Formulation & Evaluation of Neosomes

1. FORMULATION AND EVALUATION OF
ATORVASTATIN CALCIUM NIOSOMES
BY
NEHA
HIMALYAN INSTITUTE OF
PHARMACY
HPTU HAMIRPUR

2.  Chapter 3
 Material and methods
 Chapter 4
 Result and Disscussion

3. LIST OF CONTENT
 Chapter 1
 Introduction
 1.1 Novel drug delivery system
 1.2 Advantage & Disadvantage of Novel drug delivery system
 1.3 Niosomes
 1.4 Advantage & Disadvantage,Structure,Applications
 Chapter 2
 Aim and objective
 2.1 Main objective of present study
 2.2 Novelty of the work
 2.3 Plan of work

4. NOBLE DRUG DELIVERY SYSTEM
 NDDS attain regular delivery of drug and drug attain extended period of time in
circulation.
 ADVANTAGES
 1.Improve management of patient.
 2. Improve potential of respective drug.
 3.Improve patient compliance.
 4.Maximum availability with minimum dose.
 5.Protect 1st pass metabolism & GIT Degradation.
 Disadvantages
 1. Fluctuation of drug delivery may lead to precipitation

5. NIOSOMES
 Niosomes are NDDS,in which the medication is encapsulated in a
vesicle.The vesicle is composed of a bilayer of non-ionic surface active
agents.Niosomes are very small & microscopic in nature.
 Advantages
 1.Osmotically active and stable.
 2.Enhance skin permeability of drug when applid on skin topically.
 3.Subsequent decrease in the effect.
 4.Niosomes are ampiphillic.
 5.Improve oral bioavailability.

6. Disadvantages & Applications
 Disadvantages
 1.Time consuming.
 2.Required specialized equipment.
 3.Ineffecient drug loading.
 Applications
 1.Pulmonary delivery.
 2.Protein & peptide delivery.
 3.Niosomes as carrier.
 4.Transdermal delivery of drug by niosomes.
 5.In neoplasia
 6.In leishmaniasis.
 7.Immunological application.

7. STRUCTURE OF NIOSOME

8. COMPONENTS OF NIOSOMES
 1.Non Ionic surfactant.
 consisting of a hydrophilic head group and a hydrophobic tail, used in the
preparation of niosomes.
 E.g-Crown ether,tween,spans.
 2.Cholestrol.
 Steroidal derivative used for formulation of niosomes
 3.Charge inducing molecule
 Some charge molecule added to niosomes to increase stability of niosomes by
electrostatic repulsion which prevent aggregation & coalescence

9. METHODS(PASSIVE TRAPPING)
 PASSIVE TRAPPING
 Thin film hydration
 Ether injection method
 Reverse phase evaporation
 Sonication method
 Multiple membrane extrusion
 Micro Fluidization
 Bubble method
 Heating method

10. METHODS(ACTIVE TRAPPING)
 Entapment efficiency
 In vitro release
 Zero potential measurement
 Bilayer formation
 Stability study
 Kinetic Modeling
 Bilayer rigidity & homogeneity

11. NOVELTY OF DRUG
 Atorvastatin calcium is the choice of many researches at the present time,also
there is a lot of work available in literature using Atorvastatin calcium and various
other novel drug delivery systems.There is no any research work on niosomes of
atorvastatin calcium till the present date..
 This research work is nivel covering the all aspect of the new research.

12. MAIN OBJECTIVE OF PRESENT STUDY
 1.To develop a stable,reproducible & non-infringing drug delivery system of
atorvastatic calcium.
 2.Extend duration of activity that aloows greater patient compliance owing to
elimination of multiple dosing schedule.
 3. Evaluation of pharmacokinetics of atorvastatin calcium with the formulations of
this study.
 4.To judge the suitability of new formulation on the basis of results.
 5.To oviate specific problems associated with the drugs.eg Low absorption,first
pass effect.

13. PLAN OF WORK
 The aim of the present study was to develop niosomes of atorvastatin calcium.
 Physiochemical characterization:-
 Melting point, Solubility
 Partition coefficient
 Analytical Methodlogy
 Determination of UV absorption spectra.
 Preparation of calibration curve.
 Drug and surfactant compatibility study by FTIR.
 Evaluation parameter for niosomes of atorvastanin calcium.
 Vesicle size determination
 Drug content
 Shape & surface morphology
 Drug entrapment efficiency of niosomes
 Drug release kinetic data analysis.
 Stability studies,using accelatered stability study

14. MATERIAL & METHODS
 List of chemicals used
Sr No Chemicals
1 Atorvastatin calcium 8 HCL
2 Span 40, Span 60, Span 80, 9 Sodium Lauryl sulphate
3 Tween20, Tween40, Tween60, 10 Di sodium hydrogen
phosphate
4 Cholestrol 11 Ehtanol
5 Potassium dihydrogen
orthophosphate
6 Octanol
7 Sodium hydroxide

15. INSTRUMENTS
Sr No Equipments
1 Single pan electronic balance 9 Franz Diffusion Cell
2 Magnetic stirrer 10 Scanning Electron Microscope
3 p H meter 11 Dissolution Apparatus (USP XXIV, type I)
4 Hot air oven 12 Dessicator
5 Uv-Vis spectrophotometer 13 DSC instrument
6 Mehanical shaker
7 FTIR Spectrophotometer

16.  Physical Appearance: The drug was observed visually for the color and
appearance, weather amorphous or crystalline.
 5.2.1.2 Solubility analysis79
 Solubility of Atorvastatin calcium in water was determined at 370C.10gm
Atorvastatin calcium was transferred in a series of different solvent having volume
10 ml in different test tubes. These test tubes were shaken by a mechanical
shaker for 2 hrs under constant temperature. Solution was filtered by Whatman
filter paper and the clear filtrate was assayed spectrophotometrically at 244 nm
against blank solution. Available literature shows that adsorption to Filter II
(Whatman Filter) was much lower and in most cases negligible.

17.  Melting point determination79
 Melting point of Atorvastatin calcium was determined by using Thiels tube
method. 300 ml of heavy paraffin was filled in Thiels tube, the drug filled (sample
amount) in a capillary tube whose one end is sealed with the flame was tied with
thermometer and dipped into Thiels tube those filled with paraffin. The heating
was started and the point at which drug start melting was noted.

18.  Partition coefficient
 The partition coefficient study was performed using n-octanol as oil phase and
phosphate buffer pH 7.4 and water as aqueous phase. The two phases were mixed in
an equal quantity and were saturated with each other on a mechanical shaker for 24
hrs. The saturated phases were separated by separating funnel. Equal volume (10 ml
each) of the two phases were taken in conical flasks and to each, 100 mg of drug was
added. The flasks were stirred at 320C for 6 hrs to achieve a complete mixing at 100
rpm. The two phases were separated by separating funnel then the phases were
analyzed for respective drug amount by the means of UV spectrophotometer at 244
nm.
 The partition coefficient value ‘p’ was calculated by the following equation-
 Po/w =Concentration in octanol/Concentration in phosphate buffer pH 7.4

19.  Identification
 i) UV-Visible Spectroscopy
 An accurately weighed amount of the drug (10 mg) was dissolved in methanol and the
volume was made up to the mark. From the stock solution, graded dilutions were
made to obtain standard solution of the drug ranging from 2-16 μg/mL. A scan was
obtained by using UV visible spectrophotometer (UV-1700, Shimadzu, Japan) from
which λmax was interpreted and compared with the standard literature value
 ii) Preparation of Calibration Curve80
 10 mg of drug was weighed and dissolved in 10 ml of phosphate buffer 7.4 (containing
20% methanol), to give a solution of 1000 μg/ml concentration. From this solution 1 ml

20.  was taken and diluted to 10ml using Phosphate buffer 7.4 (20% methanol) to produce a stock solution of
100 μg/ml. From this stock solution different concentrations were prepared. The absorbance of these
solutions was measured at 244 nm by UV spectrophotometer. Calibration curve of Atorvastatin also
prepared in water and methanol (20%) solvent system by preparing stock solution of different
concentration ranging from 2-16 μg/ml as above. The absorbance was measured at 244 nm by UV
spectrophotometer.
 iii) Excipients compatibility studies: Fourier Transform Infra-Red (FTIR) Spectroscopy79
 The FTIR spectrum of the pure drug was taken on IR Spectrophotometer (Shimadzu FT/IR 8400, Japan)
using KBr pellet technique. Initially, the drug was mixed with KBr and grind to a fine powder. The
preparation of very finely grounded sample helps in reducing scattering losses and absorption band
distortions. The sample was then placed in between two stainless steel disks, which upon pressure
(hydraulic press) results in the formation of a thin, homogenous and transparent film. The film was placed
into the IR sample holder and the spectrum was run between the range 4000-400 cm-1. The peaks
obtained in the spectra were then compared with corresponding functional groups in structure of pure
ATR.

21.  iv) Differential Scanning Calorimetry (DSC)
 Differential scanning Calorimetry, experiments were performed with differential
Scanning Calorimeter chamber. Instrument is comprised of a calorimeter a flow
controller a thermal analyzer and an operating software. Sample of pure
Atorvastatin calcium, span 60, and lyophilized niosomal sample were weighed in
an aluminium pan and sealed with an aluminium lid. The pan was placed in the
DSC and heated from 20-350°C at a heating rate of 50°C/min in nitrogen
atmosphere. The scan was recorded and plotted showing heat flow (w/g) on the
Y-axis and temperature on the X-axis.

22. Formulation development
(Thin film hydration technique)
 Niosomes containing Atorvastatin calcium were prepared by Thin film hydration method using
nonionic surfactant and cholesterol at different concentrations. Cholesterol and surfactant were
dissolved in 6 ml diethyl ether mixed with 2ml methanol containing weighed quantity of
atorvastatin calcium. The resulting solution was slowly injected using micro syringe at a rate of
1ml/min into 15 ml of hydrating solution phosphate buffer (pH 7.4).The solution was stirred
 continuously on magnetic stirrer and temperature was maintained at 60-65ºC. As the lipid solution
was injected slowly into aqueous phase, the differences in temperature between phases
 cause rapid vaporization of ether, resulting in spontaneous vesiculation and formation of
niosomes82.
 Different batches of niosomes were prepared in order to select an optimized formula as per
general method described above and proportion of surfactant and cholesterol for the preparations
of niosomes is given in Table 5.3

23. Compositions of the Atorvastatin
calcium niosomes formulations
Sr No Formulation Code Surfactant Drug:Surfactant:Cholesterol (mg)
1 ANS1 Span 40 100:100:100
2 ANS2 Span 40 100:200:100
3 ANS3 Span 40 100:100:100
4 ANS4 Span 40 100:200:100
5 ANS5 Span 40 100:100:100
6 ANS6 Span 40 100:200:100
7 ANS7 Tween 20 100:100:100
8 ANS8 Tween 20 100:200:100
9 ANS9 Tween 40 100:100:100
10 ANS10 Tween 40 100:200:100
11 ANS11 Tween 60 100:100:100
12 ANS3 12 Tween 60 100:200:100

24. Evaluation of formulations
 Particle size
 Vesicle size determination was carried out using an optical microscopy with a calibrated eyepiece
micrometer. About 200 niosomes were measured individually, average was taken, and their size
range, mean diameter were calculated .
 Entrapment efficiency84
 The percentage entrapment efficiency of the vesicles was determined by freeze thawing
centrifugation technique. Niosomal suspension was filled in drop tubes and stored at -20ºC in a
refrigerator for 24 hours. After 24 hours niosomal suspension was taken from refrigerator and
stored at room temperature. The niosomal suspension was centrifuged at 1500 rpm for 30 minute.
Supernatant containing unentrapped drug was withdrawn and diluted with water methanol mixture
(80:20), then measured UV spectrophotometrically at 244 nm against water methanol mixture as
standard85.
 Entrapment efficiency was calculated by using following equation:-
 (%)𝐸𝐸= Total amount of drug in suspension−drug in supernant/𝑇𝑜𝑡𝑎𝑙 𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝐷𝑟𝑢𝑔 𝑝𝑟𝑒𝑠𝑒𝑛𝑡 𝑖𝑛
𝑠𝑢𝑠𝑝𝑒𝑛𝑠𝑖𝑜𝑛× 100

25.  Scanning electron microscopy (SEM)
 The shape and surface characteristics of niosomes were evaluated by scanning
electron microscopy (SEM). The lyophilized sample was mounted directly on to
the SEM sample holder
 using double-sided sticking tape and after gold coating images were recorded at
the required magnification.

26.  In-vitro drug release
 The in-vitro permeation of Atorvastatin from niosomal formulation was studied
using locally fabricated diffusion cell. The in-vitro diffusion of the drug through egg
membrane was performed. It was clamped carefully to one end of the hollow
glass tube of 17 mm (area 2.011 cm²) (dialysis cell) this acted as donor
compartment. 100 ml of phosphate buffer saline PBS 7.4 was taken in a beaker
which was used as a receptor compartment. The known quantity was spread
uniformly on the membrane. The donor compartment was kept in contact with the
receptor compartment and the temperature was maintained at 37 ± 0.1 ºC. The
solutions of the receptor side were stirred by externally driven Teflon-coated
magnetic bars.

27.  At predetermined time intervals, sample was withdrawn and replaced by 4 ml of
PBS. The drug concentrations in the aliquot were determined at 244 nm against
appropriate blank. This experiment was done in triplicate and average value was
reported. In- vitro permeation studies were conducted for different formulation and
effect of variation in composition on permeation rate. Drug release data was
normalized by converting the drug concentrations in solution to a percentage of
cumulative drug release and was shown graphically.

28. Drug release kinetic data analysis
 Release data was fitted into various drug release kinetic models like zero order, first order, Higuchi model,
Korsmeyer-peppas model.
 1. Zero order release kinetics:
 Zero order kinetics defines the system in which the release of drug is independent of its concentration.
 Zero order release kinetics expressed as:
 C = C0-K0 t
 Where,
 C = Released/ dissolved amount of drug.
 C0= Initial amount of drug.
 K0= Rate constant for zero order.
 t = Time
 Graph was plotted between cumulative amount of drug released and time.

29.  First order kinetics: Release rate = k [A] ………………………. (1)
 A is concentration of reactant
 3. Higuchi’s model:
 C= [D (2qt-Cs) Cst]1/2
 Where,
 C= Total amount of drug per unit area of matrix (mg/cm2)
 D= Diffusion coefficient
 Qt= Total amount of drug in a unit volume of the matrix
 Cs= Dimension solubility of the drug in polymer matrix
 t= Time (hr)
 Korsemeyer, s and Peppas release model:
 Mt / M∞ = K.tn………………………. (2)
 Where, Mt / M∞ = fraction of drug release, K = release constant, t = release time,
n = Diffusion exponent for the drug release that is dependent on the slop of the
matrix dosage forms.

30. Stability study of niosomes of
Atorvastatin calcium
 Stability studies carried out by storing the prepared niosomes of Atorvastatin
calcium at various temperature conditions like refrigeration on (2-80C) room
temperature (25±0.50C) and elevated temperature (45±0.50C) for a period of 12
weeks. Drug content and variation in the average vesicle diameter were
periodically monitored. ICH (International Conference on Harmonisation)
guidelines suggests stability studies for dry niosomes powder meant for
reconstitution should be studied for accelerated stability at 75% relative humidity
as per international climatic zones and climatic conditions.

31. RESULT AND DISCUSSION
 Preformulation Studies: The drug was authenticated by different test i.e. solubility, melting point,
test according to Indian Pharmacopoeia and analytical methodology was performed on sample to
justify the authenticity of sample.
 Characterization of pure drug
 i) Physical Appearance: Atorvastatin Calcium was found to be white amorphous powder.
 ii) Solubility: The solubility of drug in water was found to be 0.15mg/ml.
 iii) Partition Coefficient: Log P value was found to be 5.6.
 iv) Melting Point: The melting point of Atorvastatin calcium was found to be 161ºC by capillary
tube method.
Identification of pure drug
 I) Preparation of Calibration Curve: The calibration curve for atorvastatin (ATR) was plotted in
phosphate buffer, pH 7.4 and 20 % methanol .The graphs was plotted between concentration (X
axis) and absorbance (Y-axis).

32. Calibration curve of ATR in phosphate
buffer pH 7.4
 S.N. Concentration Absorbance (Mean±S.D), N=3
1 1 0.154±0.013
2 2 0.258±0.012
3 3 0.361±0.057
4 4 0.430±0.031
5 5 0.532±0.031
6 6 0.619±0.18
7 7 0.733±0.019
8 10 0.816±0.011

33. Calibration curve of Atorvastatin
Calcium in 20% methanol

S.N Concentration (μg/ml) Absorbance
(Mean±S.D), N=3
1 0 0
2 1 0.115±0.012
3 2 0.219±0.034
4 3 0.354±0.011
5 4 0.457±0.066
6 5 0.663±0.052
7 6 0.714±0.033
8 8 0.833±0.075

34. Fourier Transform infra-red
spectroscopy Atorvastatin Calcium

35. FTIR spectrum of mixture of
Atorvastatin Calcium, Span 40, Span
60 and Span 80

36. FTIR spectrum of mixture of
Atorvastatin Calcium, Tween 20, Tween
40 and Tween 60

37. Differential Scanning Calorimeter (DSC)

38. Evaluation parameters for Niosomes of
Atorvastatin calcium
 Particle size analysis
 The mean particle diameter of the Atorvastatin calcium niosomes was between 2.33-2.50 μm for
all twelve formulations. Particles of all formulations were smooth, oval and discrete. Niosomes
formulations of batch ANS3 prepared by span 40 shows maximum mean particle diameter i.e.
2.50 μm.
 Drug Entrapment efficiency: The entrapment efficiency of the niosomes was between 66.32-
89.42% .Higher entrapment efficiency of the vesicles of Tween 60 is predictable because of its
higher alkyl chain length. The entrapment efficiency was found to be higher with the batch ANS12
(89.42%), which may have an optimum surfactant cholesterol ratio to provide a high entrapment of
Atorvastatin calcium. The higher entrapment may be explained by high cholesterol content (~50%
of the total lipid). Study concluded that entrapment efficiency increase with increasing cholesterol
content. It was also observed that very high cholesterol content had a lowering effect on drug
entrapment to the vesicles. This could be due to the fact that cholesterol beyond a certain level
starts disrupting the regular bi-layered structure leading to loss of drug entrapment.

39. Properties of Atorvastatin calcium
Niosomes
 Properties of Atorvastatin calcium Niosomes
S.
N
Formulation Code Average mean diameter of
non sonicated niosomes (μ
m)
% Entrapment
efficacy
Viscosity
(Centipoise)
1 ANS1 2.43±0.11 66.32±0.11 2.016
2 ANS2 2.46±0.15 68.31±0.33 2.573
3 ANS3 2.51±0.19 71.12±0.19 2.157
4 ANS4 2.33±0.11 81.32±0.13 3.322
5 ANS5 2.15±0.32 84.41±0.41 2.336
6 ANS6 2.33±0.15 72.45±0.15 2.520
7 ANS7 2.47±0.22 71.42±0.09 2.167

40. S.N Formulation Code Average mean diameter of non
sonicated niosomes (μ m)
% Entrapment
efficacy
Viscosity
(Centipoise)
8 ANS 8 2.69±0.31 85.37±0.12 3.634
9 ANS 9 2.33±0.08 68.51±0.08 2.349
10 ANS 10 2.50±0.06 67.42±0.21 1.686
11 ANS 11 2.13±0.11 68.12±0.08 2.768
12 ANS 12 2.44±0.14 89.42±0.19 3.814

41. Scanning Electron Microscopy
 Morphology and internal cross-sectional structure of the microspheres were
investigated with a scanning electron microscope. SEM is one of the common
methods used owing to the simplicity of sample preparation and ease of
operation. Scanning electron photomicrographs of the selected formulation shown
in figure 6.7. SEM indicates that the niosomes were spherical with a smooth
surface; distinct pores were evident on the surface of niosomes, which will be
responsible for the release. The photomicrographs also showed presence of
loose crystals of drug on the surface of a few niosomes.

42. SEM photograph of Atorvastatin
calcium niosomes of batch ANS4 and
ANS9

43. In-vitro drug release study
 The in-vitro permeation of Atorvastatin calcium from niosomal formulation was studied using
locally fabricated diffusion cell. The cumulative percent drug release after 11 hrs of the
Atorvastatin calcium niosomes in between 32.46-85.43%
 The formulation containing tween showed less permeation compared with the preparation
containing span. It might be due to the larger size of the vesicles and less lipophilic nature of the
Tween, which makes it more difficult for these vesicles to penetrate or fuse with skin whereas, the
inclusion of span which is more lipophilic than tween further increased the lipophilicity of the drug
leading to better penetration.
 Rapid drug leakage was observed during the initial phase. However, after that a slow release
occurred. This could be because the drug is mainly incorporated between the fatty acid chains in
the lipid bilayer of niosomal vesicles which leads to rapid ionization and release upon dispersing
niosomes in large buffer (pH 7.4) volumes until reaching equilibrium.

44. Percentage of drug released from
batch ANS1 to ANS4

Time (Hrs) ANS1 ANS2 ANS3 ANS4
0 0 0 0 0
1 9.605±0.32 8.593±0.08 5.702±0.13 22.46±0.12
2 12.975±0.25 11.283±0.32 7.881±0.26 34.81±0.43
3 20.36±0.41 15.491±0.41 10.128±0.42 44.37±0.26
4 25.787±0.26 19.619±0.24 13.842±0.08 52.48±0.58
5 31.22±0.35 23.065±0.26 18.074±0.41 60.69±0.41
6 34.657±0.67 26.904±0.28 21.624±0.36 64.58±0.26
7 37.764±0.41 30.177±0.26 24.244±0.25 71.47±0.81
8 40.538±0.18 32.878±0.34 27.129±0.61 74.51±0.08
9 43.335±0.09 34.64±0.51 29.318±0.25 77.62±0.07
10 45.036±0.32 37.138±0.24 32.009±0.41 81.53±0.21
11 46.671±0.61 38.904±0.71 32.466±0.34 84.64±0.31

45. Percentage of drug released from
batch ANS5 to ANS8
Time (Hrs) A7NS5 ANS6 ANS7 ANS8
0 0 0 0 0
1 14.785±0.08 9.557±0.31 5.702±0.21 24.206±0.35
2 26.303±0.12 14.666±0.35 8.158±0.15 34.669±0.36
3 34.515±0.21 20.992±0.24 12.53±0.41 42.724±0.26
4 41.964±0.24 26.935±0.51 17.954±0.56 48.771±0.27
5 48.52±0.25 32.381±0.08 23.071±0.42 57.849±0.37
6 52.812±0.34 37.275±0.21 27.393±0.08 63.111±0.41
7 58.309±0.31 41.132±0.51 30.913±0.09 66.938±0.24
8 63.364±0.24 46.023±0.71 34.346±0.21 72.326±0.51
9 65.49±0.25 48.587±0.24 38.893±0.16 76.269±0.06
10 68.521±0.14 50.497±0.41 40.592±0.47 78.558±0.25
11 72.974±0.26 56.746±0.45 41.786±0.55 80.224±0.61

46. Percentage of drug released from
batch ANS9 to ANS12
Time (Hrs) ANS 09 ANS 10 ANS 11 ANS 12
0 0 0 0 0
1 20.26±0.21 6.93±0.12 5.72±0.24 14.26±0.09
2 34.41±0.34 12.26±0.25 8.85±0.23 25.62±0.54
3 41.36 0.35 17.65 0.32 11.94 0.35 34.89 0.26
4 47.59 0.41 20.09 0.15 15.97 0.36 39.88 0.41
5 53.63 0.09 24.71 0.67 19.88 0.46 45.56 0.15
6 62.51 0.62 28.64 0.38 22.67 0.31 48.27 0.23
7 67.32 0.31 32.52 0.09 26.47 0.33 52.51 0.42
8 71.42 0.46 35.12 0.12 29.13 0.42 56.38 0.35
9 75.52 0.71 37.34 0.23 30.68 0.41 60.46 0.14
10 80.41 0.08 38.74 0.32 31.99 0.51 63.47 0.21
11 85.43 0.52 43.89 0.41 37.32 0.26 66.47 0.62

47. Kinetic modeling for Niosomes
Formulation ANS9
 zero order kinetic model, first order kinetic model, Higuchi kinetic model, and
Korsmeyer-peppas release model.
 This indicates that the drug release is controlled by diffusion of the drug through
the pores. The formulations are best fitted into the Peppas model. The 'n' values
of these indicate that in these formulations followed Fickian controlled release
mechanism and in addition, the release appears to be also by erosion and is drug
- dissolution limited.

48. In-Vitro release kinetics of Formulation
ANS9.
time log time sq.rt of
time
%drug
release
log %drug
release
%drug
remaining
log %drug
remaining
0 0 0 0 0 100 2
1 0 1 20.21 1.305566314 79.79 1.901948465
2 0.30103 1.414214 34.44 1.537063143 65.56 1.816638945
3 0.477121 1.732051 41.36 1.61658053 58.64 1.768193962
4 0.60206 2 47.59 1.677515705 52.41 1.71941416
5 0.69897 2.236068 53.69 1.729893404 46.31 1.665674781
6 0.778151 2.44949 62.52 1.796018969 37.48 1.573799582
7 0.845098 2.645751 67.11 1.826787239 32.89 1.517063873
8 0.90309 2.828427 71.42 1.853819846 28.58 1.456062224
9 0.954243 3 75.51 1.87800447 24.49 1.388988785
10 1 3.162278 80.41 1.905310062 19.59 1.292034436
11 1.041393 3.316625 85.43 1.931610406 14.57 1.163459552

49. R2 values of all applied models
Model name Zero order First order Higuchi’s
model
Korsmeyer-
Peppas model
R2 value 0.974 0.986 0.983 0.994

50. Stability studies of batch ANS9
Weeks Refrigeration Room Oven
0 100 100 100
1 99.94±0.01 99.94±0.06 98.85±0.04
3 99.8±0.03 99.8±0.05 97.95±0.08
6 99.76±0.05 99.73±0.04 97.74±0.12
9 99.74±0.03 99.7±0.06 97.36±0.31
12 99.62±0.02 99.60±0.02 97±0.28

51.  Accelerated stability studies for 12 weeks revealed that the formulations were stable at
up to 450C. The results showed that niosomes formulation was quite stable at
refrigeration and room temperatures as not much leakage of drug was found at these
temperatures.
 Therefore, the selected Atorvastatin calcium niosomes formulations can be stored at
either refrigeration or room temperature. The pure drug shows sensitivity to light and
moisture. Therefore formulating it into niosomes can solve this problem to a large
extent. Stability studies revealed that the niosomes kept at room temperature (~25°C)
and 40°-75% RH showed the maximum stability. The values of drug content and in-
vitro studies were close to that of the initial data with only slight variations suggesting
that it has an acceptable shelf life. It should be stored in a cool, dry place.
 Niosomes formulations of Atorvastatin calcium of batch ANS9 shows good stability at
refrigeration and room temperature in comparison to other.

52. THANK
YOU