Nikhil nanoparticles and liposomes

nanoparticles and liposomes

1. Targeted Drug Delivery System NANOPARTICLES & LIPOSOMES. Presented By: Mr. Nikhil Patil. M.Pharm 1 st year. Department Pharmaceutics

2. Introduction Nanoparticles are sub- nanosized colloidal structures composed of synthetic or semi synthetic polymers. The first reported nanoparticles were based on non-biodegradable polymeric systems. e.g. polyacrylamide , polymethylmethacrylate , polystyrene etc. The possibilities of chronic toxicity due to tissue and immunological response towards these polymers had restricted their use for systemic administration.

3. This problem has been solved by using biodegradable polymers. The term particulate is suggestively generalized because they could be Nanospheres Nanocapsules Nanocrystals Nanoparticles

4. S olid core spherical particle , in which drug embedded within matrix or adsorbed on the surface . Drug is encapsulated Within central volume surrounded by embryonic polymeric sheath

5. Nanospheres and Nanocapsules

6. Natural Hydrophilic Polymers Proteins and Polysaccharides have been extensively studied and characterized. Proteins Polysaccharides Gelatin Albumin Lectins Legumin Viciline Alginate Dextran Chitosan Agarsoe Pullulan

7. Disadvantage: Batch to batch variation. Conditional biodegradability. Antigenicity .

8. Synthetic Hydrophobic Polymer : The polymer used are either pre-polymerized or polymerized in process Pre-polymerized Polymerized in process Poly ( ε - caprolactone ) (PECL) Poly (lactic acid) (PLA) Poly ( lactide -co- glycolide ) (PLGA) Polystyrene Poly ( isobutylcynoacrylates ) (PICA) Poly ( butylcynoacrylates ) (PBCA) Polyhexylcyanoacrylates (PHCA) Poly ( methacrylate ) (PMMA)

9. Preparation Techniques The appropriate method selection depends on the physicochemical characteristics of the polymer and the drug to be loaded. The preparation technique largely determine the Inner structure In vitro release profile Biological fate of the systems.

10. Preparation Techniques of Nanoparticles 1) Amphiphilic macromolecule cross linking a) Heat cross linking. b) Chemical cross linking. 2 ) Polymeriazation based method. a) Polymerization of monomers in situ. b) Emulsion ( micellar ) polymerization . c) Dispersion polymerization. d) Interfacial condensation polymerization. d) Interfacial complexation .

11. 3) Polymer precipitation methods a) Solvent extraction/evaporation b) Solvent displacement ( Nanoprecipitation ) c) Salting out

12. Nanoparticles Preparation by Cross-linking of Amphiphilic Macromolecules: Proteins and polysaccharides are used . This technique involves two steps: a) The aggregation of amphiphile (s) b) Stabilization either by heat denaturation or chemical cross-linking.

13. These processes may occur in a biphasic o/w or w/o type dispersed systems, which subdivide the amphiphile (s) prior to aggregative stabilization. It may also take place in an aqueous amphiphilic solution where on removal of the solvent by extraction or diffusion, amphiphile (s) are aggregated as tiny particles and subsequently rigidized via chemical cross-linking.

14. Cross-linking in w/o emulsion:

15. Factors governing the size and shape of the nanoparticles are mainly, - emulsification energy - temperature Alternative to this is the chemical cross-linking method. Most widely used cross-linking agent is glutaraldehyde as 3% v/v solution. The problem associated with the use of chemical as a cross-linking agent is the complete removal of the agent.

16. Emulsion chemical dehydration : Hydroxypropyl cellulose solution in chloroform was used as a continuous phase. 2,2, di -methyl propane (Dehydrating agent) was used to translate internal aqueous phase in to a solid particulate dispersion. produce nanoparticles of size ( 300 nm )

17. Phase separation in aquious medium ( Desolvation ) The protein or polysaccharide from an aqueous environment can be desolvated by pH change , temperature or by adding appropriate counter ions . Cross linking may be affected simultaneously or subsequent to desolvation technique . This proceeds via three steps Protein dissolution , protein aggregation and protein deaggregation

18. Here sodium sulphate is used as desolvating agent While Alcohol (ethanol and isopropyl alcohol) are added as desolvating or deaggregating agent . Both lipophilic and hydrophillic drugs could be entrapped in nanoparticles by this method.

19. pH Induced Aggregation Here protein phase may be seperated by change of pH. E.g.-Insulin nanoparticles Insulin precipitated redissolved nanodroplet hardened using glutaraldehyde . Eg - Gelatin & Tween 20 were dissolved in aqueous phase. pH was adjusted to optimum value . Clear solution so obtained was heated to 40 c & followed by quenching at 40 c for 24 hours & subsequently left at ambient temperature for 48 hours .

20. This lead to gelatin colloidal dispersion . Finally colloidal aggregate were cross linked using glutaraldehyde .

21. Counter Ion Induced aggregation Protein phase is separated due to presence of counter ions in aqueous phase. Aggregation of dispersed phase ( polysaccharide) can be effectively . Initiated by adding appropriate counter ions. Aggregation can be propagated by adding secondary specious of counter ions followed by rigidisation step. Eg – Alginate nanoparticles Ca +2 - Gelation inducing agent. Poly ( L lysine )- Propagation of reaction .

22. Nanoparticle -Preparation Using Polymerization Based Methods : a . Polymerization of monomers in situ: Poly acrylate derivatives are used as polymers. Two different approaches are generally adopted for the preparation of nanospheres using this technique; i ) Emulsion polymerization: The monomer to be polymerized is emulsified in a non-solvent phase.

23. ii) Dispersion polymerization: The monomer is dissolved in a solvent that is non- solvent for the resulting polymer. In emulsion polymerization method, the monomer is dissolved in an internal phase while in the case of dispersion polymerization, it is taken in the dispersed phase. In either of the cases, following polymerization, the polymer tends to be insoluble in the internal phase or dispersed phase thus results into an ordered suspension of nanospheres

24. Micellar Polymerization Mechanism :

25. Homogenous Polymerization Mechanism:

26. b) Dispersion polymerization Monomer is dissolved in aqueous medium , which act as a precipitant ,for subsequently formed polymer. Polymerization based method involve in situ polymerization method where drug to be loaded is added to formed polymeric nanoparticles .

27. c. Interfacial Polymerization:

28. d . Interfacial Complexation :

29. 3. Polymer precipitation methods: a. Solvent extraction/evaporation

30. Solvent Evaporation method

31. b. Solvent displacement ( nanoprecipitation )

32. c. Salting out

33. Novel Nanoparticulate System Solid Lipid Nanoparticles. These are colloidal carriers (50-100 nm ) which are composed of physiological lipid dispersed in water or in an aqueous surfactant solution.

34. Advantages of SLN : Small size and relatively narrow size distribution which provide biological opportunities for site specific drug delivery by SLN Controlled release of active drug over a long period can be achieved Protection of incorporated drug against chemical degradation. No toxic metabolites are produced. Relatively cheaper and stable. Ease of industrial scale production by hot dispersion technique.

35. Preparation methods of SLN Hot Homogenization Technique : Homogenization of melted lipids at elevated temperature Cold Homogenization Technique : Homogenization of a suspension of solid lipid at room temperature

36. Melting of the lipid Dissolution of the drug in the melted lipid Mixing of the preheated dispersion medium and the drug lipid melt Hot Homogenization Technique :

37. High pressure homogenization at a temperature above the lipids melting point O/W – nano emulsion Solidification of the nano emulsion by cooling down to room temperature to form SLN Premix using stirrer to form coarse pre emulsion

38. Melting of the lipid Cold Homogenization Technique : Dissolution / solubalization of the drug in the melted lipid Solidification of the drug loaded lipid in liquid nitrogen or dry ice

39. Grinding in a powder mill (50 – 100 µm particles ) Dispersion of the lipid in the cold aqueous dispersion medium Solid Lipid Nanoparticles

40. Nanocrystals : Drug Dispersion with agitation Surfactant solution Milling for few hours/day Nanocrystals

41. Nanosuspension : Drug Dispersion with high speed stirring Surfactant solution High pressure homognization 1500 bar pressure Nano – suspension

42. Pharmaceutical aspects of Nanoparticles Should be free from potential toxic impurities Should be easy to store and administer Should be sterile if parentral use is advocated Process parameters are performed before releasing them for clinical trials; Purification Freeze drying Sterilization

43. Purification of nanoparticles : Gel filtration : Remark : High molecular weight substances and impurities are difficult to remove

44. Purification of nanoparticles : Dialysis : Remark : High molecular weight impurities are difficult to remove Time consuming process

45. Purification of Nanoparticles : Ultra-centrifugation : Remark : Aggregation of particles Time consuming process

46. Purification of Nanoparticles : Cross-flow filtration technique:

47. Freeze drying of Nanoparticles This technique involves the freezing of the nanoparticle suspension and subsequent sublimation of its water content under reduced pressure to get free flowing powder material. Advantages : Prevention from degradation. Prevention from drug leakage, drug desorption . Easy to handle and store and helps in long term preservation. Readily dispersed in water without modifications in their physicochemical properties

48. Sterilization of Nanoparticles : Nanoparticles intended for parenteral use should be sterilized to be pyrogen free . Sterilization can achieved by Using aseptic technique throughout their preparation, processing and formulation. Subsequent sterilizing treatments like autoclaving, irradiation.

49. Characterization of nanoparticles Parameter Characterization method Particle size and size distribution Charge determination Laser Doppler Anemometry Zeta potentiometer Chemical analysis of surface Static secondary ion mass spectrometry Sorptometer Carrier drug interaction Differential scanning calorimetry photon correlation spectroscopy Laser diffractometry Transmission electron microscopy Scanning electron microscopy Atomic force microscopy Drug stability Bioassay of drug extracted from nanoparticles Chemical analysis of drug

50. Therapeutic application of nanoparticles Cancer therapy : Material – poly ( alkylcyanoacrylate ) nanoparticles with anticancer agents, oligonucleotides Purpose – Targeting, reduced toxicity, enhanced uptake of antitumour agents, improved in vitro and in vivo stability.

51. b) Intracellular targeting Material : Poly ( alkylcyanoacrylate ) polyester nanoparticles with anti-parasitic or antiviral agents Purpose : Targeting reticuloendothelial system for intracellular infections

52. c) Prolonged systemic circulation : Material : Polyesters with adsorbed polyethylene glycols or pluronics or derivatized polyesters Purpose : Prolong systemic drug effect, avoid uptake by the reticuloendothelial system

53. d) Occular delivery : Material : poly ( alkylcyanoacrylate ) nanoparticles with steroids, anti-inflammatory agents, anti bacterial agents for glucoma Purpose : improved retention of drug / reduced wash out.

54. e) DNA delivery : Material : DNA-gelatin nanoparticles, DNA- chitosan nanoparticles, PDNA-poly(D,L) lactic acid nanoparticles Purpose : Enhanced delivery and significantly higher expression levels.

55. Other applications: Poly ( alkylcyanocrylate ) nanoparticles with peptides Poly ( alkylcyanocrylate ) nanoparticles for transdermal application Nanoparticles with a adsorbed enzymes Nanoparticles with radioactive or contrast agents Crosses blood- brain barrier Improved adsorption and permeation Enzyme immunoassays Radio-imaging

56. Brand name Description Advantages Emend (Merck & Co. Inc.) Nanocrystal aprepiant (antiemetic) in a capsule Enhanced dissolution rate & bioavailability Rapamune (Wyeth-Ayerst Laboratories) Nanocrystallied Rapamycin (immunosuppressant) in a tablet Enhanced dissolution rate& bioavailability Abraxane (American Biosciences, Inc.) Paclitaxel (anticancer drug) bound albumin particles Enhance dose tolerance and hence effect elimination of solvent associated toxicity Rexin-G (Epeius Biotechnology corporation) A retroviral vector carrying cytotoxic gene Effective in pancreatic cancer treatment

57. Targeted Drug Delivery System LIPOSOMES

58. What are Liposomes ? They are simply vesicles or ‘bags’ in which an aqueous volume is entirely enclosed by a membrane composed of lipid (fat) molecules, usually phospholipids.

59. These vesicles can encapsulate water-soluble drugs in their aqueous spaces and lipid soluble drug within the membrane itself. Structurally , liposomes are bilayered vesicles in which an aqueous volume is entirely enclosed by a membranous lipid bilayer mainly composed of natural or synthetic phospholipids.

60. Advantages of liposome : Provides selective passive targeting to tumor tissues Increased efficacy and therapeutic index Increased stability via encapsulation Reduction in toxicity of the encapsulated agent. Improved pharmacokinetic effects Used as carriers for controlled and sustained drug delivery Can be made into variety of sizes.

61. Disadvantages of liposome : Leakage of encapsulated drug during storage. Uptake of liposomes by the reticuloendothelial system Batch to batch variation Difficult in large scale manufacturing and sterilization Once administered, liposomes can not be removed Possibility of dumping, due to faulty administration

62. Mechanism of liposome formation In order to understand why liposomes are formed when phospholipids are hydrated, it requires a basic understanding of physiochemical features of phospholipids. Phospholipids are amphipathic molecules (having affinity for both aqueous and polar moieties) as they have a hydrophobic tail is composed of two fatty acids containing 10-24 carbon atoms and 0-6 double bonds in each chain.

63. In aqueous medium the phospholipids molecules are oriented in such a way that the polar portion of the molecule remains in contact with the polar environment and at the same shields the non-polar part. They align themselves closely in planer bilayer sheets to minimize the interaction between the bulky aqueous phase and long hydrocarbon fatty acyl chains. This alignment requires input of sufficient amount of energy (in the form of shaking, sonication , homogenization, heating, etc).

64. Interactions are completely eliminated when these sheets fold over themselves to form closed, sealed and continuous bilayer vesicles.

65. Classification of liposome's 1) Based on structural parameters MLV, OLV,UV,SUV,MUV,LUV,GUV,MV. 2) Based on method of liposome preparation REV, MLV-REV, SPLV, FATMLV, VET, DRV. 3) Based on the composition and application CL, RSVE, LCL ,pH sensitive liposome, cationic liposome , immuno - liposomes .

66. Materials used in preparation of liposomes A) Phospholipids : It is the major component of the biological membrane. Two types of phospholipids are used natural and synthetic phospholipids. The most common natural phospholipid is the phospatidylcholine (PC) is the amphipathic molecule and also known as lecithin. It is originated from animal (hen egg) and vegetable ( soya bean ).

67. B. Steroids : Cholesterol is generally used steroid in the formulation of liposomes . It improves the fluidity of the bilayer membrane and reduces the permeability of bilayer membrane in the presence of biological fluids such as blood / plasma. Cholesterol appears to reduce the interactions with blood proteins.

68. Methods of liposomes preparations Passive loading technique Active loading technique Mechanical dispersion methods Solvent dispersion methods Detergent removal methods

69. Mechanical dispersion methods Lipid is solublised in organic solvent, drug to be entrapped is solubilise in aqueous solvent, the lipid phase is hydrated at high speed stirring due to affinity of aqueous phase to polar head it is entrapped in lipid vesicles. e.g. Lipid film hydration, Micro-emulsification. (Micro fluidizer ), Sonication .

70. Solvent dispersion methods In this method, lipids are first dissolved in organic solvent, which then brought in to contact with aqueous phase containing material which is to be entrapped in liposome under rapid dilution and rapid evaporation of organic solvent. E.g. Ethanol injection Ether injection De-emulsification

71. Detergent removal method In this methods, the phospholipids are brought into intimate contact with the aqueous phase via detergent which associate with phospholipids molecules and serve to screen the hydrophobic portions of the molecules from water. Detergent ( Cholate , Alkyglycolate , Triton X-100) removal from mixed micells by Dialysis Column chromatography Dilution

72. Surface charge Free-flow electrophoresis Electrical surface potential and surface pH Zeta potential measurements & pH sensitive probes Percent of free drug/ percent capture Drug release Diffusion cell/ dialysis Parameter Characterization method Vesicle shape and surface morphology Mean vesicle size and size distribution Dynamic light scattering, zetasizer , Photon correlation spectroscopy, laser light scattering, gel permeation and gel exclusion Mini column centrifugation, ion-exchange chromatography,  radiolabelling Transmission electron microscopy, Freeze-fracture electron microscopy Physical Characterization

73. Phopholipid peroxidation UV absorbance, Iodometric  and GLC Phospholipid hydrolysis, Cholesterol auto-oxidation HPLC and TLC Osmolarity Parameter Characterization method Phospholipid concentration Cholesterol concentration Cholesterol oxidase assay and HPLC Osmometer Barlett assay, stewart assay, HPLC Chemical Characterization

74. Animal toxicity Monitoring survival rates, histology and pathology Parameter Characterization method Sterility Pyrogenicity Limulus Amebocyte Lysate (LAL) test Aerobic or anaerobic cultures Biological Characterization

75. Stability Physical stability : Once liposome are formed, they behave similar to the other colloidal particles suspended in water. Neutral particles tend to aggregate or flocculate and sediment with increase in size on storage. Adding charged lipids such as stearyl amine, diactyl phosphate and phosphatidyl serine can control the aggregation. The addition of charged lipids causes repulsion and prevents major changes in the overall size of liposome.

76. Chemical stability : Phospholipids, especially those derived from natural sources, are subject to two major degradative reaction A. Lipid peroxidation : most phospolipid liposomes contain unsaturated acyl chains as part of their molecular structure and susceptible to oxidative degradation. It can be minimized by the use of animal derived lipids like egg PC, which has less saturated lipids, use of light resistant containers, use of antioxidants are useful in minimizing oxidation.

77. B. Lipid hydrolysis : hydrolysis in phospholipids results in the formation of free fatty acids and lyso -lecithin. Selecting a good source of lipid, temperature, pH, and minimizing oxidation. Biological stability : liposome's release entrapped molecules rapidly when incubated with blood or plasma. This instability is attributed to the transfer of bilayer lipids to albumin and high density liposome.

78. Therapeutic applications of liposomes Liposomes as drug / protein delivery vehicles Controlled and sustain release in situ Enhanced drug solubilization Enzyme replacement therapy and lysosomal storage disorders Altered pharmacokinetics and biodistribution Liposomes in antimicrobial, antifungal and antiviral therapy Liposomes in tumour therapy Carrier of small cytotoxic molecules Vehicle for macromolecules as cytokines and genes

79. Liposome in gene delivery Genes and antisense therapy Genetic (DNA) vaccination Liposome in immunology Liposome as radiopharmaceutical and radio diagnostic carrier Liposome in cosmetic and dermatology Liposome in enzyme immobilization and bioractor technology

80. Drug Route of administration Targeted Diseases Amphotericin -B Oral delivery Mycotic infection  Insulin Oral, Ocular, Pulmonary and Transdermal delivery Diabetic mellitus  Ketoprofen Ocular delivery Pain muscle condition Pentoxyfylline Pulmonary delivery Asthma Tobramycin Pulmonary delivery Pseudomonas infection, aeruginosa

81. Drug Route of administration Targeted Diseases Salbutamol Pulmonary delivery Asthma Benzocain Transdermal ulcer on mucous surface with pain Ibuprofen Oral delivery Rheumatoid arthritis Adrenaline Ocular delivery Glucoma, Conjectivitis Penicillin G Pulmonary delivery Meningococal, staphylococcal Methotrexate Transdermal Cancer

82. Marketed product Drug used Target diseases Company DoxilTM or CaelyxTM Doxorubicin Kaposi’s sarcoma SEQUUS, USA DaunoXomeTM Daunorubicin Kaposi’s sarcoma, breast & lung cancer NeXstar, USA AmphotecTM Amphotericin-B fungal infections, Leishmaniasis SEQUUS, USA VENTUSTM Prostaglandin-E1 Systemic inflammatory diseases The liposome company, USA ALECTM Dry protein free powder of DPPC-PG Expanding lung diseases in babies Britannia Pharm , UK

83. Reference Targeted and controlled drug delivery, S.P.Vyas and R.K.Khar , CBS Publication 2008, Nanoparticles – page no 331 to 386. Liposomes – page no 173 to 248. b) Controlled And Novel drug delivery – By N.K.Jain . c) Novel Drug Delivery system by Y.W.Chien

84. d) Text book of Industrial Pharmacy, Shobha Rani Hiremath, Orient Longman Private ltd. e) www.google.com