nanoparticles and liposomes

1. Targeted Drug Delivery
System
NANOPARTICLES &
LIPOSOMES.
Presented By:
Mr. Nikhil Patil.
M.Pharm 1st 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. Nanoparticles
Nanospheres Nanoencapsules
Solid 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
 Alginate
 Albumin
 Dextran
 Lectins
 Chitosan
 Legumin
 Agarsoe
 Viciline
 Pullulan

7. Disadvantage:
1. Batch to batch variation.
2. Conditional biodegradability.
3. 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. 1. 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 0c & followed
by quenching at 40 0c 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. 2. 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. Hot Homogenization Technique :
Melting of the lipid
Dissolution of the drug in the melted lipid
Mixing of the preheated dispersion
medium and the drug lipid melt

37. Premix using stirrer to form
coarse pre emulsion
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

38. • Cold Homogenization Technique :
Melting of the lipid
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
Surfactant solution
Dispersion with agitation
Milling for few hours/day
Nanocrystals

41. Nanosuspension :
Drug
Surfactant solution
Dispersion with high speed stirring
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
photon correlation spectroscopy
Laser diffractometry
Transmission electron microscopy
Scanning electron microscopy
Atomic force microscopy
Charge determination Laser Doppler Anemometry
Zeta potentiometer
Chemical analysis of surface
Static secondary ion mass spectrometry
Sorptometer
Carrier drug interaction Differential scanning calorimetry
Drug stability
Bioassay of drug extracted from nanoparticles
Chemical analysis of drug

50. Therapeutic application of
nanoparticles
A. 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. • 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.
These vesicles can encapsulate water-soluble drugs in
their aqueous spaces and lipid soluble drug within
the membrane itself.

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

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

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

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
1. 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
2. Liposomes in antimicrobial, antifungal and antiviral
therapy
3. Liposomes in tumour therapy
• Carrier of small cytotoxic molecules
• Vehicle for macromolecules as cytokines and genes

79. 4. Liposome in gene delivery
• Genes and antisense therapy
• Genetic (DNA) vaccination
5. Liposome in immunology
6. Liposome as radiopharmaceutical and radio
diagnostic carrier
7. Liposome in cosmetic and dermatology
8. 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
a) 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