INTRODUCTION:

Nanosuspensions are colloidal dispersions of nanosized drug particles stabilized by surfactants. It is a biphasic system compromising of pure drug particles dispersed in an aqueous vehicle in which the diameter of the suspended particle is less than 1μm in sizeSolubility of a drug is major challenge for development of formulation. A poorly water soluble drug exhibit inadequate and variable bioavailability and finally leads to gastro intestinal toxicity due to slow drug absorption, when administered orally. For such drugs, solubility is the most important parameter, to achieve their desired concentration in systemic circulation for therapeutic effect. The drugs belonging to BCS ΙΙ have these problems. The rate limiting step for BCS class ΙΙ drugs is the solubility and drug release from dosage form, So increasing the solubility of BCS class ΙΙ drugs in turn increases the bioavailability.

This can be achieved by different techniques like media milling, high speed homogenization and other techniques¹.

To produce nanosuspension of small size suitable for pharmaceutical uses most commonly used techniques high pressure homogenization and wet milling. High pressure homogenization technique has been extensively used with regard to particle size reduction efficiency and also it shows more advantages like simple, time saving over other milling techniques. The drug particles should be sufficiently small to pass through the high speed homogenizer².

Ambroxol hydrochloride is a metabolite of Bromhexine. It used as an expectorant, mucolytic agent to treat acute and chronic diseases. It has short plasma half-life 4 hours require frequent daily dosing (2-3 times)³.

Therefore Ambroxol hydrochloride was chosen as a model drug with the objective of formulation of nanosuspension by high pressure homogenization technique for improving bioavailability, therapeutic effect with lower dose and better patient compliance⁴.

MATERIALS AND METHODS:

Materials:

Ambroxol hydrochloride was obtained as gift sample from Red Cross formulation, Aurangabad, Maharashtra. Poloxamer-188 was obtained from Ansul life science, PVP k90 is collected from Nature of business manufacturer, and Methanol was purchased from Himalaya pharmaceutical, Nellore, Hyderabad, India.

Method:

Solubility study:

The solubility of Ambroxol hydrochloride was carried out to prepare, take 10gm of drug in beaker and fill the methanol in burette then continues addition drop by drop of methanol in breaker with continue shaking. After few ml of addition methanol drug will completely soluble in solvent⁵.

Melting point:

Melting point of the Ambroxol hydrochloride carried by capillary tube method, take small amount of AMB.HCL in capillary. Thiele tube fill with liquid paraffin and capillary mounted and center position. Heat the thiele tube by flame burner and record the melting point of drug⁵

Formulation of Nanosuspension with High speed homogenization:

Nanosuspension is prepared by Nanoprecipitation method. 10gm of Ambroxol Hydrochloride dissolved in 35ml Methanol. This solution is added in specific gravity bottle or vials. Bottle is place in ultrasonic bath for 12hr. After 12hr the concentrated solution is filter through whatman filter paper. 5gm of poloxamer-188 dissolved in 20ml of water in another beaker and filter through whatman filter paper. Beaker kept in ice bath for overnight. Polymer beaker place in high speed homogenizer at 1000rpm. With continue process the collect the sample in 1ml syringe from specific gravity bottle.

Table no 1. Formulation table Ambroxol Hydrochloride Nanosuspension.

Sr.No	Formulation	Drug and Polymer gm	Methanol ml
1	F1	ABH.HCL+Polo188 1:1	35
2	F2	ABH.HCL+Polo188 1:2
3	F3	ABH.HCL+Polo188+PVPK90 1:1:1
4	F4	ABH.HCL+Polo188+PVPK90 1:2:2
5	F5	ABH.HCL+Polo188 1:5
6	F6	ABH.HCL+Polo188+PVPK90 1:5:1
7	F7	ABH.HCL+Polo188+PVPK90 1:5:2
8	F8	ABH.HCL+PVPK90 1:2

With continues drop by drop addition in the side of homogenizer beaker. At the continue homogenization for 5 hr drug will precipitate in the form of crystals and nanosuspension. Prepared Suspension is added in epien drop tube for centrifugation for 10 min Separated solution and partials collect in air tight container for further experimental work.

Characterization of the Nanoparticle:

Solubility:

Table no 2. Solubility:

Drug	Methanol	0.1N HCl	Ethanol	Water
Ambroxol hydrochloride	35ml	40ml	42.5ml	50ml

Determination of the Particle Size:

Size distribution, average particle size and PDI were determined by photon correlation spectroscopy using Zeta-particle size, Model Nano ZS. The separated nanoparticles were subjected to measurement followed by dilution with distilled water. The particle size and PDI measurements were carried out at a scattering angle of 900 and at a temperature 25°C. All experiment done in triplicate⁶.

Encapsulation Efficiency (EE %) Measurements:

The encapsulation efficiency of ambroxol hydrochloride in the polymeric nanoparticle was determine by the extraction and quantification if an encapsulated ambroxol hydrochloride. Polymeric nanoparticles were applied to 25ml of methylene chloride and subjected in 12 her room temperature shaking to ensure complete particle dissolution.

The resulting solution was diluted with appropriate dilution and concentration of the drug in the methylene chloride was determine by spectrophotometric by measuring UV absorbance at 244nm.

EE of the drug= (amount of encapsulated drug)/(total amount of the drug) X 100 ……………..Equation No 1

The entrapped Ambroxol hydrochloride concentration was expressed as percentage entrapment efficiency which can be defined as the percent fraction of the total input drug encapsulated in the polymeric nanoparticles⁷.

Scanning Electron Microscopy:

The scanning electron microscopy (JEOL Model JSM - 6390LV) was used to characterize the surface morphology of nanoparticles. The nanoparticles were mounted directly on the SEM stub, using double sided, sticking tape and coated with platinum and scanned in a high vacuum chamber with a focused electron beam. Secondary electrons, emitted from the samples were detected and the image formed⁸.

Differential Scanning Calorimetry:

Differential scanning calorimetric measurements were carried out by using differential Scanning Calorimeter (DSC DA 60 Shimadzu, Japan) equipped with a liquid nitrogen subambient accessory. The DSC was performed for the pure Ambroxol, the PLA and the drug loaded nanoparticle formulation. Sample 2mg were loaded in a flat-bottomed aluminium pan and subjected to a heating cycle from 40 to 400^oC with a heating rate of 100 C/min. A stream of nitrogen gas was used to control the heating and cooling rate. The temperature and energy scales of the instrument were calibrated using purified indium as the reference material⁹.

Redispersibility of Nanoparticles:

The selected formulation was freeze-dried to obtain a dry powder for further investigation. In addition to that it was taken to study the effect of cryoprotectant on freeze-drying and dispersibility of the prepared nanosuspension. Mannitol at a concentration of 5 times the total solid contents in the formulation used as a cryoprotectant. Two samples of nanosuspension each were placed in a flask, the amount of mannitol required added to one and shaken to dissolve, the second sample left with out of cryoprotectants. These flasks were frozen in a deep freezer at –20°C for 12 h for primary freezing. Then the container was attached to the vacuum adapter of the lyophilizer. The solvent sublimed under a pressure of 80 mmHg for 48- 72 h¹⁰.

In Vitro Release Studies:

The release study of nanoparticle formulation for the release of Ambroxol hydrochloride from polymeric nanoparticles was carried out using a membrane diffusion technique. In vitro diffusion cell was made using dialysis membrane as a semi permeable membrane. To retain the nanoparticles as well as to enable the free drug to diffuse freely into the release media a dialysis membrane of 12,000 - 14,000 Molecular weight cut-off was used. Formulated nanoparticles equivalent to 1 mg of the drug were dispersed in 1mL of isotonic phosphate-buffered saline at pH 7.4. The nanoparticle dispersions were packed in dialysis membrane secured with two clamps at each end. To maintain sink condition, the dialysis bag was immersed in tightly-capped glass vials (7 × 2.8cm) containing 10 mL of 0.5% (w/v) of sodium lauryl sulphate solution in distilled water. The release test was performed by placing the glass vials in a thermostatically-controlled shaking water bath adjusted to 37±0.5°C with a constant shaking rate of 100rpm. At predetermined time points, the whole release medium was withdrawn and replaced with fresh release medium¹¹.

Sterility Testing:

All parenteral preparations should be sterile. Sterility studies were carried out to ensure the sterility of finished product. Since it is administered by parenteral route, direct inoculation method was preferred to carry out sterility testing. In this method, the specified quantity of sample under test was drawn aseptically from the containers and transferred to fluid thioglycollate medium (20mL) and Soybean-Casein digest medium (20mL), separately. Mixture of nanoparticles with the medium was incubated for not less than 14 days at 30°C–35°C in case of fluid thioglycolate medium and 20°C–25°C in case of Soybean-Casein digest medium. The growth of any microorganisms in the medium was observed¹².

RESULTS AND DISCUSSION:

Determination of the Particle Size and Encapsulation Efficiency (EE %):

When considering irritation and comfort, the particle size is an important factor in the development of an nanosuspension. The mean particle size of optimized nanoparticle formulae. The particle size varies from 225.8nm and 0.502nm

Figure no: 1 the Particle Size and Encapsulation Efficiency (EE %)

Nanoparticle encapsulation efficiency % ranged between 80.2 to 96.8%. All tested variables have a significant effect on EE%. It was observed that the increase in polymer content resulted in decrease in EE% of the nanoparticles formulae This result may be attributed to the increasing viscosity of the organic phase upon increasing polymer content.

Scanning Electron Microscopy:

The morphology of drug loaded nanoparticles was accessed using SEM and is shown in Fig. 2 This figure indicates that the nanoparticles were cylindrical in shape and their size was in the nanometer range with smooth surface essential for nanosuspension.

Differential Scanning Calorimetry:

The DSC thermogram observed a sharp Endothermic peak of optimized nanoparticle Ambroxol hydrochloride onset temp at 136.92°C with maximum peak 138.92°C and end set temp at 141.22°C depicted in figure no.7.6.Thus no significant change in the position of endothermic peaks was observed after running the drug, absence of any chemical interaction and chemical in compatibility between pure drug Ambroxol hydrochloride depicted

Redispersibility of Nanoparticle:

It was found that the dispersibility was improved when using mannitol as cryoprotectants and products were spontaneously dispersed into primary nanosuspension within 1-5 min in both media (0.1 N HCl and phosphate buffer pH 6.8). It suggested that mannitol in the products would improve the wetting of the hydrophobic drug and accelerate the penetration of water into the products. On the other hand, the products without cryoprotectants could not be dispersed well and transformed into the original nanosuspension within 13 min as expected from their agglomerated structure

In Vitro Release Studies:

The drug formulae prepared with nanoprecipitation technique were subjected to in vitro release study. The amount of Ambroxol Hydrochloride released from nanoparticle was evaluated using a dialysis technique. The release profiles of Ambroxol hydrochloride are shown in Fig. 4 Drug release from Polo-188 based nanoparticle reveals sustained release up to 1 hrs due to wetting followed by immediate release as results of rapid diffusion of drug release from swollen polymeric nanoparticle.

Figure No: - 4 In Vitro Release Studies of AMB.HCL

Sterility Testing:

During sterility testing, we found that there was no evidence of microbial growth when formulations were incubated for not less than 15 days at 30°C to 35°C in case of fluid thioglycolate medium and at 20°C to 25°C in case of Soybean-Casein digest medium demonstrating that formulation passes the test for sterility.

CONCLUSION:

Ambroxol hydrochloride was successfully suitable within biodegradable nanoparticle using nanoprecipitation technique. The formulation study using F5 batch is used for the optimized formulation to be obtained. The drug-polymer ratio and speed had a significant effect on the particle size and encapsulation efficiency of the nanoparticle. The formulated Ambroxol nanoparticles were found to be a suitable and potential natural carrier in terms of their particle size, redispersibility in vitro release characteristics, sterility. The stability study of Ambroxol from nanoparticles has shown suitable results.

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Received on 01.06.2022 Modified on 13.09.2022

Asian J. Pharm. Tech. 2023; 13(1):4-8.

DOI: 10.52711/2231-5713.2023.00002