FORMULATION AND OPTIMIZATION OF NANOSTRUCTURED LIPID
MATRICES OF REPAGLINIDE USING
FACTORIAL DESIGN
M.Pharm Dissertation Protocol Submitted to
Rajiv Gandhi University of Health Sciences, Karnataka
Bangalore– 560 041
By
Mr. MAULIK PRABHUBHAI TALSANIA B.Pharm
Under the Guidance of
Mr. SHANTHAKUMAR G.S M.Pharm (Ph.D)
Assistant Professor
Department of Pharmaceutics,
Acharya & B.M. Reddy College of Pharmacy,
Dr. Sarvepalli Radhakrishanan Road,
Soldevanahalli, Chikkabanavara (Post)
Hesaraghatta Main Road, Bangalore – 560 090.
2011-2013
1
RAJIVGANDHIUNIVERSITY OF HEALTH SCIENCES,
KARNATAKA, BANGALORE.
ANNEXURE - II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
1.
Name of the Candidate
and Address
MR. MAULIK PRABHUBHAI TALSANIA.
“NILKANTH”,
B/H DESAL BHAGAT WAV, OPP: KISHOR SOC.,
NEAR BLOCK NO.1, 80 FEET ROAD,
RAMAPEER WALI GALI,
SURENDRANAGAR-363002
GUJARAT.
2.
Name of the Institution
ACHARYA & B.M. REDDY COLLEGE OF PHARMACY,
DR.SARVEPALLI RADHAKRISHANAN ROAD,
SOLDEVANAHALLI,HESARAGHATTA MAIN ROAD,
CHIKKABANAVARA POST.
BANGALORE-560090
3.
Course of Study and Subject
M. Pharm
(Pharmaceutics)
4.
Date of Admission
16 DEC-2011
5. TITLE OF THE PROJECT:-
FORMULATION AND OPTIMIZATION OF NANOSTRUCTURED LIPID
MATRICES OF REPAGLINIDE USING
FACTORIAL DESIGN
2
6.0
BRIEF RESUME OF THE INTENDED WORK:
6.1 NEED FOR THE STUDY:
Nano drug delivery system is the latest technology employed in various pharmaceutical and
medicinal applications. This technology was adapted to avoid the conventional drug administration
due to its site specific targeting phenomena. In the middle of 1990’s, the attention of different
research groups has focused on alternative nanoparticles made from solid lipids, so called Solid
lipid nanoparticles (SLN) or Nanostructured lipid matrices/carrier (NLCs) or lipid nanoparticles.1
The solid lipid nanoparticles are sub-micron colloidal carriers (10-1000nm) which are composed
of physiological lipid, dispersed in water or in aqueous surfactant solution.2 In order to overcome
the disadvantages associated with liquid state of oil droplets of polymeric nanoparticles, liquid
state is replaced by a solid lipid which eventually transformed into solid lipid nanoparticles. Solid
lipid nanoparticles(SLN) are alternative carrier system to traditional colloidal carriers, such as
emulsions, liposomes and polymeric micro and nanoparticles but are having several disadvantages
:

Poor drug loading capacity.

Drug expulsion after polymeric transition during storage.

Relatively high water content of the dispersions (70-99.9%).

The low capacity to load hydrophilic drugs due to partitioning effects during the
production process.
SLN is limited by the solubility of drug in the lipid melt, the structure of the lipid matrix and the
polymeric state of the lipid matrix. If the lipid matrix consists of especially similar molecules (i.e.
tristearin or tripalmitin), a perfect crystal with few imperfections is formed. Since incorporated
drugs are located between fatty acid chains, between the lipid layers and also in crystal
imperfections, a highly ordered crystal lattice cannot accommodate large amounts of drug.
Therefore, the use of more complex lipids is more sensible for higher drug loading.
To overcome the potential difficulties of SLN, nanostructured lipid matrices/carriers
(NLC) were introduced. The aim was to increase the drug loading and prevent drug expulsion. The
use of spatially different lipids leads to larger distances between the fatty acid chains of the
glycerides and general imperfections in the crystal and increases accommodation of guest
molecules. The highest drug load could be achieved by mixing solid lipids with small amounts of
liquid lipids (oils). This model is called imperfect type NLC. Drugs showing higher solubility in
oils than in solid lipids can be dissolved in the oil and can be protected from degradation by the
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surrounding solid lipids. These types of NLC are called multiple types NLC. Since drug expulsion
is caused by ongoing crystallization or transformation of the solid lipid, this can be prevented by
the formation of a third type, the amorphous type NLC. Here the particles are solid but
crystallization upon cooling is avoided by mixing special lipids like hydroxyl octacosanyl,
hydroxyl stearate and isopropyl myristate.3

Advantages of Nanostructured lipid carriers :

Control and/or target drug release.

Improve stability of pharmaceuticals.

High and enhanced drug content (compared to other carriers).

Feasibilities of carrying both lipophilic and hydrophilic drugs.

Most lipids being biodegradable, NLC have excellent biocompatibility, Water based
technology (avoid organic solvents).

Easy to scale-up and sterilize, More affordable (less expensive than polymeric/surfactant
based carriers), Easier to validate and gain regulatory approval.
Diabetes mellitus is a chronic metabolic disorder characterized by high blood glucose
concentration - hyperglycemia. There are two types of diabetes mellitus. Type-1 diabetes is
managed by Insulin administration. In case of type-2 diabetes, treatment is initially dietary
although oral hypoglycemic drugs usually become necessary. There are many classes of oral antidiabetic agents. The objective of the treatment is to achieve hypoglycemia, by using an ideal
dosage regimen.
Oral hypoglycemic agent such as Repaglinide, a water insoluble drug is used in the management
of type 2 diabetes mellitus (NIDDM). It lowers blood glucose levels by stimulating the release of
insulin from pancreas. Insulin release is glucose dependent and diminishes at low glucose
concentrations. Repaglinide closes ATP-dependent potassium channels in the beta cell membrane
by binding at characterizable sites. The potassium channel blockade depolarizes the beta cell,
which leads to an opening of calcium channels. The resulting increased calcium influx induces
insulin secretion. Repaglinide has a very short half- life (1 h), low bioavailability (50%) and poor
absorption in the upper intestinal tract. It is completely metabolized by oxidative
biotransformation and direct conjugation with glucuronic acid after either an IV or oral
dose4.Hence, preparation of Repaglinide NLC will increase solubility, thereby, increasing
bioavailability.
The objective of present investigation is to formulate and evaluate nanostructured lipid
matrices of Repaglinide and subject the formulation for optimization by using factorial design.
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6.2 REVIEW OF LITERATURE:
Literature survey was carried out on the proposed topic with the facility of internet and
helinet and referring scientific journal. The survey reveals that, no work has been reported on the
proposed topic and some related research work are coated below :

Poovi G. et al., in their work, prepared repaglinide loaded chitosan polymeric nanoparticles
by solvent evaporation technique.The prepared nanoparticles showed high drug loading
capacity (11.22%w/w), encapsulation efficiency (97.0%) and nanoparticle recovery
(86.40%) with nanosize. Scanning electron microscopy revealed that nanoparticles are
spherical in a shape with smooth surface morphology. Particle size were analysed by
malvern particle size analyser having 48-100nm range. FT-IR studies showed that there
was no interaction between repaglinide and polymers. Based on the in vitro study,
repaglinide released from prepared formulation was slow and sustained over 15 days.
Application of the in vitro drug release data to various kinetic equations revealed first order
release, swelling and diffusion mechanism from repaglinide nanoparticles5.

Thatipamula RP and other coauthors., formulated domperidone solid lipid nanoparticles
and nanostructured lipid carriers by using hot homogenisation followed by ultrasonication
technique. The particles were evaluated for particle size, polydispersity index, zeta
potential and entrapment efficiency. DSC studies revealed that domperidone was in an
amorphous state and triglycerides were in the β prime form in SLN and NLC. Shape and
surface morphology determined by TEM indicated that particles were spherical in shape. In
vitro release studies demonstrated that both SLN and NLC formulations possessed a
controlled release over a period of 24 h. Stability studies revealed that there was no
significant change in particle size, zeta potential, polydispersity index and entrapment
efficiency indicating that developed SLN and NLC were fairly stable6.

Abdullah R. et al., developed nanostructured carriers stabilized with polysorbate 20 and
polysorbate 80 by high pressure homogenisation technique. TEM studies showed that these
NLCs were spherical in shape. Zeta potentialof NLC80 showed a more stable formulation
than NLC20. X-ray diffractometry and differential scanning calorimetry revealed that NLCs
were less crystalline than the bulk lipid. These findings suggest that polysorbate 80 was a
better dispersing agent for NLC than polysorbate 20. The small size and superior particle
surface to volume ratio would increase loading efficiency and bioavailability of drugs, thus
5
making NLC a promising drug delivery system7.

Gupta Jand other co-researchers, formulated solid lipid nanoparticles of nateglinide using
hot homogenization technique. Characterization and evaluation studies such as particle size
measurement, poly dispersity index, Zeta potential, entrapment and loading capacity,
Stability studies and in vitro release studies were studied for the nanoparticles. Scanning
Electron Microscopy showed that SLN particles were spherical in shape. Differential
scanning calorimetry (DSC) revealed the stability of SLNs with no tendency of
recrystallization. It was revealed that increase in concentration of lipid content has
increased the entrapment efficiency of SLN. It was concluded that SLNs with small
particle size, excellent physical stability, high entrapment efficiency, good loading capacity
for diabetic drug can maintain blood glucose level normally for a long period of time8.

Park JS et al., developed nanostructured lipid carriers (NLCs) of tacrolimus by the hot
homogenization method followed by sonication technique. The feasibility of fabricating
tacrolimus loaded NLCs were successfully demonstrated in this study. The developed
NLCs were characterized for morphology, particle size, zeta potential, and entrapment
efficiency (EE) of tacrolimus. The particle size, zeta potential, entrapment efficiency (EE)
were found to be 123±0.3nm,-24.3±6.2mV and 50 %.In vitro penetration studies indicated
that the tacrolimus-loaded NLCs have a penetration rate that is 1.64 times that of the
commercial tacrolimus ointment,Protopic®9.

Pardeike J and other coauthors., prepared itraconazole loaded NLC by hot high pressure
homogenisation technique. The tonicity of the formulation was adjusted with glycerol.
Sterility was obtained by autoclaving. Evaluation parameters such particle size, Zeta
potential, entrapment efficiency were studied. SEM images show that particles were
spherical in shape. Burst release of itraconazole from the developed carrier system was
found. Itraconazole-loaded NLC possessed good storage stability. Nebulizing itraconazoleloaded NLC with a jet stream and an ultrasonic nebulizer had no influence on the particle
size and the entrapment efficiency of itraconazole in the particle matrix, being a
precondition for pulmonary application10.

Das S. et al., formulated SLN and NLC formulations by different formulation techniques.
Stability of the formulations might increase due to the solid matrix of these lipid
nanoparticles. Scaling up of the production process from lab scale to industrial scale can be
easily achieved. Reasonably high drug encapsulation efficiency of the nanoparticles was
documented. Oral absorption and bioavailability of several drugs were improved after oral
6
administration of the drug loaded SLNs or NLCs11.

Sanad RA. et al., prepared oxybenzone nanostructured lipid carriers (NLCs) by the solvent
diffusion method. A complete 23 factorial design having variable parameters namely liquid
lipid type(Miglycol 812 and oleic acid), liquid lipid concentartion (15% and 30%), and
oxybenzone concentration (5% and 10% with respect to liquid lipids) were used to study
evaluation parameters such as particle size, entrapment efficiency (EE%) and in vitro drug
release. The prepared NLCs were spherical in shape and were below 0.8µm. Miglitol 812
and 30% liquid lipids were found to increase the EE%,when compared to oleic acid and
15% liquid lipid. Increasing oxybenzone concentration increased significantly the P.S. but
did not affect the EE%. NLCs prepared using Miglyol 812, 15% liquid lipid, and 10%
oxybenzone showed slower drug release as compared to oleic acid, 30% liquid lipid, and
5% oxybenzone. The candidate oxybenzone-loaded NLC dispersion was then formulated
into gel. In conclusion, the results of this study emphasize the potential of NLCs using
Miglyol 812 and glyceryl monostearate as a new topical drug delivery system for
enhancing the sun screening efficacy of oxybenzone by about six fold while reducing its
side effects12.

Gokce EH and other coauthors., formulated resveratrol (RSV) loaded SLN and NLC by
high shear homogenisation technique using compritol 888ATO, Myglycol, poloxamer 188
and tween 80. Nanoparticles were evalated for particle size, polydispersity index, zeta
potential, drug entrapment efficiency and production yield were determined. Entrapment
efficiency was 18% higher in NLC systems. Concentration below 50µM were determined
as suitable RSV concentrations for both SLN and NLC in cell culture studies. Ex vivo
studies revealed that NLC are more efficient in carrying RSV to the epidermis. In
conculsion, when two studies are compared, NLC penetrated deeper into the skin. RSV
loaded NLC with smaller particle size and higher drug loading capacity appears to be
superior to SLN for dermal applications13.
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6.3 - Objective of the Study
Following are the objectives of the present study
1. To carry out authentication and Preformulation study of drug and selected lipids.
2. To formulate and Optimize Nanostructured Lipid Matrices (NLC) of Repaglinide using
factorial design.
3. Evaluation of Nanostructured Lipid Matrices for various parameters like:

Particle size analysis and shape morphology.

Polydispersity Index.

Entrapment efficiency.

Physicochemical evaluation.

Content uniformity.

Drug and lipid interaction studies.

Crystallinity study.

Zeta potential.
4. To carryout in vitro dissolution studies of NLC formulations.
5. To carry out comparative antidiabetic study (in vivo) of optimized formulation and
marketed products of Repaglinide.
6. To carry out stability studies as per ICH guidelines.
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7.0
MATERIALS AND METHODS:
7.1 - Source of Data
7. Journals such as,
a. Indian Drug.
b. Indian Journal of Pharmaceutical Sciences.
c. Indian Journal of Pharmaceutical Education and research.
d. European Journal of Pharmaceutical Sciences.
e. International Journal of Pharmaceuticals.
f. Drug Development & Industrial Pharmacy.
g. Journal of Controlled Release.
h. African journal of pharmaceutical sciences
i. Journal of nanomedicine.
j. American association of pharmaceutical scientists.
8. Review articles
9. World Wide Web.
10. J-gate@Helinet.
11. Science Direct, Pub medand biomed central.
12. Library: Acharya and B M Reddy College of Pharmacy.
13. E-library: Acharya and B M Reddy College of Pharmacy.
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7.2MATERIALS AND METHODS:
Materials
Drug
: Repaglinide.
Lipids
: Soya lecithin, Trimyristin, Tripalmitin, Compritol 888 ATO,
Medium Chain Triglyceride (MCT), Oleic acid, Miglyol812, etc.
Surfactant
: kyron T314,Pluronic F 127,Pluronic F68,Cremophor EL, Tween80,
Poloxamer 188 etc.
Solvent
: Ethanol, Chloroform, Acetone, Ether, water etc.
Method of collection of data:
1. Authentication of drug :
a. Solubility studies.
b. Melting Point determination.
c. Purity of the drug
d. Compatability Studies by FTIR/DSC.
2. Preformulation studies of Repaglinide nanostructured lipid matrices.

Identification and purity of drug by FTIR/DSC.
3. Preparations of Nanostructured Lipid Matrices by Hot homogenization technique/high
shear homogenization/Ultrasonication or any suitable / developed method14.
4. Optimization of formulation was carried out by using factorial design.
5. Evaluation of the various properties of Repaglinide Nanostructured Lipid Matrices15,16

Polydispersity Index.

Entrapment efficiency.

Physicochemical evaluation /TEM.

Content uniformity.

Particle size and shape morphology by Scanning Electron Microscopy.

Crystallinity study by using XRD and DSC.

Zeta potential by using zeta sizer.
6. To carry out in vitro dissolution study of NLC formulations16.
7. To carry out comparative antidiabetic study (in vivo) of optimized formulation and
marketed product of repaglinide16.
8. To carry out Stability studies as per ICH guidelines17.
10
7.3 Does the study require any investigation or interventions to be
Conducted on patients or other humans or animals?
“YES”
7.4 Has ethical clearance been obtained from your institution in case of 7.3?
“Copy is enclosed”.
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8.0
REFERENCES:
1.Patel RP, Singhal GB, Prajapati BG, Patel NA. Solid lipid nanoparticles and nano lipid carriers:
as novel solid lipid based drug carrier. Int Res J Pharm. 2011; 2(2):40-52.
2. Shinde NC, Keskar NJ, Argade PD. Nanoparticles: Advances in drug delivery systems. Res J
Pharm Bio Chem Sci. 2012; 3(1):922-28.
3. Mukherjee S, Ray S, Thakur RS. Solid lipid nanoparticles: A modern formulation approach in
drug delivery system. Indian J Pharm Sci.2009;71(4):349-58.
4. South Thames. UK Drug Information Pharmacists Group, New medicines on the market 1999;
Monograph Number: 4/99/06:1-6.
5. Poovi.G, Dhana Lekshmi UM, Narayanan N, Reddy PN.Preparation and characterisation of
repaglinide loaded chitosan polymeric nanoparticles. Res J Nanosci Nanotechnol.2011; 1(1):1224.
6.Thatipamula RP, Palem CR., Gannu R., Mudragada S., Yamsani MR. Formulation and in vitro
characterization of domperidone loaded solid lipid nanoparticles and nanostructured lipid carriers.
Daru. 2011; 19(1): 23-32.
7.Abdullah R, How CW, Abbasalipourkabir R. Physicochemical properties of nanostructured lipid
carriers as colloidal carrier system stabilized with polysorbate 20 and polysorbate 80. Afr J
biotechnol. 2011; 10(9): 1684-89.
8. Gupta J, Rajpoot AK, Soni R, Sharma P. Formulation, development and characterization of oral
hypoglycemic agent loaded solid lipid nanoparticles. Int J Biopharm Toxico Res. 2012; 2(1): 25156.
9. Park JS, Nam HS, Ji XY. Investigation of tacrolimus loaded nanostructured lipid carriers for
topical drug delivery. Bull. Korean Chem Soc 2011; 32(3):956-60.
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10. Pardeike J, Weber S, Haber T, Wagner J, Zarfl HP, Plank H, Zimmer A. Development of an
itraconazole-loaded nanostructured lipid carrier (NLC) formulation for pulmonary application. Int
J Pharm. 2011; 419(1-2): 329-38.
11.Das S, Chaudhury A. Recent Sci Tech. 2011 advances in lipid nanoparticle formulations with
solid matrix for oral drug delivery. AAPS Pharm; 12(1): 62-76.
12.Sanad RA, AbdelMalak NS, ElBayoomy TS, Badawi AA. Formulation of a Novel
Oxybenzone-Loaded Nanostructured Lipid Carriers (NLCs).AAPS Pharm Sci Tech. 2011; 11(4):
1684-94.
13.Gokce EH, Korkmaz E, Dellera E, Sandri G, Bonferoni MC, Ozer O. Resveratrol-loaded solid
lipid nanoparticles versus nanostructured lipid carriers: evaluation of antioxidant potential for
dermal applications. Int J Nanomedicine.2012; 7:1841-50.
14. Sathali Abdul HA, Ekambaram P, Priyanka K. Solid lipid nanoparticles: A review. Sci Revs
Chem Commum.2012; 2(1):80-102.
15. Mowafy HA, Alaa Eldeen B. Yassin, Anwer MK, El-Bagory IM, Bayomi MA, Alsarra IA.
Optimization of 5-fluorouracil solid-lipid nanoparticles: a preliminary study to treat colon cancer.
Int J Med Sci.2010; 7(6):398-408.
16. Jain S, Saraf S. Repaglinide loaded long- circulating biodegradable nanoparticles: Rational
approach for the management of type 2- diabetes mellitus. J diabetes.2009;1(1):29-35.
17. ICH Guidelines [Online] [Quality Guidelines] [Cited 2012 June 21];
www.ich.org/products/guidelines/quality/article/quality-guidelines.html.
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9.
SIGNATURE OF THE
CANDIDATE
10.
REMARKS OF THE GUIDE
The topic selected for dissertation is satisfactory. Adequate
equipments and chemicals are available to carry out project
work.
11.
NAME AND
DESIGNATION OF
11.1 Guide
Mr. SHANTHAKUMAR G.S M.Pharm, (Ph.D)
Assistant Professor
11.2 Signature
11.3 Co-Guide ( If any)
11.4 Signature
11.5 Head of the Department
-NIL-
----
Dr. SHIVANANDAKALYANAPPA M.Pharm, Ph.D
Professor & Head
Department of Pharmaceutics
11.6 Signature
12.
12.1 Remarks of
the Principal
12.2 Signature
Principal
Dr. DIVAKAR GOLI M.Pharm, Ph.D
Professor and Principal
Acharya & B.M. Reddy College of Pharmacy,
Bangalore – 560 090.
14