Lectin-functionalized carboxymethylated kappa-carrageenan microparticles for oral insulin delivery

ANKIT GILANI [NIPERA1113PC03]DEPT. OF PHARMACOLOGY & TOXICOLOGY

NIPER AHMEDABAD

CONTENTS

• INTRODUCTION• ARTICLE INFO.• MATERIALS AND METHODS• RESULTS AND DISCUSSION• CONCLUSION• FUTURE PROSPECTS• REFERENCES

INTRODUCTION

• The current administration of peptide-based drugs, such as insulin, is predominately via the parenteral route, which has a number of disadvantages.

• 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.

• 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.

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

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.

• 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.

Polyelectrolyte biopolymers employed in hydrogel preparation

• Alginates• Gellan Gum• Chitosan• Carboxymethyl Cellulose• Kappa-carrageenan

MATERIALS AND METHODS

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

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

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.

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.

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).

dried in a desiccator overnight at 4◦C.

The insulin-loaded microparticles were then collected by decantation

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.

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.

Determination of insulin encapsulation efficiency and insulin load

Determination of the degree of surface lectin conjugation

• performed in parallel to determine the insulin encapsulation efficiency and insulin load.

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.

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.

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.

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

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

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

• 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

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).

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.

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

Encapsulation efficiency of insulin with increasing amounts of drug (10–35 mg).

(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).

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.

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

• 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.

In vivo studies

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.

• 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.

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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