (50 nm/min) with a 4 s response time were employed. Each spectrum obtained represents an average of three consecutive scans. Noise reduction, blank solution subtraction, and data analysis were performed using standard analysis and temperature/wavelength analysis programs (Jasco). For CD analysis, insulin was extracted from NPs using 0.1 N HCl (2 mL) and further diluted with phosphate buffer saline (PBS pH 7.4) to 10 mL. The spectra of insulin samples were compared with that of native insulin. Stability study Insulin NPs were evaluated for stability under refrigeration (2–8 C) filled in clean, dry, air tight, amber glass vials. At time intervals of 0, 3, 6 and 12 months samples were evaluated for particle size, insulin content and CD study. In-vivo evaluation of insulin nanoparticles Healthy male Wistar rats (200–280 g) were purchased from the Haffkine Institute (Mumbai, India). All animal experiments were approved by the Institutional Animal Care and Ethic Committee. The rats were housed in cages (five rats per cage) under controlled conditions of 25 C and 55% air humidity with free access to water and standard rat chow. The rats were acclimatized for at least 7 d before use. Protocol was approved by ethical committee registered (87/1999/CPCSEA), Department of Pharmaceutical Science and Technology, Institute of Chemical Technology, Mumbai, India.
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Induction of diabetes. Healthy male Wistar rats (200–280 g)
were fasted for 12 h with free access to water. Diabetes was induced by a single intraperitoneal injection of streptozotocin dissolved in 0.1 M citrate buffer (pH 4.5) at a dose of 45 mg/kg. Blood samples were withdrawn from the retroorbital plexus using heparinized capillaries and centrifuged at 10 000 rpm for 5 min to separate the serum. Serum glucose level was measured using a GOD-POD Autopac kit (Accurex Biomedical, Mumbai, India) according to the manufacturer’s kit procedure. The rats with fasting serum glucose level higher than 300 mg/dL were considered as diabetic and used for further studies. Evaluation of the hypoglycemic effect and serum insulin levels in rats. Diabetic rats were divided into three groups (n ¼ 6).
Rats were fasted for 12–16 h prior to the experiment. Group I received insulin (1.0 IU/kg) as a solution (0.5 mL, pH 7.4) by subcutaneous injection. Nanoparticulate formulations were dispersed in distilled water such that 25 ml contained 1.25 IU of insulin. The insulin NPs (5 IU/kg) without and with nicotinamide were sublingually administered to rats of Group II and Group III using micropipette, respectively. Blood samples were collected from the retro-orbital plexus at 0, 1, 3, 5, 8, 12 and 24 h using a heparinized capillary and centrifuged at 10 000 rpm for 5 min to separate the serum. Serum glucose levels using the GOD-POD Autopac kit and serum insulin was measured using the ELISA Rat Insulin Kit (Mercodia, Sweden) according to the manufacturer’s kit procedure. Basal insulin was corrected by calibration. The area above the curve (AAC) for serum glucose level and area under the curve (AUC) for serum insulin level were calculated using BASICA software. Cmax and tmax were determined from AAC and AUC. The pharmacological availability (PA) and relative bioavailability (BA) were calculated. Student’s t-test and ANOVA were used to determine statistical significance. Differences were considered to be significant for values of p50.05. Insulin NPs containing nicotinamide (5 IU/kg) as a dispersion (&25 mL) was administered sublingually in one group of rats (n ¼ 4) and the other group was administered with water as vehicle control. Rats were observed for mortality upto 24 h. After 24 h the rats were sacrificed, and the sublingual mucosae excised inflated with a formaldehyde solution 4% (v/w) in physiological saline, fixed and stored at 4 C until further histological examination. The formaldehyde-treated mucosae were dehydrated and embedded in paraffin. Slices of 5 mm thickness were sectioned and stained with a regular hematoxylin/eosin staining. Histological evaluation was performed by using an Olympus BX50 light microscope equipped with a Leica DFC 320 camera. Photographs were taken using a 40 magnification objective.
Histopath study of sublingual mucosa.
Results and discussion Bioadhesive NPs by enabling prolonged retention in the sublingual cavity present a promising strategy for enhanced insulin absorption. Alginic acid was selected as the polymer due to its bioadhesive property. Insulin-loaded alginic acid
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NPs were prepared by a mild and aqueous process (Cheng et al., 2012; Liu et al., 2013). This was important as processes involving high shear or the use of organic solvents could be detrimental to insulin stability. Alginic acid NPs of around 220 nm with low PI were readily prepared by nanoprecipitation induced by simple pH change, without the need for high shear homogenization. A marginal increase in particle size observed with enhancer was considered insignificant (Table 1). SEM photographs of insulin-loaded alginic acid NPs revealed spherical NPs (Figure 1). NPs were strongly negatively charged with zeta potential values ranging from 32 ± 2.5 mV, attributed to carboxyl group of alginic acid. Bioadhesion is based on the measurement of shear stress required to break the adhesive bonds between a mucosal membrane and the formulation. Alginic acid NPs revealed strong bioadhesion compared to insulin solution (Table 1). Inclusion of nicotinamide did not influence bioadhesion of alginic acid NPs. The bioadhesive property of alginic acid is reported earlier (Cheng et al., 2012; Liu et al., 2013). Insulin entrapment efficiency of 495% suggested high association of insulin with alginic acid. Alginic acid has pKa of 3.5 and remains in the ionized form (COO) at pH 43.5.
Figure 1. Scanning electron microscopy (SEM) images of insulin-loaded alginic acid nanoparticles.
Table 1. Physico-chemical properties of insulin-loaded alginic acid nanoparticles with/without nicotinamide.
Formulation Insulin solution Alginic acid nanoparticles Alginic acid nanoparticles with nicotinamide
Particle size (nm)
Polydispersity index
Entrapment efficiency (%)
Zeta potential
Bioadhesive force (N)
Ratio of molar ellipticity as 208/222
– 210 ± 2.4 nm 225 ± 3.5 nm
– 0.15 ± 0.3 0.23 ± 0.4
– 96 ± 5.5 % 97 ± 3.3 %
– 32 ± 2.5 30 ± 3.6
0.005 ± 0.025 0.423 ± 0.15 0.436 ± 0.23
1.1 1.2 1.1
Figure 2. FTIR spectra of (a) alginic acid, (b) insulin and (c) alginic acid nanoparticles of insulin.
Insulin-loaded alginic acid NPs
DOI: 10.3109/10717544.2014.916769
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Addition of 0.025 N HCl to a solution of alginic acid and insulin in 0.025 N NaOH (pH 11.3) resulted in a gradual drop in pH, favouring insulin–alginic acid interactions at pH 5, as isoelectric point of insulin (pI: 5.3). Such high entrapment efficiency reflects minimal insulin loss, a major plus point for process scale-up. The FTIR spectrum of alginic acid revealed a weak band at 1226 cm1 from C–O stretching and a strong peak near 1033 cm1 attributed to C–O–C symmetric stretching (Figure 2). The alginic acid carboxyl peaks are seen near 1728 cm1 (symmetric COO stretching vibration) and 1404 cm1 (asymmetric COO stretching vibration). The spectra of insulin presented the pattern of amino acids characterized by asymmetrical N–H bending band near 1643 cm1, a symmetrical bending band near 1519 cm1, and a weak band from symmetrical C¼(O)2 stretching of the carboxylate ion group near 1450 cm1. Insulin contains six
5
amino acid residues which could acquire a positive charge enabling interaction with alginic acid (Bouchard et al., 2002; Bruno et al., 2006). FTIR spectra of insulin-loaded NPs confirmed insulin entrapment in the NPs as two sharp bands of Amide I (1643 cm1) and Amide II (1527 cm1) were seen (Bruno et al., 2006). The shape and the position of the amide I band is consistent with the presence of a largely a-helical structure. This result suggests that a-helical structure of insulin was not affected under the conditions studied. The interaction between insulin and alginic acid was further confirmed by DSC. Pure insulin revealed a melting endotherm at 64 C and a tiny and broad exothermic peak at 278 C indicating crystalline nature. The thermograms of alginic acid showed an initial endothermic peak at 77 C and a higher exothermic peak at 248 C. Endothermic peaks are correlated with the loss of water associated to hydrophilic groups of polymers while exothermic peaks resulted from
Figure 3. Thermograms of (a) insulin, (b) alginic acid, and (c) insulin nanoparticles.
Figure 4. Stability of insulin nanoparticles at the end of 12 months evaluated by (a) CD spectra, (b) drug content (c) particle size.
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degradation of polyelectrolytes due to dehydration and depolymerisation reactions most probably to the partial decarboxylation of the protonated carboxylic groups and oxidation reactions of the polyelectrolytes. The disappearance of insulin and alginic acid peaks in the NPs suggested their association. The endothermic and exothermic peaks associated with insulin, which are attributed to denaturation process and water loss, became indistinct after insulin entrapment into the alginic acid NPs (Figure 3). Conservation of the structural integrity of a protein drug after release is crucial for its biological activity. The CD spectra of insulin recovered from NPs immediately after preparation and at 12 months showed typical double minima at 208 nm and 222 nm indicative of substantial alpha-helical
Figure 5. Effect of single administration of insulin on (a) serum insulin level and (b) serum glucose level in streptozotocin-induced diabetic rats, (——) insulin solution (subcutaneous-1 IU/kg), (—˙—) insulin nanoparticles (sublingual-5 IU/kg), (—m—) insulin nanoparticles with nicotinamide and (——)blank nanoparticles with nicotinamide (n ¼ 6).
structure, and were in close agreement with the spectra of native insulin (Figure 4a). This indicated that there was no change in random coil structure of insulin, suggesting good physico–chemical stability of insulin in the NPs. HPLC analysis confirmed chemical stability of insulin up to 12 months (Figure 4b). No significant change in particle size (p50.05) was observed on storage (Figure 4c). Insulin release from alginic acid NPs followed first-order kinetics. The release profile of insulin from NPs over a period of 12 h indicated very rapid initial burst (65%) in barely 2 h followed by slow release. Inclusion of nicotinamide did not influence in-vitro release of insulin from NPs (F2 ¼ 69.15). Alginic acid NPs with nicotinamide revealed significant decrease in serum glucose level (p50.05) compared to alginic acid NPs without nicotinamide although Cmax with the insulin-loaded NPs was lowered with subcutaneous injection. Insulin-loaded NPs revealed sustained lowering compared to the relatively short hypoglycaemic effect following subcutaneous injection. Blank NPs with nicotinamide showed no reduction in serum glucose levels (Figure 5). Pharmacological availabilities in literature were compared to subcutaneous administration at doses 5–10 IU/kg following nasal administration for typically in the range 8–13%, while similar pharmacological availabilities in the range 3–16% following oral administration are reported at significant larger doses of 12.5–20 IU/kg (Wang et al., 2009; Makhlof et al., 2011; Jose et al., 2012; Zhang et al., 2012). In contrast, our NPs revealed high pharmacological availability of 100.2% and 125.1%. The dose-corrected bioavailability with reference to subcutaneous injection (1 IU/kg) is also significantly higher (20% and 25%), respectively. Our findings appear promising as large doses of insulin could induce adverse mitogenic changes and moreover insulin being a protein could also trigger immunological reactions (Bellary & Barnett, 2006). Subcutaneous injection revealed a high Cmax (97 mIU/mL), however a low tmax (1 h) and a rapid decline in insulin concentration (Figure 5). NPs revealed slower absorption, with tmax between 1 and 2 h and lower Cmax (Table 2). A good correlation (r2 ¼ 0.909) was seen between decrease in serum glucose and increase in serum insulin level. Nevertheless, bioavailability obtained with the alginic acid NPs containing nicotinamide (5 IU/kg) was comparable with subcutaneous injection.
Table 2. Pharmacodynamic and pharmacokinetic parameters obtained by administration of insulin solution or nanoparticles to the diabetic rats.
Formulation Insulin dose (IU/kg) Maximum reduced glucose level (%) Time point of maximum reduced glucose level (h) AAC PA Cmax (mIU/mL) tmax (h) AUC0!24/rat BA (%)
Subcutaneous injection
Sublingual alginic acid nanoparticles
Sublingual alginic acid nanoparticles-nicotinamide
1 68.2 ± 2.3 2 562.3 ± 1.6 – 97.2 ± 2.1 1 244.3 ± 7.1 –
5 38.1 ± 4.2 2 559.3 ± 4.2 100.2 ± 4.9 17.4 ± 2.1 2 201. ± 5.5 80.1 ± 2.5
5 49.2 ± 1.3 2 691.2 ± 6.3 125.1 ± 5.7 25.3 ± 3.4 2 292.2 ± 4.3 96.3 ± 2.1
Each data represents the mean ± SD (n ¼ 4). Cmax, the maximum concentration; tmax, the time to reach the Cmax; AAC, the area above the curve; AUC, the area under the curve; PA, pharmacological availability compared with s.c.; BA, relative bioavailability compared with s.c.
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DOI: 10.3109/10717544.2014.916769
Insulin-loaded alginic acid NPs
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Figure 6. Light micrographs of the sublingual rat mucosal membranes following administration of INS-NPs (5 IU/kg) containing nicotinamide (a) and distilled water as control (b).
Microscopic evaluation of mucosal cross sections has been widely used as an easy and practical method for assessing any damaging effect associated with exposure to enhancer. The histopathology of sublingual mucosa after administration of insulin NPs with nicotinamide showed that membrane integrity appeared to remain undamaged (Figure 6), suggesting nicotinamide as relatively safe permeation enhancer. Nevertheless, changes following chronic administration need to be evaluated.
Conclusion In this article, an attempt has been made to develop a sublingual insulin delivery formulation with bioadhesive alginic acid polymer and nicotinamide as permeation enhancer. Particles with a small size 200 nm and high entrapment efficiency (96%) were obtained. Stability of insulin in NPs was confirmed by HPLC and CD analysis. The high pharmacological availability and bioavailability of insulin from insulin-loaded alginic acid NPs with nicotinamide in the diabetic rat model suggested promising for sublingual delivery.
Acknowledgements The authors would like to acknowledge USV Ltd., India, for gift sample of insulin. We are also thankful to TIFR, India, for helping in CD spectra study.
Declaration of interest The authors report no declarations of interest. Authors thank University Grant Commission for grant and fellowship to Nilam Patil.
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RESEARCH ARTICLE
Insulin-loaded alginic acid nanoparticles for sublingual delivery Nilam H. Patil and Padma V. Devarajan
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Department of Pharmaceutical Science and Technology, Institute of Chemical Technology, Mumbai, Maharashtra, India
Abstract
Keywords
Alginic acid nanoparticles (NPs) containing insulin, with nicotinamide as permeation enhancer were developed for sublingual delivery. The lower concentration of proteolytic enzymes, lower thickness and enhanced retention due to bioadhesive property, were relied on for enhanced insulin absorption. Insulin-loaded NPs were prepared by mild and aqueous based nanoprecipitation process. NPs were negatively charged and had a mean size of 200 nm with low dispersity index. Insulin loading capacities of 495% suggested a high association of insulin with alginic acid. Fourier Transform Infra-Red Spectroscopy (FTIR) spectra and DSC (Differential Scanning Calorimetry) thermogram of insulin-loaded NPs revealed the association of insulin with alginic acid. Circular dichroism (CD) spectra confirmed conformational stability, while HPLC analysis confirmed chemical stability of insulin in the NPs. Sublingually delivered NPs with nicotinamide exhibited high pharmacological availability (4100%) and bioavailability (480%) at a dose of 5 IU/kg. The high absolute pharmacological availability of 20.2% and bioavailability of 24.1% in comparison with subcutaneous injection at 1 IU/kg, in the streptozotocin-induced diabetic rat model, suggest the insulin-loaded alginic acid NPs as a promising sublingual delivery system of insulin.
Circular dichroism, diabetic rats, FTIR, insulin, nanoparticles, permeation enhancer, stability, sublingual delivery
Introduction Insulin is currently the only drug for the treatment of insulindependent diabetes, which when genetic, afflicts even infants. Current therapy, namely the subcutaneous injection presents clear disadvantages ranging from severe patient discomfort and pain, significant variations in absorption and the possibility of hyperinsulinemia. Mucosal routes of administration have been extensively researched as alternatives and among the various mucosal routes the oral route, although biomimetic presented serious challenges, due to high concentration of proteolytic enzymes (Aungst et al., 1988). The nasal route appeared most promising, and triggered significant interest (Hinchcliffe & Illum, 1999; Sintov et al., 2010). Nasally delivered bioadhesive nanoparticles (NPs), enabled enhanced residence and hence increased bioavailability (Illum et al., 2001; Varshosaz et al., 2006; Jain et al., 2008). Polymeric NPs and microparticles exhibited enhanced insulin absorption, which was further increased by the inclusion of permeation enhancers (Krauland et al., 2006; Bhumkar et al., 2007; Wang et al., 2009). Nevertheless, mucociliary clearance, nasal irritation, inconsistent absorption and enzymatic degradation by proteases in the nasal mucosa, proved to be practical limitations of nasal delivery (Hinchcliffe & Illum, 1999; Sintov et al., 2010).
Address for correspondence: Padma V. Devarajan, Head, Department of Pharmaceutical Science and Technology, Institute of Chemical Technology, Mumbai 400019, Maharashtra, India. Tel: +91 22 33612201. Fax: +91 22 3361 1020. Email: [email protected]
History Received 18 March 2014 Revised 12 April 2014 Accepted 16 April 2014
The lower concentration of proteolytic enzymes compared to the nasal mucosa presents the buccal and sublingual route as a more viable option (Aungst et al., 1988; Aungst & Rogers, 1989; Oh & Ritschel, 1990). A novel insulin buccal spray containing soy lecithin and propranediol showed good hypoglycaemic effect in rabbits (Xu et al., 2002). Enhanced insulin bioavailability using permeation enhancers was achieved following buccal administration in rats and rabbits (Aungst & Rogers, 1989; Oh & Ritschel, 1990). Insulinloaded pelleted bioadhesive NPs exhibited significant glucose lowering following buccal administration (Venugopalan et al., 2001). A micellar insulin formulation Oral-Lyn has been commercialized. Although the buccal mucosa (500–600 mm) in thickness, presents a greater expanse, the lower thickness of the sublingual mucosa (100–200 mm) could provide improved permeation of insulin. Nevertheless, inclusion of permeation enhancers is essential to maximize insulin absorption. Among the enhancers reported to increase mucosal permeation, most were surfactants, known to disrupt the mucosa although temporarily (Cui et al., 2005). However, for administration of insulin which would be long term, safer permeation enhancers are imperative. Nicotinamide, a vitamin, is reported to enhance bioavailability of drug by increasing blood flow at the site of action, provides a promising alternative (Gupta et al., 2006). The highly vascularised sublingual mucosa prompted us to evaluate nicotinamide as a promising and safe permeation enhancer for effective sublingual delivery. A major requirement for sublingual delivery of insulin is retention of the system at the absorption site coupled with enhanced permeation. Insulin-loaded NPs using bioadhesive
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polymer alginic acid with nicotinamide as enhancer was designed to simultaneously address both issues. The aim of the study was the design stable insulin-loaded alginic acid NPs, with the objectives of achieving good decrease in serum glucose level and increase in serum insulin level in diabetic rats.
Materials and methods
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Materials Recombinant human insulin (28.7 IU/mg) was a gift sample from USV Ltd, Mumbai, India. Streptozotocin (STZ, MW 265.2) was purchased from Sigma-Aldrich, Mumbai, India. Alginic acid was a gift from Signet Co. and Pluronic F 68 from BASF Ltd., Mumbai, India. Nicotinamide was purchased from SD Fine Chemicals Ltd, Mumbai, India. GOD-POD Autopac kit was purchased from Accurex Biomedical, Mumbai, India. ELISA Rat Insulin Kit (Mercodia) was purchased from R. K. Diagnostics, Mumbai, India. All other chemicals were of analytical grade and commercially available. Methods Nanoparticles preparation Recombinant human insulin NPs (Rh-INS-NPs) were prepared by nanoprecipitation. Alginic acid (10 mg), pluronic F 68 (2.5 mg) and insulin (3 mg) were dissolved in sodium hydroxide 0.025 N (3 mL). Hydrochloric acid 0.025 N (2.5 mL) was subsequently added to the above solution under stirring using a mechanical stirrer and stirring was continued for 15 min followed by probe sonication for 5 min. The nanoparticulate dispersions were centrifuged at 15 000 rpm for 30 min at 25 C. Insulin in the supernatant was analyzed by HPLC at lmax of 214 nm to monitor entrapment efficiency. Nicotinamide (2 mg) was dissolved in double-distilled water (5 mL) using a vortex mixture. The pellet was re-dispersed in 5 mL solution of the nicotinamide. Albumin was added as cryoprotectant such that the ratio of solid nanoparticulate content: albumin was 1:2. Samples were frozen at 80 C in amber glass vials, and dried in a chamber at 0 C for 48 h at 0.133 mbar, corresponding to a condenser temperature of 50 C using a Lyph-lock 6 apparatus (Labconco, Boston, MA). Particle size determination Particle size measurements were performed by dynamic light scattering (photon correlation spectroscopy (PCS)) using an N4 Particle Analyser (Beckman Coulter Inc., Mumbai, India). Measurements were carried out in triplicate at a detection angle of 90 . Each sample was analyzed in triplicate and average particle size and polydispersity index (PI) measured. Zeta potential analysis Zeta potential measurements were performed by laser Doppler electrophoresis using a Zetasizer Nano ZS (Malvern Instruments Ltd., UK). Measurements were carried out in a folded capillary electrophoresis cell (Malvern Aimil Instruments, Mumbai, India) using ultrapure water as diluent. Data are presented as mean and standard deviation of triplicate runs.
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Drug loading Accurately weighed freeze-dried NPs (10 mg) were suspended in 2 mL of 0.1 N HCl and sonicated for 5 min to dissolve the loaded insulin. Aliquot of 1 mL was collected and further diluted with phosphate buffer saline (pH 7.4) to 5 mL and centrifuged for 15 min at 5000 rpm. The supernatant containing insulin was separated and analyzed by a developed HPLC method. HPLC (Shimadzu Co., Japan) equipped with a quaternary pump, a UV detector set at max of 214 nm, a reversed-phase X-Terra RP 18 column 5 mm, 4.6 250 mm (Merck, Darmstadt, Germany). The mobile phase comprised phosphate buffer pH 2.5 and acetonitrile (72:28), degassed and isocratically run at a flow rate of 1 mL/min. The injection volume was 100 mL. Percent Drug loading (DL %) was calculated using the equation: DL ð%Þ ¼ WDL =WNP 100
ð1Þ
where, WDL ¼ weight of drug in Np and WNP ¼ weight of Np. In-vitro release study of insulin nanoparticles Insulin NPs (10 mg) were suspended in PBS (pH 6.8, 1 mL), sealed in dialysis bags (M.W. 12 000 cut-off, Sigma) and immersed in 25 mL of PBS (pH 6.8) at 37 C under magnetic stirring. Samples were withdrawn at scheduled intervals of 1, 2, 4, 6, 8 and 12 h replaced with equivalent amount of dissolution medium. Insulin content was estimated by reverse phase HPLC method. The release study was performed in triplicate. Scanning electron microscopy The morphology/shape of NPs was determined by scanning electron microscopy (SEM) (JSM-6380-LA, JEOL, Tokyo, Japan). A drop of colloidal dispersion was deposited onto a carbon tape and dried under vacuum. The samples were sputtered with platinum using an auto fine coater prior to analysis (JFC-1600, JEOL, Tokyo, Japan). Ex-vivo bioadhesion studies Mucoadhesive properties of insulin-loaded NPs were evaluated by Texture analyzer (CT3 TA) using porcine sublingual mucosa. Sections of sublingual mucosa from healthy porcine were obtained from slaughter house. The underlying connective tissue was removed carefully using a surgical scissor, making sure that the basal membrane was not damaged. Membrane of a 1.6 cm diameter (2 cm2 area) were cut using the surgical scissor and equilibrated in phosphate buffer (pH 6.8) for 30 min before mounting to ensure removal of the soluble components present. The membranes were then attached on the base of texture analyzer in a holder and to the upper probe using two sided adhesive tape. The probe was fixed to the mobile arm of the texture analyzer. Freeze dried NPs (10 mg) were transferred onto the centre of the membrane placed in the holder, and moistened with 1 mL of phosphate buffer (pH 6.8). The mobile arm was lowered at a rate of 0.5 mm s1 until contact with the formulation was made. A contact force of 10 g was maintained for 500 s, after which the probe was withdrawn from the membrane.
Insulin-loaded alginic acid NPs
DOI: 10.3109/10717544.2014.916769
The force (N) required to separate the adhesive bond was recorded as mucoadhesive strength. Insulin solution prepared using phosphate buffer saline (pH 7.4) was used as control (Thirawong et al., 2007). Fourier transform infra-red spectroscopy FTIR studies was performed on Perkin Elmer RX1 (Mumbai, India) using a KBr pellet to determine drug excipient interaction. Briefly insulin, alginic acid and lyophilized insulin NPs were crushed to a fine powder, mulled with KBr and compressed to form a thin transparent pellet at a force of 10–11 KN for 2 min using a KBr press model no. H-15 (Technosearch Instruments, Mumbai, India). The IR spectra were recorded over wavenumber range of 4000 to 500 cm1
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Differential scanning calorimetry Thermal behavior of insulin NPs was determined by DSC. Samples were accurately weighed (5 mg) in aluminium pans, crimped and subjected to DSC under constant purging of nitrogen at 20 mL/min using a Perkin Elmer Pyris 6 (Mumbai, India) DSC thermal analysis instrument. Thermograms were recorded by heating samples from 40 C to 350 C at a heating rate of 10 C min1 with empty aluminium pan as the reference. Circular dichroism spectroscopy Circular dichroism (CD) spectra were obtained with a Jasco J-810 (Easton, MD) spectropolarimeter equipped with a temperature controller to examine the secondary structure of insulin in particles. Spectra were collected at 25 C using a 0.1 cm cell over the wavelength range of 200–250 nm. A resolution of 0.2 nm and scanning speed (50 nm/min) with a 4 s response time were employed. Each spectrum obtained represents an average of three consecutive scans. Noise reduction, blank solution subtraction, and data analysis were performed using standard analysis and temperature/wavelength analysis programs (Jasco). For CD analysis, insulin was extracted from NPs using 0.1 N HCl (2 mL) and further diluted with phosphate buffer saline (PBS pH 7.4) to 10 mL. The spectra of insulin samples were compared with that of native insulin. Stability study Insulin NPs were evaluated for stability under refrigeration (2–8 C) filled in clean, dry, air tight, amber glass vials. At time intervals of 0, 3, 6 and 12 months samples were evaluated for particle size, insulin content and CD study. In-vivo evaluation of insulin nanoparticles Healthy male Wistar rats (200–280 g) were purchased from the Haffkine Institute (Mumbai, India). All animal experiments were approved by the Institutional Animal Care and Ethic Committee. The rats were housed in cages (five rats per cage) under controlled conditions of 25 C and 55% air humidity with free access to water and standard rat chow. The rats were acclimatized for at least 7 d before use. Protocol was approved by ethical committee registered (87/1999/CPCSEA), Department of Pharmaceutical Science and Technology, Institute of Chemical Technology, Mumbai, India.
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Induction of diabetes. Healthy male Wistar rats (200–280 g)
were fasted for 12 h with free access to water. Diabetes was induced by a single intraperitoneal injection of streptozotocin dissolved in 0.1 M citrate buffer (pH 4.5) at a dose of 45 mg/kg. Blood samples were withdrawn from the retroorbital plexus using heparinized capillaries and centrifuged at 10 000 rpm for 5 min to separate the serum. Serum glucose level was measured using a GOD-POD Autopac kit (Accurex Biomedical, Mumbai, India) according to the manufacturer’s kit procedure. The rats with fasting serum glucose level higher than 300 mg/dL were considered as diabetic and used for further studies. Evaluation of the hypoglycemic effect and serum insulin levels in rats. Diabetic rats were divided into three groups (n ¼ 6).
Rats were fasted for 12–16 h prior to the experiment. Group I received insulin (1.0 IU/kg) as a solution (0.5 mL, pH 7.4) by subcutaneous injection. Nanoparticulate formulations were dispersed in distilled water such that 25 ml contained 1.25 IU of insulin. The insulin NPs (5 IU/kg) without and with nicotinamide were sublingually administered to rats of Group II and Group III using micropipette, respectively. Blood samples were collected from the retro-orbital plexus at 0, 1, 3, 5, 8, 12 and 24 h using a heparinized capillary and centrifuged at 10 000 rpm for 5 min to separate the serum. Serum glucose levels using the GOD-POD Autopac kit and serum insulin was measured using the ELISA Rat Insulin Kit (Mercodia, Sweden) according to the manufacturer’s kit procedure. Basal insulin was corrected by calibration. The area above the curve (AAC) for serum glucose level and area under the curve (AUC) for serum insulin level were calculated using BASICA software. Cmax and tmax were determined from AAC and AUC. The pharmacological availability (PA) and relative bioavailability (BA) were calculated. Student’s t-test and ANOVA were used to determine statistical significance. Differences were considered to be significant for values of p50.05. Insulin NPs containing nicotinamide (5 IU/kg) as a dispersion (&25 mL) was administered sublingually in one group of rats (n ¼ 4) and the other group was administered with water as vehicle control. Rats were observed for mortality upto 24 h. After 24 h the rats were sacrificed, and the sublingual mucosae excised inflated with a formaldehyde solution 4% (v/w) in physiological saline, fixed and stored at 4 C until further histological examination. The formaldehyde-treated mucosae were dehydrated and embedded in paraffin. Slices of 5 mm thickness were sectioned and stained with a regular hematoxylin/eosin staining. Histological evaluation was performed by using an Olympus BX50 light microscope equipped with a Leica DFC 320 camera. Photographs were taken using a 40 magnification objective.
Histopath study of sublingual mucosa.
Results and discussion Bioadhesive NPs by enabling prolonged retention in the sublingual cavity present a promising strategy for enhanced insulin absorption. Alginic acid was selected as the polymer due to its bioadhesive property. Insulin-loaded alginic acid
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NPs were prepared by a mild and aqueous process (Cheng et al., 2012; Liu et al., 2013). This was important as processes involving high shear or the use of organic solvents could be detrimental to insulin stability. Alginic acid NPs of around 220 nm with low PI were readily prepared by nanoprecipitation induced by simple pH change, without the need for high shear homogenization. A marginal increase in particle size observed with enhancer was considered insignificant (Table 1). SEM photographs of insulin-loaded alginic acid NPs revealed spherical NPs (Figure 1). NPs were strongly negatively charged with zeta potential values ranging from 32 ± 2.5 mV, attributed to carboxyl group of alginic acid. Bioadhesion is based on the measurement of shear stress required to break the adhesive bonds between a mucosal membrane and the formulation. Alginic acid NPs revealed strong bioadhesion compared to insulin solution (Table 1). Inclusion of nicotinamide did not influence bioadhesion of alginic acid NPs. The bioadhesive property of alginic acid is reported earlier (Cheng et al., 2012; Liu et al., 2013). Insulin entrapment efficiency of 495% suggested high association of insulin with alginic acid. Alginic acid has pKa of 3.5 and remains in the ionized form (COO) at pH 43.5.
Figure 1. Scanning electron microscopy (SEM) images of insulin-loaded alginic acid nanoparticles.
Table 1. Physico-chemical properties of insulin-loaded alginic acid nanoparticles with/without nicotinamide.
Formulation Insulin solution Alginic acid nanoparticles Alginic acid nanoparticles with nicotinamide
Particle size (nm)
Polydispersity index
Entrapment efficiency (%)
Zeta potential
Bioadhesive force (N)
Ratio of molar ellipticity as 208/222
– 210 ± 2.4 nm 225 ± 3.5 nm
– 0.15 ± 0.3 0.23 ± 0.4
– 96 ± 5.5 % 97 ± 3.3 %
– 32 ± 2.5 30 ± 3.6
0.005 ± 0.025 0.423 ± 0.15 0.436 ± 0.23
1.1 1.2 1.1
Figure 2. FTIR spectra of (a) alginic acid, (b) insulin and (c) alginic acid nanoparticles of insulin.
Insulin-loaded alginic acid NPs
DOI: 10.3109/10717544.2014.916769
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Addition of 0.025 N HCl to a solution of alginic acid and insulin in 0.025 N NaOH (pH 11.3) resulted in a gradual drop in pH, favouring insulin–alginic acid interactions at pH 5, as isoelectric point of insulin (pI: 5.3). Such high entrapment efficiency reflects minimal insulin loss, a major plus point for process scale-up. The FTIR spectrum of alginic acid revealed a weak band at 1226 cm1 from C–O stretching and a strong peak near 1033 cm1 attributed to C–O–C symmetric stretching (Figure 2). The alginic acid carboxyl peaks are seen near 1728 cm1 (symmetric COO stretching vibration) and 1404 cm1 (asymmetric COO stretching vibration). The spectra of insulin presented the pattern of amino acids characterized by asymmetrical N–H bending band near 1643 cm1, a symmetrical bending band near 1519 cm1, and a weak band from symmetrical C¼(O)2 stretching of the carboxylate ion group near 1450 cm1. Insulin contains six
5
amino acid residues which could acquire a positive charge enabling interaction with alginic acid (Bouchard et al., 2002; Bruno et al., 2006). FTIR spectra of insulin-loaded NPs confirmed insulin entrapment in the NPs as two sharp bands of Amide I (1643 cm1) and Amide II (1527 cm1) were seen (Bruno et al., 2006). The shape and the position of the amide I band is consistent with the presence of a largely a-helical structure. This result suggests that a-helical structure of insulin was not affected under the conditions studied. The interaction between insulin and alginic acid was further confirmed by DSC. Pure insulin revealed a melting endotherm at 64 C and a tiny and broad exothermic peak at 278 C indicating crystalline nature. The thermograms of alginic acid showed an initial endothermic peak at 77 C and a higher exothermic peak at 248 C. Endothermic peaks are correlated with the loss of water associated to hydrophilic groups of polymers while exothermic peaks resulted from
Figure 3. Thermograms of (a) insulin, (b) alginic acid, and (c) insulin nanoparticles.
Figure 4. Stability of insulin nanoparticles at the end of 12 months evaluated by (a) CD spectra, (b) drug content (c) particle size.
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degradation of polyelectrolytes due to dehydration and depolymerisation reactions most probably to the partial decarboxylation of the protonated carboxylic groups and oxidation reactions of the polyelectrolytes. The disappearance of insulin and alginic acid peaks in the NPs suggested their association. The endothermic and exothermic peaks associated with insulin, which are attributed to denaturation process and water loss, became indistinct after insulin entrapment into the alginic acid NPs (Figure 3). Conservation of the structural integrity of a protein drug after release is crucial for its biological activity. The CD spectra of insulin recovered from NPs immediately after preparation and at 12 months showed typical double minima at 208 nm and 222 nm indicative of substantial alpha-helical
Figure 5. Effect of single administration of insulin on (a) serum insulin level and (b) serum glucose level in streptozotocin-induced diabetic rats, (——) insulin solution (subcutaneous-1 IU/kg), (—˙—) insulin nanoparticles (sublingual-5 IU/kg), (—m—) insulin nanoparticles with nicotinamide and (——)blank nanoparticles with nicotinamide (n ¼ 6).
structure, and were in close agreement with the spectra of native insulin (Figure 4a). This indicated that there was no change in random coil structure of insulin, suggesting good physico–chemical stability of insulin in the NPs. HPLC analysis confirmed chemical stability of insulin up to 12 months (Figure 4b). No significant change in particle size (p50.05) was observed on storage (Figure 4c). Insulin release from alginic acid NPs followed first-order kinetics. The release profile of insulin from NPs over a period of 12 h indicated very rapid initial burst (65%) in barely 2 h followed by slow release. Inclusion of nicotinamide did not influence in-vitro release of insulin from NPs (F2 ¼ 69.15). Alginic acid NPs with nicotinamide revealed significant decrease in serum glucose level (p50.05) compared to alginic acid NPs without nicotinamide although Cmax with the insulin-loaded NPs was lowered with subcutaneous injection. Insulin-loaded NPs revealed sustained lowering compared to the relatively short hypoglycaemic effect following subcutaneous injection. Blank NPs with nicotinamide showed no reduction in serum glucose levels (Figure 5). Pharmacological availabilities in literature were compared to subcutaneous administration at doses 5–10 IU/kg following nasal administration for typically in the range 8–13%, while similar pharmacological availabilities in the range 3–16% following oral administration are reported at significant larger doses of 12.5–20 IU/kg (Wang et al., 2009; Makhlof et al., 2011; Jose et al., 2012; Zhang et al., 2012). In contrast, our NPs revealed high pharmacological availability of 100.2% and 125.1%. The dose-corrected bioavailability with reference to subcutaneous injection (1 IU/kg) is also significantly higher (20% and 25%), respectively. Our findings appear promising as large doses of insulin could induce adverse mitogenic changes and moreover insulin being a protein could also trigger immunological reactions (Bellary & Barnett, 2006). Subcutaneous injection revealed a high Cmax (97 mIU/mL), however a low tmax (1 h) and a rapid decline in insulin concentration (Figure 5). NPs revealed slower absorption, with tmax between 1 and 2 h and lower Cmax (Table 2). A good correlation (r2 ¼ 0.909) was seen between decrease in serum glucose and increase in serum insulin level. Nevertheless, bioavailability obtained with the alginic acid NPs containing nicotinamide (5 IU/kg) was comparable with subcutaneous injection.
Table 2. Pharmacodynamic and pharmacokinetic parameters obtained by administration of insulin solution or nanoparticles to the diabetic rats.
Formulation Insulin dose (IU/kg) Maximum reduced glucose level (%) Time point of maximum reduced glucose level (h) AAC PA Cmax (mIU/mL) tmax (h) AUC0!24/rat BA (%)
Subcutaneous injection
Sublingual alginic acid nanoparticles
Sublingual alginic acid nanoparticles-nicotinamide
1 68.2 ± 2.3 2 562.3 ± 1.6 – 97.2 ± 2.1 1 244.3 ± 7.1 –
5 38.1 ± 4.2 2 559.3 ± 4.2 100.2 ± 4.9 17.4 ± 2.1 2 201. ± 5.5 80.1 ± 2.5
5 49.2 ± 1.3 2 691.2 ± 6.3 125.1 ± 5.7 25.3 ± 3.4 2 292.2 ± 4.3 96.3 ± 2.1
Each data represents the mean ± SD (n ¼ 4). Cmax, the maximum concentration; tmax, the time to reach the Cmax; AAC, the area above the curve; AUC, the area under the curve; PA, pharmacological availability compared with s.c.; BA, relative bioavailability compared with s.c.
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DOI: 10.3109/10717544.2014.916769
Insulin-loaded alginic acid NPs
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Figure 6. Light micrographs of the sublingual rat mucosal membranes following administration of INS-NPs (5 IU/kg) containing nicotinamide (a) and distilled water as control (b).
Microscopic evaluation of mucosal cross sections has been widely used as an easy and practical method for assessing any damaging effect associated with exposure to enhancer. The histopathology of sublingual mucosa after administration of insulin NPs with nicotinamide showed that membrane integrity appeared to remain undamaged (Figure 6), suggesting nicotinamide as relatively safe permeation enhancer. Nevertheless, changes following chronic administration need to be evaluated.
Conclusion In this article, an attempt has been made to develop a sublingual insulin delivery formulation with bioadhesive alginic acid polymer and nicotinamide as permeation enhancer. Particles with a small size 200 nm and high entrapment efficiency (96%) were obtained. Stability of insulin in NPs was confirmed by HPLC and CD analysis. The high pharmacological availability and bioavailability of insulin from insulin-loaded alginic acid NPs with nicotinamide in the diabetic rat model suggested promising for sublingual delivery.
Acknowledgements The authors would like to acknowledge USV Ltd., India, for gift sample of insulin. We are also thankful to TIFR, India, for helping in CD spectra study.
Declaration of interest The authors report no declarations of interest. Authors thank University Grant Commission for grant and fellowship to Nilam Patil.
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