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Fate and Effects of Nanoparticles in Lungs

Fate and Effects of Nanoparticles in Lungs. Md. Faiyazuddin , M. Pharm., Ph.D. Principal Investigator Nanopharmaceutical & Drug Delivery Research Lab Division of Pharmaceutics, Faculty of Pharmacy INTEGRAL UNIVERSITY, India. Liquid Nanoformulations. Solid Nanoformulations. Nanoclusters

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Fate and Effects of Nanoparticles in Lungs

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  1. Fate and Effects of Nanoparticles in Lungs Md. Faiyazuddin,M. Pharm., Ph.D. Principal Investigator Nanopharmaceutical & Drug Delivery Research Lab Division of Pharmaceutics, Faculty of Pharmacy INTEGRAL UNIVERSITY, India

  2. Liquid Nanoformulations

  3. Solid Nanoformulations Nanoclusters Semi crystalline nanostructures 1-10 nm Nanocrystals Single crystalline nanomaterial <100 nm Nanopowders Noncrystalline agglomerates <100 nm Nanotriangles Trigonal NP; <100 nm Nanospheres Nanocups Nanorods

  4. Macro versus Nano

  5. Particle Scale • Ultrafine • Respirable • PM 2.5 • Nanoparticles • 10 mm • 1 mm • 1 nm • 10 nm • 100 nm

  6. Definitions- Particle Size • 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

  7. Forces & Surface Chemistry Single particle Mechanical interlocking Capillary (surface tension) Van der Waals (cohesive force) Chemical bonds Equivalent dia. ~2 x Settling velocity ~3-4 x Equivalent diameters of 10-1000x are common

  8. Properties influence lung deposition • 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

  9. 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).

  10. Potential Pathways

  11. Current Inhalers in Market

  12. Nanoparticle formation

  13. Particle Engineering

  14. Lungs drug delivery System DPI Nebulizer MDI Novel Conventional (Drug + Lactose) Standard inhalers Breath activated inhalers • Liposomes • Nanoparticles • Low density particles • Low targeting • High and frequent drug dosing • No propellants • Drug stability advantages • High drug dose carrying capacity • Minimal extrapulmonary loss • Low exhaled loss

  15. Pharmaceutical Research in Drug Delivery to Lungs An experimental approach in formulation design

  16. Drug Candidate DSC Structure FTIR Terbutaline sulfate (C12H19NO3) HPTLC 1HNMR

  17. HPTLC Method HPTLC condition: 100–1000 ng spot–1 spotted on Silica gel plates 60F254 (Chloroform-methanol; 9:1, v/v) withRF: 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).

  18. Forced degradation studies 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).

  19. UHPLC-ESI-qTOF/MS • 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).

  20. Fragments & Chromatograms 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).

  21. Analytesstability in UHPLC

  22. UHPLC-qTOF/MS • 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 ngmL) and simple extraction procedure.

  23. Formulation Development • If Particles are: • Small: <0.3 μ are exhaled • Large: >1.5 μ are lost in epiglottis/ GIT • Intermediate: 0.5 - 1.5 μ goes deep into lungs OPTIMIZATION

  24. Simple stirring method • 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). 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.

  25. Ultrasonication 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) Effect of stabilizers in ACN Effect of stabilizers in Ethanol 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.

  26. High Pressure Homogenization Probe Sonication • 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. • 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.

  27. (b) (a) Particles (Probe Sonication) Raw TBS particles before nanosizing • 10X magnification • 40X magnification 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

  28. Nanoprecipitation method Sol/Antis Homogenize • 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. Evaporation Stabilizer Nanoprecipitation Particles Droplet

  29. Effect of Surfactant

  30. (b) (a) Effect of stirring TEM images of TBS particles precipitated out at High stirring rate: 2000 rpm Low stirring speed; 1000 rpm (c) (b) 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).

  31. Effect of Drying AA3225.8 AB10278.8 AC3187.4 AD1186.1 AG11122.5 AG1689.65

  32. Spray dried particles AS16 (789.55 nm)

  33. Lyophilized particles Effect of Cryoprotectants AF16 (612.22 nm)

  34. Optimised particles & Carriers AG16 89.65 nm AS16 789.55 nm Raw TBS 16.3µ AF16 612.22 nm Lactose (4-25µ) Dextrose (4.5-24µ) Sorbitol (20-43µ) Mannitol (10-26µ) On performance basis Lactose was selected as carrier for pulmonary delivery submicron TBS particles.

  35. Characterization FTIR AF16 FTIRAS16 PXRD AS3 DSC AS3 AS16 AF16 TBS AS16 AS16 AF16

  36. Stability evaluation 250±20 μg TBS filled into HPMC Cap#2 packed in HDPE bottles sealed with PVC coated aluminum foil, loaded to Stability Chamber

  37. Andersen Cascade Impaction AS16 789.55 nm AF16 612.22 nm Dissolution study

  38. Inhalation device

  39. In-vivo estimations

  40. Proof of drug delivered to Lungs

  41. Expert Opinion Each Capsule contains Terbutaline Sulphate 0.25mg Excipientq.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 Warning To be sold by retail on the prescription of a RMP only Terbohale Rs. Batch No. C20240 Mfd. Date Jan 2012 Exp. Date Feb 2014 Mfd. By Jamia Hamdard 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.

  42. Summary Nanomission: Save Lungs

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

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