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A.J. Almeida, 2007
SOLID LIPID NANOPARTICLES AS COLLOIDAL DRUG CARRIER SYSTEMS
António J. Almeida
Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculdade de FarmáciaUniversidade de Lisboa
Universidad de Sevilla, Facultad de Farmacia, November 2007
A.J. Almeida, 2007
MICROPARTICLES AND NANOPARTICLES:
THE BASIC CONCEPTS
2
A.J. Almeida, 2007
“The recent tremendous advances in health science technology and inexorable
progress in related scientific innovations, coupled with changes in population
demographics and the colouring of political agendas with the economics of
disease, are all acting in concert to take the pharmaceutical industry into
exciting and challenging new dimensions”.
G. Gregoriadis (2002)
INOVATIVE DRUGS
A.J. Almeida, 2007
InovativeDrugs
AIDSCancer
EbolaTuberculosis
AlzheimerSARS
EconomicsDemographic changes
Globalization
New drug delivery systemsGenomicsBiotechnologyNeurosciencesGene TherapyBioinformaticsProteomicsMetalolomics
INOVATIVE DRUGS
3
A.J. Almeida, 2007
PHYSICAL APPROACH
• Macroscopic systems activated by physical processes (capsules, granules, etc)
CHEMICAL APPROACH
• Chemical modification of drugs (derivative preparation, salts, pro-drugs, association to polymers, etc.)
BIOCHEMICAL / BIOTECHNOLOGICAL APPROACH
• Naturally occurring macromolecules
• Cells
• Colloidal polymeric or lipid systems
DIFFERENT APPROACHES
PHARMACEUTICALAPPROACH
(pharmaceutical formulation)
A.J. Almeida, 2007
in vitro
in vivo
C
Time
0 6 12 18
Time (hour)
Dru
g co
ncen
trat
ion
in s
erum
t
GENERAL CONCEPTS IN MODIFIED DRUG RELEASE
4
A.J. Almeida, 2007 Chien (1992). Novel Drug Delivery Systems
GENERAL CONCEPTS IN MODIFIED DRUG RELEASE
Convencional Release
Modified Release
Delayed Release
Prolonged Release
Controlled Release
Drug Targeting
A.J. Almeida, 2007
“NEW DRUG DELIVERY SYSTEMS”
SKF
Spansule
ALZA
Oros OcusertProgestasert
5
A.J. Almeida, 2007
Physicochemical problemsMolecular weight and sizeComplex strutureConformational stabilitySolubilitySensitivity (light, temperature, pH)CrystallizationMolecularar interactionsAdsorptionAggregationPresence of protein impurities
Biological problemsBiodegradation by digestive enzymesShort in vivo half-lifeImunogenicityComplex metabolismDificulty in crossing mucosal barriersNo access to some compartments
Pharmacokinetic ProblemsAnalytical Problems
FORMULATION OF NEW DRUG MOLECULES
PROTEIN DRUGS
A.J. Almeida, 2007
Investigation of alternative administration routes (nasal, rectal, pulmonary, etc.);
Investigation of alternative prolonged or pulsed release drug delivery systems;
Investigation of new dosage forms that can provide protection against premature degradation;
Invstigation of new site specific drug delivery systems.
PROTEIN DRUGS
6
A.J. Almeida, 2007
PARTICULATE SYSTEMS
liposomes
microemulsions
nanosuspensions
microspheres
nanoparticles
niosomes
virosomes
cubosomesParticulate
Systems
iscoms
cochleates
nanocapsules
micelles
transfersomes
A.J. Almeida, 2007
Definition IUPAC
COLLOIDAL
The term refers to a state of subdivision, implying that the molecules
or polymolecular particles dispersed in a medium have at least in
one direction a dimension roughly between 1 nm and 1 µm, or that in
a system discontinuities are found at distances of that order.
COLLOIDAL SYSTEMS
7
A.J. Almeida, 2007
Microparticles
Solid particles made of polymeric or solid lipid materials
Podem conter o fármaco disperso, encapsulado ou adsorvido à superfície.
Microspheres (≥1 μm)
• Microcapsules (reservoir systems)
• Microparticles (matrix systems)
Nanospheres (<1 μm)
• Nanocapsules (reservoir systems)
• Nanoparticles (matrix systems)
COLLOIDAL SYSTEMS
A.J. Almeida, 2007
MAIN POLYMERS
Biocompatibility versus Biodegradation
Biodegradation mechanisms: Type I, II e III
poly(lactide-co-glycolide) (PLGA)poly-ε-caprolactone (PCL)poly(hydroxibutiric acid) (PHB)polyorthoesterspolyanhydridespolyphosfazenespolyalkylcianoacrylates (PIBCA ou PHCA)proteins (albumine, gelatine, gliadines)starchchitosanalginate
POLYMERIC MICRO- AND NANOPARTICULATE SYSTEMS
8
A.J. Almeida, 2007
Biodegradation Mechanisms
Type I
Type II
A B A C
Type III
POLYMERIC MICRO- AND NANOPARTICULATE SYSTEMS
A.J. Almeida, 2007
BIODEGRADABLE IMPLANTS
POLYMERIC MICRO- AND NANOPARTICULATE SYSTEMS
Poly(lactide-co-glycolide) (PLGA)
9
A.J. Almeida, 2007
Drug release by polymer enzymatic degradation.
Drug release rate is controlled by the rate of the enzymatic reaction.
Predominat use in controlled release from biodegradable polymers often used in particulate carriers (e.g. albumine, gelatine, polyalkylcianoacrylates).
F F FF
POLYMERIC MICRO- AND NANOPARTICULATE SYSTEMS
A.J. Almeida, 2007
Main Technologic and Therapeutic Uses
• Diagnostic systems
• Controlled drug delivery systems
• Implants• Aerosols• Solid dosage forms• Vaccines• Site-specific drug delivery
POLYMERIC MICRO- AND NANOPARTICULATE SYSTEMS
10
A.J. Almeida, 2007
MAIN MICRO / NANOENCAPSULATION TECHNIQUES
1. Spray coating; Pan-coating, Spray-drying
2. Droplet extrusion
3. Coacervation / Phase separation
4. Emulsion solidification
4.1. Solvent evaporation (simple or multiple emulsion)
4.2. Solvent extraction (simple or multiple emulsion)
4.3. Melting and solidification (simple or multiple emulsion)
5. Polymerization methods
5.1. Interfacial polymerization
5.2. Emulsion polymerization
Colloidal Systems
A.J. Almeida, 2007
A
Aqueous phase+
surfactant
Polymer in organic solvent
Drug suspended in polymer solution
Drug
Evaporation or Extractionof organic solvent
Particle isolation by filtration or centrifugation
simple [A] or multiple [B]
emulsion
w/o
emulsionAqueous phase
+ surfactant
Polymer in organic solvent Drug + water +
emulsifying agent
B
Solvent Evaporation/ Extraction
simple emulsion multiple emulsion
MAIN MICRO / NANOENCAPSULATION TECHNIQUES
11
A.J. Almeida, 2007
A
Aqueous phase heated at same
temperature
Melted lipid
Drug suspended in melted lipid
Drug
Cooling to solidify disperse phase
Particle isolation by filtration or centrifugation
Simple [A] or multiple [B]
emulsion
w/o
emulsion Aqueous phase heated at same
temperature
Melted lipidDrug+ Water +
Emulsifying agent
B
Melting and Solidification (1)
MAIN MICRO / NANOENCAPSULATION TECHNIQUES
simple emulsion multiple emulsion
A.J. Almeida, 2007
Cooling
Particle isolation by filtration or centrifugation
Emulsion 2
Drug
Oil
Emulsion 1
Protein Solution (albumin or
gelatine)
Oil at 180ºC + surfactant
Melting and Solidification (2)
MAIN MICRO / NANOENCAPSULATION TECHNIQUES
12
A.J. Almeida, 2007
Emulsion Polymerization
MAIN MICRO / NANOENCAPSULATION TECHNIQUES
A single liquid (or disolved) reacting monomerDrug disolved or suspendedPolymerisation started by chemical agent or radiationPolymer MW and particle size dependent on:
monomer concentrationInitiator concentrationTemperature
Emulsion stabilizer neededParticles >20 nm
Ex.: PIBCA; polyacrylamide
A.J. Almeida, 2007
CaCl2
Ferreira Almeida and Almeida (2004). J Control Release.
Gelatine + alginate + drug
Washing
Fluid-bed drying
Droplet Extrusion
MAIN MICRO / NANOENCAPSULATION TECHNIQUES
13
A.J. Almeida, 2007
CONTROL OF FORMULATIONS
Particle sizeElectron microscopyElectric sensing zoneLaser diffractionPhoton correlation spetroscopy (PCS)Optical sensing zone
Encapsulation efficiencyDrug loadingSurface charge (zeta potential)Hydrofobicity
Contact anglePartition coeficientHydrophobic interaction chromatography
Release profile
Sterilization
Stability
MICRO- AND NANOPARTICULATE SYSTEMS
A.J. Almeida, 2007
General drug release kinetics
Dependent on:
Drug location inside the particle
Type and amount of polymer
Particle size and density
Cross-linking
Physicochemical properties of drug and polymer
Drug MW and concentration
Presence of excipients
Release medium
DRUG RELEASE
MICRO- AND NANOPARTICULATE SYSTEMS
14
A.J. Almeida, 2007
PARTICULATE CARRIERS IN THE PHARMACEUTICAL MARKET
LIPOSOMES
physical and chemical stability problems
no “cheap” liposome available
marketed products but behind expectations
POLYMERIC NANOPARTICLES
input: 30 years of research
no output: no products on the market
A.J. Almeida, 2007
SITE-SPECIFIC DRUG DELIVERY
15
A.J. Almeida, 2007
SITE-SPECIFIC DRUG DELIVERY
The magic bullet concept
The term used to describe a specific cure for syphilis, which would attack the syphilis spirochaete while having no effect whatsoever on human tissue.
Paul Ehrlich (1854-1915)
A.J. Almeida, 2007
RATIONALE FOR SITE-SPECIFIC DRUG DELIVERY
Target site
Other sites
Carrier
Drug
Specificity Translocation across biological barriers Protection against inactivation
Puisieux and Roblot-Treupel (1989) STP Pharma
16
A.J. Almeida, 2007
• Exclusive delivery to specific compartments (and/or diseases)
• Access to previously inaccessible sites (e.g. intracellular infections)
• Protection of drug and body from unwanted deposition, which could lead to unwanted reactions and metabolism, etc.
• Controlled rate and modality of delivery to pharmacological receptor
• Reduction in the amount of drug employed
RATIONALE FOR SITE-SPECIFIC DRUG DELIVERY
↑ Drug safety
↑ Drug efficacy
↑ Patient compliance
A.J. Almeida, 2007
ACTIVE TARGETING
Magnetic fieldsLynphotropic adjuvants
MPS blockingReceptor mediated processes
Immunotherapy
0,1 4
0,01
PASSIVE TARGETING
Uptake by MPS and tropism for the lysosomes•macrophages•Kupffer cells
Translocation to the tissues• hepatocytes•bone marrow•splenocites•tumors (?)
Capillar embolismlung targeting (iv)• specific regions (ia)
4 7 10 100
Diametre (μm)
SITE-SPECIFIC DRUG DELIVERY: DRUG TARGETING
17
A.J. Almeida, 2007
Utilização de lipossomas como transportadores de fármacos
Crommelin et al. (http://www.drugdeliverypartnerships.com/lip.html)
DRUG TARGETING
A.J. Almeida, 2007
• Soluble carriers (monoclonal antibodies, dextrans, soluble synthetic polymers)
• Particulate carriers (liposomes, microspheres, nanoparticles, etc.)
• Target-specific recognition moieties (monoclonal antibodies, carbohydrates)
• Antibody-directed enzyme/prodrug therapy
• Virus-directed enzyme/prodrug therapy
MAIN DRUG TARGETING SYSTEMS
18
A.J. Almeida, 2007
SOLID LIPID NANOPARTICLES
A.J. Almeida, 2007
LIPID NANOPARTICLES
Lipophilic colloidal delivery system (R.H. Müller, Berlin; M.R. Gasco, Turin);
Efficient and non-toxic drug carrier specially for lipophilic drug molecules;
Composed of physiological / well tolerated excipients e.g. GRAS (= similar to emulsions and liposomes);
Possess solid matrix (= similar to polymeric nanoparticles);
Protective properties
Controlled release properties
Colloidal dimensions and controlled release behaviour enable drug protection and administration by parenteral and non-parenteral routes.
19
A.J. Almeida, 2007
LIPID NANOPARTICLES
Versatility
Parenteral administration (Fundarò et al, 2000)
Brain delivery (Fundarò et al, 2000)
Ocular delivery (Cavalli et al, 2002)
Rectal delivery (Sznitowska et al, 2001)
Oral delivery (García-Fuentes et al, 2002)
Topical delivery (Souto et al, 2004)
Potential vaccine delivery systems (Almeida et al, 1997)
Large scale production possible using lines available in pharmaceutical plants (i.e. lines for parenteral emulsions (Müller et al, 2001)
A.J. Almeida, 2007
SLN PREPARATION
High Pressure Homogenization (R.H. Müller et al.)
-SkyePharma PLC, Berlin, Germany
-PharmaSol GmbH, Berlin, Germany
Microemulsion Technology (M.R. Gasco)
- Vectorpharma/Eurand Spa, Turin, Italy
Solvent Evaporation
Phase Inversion Type
20
A.J. Almeida, 2007
A
Aqueous phase at same
temperature
Melted Lipid
Drug solution in melted lipid
DrugEmulsion
w/o Aqueous phase at same
temperature
Melted LipidDrug + Water + surfactant
B
Cooling for solidification and particle hardening
SLN suspension
High-Pressure Homogenisation
Emulsification using Ultra-turrax
single [A] or double [B]
SLN PREPARATION BY HIGH PRESSUE HOMOGENIZATION (HPH)
HOT HOMOGENIZATION PROCESS
A.J. Almeida, 2007
HIGH PRESSURE HOMOGENIZATION (HPH)
E. Souto (2005) PhD Thesis Dept. Pharmaceutics, Biopharmaceutics and Biotechnology, Free University of Berlin
APV Gaulin LAB40 discontinuous(40 g batch)
21
A.J. Almeida, 2007
SLN PREPARATION BY HPH
HOT HOMOGENIZATION PROCESS
Basic principles:
equipment can be qualified and validated
accepted by regulatory authorities in production lines used for parenterals
existing industrial production lines for i.v. parenteral emulsions can be used
all production lines developed for SLN are usable
A.J. Almeida, 2007
SLN PREPARATION BY HPH
APV Gaulin LAB40 continuous(0.5-2 Kg batch)
APV Gaulin LAB60(2-10 Kg batch)
APV Gaulin 5.5(150 Kg/h)
Dept. Pharmaceutics, Biopharmaceutics and Biotechnology, Free University of Berlin
22
A.J. Almeida, 2007
SLN PREPARATION BY HPH
COLD HOMOGENIZATION PROCESS
Melted Lipid
Drug suspensionin melted lipid
Drug
SLN suspension
High-Pressure Homogenisation
Suspension in water using Ultra-turrax
Liquid nitrogen
Micronization
Dept. Pharmaceutics, Biopharmaceutics and Biotechnology, Free University of Berlin
A.J. Almeida, 2007 Müller et al (2000). Eur J Pharm Biopharm
DRUG ENTRAPMENT INTO SLN
A m.p. drug ≈ m.p. lipid homogeneous matrix
B m.p. drug < m.p. lipid lipid-enriched core
C m.p. drug > m.p. lipid drug-enriched core
23
A.J. Almeida, 2007
PROBLEMS ASSOCIATED WITH SLN
Formation of “perfect” crystalline structure during storage (β modification) ⇒ drug expulsion
DRUG ENTRAPMENT INTO SLN
Souto (2007)
A.J. Almeida, 2007
Physical stability of aqueous solution
Gel formation
Particle aggregation
High water content of dispersions (70 - 95%)
Need to remove too much water in tablet / pellet production
Dosing problems (e.g. dispersion for soft gelatin capsules, lipid
particle content max. 30%)
SLN PROCESSING DISADVANTAGES
Souto (2007)
24
A.J. Almeida, 2007
SLN PROCESSING DISADVANTAGES
SLN – Solid Lipid Nanoparticles
Produced from solid lipids
Tend to form perfect crystals ⇒ drug expulsion
NLC – Nanostructured Lipid Carriers
Produced from blend of solid and liquid lipids
Particles are in solid state at body temperature
Inhibit crystallization process by mixing “spatially” very different molecules ⇒ imperfections in lattice
• not “just mixing” solid lipids but:• controlled “nanostructuring” of lipid matrix
to accommodate drugto control releaseto trigger release
Souto (2007)
A.J. Almeida, 2007
LIPID NANOPARTICLES
Solid lipid particle
Oil-loaded particle
Souto et al (2007) Pharm Tech Europe (in press)
NLC – a “multiple” carrierSLN: one phase (= solid lipid)
NLC: multiple O/F particle (oil nanodroplets in solid fat nanoparticles)
Advantages:higher drug load, firm incorporation (e.g. 1% Retinol in SLN, 6% in NLC)
25
A.J. Almeida, 2007
LN AS A POTENTIAL PROTEIN / ANTIGEN
DELIVERY SYSTEM
A.J. Almeida, 2007
• Lipid compositionHard fats (Softisan 142® and Witepsol® E85)Cetyl alcoholPropyleneglycol palmitostearate (Monosteol®)PEG 300 mono-, di-stearate (Superpolystate®)
Surfactant Poloxamer 188Tween 80
PROTEIN NANOENCAPSULATION IN SLN
Lipid + poloxamer 188
Aqueous Lysozyme
melting
Solubilisation using a rotary evaporator
Micronisation in a powder-mill
Pre-mix using a high-speed homogeniser
HPH at room temperature
Solidification in liquid nitrogen
Almeida et al (1997). Int J Pharm
26
A.J. Almeida, 2007
0
100
200
300
400
500
Distilled water
3% Tween 80
3% NaCholate
3% Poloxamer188
Lyso
zym
e ac
tivity
(Uni
ts/m
l)
Day 1Day 10
Almeida et al (1997). Int J Pharm
STABILITY OF LYSOZYME THROUGHOUT FORMULATION
MW rt 0,5h 1h 2h 3h 4h 5h
Influence of temperature (50º)
MW Ct 3 4 5 6 7 8 9 10
Influence of HPH treatment(Nº cycles/Pressure)
500 bar/1 cy/ 25ºC 1000 bar/3 cy/ 50ºC
A.J. Almeida, 2007
MW Water S/CA W/CA Mono Sup
NANOENCAPSULATION OF LYSOZYME IN LN
Almeida et al (1997). Int J Pharm.
0
1000
2000
3000
4000
5000
Softisan142/Cetylalcohol
WitepsolE85/Cetyl
alcohol
Monosteol Superpolystate
Lyso
zym
e ac
tivity
(Uni
t/gof
lipi
d)
27
A.J. Almeida, 2007
0
20
40
60
80
100
120
140
Lyso
zym
e ac
tivity
(%)
Wit/CASoft/CA
Initial Sup LN Initial Sup LN
MW Ct 3 4 5 6 7 897.0 KDa66.2 KDa45.0 KDa
31.0 KDa
21.5 KDa14.4 KDa
NANOENCAPSULATION OF LYSOZYME IN LN
Almeida et al (1997). Int J Pharm
-9.8±0.7549±7.0D50 0.58±0.00D90 0.93±0.01
0.30W E85/CA
-11.0±0.2644±12.9D50 0.60±0.00D90 1.86±0.02
0.22S 142/CA
Zeta Potential(mV ±sd)
Particle Size PCS
(nm ±sd)
Particle SizeLaser diffraction
(μm ±sd)
Lys Content
(%)
Lipid matrix
A.J. Almeida, 2007 Videira and Almeida (1998). Proceed III SPLC-CRS Conf
• Lipid compositiongliceryl behenate (Compritol® 888 ATO)gliceryl palmitosterarate (Precirol ® ATO 5)cetyl Alcohol
• Surfactant Tween 80
• Speed rate8 000 - 10 000 rpm
Lipid
Aq.phase
melting
heatingheatingemulsification
high-speed homogenization8 000 - 10 000 rpm
solidification
DEVELOPMENT OF OVA-CONTAINING LN
Particle size (PCS)100-400 nm
PI - 0,250
28
A.J. Almeida, 2007
SLN (nm) OVA –SLN (nm)
Lipid (%) dmt=3d dmt=30d dmt=3d dmt=30d
Pr 103 115 128 1536 Cp 171 168 196 205
W/AC 284 280 314 320
Pr 196 205 214 2068 Cp 240 253 280 305
W/Ac 252 265 290 312
Pr 230 200 300 nd10 Cp 373 378 390 nd
W/Ac 307 289 400 nd
NANOPARTICLE CHARACTERISATION: PHYSICAL STABILITY
Videira et al. (2002). Proceed V SPLC-CRS Conf
A.J. Almeida, 2007
330330330
330330330
330330330
Lipid
10%Pr
Cp
W/CA
8%Pr
Cp
W/CA
Pr
Cp
W/CA6%
Tween 802%
60% 100%
OVA-6SLN 92% EE with 2% surfactant
ENTRAPMENT EFFICIENCY
Videira et al (2002). Proceed V SPLC-CRS Conf
29
A.J. Almeida, 2007
Protein Integrity
97.0 KDa66.2 KDa
45.0 KDa
31.0 KDa
21.5 KDa14.4 KDa
MW LNsup 1W 2W 3W
0
10
20
30
40
50
60
70
80
0 5 10 15 20 25 30 35Time (day)
OVA
rele
ased
(%)
FORMULATION STUDIES OF OVA OF LYSOZYME IN LN
0
10
20
30
40
0 50 100 150 200 250 300 350 400time (min)
Con
c.O
VA (%
)
Videira et al (2002). Proceed V SPLC-CRS Conf
A.J. Almeida, 2007
ADSORPTION STUDIES
Desorption profiles
OVA/lipid (%) 4 7 10
25ºC 90 80 82
15ºC 80 70 79
A.E
(%)
0
20
40
60
80
100
0 250 500 750 1000 1250 1500
time (min)
Con
c. O
VA (%
)
25ºC15ºC
Videira et al (2002). Proceed V SPLC-CRS Conf
30
A.J. Almeida, 2007
LN are potential protein carriers with high protein binding
Formulations are stable
Partial adsorption of protein may not be precluded
Production conditions seem not to damage protein integrity
Following a burst effect, a sustained release profile is obtained
Loading capacity is suitable to the usual antigen doses
LN AS A POTENTIAL PROTEIN / ANTIGEN DELIVERY SYSTEM
Videira et al (2002). Proceed V SPLC-CRS Conf
A.J. Almeida, 2007
PULMONARY DELIVERY OF SLN
31
A.J. Almeida, 2007
PULMONARY ADMINISTRATION
The Lung
Unique features that can facilitate systemic delivery via pulmonary administration of drugs:
• Large surface área (≈75 m2).
• Good vascularisation.
• Large capacity for solute exchange.
• Ultra-thineness of the alveolar epithelium (0.1-0.5 mm).
• First-pass metabolism is avoided.
A.J. Almeida, 2007
PULMONARY DELIVERY USING PARTICULATE CARRIERS
Advantages
• An alternative non-invasive means for both local and
systemic drug delivery using particulate carriers.
• Avoids instability in blood stream and uptake by the MPS.
• Allows high concentrations of drug in the lungs
minimizing side toxic effects.
• A potential route for drug therapy and immunisation.
Poyner et al (1995). J Controlled Release
FreeLiposomal (0,7 μm)PLGA Microencapsulated (0,7 μm)
Lung
020406080
100
Kidney
020406080
6 24Time (hour)
Tobr
amyc
in(%
)
Blood
020406080
32
A.J. Almeida, 2007
LYMPHATIC TARGETING AND LUNG CANCER THERAPY
• A leading cause of death around the world, accounting for ≈13% of newly diagnosed cancers and ≈30% of all cancer deaths.
• Very low survival rate (≤14% in 5 years; mean survival time 14 months).
• Metastatic disease starts at very early stages, particularly in SCLC, the majority of patients with lung cancer having metastatic disease at the time of diagnosis.
• Metastasis spread mainly via the lymphatics to the lymph nodes, liver, brain, bones and adrenal glands.
• Early diagnosis is crucial to increase survival rate and stagingis critically dependent upon the involvement of the lymph nodes that drain the region containing the tumour.
• Chemotherapy plays an important role alongside surgery and radiation therapy, but lung cancer is usually diagnosed at an incurable stage.
A.J. Almeida, 2007
IMAGING USING PARTICULATE CARRIERS
• Lung perfusion imaging is based on the trapping of i.v. injectedradiolabelled albumin microspheres (10-50μm) in the capillary bed of the lung: Technetium [99m Tc] Microspheres Injection (Ph Eur).
• I.v. injected radiolabelled PLGA microspheres have also been proposed for lung imaging (Delgado et al. 2000).
• Several particulate carriers have been used for lymphoscintigraphy, such as liposomes, sulfur colloid, PMMA and PHCA nanoparticles.
33
A.J. Almeida, 2007
PULMONARY ADMINISTRATION OF LIPID NANOPARTICLES
• To investigate LN as a potential drug carrier to the lungs and, through alveolar airways, to the lymphatic system, thus optimising concentration at the tumour site or at distant metastasis sites.
• To evaluate LN in vivo fate after pulmonary absorption upon nebulisation and delivery to laboratory animals.
Lipid Nanoparticles
• Administration have been studied by parenteral and non-parenteral routes.
A.J. Almeida, 2007
NANOPARTICLE PRODUCTION
• Lipid compositiongliceryl behenate (Compritol® 888 ATO)gliceryl palmitosterarate (Precirol ® ATO 5) cetyl Alcohol
• Surfactant Tween 80
• Speed rate8 000 - 10 000 rpm
Lipid
Aq.phase
melting
heatingheatingemulsification
high-speed homogenization8 000 - 10 000 rpm
solidification
Videira et al (2002). J Drug Targeting
34
A.J. Almeida, 2007
D,L-hexamethylpropyleneamine oxime(HMPAO)
N
NH
N
NH
OH OH
MeMe
Me
Me
Me
Me
+99mTcO4-
NaCl
Radiolabelled LN
LABELLING AND ADMINISTRATION
Lipid Nanoparticles
Ultrasonicnebulization
Labelling efficiency
Particle size
γ - Scintigraphy
anaesthetised male Wistar rats
Videira et al (2002). J Drug Targeting
A.J. Almeida, 2007
CHARACTERISATION OF 99mTc-HMPAO-LN
Before aerosolisation
After aerosolisation
D50 (nm) PI D50 (nm) PI
197.5 0.231 218.3 0.378
194.8 0.291 220.3 0.379
196.1 0.261 219.3 0.379
PARTICLE SIZE
Videira et al (2002). J Drug Targeting
ASPECT
35
A.J. Almeida, 2007
CHARACTERISATION OF 99mTc-HMPAO-LN
Labelling efficiency: 97±2%
Radiochemical Purity (TLC)
Sys I Sys II
t0’
t30’
99mTc-HMPAO
butanone 0.9% aq. NaCl
Sys I
t0’
t30’Sys II
99mTc-HMPAO-LN
butanone 0.9% aq. NaCl
99mTc-HMPAO reduced-hydrolysed 99mTc
Na 99mTcO4
Other hydrophilic species
A.J. Almeida, 2007
99mTc-HMPAO-LN99mTc-HMPAO
BIODISTRIBUTION
Videira et al (2002). J Drug Targeting
36
A.J. Almeida, 2007
3 min 8 min 60 min
99mTc-HMPAO
99mTc-HMPAO-LN
BIODISTRIBUTION
Videira et al (2002). J Drug Targeting
A.J. Almeida, 2007
LYMPHATIC DISTRIBUTION OF 99mTc-HMPAO-LN
Videira et al (2002). J Drug Targeting
37
A.J. Almeida, 2007
Periaortic nodes Inguinal nodes
Axillary nodes
LYMPHATIC DISTRIBUTION
Videira et al (2002). J Drug Targeting
T1/2 ≈ 10 min
Lungs
A.J. Almeida, 2007
BIODISTRIBUTION IN RATS 4h AFTER INHALATION
0
5
10
15
20
25
30
35
Lung
s
Serum
Periao
rtic ly
mph no
des
Axillar
y lym
ph no
des
Inguin
al lym
ph no
des
Liver
Spleen
Thyroi
d
Kidney
sHea
rtBrai
n
Small in
testin
e
Large
intes
tine
Testic
lesUrin
e
% a
ctiv
ity/g
of t
issu
e
Videira et al (2002). J Drug Targeting
38
A.J. Almeida, 2007
BIODISTRIBUTION OF 99mTc-HMPAO IN RATS AFTER i.v. INJECTION
Sharp et al (1986). J Nucl Med
21.5±1.519.8±0.315.8±0.215.4±1.7Intestines
8.6±1.010.4±0.410.2±0.413.9±1.9Liver
14.6±0.39.5±0.13.2±0.30.4±0.4Urine
8.6±0.39.4±0.210.5±1.111.0±0.4Blood
2.8±0.33.1±0.13.1±0.23.7±0.8Lungs
6.4±1.26.6±0.46.3±0.26.5±1.1Kidneys
% Dose in OrganTissue
2.0±0.21.8±0.22.1±0.22.1±0.1Brain
60 min30 min10 min2 min
A.J. Almeida, 2007
RADIOACTIVITY IN RAT LUNGS AFTER ENDOTRACHEAL ADMINISTRATION
(n=4; mean±sd)
0
20
40
60
80
100
120
3 8 15 30 60
Time (min)
% a
ctiv
ity/g
% A
ctiv
ity/g
Videira et al (2006).J Microencapsulation
39
A.J. Almeida, 2007
LYMPHATIC DISTRIBUTION IN RATS AFTER ENDOTRACHEAL ADMINISTRATION
0
10
20
30
40
50
60
70
80
90
3 8 15 30 60
Time (min)
% a
ctiv
ity/g
StomachLiverAortic lymph nodesAxillary lymph nodesInguinal lymph nodes
(n=4; mean±sd)
99mTc-HMPAO-LN
Videira et al (2006).J Microencapsulation
A.J. Almeida, 2007
LYMPHATIC DISTRIBUTION IN RATS AFTER ENDOTRACHEAL ADMINISTRATION
(n=4; mean±sd)
99mTc-HMPAO
Videira et al (2006).J Microencapsulation
0102030405060708090
3 8 15 30 60Time (min)
% a
ctiv
ity/g
StomachLiverAxillary lymph nodesInguinal lymph nodes
40
A.J. Almeida, 2007
ALVEOLAR CLEARANCE
Uptake of 1.1 μm fluorescent polystyrene particles administered i.n. to miceEyles et al (2001). Vaccine
24 h15 min
Alveolar clearance of 400 nm fluorescent polystyrene particles administered i.n. to miceByersdorfer et al (1995). J Immunol
6 h
Dendritic cells
24 h
Peribronchial LN
A.J. Almeida, 2007 Valentine and Kennedy (2001) In: Principles and Methods of Toxicology
PARTICLE UPTAKE AT THE LUNGS
LN
BALT
Blood circulation
Lymphatics
Alveolus
Interstitium
Pleura
M
Possible Mechanisms
• Dissolution• Macrophage phagocytosis• Direct passage• Uptake by vascular system• Movement into the lymphatic system• Axonal translocation to CNS
Elimination• Mucociliary escalator• Exhaled air
41
A.J. Almeida, 2007
TREATMENT OF BREAST CANCER LUNG METASTASIS
non-treated
LN-paclitaxel aero (last 15 days)
Taxol® i.v. (last 15 days)
LN aero control
LN-paclitaxel aero (first 15 days)
Taxol® i.v. (first 15 days)
LN iv control 100
100
25
100
100
75
100
% mice metastised per group
PRELIMINARY STUDY
Videira (2007). PhD Thesis
A.J. Almeida, 2007
LIPID NANOPARTICLES AS TOPICAL
DELIVERY SYSTEMS
42
A.J. Almeida, 2007
LIPID NANOPARTICLES AS TOPICAL DELIVERY SYSTEMS
BACKGROUND
• Topical application of LN have been used with promising results either for therapeutic or cosmetic purposes (Jenning et al. 2000; Müller et al. 2002).
• SLN have shown some protective activity on skin surface, such as a UV-blocking potential (Wissing et al. 2003).
• SLN may be formulated in creams, gels,sprays.
• SLN allow modulated drug release.
A.J. Almeida, 2007
MECHANISMS OF UV PROTECTION
Background:• Topical application of SLN have been used for therapeutic or cosmetic
purposes.
• SLN act as an efficient particulate UV blocker (scattering effect) SLN and molecular sunscreens have synergistic effect.
• Reduced release of sunscreens from SLN when compared to emulsions
• Reduced side effects
• SLN may be formulated in creams, gels, sprays, allowing modulated drug release.
Is it so ?
UV
Particulate sunscreen
Jenning et al. (2000); Müller et al. (2002); Wissing et al. (2003)
Molecular sunscreenheath, light
43
A.J. Almeida, 2007
Lipid compositiongliceryl behenate (Compritol® 888 ATO)gliceryl palmitosterarate (Precirol ® ATO 5)
Surfactant Tween 80
Speed rate8 000 - 10 000 rpm
Model DrugsOctylmethoxycinamate (OMC)
Emulsification
Lipid
Aq.phase
meltingmelting
heatingheating
solidification
Homogenization
NANOPARTICLE PRODUCTION
A.J. Almeida, 2007
Formulation Time(months)
PI D50 D90 D95
1 147,0 174,3 206,6 0,516C (empty) 12 80,4 320,4 320,4 0,546
16 102,2 201,8 201,8 0,5291 16,5 83,6 105,3 0,541
C-OMC 12 33,9 41,4 41,4 0,44316 14,9 49,5 49,5 0,4181 221,6 247,3 247,3 0,541
P (empty) 6 269,4 269,4 269,4 0,17616 289,1 289,1 289,1 0,6141 144,7 203,3 241,0 0,204
P-OMC 6 59,3 139,8 186 0,58916 87,2 202,4 268 0,586
Particle size (nm)
NANOPARTICLE CHARACTERISATION: PHYSICAL STABILITY
Toscano et al. (2004) Eur J Pharm Sci, 23 (Suppl. 1)
44
A.J. Almeida, 2007
OMC RELEASE STUDIES
Membrane: SiliconeReceiving medium: 2% albumin in phosphate buffer (pH=7.4)
0
50
100
150
200
250
300
350
0,0 0,5 1,0 1,5 2,0 2,5 3,0
SQRT time (h)
Am
ount
rele
ased
(g/c
m2 )
NL PR/OMC NL CP/OMC
Toscano et al. (2004) Eur Conf Drug Delivery Pharm Technol
A.J. Almeida, 2007
0
3
6
9
12
15
0,0 3,0 6,0 9,0 12,0
Time (h)
OM
Cam
ou
nt
inre
cep
tor
ph
ase
(u
g/
cm2)
NL CP/OMC NL PR/OMC
IN VITRO PERCUTANEOUS ABSORPTION OF OMC
Membrane: Human skinReceiving medium: 2% albumin in phosphate buffer (pH=7.4)
Toscano et al. (2004) Eur Conf Drug Delivery Pharm Technol
45
A.J. Almeida, 2007
IN VIVO STUDIES
• 16 female volunteers (24 to 54 years old; phototypes II and III
• 10-day application trial on the antero-external surface of both legs
• 3 different areas studied corresponding:
• LN-OMC formulation
• LN blank formulation
• non-treated area (1 negative control per leg).
• Experimental challenge: controlled exposure of volunteers to a UV radiation source (Sunny HB-406 Solarium, Phillips)
• Parameter measured: skin colorimetry (erythema and melanisation indexes), TEWL and elasticity.
Fonte 50 cm de
Radiação
A.J. Almeida, 2007
Erythema
0123456789
10
0 3 5 7 10
Time (days)
Eryt
hem
a (A
U)
LNNeg ContLN-OMCNeg Cont
Toscano et al. (2004) Eur J Pharm Sci
56
58
60
62
64
66
68
70
0 3 5 7 10
Time (days)
Mel
anis
atio
n (A
U)
LNNeg contLN-OMCNeg Cont
Melanisation
IN VIVO RESULTS
46
A.J. Almeida, 2007
Cutanova ®
Dr. Rimpler GmbH/Germany
Nanobase ®
Yamanouchi/Poland
MARKETED PRODUCTS
A.J. Almeida, 2007
PRODUCTION OF LIPID MICRO- OR NANOPARTICLES
USING SUPERCRITICAL FLUIDS
47
A.J. Almeida, 2007
AIM
Production of drug-lipid micro- and nanoparticles by a PGSS (particles from gas saturated solutions) process, as an alternative method.
MAIN ADVANTAGES
Avoid the use of solventsParticles are obtained as a dry powders, instead of suspensionsMild pressure and temperature conditions
LIPID MICROPARTICLES USING SUPERCRITICAL FLUIDS
A.J. Almeida, 2007
LIPID MICROPARTICLES USING SUPERCRITICAL FLUIDS
PGSS System
(A) CO2 supply cylinder(B) Gas compressor (C) CO2 storage cylinder (D) Mixing cell (E) Collecting chamber
Lipid compositionTripalmitin
Temperature< 60ºC
Pressure120-180 Bar
Model drugtheophiline
Rodrigues et al (2004). J Supercrit Fluid
48
A.J. Almeida, 2007
LIPID MICROPARTICLES USING SUPERCRITICAL FLUIDS
0
20
40
60
80
100
0 6 12 18 24Time (hours)
% R
elea
sed
T F L co mmercia l160 bar180 bar
0,100,46
2,210
46 120140
160180
02468101214
Per
cent
age
of p
artic
les
Diameter (μm) Pressure (bar)
Rodrigues et al (2004). J Supercrit Fluid
A.J. Almeida, 2007
LYSOZYME-CONTAINING LN PREPARED USING SUPERCRITICAL CO2
Rodrigues et al (2007). submitted
Lysozyme Biological Activity
Lysozyme particles produced fromsolutions with an ethanol mole fractionof C = 0.28 showed no significant lossof activity.
49
A.J. Almeida, 2007
• LN show suitable properties to become one of the most versatile nanoparticulatecarriers for drug and protein delivery.
• Several drug incorporation techniques are applicable, including protein adsorption.
• Inhalation may be an effective route to deliver LN, representing an alternative to the intraperitonial route for targeting colloidal carriers to the lymphatics.
• LN present lymphatic tropism, thus providing the possibility of using radiolabelled LN as a lymphoscintigraphic agent, and allowing the direct delivery of cytotoxic drugs to lung cancer in limited or extensive stage.
• The limited amount of particle retention in lungs may allow the use of LN as a sustained release formulation in chronic lung therapy.
• LN show an effective control release of lipophilic drugs, whici is suitable for percutaneous absorption, with a minimum permeation thus making LN a safe vehicle for sunscreen agents.
CONCLUSIONS (I)
A.J. Almeida, 2007
CONCLUSIONS (II)
• In vivo results do not confirm early reports, i.e. LN on their own no not improve significantly TEWL and elasticity (results not shown).
• Melanisation index was the only parameter that showed a trend, demonstrating that LN present some protective capacity against UV radiation in vivo.
• SCF techniques can be successfully applied to the formulation of solid lipid micro-and nanoparticles with controlled-release characteristics.
• Using a modified SCF technique, it is possible to produce LN containing intact and biologically active protein molecules.
50
A.J. Almeida, 2007
ACKNOWLEDGEMENTS
Faculdade de Farmácia, LisboaAna CerdeiraAna Filipa AzevedoHelena FlorindoJorge GalhardasJorge MoitaJosé A. G. Morais Luís F. GouveiaLuís M. RodriguesMafalda VideiraPaulo Ferreira Almeida
Freie Universtät, BerlinRainer H. MüllerStephan Runge
IBILI, CoimbraAna Cristina SantosFilomena BotelhoJ.J. Pedroso de Lima
Institut de Recerca Oncològica, BarcelonaAngels Fabra
UNFAB, INETI, LisboaEugénia CruzLuísa CorvoManuela Gaspar
IBET, OeirasManuel CarrondoAntónio CunhaLídia Gonçalves
IST, LisboaEdmundo Gomes de AzevedoHenrique MatosJun LiMiguel Ângelo Rodrigues
LEF, LisboaAscensão FarinhaCristina Toscano
Instituto Tecnológico Nuclear, LisboaMaria dos Anjos NevesLurdes Gano
Laboratório Sorológico, SAPatrícia Cruz
London School of Pharmacy (CDDR)H.Oya AlparS. Pandit
Fundação para a Ciência e a TecnologiaTecnimede, SALaboratório Sorológico, SASINDEPEDIP- IMUNOPORFundação C. GulbenkianDAAD, GermanyINVOTAN
A.J. Almeida, 2007
OLD LISBON
51
A.J. Almeida, 2007
MODERN LISBON
A.J. Almeida, 2007
Thank you for your attention !