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 Material

Solid Liquid Gas

Nanoformulations

Liquid/
Solid Aerosol
Semisolid
Nanopowder Nanoemulsion NanoDPI
Nanocrystals NanoMDI
Nanosuspension Nanaerosols
Nanorods
SLN/Nanoshells Lipid nanocarriers Nebulizers
 Solid Nanoformulations

Nanoclusters Nanocrystals Nanopowders Nanotriangles

Semi crystalline nanostructures Single crystalline nanomaterial Noncrystalline agglomerates Trigonal NP; <100 nm
1-10 nm <100 nm <100 nm

Nanocups Nanospheres Nanorods

 Ultrafine Respirable

PM 2.5

Nanoparticles

1 nm 10 nm 100 nm 1 mm 10 mm

 Nano = Ultrafine = < 100 nm (Conventional)
 Nano = <10 nm (suggested by unique
quantum and surface-specific functions)
 Fine = 100 nm - 3 mm
 Respirable (human) = < 5 mm (max = 10 mm)
 Inhalable (human) = ~ 10 - 50 mm
 Mechanical Single particle
interlocking

Van der Waals Capillary

(cohesive force) (surface tension)

Chemical
bonds

Equivalent dia. ~2 x Equivalent diameters of 10-1000x

Settling velocity ~3-4 x are common
 Ultra-fine/ nanoparticles may deposit as
aggregates due to high Van Der Waals forces,
rather than discrete particles
 If an inhaled particle with a diameter of 50–100
nm forms an aggregate of 5–10 particle types, in
terms of deposition it may have the properties of a
200–500 nm particle
 Inhaled agglomerates may dissociate when in
contact with lung surfactants
 Inertial impaction: Airborne
particles possess enough
momentum to keep its trajectory
despite changes in direction of
the air stream colliding with walls
of respiratory tract.
Sedimentation: Time-dependent
particles settling due to the
influence of gravity. Breathing
maneuvers (holding) allows
particles to sediment & increase
lung deposition.
Diffusion: It occurs when
particles are sufficiently small to
undergo a random motion due to
molecular bombardment.
(d: particle diameter; Stk: Stokes number; ρp: particle density; V: air velocity; η: air viscosity; R: airway
radius; Vts: terminal settling velocity; ρa: air density; g: gravitational acceleration; Dif: diffusion coefficient;
k: Boltzmann’s constant; T: absolute temperature; dae: aerodynamic diameter; ρ0: unity density).
 Lungs drug delivery System
DPI Nebulizer MDI

Conventional Novel
(Drug + Lactose) Standard Breath activated
inhalers inhalers

o Liposomes
o Nanoparticles
o Low targeting
o Low density particles
o High and frequent drug dosing
o No propellants
o Drug stability advantages
o High drug dose carrying capacity
o Minimal extrapulmonary loss
o Low exhaled loss
An experimental approach in formulation design
 D
S
Structure C FTIR

HPTLC 1HNMR
 HPTLC condition: 100–1000 ng spot–1
spotted on Silica gel plates 60F254
(Chloroform-methanol; 9:1, v/v) with
RF: 0.34 at 366 nm.
 Validation: Precision/ Accuracy at
200, 400 and 800 ng spot−1, n=6); Intra
day precision was ≤1.91%; Inter day
precision<2.15; Intra day accuracy=99.30–
100.63; Inter day accuracy = 98.09–
99.29%.
 Robustness: small change in mobile phase compositions/volume and saturation
time, drying of plates were monitored. Low values of SD (<3.0) and % RSD (<1.2)
 Sensitivity: Blank methanol spotted 6 times (scanned) and s.d. of analytical
response magnitude was determined. LOD (3.3σ/slope): 9.41ng spot−1;
LOQ=10σ/slope: 28.35 ng spot−1 (Calibration curve).
Acid induced degradation (2N HCl) Base induced degradation (2N NaOH)
50 mg TBS
in 50 mL
methanol

UV induced degradation Photochemical degradation (Day light)

Result: Acid degradation: 4 (TBS)/3 (Sµ-TBS); Base degradation: 2 (TBS)/3 (Sµ-TBS); UV degradation: 2
(TBS)/3 (Sµ-TBS); Photochemical degradation: 2 (TBS)/1 (Sµ-TBS).
 UHPLC/MS condition: Flow rate: 0.25
mL min-1; Runtime: 3.0 min; m/z
226.19→152.12 (TBS) and m/z
260.34→183.11 (IS); Column: BEH C18;
Mobile phase: Acetonitrile–2 mM
Ammonium acetate (1/9)
 Precision for Intra-batch: 3.1-4.3% and
Inter-batch: 4.8-6.8%; Accuracy:
94.50−99.35%.
 Stability study (as %Recovery): Long term
stability (1 month,-80ºC): 95.23 (L) & 95.78
(H); Freeze–thaw stability (-80ºC to 25ºC):
Pharmacokinetics: Rodents Oral dose: 5mg kg-1; Blood sample collection: (0.083,
0.166, 0.25, 0.5, 1-4, 6, 8, 12& 16 h); AUC0−t (735.1±102.3 h.ng mL-1); Cmax
(258.0±15.3 ng mL-1); Tmax (1.0±0.2 h); T0.5 (4.3±0.3 h).
 TBS: (a) protonated precursor ions at m/z 226.19; and (b) Propranolol (IS): (a) precursor ion peaks at m/z 260.34; and (b)
major fragmentated product ion mass spectra at m/z 152.12). major fragmented product ions at m/z 183.11).

Parameter Mean value (±SD)

AUC0−t (h.ng mL-1) 735.10±102.33
Cmax (ng mL-1) 258.00±15.32
Tmax (h) 1.00±0.18
T0.5 (h) 4.34±0.32

TBS Chromatograms: (a) extracted TBS (50 ng mL-1); (b) IS

(100 ng mL-1); (c) Extracted blank plasma (d) extracted TBS
spiked plasma sample (1 ng mL-1).
Condition LQC (2ng mL-1) HQC (800ng mL-1)
Long term stability; recovery (ng) after storage (−80 ◦C)
Initial 1.89±0.01 742.5±10.02
1 month 1.81±0.03 (95.23 %) 711.2±12.51 (95.78%)
Freeze–thaw stability; recovery (ng) after freeze–thaw cycles (−80 ◦C to 25 ◦C)
Cycle 0 1.89±0.01 742.50±10.02
Cycle 1 1.87±0.01 (98.94%) 740.11±12.00 (99.76%)
Cycle 2 1.86±0.01 (98.41%) 731.32±10.17 (98.49%)
Cycle 3 1.85±0.01 (97.88%) 712.61±14.15 (95.97%)
Bench top stability; recovery (ng) after storage at optimized condition
0h 1.89±0.01 742.51±10.02
24 h 1.84 ± 0.01 (97.35%) 720.90±8.25 (97.09%)
Post processing stability; recovery (ng) after storage in autosampler (4 ◦C)
0h 1.89±0.01 742.51±10.02
24 h 1.85±0.02 (97.88%) 723.62±11.05 (97.45%)
 UHPLC-ESI/q-TOF-MS method for the determination of
TBS was developed & validated.
 The method was successfully implicated for PK studies.
 Advantages: Short analysis time (3 min), high sensitivity
(LLOQ: 1.0 ng mL) and simple extraction procedure.
 Simple stirring
 If Particles are: (2000 rpm, 2-4h)

i) Small: <0.3 μ are exhaled Ultrasonication

(25ºC, 15 min)
ii) Large: >1.5 μ are lost in
HPH
epiglottis/ GIT (15000 psi, 1-6 cycle)

iii) Intermediate: 0.5 - 1.5 μ Probe Sonication

goes deep into lungs (250 W, 10 min)

Nanoprecipitation
(solvent/antisolvent)
 The weighed amount of drug (250 Antisol Stabilizer Conc Size (µ)
mg) was passed through 400-mesh ACN Leucine 1-20 >6
sieve and slowly added in different Tween 80 1-20 >8.5
antisolvent containing different Pluronic F68 1-10 2.3
stabilizers (10% w/w), placed over
magnetic stirrer (2000 rpm; 2-4 h). IPA Leucine 20 >7.4
Tween 80 1-20 >10
Pluronic F68 1-20 >10
Ethanol Leucine 1-20 >10
Tween 80 1-20 >10
Pluronic F68 1-20 >10

Particles obtained in all batches were large

(2.3 to >10.0 μ), it was concluded that stirring
method was insufficient enough to produce
nanosized/submicronized particles.
Weighed amount of drug was passed through 400-mesh sieve and dropped
slowly into solution of stabilizer placed on bath sonicator (25°C; 15 min)
Effect of stabilizers in Ethanol Effect of stabilizers in ACN
Code Stabilizer Conc. Size (nm) Code Stabilizer Conc. Size* (nm)
AB1 Leucine 5 1879.12±31.78 AA1 Leucine 5 1421.20±39.61
AB2 10 1042.90±26.41 AA2 10 741.91±23.56
AB3 20 823.61±16.21 AA3 20 225.89±18.09*
AB4 Tween 80 5-20 Aggregates AA4 Tween 80 5-20 Aggregated
AB5 Pluronic F68 1 3559.10±21.10 AA5 Pluronic F68 5 3319±11.29
AB6 10 1119±25.29 AA6 10 728±33.17
AB7 20 728±33.17 AA7 20 515±24.12
AB8 PVA 5 783.48±16.73
AB9 10 545.12±19.18
AB10 20 278.70±8.42*
Conclusion: Ultrasonication method was found to
produce smaller droplets. Best size achieved was 278.70
nm with 20% of PVA (AB10) in ethanol and 225.89
nm with 20% Leucine (AA3) in ACN.
 Ultrasonically induced particles  TBS (400-mesh sieved) poured slowly
were further subjected to into antisolvent containing stabilizer
homogenization (10000-15000 and irradiated with ultrasonic energy by
psi/1-5 cycles). probe and sonifier device (20 kHz; 250 W
for 10 min).
 Increasing homogenization cycle
 Stabilizing effect= ACN: Pluronic
(1-3), particle size reduced [(278.70 F68<Leucine<PVA<Tween80; Ethanol:
nm (AC1) to 187.44 nm (AC3)]. PVA<Pluronic F68<Leucine<Tween 80;
Particle size remained unchanged after IPA:Pluronic F68<PVA<Leucine<Tween80.
further treatment. Code Antisol Stabilizer Size
Code Cycles Size AD1 ACN Pluronic F68 186.15*
AD2 Leucine 217.61
AC1 0 278.70 AD3 PVA 243.57
AD4 Tween 80 746.33
AC2 1 215.35 AD5 Ethanol Pluronic F68 242.26
AD6 Leucine 419.47
AC3 3 187.44* AD7 PVA 211.58*
AD8 Tween 80 862.13
AC4 5 185.74 AD9 IPA Pluronic F68 267.15*
AD10 Leucine 389.11
AD11 PVA 314.45
AD12 Tween 80 654.00
 Particles (Probe Sonication)
Raw TBS particles before
nanosizing
(a) 10X magnification
(b) 40X magnification

TEM images of TBS NP

produced in ACN with
different stabilizers (a)
Pluronic F68: AD1 (b)
Tween 80: AD4

(a) (b) SEM images of TBS NP

particles produced in
ACN by probe sonicator
using (a) Pluronic F68:
AD1 (b) Tween 80: AD4
 Sol/Antis Homogenize

Stabilizer Evaporation

Nanoprecipitation Droplet Particles

Solvent Solubility
 The drug was dissolved in water Water Freely soluble

(HPLC grade) and passed through ACN Insoluble

0.22 µ pore size filter to remove Chloroform Insoluble

DCM Insoluble
particulate impurities. The solution
Methanol Slightly soluble
was then drop wise added into
IPA Insoluble
different organic solvents n-Hexane Insoluble
containing stabilizer. DMSO Insoluble
Acetone Insoluble
Ethanol Insoluble
Code Stabilizer Conc. Size (nm)
AG1 Nil Nil Aggregates AG16 89.65

AG2 PVA 5 1095.17±29.45 AG15 95.86

AG3 10 568.53±11.38
AG14 195.3
AG4 20 Aggregates
AG13 221.7
AG5 Tween 80 1-20 Sticky aggregates
AG6 5 595.20±18.29 AG11 122.5
Leucine

Formulation Code
AG7 10 222.70±19.24 AG10 216.51

AG8 20 198.20±22.17 AG9 1419.09

AG9 Pluronic F68 1 1419.09±31.94

AG8 198.2
AG10 10 216.51±15.07
AG7 222.7
AG11 20 122.50±21.35
AG12 PVA+Tween 80 Aggregates AG6 595.2

10+10
AG13 PVA+PL F68 221.70±17.87 AG3 568.53

AG14 PVA+Leucine 195.30±11.79

AG2 1095.17
AG15 Leucine+ 10+10 95.86±15.19
0 200 400 600 800 1000 1200 1400 1600
AG16 PL F68 15+15 89.65±10.58* Particle size (nm)
 TEM images of TBS particles
precipitated out at
High stirring rate: 2000 rpm
Low stirring speed; 1000 rpm

(a) (b)
(b) (c)

TBS Submicron particles SEM images (a) Raw TBS (b) without stabilizer (c)
with 15% Leucine+Pluronic F68
Raw TBS: large sized particles; Without stabilizer: needle shaped, aggregated
and large in size; With Leucine+Pluronic F68: Nanoparticle, spherical, Leucine
coating: feather like (pollen shape).
AA3 225.8 AB10 278.8 AC3 187.4 AD1 186.1 AG11 122.5 AG16 89.65

Spray drying Form. Technique Initial Hot plate Vacuum Freeze Spray Rotary
AA3 Ultrasonication 225.89 >3000 >2500 1080.31 1441.06 1539.46
Freeze drying
AB10 Ultrasonication 278.71 >3000 >2500 1305.77 1826.14 1950.91
Vacuum drying AC3 US-HPH 187.44 >3000 >2500 956.89 1244.65 1529.08
AD1 Probe sonication 186.15 >3000 >2500 911.37 1179.17 1345.71
Rotary
evaporator AG11 Nanoprecipitation 122.50 >3000 >2500 897.03 1018.94 1245.01
AG15 Nanoprecipitation 95.86 >3000 >2500 620.81 993.04 1092.49
Hot plate
AG16 Nanoprecipitation 89.65 >3000 >2500 612.22 789.55 1025.25
AS16 (789.55 nm)
Code Stabilizer concentration (%) Size (nm) Effect of Cryoprotectants
Pluronic F68 PVA Leucine Tween 80
AF11 2.0 121.92 Concentration (%) Before Scale After
Lactose Sorbitol Mannitol drying drying
AF3 1.0 568.31
0.5 - - 89.65 +++ 1224.33
AF6 2.0 198.84
1 - - - ++ 1188.12
AF5 1.0 Aggreg.
1.5 - - - + 1085.06
AF15 1.0 1.0 95.31
AF16 1.5 1.5 89.65 2.0 - - - + 991.41
1.5 0.5 - - + 905.42
1.5 1.0 - - ++ 866.59
1.5 0.5 0.5 - + 815.03
AF16 1.5 0.5 1.0 - + 729.53
(612.22 1.5 0.5 1.5 - * 685.43
nm) 1.5 0.5 2.0 - * 620.81
1.5 0.5 2.5 - * 612.22
(*)Dry product; (+++) formation of sticky mass; (++)
High Aggregation; (+) Low aggregation.
 Raw TBS AG16 AS16 AF16
16.3µ 89.65 nm 789.55 nm 612.22 nm

Lactose Sorbitol Dextrose Mannitol

(4-25µ) (20-43µ) (4.5-24µ) (10-26µ)

On performance basis Lactose was selected as carrier for pulmonary

delivery submicron TBS particles.
 FTIR AF16 FTIR AS16

PXRD AS3 DSC AS3

AS16
AF16
TBS
AS16 AS16

AF16
 Characters AF16 (Stability condition)
250C/60%RH (Controlled) 400C/75%RH (Accelerated)
Sampling Initial 3 6 12 3 6
Appearance Free flow
• AF

Freeze
16
Size (nm) 612.22±8.3 628.4±12.8 639.8±11.4 654.3±13.4 834.3±14.6 874.2±12.3
Dried Drug (%) 99.80±2.60 98.50±1.80 98.30±2.90 97.30±2.60 97.40±2.20 96.10±2.40
sample MC (%) 2.1±0.10 1.8±0.07 1.5±0.04 1.3±0.02 1.5±0.10 1.3±0.03
FPF (%) 78.57±3.08 75.58±1.82 73.63±2.44 72.16±2.31 65.61±2.64 59.87± 1.41
• AS
16 ED (%) 84.68±2.11 84.34±1.30 83.28±1.78 82.51±1.94 78.39±2.32 74.66±1.87
Spray
dried Characters AS16 (Stability condition)
sample 250C/60%RH (Controlled) 400C/75%RH (Accelerated)
Sampling Initial 3 6 12 3 6
250±20 μg TBS
Appearance Free flow
filled into HPMC
Cap#2 packed in Size (nm) 789.55±6.41 793.24±14.32 799.11±16.44 815.26±19.81 838.43±14.61 869.37±20.23
HDPE bottles
sealed with PVC Drug (%) 99.84±1.91 98.71±2.12 98.15±2.35 97.30±2.60 97.89±3.12 96.56±3.34
coated aluminum MC (%) 1.71±0.05 1.53±0.05 1.33±0.04 1.17±0.02 1.49±0.04 1.12±0.01
foil, loaded to
Stability Chamber FPF (%) 82.06±2.19 81.58±1.92 79.63±2.38 75.34±2.63 68.53±2.14 62.28±1.95
ED (%) 88.25±1.95 87.42±2.51 86.72±3.35 86.51±3.94 81.44±3.32 78.12±3.87
 18 AS16 20 AF16
16
789.55 nm 612.22 nm

Percentage deposition
Percentage deposition
14 15
12
10 10
8
6
5
4
2
0
0 I.P. P.S. 0 1 2 3 4 5 6 7 filter
I.P. P.S. 0 1 2 3 4 5 6 7 filter
Part of ACI
Part of ACI

Form. ACI inhalation data

ED (%) FPF (%) MMAD (μ)

Raw TBS 49.87±3.81 38.19±2.21 4.98±1.21 Dissolution

study
AF16 84.68±2.11 78.57±3.08 1.43±0.59

AS16 88.25±1.95 82.06±2.19 1.61±0.73

Wistar rats (n=6; 200–250g); Dose: 25 mg for 30 min UHPLC peaks in Plasma, BAL, Alveolar tissue

Parameters Oral Inhalation

AF16 AS16 AF16 AS16
Cmax (ng/mL) 657.73±58.00 905.15±86.14 713.36±0.98 978.67±105.30
AUC0−t [(ng/mL)/h] 4450.53±125.86 3855.21±152.07 6950.11±217.26 10178.34±392.67
T0.5 (h) 3.67±0.71 3.89±1.04 3.62±0.84 5.06±1.46
MRT (h) 5.08±0.89 6.33±1.17 5.92±1.00 8.13±1.96
 NP were successfully produced from freeze and spray drying methods.
 Both particles behave good aerosol effects and deposition..
 In vitro and in vivo data confirmed the potential of NP in achieving
better pulmonary targeting.

FOR USE WITH REVOLIZER ONLY

Each Capsule contains
Terbutaline Sulphate 0.25mg
Excipient q.s.
Approved colours used in empty Warning
Capsule To be sold by retail on the
prescription of a RMP only
Terbohale
Direction for use
Refer to the enclosed leaflet before use.
Do not exceed the recommended dose.
Rs.
Keep the container tightly closed.
Batch No. C20240
Caution
Mfd. Date Jan 2012
Capsules are intended for use through
Exp. Date Feb 2014
Revolizer only and are not to be swallowed.

Mfd. By Jamia Hamdard

Nanomission:
Save Lungs
 Queries?

Principal Investigator
Nanopharmaceutical & Drug Delivery Research Lab
Division of Pharmaceutics, Faculty of Pharmacy
INTEGRAL UNIVERSITY, India
Email: md.faiyazuddin@gmail.com
 Journal of Nanomedicine &
Biotherapeutic Discovery

 Journal of Nanomaterials & Molecular

Nanotechnology
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