Swati Nagar 12032019

FORMULATION AND DEVELOPMENT OF NANO DRUG
DELIVERY SYSTEM OF CILNIDIPINE FOR IN-VITRO

DISSOLUTION ENHANCEMENT
Presented to
R K UNIVERSITY
Prepared by: Guided by:

Nagar Swati k dr. m. m. soniwala
F & D scientist M.Pharm. Ph. D.
FTF Pharma Pvt Ltd, Associate Professor
Ahmedabad B K Mody government pharmacy
114060 college,
Rajkot
R K UNIVERSITY, RAJKOT 1
Index
1.Abstract
2.Introduction
3.Review of literature
4.Research problem
5. Aim &Objectives
6. Formulation and development
7.Previous comments
8.Publications
9.Possible outcome
10. Impact on science/industry/society
11.References
1. ABSTRACT
• Background: Nanosuspension, solid lipid nanoparticle and self nano-
emulsifying drug delivery system is an emerging and promising approach
for the increasing solubility and dissolution rate. The present study aims at
producing nanosuspension of Cilnidipine, a poorly water soluble
antihypertensive drug with a view to enhance its dissolution and saturated
solubility. Hypertension (HTN or HT), also known as high blood
pressure (HBP), is a long-term medical condition in which the blood
pressure in the arteries is persistently elevated.
• Aim: The purpose of present research work was to formulate and evaluate
Nanosuspension, Self nano-emulsifying drug delivery system and solid
lipid nanoparticle of Cilnidipine and comparison with marketed
preparation with a view to enhance its dissolution, saturated solubility and
bioavailability
Materials and methods:
• Nanosuspension: Drug solution of Cilnidipine in acetone was added to
solution of stabilizer (antisolvent system) under continuous
homogenization. Various process and formulation parameters were
screened like homogenization speed, homogenization time, type of
stabilizer, solvent to antisolvent ratio, drug concentration and stabilizer
concentration. With a view to enhance physical stability of this colloidal
system, nanosuspensions were freeze dried using D- mannitol. They were
characterized for particle size, XRD and DSC studies, SEM, drug content,
saturation solubility, dissolution studies and FTIR studies and compare
pharmacokinetic parameter of lyophilize nanosuspension with RLD.
Nanosuspension prepared by solvent antisolvent precipitation method using
Tween 80 as a stabilizer was selected for further in vivo study.
• SNEDDS-Solubility of Cilnidipine was determined in various oils,
surfactants and cosurfactant by using Spectrophotometric method.
Pseudoternary phase diagrams were constructed to study the phase
behavior and most efficient self emulsifying region. The Solid Self nano-
Emulsifying Drug Delivery System (S-SNEDDS) was prepared using
adsorbent. The prepared S-SNEDDS were filled in hard gelatin capsule and
evaluated for various physicochemical
R K UNIVERSITY,parameters.
RAJKOT 5
• Solid lipid nanoparticle- Lipid nanoparticle prepared by microemulsion
technique and compared on basis of three parameters (particle size<200 nm,
Zeta potential<-20 mV, entrapment efficiency>60%). Optimized by Box-
benhken design batches characterized by X-ray Diffractometry study (XRD),
Differential Scanning Calorimeter study (DSC)
• Results and Discussion:

Nanosuspension: Nanosuspensions were successfully prepared using
solvent antisolvent precipitation using high speed homogenizer. Seven
different stabilizers were tried. Among them Poloxamer 407, Poloxamer
188, PVA and Tween 80 yielded nanosuspension in range of 90-350 nm. In
XRD and DSC studies, it was revealed that physical state of drug was not
changed but decreased in crystallinity was observed.
Freeze dried nanosuspension showed drastic increase in saturation
solubility & dissolution rate as compared to pure drug. Moreover, freeze
dried nanosuspensions were found to be stable over a period of one month.
Freeze dried nanosuspensions were filled in capsules to make a deliverable
dosage form and almost 90% drug dissolved in 5 mins. The in vivo test
demonstrated that lyophilize nanosuspension of cilnidipine were tmax of 10
min compared to RLD(Cilacar) have tmax of 60min.
• SNEDDS-The composition of optimized formulation consisted of 10 mg of
Cilnidipine, Capmul MCM (oil), Cremophor ELP (surfactant) and
Propylene glycol (cosurfactant). The optimized formulation showed
optimal particle size (10.59 nm), negative zeta potential (-22.11 mv) and
exhibited good self emulsifying ability with highest Solubilizing capacity.
The optimized liquid SNEDDS was further used for the preparation of S-
SEDDS formulations which revealed highest release rate (97.7%) among
all the S-SEDDS formulation and marketed(Cilacar) formulation.
• Solid lipid nanoparticle-Lipid nanoparticles achieved satisfactory results

on the quality examination (particle size<200 nm, Zeta potential<-20 mV,
entrapment efficiency>60%).Parameters for Microemulsion technique were
66 nm, -25 mV and 86%. XRD studies suggested conversion of crystalline
structure of drug molecules into amorphous structure. In vitro release study
revealed 90% release of drug over 5 min.
• Conclusion: The outcome of this study reveals the immense
potential of nanosuspension, self nanoemulsifying drug delivery,
solid lipid nanoparticle for delivery of Cilnidipine by improving its
saturation solubility, enhancement of dissolution rate and
bioavailability.
• Keywords: “Nanosuspension”, “Quality by design”, “Cilnidipine”,

“Dissolution enhancement”, “SNEEDS”, “Lipid nanoparticle”,
“Anti- Hypertensive”
Literature search API Procurement, Literature review, Patent
search, Drug profile, Excipient profile, RLD
Characterization
API, Material Procurement and method
development
Nanosuspension Self nanoemulsifying drug delivery Solid lipid nanoparticle
Screening of oils, surfactant, co- Screening of solid lipid, Liquid lipid ,

Screening of stirring speed, time, surfactant concentration surfactant, co-surfactant concentration
surfactant concentration
Feasibility trail Feasibility trail Feasibility trail
Design trials, ANOVA, Physical and Design trials, ANOVA, Physical and Design trials, ANOVA, Physical and
chemical evaluation chemical evaluation chemical evaluation
Optimize Formulation Optimize Formulation Optimize Formulation
Characterization of optimize Characterization of optimize formulation Characterization of optimize formulation

formulation
Stability studies Stability studies RAJKOT

R K UNIVERSITY, Stability studies 9
2. INTRODUCTION
• In recent years, the formulation of poorly soluble compounds
presented interesting challenges for formulation scientists in the
pharmaceutical industry. Up to 40% of new chemical entities
discovered by the pharmaceutical industry are poorly soluble or
lipophilic compounds, which lead to poor oral bioavailability,
high intra and inter subject variability and lack of dose
proportionality.
• A drug having a poor aqueous solubility has a low saturation
solubility which is typically correlated with the low dissolution
velocity, resulting in poor oral bioavailability [1].
• Bio-pharmaceutically acceptable formulations for drugs with low
solubility in aqueous solvents are a challenge since slow and
erratic dissolution is preventing the rapid and complete
absorption of these compounds from GI tract [2-3].
• Due to certain limitations of various solubility enhancement
techniques, the nanonization of the insoluble particles may prove to
be a suitable method in order to enhance the solubility with the
least possible limitations [4-5].
• The nanoparticulate drug delivery systems consist of various
formulations like nanocrystals, nanosuspension, nanoemulsion,
niosomes, solid-lipid nanoparticles, Self nanoemulsifying drug
delivery, erythrosomes, nanogel, etc.
• Therefore, poor aqueous solubility of drugs is a major limiting
factor in their successful launch in market in spite of their potential
pharmacokinetic activity [6-7].
• In this project work we have focus on

1. Nanosuspension,
2. Self nanoemulsifying drug delivery
3. Solid lipid nanoparticles
3. Review of literature
AUTHOR TITLE DESCRIPTION
O Kayser et A new They have the ability to reside in the gastrointestinal tract
al(2001) [8] approach for for an extended period. The hydrogel contained bupravaquo
targeting to ne nanosuspensions and an adhesive polymer (chitosan)
Cryptosporidi powder dispersed in water. By the development of
m parvum mucoadhesive nanosuspensions, a potential drug delivery
using system for poorly soluble drugs has been investigated to
mucoadhesive overcome bioavailability problems caused by the
nanosuspensi pathophysiological diarrhoeic situation in patients suffering
ons: research from cryptosporidiosis. Adapting drug delivery systems to
and the situation of Cryptosporidium parvum infections in man
applications allows increased retention times with a prolonged action at
reduced elimination in the gastrointestinal tract.
Rajalakshmi. Design and Poorly water soluble compounds are difficult to develop as
R et al characterizati drug products and conventional formulation techniques are
(2012) [9]
on of frequently abandoned early in discovery. The aim of the
valsartan present study was to improve the dissolution rate of a
nano poorly water soluble drug, valsartan, by a high pressure
suspension homogenization technique.
Six different formulations were prepared by optimizing
various parameters using different polymers like
polaxamer, soya lecithin, PVP and PVA, Formulations.
Ghosh I, et Nanosuspensio A promising nanosuspension was developed with Vitamin
al (2011)[10] n for improving E TPGS based formulation with particle size in the nano
the range. Although the formulation showed significant
bioavailability improvement during in vitro dissolution and in vivo
of a poorly plasma level, probably due to the strong hydrophobic
soluble drug interaction between Vitamin TPGS and the drug
and screening molecule, crystal growth was observed during stability
of stabilizing studies. A systematic study was done with different
agents to inhibit combinations of solubilizer /stabilizer system in order to
crystal growth obtain a more stable nanosuspension. Hydroxypropyl
methylcellulose (HPMC 3 cps).
Wei L, Preparation and Revaprazan hydrochloride (RH) is a new reversible
Yonggang in vitro/in vivo proton pump inhibitor. However, due to poor water
et al evaluation of solubility, oral bioavailability of the drug was relatively
(2011)[11] revaprazan low. To investigate the particle size reduction effect of
hydrochloride RH on dissolution and absorption, three suspensions that
nanosuspension containing different sized particles were prepared by high
pressure, cycle numbers and crushing principles on the mean
particle size, 99% diameter and polydispersity of the
nanosuspension were investigated. Characterization of the
product was performed by scanning electron microscope
(SEM) and differential scanning calorimeter (DSC). The safety
of the nimodipine nanosuspension was discussed with special
attention to contamination by microparticles and the increase
in saturation solubility Cs.
Villar Design and Preliminary screening was performed to select proper
AMS et optimization components combination. Box–Behnken experimental design
al(2012) of self was employed as statistical tool to optimize the formulation
[12] nanoemulsifyi variables, X1 (Cremophor® EL), X2 (Capmul® MCM-C8),
ng drug and X3 (lemon essential oil). Systems were assessed for visual
delivery characteristics (emulsification efficacy), turbidity, droplet size,
systems polydispersity index and drug release. Different pH media
(SNEDDS) for were also assayed for optimization. Following optimization,
enhanced the values of formulation components (X1, X2, and X3) were
dissolution of 32.43%, 29.73% and 21.62%, respectively (16.22% of
gemfibrozil gemfibrozil). Transmission electron microscopy demonstrated
spherical droplet morphology.
SNEEDS release study was compared to commercial
tablets. Optimized SNEDDS formulation of gemfibrozil
showed a significant increase in dissolution rate compared
to conventional tablets. Both formulations followed
Weibull mathematical model release with a significant
difference in parameter in favor of the SNEDDS.
Kassem AA Preparation Based on solubility studies, oil phase (oleic acid without
et al and in vitro or with coconut oil), surfactant (Tween 20), and co-
(2010)[13] evaluation surfactants (PEG 200 and n-butanol) were selected and
of self- grouped in two combinations for phase studies. Based on
nanoemulsif the results with regard to droplet size, turbidity values,
ying drug and complete drug release after 3 h, three optimized
delivery formulations were selected; each contained oleic acid/
systems coconut oil/ Tween 20 / PEG 200 / n-butanol in ratios of
(SNEDDS) 10:0:60:15:15 (%, w/w), 7.5:2.5:53.5:13.3:13.3 (%, w/w),
containing and 6.7:3.3:60:10:10 (%, w/w), respectively. Results
clotrimazole suggested that the prepared SNEDDS formulations
produced acceptable properties in terms of immediate
drug release and could increase the bioavailability of CT.
Saijie Z et Application A three-factor, three-level Box-Behnken design was used to
al (2009)[14] of Box- explore the main and interaction effect of several
Behnken independent formulation variables including the amount of
design in Maisine 35-1 and Labrafac Lipophile WL 1349 (1:1, w/w)
understandin (X1), Cremophor EL and Labrasol (3:1, w/w) (X2), and
g the quality Transcutol P (X3). Droplet size (Y1), turbidity (Y2), and
of genistein dissolution percentage of GN after 5 (Y3) and 30 (Y4) min
self- were the dependent variables. A mathematical relationship,
nanoemulsifi Formulation optimization was then performed to maximize
ed drug dissolution percentage of GN at 5 min (Y3). The optimized
delivery formulation was predicted to dissolution 93.34% of GN at 5
systems and min, when X1, X2 and X3 values were 37.1, 101.7 and
optimizing its 77.3 mg, respectively. A new batch was prepared according
formulation to the optimized formulation, and the observed and
predicted values of Y3 were in close agreement.
Faiyaz S, Ultrafine Thermodynamically stable SNEDDS was selected for self-

Haq N et al super nanoemulsification efficiency test. Selected formulations
(2013)[15] were characterized in terms of droplet size distribution,
viscosity and refractive index.
Self nano Finally, selected SNEDDS (F1–F9) were subjected to
emulsifying in vitro dissolution/drug release studies. Droplet size
drug delivery and viscosity of formulation F1 was found to be lowest
system as compared to other formulations. The results of zeta
(SNEDDS) potential indicated the formation of stable SNEDDS.
enhanced In vitro drug release studies showed 98.4% release of
solubility and IND from optimized formulation F1.
dissolution of
indomethacin
Janga KY et In situ absorption Preformulation studies including screening of
al (2013)[16] and relative excipients for solubility and pseudoternary phase
bioavailability diagrams suggested the suitability of Capmul MCM as
studies of lipid, Labrasol as surfactant, and Tween 20 as
zaleplon loaded cosurfactant for preparation of self-emulsifying
self nano formulations. Preliminary composition of the
emulsifying SNEDDS formulations were selected from the phase
powders. diagrams and subjected to thermodynamic stability
studies and dispersibility tests. The prepared liquid
SNEDDS formulations were characterized for
viscosity, refractive index, droplet size and zeta
potential.
Dinda, et Formulation SLNs have been reported as an alternative drug delivery
al Development device to traditional polymeric nanoparticles. SLNs are in
(2013)[17] and submicron range (50- 1000nm) and are composed of
Evaluation of physiologically tolerated lipid components. Paclitaxel is a Di-
Paclitaxel terpenoid Pseudo-alkaloid having anti-neoplastic activity
Loaded Solid particularly against primary epithelial, ovarian carcinoma,
Lipid Breast cancer, Colon Cancer, Brain Cancer, Lungs cancer and
Nanoparticles AIDs Related Kaposi’s Sarcoma. The present study is to
Using investigate the probability of incorporating paclitaxel in
Glyceryl SLNs using Glyceryl Mono-stearate (GMS) as a lipid matrix,
Monostearate
Rawia preparation Hot homogenization and ultrasonication were applied in the
Khalil and production of (Nyst-SLNs). Different matrix material and
etal characterizati stabilizers were tested. All the formulations were subjected to
(2013)[18] on of particle size analysis, zeta potential, drug entrapment
nystatin- efficiency and in-vitro release studies. Transmission electron
loaded solid microscopy was conducted to investigate the morphology of
lipid nystatin loaded nanoparticles. The differential scanning
nanoparticles calorimetry visualized the dispersed amorphous state of
for topical nystatin in the SLN.
delivery
Liandong Preparation, Solid dispersion technique has been developed many years
Hu et al characterizati for improving solubility of water-insoluble drugs, aiming to
(2013)[19] on and achieve a better oral bioavailability. However, this technique
tableting of exhibits many inconveniences when used for large-scale
cilnidipine tableting procedures. The objective of current research work
solid was to develop cilnidipine solid dispersions (SDs) to improve
dispersions the dissolution behaviors of this water-insoluble drug.
Moreover, an innovative granulation method was designed to
simplify the traditional tableting technology used in solid
dispersion technique. Three different kinds of polymers,
polyethylene glycol (PEG), polyvinylpyrrolidone (PVP) and
poloxamer, were used as carriers to prepare solid dispersions.
Pankaj P Method Present study describes accurate, precise and reproducible

et al validation for spectrophotometric method for estimation of Cilnidipine. The
(2013)[20] spectrophoto method was validated by using various parameters as per
metric ICH guidelines. Cilnidipine is a new dihydropyridine (DHP)
estimation of calcium channel antagonist used as antihypertensive agent.
cilnidipine Method was validated by checking parameters like linearity,
precision, accuracy, sensitivity, recovery study. Cilnidipine
showed maximum absorbance at 240 nm.
Upadhay Preparation Cilnidipine, a calcium channel blocker having
M et al and neuroprotective action and BCS Class II drug, hence
(2012)[21] evaluation of formulating in Microemulsion will increase solubility. The
cilnidipine formulation was prepared using titration method by
microemulsio tocotrienol, tween 20 and transcutol HP as oil, surfactant and
n co-surfactant and characterized for dilutability, dye solubility,
assay (98.39±0.06), pH (6.6±1.5), Viscosity (98±1.0 cps) and
Conductivity (0.2±0.09 μS/cm). The formulation was
optimized on basis of percentage transmittance (99.269±0.23
at 700 nm), Globule size (13.31±4.3 nm) and zeta potential (–
11.4±2.3 mV). Cilnidipine microemulsion was found to be
stable for 3 months.
Safhi Spectrophoto developed a new, simple and sensitive Spectrophotometric
MM et al metric Method in ultraviolet region has been developed for the
(2013)[22] Method for determination of cilnidipine in bulk and in pharmaceutical
the formulations. Cilnidipine exhibits absorption maxima at 240
Estimation of nm. The method obeys the Beer's law in the concentration
Cilnidipine in range of 2 - 30 μg/ml. The method is accurate, precise and
Bulk and economical. The % recovery is greater than 99.86 - 100.67%.
Pharmaceutic This shows that the method was free from the interference of
al Dosage excepients.
forms
4. RESEARCH PROBLEM
• The main aim of this project work is to enhance dissolution

rate of poorly soluble drug- Cilnidipine
• The poorly soluble drugs (Cilnidipine) have limited

solubility(0.000566 mg/ml) and bioavailability (20%) so it
have limited dissolution rate.
• By preparing nano-particulate drug delivery system, the

solubility of Cilnidipine can be enhanced which is believed to
be the main reason for its enhance dissolution rate [23].
Drug Cilnidipine
Chemical Name 1,4-Dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-
pyridinedicarboxylic acid 2-methoxyethyl (2E)-3-phenyl-
2-propenyl ester
Molecular weight 492.52
Formula C27H28N2O7
Solubility Soluble in DMSO (> 25 mg/ml), ethanol (20 mg/ml),
water (0.000566mg/ml), and soluble in methanol
CAS NO 132203-70-4
Melting Point: 130-140 °C
Class Calcium channel blocker
Biological Activity Dual L- and N-type calcium channel blocker that displays
antihypertensive, sympatholytic and neuro protective
activity. Lowers mean blood pressure and reduces the size
of cerebral infarction in the rat model of focal brain
ischemia.
Structure
BCS Class 2
Bioavailability 20%
Oral
Indication & Hypertension
Dosage Adult: 5-10 mg once daily, increase to 20 mg once daily if
necessary.
Contraindications Cardiogenic shock; recent MI or acute unstable angina;
severe aortic stenosis.
Hypotension, poor cardiac reserve, heart failure. Sudden
Special withdrawal may exacerbate angina. Discontinue in
Precautions patients who experience ischemic pain following
administration. Pregnancy, lactation.
Dizziness; flushing; headache; hypotension; peripheral
oedema; tachycardia; palpitations; GI disturbances;
Adverse Drug increased micturition frequency; lethargy; eye pain;
Reactions depression; ischaemic chest pain; cerebral or myocardial
ischaemia; transient blindness; rashes; fever; abnormal
liver function; gingival hyperplasia; myalgia; tremor;
impotence.
Other antihypertensive; aldesleukin; antipsychotics that cause hypotension;
Drug may modify insulin and glucose responses; quinidine; carbamazepine;
Interactions phenytoin; rifampicin; cimetidine; erythromycin. Potentially Fatal: Other
antihypertensive; aldesleukin; antipsychotics that cause hypotension; may
modify insulin and glucose responses.
Cilnidipine is a dihydropyridine calcium-channel blocker. It inhibits cellular
Mechanism influx of calcium, thus causing vasodilatation. It has greater selectivity for
of Action vascular smooth muscle. It has little or no action at the SA or AV nodes and -
ve inotropic activity is rarely seen at therapeutic doses.
Cilacar 10 mg tablets
Company- J B Chemicals and pharmaceuticals limited
Excipients- Microcrystalline cellulose, maize starch, purified water,
RLD Details magnesium stearate, talc, Croscarmellose sodium
Process- Wet granulation
Batch NO- CTS0215
Exp. Date- Feb, 2018
5. Aim &objectives
• To perform preformulation study of drug and development of
various analytical methods.
• To perform drug-excipients compatibility study.
• To formulate & optimize Nanosuspension of cilnidipine
• To perform characterization of Nanosuspension by, Mean
particle size and size distribution, particle charge (Zeta
potential), crystalline state and particle morphology, In vitro
drug release study, stability, drug content
• To study saturation solubility and dissolution rate of
nanosuspension and compare with marketed product.
• To formulate and optimization of self-micro/nano emulsifying
drug delivery system.
• To perform characterization of self-micro/nano emulsifying
drug delivery by Visual assessment, Refractive index and
percentage transmittance, Turbidity measurement, Zeta
potential measurement, X-ray scattering, Thermodynamic
stability studies, Drug content
• To perform In-vitro dissolution study and compression with
marketed product.
• To formulate and optimize solid lipid nanoparticles
cilnidipine.
• To perform characterization of solid lipid nanoparticles by,
Particle size, Zeta potential, % Entrapment Efficiency, X-Ray
Diffraction Study (XRD), Differential scanning Calorimetry
(DSC)
• To study in-vitro drug release of solid lipid nanoparticles
cilnidipine and compare with marketed product.
• To perform stability study of optimize nanosuspension, solid
lipid nanoparticle and self-nano-emulsifying drug delivery.
• In-vivo study of nanosuspension with marketed drug(Cilacar) .
6. FORMULATION AND DEVELOPMENT
Table 6.1: List of Material
Sr. No. Materials/ Chemicals Sources
1 Cilnidipine J B Chemical and pharmaceutical limited
2 Poly (vinyl pyrollidone) PVP K29/32, PVA International Speciality Products, Singapore.
3 Poloxamer 407,188 Signet Chemicals Pvt. Ltd, Mumbai.

4 HPMC E5 Colorcon, India
5 Tween 80
6 SLS
Merck
7 Acetone
8 Methanol
9 D-mannitol FMC Biopolymer
10 Microcrystalline Cellulose FMC Biopolymer
11 Capmul MCM
12 Capmul MCM PG8
13 Capmul GMO Abitec Corporation, USA
14 Captex 335
15 Captex 200 R K UNIVERSITY, RAJKOT 28
Continue….
Sr. No. Materials/ Chemicals Sources

16 Acconon MC8-2EP Abitec Corporation, USA
17 Labrasol
18 Compritol 888 ATO
19 Geleol
20 Gelucire 43/01, Gelucire 44/14, 50/13
21 Labrafec PG
22 Labrafac Lipophile Wl 1349 Gattefosse, France
23 Labrafec lipophile WL1349
24 Labrafil 1944 CS
25 Labrafil M 1944
26 Plurol Diisosterique CG
27 Peceol, Capryol 90
28 Miglyol 810,812,840,829 Sasol, Germany
29 Cremophore RH40 BASF, Germany
30 Cremophore ELP BASF, Germany
31 Tween 80,20 Merck
32 Span 80, 20 Merck
Table 6.2: List of Equipments
Sr. No Equipments Source
1 Melting point Apparatus Electro Quip
2 Fourier Transform Infra-Red Shimadzu 1800, Japan
UV/VIS Double beam
3 Shimadzu UV 1800 corporation, Japan
Spectrophotometer
4 Differential Scanning Calorimeter Perkin Elmer, Model DSC 7, USA
5 Bath Sonicator Electro Quip

6 pH meter Systronics
OMNI International, USA. Model : OMNI PDH
7 High Speed Homogenizer
Variable Motor Speed: 1,000- 28, 000 rpm
8 Particle size analyzer Sympatec, Germany. Model: Helo
Malvern Instruments Ltd, USA. Model: Malvern
9 Zetasizer
Zetasizer Nano S90, Software version: Ver.6.01
10 Lyophilizer Christ, Germany.
11 Dissolution apparatus TDT-081, Electrolab, India
6.1 Preliminary experimental work
• Identification of drug:
Identification of drug was carried out by melting point study,
FTIR study, DSC and XRD study
• Melting point study:

Melting point of Cilnidipine was determined using melting
point apparatus. Melting point of Cilnidipine was found to be
130-1400 C which in accordance with the reported melting
point 1390 C for the drug.
Figure 6.1: FTIR study
Table 6.3: FTIR spectra of Cilnidipine
Peaks at wave number (cm-1 ) Interpretation

2960-2872 cm-1 Alkane (H-C-H Asymmetric and symmetric stretch)
3059 cm-1 Aromatic ring (C=C-H Asymmetric stretch)
1597cm-1 (C-C=C Symmetric stretch)
1480 cm-1 (C-C=C Asymmetric stretch)
• DSC study
• Figure shows the DSC thermograms of Cilnidipine which
shows a sharp peak at 139 0C corresponding with a signal
value at which is in accordance with the reported melting point
of drug. Moreover, the result of DSC study was found to be
comparable with the melting point study of the drug.
Figure 6.2:DSC spectra of Cilnidipine
6.2 Formulation and development of Cilnidipine liquisolid compact
6.2.1 Spectroscopy estimation of Cilnidipine (CLD)

The calibration curve of Cilnidipine (CLD) was taken in two different media
Viz: Methanol and Phosphate buffer pH 6.8+ 1.0 % SLS
Selection of buffer media for in vitro dissolution study of the respective drugs
was based on the OGD media given by USFDA.
• Preparation of standard stock solution

• For Methanol- An accurately weighed 100 mg of CLD was added in a 100
mL volumetric flask and volume was adjusted up to the mark with methanol
to obtain a standard solution of 1000 µg/mL. Withdrawn 10 mL solution from
that and added in 100 mL flask and volume was adjusted up to the mark with
methanol to obtain a standard stock solution of 100µg/ mL
• For CLD buffer media – Take 10 mg of CLD added in a 100 mL volumetric
flask add 20 ml methanol and volume was adjusted up to the mark with
Phosphate buffer pH 6.8+ 1.0 % SLS
• Preparation of calibration curve of Cilnidipine
Aliquots of 0.2 to 1.0 mL stock solution were transferred to a series of 10
mL volumetric flasks and volume in each flask was adjusted to 10 mL with
methanol to obtain concentration of 2-10 µg/mL. Same as above procedure
aliquots of 0.8 to 4.0 mL of stock solution were transferred to a series of 10
mL volumetric flasks and volume in each flask was adjusted to 10 mL with
Phosphate buffer pH 6.8+ 1% SLS to obtain concentration of 8-40 µg/mL.
The solution with suitable concentration was scanned in UV range 200-400
nm using Phosphate buffer pH 6.8+ 1% SLS as a blank and absorption
maxima (λmax) of CLD was recorded.
Development of UV Spectrophotometric Assay Method for
Cilnidipine in methanol at 240 nm
Table 6.4:Assay of cilnidipine in
methanol
Concentration Absorbance
(µg/ml)
0 0
2 0.204±0.0025
4 0.373±0.0015
6 0.578±0.0010
8 0.740±0.0026
10 0.934±0.0012
(n= 3 determination)
Figure 6.3:Assay of cilnidipine in methanol
Development of UV Spectrophotometric Assay Method for
Cilnidipine in Phosphate buffer pH 6.8 + 1% SLS at 243 nm.
Table 6.5:Assay of cilnidipine in

Phosphate buffer 6.8 +1%SLS
Concentration Absorbance
(µg/ml)
0 0
8 0.235 ± 0.002867
12 0.32 ± 0.001633
16 0.41 ± 0.003266
20 0.496 ± 0.006944
24 0.542 ± 0.006236
28 0.609 ± 0.006976
32 0.721 ± 0.006236
36 0.82 ± 0.006976
40 0.895 ± 0.004643
Figure 6.4:Assay of cilnidipine in Phosphate

(n= 3 determination)
buffer 6.8 +1%SLS
6.3 Formulation of Nanosuspension
Cilnidipine dissolve in
Acetone
Stabilizer in water
High speed homogenizer
6.3.1Optimization of process and formulation parameters
• Screening of stirring speed
100 mg of drug dissolve
in 10 ml Acetone
500 mg Poloxamer 188

dissolve in 100 ml water

For different homogenization speed and
different homogenization time
Table 6.6: Protocol of experiments for optimization stirring speed
Preliminary Stirring speed (rpm) Stirring Time (min) Initial Liquid state
observation Stability
trial batch
P1 5000 10 Aggregates --
P4 10000 10 Bluish tinge 1 Day
P5 10000 20 Bluish tinge 2 Days
P6 10000 30 watery 2 Days
P9 15000 30 watery 2 Days
P10 20000 10 Bluish tinge >15 days
P11 20000 20 Bluish tinge >15 days
P12 20000 30 Orange yellow >15 days
At 5000 rpm, nanosuspension was not formed after stirring for 10, 20 or 30 mins.
Homogenization at 10,000 rpm and for 10, 20 and 30 mins produced
nanosuspensions initially but they possessed very low liquid state stability.
At 20,000 rpm, stirring for 10, 20 and 30 mins produced nanosuspensions with
stability of more than 15 days.
Figure 6.5: Nanosuspensions prepared by stirring at 5000 rpm
Figure 6.6: Nanosuspensions prepared by stirring at 10,000 rpm
Figure 6.7:Nanosuspensions after 15

R Kdays (prepared
UNIVERSITY, RAJKOT by stirring at 10,000 rpm) 41
Figure 6.8: Nanosuspensions prepared by stirring at 15,000 rpm
Figure 6.9:Nanosuspensions after 15 days (prepared by stirring at 15,000 rpm)

Figure 6.10:Nanosuspensions prepared by stirring at 20,000 rpm
Figure 6.11:Nanosuspensions after 15 days of (prepared stirring at 20,000 rpm)
Screening of stirring time and stabilizers

in 10 ml Acetone
Different concentration of
stabilizer dissolve in 100
ml water

For 20000 RPM homogenization speed
and different homogenization time
Table 6.7:optimization of stirring time and selection of stabilizers
Batch Stabilizer concentration Stirring Time Initial Liquid state
observation stability(days)
(0.5% w/v) (min)
T1 PVP K30 10 watery 1
T3 Poloxamer 407 10 Bluish tinge 5
T4 Poloxamer 407 20 Bluish tinge >15
T5 Poloxamer 188 10 Bluish tinge >15 (no bluish tinge)
(distinct)
T7 HPMC E5 10 aggregates -
T8 HPMC E5 20 Bluish tinge 2
T9 PVA 10 Bluish tinge 5
T10 PVA 20 Bluish tinge >15
T11 Tween 80 10 Bluish tinge 4
T12 Tween 80 20 Bluish tinge >15
T13 SLS 10 aggregates --
T14 SLS 20 RAJKOT
R K UNIVERSITY, watery 1 hour only 45
• From the above results it was concluded that homogenization at 20000 rpm
and for 20 mins with stabilizer concentration (0.5% w/v) would produce
nanosuspension of good quality in terms of particle size and liquid state
stability. To check whether increasing the stabilizer concentration (1% w/v)
would yield nanosuspensions after homogenization at 20,000 rpm and for
10 mins, further experiments were carried out.
Table 6.8:Optimization of stirring time and selection of stabilizers
Batch Stabilizer concentration Stirring Time Initial Liquid state
observation stability(days)
(1% w/v) (min)
T15 PVP K30 10 Watery 1

T17 Poloxamer 407 10 Bluish tinge 6
T19 Poloxamer 188 10 Bluish tinge >15 (no bluish tinge)
(Distinct)
T21 HPMC E5 10 Aggregates --
T22 HPMC E5 20 Bluish tinge 3
T23 PVA 10 Bluish tinge 5
T24 PVA 20 Bluish tinge >15
T25 Tween 80 10 Bluish tinge 5
T26 Tween 80 20 Bluish tinge >15
T27 SLS 10 Aggregates --
T28 SLS 20 watery 4 hours
• From the above results it was concluded that, homogenization
at 20,000 rpm and for 20 mins is crucial for preparation of
nanosuspension. Moreover, nanosuspensions were not
obtained with PVP K30, SLS and HPMC E5 while Poloxamer
407, Poloxamer 188, PVA and Tween 80 yielded
nanosuspensions
Selection of stabilizer

in 10 ml Acetone
Different concentration of
stabilizer dissolve in 100
ml water

For 20000 RPM homogenization speed
and homogenization 20 min time
Prepared batches analyze for particle

size analysis by zetasizer, Drug content,
centrifugation study
Table 6.9:Protocol of experiments for selection of stabilizers
Batch Stabilizer concentration Particle PDI Centrifugation Drug

size nm study Content %
(0.5% w/v)
B1 Poloxamer 407 92.12 0.101 No settling 98.98 %

B2 Poloxamer 188 140.0 0.138 No settling 99.12 %
B3 PVA 350.4 0.062 No settling 99.45%
B4 Tween 80 317.5 0.091 No settling 99.78%
Figure 6.12:Particle size analysis of B1 (stabilizer Poloxamer407)
Figure 6.13:Particle size analysis of B2 (stabilizer Poloxamer 188)
Figure 6.14: Particle size analysis of B3 (stabilizer PVA)
Figure 6.15:Particle size analysis of B4 (stabilizer Tween 80)
• Nanosuspensions of Cilnidipine were obtained successfully
with Poloxamer 188 & 407, PVA and Tween 80 while PVP
K30, SLS and HPMC E5 were unable to produce
Nanosuspensions.
• From the above result nanosuspension produce with 20000
RPM homogenization speed and 20 minute homogenization
time and 0.5% concentration of stabilizer Poloxamer 407.
• Now further trial will taken using the central composite
design.
6.3.2 Optimization of prepared nanosuspension by central
composite design
• Central composite design (DESIGN EXPERT® 7.1.5) with 2 factors,
5 levels, and 13 runs was selected for the optimization study. The
independent and dependent variables are listed in table.
Table 6.10:Factors and their different levels for Central composite design for
preparation nanosuspension
Independent Variables Levels
Lowest Low Medium High Highest
(-α) (-1) (0) (+1) (+α)
Poloxamer 407 concentration
0.36 0.4 0.5 0.6 0.64
(%) (X1)
Stirring time (X2) Min 12.93 15 20 25 27.07
Transformed values -1.414 -1 0 +1 +1.414
Y1 particle size (nm)
Dependent variables Y2 saturation solubility study (mg/ml)
Y3 cumulative percentage release at 5 min (CPR 5min). (%)
Table 6.11:Experimental matrix and results
RUN Independent Variables Responses
X1 (Poloxamer407 X2 Y1 particle Y2 saturation Y3 cumulative
concentration %) (Stirring time ) size (nm) solubility study percentage release
(mg/ml) at 5 min
NS1 0.00 1.414 99.12 45.12 95.11
NS2 -1.414 0.00 195.10 30.75 70.12
NS3 0.00 0.00 92.12 50.12 99.85
NS4 0.00 0.00 94.52 49.12 100.11
NS5 0.00 0.00 91.45 51.48 98.74
NS6 0.00 0.00 93.11 50.14 99.85
NS7 -1.00 1.00 145.45 39.21 79.15
NS8 1.00 -1.00 120.35 40.87 72.98
NS9 0.00 0.00 92.45 50.22 101.11
NS10 1.00 1.00 105.12 42.11 85.09
NS11 1.414 0.00 115.15 46.29 84.10
NS12 0.00 -1.414 160.25 32.12 86.12
NS13 -1.00 -1.00 160 45.11 76.64
Table 6.12:Dependent variables with constraints in Central Composite Design
Response variables Constraints

Y1 particle size (nm) 91 to 95
Y2 saturation solubility study (mg/ml) 45 to 51
Y3 cumulative percentage release at 5 min (CPR 5min). (%) 95 to101
Table 6.13:Regression analysis of central composite design batches
Model Coefficient Y1 particle size Y2 saturation solubility Y3 cumulative percentage
(nm) study (mg/ml) release at 5 min (CPR 5min). (%)
β0 +92.71 +50.07 +99.55

β1 (X1) -28.28 +5.66 +4.91
β2 (X2 ) -21.57 +4.60 +3.18
β12 (X1X2) +0.00 -0.50 +2.50
β3 (X12) +28.71 -5.85 -12.69
β4 (X22) +15.86 -5.60 -5.97
Β5 (X12X2) +14.07 -3.10 +0.82
Β6 (X1X22) +8.28 -3.66 -4.41
Cubic R2 0.9859 0.9955 0.9546

Adjusted R2 0.9662 0.9861 0.8909
PRESS 12176.38 74.81 4281.69
The approximations of response values Y1 particle size (nm), Y2 saturation

solubility study (mg/ml), Y3 cumulative percentage release at 5 min (CPR 5min).
based on the cubic model was most suitable because its PRESS was smallest. 57
R K UNIVERSITY, RAJKOT
Table 6.14:ANOVA table
Source D.F. Sum Square Mean Square F Value p value
Particle size(Y1) (nm)
Model 7 13674 1953.52 49.97 0.0002
X1 1 3200 3200.00 81.85 0.0003
X2 1 1860.50 1860.00 47.59 0.0010
X1X2 1 0.000 0.000 0.000 1.0000
X12 1 5733.41 5733.41 146.64 <0.0001
X22 1 1771.64 1771.64 45.31 0.0011
X12 X2 1 395.75 395.75 10.12 0.0245
X1 X22 1 137.26 137.26 3.51 0.1198
Y2 saturation solubility study (mg/ml)
Model 7 641.65 91.66 156.71 <0.0001
X1 1 128.00 128.00 218.83 <0.0001
X2 1 84.50 84.50 177.46 <0.0001
X1X2 1 1.00 1.00 1.71 <0.0001
X12 1 237.78 237.78 406.51 <0.0001
X22 1 217.88 217.88 372.49 <0.0001
X12 X2 1 19.17 19.17 32.78 0.0023
X1 X22 1 26.75 26.75
R K UNIVERSITY, RAJKOT 45.72 0.0011 58
Continue…
Source D.F. Sum Square Mean Square F Value p value

Cumulative percentage release at 10 min (CPR10 min) (Y3) (%)
Model 7 1478.60 211.23 15.01 0.0044
X1 1 96.33 96.33 6.84 0.0473
X2 1 40.50 40.50 2.88 0.0006
X1X2 1 25.00 25.00 1.78 0.0401
X12 1 1120.07 1120.07 79.57 0.0003
X22 1 247.85 247.85 17.61 0.0085
X12 X2 1 1.34 1.34 0.095 0.7703
X1 X22 1 38.85 38.85 2.76 0.1575
Influence of formulation composition factor on particle size
For dependent variable Y1 if, X1 from −α to +α level increased and keeping X2 at
lower level particle size decreases from 195 to 114 nm. If keeping X1 constant
and X2 level increased from -α to +α angle of repose will decreases up to 160 to
99 nm. A lowest particle size of 91.45 was observed with Poloxamer
concentration 0.50 and stirring time-20 min (Batch 5) which suggests good
particle size.
Figure 6.16: Response surface plot and contour plot showing the effect of X1 and X2 on
particle size
Influence of formulation composition factor on saturation solubility study
lower level solubility increases from 30 to 41 mg/ml. If keeping X1 constant and
X2 level increased from -α to +α angle of repose will increases up to 32 to 48
mg/ml. A highest saturation solubility of 51 mg/ml was observed with Poloxamer
concentration 0.50 and stirring time-20 min (Batch 5) which suggests good
saturation solubility.
Figure 6.17:Response surface plot and contour plot showing the effect of X1 and X2 on
saturation solubility
Influence of formulation composition factor on cumulative percentage
release at 5 min (CPR 5min). (%)
lower level cumulative percentage releases increases from 70 to 84 %. If keeping
X1 constant and X2 level increased from -α to +α angle of repose will increases up
to 86 to 95% A highest cumulative percentage release at 5 min of 100% was
observed with Poloxamer concentration 0.50 and stirring time 0 min (Batch 5)
which suggests cumulative percentage release at 5 min.
Figure 6.18:Response surface plot and contour plot showing the effect of X1 and X2 on cumulative
percentage release at 5 min (CPR 5min). (%)
Optimization of formula and Validation of CCD
Design-Expert® Software
25.00
Overlay Plot
Overlay Plot
Particle size: 95
Particle size
saturation solubility study
Cumulative percentage release at 5 min 22.50 Particle size: 92.9655
Design Points saturation solu 50.0139
Cumulative perc 99.5079
X1 0.50
X1 = A: A
X2 20.00
X2 = B: B
B : B
20.00 5
Particle size: 95 saturation solubility study: 51
Particle size: 91
saturation solubility study: 45
17.50
Cumulative percentage release at 5 min: 95
15.00
0.40 0.45 0.50 0.55 0.60
A: A
Figure 6.19:Overplay plot showing combined effects of factors X1 and X2 on Y1, Y2 and Y3
25.00 Particle size: 92.6763 Overlay Plot
Overlay Plot saturation solu 49.7448
Cumulative perc 98.0926 Particle size: 95
Particle size X1 0.48
saturation solubility study X2 22.54
Cumulative percentage release at 5 min 22.50
Design Points
X1 = A: A
X2 = B: B
B : B
20.00 5
Particle size: 91
17.50
15.00
0.40 0.45 0.50 0.55 0.60
A: A
Figure 6.20: Overplay plot showing combined effects of factors X1 and X2 on Y1, Y2 and Y3
25.00
Overlay Plot
Overlay Plot
Particle size: 95
Particle size
saturation solubility study
Cumulative percentage release at 5 min 22.50
Design Points
Particle size: 92.6934

X1 = A: A saturation solu 49.8566
X2 = B: B Cumulative perc 98.4193
B : B
20.00 X1 5 0.53
X2 18.78
Particle size: 91
17.50
15.00
0.40 0.45 0.50 0.55 0.60
A: A
Figure 6.21: Overplay plot showing combined effects of factors X1 and X2 on Y1, Y2 and Y3
Table 6.15:Results of optimized batches
Sr.No. Responses Experimental Values Predicted Values %Relative
Error*
Y1 particle size (nm) 92 92.96 1.03
Y2 saturation solubility study 49.50 50.01 1.01
CPB1
Y3 cumulative percentage release at 5 min 99 99.50 0.50
Y1 particle size (nm) 92.00 92.67 0.72
CPB2
CPB3
* Relative Error (%) = (predicted value - Experimental value)/predicted value×100 %.

The optimum formulation was prepared according the above values of the factors and
subjected to the evaluation test of responses. The predicted batch shows significant
reproducibility within the percentage deviation. From the result shows that the predictive
value close to the experimental value so design is significant.
6.3.3 Lyophilization of optimized nanosuspensions
• Further transformation into solid products is often required for
physical stability and/or patient convenience reasons.
Generally, there are two methodologies to convert aqueous
dispersions to dry powders, i.e. lyophilization and spray
drying.
• Due to low melting point of Cilnidipine, lyophilization was

chosen in the present study to avoid the higher temperatures
applied during spray drying.
• Nanosuspensions contained in a petridish with the addition of

cryoprotectant (100 %w/w of drug) were frozen in deep
freezer at -40°C for 2 hr for primary freezing. The petridish
were then transferred to freeze dryer and the lyophilization
was carried out under a vacuum at 15 mTorr -54 0 C for 24 hr.
Selection of cryoprotectant
• Three different cryoprotectant D-mannitol, Sucrosse, MCC were
tried. Cryoprotectant selection was carried out using CPB1.
Lyophillization was carried out according to procedure described in.
Based on visual inspection of quality and quantity of the lyophilized
product, cryoprotectant was selected
Table 6.16: Selection of cryoprotectant
Batch Cryoprotectant Appearance of freeze dried product
L1 D-mannitol Fluffy powder

L2 Sucrose Waxy film
L3 MCC Brittle film
Optimization of cryoprotectant concentration
• On the basis of results of batches L1, L2 and L3, cryoprotectant was selected.
For optimization of the cryoprotectant concentration, three different
concentrations of cryoprotectant (50 %, 100 % and 250% w/w of drug) were
taken. From the above results, it was concluded that D- Mannitol 100% w/w of
drug would be sufficient for cryoprotectant effect.
Table 6.17:Optimization of cryoprotectant concentration
Batch Code Optimize batch Cryoprotectant Conc (%) Appearance
L4 OP1 50 Film
L5 OP1 100 Fluffy powder
L7 OP2 50 Film
L10 OP3 50 Film
L11 OP3 100 Film
L12 OP3 250 Film
Final formulation of nanosuspension
Ingredients Qty in 100 ml
Cilnidipine 100.0 mg
Poloxamer 407 500.0 mg
Acetone 100 ml
Purified water 100 ml
Lyophilization
D-mannitol 10 gm
Each capsule (0 size) contains 1.06 gm of lyophilize powder
6.3.4 Characterization of nanosuspension
• 1. Average particle size
Figure 6.22: Particle size analysis of NS 1 to NS 3

Figure 6.23:Particle size analysis RofK UNIVE
NS 4RSto
ITY, NS 6
RAJKOT 72
Figure 6.24:Particle size analysis of NS 7 to NS 9 73
Figure 6.25:Particle size analysis of NS 10 to NS 12 74
Figure 6.26:Particle size analysis of NS 13 to CPB1
Figure 6.27:Particle size analysis of CPB 2 to CPB 3
Table 6.18:Particle size of nanosuspension
Batch Z. Avg (d.nm) Dia. (nm) PDI
NS1 99.00 100.12 0.120
NS2 195.00 200.15 0.138
NS3 92.12 90.12 0.101
NS4 94.52 95.45 0.105
NS5 91.45 92.00 0.109
NS6 93.00 95.00 0.108
NS7 145.00 160.12 0.138
NS8 120.00 125.45 0.128
NS9 92.45 93.52 0.101
NS10 105.00 110.00 0.108
NS11 115.00 116.12 0.184
NS12 160.00 165.45 0.085
NS13 160.00 168.23 0.086
CPB1 92.33 93.23 0.102
CPB2 92.03 92.12 0.108
CPB3 92.70 R K UNIVERSITY, RAJKOT92.15 0.102 77
2. XRD analysis
Figure 6.28:XRD analysis of pure drug, optimized nanosuspension, optimized

lyophilized suspension
XRD study was performed to determine the change in crystalline nature of the pure
drug after formulation of lyophilized nanosuspension. From the data obtained from
XRD analysis, decrease in peak height was observed.
3. DSC studies
Figure 6.29:DSC thermograms of Pure drug optimized nanosuspension, optimized lyophilized

suspension
The pure drug shows sharp peak at 139.30 C corresponding to its melting point.
On the other hand, CPB1 showed peak at 136.70 C. This reduction in enthalpy
and melting point can be due to decreased crystallinity of pure drug which is
supported by results of XRD analysis.
5. Drug content
• Lyophilized nanosuspensions equivalent to 10 mg of drug was taken in 100 ml
volumetric flask and diluted up to 100 ml with methanol. The absorbance of resulting
solution was measured at 240.0 nm and drug content was calculated.
Table 6.19: Drug content of nanosuspension
Batch Drug content (%)
NS1 98.98±0.12
NS2 99.16±0.13
NS3 99.45±0.56
NS4 99.12±0.01
NS5 102.01±0.04
NS6 101.01±0.1
NS7 100.02±0.4
NS8 99.12±0.09
NS9 100.14±0.2
NS10 98.23±0.5
NS11 100.24±0.2
NS12 98.56±0.1
NS13 99.67±0.13
CPB1 101.01±0.1
CPB2 100.02±0.4
CPB3 99.12±0.09
L5 101.01±0.1
L8 100.02±0.4
L11 101.01±0.1
6. Saturation solubility determination
• Saturation solubility of lyophilized nanosuspensions were carried out in
distilled water known excess of lyophilized nanosuspension (200 mg) were
added to 10 ml distilled water. The sample was rotated at 20 rpm in an
orbital shaker at 25± 0.50 C for 24 hr. The stirred samples were further
taken in test tubes and centrifuged at 10,000 rpm for 15 minutes.
Supernants was then filtered (0.45 μm, Gelman, Mumbai) suitably diluted
and analyzed spectrophotometrically at 243 nm. Triplicate determination
was perform.
Table 6.20:Saturation solubility study
BATCH Saturation solubility determination (mg/ml)

L5 10.00
L8 9.52
L11 9.81
Pure drug 0.000566
7. In vitro Dissolution
• Dissolution studies of nanosuspensions were performed in triplicate
using USP Type II dissolution apparatus. nanosuspensions
equivalent to 10 mg of cilnidipine were taken and placed in
dissolution vessels containing 900 ml of Phosphate buffer 6.8+1%
SLS in maintained at 37 ± 0.50 C and stirred at 75 rpm.
• Samples were withdrawn using 0.22µ nylon merck filter at 1 to 10
mins and replaced with fresh dissolution medium. Samples were
suitably diluted and concentration of cilnidipine was determined
spectrophotometrically at 243 nm.
• For lyophilized nanosuspension equivalent to 10 mg of cilnidipine
were taken and placed in dissolution vessel.
Figure 6.30: Dissolution profile of nanosuspension NS1 to NS13
Figure 6.31:Dissolution profile of nanosuspension CPB1 to CPB3
From the dissolution studies it is evident that dissolution velocity of

Cilnidipine was dramatically increased in case of freeze dried
nanosuspensions. The % drug released in 5 mins was 90% CPB1 as compared
to RLD. Almost 100% drug dissolved from freeze dried nanosuspensions in 5
mins while only 45% of pure drug got dissolved in 60 mins. So it is clear that
by freeze dried nanosuspension increased rate and extent of dissolution of
Cilnidipine. This may be due to increased saturation solubility, decrease in
particle size to nano size and subsequent increase in surface area of particles
and decreased crystallinity of particles.
6.3.4 Development of dosage form
• Lyophilized nanosuspension equivalent to 10 mg of drug was filled in hard
gelatin capsule shells and they were evaluated for following parameters.
1. Disintegration time
• Place 1 capsule in each of the six tubes of the basket and, place a disc.
Operate the apparatus, using purified water as media maintained at 37 ± 2
°C. The disintegration time was noted when there was no residue on the
screen of apparatus.
Table 6.21:Disintegration time of finish product
Batch Disintegration time (min)
C1 2 min 30 sec
C2 1 min 59 sec
C3 2 min 10 sec
2. Dissolution studies
• Dissolution studies of nanosuspensions were performed in

triplicate using USP Type II dissolution apparatus. 1 capsule
were taken and placed in dissolution vessels containing 900 ml
of 1% SLS in maintained at 37 ± 0.50 C and stirred at 75 rpm.
• Samples were withdrawn at 1 to 10 mins and replaced with
fresh dissolution medium. Samples were suitably diluted and
concentration of cilnidipine was determined
spectrophotometrically at 243 nm
100
90
80
70
CPR (%)
60
50 C1
40 C2
30 C3
20
10
0
0 2 4 6 8 10 12
Time (min)
Figure 6.32: Dissolution profile of capsule
6.4 Formulation and development of
self nanoemulsifying drug delivery
system
6.4.1 Solubility determination of Cilnidipine in oils
Add an excess amount of drug in 2mL
of selected oils and mixed using a
vortex mixer for 10 min
kept for 72 h at room

temperature to reach
equilibrium
Samples were centrifuged at 3000 rpm

for 15 min then supernatant was taken
Cilnidipine was determined

using UV spectrophotometer
Table 6.22: Solubility of Cilnidipine in various oils
Sr. Oils Solubility (mg/mL) Mean

No. I II III
1. Capmul MCM 288 286.5 288.6 287.7 ± 1.08
2. Capmul MCM PG8 178 177.2 178.4 177.8 ± 0.61
3. Capmul GMO 50 52 50.7 50.9 ± 1.01
4. Captex 335 6.3 5.8 5.5 5.86 ± 0.44
5. Captex 200 6.8 6.0 7.1 6.63 ± 0.56
6. Acconon MC8-2EP 28 .0 27.6 28.9 28.16 ± 0.66
7. Labrafec PG 10 0 11.4 10.3 10.56 ± 0.73
8. Labrafec lipophile 4.9 5.3 5.8 5.33 ± 0.45
WL1349
9. Labrafil 1944 CS 15 0 16.2 15.9 15.7 ±0.625
10. Capryol 90 40.0 42.0 41.6 41.2 ±1.058
11. Miglyol 810 2.0 2.4 2.7 2.36 ± 0.351
12. Miglyol 812 2.3 3.1 2.6 2.66 ±0.404
13. Miglyol 840 2.8 3.5 3.3 3.2 ±0.36
Solubility of Cilnidipine in various oils
Solubility (mg/ml)
300
250
200
150
100
50
0
Labrafil 1944 CS
lipophileWL134
Captex 335
Captex 200
Miglyol 810
Miglyol 812
Miglyol 840
Capmul GMO
Capmul MCM
Capmul MCM
Capryol 90
Labrafec PG
Acconon MC8-
Labrafec
PG8
2EP
Oils
Figure 6.33: Solubility of cilnidipine in various oils

Solubility of Cilnidipine was determined in thirteen different oils to find out
highest solubility of Cilnidipine. The highest solubility of Cilnidipine was
observed in Capmul MCM as showed in Figure 6.33. The highest solubility
was obtained in Capmul MCM. Hence, these were chosen as oil phase for
preparation of micro emulsion.
6.4.2 Solubility determination of Cilnidipine in Surfactants
and Co-Surfactants
Add an excess amount of drug in 2 mL of
selected Surfactant & Co-surfactant and
mixed using a vortex mixer for 10 min

equilibrium


Table 6.23: Solubility of Cilnidipine in Surfactant & Co- surfactant
Sr. No. Oils Solubility (mg/mL) Mean
I II III
Surfactant
1. Cremophore RH40 28.0 28.6 29.4 28.66 ± 0.70
2. Cremophore ELP 130.0 128.7 128.3 129 ± 0.88
3. Tween 80 97.7 98.3 97.0 97.66 ± 0.65
4. Tween 20 62.7 61.4 62.0 62.03 ± 0.65
5. Span 80 39.5 40.0 40.7 40.06 ± 0.60
6. Span 20 26.4 27.9 25.8 26.7 ± 1.08
7. Labrasol 81.3 80.1 80.7 80.7 ± 0.6
Co- Surfactant
1. Propylene glycol 187.0 186.6 188.1 187.2 ± 0.77
2. PEG 400 163.0 164.3 163.5 163.6 ± 0.65
Figure 6.34: Solubility of cilnidipine in surfactant & co- surfactant
Satisfactory solubility of Cilnidipine was observed in Tween 80 &

Cremophore ELP and PG as showed in Figure 6.34. Hence, Tween 80 &
Cremophore ELP and PG were chosen as surfactant and co surfactant
respectively for preparation of micro emulsion.
6.4.3 Construction of Pseudo ternary Phase diagram
• Surfactant and co surfactant (Smix) in each group were mixed in different
weight ratios (1:1, 2:1, and 3:1).
• These Smix ratios were chosen in increasing concentration of surfactant
with respect to co surfactant. Based on solubility study Capmul MCM was
selected as oil phase, tween 80 and Cremophore ELP as surfactant,
propylene glycol as co surfactant.
• For each phase diagram, oil and specific Smix ratio was mixed thoroughly
in different weight ratios from 1:9 to 9:1 in different glass vials, a
transparent and homogenous mixture of oil and S/CoS was formed by
vortexing for 5 min.
• Then each mixture was titrated with water and visually observed for phase
clarity and flowability. The concentration of water at which turbidity-to-
transparency and transparency-to-turbidity transitions occurred was derived
from the weight measurements.
• Phase diagrams were then constructed using CHEMIX-School 3.51
software.
• The following different combination of oil, surfactant and co
surfactant were screened.
• 1. Capmul MCM, Cremophore ELP, Propylene glycol
• 2. Capmul MCM, Tween 80, Propylene Glycol
Figure 6.35: A, B & C Pseudo ternary phase diagram of SNEDDS containing Capmul
MCM, Cremophore ELP, PG with Smix ratio 1:1, 2:1, 3:1 respectively
Figure 6.36: A, B &C pseudo ternary phase diagram of SNEEDS containing
Capmul MCM, Tween 80 and PG with Smix ratio 1:1, 2:1, 3:1 respectively.
• Pseudo ternary phase diagram of system consisting of Capmul
MCM, Tween 80 and propylene glycol as oil, surfactant and co
surfactant respectively was plotted. The micro emulsion
existence region is showed in fuchsia color and as the ratio of
surfactant to co surfactant increases the micro emulsion region
was increased.
• The micro emulsion regions observed for second combinations
were lesser than the first combination. The combination of
Capmul MCM, Cremophore ELP and propylene glycol was
selected for formulation.
6.4.4 Preparation of SNEDDS formulations
Take weighed quantity of Cremophore ELP as

Surfactant and propylene glycol as Co-surfactant
heat it at 37˚C mix properly
Add weighed quantity of Capmul

MCM as oil heat it at 37˚C mix
properly
Add 10 mg of Cilnidipine in previous

mixture mix it properly through vortex
mixture and store at room temperature

Table 6.24: Formulations of Cilnidipine SNEDDS (Trial batch)
Batch No. Drug (mg) Oil % Surfactant % Co surfactant % S mix

F1 10 10 45 45 1:1
F2 10 15 42.5 42.5 1:1
F3 10 20 40 40 1:1
F4 10 25 32.5 32.5 1:1
F5 10 30 35 35 1:1
F6 10 10 60 30 2:1
F7 10 15 56.6 28.3 2:1
F8 10 20 53.3 26.6 2:1
F9 10 25 50 25 2:1
F10 10 30 46.6 23.3 2:1
F11 10 10 67.5 22.5 3:1
F12 10 15 63.7 21.3 3:1
F13 10 20 60 20 3:1
F14 10 25 56.2 18.7 3:1
F15 10 30 52.5 17.75 3:1
From the result of trial batches design batches will be prepared by simplex
lattice design.
6.4.5 Preparation of solid SNEDDS
• Solid SNEDDS were prepared by use of Lactose as adsorbent
to load Cilnidipine SNEDDS.
• The lipid formulations were added in increments and blended
with the adsorbent at the following fixed SNEDDS to
adsorbent ratios by weight: 0.25:1, 0.5:1, 0.75:1, 1:1, 1.25:1,
1.5:1, 2:1, 2.25:1, 2.5:1, 3:1, 3.25:1.
• Briefly, a constant aliquot of SNEDDS was initially added to
and mixed with the adsorbent in a mortar. Addition of
SNEDDS to the adsorbent however was discontinued once a
non-flowing cohesive mass is formed.
• Among all the different ratios, 1.5:1 form free flowing
granules was selected for solidification of SNEDDS.

6.4.6Optimization of prepared SNEEDS by Simplex lattice
design design
• Simplex lattice design (DESIGN EXPERT® 7.1.5) with 3 factors, 6
levels, and 14 runs was selected for the optimization study. The
independent and dependent variables are listed in table.
Table 6.25: Factors and their different levels for Central composite design
Independent Levels
Variables
Transformed values (0.00) (0.167) (0.333) (0.50) (0.667) (1.00)
Oil % (X1) 10 15 20 25 30 35
Surfactant % (X2) 60 56.6 53.3 50 46.6 32.5
Co surfactant % ( X3 ) 30 28.3 26.6 25 23.3 32.5
Dependent variables Y1 particle size (nm)
Y2 cumulative percentage release at 5 min (CPR 5 min). (%)
Y3 cumulative percentage release at 10 min (CPR 10 min). (%)

Table 6.26:Experimental matrix and results
RUN Independent Variables Responses
X1 X2 X3 Y1 particle Y2 cumulative Y3 cumulative
Oil % Surfactant Co size (nm) percentage release at percentage release at
% surfactant 5 min (CPR 5min). (%) 10 min
% (CPR 10min). (%)
SN1 0.500 0.000 0.500 25.12 13 78
SN2 1.000 0.000 0.000 55.10 24 55
SN3 1.000 0.000 0.000 54.15 24 54
SN4 0.500 0.500 0.000 25.01 22 85
SN5 0.000 1.000 0.000 100.01 42 95
SN6 0.167 0.667 0.167 85.56 25 87
SN7 0.000 0.000 1.000 99.23 30 52
SN8 0.667 0.167 0.167 45.22 30 68
SN9 0.500 0.500 0.000 25.12 25 83
SN10 0.333 0.333 0.333 10.59 48 99
SN11 0.167 0.167 0.667 23.02 35 80
SN12 0.000 1.000 0.000 100.02 45 94
SN13 0.000 0.000 1.000 10005 31 55
SN14 0.000 0.500 0.500 66.22 10 54
Table 6.27:Dependent variables with constraints in Simplex lattice design
Response variables Constraints

Y1 particle size (nm) 10 to 25
Y2 cumulative percentage release at 5 min 35 to 48
(CPR 5 min). (%)
Y3 cumulative percentage release at 10 min ≥ 85
(CPR 10 min). (%)

Table 6.28:Regression analysis of simplex lattice batches
Model Coefficient Y1 particle Y2 Cumulative Y3 Cumulative
size (nm) percentage percentage release
release at 5 min at 10 min (CPR
(CPR 5min). (%) 10min). (%)
β1 (X1) + 55.34 +23.79 +54.21

β2 (X2 ) + 100.84 +43.29 +94.21
Β3 (X3) + 100.34 +30.29 +53.21
β12 (X1X2) -205.64 -41.83 +36.85
β13 (X1X3) -197.92 -59.48 +92.54
β23 (X2X3) -124.92 -110.48 -83.46
Β123(X1X2X3) -29.10 +954.38 +580.49
Β4 X1 X2 (X1 -X2 ) -441.24 +170.83 -28.85
Β5 X1 X3 (X1 –X3 ) + 681.48 -152.66 -188.30
Cubic r2 0.9614 0.9722 0.9812
Adjusted r2 0.8997 0.9276 0.9510
PRESS 67617.37 4143.55 7980.82
the approximations of response values Y1 particle size (nm), Y2 cumulative

percentage release at 5 min (CPR 5min), Y3 cumulative percentage release at 10
min (CPR 10 min). Based on the cubic model was most suitable because its PRESS
was smallest. R K UNIVERSITY, RAJKOT 106
Table 6.29: ANOVA table of simplex lattice batches
Source Sum Square D.F. Mean Square F Value p value
Particle size (Y1) (nm)
Model 14131.49 8 1766.44 15.58 0.0039
X1 136.41 1 136.41 1.20 0.0227
X2 24.22 1 24.22 0.21 0.633
X3 1123.17 1 1123.17 9.91 0.0254
X1 X2 3529.86 1 3529.86 31.14 0.0025
X1 X3 1976.38 1 1976.38 17.43 0.0087
X2 X3 787.33 1 787.33 6.95 0.0462
X1 X2 X3 0.91 1 0.91 8.056E-003 0.9320
X1 X2 (X1- X2) 778.83 1 778.83 6.87 0.0470
X1 X3 (X1 -X3) 1903.96 1 1903.96 16.80 0.0094

Model 1531.86 8 191.48 21.83 0.0018
X1 203.44 1 203.44 23.19 0.0048
X2 0.065 1 0.065 7.432E-003 0.0046
X3 134.83 1 134.83 15.37 0.0011
X1 X2 146.04 1 146.04 16.65 0.0095
X1 X3 178.52 1 178.52 20.35 0.0063
X2 X3 615.86 1 615.86 70.21 0.0004
X1 X2 X3 982.41 1 982.41 112.00 0.0001
X1 X2 (X1- X2) 116.74 1 116.74 13.31 0.0148
X1 X3 (X1 -X3) 95.54 1 95.54 10.89 0.0215


Model 3856.32 8 482.04 32.55 0.0007
X1 2.49 1 2.49 017 0.0086
X2 74.31 1 74.31 5.02 0.0752
X3 402.50 1 402.50 27.18 0.0034
X1 X2 113.34 1 113.34 7.65 0.0395
X1 X3 432.10 1 432.10 29.18 0.0029
X2 X3 351.40 1 351.40 23.73 0.0046
X1 X2 X3 363.44 1 363.44 24.55 0.0043
X1 X2 (X1- X2) 3.33 1 3.33 0.22 0.6554
X1 X3 (X1 -X3) 145.37 1 145.37 9.82 0.0259

Influence of formulation composition factor on particle size
For dependent variable Y1 if, X1 from 0.00 to +1.00 level increased and keeping
X2 & X3 at constant particle size decreases from 100 to 55 nm. If keeping X1, X3,
constant and X2 level increased from 0.00 to +1.00 particle size will decreases up
to 100 to 55 nm. If keeping X1, X2, constant and X3 level increased from 0.00 to
+1.00 particle size will decreases up to 100 to 25 nm. A lowest particle size of
10.59 was observed with oil % 20, surfactant % 53.3, Co-surfactant % 26.6 (Batch
10) which suggests good particle size.
Figure 6.37:Response surface plot and contour plot showing the effect of X1, X2 and
X3 on particle size R K UNIVERSITY, RAJKOT 110
Influence of formulation composition factor on Cumulative percentage
release at 5 min (CPR5 min) (Y2) (%)
For dependent variable Y2 if, X1 from 0.00 to +1.00 level increased and keeping X2 & X3 at
constant particle Cumulative percentage release at 5 min will decreases from 45 to 24 %. If
keeping X1, X3, constant and X2 level increased from 0.00 to +1.00 Cumulative percentage
release at 10 min will increases up to 24 to 45%. If keeping X1, X2, constant and X3 level
increased from 0.00 to +1.00 Cumulative percentage release at 5 in will increases up to 22
to 31 %. A highest Cumulative percentage release at 5 min of 48% was observed with oil %
20, surfactant % 53.3, Co-surfactant % 26.6 (Batch 10) which suggests higher Cumulative
percentage release at 5 min.
X3 on Cumulative percentage release at 5 min (CPR
R K UNIVERSITY, RAJKOT 5 min
) (Y2) (%) 111
Influence of formulation composition factor on Cumulative percentage
release at 10 min (CPR10 min) (Y3) (%)
For dependent variable Y2 if, X1 from 0.00 to +1.00 level increased and keeping X2 & X3 at
constant particle Cumulative percentage release at 10 min will decreases from 95 to 55 %.
If keeping X1, X3, constant and X2 level increased from 0.00 to +1.00 Cumulative
percentage release at 10 min will increases up to 54 to 95%. If keeping X1, X2, constant and
X3 level increased from 0.00 to +1.00 Cumulative percentage release at 10 min will
decreases up to 85 to 55 %. A highest Cumulative percentage release at 10 min of 99% was
observed with oil % 20, surfactant % 53.3, and Co-surfactant % 26.6 (Batch 10) which
suggests higher Cumulative percentage release at 10 min.
X3 on Cumulative percentage release at 10 min (CPR10 min) (Y3) (%)
Optimization of formula and Validation of Simplex lattice
design
A: OIL (%)
Design-Expert® Software 1.000
2
Overlay Plot
PARTICLE SIZE (nm)

CUMULATIVE PERCENTAGE RELEAGE AT 5min
CUMULATIVE PERCENTAGE RELEASE AT 10 min
Design Points PARTICLE SIZE (nm): 10
PARTICLE SIZE ( 25.516

CUMULATIVE PE PARTICLE SIZE (nm): 28
44.3309
Std # 10 Run # 10
CUMULATIVE PE 93.7617
2 CUMULATIVE PERCENTAGE RELEAGE AT 5min: 34
X1 = A: OIL (%) = 0.333 X1 0.333 CUMULATIVE PERCENTAGE RELEASE AT 10 min: 85
0.000 0.000
X2 = B: SURFACTANT (%) = 0.333 X2 0.333
X3 = C: CO-SURFACTANT(%) = 0.333 X3 0.333
PARTICLE SIZE (nm): 28

CUMULATIVE PERCENTAGE RELEAGE AT 5min: 34

CUMULATIVE PERCENTAGE RELEASE AT 10 min: 85
2 2
1.000 0.000 1.000
B: SURFACTANT (%) C: CO-SURFACTANT(%)
Overlay Plot
Figure 6.40: Overplay plot showing combined effects of factors X1 , X2 and X3 on

Y1, Y2 and Y3 R K UNIVERSITY, RAJKOT 113
A: OIL (%)
2
Overlay Plot
PARTICLE SIZE (nm)

CUMULATIVE PERCENTAGE RELEASE AT 10 min
Design Points PARTICLE SIZE (nm): 10

X1 = A: OIL (%)
X2 = B: SURFACTANT (%) PARTICLE SIZE ( 14.5689
2 CUMULATIVE PERCENTAGE RELEAGE AT 5min: 34
X3 = C: CO-SURFACTANT(%)
0.000 CUMULATIVE PECUMULATIVE
41.6789PERCENTAGE RELEASE0.000
AT 10 min: 85
X1 0.227
X2 0.250
X3 0.522

2 2
1.000 0.000 1.000
Overlay Plot
Figure 6.41:Overplay plot showing combined effects of factors X1 , X2 and X3 on

Y1, Y2 and Y3 R K UNIVERSITY, RAJKOT 114
A: OIL (%)
2
Overlay Plot
PARTICLE SIZE (nm)

CUMULATIVE PERCENTAGE RELEASE AT 10 min PARTICLE SIZE ( 22.3701
Design Points CUMULATIVE PE 41.1351
X1 0.452 SIZE (nm): 28
PARTICLE
X1 = A: OIL (%)
X2 0.211
X2 = B: SURFACTANT (%)
X3 2 0.336 PERCENTAGE RELEAGE AT 5min: 34
CUMULATIVE
X3 = C: CO-SURFACTANT(%)
0.000 CUMULATIVE PERCENTAGE RELEASE0.000
AT 10 min: 85


2 2
1.000 0.000 1.000
Overlay Plot
Figure 6.42:Overplay plot showing combined effects of factors X1 , X2 and X3 on

Y1, Y2 and Y3
Sr. Responses Predicted Experiment %Relative
No. Values al Values Error*
CPB1 Y2 cumulative percentage release at 5 min (CPR 5min) (%) 44.33 45 1.48
Y3 cumulative percentage release at 10 min (CPR 10min) (%) 93.76 94 0.25
Y1 particle size (nm) 14.56 15 2.00
Y3 cumulative percentage release at 10 min (CPR 10min) (%) 89.77 89 0.85
Y1 particle size (nm) 22.37 23 2.10
Y3 cumulative percentage release at 10 min (CPR 10min)(%) 88.68 88 0.76
* Relative Error (%) = (predicted value - Experimental value)/predicted value×100 %.7

The optimum formulation was prepared according the above values of the factors and
subjected to the evaluation test of responses. The predicted batch shows significant
reproducibility within the percentage deviation. From the result shows that the predictive
value close to the experimental value so design is significant.

6.4.7 CHARACTERIZATION
• Characterization of SNEDDS formulation.
One ml of SNEDDS formulation was diluted with 500ml
distilled water, this micro emulsion solution considered for
further assessment of various in vitro parameters.
• 1 Appearance.
Appearance of fifteen trial batches (F1-F15) design batches
SN1 to SN14 was tested against white & black background &
turbidity were checked, the test was carried out described in
United States pharmacopeia.
All the formulations were observed for 24 h for clarity,
phase separation and precipitation of drug. The formulations
were categorized as clear (transparent or transparent with
bluish ting), non-clear (turbid or milky), stable (no
precipitation at the end of 24 h) and unstable (phase
separation or precipitate with 24 h).
Final formulations of solid SNEDDS
Ingredients Formula for 100 ml
Cilnidipine 1.0 gm
Capmul MCM 20 gm (20%)
Tween 80 53.3 gm (53.3%)
Propylene glycol 26.6 gm (26.6%)
Total 100.9 gm
SNEEDS to adsorbent ratio 1.5:1 finalized
Lactose 67.26 gm
Each capsule (0 size) contains solid SNEDDS powder 1.68 gm

Table 6.33:Visual Observation of trial batches
Batch No. Precipitation Phase separation Clarity
F1 √ √ √
F2 √ √ √
F3 √ √ ×
F4 √ √ ×
F5 √ √ ×
F6 √ √ √
F7 √ √ √
F8 √ √ √
F9 √ √ ×
F10 √ √ ×
F11 √ √ √
F12 √ √ √
F13 √ √ √
F14 √ √ ×
F15 √ √ ×
Where, √ = batch passed and × = batch failed
Table 6.34:Visual observation of design batches
Batch No. Precipitation Phase separation Clarity
SN1 √ √ √
SN2 √ √ √
SN3 √ √ √
SN4 √ √ √
SN5 √ √ √
SN6 √ √ √
SN7 √ √ √
SN8 √ √ √
SN9 √ √ √
SN10 √ √ √
SN11 √ √ √
SN12 √ √ √
SN13 √ √ √
SN14 √ √ √
Where, √ = batch passed and × = batch failed
• 2. Refractive Index
Refractive index measures clarity of prepared system. Abbes Refractometer
(1310 E- 20 Atago, Japan) was used to determine refractive index of 15
fifteen batches (F1-F15), refractive index of distilled water was considered
as standard to compare the values obtained sample. Refractive index
measures the clarity of nanoemulsion.
• 3. pH
pH values of SEDDS (F1-F15) were determine using pH meter calibration
of pH meter was conducted before tablets of pH 4 & pH 7. Each
measurement was carried out in triplicate & results were presented as mean
± standard deviation. The change in the pH may affect the zeta potential of
the formulation which in turn can affect the stability of preparation. All the
formulations showed similar pH values in the range of 7.20 to 7.73.

• 4 . Conductance
Type of emulsion (o/w or w/o) can be determined by measurement of conductance,

it was measured by conductivity meter. conductivity measurement the tested micro
emulsion where prepared with a 0.01 N aqueous solution of sodium chloride.
Which measurement was carried out in triplicate & the results were presented as
mean ± standard deviation.
• 5 . % Transmittance
The clarity of nanoemulsion can be observed by transparency, measured in form of

% transmittance (% T). SEDDS form o/w nanoemulsion since water is external
phase. Formulations having % T greater than 96% indicated high clarity of
nanoemulsion while formulations having % T less than 96% suggested less clarity
of nanoemulsion.
In the present study formulation F8 showed highest transmittance compared to

other formulations.
Table 6.35:Characterization of trial batches
Batch no Refractive Index pH Conductance % Transmittance
F1 1.3330 6.41 96.8 92.5
F2 1.3327 6.53 94.6 96.2
F3 1.3356 6.36 93.4 90.3
F4 1.3358 6.51 92.6 85.7
F5 1.3372 6.68 91.4 81.6
F6 1.3331 6.51 97.2 78.4
F7 1.3334 6.48 97.3 97.2
F8 1.3332 6.52 98.7 98.8
F9 1.3362 6.36 94.7 98.6
F10 1.3369 6.84 92.4 98.6
F11 1.3365 6.75 96.9 92.3
F12 1.3335 6.71 96.0 96.9
F13 1.3364 6.85 96.7 98.4
F14 1.3362 6.74 95.1 92.6
F15 1.3332 6.83 91.6 90.5

Table 6.36:Characterization of design batches
Batch no Refractive Index pH Conductance % Transmittance
SN1 1.3372 7.75 96.9 96.9
SN2 1.3331 7.71 96.0 98.4
SN3 1.3362 7.36 96.7 92.6
SN4 1.3369 7.36 94.7 90.5
SN5 1.3365 7.84 92.4 92.5
SN6 1.3335 7.51 97.2 96.2
SN7 1.3364 7.83 97.3 90.3
SN8 1.3362 7.41 98.7 98.8
SN9 1.3332 7.53 91.6 98.6
SN10 1.3330 7.51 96.8 98.9
SN11 1.3327 7.48 94.6 92.3
SN12 1.3356 7.52 93.4 81.6
SN13 1.3358 7.85 95.4 78.4
SN14 1.3322 7.74 95.1 97.2

6.4.8 Characterization of Solid SNEDDS
1 Visual observation
To assess emulsification properties each formulation (F1-F15) & (SN1- SN14)
was introduced into 100 ml of distilled water in glass beaker at room
temperature thee content was gently stirred manually. The tendency to form
clear or transparent emulsion was judged as good & it was judge bad when
the formulation was poor or milky in appearance.
2 Robustness on dilution.
Robustness to dilution was studied by diluting it 10, 100 and 1000 times
with various dissolution media viz. distilled water, 0.1N HCL and pH 6.8
phosphate buffer. The diluted micro emulsions were stored for 12 h and
observed for any signs of phase separation or drug precipitation.
The formulations F1, F2, F6, F7, F8, F11, F12 and F13 showed no signs of
precipitation, cloudiness or phase separation for 24 h. On the contrary,
formulations F3, F4, F5, F9, F10, F14 and F15 formed turbid dispersions
immediately with 100 and 1000-fold dilutions.

3. Drug content
Assay of weighed amount of formulations were carried out to determine the
drug content.
The weighed samples were dissolved in 10 ml of methanol and the
solutions were filtered, using whatman filter paper.
The content was estimated spectrophotometrically The percentage drug
content was determined by considering 10 mg of Cilnidipine as a100%.
All the formulations are within the specified limit (90-110%)
4. Uniformity of weight of capsule

Uniformity of weight of capsule was determined for all S-SNEDDS
formulation.
The value of average weight of capsules ranges from (310-330 mg). The
weight variation was observed within acceptable limit i.e. less than ± 7.5%
according to IP 2014.

Table 6.37:Drug content of design batches & trial batches
Batch No Drug content Batch No Drug content
F1 99.2 SN1 96.9
F2 96.3 SN2 96.0
F3 95.4 SN3 96.7
F4 98.7 SN4 94.7
F5 95.2 SN5 92.4
F6 97.5 SN6 97.2
F7 94.2 SN7 97.3
F8 98.1 SN8 98.7
F9 92.5 SN9 91.6
F10 98.4 SN10 96.8
F11 98.5 SN11 94.6
F12 98.4 SN12 93.4
F13 97.4 SN13 95.4
F14 96.3 SN14 95.1
F15 93.2
• Dissolution studies of SNEDDS were performed in triplicate
using USP Type II dissolution apparatus. nanosuspensions
equivalent to 10 mg of cilnidipine were taken and placed in
dissolution vessels containing 900 ml of 1% SLS in
maintained at 37 ± 0.50 C and stirred at 75 rpm.
• Samples were withdrawn using 0.22µ nylon merck filter at 1
to 10 mins and replaced with fresh dissolution medium.
Samples were suitably diluted and concentration of cilnidipine
was determined spectrophotometrically at 243 nm.
• For solid- SNEDDS equivalent to 10 mg of cilnidipine were
taken and placed in dissolution vessel.

Figure 6.43:Dissolution profile of F1 to F10 batches
Figure 6.44:Dissolution profile of F11to F15batches
From the above dissolution graph conclude that F8 batch release the 100 % drug with
in the 10 mins.

Figure 6.45:Dissolution profile of SN11to SN14batches
From the dissolution profile of design batches SN 10 Batch release 100 % drug
with in the 10 mins which is optimized batch in trial design and check point batch.
Comparison of design and MKT batches
Figure 6.46:Comparision of dissolution profile
From the above graph concluded that the MKT formulation Cilacar tablets
10 mg dissolve 100 % drug with in 60 mins and SNEDDS formulation
dissolve drug 100 % with in 10 mins.

• Particle size and zeta potential analysis
The droplet size of the emulsion is a crucial factor in self-

emulsification performance because it determines the rate and extent
of drug release as well as drug absorption.
Also, it has been reported that the smaller particle size of the
emulsion droplets may lead to more rapid absorption and improve
the bioavailability.
Zeta potential is a measure of the magnitude of the repulsion or
attraction between particles. This could be because the emulsifier
used in the formulation is a nonionic-surfactant.

Table 6.38: Particle size of Trial and design batches
Batch No Particle size Zeta potential Batch No Particle size Zeta potential
F1 106.3 -6.67 SN1 25.12 -18.35
F2 88.12 -10.28 SN2 55.10 -10.45
F3 90.45 -18.45 SN3 54.15 -10.78
F4 80.12 -17.22 SN4 25.01 -17.00
F5 70.14 -15.45 SN5 100.01 -6.00
F6 25.41 -17.25 SN6 85.56 -8.45
F7 29.47 -18.20 SN7 99.23 -5.78
F8 10.59 -22.11 SN8 45.22 -12.45
F9 26.93 -18.35 SN9 25.12 -18.45
F10 67.49 -12.86 SN10 10.59 -22.11
F11 45.26 -16.42 SN11 23.02 -19.45
F12 33.68 -13.89 SN12 100.02 -16.45
F13 25.45 -17.26 SN13 10005 -15.48
F14 35.46 -13.25 SN14 66.22 -12.45
F15 66.25 -12.45 CPB1 10.60 -22.20
CPB2 10.10 -21.22 CPB3 10.20 -22.31
Figure 6.47: Particle size and zeta potential of optimized formulation

6.5 Formulation development of lipid
nanoparticles of cilnidipine by micro
emulsion technique

6.5.1 Solubility determination of Cilnidipine in Solid lipid
50 mg of Drug was weighed accurately.
The weighed quantity of the solid lipid was

taken and added in the small increment to the
Drug.
The mixture was heated using Water bath and

mixed using Cyclone vortex mixer. The solid
lipid was added till the clear melt achieved.
The remaining amount of the solid lipid is

weighed again and by that way the quantity
required to dissolve 50 mg Drug was found
out.

Table 6.39:Solubility Study of Cilnidipine in Different Solid Lipids
Sr. No. Solid lipid Amount of liquid required

to dissolve 50 mg of drug
1. Glyceryl monostearate (GSM) 1120±13mg
2. Stearic acid (SA) 1024±9mg
3. Compritol 888 ATO (Com888 ATO) 1307±6mg
4. Geleol 1087±7mg
5. Gelucire 43/01 (Gel 43/01) 961±5mg
6. Gelucire 44/14 (Gel 44/14) 525±9mg
7. Gelucire 50/13 (Gel 50/13) 520±3mg
8. Gel 43/01:Gel 50/13 (1:1) Two layers seperated
9. Gel 43/01:Gel 44/14 (1:1) Two layers seperated
10. Gel 44/14:Com 888 ATO (1:1) 750±10mg
11. Gel 50/13:Com 888 ATO (1:1) 700±10mg
12. Gel 50/13:Com 888 ATO (2:1) 663±9mg
13. Gel 50/13:Com 888 ATO (3:1) 590±12mg
14. Gel 50/13:Com 888 ATO (3:2) 506±10mg

Figure 6.48: Solubility of cilnidipine in different solid lipids
Gelucire 50/13: Compritol 888 ATO (3:2) and that lipid mixture has been found to
be best protective lipid combination which is essential features required for Other
properties of the Gelucire 50/13: Compritol 888 ATO (3:2) like lack of toxicity,
approved regulatory status, cost etc. were also favours its choice as solid lipid.
Therefore, Gelucire 50/13: Compritol 888 ATO (3:2) was selected as a suitable solid
lipid for Cilnidipine.
6.5.3 Solubility Study of Cilnidipine in Different liquid
Lipids/ Surfactant- Co-surfactant
Add an excess amount of drug in 2 mL of selected
Liquid lipid Surfactant & Co-surfactant and mixed
using a vortex mixer for 15 min

equilibrium



Table 6.40:Solubility of Cilnidipine in Liquid lipid
Sr. No. Liquid lipid Solubility (mg/ml)

1. Maisine 35-1 7.1
2. Labrafac pg 7.9
3. Labrafac lipophile wl 1349 1.7
4. Labrafil m 1944 10.8
5. Captex 200 6.5
6. Captex 355 4.3
7. Miglyol 810 12.5
8. Miglyol 812 16.8

Figure 6.49: Solubility of cilnidipine in different liquid lipids
Result of solubility study showed that Cilnidipine had highest solubility in

Miglyol 812 among the liquid lipid tested. Its desirable properties like absence
of inhalation toxicity, high stability against made it best option as liquid lipid.

Table 6.41:Solubility of Cilnidipine in Surfactant
Sr. No. Surfactant Solubility (mg/ml)

1. Plurol diisosterique CG 12.25
2. Labrasol 20.15
3. Peceol 21.62
4. Capmul GMO-50 37.
5. Acconan CCG 60.23
6. Lutrol F 68 42
7. Lutrol F 127 31
8. Cremophore A 25 112
9. Cremophore EL 34.75
10. Cremophore ELP 37.82
11. Cremophore RH 40 132
12. Solutol HS 15 104
13. Tween 80 102

Solubility (mg/ml)
140
120
100
80
60
40
20
0
Cremophore EL
Peceol
Solutol HS 15
Capmul GMO-50
Lutrol F 68
Lutrol F 127
Cremophore RH 40
Labrasol
Acconan CCG
Plurol diisosterique CG
Tween 80
Cremophore A 25
Cremophore ELP
Surfactant
Figure 6.50: Solubility of cilnidipine in different surfactants
Result of solubility study showed that Cilnidipine had highest solubility in

Cremophore RH 40 among the surfactant tested. Its desirable properties like
absence of inhalation toxicity, high stability against made it best option as
surfactant.

Table 6.42:Solubility of Cilnidipine in Co-Surfactant
Sr. No. Co-Surfactant Solubility (mg/ml)
1. Plurol-oleique cc 497 2.4
2. Lauroglycol 90 55
3. Capmul mcm 11.14
4. Capmul mcm c8 13.5
5. Caproyl 90 80.5
6. Propylene glycol 57
7. PEG-400 102
Figure 6.51: Solubility of cilnidipine in different co-surfactants

6.5.4 Construction of Pseudo ternary Phase diagram
• The lipid phase was taken in 9 test tubes such that 1st tube contain 1
portion of lipid and 9 portion of surfactant and 9th All the tubes
were maintained at 80-90˚C tube contains 9 portions of lipid and
one portion of surfactant.
• In the remaining seven tubes lipid and surfactant ratio was
maintained as 2:8, 3:7, 4:6, 5:5, 6:4,7:3,8:2 respectively. All the
tubes were maintained at 70˚C and water to be titrated was also
maintained at same temperature separately. Resultant blends of lipid
and surfactant were titrated with water carefully wherein water was
added drop wise to each lipid surfactant mixture with vigorous
stirring.
• A pseudo-ternary plot was developed with the composition of lipid
[Solid Lipid (Compritol 888 ATO: Gelucire 50/13 (2:3)): Liquid
Lipid (Miglyol 812)] surfactant (Cremophor RH 40), Co-surfactant
(PEG 400) and water till it gave clear solution and region of micro
emulsion was determined in Various Ratio Surfactant: Co-surfactant
(1:1, 2:1, 3:1)
Figure 6.52:Represented Pseudoternary phase diagram showing light blue
colour microemulsion region.
(A) Lipid, surfactant: Cremophor RH 40, co-surfactant: PEG 400 (S/Cos = 1:1)
(B) Lipid, surfactant: Cremophor RH 40, co-surfactant: PEG 400 (S/Cos = 2:1)
(C) Lipid, surfactant: Cremophor RH 40, co-surfactant: PEG 400 (S/Cos = 3:1)
• Where, Lipid = [Solid Lipid (Compritol 888 ATO: Gelucire 50/13
2:3): Liquid Lipid (Miglyol 812)]. Pseudoternary phase diagrams of
system consisting of lipid, Cremophor RH 40 and PEG 400
represent the microemulsion region in light blue colour, the
microemulsion region increase with increasing the concentration of
surfactant as compare to co-surfactant. So, optimize ratio of
Cremophore RH 40 and PEG 400 was selected as 2:1

6.5.5 Preparation of Solid lipid nanoparticle
Lipids Solid Lipid (Compritol 888 ATO: Gelucire 50/13 2:3) : Liquid
Lipid (Miglyol 812)] were heated to 80- 90°C on heating mantle and 10
mg of drug was solubilized to it.
Aqueous phase containing surfactants and Co-Surfactant were heated to

the same temperature 80- 90°C and then lipid phase was added to
aqueous phase and Mix on Magnetic Stirrer or vortexed to form
microemulsion.
The hot microemulsion was dispersed in cold water (20C) under stirring.
Typical volume ratios of the hot microemulsion to cold water were in the
range of 1:25.

6.5.6 Screening Trials
Table 6.43: Characterization of LNP with respect to Solid Lipid: Liquid
Lipid ratio
Solid lipid : liquid Particle size Zeta Entrapment

lipid Potential efficiency
1:1(10mg:10mg) Not formed
2:1(20:10) Not formed
3:1(30:10) Not formed
4:1(40:10) 89 -23 86
5:1(50:10) 90 -4 74
6:1(60:10) 100 -7 60
7:1(70:10) 118 -22 53

Table 6.44:Characterization of LNP with respect of Lipid: Drug ratio
Lipid: Drug ratio Particle size Zeta Entrapment
Potential efficiency
1:1(10:10mg) Not formed
3:1(30:10 mg) Not formed
6:1(60:10mg) Not formed
9:1(90:10mg) 89 -23 86
12:1(120:10mg) 90 -25 99
15:1(150:10mg) 265 -21 90
Table 6.45:Characterization of LNP with Respect to Lipid: S/Cos Ratio

Lipid: S/Cos Particle size Zeta Entrapment
Ratio Potential efficiency
1:2 123 -8 83
1:5 89 -23 86
1:8 52 -31 90

Table 6.46:Characterization of LNP with respect to Hot microemulsion:
Cold water Ratio
Hot microemulsion: Particle size Zeta Entrapment

Cold water Ratio Potential efficiency
1:10 143 -23 83
1:25 103 -21 81
1:40 120 -21 90
From the results obtained, the Hot Microemulsion : Cold Water ration of 1:25
was finalized due to very minor difference in particle size between 1:25 and
1:40. Based on Minimum Particle Size, Minimum Zeta potential and Entrapment
efficiency Criteria; the following Levels were selected for Box-Behnken Design.

6.5.7 Optimization of prepared solid lipid nanoparticle by Box
Behenken design
• For the optimization of the formula, design of experiment concept

was used. As there are three major factors affecting the formulation
viz, Solid lipid: Liquid Lipid, Total Lipid : Drug, Oil : (Surfactant:
Co-Surfacatnt Mixture), The Box Behnken design of experiment fits
suitable. This design analyses the factors at four different levels.
Table 6.47: Factors and their different levels for Central composite design
Sr. No Parameter Level

-1 0 +1
1 Solid lipid : Liquid Lipid 3:1 4:1 5:1
2 Lipid : Drug 9:1 11:1 13:1
3 Oil :(Surfactant: Co- Surfacatnt Mixture) 1:4 1:5 1:6

Table 6.48: Experimental matrix and results
Run Solid Lipid Lipid Particle Zeta Entrapment Dissolutio
Lipid to to to(surfactant: Size Potential Efficiency n at 10
Liquid Drug co-surfactant) (nm) (mV) (%) min
Lipid Ratio
1 5:1 13:1 1:5 162 -23 93 75
2 4:1 11:1 1:5 66 -25 86 100
3 5:1 11:1 1:4 126 -18 87 80
4 4:1 13:1 1:4 183 -18 88 80
5 3:1 11:1 1:6 121 -17 66 74
6 3:1 11:1 1:4 112 -21 76 55
7 4:1 9:1 1:4 107 -20 61 50
8 4:1 11:1 1:5 66 -25 86 100
9 4:1 11:1 1:5 66 -25 86 100
10 4:1 9:1 1:4 107 -20 61 90
11 3:1 13:1 1:5 168 -20 52 80
12 4:1 9:1 1:6 76 -29 63 75
13 4:1 11:1 1:5 66 -25 86 100
14 4:1 11:1 1:5 66 -25 86 100
15 3:1 9:1 1:5 53 -24 56 60
16 5:1 9:1 1:5 108 -19 76 80
17 5:1 11:1 1:6 113 -27 89 60

A. SIZE
• In the Box Behnken design the size, for the size models the P –Value
for the linear, quadratic and cubic model is less than 0.0001.
• This Small P-value indicates that there is very less probability to
deviate from the given results. From the various model Quadratic
model was selected on the basis of p-value, R2 value, Difference
between adjusted and predicted R² value and Adequate precision.
• Final Equation in term of Actual factor

• R1= +66.00 +1.50 * A +26.75 * B -26.75* C -15.25 * A * B -5.50
*A*C -11.25* B *C+28.25 * A2 +28.50 * B2 +23.75 * C2
+15.50 * A2 * B +25.75* A2 * C +10.75 * A* B2

Table 6.49:ANOVA Table for Particle size
Source Sum Square D.F. Mean Square p value
Particle size(Y1) (nm)
Model 25257.76 12 2104.81 < 0.0001
A 9.00 1 9.00 < 0.0001
B 2862.25 1 2862.25 < 0.0001
C 2862.25 1 2862.25 < 0.0001
AB 930.25 1 930.25 < 0.0001
AC 121.00 1 121.00 < 0.0001
BC 506.25 1 506.25 < 0.0001
A2 3360.26 1 3360.26 < 0.0001
B2 3420.00 1 3420.00 < 0.0001
C2 2375.00 1 2375.00 < 0.0001
A2B 480.50 1 480.50 < 0.0001
A2C 1326.13 1 1326.13 < 0.0001
AB2 231.12 1 231.12 < 0.0001
F value-6.366E+007
Design-Expert® Software Predicted vs. Actual
Particle size
183.00
Color points by value of
Particle size:
183
53 150.50
P r e d ic te d
118.00
85.50
53.00
53.00 85.50 118.00 150.50 183.00
Actual
Figure 6.53: Predicted Vs. Actual graph of size
From the above Predicted Vs. Actual graph it was conclude that there is
almost linear relationship between the Size values predicted by and the
Actual values of the size

1.00
Particle size
Particle size 148.833
110.5 129.667
Design Points 129.667
183
91.3333
53
B : L ip id t o d r u g r a t io
0.50
X1 = A: Solid lipid to liquid lipid ration

X2 = B: Lipid to drug ratio 72.1667
Actual Factor 0.00 5

C: Lipid to surfactant co surfactant ratio = 0.00
-0.50
91.3333
-1.00
-1.00 -0.50 0.00 0.50 1.00
A: Solid lipid to liquid lipid ration
Figure 6.54: Contoured plot of size
• From the above contoured plots it was concluded that as we are

increases the solid lipid: Liquid lipid ratio the size of the size of the
Lipid nanoparticals decreases. (Blue shade shows the size towards
the lower limit and red colour shows the upper limit. The liquid lipid
particles may occupy the space in the lipid matrix that might be the
reason for the higher size with higher amount of liquid lipid.
Particle size
183
53
170
P a rtic le s iz e
X2 = B: Lipid to drug ratio
140
Actual Factor
110
80
50
1.00 1.00
0.50 0.50
0.00 0.00
-0.50 -0.50
B: Lipid to drug ratio A: Solid lipid to liquid lipid ration
-1.00 -1.00
Figure 6.55: response surface plot of size
• The response surface plots were plotted to check the effect of various
factors on size. Here one of the three factors was kept constant and the
combined effect of rest two was analyzed using the response surface
curves. Here it was conclude that the size of Lipid nanoparticle decreases
as the surfactant: co-surfactant concentration increases. For the size model
various factors may affect but to find out the significant factor which can
affect the size of the lipid nanoparticles is found out by the following
graph.
Table 6.50:ANOVA Table for Zeta potential
Zeta potential (Y2)
Model 200.12 12 16.68 < 0.0001
A 12.25 1 12.25 < 0.0001
B 30.25 1 30.25 < 0.0001
C 30.25 1 30.25 < 0.0001
AB 16.00 1 16.00 < 0.0001
AC 42.25 1 42.25 < 0.0001
BC 12.25 1 12.25 < 0.0001
A2 21.32 1 21.32 < 0.0001
B2 6.58 1 6.58 < 0.0001
C2 16.84 1 16.84 < 0.0001
A2B 15.12 1 15.12 < 0.0001
A2C 4.50 1 4.50 < 0.0001
AB2 10.12 1 10.12 < 0.0001
F value-6.366E+007
• For zeta potential the P-value of the Linear, Quadratic and cubic model is
0.2730, 0.0308 and <0.0001 respectively .These P – values indicates that
the probability to deviate from the given model results are very less. From
the various model Quadratic model was selected on the basis of p-value, R2
value, Difference between adjusted and predicted R2 value and Adequate
precision.
• Zeta potential = -25.00 -1.75 * A +2.75 * B -2.75* C -2.00 * A * B-3.25* A
* C+1.75 * B * C+2.25 * A2 +1.25 * B2 +2.00 * C2 -2.75 * A2 * B +1.50*
A2 * C +2.25 * A * B2
Design-Expert® Software Predicted vs. Actual
Zeta potential
-17.00
Color points by value of
Zeta potential: 2
-17
-29 -20.00 3
P r e d ic t e d
-23.00
-26.00
-29.00
-29.00 -26.00 -23.00 -20.00 -17.00
Actual
Figure 6.56: Predicted Vs. Actual graph of zeta potential

1.00
Zeta potential
Zeta potential
Design Points -20.3021
-17
-21.6042
-29
B : L ip id to d r u g r a tio
0.50
-22.9063

-24.2083

C: Lipid to surfactant co surfactant ratio = 0.00 -25.5104
-0.50 -24.2083
-22.9063
-21.6042
-20.3021
-1.00
-1.00 -0.50 0.00 0.50 1.00
Figure 6.57: Contoured plot of Zeta potential
• From the above contoured plots it was concluded that as the

surfactant: co-surfactant concentration increases the zeta potential
also increases in the negative side. We can also conclude that the
higher level of lipid: Drug ratio the Surfactant : co-surfactant
concentration favours more negative zeta potential.
Figure 6.58: Response surface plot of Zeta potential
The surfactant used here is Poloxamer that is the negatively charged surfactant.
During formulation, the surfactant forms the coat around the particles that may
impart the negative charge to the particles. The response Surface curves for the
zeta potential is shown here.

Design-Expert® Software Cube
Zeta potential
Zeta potential
X2 = B: Lipid to drug ratio -14.25 -23.75
X3 = C: Lipid to surfactant co surfactant ratio
2
B+: 1.00 -21.75 -18.25
5
2
-21.75 -23.25 C+: 1.00
C: Lipid to surfactant co surfactant ratio
B-: -1.00 -22.25 -10.75 C-: -1.00

A-: -1.00 A+: 1.00
Figure 6.59: Cube plot of Zeta potential

• Effect of surfactant concentration and cube for zeta potential
The above cube states the value of zeta potential at the different boundaries
of the Experiment.

Table 6.51: ANOVA table for % Entrapment efficiency
Entrapment efficiency (Y3)

Model 2915.88 12 242.99 < 0.0001
A 289.00 1 289.00 < 0.0001
B 156.25 1 156.25 < 0.0001
C 156.25 1 156.25 < 0.0001
AB 110.25 1 110.25 < 0.0001
AC 36.00 1 36.00 < 0.0001
BC 210.25 1 210.25 < 0.0001
A2 31.84 1 31.84 < 0.0001
B2 825.26 1 825.26 < 0.0001
C2 59.21 1 59.21 < 0.0001
A2B 18.00 1 18.00 < 0.0001
A2C 36.13 1 36.13 < 0.0001
AB2 91.13 1 91.13 < 0.0001
F value-6.366E+007
• From the various model cubic model was selected on the basis of p-
value, R² value, Difference between adjusted and predicted R² value
and Adequate precision. The Model F-value of 11.52 implies the
model is significant. There is only a 0.20% chance that a "Model F-
Value" this large could occur due to noise. Values of "Prob > F" less
than 0.0500 indicate model terms are significant. In this case A, B,
B² are significant model terms. Values greater than 0.1000 indicate
the model terms are not significant.
• Entrapment efficiency = +86.00 +8.50 * A +6.25 * B -6.25* C +5.25

* A * B +3.00* A * C -7.25 * B * C -2.75* A2 -14.00 * B2 -3.75 * C2
-3.00 * A2 * B +4.25 * A2 * C +6.75 * A * B2

1.00
Entrapment efficiency
59.0401
Design Points 66.0802
93 73.1202
52
0.50


80.1603
-0.50
73.1202
66.0802
59.0401
-1.00
-1.00 -0.50 0.00 0.50 1.00
E n t r a p m e n t e f f ic ie n c y
93
52
95
84
Actual Factor
73
62
51
1.00 1.00
0.50 0.50
0.00 0.00
-0.50 -0.50
-1.00 -1.00
Figure 6.60: Contoured and response surface plot of % Entrapment efficiency

X2 = B: Lipid to drug ratio 36 83
2
B+: 1.00 60.5 95.5
5
2
54.5 80.5 C+: 1.00
B-: -1.00 50 64 C-: -1.00

A-: -1.00 A+: 1.00
Figure 6.61: Cube plot of % Entrapment efficiency
• From the above graphs it can be concluded that the Lipid:

Drug ratio and the Solid lipid: liquid lipid are the major factors
which can contribute the % Entrapment Efficiency.

Table 6.52: ANOVA table for dissolution at 10 min (%)

Dissolution at 10 Min. (Y4)
Model 4870.94 12 405.91 < 0.0001
A 156.25 1 156.25 < 0.0001
B 156.25 1 156.25 < 0.0001
C 56.25 1 56.25 < 0.0001
AB 49.00 1 49.00 < 0.0001
AC 56.25 1 56.25 < 0.0001
BC 156.25 1 156.25 < 0.0001
A2 286.58 1 286.58 < 0.0001
B2 1402.37 1 1402.37 < 0.0001
C2 1364.21 1 1364.21 < 0.0001
A2B 55.13 1 55.13 < 0.0001
A2C 0.000 1 0.000 < 0.0001
AB2 10.13 1 10.13 < 0.0001
F value-6.366E+007
• From the various model cubic model was selected on the basis
of p-value, R² value, Difference between adjusted and
predicted R² value and Adequate precision. The Model F-value
of 11.52 implies the model is significant. There is only a
0.20% chance that a "Model F-Value" this large could occur
due to noise. Values of "Prob > F" less than 0.0500 indicate
model terms are significant. In this case A, B, B² are
significant model terms. Values greater than 0.1000 indicate
the model terms are not significant.
• Dissolution at 10 min = +100.00 +6.25 * A +6.25* B -3.75* C
-3.50* A * B +3.75 * A * C -6.25 * B * C -8.25* A2 -18.25 *
B2 -18.00 * C^2 +5.25* A2 * B +0.000 * A2 * C +2.25 * A *
B2

1.00
Dissolution at 10 min
84.4296
Design Points
100
50
0.50


-0.50
84.4296
75.8222
67.2148
58.6074
75.8222
-1.00
-1.00 -0.50 0.00 0.50 1.00
D is s o lu tio n a t 1 0 m in
100
50
102
88.75
Actual Factor
75.5
62.25
49
1.00 1.00
0.50 0.50
0.00 0.00
-0.50 -0.50
-1.00 -1.00
Figure 6.62: Contoured and response surface plot of dissolution at 10min

X2 = B: Lipid to drug ratio 48.25 65.75
2
B+: 1.00 75.75 78.25
5
2
30.75 62.25 C+: 1.00
B-: -1.00 33.25 49.75 C-: -1.00

A-: -1.00 A+: 1.00
Figure 6.63: Cube plot of dissolution at 10 min
• From the above graphs it can be concluded that the Lipid:

Drug ratio and the Solid lipid: liquid lipid are the major factors
which can contribute the dissolution at 10 min

Optimization of formula and Validation of box benhken design
1.00
Overlay Plot
Overlay Plot
Particle size
Zeta potential
Entrapment efficiency 0.50
Design Points Particle size: 66.0352 Particle size: 80
Zeta potential: -25.6451
Entrapment effi 85.8967Zeta potential: -25
X1 = A: Solid lipid to liquid lipid ration Dissolution at 98.6079
X2 = B: Lipid to drug ratio 0.00 Dissolution at 10 min: 90 X1 5 0.35
Dissolution at 10 min: 100
X2 -0.25
Actual Factor Entrapment efficiency: 80
-0.50
-1.00
-1.00 -0.50 0.00 0.50 1.00
Figure 6.64: Overlay plot showing combined effects of factors X1, X2 and X3 on Y1, Y2,
Y3 and Y4

Sr. Responses Predicted Experimental %Relative
No. Values Values Error*
Particle Size (nm) 66.0352 66.0 0.10%
CPB1 Zeta Potential (mV) -25.02 -25 0.07%
Entrapment Efficiency (%) 85.89 86 0.17%
Dissolution at 10 min (%) 98.60 99 0.12%
•Relative Error (%) = (predicted value - Experimental value)/predicted

value×100 %.
The predicted batch shows significant reproducibility within the percentage

deviation. From the result shows that the predictive value close to the
experimental value so design is significant.

6.5.8 Lyophillization of drug loaded Lipid nanoparticles
• Selection of cryoprotectant for lyophilization The study was conducted

using Lactose, mannose, each with the concentrations 2.5 %, 5 % and 7.5
%. In general, the increase in particle size was accessed. Looking at the
result of cryoprotectant study Table, it was found that a high concentration
was most effective to produce lower particle size. Lactose was select for
our studies as it produced lowest particle size of lyophilized Lipid
nanoparticles.
Table 6.54:Particle size of lyophilization batches
Cryoprotectant Size after lyophilization (nm)
2.5% 5.0% 7.5%
Lactose 91 80 73.46
Mannose 110 134 90
Looking at the result of cryoprotectant study it was found that high
concentrations was most effective to produce lower particle size. Glucose
with 7.5% was select for our studies as it produced lowest particle size of
lyophilized Lipid nanoparticles.
Final formula of solid lipid nanoparticle
Ingredients Formula for 100 ml
Solid lipid: Liquid lipid (4;1)
Lipid: drug (11:1)
Oil (surfactant: co surfactant mixture)(1:5)
Hot microemulsion: Cold water ration (1:25)
Cilnidipine 1.0 gm
Gelucire 50/13:compritol 888ATO (3:2) 8.8 gm
Miglyol 812 2.2 gm
surfactant: co surfactant mixture 55 gm
Total weight 67 gm
Purified water 45 gm
Cold water ratio 1675 gm
Lyophilization using lactose 7.5%
Lactose 5.025 gm
Total 72.025 gm
6.5.9Characterization of optimization batch of various methods
Figure 6.65: Particle size of optimize batch

2. Zeta-potential
Figure 6.66: Zeta potential of optimize batch

As shown figure, the Zeta potential values of LNPs are below -
20 mV. This indicates that the dispersions of LNP will remain
deflocculated and would be physically stable.
• Dissolution studies of nanoparticles were performed in
triplicate using USP Type II dissolution apparatus.
nanoparticles equivalent to 10 mg of cilnidipine were taken
and placed in dissolution vessels containing 900 ml of 1% SLS
in maintained at 37 ± 0.50 C and stirred at 75 rpm.
• Samples were withdrawn using 0.22µ nylon merck filter at 1
to 10 mins and replaced with fresh dissolution medium.
Samples were suitably diluted and concentration of cilnidipine
was determined spectrophotometrically at 243 nm.
• For lyophilized nanoparticles equivalent to 10 mg of
cilnidipine were taken and placed in dissolution vessel.

Figure 6.67: Dissolution profile of LNP 1 to LNP10 batch
Figure 6.68: Dissolution profile of LNP 11 to LNP17 batch
Figure 6.69: Dissolution profile of CPB batch and MKT batch
The prepared final formulation CPB1 was compared with

marketed product (Cilacar) with respect to drug dissolution
profile. CPB release 100% drug within 10 mins and marketed
product (Cilacar)release 100% within 60 minutes.

4. X-ray Diffractometry
Figure 6.70: X-ray diffractograms of Cilnidipine
Figure 6.71: X-ray diffractograms of Cilnidipine loaded LNP

• XRD studies were performed to understand the crystalline
property of the formulation and the bulk drug. It is
hypothesized that during the formulation of the nanoparticles
by homogenization technique, the drug is converted in to
amorphous form. The Lipids are also converted in to its
unstable α-form. Here the XRD study of the plain drug and the
lyophilized formulations were compared. From the below data
it was concluded that the after incorporation in lipid
nanoparticles the drug loses its crystalline behavior. As the
intensity of the XRD peaks decreases significantly

6.6 Method development for related substances
determination in cilnidipine
Table 6.55: Specification of Related Substances for Cilnidipine
Related substances JP limit for Cilnidipine JP limit for Cilnidipine tablet
Related substance
Individual impurities NMT 0.1% NMT 0.2%
specifications as
Unknown maximum NMT 0.3% NMT 0.3%
per Japanese
impurity
Pharmacopoiea
Total impurities NMT 0.5% NMT 0.5%
• Test solution-. Disperse a quantity of the powdered tablets

containing about 20mg of Cilnidipine in 10 mI of Acetonitrile
with the aid of ultrasound for 5 minutes, filter. Dilute 1ml of
this solution to 4 ml with 5 per cent v/v of Acetonitrile.
• Reference solution (a), A 0.00025 per cent w/v of Cilnidipine
RS in 5 per cent v/v of Acetonitrile.
• Reference solution (b), A0.0005 per cent w/v of thiamazole in
5 per cent v/v of Acetonitrile.
• Reference solution (c). A solution containing 0.01 per cent w/v
of Cilnidipine RS and 0.0005 per cent w/v of thiamazole in 5
per cent v/v of Acetonitrile.
• Chromatographic system
- a stainless steel column 15 cm x 3.9 mm packed with
octadecylsilane bonded to porous silica (5 µm),
- Mobile phase: A. 5.0 per cent v/v solution of Acetonitrile,
- Mobile phase: B. 20.0 per cent v/v solution of Acetonitrile,

• Related substance is a major part for formulation and
development. Its describe safety and efficacy of particular drug
product.
Table 6.56: Related Substances of Cilnidipine product
Related Criteria Nanosuspension SNEDDS Solid lipid

substance nanoparticle
Individual NMT 0.1% 0.01% 0.01% 0.01%

impurities
Unknown NMT 0.3% ND ND ND

maximum
impurity
Total impurities NMT 0.5% 0.01% 0.01% 0.01%

6.7 Stability studies on the optimized lyophilized
nanosuspensions, SNEDDS and solid lipid nanoparticle
• Stability study of lyophilized nanosuspension, SNEEDS and
solid lipid nanoparticle was assessed as per ICH specified long
term storage conditions (25°C ± 2°C/60% RH ± 5% RH),
accelerated condition(40°C ± 2°C/75% RH ± 5% RH) The
samples were packed in an aluminum pouch and stored in a
humidity chamber for one month. At regular intervals, sample
was withdrawn and analyzed for the following:
• Nanosuspension- Drug content, Saturation solubility,
Dissolution studies, related substances .
• SNEEDS-percentage drug content, particle size, zeta potential
and in vitro drug release, related substances
• Solid lipid nanoparticle- Particle size, Zeta potential, %
Entrapment efficiency, related substances.
Table 6.57: Stability study of Nanosuspension CPB1 batch
Parameter Initial 40/75-1M 25/60-1M 40/75-3M 25/60-3M
Drug content 101.0 mg 100.0 mg 100.0 mg 99.0mg 100.0mg
Saturation
10mg/ml 10mg/ml 9.98mg/ml 9.50mg/ml 9.90mg/ml
solubility
100% 100% 100%
Dissolution 100% within 100% within
within 5 within 5 within 5
profile 5 min 5 min
min min min
Related substances
Individual
0.01% 0.02% 0.01% 0.03% 0.01%
impurities
Unknown
maximum ND ND ND ND ND
impurity
Total impurities 0.01% 0.02% 0.01% 0.03% 0.01%
Figure 6.72:Dissolution profile of nanosuspension at stability condition
Decrease in drug content at the end of 90 days is negligible.
Hence formulation is quite stable. From above study conclude
that there is no change in parameters of nanosuspension at 90
days and product is stable.

Table 6.58:Stability study of SNEDDS CPB 1 batch
Drug content 98.9 98.00 97.00 96.00 98.00
Zeta potential -22.20 -20.00 -22.00 -19.00 -22.00
Particle size 10.60 11.90 11.00 12.00 10.90
100% 100% 100% 100%
Dissolution 100% within
within 10 within 10 within 10 within 10
profile 10 min
min min min min
Related substances
Individual
0.01% 0.02% 0.01% 0.03% 0.01%
impurities
Unknown
impurity
Figure 6.73: Dissolution profile of SNEDDS at stability condition

Hence formulation is quite stable. At 40/75-1M particle size
increases and zeta potential decreases. There is no change in
related substance and product is stable.

Table 6.59:Stability study of Solid lipid nanoparticle CPB 1 batch

Zeta potential -25.00 -24.00 -25.12 -24.00 -23.00
Particle size 66.00 70.00 65.00 72 69
% Entrapment
85.59 85.59 86.00 85.00 85.00
efficiency
Related substances
Individual
0.01% 0.02% 0.01% 0.03% 0.01%
impurities
Unknown
impurity

Hence formulation is quite stable. At 40/75-1M particle size
increases and zeta potential decreases. There is no change in
related substance and product is stable.

6.8 In- vivo pharmacokinetic study
• 6.8.1 Preparation of calibration curve of Cilnidipine by HPLC method
• Selection of mobile phase
Mobile phase of different composition of Acetonitrile to water was
evaluated for quantitative and qualitative estimation of Cilnidipine. Initially
different ratios of Acetonitrile (HPLC grade) to water (60:40, 70:30, 80:20
and 90:10) were taken.
• Standard stock solution (1000 ng/ml)
Accurately weighed quantity of 10 mg Cilnidipine was transferred into
100ml volumetric flask and diluted up to 100ml using mobile phase
(Acetonitrile: water in 80:20 ratio) to give a solution having strength
100μg/ml. 0.1ml from stock solution was taken in 10ml volumetric flask
and diluted up to the mark with mobile phase to obtain stock solution
having strength of 1000 ng/ml.
• Working standard solution
0.1ml of stock solution was taken in 10 ml volumetric flask and diluted
with mobile phase (Acetonitrile: water in 80:20 ratio) to get the
concentration 10 ng/ml.
• Calibration curve of Cilnidipine
From the working solution, aliquots of 2.0, 4.0, 6.0, 8.0,
10.0 ml were taken to 10ml volumetric flask and diluted
up to the mark with mobile phase to obtain concentration
in the range of 2, 4, 6, 8, 10 ng/ml respectively. 10μL of
the above aliquots were injected into the HPLC system to
get the retention time and peak area. The values of peak
area were plotted against concentration to get the standard
curve. The values were subjected to regression and
linearity. The procedure was repeated thrice and means
were taken.

Table 6.60: Calibration curve of Cilnidipine in acetonitrile: water (80:20 v/v)
Sr. No. Concentration (ng/ml) Peak area ± SD (n=3) (μV*sec) % RSD

1 0 0 0
2 2 1212 ± 38.90 1.70
3 4 2514 ± 62.14 1.62
4 6 3915 ± 85.17 1.25
5 8 4943 ± 89.15 1.12
6 10 5903 ± 91.52 1.12
Figure 6.74:Calibration curve of Cilnidipine in acetonitrile: water (80:20 v/v)

Table 6.61:HPLC instrumentation and Chromatographic conditions
System Waters 2998

Stationary phase RP C18 2.27μm size, 250 mm 4.6 mm
Mobile phase Acetonitrile: water (80:20 v/v)
Flow rate 1.0 ml/min
Elution mode Isocratic
Detector Photodiode array detector
Detection wavelength 262 nm
Injection volume 10 μl
Run time 4 Min

6.8.2 Preparation of calibration curve of Cilnidipine in
extracted rat plasma by HPLC method
• Standard stock solution (1000 ng/ml)
Accurately weighed quantity of 10 mg Cilnidipine was transferred into
100ml volumetric flask and diluted up to 100ml using mobile phase
(Acetonitrile: water in 80:20 ratio) to give a solution having strength
100μg/ml. 0.1ml from stock solution was taken in 10ml volumetric
flask and diluted up to the mark with mobile phase to obtain stock
solution having strength of 1000 ng/ml.
• Working standard solution

0.1ml of stock solution was taken in 10 ml volumetric flask and diluted
with mobile phase (Acetonitrile: water in 80:20 ratio) to get the
concentration 10 ng/ml. From the working solution, aliquots of 2.0, 4.0,
6.0, 8.0, 10.0 ml were taken to 10ml volumetric flask and diluted up to
the mark with mobile phase to obtain concentration in the range of 2, 4,
6, 8, 10 ng/ml respectively.

Calibration curve of Cilnidipine in extracted rat
plasma by HPLC method
From the each working standard solution 100μL was spiked

with 0.9 ml of blank rat plasma and homogenously mixed by
vortexing for 5 minutes to get drug concentration in the range
of 2, 4, 6, 8 and 10ng/ml. For preparation of calibration curve
300 μL of the deprotinizeing agent (Acetonitrile) was added to
200 μL aliquot of plasma and the dispersion was vortexed for
2 minutes. The samples were then centrifuged at 5000 rpm for
15 minutes at temperature 0-4°C. The supernatant was then
collected and diluted with 200μl of Acetonitrile: water (80:20).
The samples were then filtered (0.22μ nylon syringe filters)
and 10μl was injected into the HPLC system.

Table 6.62:Calibration curve of Cilnidipine in rat plasma using acetonitrile:
water (80:20 v/v)
Sr. No. Concentration (ng/ml) Peak area ± SD (n=3) (μV*sec) % RSD
1 0 0 0
2 2 1312 ± 11.20 1.14
3 4 2615 ± 15.14 1.15
4 6 3845 ± 14.15 1.52
5 8 4847 ± 22.12 1.45
6 10 6012 ± 12.25 1.72
Figure 6.75: Calibration curve of cilnidipine in rat plasma using acetonitrile: water
(80:20 v/v)
• Accuracy and precision
Aliquots of rat plasma spiked with different concentration of
cilnidipine. The concentrations were injected in triplicates and
area responses were compared to the reference standard. The
value of average deviation and standard deviation were
calculated. Accuracy was determined at three different
concentrations by comparing the measured value with that of
the standard.
• Interday
For the assay of the inter-day precision, three consecutive
batches of samples were made by the same procedure on three
(3) different days. The precision was reported as the Relative
Standard Deviation (RSD, %). The quality control samples
were prepared with (2 ng/ml) as low, (6 ng/ml) as intermediate
and (10 ng/ml) as high concentration.
• Intraday
To evaluate the intraday precision and accuracy, cilnidipine
samples at low, middle and high concentrations (as mentioned
earlier) were extracted and triplicate determinations within the
same QC sample was performed.
• Limit of detection (LOD) and limit of quantification (LOQ)
The limit of detection (LOD) is the lowest level of the drug
that can be detected in sample. The limit of quantification
(LOQ) was defined as the lowest concentration at which the
coefficient of variation (RSD) and deviation from the nominal
concentration were less than 20%.
Table 6.63: Intraday & Interday precisions for cilnidipine in rat plasma
Sr. Concentration Intraday Interday

No. (ng/ml) Peak area % RSD Peak area %
± SD (n=3) ± SD (n=3) RSD
(μV*sec) (μV*sec)
1 2 2045 ± 30.56 1.12 2358 ± 31.25 1.13
2 6 4358 ± 78.01 1.42 4468 ± 73.21 1.23
3 10 5545 ± 91.41 1.15 5895 ± 87.45 1.45
Figure 6.76: Typical HPLC chromatogram of blank plasma
Figure 6.77:Typical HPLC chromatogram of Cilnidipine in rat plasma.

6.8.3 Study design for in-vivo pharmacokinetic study
Details of animal taken as model animal and IAEC approval for this study
 Species/Common name : Wister albino rats

 Age / weight / size : 3-4 months/200-300 g
 Gender : Male
 Animals were procured from : Zydus Research Center, Ahmedabad
 IAEC approved protocol no. :CPCSEA/IAEC/IICP/2017/01

Instillation method for drug administration
• All the rats were fasted for 12h before the experiment but had free access to
water. A formulation of (suspension of RLD and Lyophilize
nanosuspension CPB1) was given orally by syringe equivalent 10 mg of
cilnidipine.
• Blood samples of 0.5 ml was collected from the caudal vein at
predetermined time points (15, 30, 45, 60, 90, 120, 150, 180, 210, 240 and
360 Minutes) Blood samples were cold centrifuged for 30 minutes at 5000
rpm below 4 º C. The drug concentration in plasma was determined by
method as described by Bhandari R et al. with slight modification. 100μL
of plasma, 700 μL of acetonitrile & 200 μL of water (HPLC Grade) were
added as deprotinizeing agent and the dispersion was vortexed for 2
minute. The supernants was further centrifuged at 10000 rpm for 15
minutes at 4 ºC. The supernants was then collected and re-centrifuged at
10000 rpm for 10min for removal of traces of precipitates from sample.
The clear supernants was taken and diluted up to 10ml in volumetric flask
(10ml). The drug concentration in diluted samples was determined by
HPLC method at predetermined chromatographic conditions.
Table 6.64:Experimental design for In-vivo pharmacokinetic study
In-vivo pharmacokinetic study
Group I II III
Treatment Normal Drug formulation(RLD) Drug formulation(Lyophilized
Control via oral route nanosuspension) via Oral route
Model animal Wister albino rats (200-300g)
Dose (mg/kg) 0 10 10
Number of 6 6 6
Animals
Duration of 1 Day 1 Day 1 Day
Treatment
Route of - Oral route Oral route
administration
Termination Blood was collected from caudal vein at different time interval
(15, 30, 45, 60, 90, 120, 150, 180, 210,240 and 360 Minutes)
Parameters to Cmax, Tmax, AUC, mean residence time (MRT) and KE
be evaluated
Table 6.65:Plasma drug concentration after oral administration of RLD
and Nanosuspension
Sr. Time Plasma Concentration ± SD (μg/ml)

No. (Min) Drug formulation(RLD) Lyophilized nanosuspension(CPB1)
1 15 110.0±0.25 600.01± 0.12
2 30 225.0±2.25 980.12±0.02
3 45 420.00±0.12 950.01±0.01
4 60 580.00±1.45 840.12±0.15
5 90 690.00±2.40 720.15±1.12
6 120 780.00±3.15 610.11±1.15
7 150 640.00±0.25 505.02±0.01
8 180 505.12±0.02 440.05±0.23
9 210 312.00±0.15 310.02±0.05
10 240 220.00±2.15 205.15±0.12
11 360 109.12±3.15 105.11±0.15
Figure 6.78: Plasma drug concentration versus time profile of RLD and
lyophilized nanosuspension

Table 6.66:Pharmacokinetic parameters after oral administration of RLD
& Lyophilized nanosuspension
Pharmacokinetic Lyophilized
RLD
Parameter nanosuspension
Cmax (μg/ml) 780.00 980.0
Tmax (hr) 2 30 min
AUC 0-t (μg•hr/ml) 2253.75 2601.25
AUC 0-∞ (μg•hr/ml) 2646.97 2980.03
KE (hr-1) 0.2772 0.2772

t1/2 (hr) 2.5 2.5
AUMC0-t (μg•hr2/ml) 38087 60991
AUMC 0-∞ (μg•hr/ml) 41865 64630
MRT 0-t (hr) 16.89 23.44

• From the graph of plasma drug concentration versus time, it was
found that the drug release is continued with high release manner.
The half-life of lyophilize nanosuspension was found to be 2.5 hr
whereas RLD having 2.5 hr. The lower plasma retention of drug
from lyophilize nanosuspension compared with RLD might be
attributed towards the higher drug release form the nanosuspension

7.Previous comment

Compliance report
Recommendations Student Compliance
Author has described many  Nanoprecipitation method selected for
methods to prepare nanosuspension nanosuspension preparation hense this method in
(1.3.3) and lipid nanoparticles which simple machinery used as compare to high
(1.5.4) in introduction section. But pressure homogenizer and it’s a laboratory scale
author has selected nano- method. Using this method scale up can be
precipitation (for nanosuspension) perform at plant and it’s a low cost method.
and microemulsion (for lipid  Microemulsion method used to prepare lipid
nanopaticles). Justification for nanoparticle This method has the potential to be
selection of methods or superiority easily scaled from laboratory batch to industrial-
of these methods should be sized batch since there is no need for the high
described in materials and methods. shear machinery. Same like high pressure
homogenizer method costly equipment require so
microemulsion
2 Author has selected 900 ml 1%  Cilnidipine is a BCS class II drug which
SLS as dissolution medium for have low solubility and permeability and
formulation with enhanced 1% SLS added to enhance solubility of
solubility. Addition of SLS in drug in dissolution media and to achieve
dissolution medium should be 100% drugs release in recovery. As
justified. dissolution methods given by USFDA in
many BCS class ii drugs dissolution
methods 1% SLS is used.
3 Author should give details about  Excipients- Microcrystalline cellulose,
brand product on page no. 28 maize starch, purified water, magnesium
(At least tentative excipients, stearate, talc, Croscarmellose sodium
process, any technology if used  Process- Wet granulation
etc. from Innovator leaflet).
4 In section 4.9.2.2, author should RLD Name- Cilacar 10 mg tablets
write details of RLD (name of Batch no- CTS0215
RLD, Batch no./ Lot No., Manufacturer- J B Chemicals &
manufacturer, exp.date) pharmaceuticals
Exp. Date- Feb, 2018
5 4.3.2 Please Justify the use of Cilnidipine is a BCS class II drug which have
SLS in dissolution media page low solubility and permeability and 1% SLS
55 added to enhance solubility of drug in
dissolution media and to achieve 100% drugs
release in recovery. As dissolution methods
given by USFDA in many BCS class ii drugs
dissolution methods 1% SLS is used.

8. Publications
1. Research article

2. Research article

3. CPCSEA Certificate

9.Possible Outcome
• Possibility of large-scale production, the pre-requisite for the

introduction of a delivery system to the market.
• Improvement in biological performance due to high
dissolution rate and saturation solubility of the drug.
• Ease of manufacture and little batch-to-batch variation.
• Long term physical stability.

10. Impact on science – industry – society
• The main goal of the present work is to enhance dissolution
rate by applying nanosuspension, solid lipid nanoparticle and
smedds / snedds of poorly soluble drugs.
• Many pharmaceutical company developing the nano drug
delivery systems now a days.
• so if the dissolution rate of poorly soluble drug increases then
patient compliance is more and so its useful to the industry and
also to the science and industry.
• If dissolution rate increases than increase in solubility of
poorly soluble drug so we can patent the drug solubility
enhancement study.

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FORMULATION AND DEVELOPMENT OF NANO DRUG

DELIVERY SYSTEM OF CILNIDIPINE FOR IN-VITRO

Prepared by: Guided by:

6. Formulation and development

10. Impact on science/industry/society

• Results and Discussion:

• Solid lipid nanoparticle-Lipid nanoparticles achieved satisfactory results

• Keywords: “Nanosuspension”, “Quality by design”, “Cilnidipine”,

Nanosuspension Self nanoemulsifying drug delivery Solid lipid nanoparticle

Screening of oils, surfactant, co- Screening of solid lipid, Liquid lipid ,

Feasibility trail Feasibility trail Feasibility trail

Optimize Formulation Optimize Formulation Optimize Formulation

Characterization of optimize Characterization of optimize formulation Characterization of optimize formulation

Stability studies Stability studies RAJKOT

• In this project work we have focus on

Faiyaz S, Ultrafine Thermodynamically stable SNEDDS was selected for self-

Pankaj P Method Present study describes accurate, precise and reproducible

• The main aim of this project work is to enhance dissolution

• The poorly soluble drugs (Cilnidipine) have limited

• By preparing nano-particulate drug delivery system, the

3 Poloxamer 407,188 Signet Chemicals Pvt. Ltd, Mumbai.

Sr. No. Materials/ Chemicals Sources

4 Differential Scanning Calorimeter Perkin Elmer, Model DSC 7, USA

5 Bath Sonicator Electro Quip

• Melting point study:

Peaks at wave number (cm-1 ) Interpretation

Figure 6.2:DSC spectra of Cilnidipine

6.2.1 Spectroscopy estimation of Cilnidipine (CLD)

• Preparation of standard stock solution

Table 6.5:Assay of cilnidipine in

Figure 6.4:Assay of cilnidipine in Phosphate

High speed homogenizer

500 mg Poloxamer 188

High speed homogenizer

Figure 6.6: Nanosuspensions prepared by stirring at 10,000 rpm

Figure 6.7:Nanosuspensions after 15

Figure 6.9:Nanosuspensions after 15 days (prepared by stirring at 15,000 rpm)

Figure 6.11:Nanosuspensions after 15 days of (prepared stirring at 20,000 rpm)

100 mg of drug dissolve

High speed homogenizer

T15 PVP K30 10 Watery 1

100 mg of drug dissolve

High speed homogenizer

Prepared batches analyze for particle

Batch Stabilizer concentration Particle PDI Centrifugation Drug

B1 Poloxamer 407 92.12 0.101 No settling 98.98 %

Figure 6.13:Particle size analysis of B2 (stabilizer Poloxamer 188)

Figure 6.15:Particle size analysis of B4 (stabilizer Tween 80)

Response variables Constraints

β0 +92.71 +50.07 +99.55

Β6 (X1X22) +8.28 -3.66 -4.41

Cubic R2 0.9859 0.9955 0.9546

PRESS 12176.38 74.81 4281.69

The approximations of response values Y1 particle size (nm), Y2 saturation

Source D.F. Sum Square Mean Square F Value p value

saturation solubility study: 45

saturation solubility study: 45

Particle size: 92.6934

saturation solubility study: 45

* Relative Error (%) = (predicted value - Experimental value)/predicted value×100 %.

• Due to low melting point of Cilnidipine, lyophilization was

• Nanosuspensions contained in a petridish with the addition of

Batch Cryoprotectant Appearance of freeze dried product

L1 D-mannitol Fluffy powder