"Formulation and Evaluation of Riboflavin

Microsphere by Ionotropic Gelation Method"

A report submitted to

Savitribai Phule Pune University

IN PARTIAL FULFILLMENT
OF THE REQUIRMENT FOR THE
DEGREE OF

BACHELOR OF PHARMACY
UNDER THE FACULTY OF
SCIENCE AND TECHNOLOGY

By
Miss. Jagtap Sayali Bharat
Final Year B. Pharm
Under the guidance of
Prof. S . J . Sanghavi.
(M. Pharm, Pharmaceutics),
Assistant Professor,
Department of Pharmaceutics.

Shivnagar Vidya Prasarak Mandal's

College of Pharmacy, Malegaon (Bk), Baramati.
2023-24
 Endorsement by the Principal of the Institution.

This is to certify that, the practice school report entitled

“Formulation and Evaluation of Riboflavin Microsphere by Ionotropic

Gelation Method" has been carried out by Miss. Jagtap Sayali Bharat student of

Final Year B. Pharm under the guidance of Prof. Sanghavi. S. J in partial

fulfillment to their requirement for the award of the Degree of Bachelor of

Pharmacy under the faculty of Science and Technology of Savitribai Phule Pune

University, Pune. The work has been carried out at Shivnagar Vidya Prasarak

Mandal’s College of Pharmacy, Malegaon BK during the academic year 2023-24.

Place: Malegaon (BK) Dr. Jadhav. R. B

(M. Pharm, Ph. D)
Date: Principal,
SVPMCOP
Malegaon (BK).
 Certificate of the Guide.
This is to certify that, the practice school report

entitled "Formulation and Evaluation of Riboflavin Microsphere by Ionotropic

Gelation Method” is submitted by MISS. Jagtap Sayali Bharat in partial fulfillment of

their requirement for the degree of pharmacy under the faculty of Science and

Technology of Savitribai Phule Pune University, Pune, was carried out in Vidya Prasarak

Mandal’s College of Pharmacy, Malegaon (BK). This work present editing this project

was carried out under my supervision and guidance in academic year 2023-24.

Place: Malegaon
Prof. Sanghavi. S. J
(BK). Date: (M. Pharm, Pharmaceutics)
Department of Pharmaceutics,
SVPMCOP
Malegaon (BK).
 Declaration by the Candidate.

I under signed here by declare that, the practice school report entitled
“Formulation and Evaluation of Riboflavin Microsphere By Ionotropic
Gelation Method" Completed and written by me and it is based on my
own work carried out during the course of my study under the guidance of
Prof. Sanghavi. S. J. I further declare that; I will not utilize any part or full
part of work for any reason in any University of India or abroad without
consent of my guide.

Place: Malegaon Miss. Jagtap Sayali Bharat

Final Year B. Pharmacy SVPM COP,
(BK) Date: Malegaon (BK)
 Acknowledgement
It gives me an immense pleasure to acknowledge everyone who have
supported and helped me during the course of my practice school report. I express my
deepest sense of gratitude from the bottom of my heart to my respected, beloved guide
Prof. Sanghavi. S. J, Assistant Professor, Department of Pharmaceutics, SVPM’S
College of Pharmacy, Malegaon (BK), for his excellent guidance, critical supervision
and keen interest and continuous encouragement throughout the study. His active
guidance helped me to develop skills and in sight in research and scientific
presentation.

I would like to express my profound gratitude and sincere and respectful thanks
to Prof. (Dr) R. B. Jadhav, Principal, SVPM’S College of Pharmacy, Malegaon, for
giving a great guidance in research as well as moral support like further during critical
conditions. I especially thankful to Miss.Jagtap Nikita & Miss. Gaikwad Shruti for
their assistance, support, timely help and continuous encouragement during my typing
work.

It is indeed a difficult task to acknowledge the service of all those gentlepeople

who have extended their valuable suggestions and support directly or indirectly whose
name have been unable to mention. Besides this, I am thankful to all those who have
knowingly and unknowingly helped me in the successful completion of this report.

Miss. Jagtap Sayali Bharat

 INDEX

Chapter No. Chapter Name Page No.

List of tables and Abbreviations I-II

Abstract III

I. Introduction 1-10

11-12
II. Literature Survey

III. Need of work 13

IV. Aim and Objective 14

V. Methodology 15-16

VI. Results and Discussion 17-24

VII. Conclusion and Outcomes 25

VIII. References 26-28

 LIST OF TABLES
Sr No Table Title Page No
No

1 1 Drug Profile 9

2 2 Preparation of microsphere formulation 16

3 3.1 Relationship between angle of repose and flowability properties 17

4 3.2 Relationship between angle of repose and flowability properties 18

5 3.3 Correlation between % compressibility and flowability properties 19

6 3.4 Micromeritic properties of prepared Riboflavin 19

microsphere

7 3.5 Percentage yield of prepared Riboflavin microsphere 20

8 3.6 Particle size of prepared Riboflavin microsphere 21

9 3.7 Swelling index (micrometer) of prepared Riboflavin 24

microsphere

10 3.8 Dissolution profile of prepared mi Riboflavin microsphere 24

I
 ABBREVIATIONS

Sr.no Abbreviations Meaning

1 SR Swelling ratio

2 DCM Dichloromethane

3 O/W Oil in water

4 Mg Milligram

5 AT1 Angiotensin 1

6 AT2 Angiotensin 2

7 % w/v Percentage weight by volume

8 I Carr's index

9 Θ Angle of repose

10 EVA Ethylene vinyl acetate

II
 ABSTRACT

Abstract-

The present work is perfomed for "Formulation and Evaluation of Riboflavin microspheres by Ionotropic Gelation

Method" using calcium chloride, sodium alginate, ethyl cellulose. A 3 2 full factorial design is fixed to elucidate the

effect of variables via the amount of drug and amount of polymer. The evaluation parameter performed for Riboflavin

microsphere are Bulk density, Tap density, % Carr's index, Hausner's ratio, Angle of repose, in vitro relase, particle

size, etc. The microspheres were found to be discrete, spherical with free flowing properties. Hence, it is concluded that

the microspheres are successfully prepared by ionotropic gelation method & has potential to deliver Riboflavin In a

controlled manner in a regular fashion over extended period of time can be used for a successful oral delivery of

Riboflavin for safe management Vitamin deficiencies.

Keywords:

Microsphere,Riboflavin, ethy celluose, evaluation parameters, etc

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CHAPTER I: INTRODUCTION
A) Microsphere

a) Introduction:

Microspheres are characteristically free flowing powders consisting of proteins or synthetic polymers

which are biodegradable in nature. and has diameter having between 1–1000μm.there are various types of

microspheres explain in detail. These microspheres prepared and fill them in a hard gelatin capsule or

compress directly.

b) Advantages

 Potential use of microspheres in the pharmaceutical industry

 Taste and odor masking

 Conversion of oils and other liquids to solids for ease of handling

 Protection of drugs against the environment (moisture, light etc.)

 Separation of incompatible materials (other drugs or excipients)

 Improvement of flow of powders

 Aid in dispersion of water-insoluble substances in aqueous media.

 Production of SR, CR, and targeted medications.

c) Disadvantages

 The costs of the materials and processing of the controlled release preparation are substantially higher

than those of standard formulations.

 The fate of polymer matrix and its effect on the environment.

 The fate of polymer additives such as plasticizers, stabilizers, antioxidants and fillers.

 Reproducibility is less.

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Process conditions like change in temperature, pH solvent addition, and evaporation.

d) Application

1. Microspheres in Vaccine Delivery:

The requirement of a vaccine is protection against the microorganism or its toxic product. An ideal

vaccine must fulfill the requirement of efficacy, safety, use in application and cost. The aspect of safety

and minimization of adverse reaction is a complex issue . The characteristics of safety and the degree of

the production of antibody responses are closely related to mode of application. Biodegradable delivery

systems for vaccines that are given by parenteral route may control the shortcoming of the conventional

vaccines. The interest in parenteral (subcutaneous, intramuscular, intradermal) carrier lies since they offer

specific advantages including:

1. Improved antigenicity by additional action.

2. Modulation of antigen release

3. Stabilization of antigen.

2. Targeting using Microparticulate Carriers:

The concept of targeting, i.e., site specific drug delivery is a well-accepted rule, which is get full

attention. The therapeutic efficacy of the drug depends on its entry and specific interaction with its

candidate receptors. The ability to leave the pool in reproducible, efficient and specific manner is center to

drug action mediated by use of a carrier system. Placement of the particles indiscrete anatomical

compartment leads to their retention either because of the physical properties of the environment or

biophysical interaction of the particles with the cellular content of the target tissue.

3. Monoclonal Antibodies Mediated Microspheres Targeting:

Monoclonal antibodies targeting microspheres are immune microspheres. This targeting is a

method used to achieve selective targeting to the specific sites. Monoclonal antibodies are extremely

specific molecules. This extreme specificity of monoclonal antibodies (Mabs) can be used to target

microspheres loaded bioactive molecules to selected sites. Mabs can be directly attached to the

microspheres by means
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of covalent coupling. The free aldehyde groups, amino groups or hydroxyl groups on the surface of the

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microspheres can be linked to the antibodies. The Mabs can be attached to microspheres by any of the

following methods

1. Nonspecific adsorption

2. Specific adsorption

3. Direct coupling

4. Coupling via reagents

4. Chemoembolization:

Chemoembolization is an endovascular therapy, which involves the selective arterial embolization

of a tumor together with simultaneous or subsequent local delivery the chemotherapeutic agent. The

theoretical advantage is that such embolization will not only provide vascular occlusion but will conduct

about sustained therapeutic levels of chemotherapeutics in the areas of the tumor. Chemoembolization is

an addition of traditional percutaneous embolization techniques.

5. Imaging:

The microspheres have been broadly studied and used for the targeting purposes. Various cells,

cell lines, tissues and organs can be imaged using radio labelled microspheres. The particle size range of

microspheres is an important factor in determining the imaging of particular sites. The particles injected

intravenously apart from the portal vein will become entrapped in the capillary bed of the lungs. This

phenomenon is utilized for the scintiographic imaging of the tumor masses in lungs using labelled human

serum albumin microspheres.

6. Topical Porous Microspheres:

Micro sponges are porous microspheres having myriad of interconnected voids of particle size

range 5300 μm. These micro sponges having capacity to entrap wide range of active ingredients such as

emollients, fragrances, essential oils etc., are used as the topical carries system further, these porous

microspheres with active ingredients can be incorporated into formulations such as creams, lotions and

powders. micro sponges consist of non-collapsible structures with porous surface through which active

ingredients are released in a controlled manner.

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7. Surface Modified Microspheres:

Different approaches have been utilized to change the surface properties of carriers to protect them

against phagocytic clearance and to alter their body distribution patterns. The adsorption of the poloxamer

on the surface of the polystyrene, polyester or poly methyl methacrylate microspheres provide them more

hydrophilic and hence decrease their MPS uptake. Protein microspheres covalently modified by PEG

derivatives show decreased immunogenicity and clearance. The most studied surface modifiers are:

1. Antibodies and their fragments

2. Proteins

3. Mono-, oligo- and polysaccharides

4. Chelating compounds (EDTA, DTPA or Deferoxamine)

5. Synthetic soluble polymers Such modifications are provided surface of microspheres in order to

achieve the targeting to the separate organs and to avoid rapid clearance from the body.

8. Medicinal application:

 Release of proteins, hormones, and peptides over extended period of time.

 Treatment of leishmaniasis.

 Tumor targeting. [31]

e) Types of microspheres

 Bio adhesive microspheres

The bio adhesion describes that the materials having the property to adhere at biosubstrate; such as

mucosal membranes. Adhesions of bioadhesive drug delivery devices to mucosal tissue provide prolong

contact of device with the site of administration. This prolonged contact time increase the absorption of

drug from its device and controlled release of drug from device also maintained it improved patient

compliance by reducing the frequency of administration. Carrier system of drug delivery gives an

approach pair the drug to a carrier system such as microspheres. Microspheres play an important role in

these types
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of drug delivery systems due to their small size and efficient carrier capacity. Nasal - Gentamycin.

(SubbiahGaneshetal.2010)

 Magnetic microspheres

This type of delivery system is very much important which localize the drug to the disease site. In this

larger amount of freely circulating drug can be replaced by smaller amount of magnetically targeted

drug. The therapeutic magnetic microspheres are used to deliver chemotherapeutic agent. These

microspheres also have the capacity to deliver drugs like proteins and peptides to the targeted site. This

system is based on the two facts

1. Drug should be encapsulated into a magnetic microsphere

2. Drug should conjugate on the surface of the microspheres.

The accumulation of the carrier at the target site allows them to deliver the drug locally.

(FarahHamadFarahetal2016).

 Floating microsphere

Floating type microspheres are the preparation with less bulk density than the gastric fluid and so

remains floating in stomach without disturbing gastric emptying time. From the floating microspheres

drug is released slowly at the preferred rate, if the system is buoyant on gastric content, it increases

gastric residence time and difference in plasma concentration. It also decreases the chance of striking

and dose dumping and produces extended therapeutic effect. g. NSAIDS, Antibiotics.

(KumarSarvanK.etal.2017)

 Radioactive microspheres

They are series from 10-30 nm in dimension. The bigger than capillary Gets tap in primary capillary

bed when they come across. They inject into the arteries that lead to the tumor of importance. These

radioactive microspheres provide the delivery of high radiation dose at the site of action without

harming the normal tissues. This is another type of drug delivery system, the radioactivity property of

the radioactive substance is avoided to release from microspheres, but acts from with a radioisotope

typical distance and the different type of radioactive microspheres are α emitters, β emitters, γ emitters.

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(GhahramaniMRetal.2014).

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 Mucoadhesive microspheres

These are of size from 1-1000mm in diameter and constituting either totally of Mucoadhesive

polymer or have an external layer of it and combination of Mucoadhesive properties to microspheres

has added advantages, e.g., efficient absorption and improved bioavailability of drug due to a high

surface to volume ration, a much more intimate contact with mucus layer, specific targeting of drug to

the absorption on the surface of the microspheres. These are able to be customized to attach to any

mucosal tissue therefore offering the potential of localized as well as a systemic controlled release of

drugs. (Rao Shrinivas Detal.2014).

f) Method used in microsphere formulation

 Emulsion solvent evaporation technique

 Emulsion cross linking method

 Coacervation method

 Spray drying technique

 Emulsion-solvent diffusion technique

 Multiple emulsion method

 Ionic gelation

 Hydroxyl appetite (HAP) microspheres

 Emulsion solvent evaporation technique

In this method the drug is dissolved in polymer which was previously dissolved in chloroform and the

resulting solution is added to aqueous phase containing 0.2 % sodium of PVP as emulsifying agent.
The
above mixture was agitated at 500 rpm then the drug and polymer (eudragit) were transformed into
fine
droplet which solidified into rigid microspheres by solvent evaporation and then collected by filtration

and washed with demineralized water and desiccated at room temperature for 24 hrs. Aceclofenac

microspheres were prepared by this technique.

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 Emulsion cross linking method

In this method drug was dissolved in aqueous gelation solution which was previously heated for1hr.at

40oC. The solution was added drop wise to liquid paraffin while stirring the mixture at 1500 rpm for

10 min at 35oC, results in w/o emulsion then further stirring is done for 10 min at 15oC. Thus, the

produced microspheres were washed respectively three times with acetone and isopropyl alcohol
which
then air dried and dispersed in 5mL of aqueous glutaraldehyde saturated toluene solution at room

temperature for 3 hrs. for cross linking and then was treated with 100mL of 10mm glycine solution

containing 0.1%w/v of tween 80 at 37oC for 10 min to block un reacted glutaraldehyde.

 Coacervation method

In this method straight forward separation of macromolecular fluid into two immiscible types of material,

a thick coacervate layer, comparatively condensed in macromolecules, and a distilled layer of equilibria.

This method is referred to as basic coacervation, in the presence of just one macromolecule. If two or
more
opposite-charge macromolecules are involved, they are considered complex coacervation. The former is

caused by specific factors including temperature shift, using non-solvent or micro-ions contributing to

dehydration in mac since they facilitate interactions between polymer and polymer through polymer solvent

interactions. This can be engineered to generate different properties on microspheres.

 Spray drying technique:

This method was used to prepare polymer microsphere mixed charged with drug. This requires
dispersing
the raw substance into liquefied coating liquid, and then spraying the mixture into the air for surface

solidification accompanied by rapid solvent evaporation. Organic solvent and polymer solution are

formulated and sprayed in various weight ratios and drug in specific laboratory conditions producing

microspheres filled with medications. This is fast but may lose crystallinity due to rapid drying.

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 Emulsion-solvent diffusion technique

In this method drug polymer mixture was dissolved in a mixture of ethanol and dichloromethane (1:1) and

then the mixture was added drop wise to sodium lauryl sulphate (SLS) solution. The solution was stirred

with propeller type agitator at room temperature at 150 rpm for 1 hr. Thus, the formed floating
microspheres
were washed and dried in a desiccator at room temperature. The following microparticle pass through
sieve
and collected.

 Multiple emulsion method:

In this method beginning powder drug was dispersed in solution (methyl cellulose) followed by

emulsification in ethyl cellulose solution in ethyl acetate. The primary emulsion was then re emulsified in

aqueous medium. Under optimized condition discrete microspheres were formed during this phase

 Ionic gelation:

In this method 25% (w/v) of diclofenac sodium was added to 1.2% (w/v) aqueous solution of sodium

alginate. In order to get the complete solution stirring is continued and after that it was added drop wise to

a solution containing Ca2+ /Al3+ and chitosan solution in acetic acid. Microspheres which were formed

were kept in original solution for 24 hr. for internal jellification followed by filtration for separation. The

complete release was obtained at pH 6.4-7.2 but the drug did not release in acidic pH.

 Hydroxyl appetite (HAP) microspheres

In this method microspheres were prepared by o/w emulsion followed by solvent evaporation. At first o/w

emulsion was prepared by dispersing the organic phase (Diclofenac sodium containing 5% w/w of EVA

and appropriate amount of HAP) in aqueous phase of surfactant. The organic phase was dispersed in the

form of tiny droplets which were surrounded by surfactant molecules this prevented the droplets from co-

solvencing and helped them to stay individual droplets. While stirring the DCM was slowly evaporated
and
the droplets solidify individual to become microspheres. [31]

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B) Drug profile

1) Losartan potassium:

a) Introduction

Losartan potassium is an effective antihypertensive drug but is extensively bound to plasma

proteins and also causes gastrointestinal disorders, neutropenia, acute hepatotoxicity, migraine and

pancreatitis. It may therefore be more desirable to deliver this drug in sustained release dosage

form. The present study was focused on development of sustained release Losartan microspheres

using injection method. [4]

Table no:1. Drug profile [2]

Sr.no Parameters of Drug Remark

1 Name of drug Riboflavin

2 Category Vitamin B complex

3 BCS Class III

4 pka 9.73

5 Solubility Water

6 Dose 10mg

7 Half life 66-84 Min

8 Protein binding Less than 5 %

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Figure: Riboflavin structure

b) Mechanism of action:

Riboflavin is d-Ribitol in which the hydroxy group at position 5 is substituted by a 7,8-

dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl moiety. It is a nutritional factor found

in milk, eggs, malted barley, liver, kidney, heart, and leafy vegetables, but the richest natural

source is yeast.

C) Polymer & other additives-

Name of excipient Solubility Category

Sodium alginate Water Gelling agent, Stabilizer,

Thickening agent

Calcium chloride Water Cross linking agent

Ethyl cellulose Ethanol To achieve sustained release

of drug

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Chapter II: Literature Survey

1) Ankita Gupta (2018): This article gives information about. A systemic study using a 32Factorial

design. The independent variables had significant influence on dependent variables. Concentration of

polymer ratio influence drug release profile & entrapment efficiency of microspheres.

2) International Journal of Applied Biology and Pharmaceutical Technology (April -June 2011):

This Review gives information about losartan microsphere and evaluation parameters of microspheres.

3)Microspheres: A Review by R. D. Salunke*, M. T. Deshmukh, R. V. Shete, R. S. Solunke: This

review highlights the Method of preparation, advantages, disadvantages, evaluation parameters of

microspheres.

4) Journal of Drug Delivery & Therapeutics. 2019: This journal gives information about preparation

of Losartan potassium microsphere by using natural polymer.

5) Arch Pharm Sci & Res Vol No 1 166 - 170 October 2009: This review gives the information about

the Losartan potassium microsphere using sodium alginate polymer by using non aqueous solvent

evaporation technique

6) LejunWu: Objective To develop poly (D, L-lactide-co Glycolides) (PLGA) microspheres to achieve

controlled and sustained release of brexpiprazole in vivo, Methods. Brexpiprazole Microspheres were

prepared by oil-in-water emulsion-solvent evaporation method and evaluated form morphology,

particle size, encapsulation efficiency, drug loading, conformation and compatibility of drug and

polymer, in vitro release and in vivo pharmacokinetics.

7) Shaveta Ahalvat: - The experimental design matrix was prepared by using a central composite design

to study the effect of various parameters over response variables. The experimental responses are good

harmony with the predicted values.

8) N K Varde: Polymeric microspheres are ideal vehicles for many controlled delivery applications due

to their ability to encapsulate a variety of drugs, biocompatibility, high bioavailability and sustained drug
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release characteristics. Research discussed in this review is focused on improving large-scale

manufacturing, maintaining, drug stability and enhancing control of drug release rates.

9) Rout. P. K, Nayak B. S. Formulation Design, Preparation of Losartan Potassium Microspheres

by Solvent Evaporation Method and Its In Vitro Characterization: Gives information about

formulation losartan potassium microsphere by solvent evaporation technique.

10) T.S. Keerthi, S.K.S Kumar , Formulation and evaluation of microspheres of losartan

potassium using biodegradable natural polymers: Present investigation describes preparation of

microspheres by solvent evaporation followed by in vitro characterization of microspheres to evaluate

the effect of method of preparation on physical properties and drug release profile of microspheres. The

microspheres were found to be discrete, spherical with free-flowing properties. The morphology

(Scanning Electron Microscopy), particle size distribution, entrapment efficiency and their release

profiles were investigated. The yield was found to be maximum in case of solvent evaporation method.

11) G Navneet, G Akanksha, J Neetesh - technique: This research gives information of Metformin

hydrochloride microspheres were prepared by ionotropic gelation method using non-ionic (ethyl cellulose

and HPMC), anionic (Carbopol 934P) and cationic (chitosan) polymers, respectively. The effect of

varying calcium chloride concentration and drug-polymer ratios was studied. The prepared microspheres

were studied for their morphology, average particle size, % drug content, % drug entrapment, % yield and

micromeritic properties. Ethyl cellulose microspheres showed least swelling and mucoadhesion property

in both simulated gastric pH and simulated intestinal pH medium depicting its non-mucoadhesive nature.

The microspheres prepared at 10% calcium chloride concentration and 1:3 drug: polymer ratio showed

the most sustained effect.

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Chapter III: Need of work

The goal of any drug delivery system is to supply therapeutic amount of drug to the right site within the

body to take care of desire drug concentration. The foremost common route for delivery of drug is that

the oral route which is effective for administration of the drug.

The limitations of the most obvious and trusted drug delivery techniques, such as conventional drug

delivery system (DDS), have been recognized for some time now, the most important limitation of them

being the patient incompliance due to frequent medication. This limitation can be overcome by modifying

existing DDS. An appropriately designed sustained release (SR) or controlled release DDS can be a major

step toward solving the problem associated with conventional DDS. The SR DDS also have solutions for

other limitations of the conventional DDS such as undesirable side effects due to fluctuating plasma drug

level, inability to maintain adequate drug concentration in plasma for therapeutic effect, larger doses than

those required by the cells have to be administered in order to achieve the therapeutic concentration,

causing the undesirable, toxicological and immunological effects in non-target tissues.

Microspheres provide a constant & prolonged therapeutic effect which will reduce dosing frequency.

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Chapter IV: Aim and Objective

Aim: The main aim of present work is to formulate and evaluate Riboflavin microspheres

incarporeted by ionotropic gelation method.

Objective:

1. To formulate microspheres of Riboflavin with Sodium alginate alone and with varying

concentrations of ethyl cellulose.

2. To evaluate the prepared microspheres for micrometric properties bulk density, tapped density,

compressibility index, hausner's ratio and angle of repose.

3. To evaluate the prepared microspheres for percentage yield, particle size, Swelling index, in-

vitro drug release study and in-vitro drug release data analysis for the prepared formulation

batches.

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Chapter V: Methodology
A) Ionotropic gelation method.

B) Material: Riboflavin combination of polymers sodium alginate, calcium chloride, ethyl cellulose

Solvent: Distilled water.

C)Procedure:

1. Riboflavin microspheres are prepared by ionotropic gelation method.

2. In this method sodium alginate and calcium chloride dissolved separately in 100ml of distilled water.

3.Dissolve sodium alginate in 100ml distilled water which is solution A.

4.Dissolve calcium chloride in 100ml distilled (solution B).

5. Solution A is stirred continuously on magnetic stirrer then add Riboflavin drug slowly while continue

stirring then after dispersion of drug add ethyl cellulose in it.

6. Take the above solution in a syringe and inject the solution in calcium chloride solution drop wise by

using 26 gauge syringe 2 to 3 drops per minute then microspheres are formed.

7. Filter the microspheres and dry at room temperature for 24 hours and store it into desicator.

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Table no: 2. Preparation of microsphere formulation

Formulation Drug(mg) Ethyl cellulose HPMC Sodium Calcium

Code (mg) (mg) alginate chloride

(%w/v) (%w/v)

F1 10 2 40 4 5

F2 10 4 80 4 5

F3 10 6 120 4 5

F4 10 8 160 4 5

Prepared microsphere Formulation:

F1 F2

F3 F4

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Chapter VII: Result and Discussion (Evaluation parameters)-

Evaluation of microspheres is done by various parameters like Bulk density, tap density, Hausner’ s

ratio, Carr's index, Angle of repose, particle size measurements, swelling index and dissolution etc.

1) Angle of repose

It is defined as the maximum angle possible between the surface of the pile of the powder and the

horizontal plane. Fixed funnel method was used. A funnel was fixed with it strip at a given height ‘h,

above a flat horizontal surface to which a graph paper was placed. Powder was carefully poured

through a funnel till the apex of the conical pile just touches the tip of the funnel. The angle of repose

was then calculated using following equation,

θ=Tan-1(h/r)

Where θ= Angle of

repose, h = Height of pile,

r=Radius of the base of the pile.[21]
Table no: 3.1: Relationship between angle of repose and flowability properties.

Angle of Repose Flowability

<20 Excellent

20 - 30 Good

30 - 40 Acceptable

>40 Very Poor

2) Bulk density

Bulk density is measured by 20 gm of microspheres are placed in measuring cylinder and

measuring the volume of microspheres. The bulk density is measured by using the following

formula

Bulk density = Mass of powder/Bulk volume

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3) Tap density

The tap density is measured by placing 20 gm of microspheres in measuring cylinder and tapping

for 50 times then tap volume is obtained. The Tap density is measured by using following formula

Tap density = Mass of powder / Tap volume

4) Hausner’s ratio

It is the ratio of tap density to the bulk density used to measure the flowability of microspheres.

Hausner ratio= Tapped density/ bulk density

Table no: 3.2: Relationship between hausner’s ratio and flowability.

Hausner’s Ratio Flowability

<1.18 Excellent

1.19 – 1.25 Good

1.3 – 1.5 Acceptable

>1.5 Very Poor

5) Carr's index

Carr’s index is an indication of the compressibility of a powder. It is expressed in percentage and is given

by,

I=Dt–Db/Dtx100

Where, Dt= Tapped density,

Db=Bulk density.

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Table no 3.3: Correlation between % compressibility and flowability properties

% Compressibility Flowability

5 – 15 Exellent

12 – 16 Good

18 – 21 Fair to passable

23 – 35 Poor

33 – 38 Very Poor

>40 Extremely Poor

Table no: 3.4: Micromeritic properties of prepared Losartan potassium microsphere.

Micromeritic properties F1 F2 F3 F4

Bulk Density 0.98 0.88 0.81 0.91

Tap Density 1.01 0.97 0.92 0.95

Angle of repose 20.80 21.30 20.80 21.80

Hausner ratio 1.03 1.10 1.13 1.03

Carr’s index% 2.97 9.27 11.9 4.21

6) Percentage yield: The prepared microspheres weighed from different formulations the measured

weight was divided by the total amount of all non -volatile components which were used for the

preparation of microspheres.[5]

Percentage yield = actual yield of drug /total weight of drug and polymer X 100
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Table no: 3.5: Percentage yield of prepared losartan microsphere.

Formulation code F1 F2 F3 F4

Percentage yield %

89.26 92.10 94.75 97.19

7) Particle Size:

Particles size is the important parameter for characterization of microspheres because particle size

affects the release of drug from microspheres. Smaller size particles have large surface area which

results in fast drug release while in larger particles the release of drug is slow. The polymer degradation

also increases within increase in particle size. The various techniques used for determination of particle

size of microsphere. Particle size is measured by electron microscope.[9]

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particle size images

F1 Batch F2 Batch

F3 Batch F4 Batch

Table no: 3.6: Particle size of prepared losartan microsphere

Formulation code F1 F2 F3 F4

Particle

size(micrometer) 642 652 738 781

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8) Swelling index:

This technique was used for Characterization of sodium alginate microspheres were performed with

swelling index technique. Take the phosphate buffer solution of pH 6.8. And kept the microsphere in

above solution and kept the temp 37 and changes in initial particle size and final particle size due to

swelling was measured by microscope and measure the particle size periodically.[29]

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Swelling index images
Time

(min) F1 Batch F2 Batch F3 Batch F4 Batch

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Table no:3.7: swelling index (micrometer) of prepared losartan microsphere.

Time(min) F1 F2 F3 F4 Batch

10 823 856 877 909

9) Dissolution study

The microspheres samples will be placed in each of six basket dissolution apparatus. The assembly is

maintained at 37°C in phosphate buffer pH 6.8 Sample is withdraw at definite time interval and replaced

by fresh medium. The absorbance of sample is measured from UV spectrophotometer.

Table no:3.8: Dissolution profile of prepared Losartan potassium microsphere.

Formulation F1 F2 F3 F4

code

Dissolution

study (%) 55 61 74 83

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Chapter VII: Conclusion and Outcomes

 Riboflavin microspheres were prepared by ionotropic gelation method. Sodium alginate was

selected as thickening agent, gelling agent & stabilizer along with calcium chloride as cross

linking agent for the preparation of microsphere. sodium alginate is converted into calcium

alginate an insoluble polymer and forms spherical microspheres. And ethyl cellulose is used as

polymer.

 Flow properties of all batches are also excellent.

 In case of percentage yield, concentration of polymer increases percentage yield also increases.

 In case of particle size, concentration of polymer increases particle size also increases.

 In swelling index, phosphate buffer of PH 6.8 is used and in this evaluation, polymer increases

swelling index also increases.

 Formulation F4 microspheres were selected as best formulation for preparation of sustained drug

delivery system.

 The data obtained shows that, microspheres are successfully prepared and evaluated also showing

sustained delivery of losartan potassium which will improve patient compliance.

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Chapter VIII: References

1. Sachan A.K., Gupta A., Kumari K., Ansari A., Formulation and characterization of

microspheres of nitazoxanide by chemical cross-linking method, Journal of Drug Delivery and

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2. IP 2014, volume III, page no. 2122

3. International Journal of Applied Biology and Pharmaceutical Technology.

4.Rout. P. K, Nayak B. S. Formulation Design, Preparation of Losartan Potassium Microspheres

by Solvent Evaporation Method and It’s in Vitro Characterization.

5. Mr. Purohit. k. P, Dr. Garud. N. Formulation and evaluation of floating microspheres of

losartan potassium using sodium alginate and HPMC by solvent evaporation method.

6.Microspheres: A Review by R. D. Salunke*, M. T. Deshmukh, R. V. Shete, R. S. Solunke

7.Https: //www ncbi nlm nih gov>pmc

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9. International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 04

Issue 08 August 2021

10. International Journal of Recent Scientific Research International Journal of Recent Scientific

Research Vol. 9, Issue, 5(E), pp. 26787-26790, May, 2018.

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local antibiotic therapy administered per-operatively. I. The case of gentamycin base and

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