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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

Liquid Nanoformulations

Nanoemulsion(<50 nm)

Emulsion(>5000 nm)

Nanosuspensions

NanoclustersSemi crystalline nanostructures

1-10 nm

Nanocups Nanospheres Nanorods

NanocrystalsSingle crystalline nanomaterial

<100 nm

NanopowdersNoncrystalline agglomerates

<100 nm

NanotrianglesTrigonal NP; <100 nm

Solid Nanoformulations

Macro versus Nano

M

acro

particl

es

Nano

particl

es

Particle Scale

Nanoparticles

Ultrafine Respirable

1 nm 10 nm 100 nm 1 mm 10 mm

PM 2.5

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).

Potential Pathways

Current Inhalers in Market

Nanoparticle formation

Particle Engineering

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

Pharmaceutical Research in Drug Delivery to Lungs

An experimental approach in formulation design

Structure

FTIR

1HNMR

HPTLC

Terbutaline sulfate

(C12H19

NO3)

DSC

Drug Candidate

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).

HPTLC Publicatio

n

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

AS16 (789.55 nm)

Spray dried particles

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

FTIR AS16

FTIR AF16

AF1

6

AS1

6

TBS

PXRD DSC

AS16

AS3 AS3

AS16

AF16

Characterization

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

Inhalation device

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

Proof of drug delivered to Lungs

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.

Summary

Nanomission: Save Lungs

DR. MD. FAIYAZUDDIN

Queries?

Principal InvestigatorNanopharmaceutical & Drug Delivery

Research LabDivision of Pharmaceutics, Faculty of

Pharmacy

INTEGRAL UNIVERSITY, India

Email: md.faiyazuddin@gmail.com

Journal of Nanomedicine & Biotherapeutic Discovery

Journal of Nanomaterials & Molecular Nanotechnology

Journal of Nanomedicine & Nanotechnology

Nano Research & Applications

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