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Md. Faiyazuddin , M. Pharm., Ph.D.
Principal InvestigatorNanopharmaceutical & Drug Delivery
Research LabDivision of Pharmaceutics, Faculty of
PharmacyINTEGRAL UNIVERSITY, India
Fate and Effects of
Nanoparticles in Lungs
N
A
N
O
Material
Solid Liquid Gas
Nanoformulations
SolidLiquid/ Semisoli
dAerosol
Nanopowder Nanocrystals
Nanorods SLN/Nanoshells
Nanoemulsion Nanosuspensio
n Lipid
nanocarriers
NanoDPI NanoMDI
Nanaerosols Nebulizers
NanoclustersSemi crystalline nanostructures
1-10 nm
Nanocups Nanospheres Nanorods
NanocrystalsSingle crystalline nanomaterial
<100 nm
NanopowdersNoncrystalline agglomerates
<100 nm
NanotrianglesTrigonal NP; <100 nm
Solid Nanoformulations
Nano = Ultrafine = < 100 nm (Conventional) Nano = <10 nm (suggested by unique
quantum and surface-specific functions) Fine = 100 nm - 3 m Respirable (human) = < 5 m (max = 10 m) Inhalable (human) = ~ 10 - 50 m
Definitions- Particle Size
Forces & Surface Chemistry
Equivalent dia. ~2 x
Settling velocity ~3-4 x
Mechanical interlocking
Capillary
(surface tension)Van der Waals
(cohesive force)
Chemical bonds
Single particle
Equivalent diameters of 10-1000x 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
Properties influence lung deposition
Mechanism in Lungs deposition
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).
DPI
MDI
Conventional (Drug + Lactose)
Standard inhalers
Breath activated inhalers
Novel
o Liposomeso Nanoparticleso Low density
particleso Low targeting
o High and frequent drug dosing
Lungs drug delivery System
o No propellants
o Drug stability advantages
o High drug dose carrying capacity
o Minimal extrapulmonary loss
o Low exhaled loss
Nebulizer
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%.
HPTLC Method
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).
Forced degradation studies
Acid induced degradation (2N HCl)
Base induced degradation (2N NaOH)
UV induced degradation
Photochemical degradation (Day light)
50 mg TBS in 50 mL
methanol
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):
UHPLC-ESI-qTOF/MS
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) major fragmentated
product ion mass spectra at m/z 152.12).
Propranolol (IS): (a) precursor ion peaks at m/z 260.34; and (b) major fragmented
product ions at m/z 183.11).
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).
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
Fragments & Chromatograms
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
0 h 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)
0 h 1.89±0.01 742.51±10.02
24 h 1.85±0.02 (97.88%) 723.62±11.05 (97.45%)
Analytes stability in UHPLC
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.
UHPLC-qTOF/MS
UHPLC/MS
Publication
If Particles are: i) Small: <0.3 μ are exhaledii) Large: >1.5 μ are lost in
epiglottis/ GITiii) Intermediate: 0.5 - 1.5 μ
goes deep into lungs
Simple stirring (2000 rpm, 2-4h)
Ultrasonication (25ºC, 15 min)
HPH (15000 psi, 1-6
cycle)
Probe Sonication (250 W, 10 min)
Nanoprecipitation (solvent/antisolvent)
OP
TI
MI
ZA
TI
ON
Formulation Development
The weighed amount of drug (250 mg) was passed through 400-mesh sieve and slowly added in different antisolvent containing different stabilizers (10% w/w), placed over magnetic stirrer (2000 rpm; 2-4 h).
Antisol Stabilizer Conc Size (µ)
ACN LeucineTween 80
Pluronic F68
1-201-201-10
>6>8.5 2.3
IPA LeucineTween 80
Pluronic F68
201-201-20
>7.4>10>10
Ethanol LeucineTween 80
Pluronic F68
1-201-201-20
>10>10>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.
Simple stirring method
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)
Code Stabilizer Conc. Size* (nm)
AA1
AA2
AA3
Leucine 51020
1421.20±39.61741.91±23.56
225.89±18.09*
AA4 Tween 80 5-20 Aggregated
AA5
AA6
AA7
Pluronic F68 51020
3319±11.29728±33.17515±24.12
Code Stabilizer Conc. Size (nm)
AB1
AB2
AB3
Leucine 51020
1879.12±31.781042.90±26.41 823.61±16.21
AB4 Tween 80 5-20 Aggregates
AB5
AB6
AB7
Pluronic F68 11020
3559.10±21.101119±25.29728±33.17
AB8
AB9
AB10
PVA 51020
783.48±16.73545.12±19.18278.70±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.
Effect of stabilizers in Ethanol
Effect of stabilizers in ACN
Ultrasonication method
Ultrasonically induced particles were further subjected to homogenization (10000-15000 psi/1-5 cycles).
Increasing homogenization cycle (1-3), particle size reduced [(278.70 nm (AC1) to 187.44 nm (AC3)]. Particle size remained unchanged after further treatment.
Code Cycles Size
AC1 0 278.70
AC2 1 215.35
AC3 3 187.44*
AC4 5 185.74
TBS (400-mesh sieved) poured slowly into antisolvent containing stabilizer and irradiated with ultrasonic energy by probe and sonifier device (20 kHz; 250 W for 10 min).
Stabilizing effect= ACN: Pluronic F68<Leucine<PVA<Tween80; Ethanol: PVA<Pluronic F68<Leucine<Tween 80; IPA:Pluronic F68<PVA<Leucine<Tween80.
Code Antisol Stabilizer Size
AD1
AD2
AD3
AD4
ACN Pluronic F68Leucine
PVATween 80
186.15*
217.61243.57746.33
AD5
AD6
AD7
AD8
Ethanol Pluronic F68Leucine
PVATween 80
242.26419.47211.58*
862.13
AD9
AD10
AD11
AD12
IPA Pluronic F68Leucine
PVATween 80
267.15*
389.11314.45654.00
High Pressure Homogenizatio
n
Probe Sonication
(b)(b)(a)(a)
TEM images of TBS NP produced in ACN with different stabilizers (a) Pluronic F68: AD1 (b) Tween 80: AD4
SEM images of TBS NP particles produced in ACN by probe sonicator using (a) Pluronic F68: AD1 (b) Tween 80: AD4
Raw TBS particles before nanosizing
(a) 10X magnification(b) 40X magnification
Particles (Probe Sonication)
The drug was dissolved in water (HPLC grade) and passed through 0.22 µ pore size filter to remove particulate impurities. The solution was then drop wise added into different organic solvents containing stabilizer.
Solvent Solubility
Water Freely soluble
ACN Insoluble
Chloroform Insoluble
DCM Insoluble
Methanol Slightly soluble
IPA Insoluble
n-Hexane Insoluble
DMSO Insoluble
Acetone Insoluble
Ethanol Insoluble
Stabilizer
Evaporation
Droplet
Nanoprecipitation
Particles
Homogenize
Sol/Antis
Nanoprecipitation method
Code Stabilizer Conc. Size (nm)
AG1 Nil Nil Aggregates
AG2 PVA 5 1095.17±29.45
AG3 10 568.53±11.38
AG4 20 Aggregates
AG5 Tween 80 1-20 Sticky aggregates
AG6 Leucine 5 595.20±18.29
AG7 10 222.70±19.24
AG8 20 198.20±22.17
AG9 Pluronic F68 1 1419.09±31.94
AG10 10 216.51±15.07
AG11 20 122.50±21.35
AG12 PVA+Tween 8010+10
Aggregates
AG13 PVA+PL F68 221.70±17.87
AG14 PVA+Leucine 195.30±11.79
AG15 Leucine+PL F68
10+10 95.86±15.19
AG16 15+15 89.65±10.58*
1095.17
568.53
595.2
222.7
198.2
1419.09
122.5
221.7
195.3
95.86
89.65
216.51
0 200 400 600 800 1000 1200 1400 1600
AG2
AG3
AG6
AG7
AG8
AG9
AG10
AG11
AG13
AG14
AG15
AG16
Fo
rmu
lati
on
Co
de
Particle size (nm)
Effect of Surfactant
TEM images of TBS particles precipitated out at
High stirring rate: 2000 rpm Low stirring speed; 1000 rpm
(a) (b)
TBS Submicron particles SEM images (a) Raw TBS (b) without stabilizer (c) with 15% Leucine+Pluronic F68
(b) (c)
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).
Effect of stirring
AA3 225.8 AB10 278.8 AC3 187.4 AD1 186.1 AG11 122.5 AG16 89.65
Spray drying
Freeze drying
Vacuum drying
Rotary evaporator
Hot plate
Form. Technique
Initial
Hot plate
Vacuum
Freeze Spray Rotary
AA3Ultrasonicati
on
225.89
>3000
>2500
1080.31
1441.06
1539.46
AB10Ultrasonicati
on
278.71
>3000
>2500
1305.77
1826.14
1950.91
AC3 US-HPH
187.44
>3000
>2500
956.89
1244.65
1529.08
AD1
Probe sonicati
on
186.15
>3000
>2500
911.37
1179.17
1345.71
AG11Nanoprecipit
ation
122.50
>3000
>2500
897.03
1018.94
1245.01
AG15Nanoprecipit
ation 95.8
6>300
0>250
0620.8
1993.0
41092.4
9
AG16Nanoprecipit
ation 89.65>300
0>250
0 612.22 789.55 1025.25
Effect of Drying
Cod
e
Stabilizer concentration (%) Size
(nm)Pluronic
F68
PV
A
Leuci
ne
Tween
80AF1
12.0 121.92
AF3 1.0 568.31
AF6 2.0 198.84
AF5 1.0 Aggreg
.
AF1
5
1.0 1.0 95.31
AF1
6
1.5 1.5 89.65
Concentration (%) Befor
e
dryin
g
Scal
e
After
dryingLacto
se
Sorbi
tol
Mannit
ol
0.5 - - 89.6
5
+++ 1224.
33
1 - - - ++ 1188.1
2
1.5 - - - + 1085.0
6
2.0 - - - + 991.41
1.5 0.5 - - + 905.4
2
1.5 1.0 - - ++ 866.59
1.5 0.5 0.5 - + 815.0
3
1.5 0.5 1.0 - + 729.53
1.5 0.5 1.5 - * 685.43
1.5 0.5 2.0 - * 620.81
1.5 0.5 2.5 - * 612.2
2(*)Dry product; (+++) formation of sticky mass; (++) High Aggregation; (+) Low aggregation.
AF16
(612.22 nm)
Effect of Cryoprotectants
Lyophilized particles
Lactose
(4-25µ)
Sorbitol (20-43µ)
Dextrose
(4.5-24µ)
Mannitol (10-26µ)
AF16 612.22
nm
AS16 789.55
nm
AG16 89.65 nm
Raw TBS
16.3µ
On performance basis Lactose was selected as carrier for pulmonary delivery submicron TBS particles.
Optimised particles & Carriers
Characte
rs
AF16 (Stability condition)
250C/60%RH (Controlled) 400C/75%RH
(Accelerated)
Samplin
g
Initial 3 6 12 3 6
Appeara
nce
Free flow
Size
(nm)
612.22±8
.3
628.4±12
.8
639.8±11.
4
654.3±1
3.4
834.3±14
.6
874.2±12.3
Drug (%) 99.80±2.
60
98.50±1.
80
98.30±2.9
0
97.30±2.
60
97.40±2.
20
96.10±2.40
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±2.4
4
72.16±2.
31
65.61±2.
64
59.87± 1.41
ED (%) 84.68±2.
11
84.34±1.
30
83.28±1.7
8
82.51±1.
94
78.39±2.
32
74.66±1.87
Characte
rs
AS16 (Stability condition)
250C/60%RH (Controlled) 400C/75%RH
(Accelerated)
Sampling Initial 3 6 12 3 6
Appeara
nce
Free flow
Size
(nm)
789.55±6.
41
793.24±1
4.32
799.11±1
6.44
815.26±1
9.81
838.43±14.
61
869.37±2
0.23
Drug (%) 99.84±1.9
1
98.71±2.1
2
98.15±2.3
5
97.30±2.6
0
97.89±3.12 96.56±3.3
4
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
FPF (%) 82.06±2.1
9
81.58±1.9
2
79.63±2.3
8
75.34±2.6
3
68.53±2.14 62.28±1.9
5
ED (%) 88.25±1.9
5
87.42±2.5
1
86.72±3.3
5
86.51±3.9
4
81.44±3.32 78.12±3.8
7
• AF16
Freeze
Dried samp
le• AS
16
Spray
dried samp
le
250±20 μg TBS filled into HPMC Cap#2 packed in HDPE bottles sealed with PVC coated aluminum foil, loaded to Stability Chamber
Stability evaluation
0
2
4
6
8
10
12
14
16
18
Perc
enta
ge de
posit
ionI.P. P.S. 0 1 2 3 4 5 6 7 filter
Part of ACI
0
5
10
15
20
Perce
ntage
depo
sition
I.P. P.S. 0 1 2 3 4 5 6 7 filter
Part of ACI
AF16 612.22
nm
AS16 789.55
nm
Dissolution study
Andersen Cascade Impaction
Form. ACI inhalation data
ED (%) FPF (%) MMAD
(μ)
Raw
TBS
49.87±3
.81
38.19±2.
21
4.98±1.2
1
AF16 84.68±2
.11
78.57±3.
08
1.43±0.5
9
AS16 88.25±1
.95
82.06±2.
19
1.61±0.7
3
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
In-vivo estimations
Each Capsule containsTerbutaline Sulphate 0.25mg
Excipient q.s.
Approved colours used in empty
Capsule
Direction for use
Refer to the enclosed leaflet before use.
Do not exceed the recommended dose.
Keep the container tightly closed.
Caution
Capsules are intended for use through
Revolizer only and are not to be swallowed.
FOR USE WITH REVOLIZER ONLY
Terbohale
Warning
To be sold by retail on the
prescription of a RMP only
Rs.
Batch No. C20240
Mfd. Date Jan 2012
Exp. Date Feb 2014
Mfd. By Jamia Hamdard
Expert Opinion 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.
DR. MD. FAIYAZUDDIN
Queries?
Principal InvestigatorNanopharmaceutical & Drug Delivery
Research LabDivision of Pharmaceutics, Faculty of
Pharmacy
INTEGRAL UNIVERSITY, India
Email: [email protected]
Journal of Nanomedicine & Biotherapeutic Discovery
Journal of Nanomaterials & Molecular Nanotechnology
Journal of Nanomedicine & Nanotechnology
Nano Research & Applications
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