Lectin-functionalized carboxymethylated kappa-carrageenan microparticles for oral insulin delivery
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Transcript of Lectin-functionalized carboxymethylated kappa-carrageenan microparticles for oral insulin delivery
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Lectin-functionalized carboxymethylated kappa-carrageenan microparticles for oral insulin delivery
ANKIT GILANI [NIPERA1113PC03]DEPT. OF PHARMACOLOGY & TOXICOLOGY
NIPER AHMEDABAD
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CONTENTS
• INTRODUCTION• ARTICLE INFO.• MATERIALS AND METHODS• RESULTS AND DISCUSSION• CONCLUSION• FUTURE PROSPECTS• REFERENCES
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INTRODUCTION
• The current administration of peptide-based drugs, such as insulin, is predominately via the parenteral route, which has a number of disadvantages.
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• The recent introduction of an inhaled delivery system for insulin was short-lived and resulted in the withdrawal of the product from the market by pharmaceutical companies.
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• Therefore, the oral delivery of insulin still remains an attractive alternative delivery route.
• But there are two main hurdles : 1) enzymatic degradation and 2) their inability to transverse the biological
barriers of the gastrointestinal tract.
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Techniques to manufacture microcapsules
(1) Physical methods * Pan coating * Air-suspension coating * Centrifugal extrusion * Vibrational Nozzle * Spray–drying
(2) Physico-chemical methods * Ionotropic gelation * Coacervation
(3) Chemical methods * Interfacial polycondensation * Interfacial cross-linking * In-situ polymerization * Matrix polymerization
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Ionotropic gelation technique
• Ionotropic gelation is based on the ability of polyelectrolytes to cross link in the presence of counter ions to form hydrogels.
• The natural polyelectrolytes inspite, having a property of coating on the drug core and acts as release rate retardants contains certain anions on their chemical structure.
• These anions forms meshwork structure by combining with the polyvalent cations and induce gelation by binding mainly to the anion blocks.
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• The hydrogel beads are produced by dropping a drug-loaded polymeric solution into the aqueous solution of polyvalent cations.
• The cations diffuse into the drug-loaded polymeric drops, forming a three dimensional lattice of ionically crossed linked moiety.
• Biomolecules can also be loaded into these hydrogel beads under mild conditions to retain their three dimensional structure.
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Polyelectrolyte biopolymers employed in hydrogel preparation
• Alginates• Gellan Gum• Chitosan• Carboxymethyl Cellulose• Kappa-carrageenan
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MATERIALS AND METHODS
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MATERIALS
• kappa-Carrageenan• Human recombinant insulin• lectin from Triticum vulgaris (WGA)• 200 mM L-glutamine, fetal bovine serum (FBS), 0.25%
trypsin-EDTA• phosphate-buffered saline (pH 7.4) tablets• Potassium chloride, sodium hydroxide, potassium
dihydrogen orthophosphate.• 37% fuming hydrochloric acid and acetonitrile • Glutaraldehyde 25% (v/v), orthophosphoric acid, sodium
acetate salt
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MATERIALS (cont..)
• Chromolith Performance RP-18e HPLC columns (4.6 m × 100 mm)
• Syringes (1 mL) and needles with diameters of 25G, 26G and 27G
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Synthesis and characterization of carboxymethylated kappa-carrageenan
The reaction mixture was heated to 50 ◦C with continuous stirring for 4 h to drive the reaction process to completion.
Monochloroacetic acid (5.3 g) was then added portionwise to the reaction mixture over a period of 20 min.
Next, 5 mL of 16 N sodium hydroxide was added at a rate of 1 mL per 15 min with continuous stirring at room temperature.
5 g of powdered kappa-carrageenan was suspended in 100 mL of 2-propanol and stirred for 30 min at room temperature.
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Sulfate content was determined using ion chromatography.
Molecular weight was measured using size-exclusion liquidchromatography
The degree of carboxymethylation on the modified kappa-carrageenan was determined using NMR
The product was recovered through vacuum filtration and washed alternately with 50 mL of ethanol–water (4:1) and 50 mL of
ethanol three times. The modified carrageenan was oven dried at 70 ◦C overnight and powdered in a glass mortar.
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Preparation of insulin-loaded microparticles
The resulting dispersion was then loaded into a 1 mL syringe and extruded dropwise through needles of varying sizes (internal diameter, 0.4–0.5 mm) into 20 mL of 1.5 M potassium chloride–HCl solution (pH 1.2) with constant stirring (50 rpm) under a constant stream of air blown perpendicular to the tip of the needle.
1 mL of human insulin (10–30 mg/mL) was addedand mixed thoroughly to form a viscous dispersion.
125–175 mg powdered carboxymethylated kappacarrageenan was dissolved in 1 mL of pH-adjusted (pH 4.0–7.0) deionized water with 0.3 M sodium acetate buffer (pH 4.0).
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dried in a desiccator overnight at 4◦C.
The insulin-loaded microparticles were then collected by decantation
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Preparation of insulin-loaded lectin surface-conjugated microparticles
microparticles were first surface activated with polyglutaraldehyde, followed by conjugation to lectin
soaked in 10 mL of polyglutaraldehyde
solution(0.1–5.0%, v/v) for
1 h.
rinsed four successive times with 5 mL of 1.5 M potassium chloride–HCl solution (pH 1.2) to remove excess crosslinking reagent, followed by washing with deionized water.
immersed in a lectin solution
(0.25–1.25 mg/mL PBS, pH 7.4) for
30 min.
Washed and then dried in a desiccator
overnight at 4 ◦C.
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Determination of insulin encapsulation efficiency and insulin load
• an indirect method to measure the insulin content in the 1.5 M potassium chloride–HCl solution (hardening solution) using an established HPLC protocol was performed
• the AUC values were used to construct the standard curve for the estimation of insulin content in the hardening solution.
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Determination of insulin encapsulation efficiency and insulin load
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Determination of the degree of surface lectin conjugation
• performed in parallel to determine the insulin encapsulation efficiency and insulin load.
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Microparticle size and surface characteristics determination
• The diameters of freshly prepared (wet) and dried microparticles were estimated using a microscope (CX31, Olympus Optical Co., Ltd., Tokyo, Japan).
• The size and surface characteristics were assessed using a field emission scanning electron microscope (Quanta 200 FESEM, FEI, Oregon, USA) in a low-vacuum mode with 50×, 2000×, 8000× and 50000× magnifications.
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In vitro insulin release kinetics
simulated gastric fluid (SGF) (pH 1.2) with stirring (100
rpm) at 37 ◦C for 2 h.
After 2 h, the SGF was carefully removed, replaced and incubated with 20 mL of SIF (pH 7.4) with stirring at
37 ◦C for 8 h.
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In vitro insulin release kinetics
• Insulin profiles from the encapsulated microparticles were fitted to the power law equation to calculate n and determine the insulin release kinetics:
Where,Mt is the amount of insulin released up to a specified time, t;M∞ is the final amount of insulin released; k is the structural/geometric constant for a particular system; t is the sampling time and n represents the release exponent of the release mechanism.
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In vivo studyInduction of diabetes
male Sprague–Dawley rats.
i.p. administration of45 mg/kg of
streptozocin in 0.1 M sodium citrate buffer
(pH 4.0).
After two weeks, rats with fasting blood
glucose levels above300 mg/dL
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In vivo study
The microparticles were pre-packed in hard gelatin capsules (size 9, Qualicaps® capsule) and administered orally at 25, 50 and 100 IU insulin/kg usinga bulb-tipped gavage needle
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In vivo studyGROUP DOSE ROUTE
TEST (1) LECTIN FUNCTIONALIZED MICROPARTICLES
25, 50 and 100 IU insulin/kg
ORAL
(2) NON-LECTIN FUNCTIONALIZED MICROPARTICLES
50 and 100 IU insulin/kg ORAL
POSITIVE CONTROL INSULIN SOLUTION 2 IU insulin/kg SC
INSULIN SOLUTION 100 IU/kg ORAL
NEGATIVE CONTROL UNTREATED
EMPTY CAPSULES ORAL
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• Blood was collected from the tail vein immediately before treatment and 0.5, 1, 1.5, 2, 4,6, 8, 12, 24 and 36 h after administration. Blood glucose levels were measured using an Accu-Check Active blood glucose meter. The post-treatment blood glucose levels were expressed as the percent of pre-treatment blood glucose.
• quantitative serum insulin determination
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RESULTS AND DISCUSSION• Preparation of insulin-loaded microparticles
Encapsulation efficiency of insulin using various polymer weights (125–175 mg) and needle sizes (0.4–0.5 mm).
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The effect of pH on the encapsulation efficiency of insulin by the addition of 0.3 M sodium acetate buffer into the insulin–polymer mixture.
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Above pH 5.4, insulin is negatively charged. Encapsulation improvement occurs (97.7±2.2%) when insulin has a net positive charge of less than 1
At a pH lower than 4.8, insulin has a net positive charge of more than 1
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Encapsulation efficiency of insulin with increasing amounts of drug (10–35 mg).
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(E) Percentage of insulin release in simulated intestinal fluid (SIF) (pH 7.4) after crosslinking with polyglutaraldehyde (0.1–1.0% v/v). (F) Percentage of microparticles by weight adhered to short segments (6 cm) of rat intestine after lectin functionalization with increasing amount of lectin (0.25–1.25 mg).
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In vitro studies
Dissolution profile of non-functionalized and lectin-functionalized carboxymethylated kappa-carrageenan microparticles in simulated gastric fluid (SGF) at 37 ◦C for 2 h, followed by simulated intestinal fluid (SIF) at 37 ◦C for 8 h.
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In vitro insulin release kinetics
• parameter n was calculated by fitting the release data into the power law, the average n value
1) for the non-functionalized microparticles is 0.45±0.06
2) for lectin-functionalized microparticles is 0.36±0.10
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• For a spherical system, when (1) n ≤ 0.43, the release mechanism is diffusion-controlled (CaseI), (2) n ≥ 0.85, the release mechanism is swelling controlled (Case II)
and (3) values between 0.43 and 0.89 present a mixed mode of a both
diffusion- and swelling-controlled mechanisms (anomalous transport)
• Thus, the release mechanism of insulin from nonfunctionalized microparticles is a mixed mode but is predominantly diffusion-controlled, whereas for lectin-functionalized microparticles, it is diffusion-controlled.
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In vivo studies
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CONCLUSION
• This study clearly shows that insulin entrapped in lectin functionalized carboxymethylated kappa-carrageenan microparticles was protected from hydrolysis and proteolysis by stomach acids and enzymes.
• Grafting of lectin (WGA) on the surface of the microparticles improves the interactions of these microparticles with the intestinal wall and enhances the absorption of insulin compared to the non-functionalized microparticles.
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• The covalently bound, negatively charged sulfate groups in carboxymethylated kappa-carrageenan interact with the amino groups of the amino acid residues in insulin via ionic interactions that prevented the bulk release of insulin in the intestine, and these interactions imparted a sustained release of up to 12–24 h for the insulin entrapped in the microparticles in contrast to the rapid dissipation of the hypoglycemic effect of insulin via the parenteral route.
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FUTURE PROSPECTS
• lectin-functionalized oral formulation might serve as a promising alternative or as a complementary therapy to parenteral administration to provide better basal and prolonged hypoglycemic control.
• Currently, oral insulin formulations such as Insugen (available commercially), and 4-CNAB and Capsulin (in clinical trials) show rapid insulin action that last no longer than 6–8 h.
• While the insulin-loaded microparticles in this study showed prolonged glycemic control that lasted 12–24 h, and hence complement existing insulin formulations to provide for the basal insulin needs of the patient.
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REFERENCES 1. Andrews, G. P., Laverty, T. P., & Jones, D. S. (2009). Mucoadhesive polymeric platforms for
controlled drug delivery. European Journal of Pharmaceutics and Biopharmaceutics, 71, 505–518.
2. Bies, C., Lehr, C. M., & Woodley, J. F. (2004). Lectin-mediated drug targeting: History and applications. Advanced Drug Delivery Reviews, 56, 425–435.
3. Chowdary, K. P. R., & Rao, Y. S. (2004). Mucoadhesive microspheres for controlled drug delivery. Biological & Pharmaceutical Bulletin, 27, 1717–1724. Clark, M. A., Hirst, B. H.,
& Jepson, M. A. (2000). Lectin-mediated mucosal delivery of drugs and microparticles. Advanced Drug Delivery Reviews, 43, 207–223.
4. Clement, S., Dandona, P., Still, J. G., & Kosutic, G. (2004). Oral modified insulin (HIM2) in patients with type 1 diabetes mellitus: Results from a phase I/II clinical trial. Metabolism, 53, 54–58.
5. Deeb, S. E., Preu, L., & Wätzig, H. (2007). Evaluation of monolithic HPLC columns for various pharmaceutical separations: Method transfer from conventional phases and batch to batch repeatability. Journal of Pharmaceutical & Biomedical Analysis, 44, 85–95.
6. Gabor, F., Bogner, E., Weissenboeck, A., & Wirth, M. (2004). The lectin-cell interaction and its implications to intestinal lectin-mediated drug delivery. Advanced Drug Delivery Reviews, 56, 459–480.
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