Compressed Fluids: Unique media for preparing nanovesicles with

Compressed Fluids: Unique media for preparing nanovesicles with

NANO4, La Habana, Cuba, September 2012
Compressed Fluids: Unique media for preparing
nanovesicles with high structural homogeneity
100 nm
Nora Ventosa, * Santi Sala, Elisa Elizondo, Ingrid Cabrera, Evelyn Moreno, Alba
Córdoba, Mildrey Sánchez, Lidia Ferrer, Jaume Veciana *
[email protected], [email protected];
Reversible self-assembling of amphiphilic molecules
through non-covalent weak interactions
50nm – 1 µm
• Biocompatible
• They can incorporate both
hydrophobic and hydrophilic
drugs
• Minimize drug degradation and
undesired side reactions (reduce
toxicity)
• Size, charge and surface
properties easily changed
• Improve intracellular penetration
and cellular distribution
V.P. Torchilin, Nature Reviews 2005, 4, 145
d ≤ 200 nm
d > 200 nm
SUV : Small Unilamellar Vesicle
LUV: Large Unilamellar Vesicle
MLV: Multi Lamellar Vesicle
MVV: Multi Vesicular Vesicle
d ≤ 200 nm
EPR
effect
Blood
vessel
• Avoid first-pass
elimination by the kidney
(>10 nm)
• Enhance Permeability
and Retention (EPR)
effect (< 200nm)
• Robust surface
functionalization
R. Langer et col., Nature Nanotechnology 2007, 2, 751
Small Unilamellar Vesicles (SUVs) as drug carrires
for the improvement of efficacy and reduction of toxicity
Keys: characterization and preparation at the nanoscale
Hubbell, J.A. Science 2003, 300, 595
pH induced vesicle destruction
Nano-structure determines drug release
Healthy cell
Sick cell
Healthy cell
Homogeneity at supramolecular level
guarantees robustness of nanomedicines targeting
Conventional methodologies to prepare SUVs
Drawbacks
•Multistep, time consuming and complex procedure
•Low reproducibility. Not easily scalable
•High risk of bioactivity loss during processing
•Low “final product/raw material” ratio
•High environmental impact
~100 n
‰ GREEN
PROCESSES
FOR
THE
REPRODUCIBLE
PREPARATION OF SMALL UNILAMELLAR VESICLES (SUVs)
WHICH
FULFILL
STANDARDIZATION
AND
QUALITY
REQUIREMENTS IMPOSED BY REGULATORY AGENCIES.
• Small batches (5-10Kg) for test clinical phases
• Large scale production
Compressed CO2 a green solvent for large scale
production of nanostructured materials
‰ Non-toxic
‰ Non-flammable
‰ Cheap
‰ Easy separated from products
‰ Suitable for fragile molecules
‰ Easy scale-up
L. Perrut et al., Ind. Eng. Chem. Res. 2003, 42, 6375
Precipitation from compressed fluids
Compressed CO2 a green solvent
T1, P1, X1
CO2
dT/dt
dP/dt
SINGLE
PATH
dX/dt
T2, P2, X2
HOMOGENEOUS particles
P.G. Jessop and B. Subramanian , Chem. Rev. 2007, H. Okamoto et al., Adv. Drug. Deliv. Rev. 2008
DELOS precipitation
Depressurization of an Expanded Liquid Organic Solution
Depressurization
Material
+
Organic solvent
Solution
Material in CO2-expanded
solvent (XCO2>0.5)
CO2=co-solvent
Large, fast and
homogeneous
solution cooling
∆T
Protected under international patents
Cryst. Growth Des. 2001, 1, 299; Cryst. Growth Des. 2006, 6, 23; Cryst. Growth Des. 2012, 12, 1717.
DELOS: precipitation from CO2 expanded solvents
Large, fast and ideally homogeneous solution cooling
3:Depressurization
β
Homogenous β profile
T
L
Homogeneous supramolecular
organization
Micro and nanoparticulate solids
200nm
Molecular Pharmaceutics 2011, 8, 395–404
Precipitation of ibuprophen nanoparticles
Highly crystalline powders
2 µm
15000
Heat Flow (mW)
Lin (counts)
PXR diffractogramms
Processed
Unprocessed
10
20
30
2θ
40
50
Differential scanning calorimetry
10000
5000
Processed
Unprocessed
0
80
Temperature (ºC)
90
Precipitation of compounds with a rich polymorphism
fatty acid long chain compound
HO
CH3
≡
O
• High nº of crystalline forms
• High similarity of different structures
• Difficulties in obtaining isolated forms
Stable (T<32ºC)
Meta-stable
dd 1
Stable (T>32ºC)
dd2
1
Meta-stable
Meta-stable
2
d1< d2
d1 < d 2
Bo
Bo
Bo
Bm
BBmm
E
oEo
Eo
Em
Em
E
m
C
C
C
E. Moreno et al., New Journal of Chemistry, 2007
As u p er
super
AAsuper
A1
AA11
Precipitation of pure polymorphic phases
from CO2-expanded solvents
With Prof. Angel Cuevas group
S. Sala et al., Cryst. Growth and Design, 2010, 10, 1226
Results
DELOS-susp: One-step procedure for the
preparation of SUVs
PT
High structural
homogeneity in particle
size and vesicular
Lipidic molecules in
membrane morphology
CO2-expanded solvent
SUVs
Water flow
SUVs
100nm
M. Cano et al, Langmuir 2008
Patents: WO2006079889; EP 1843836 ; US2007259971 ; CA 2566960
DELOS-susp: Preparation of uniform SUVs
Cholesterol
+
Cationic surfactants
DELOS-susp
Volume (%)
10
Hydration
method
8
6
4
2
0
10
100
1000
10000
Cryo-TEM images
Thin Film Hydration
12
DELOS-susp
Highly stable
200nm
200nm
Particle Diameter (nm)
Nanoscopic SUVs (~100 nm)
Langmuir, 2008, 24, 2433; J. Colloids and interfaces, 2010, 350, 10-15
¿ Could DELOS decrease hetereogeneity in
Vesicle toorganization
vesicle lipid
composition
supramolecular
of lipids
in a vesicular
hetereogeneity
system?
Elisa Elizondo
Doctoral thesis
Lipid A
Lipid B
AB vesicles
Elisa Elizondo Doctoral Thesis
Larsen, J. et al, JACS, 2011, 133, 10685-10687
Influence of the preparation route on the
supramolecular organization of lipids
Thin Film Hydration
DELOS‐susp
DPPC CTAB
Solvent evaporatio
n
hydration
cholesterol
1. Freeze/thaw (x10)
2. Extrusion
Fluorescent dyes were used as molecular probes for studying
variations in the membrane supramolecular structure of
individual vesicles
ClO4‐
DiD
Group of Prof. Stamou
University of Copenhagen
NBD‐6‐cholesterol
[Chol:CTAB:DSPE‐PEG2000‐biotin:DiD:NBD6Chol] [49.35 : 49.6 : 0.3: 0.5 : 0.25]
Confocal fluorescence microscopy study: METHODOLOGY
2 different images for each analysed area of the samples:
Zoom of typical confocal fluorescence images of vesicles
1, 2 and 3 are individual vesicles
Confocal fluorescence microscopy study: METHODOLOGY
INBD/IDiD
Vesicle membrane homogeneity
INBD/IDiD Gives information about the vesicle to vesicle membrane homogeneity
NON‐IDEAL system
Number of vesicles
Number of vesicles
IDEAL system
INBD/IDiD
INBD/IDiD
Impact of the preparation route on
cholesterol-CTAB vesicular systems
DELOS-susp
Hydration
CFs provide more homogeneous vesicular systems
regarding the assembly of lipids forming the membrane
J Am Chem Soc 2012, 134, 1918
Impact of the preparation route on
cholesterol-DOPC vesicular systems
DELOS-susp
Hydration
Non significative differences regarding vesicle to vesicle
lipid composition homogeneity
J Am Chem Soc 2012, 134, 1918
Possible explanation
Film hydration procedure
Water addition
Cholesterol and
CTAB phase
separate
Water
Cholesterol and
DO PC have
total miscibility
Water
Heterogeneous system
Homogeneous system
Possible explanation
DELOS-SUSP
PT
9 Solvent-free
intermediate state is
Water flow
avoided.
9 Lipids are kept in
Homogeneous system
solution during
vesicle formation
100 nm
9 Lipid demixing is
avoided
Proposed explanation:
Formation of a bimolecular amphiphile
Cholesterol and CTAB need to form a bimolecular agregate with
structural characteristics similar to a two-chain phospholip before
forming the vesicle bilayer
CHO
CHO ‐CTAB
Amphiphilic two‐chain moiety
Vesicular self‐assembling
Theoretical calculations made by Dr. Jordi Faraudo (ICMAB‐CSIC)
Results submitted for publication
DELOS-susp enhances homogeneity regarding
supramolecular organization in the vesicle membrane
Vesicles made of Cholesterol (
) and CTAB (
) (1:1)
DELOS-susp
Hydration
Group of Prof. Stamou
E. Elizondo et al, JACS, 2012, 134, 1918
Conjugation of biomolecules to SUVs by
DELOS-susp
Cholesterol rich SUVs functionalized with RGD peptide
Cryo-TEM images
Cellular up-take
In collaboration with University of Barcelona (Dr. M. Royo and Prof. F. Albericio)
and IBEC (Dr. M. Parajo)
Spanish patent submitted, July 2012
Integration of EGF protein to SUVs
Size: 145,4 nm
PDI: 0,390
Zp: +74,8 (±6,3)
=
+
EGFprotein (6kDa)
% EE: 90 %
EGF/colesterol : 25 µM:1 M
Promising nanomedicine for the topical treatment of
complex wounds
200 nm
Immunoelectron Microscopy
100 nm
Cryoelectron Microscopy
In collaboration with CIGB and CEAC (Centers of the Cuban Scientific Pole)
Hector Santana, Dr. Eduardo Martínez
Cuban Patent submitted, August 2012
Scaling-up of DELOS-susp
6 mL system
25mL
300 mL system
X 50
1250mL
High batch-to-batch structural consistency upon scaling-up
Encapsulation of gentamicine in SUVs
6 mL
300 mL
unilamellar
unilamellar
100 nm
100 nm
X 50
Nanomedicine, 2011
Compressed fluids based processes:
technological platform for the preparation of
vesicle based nanomedicines
Easy scale-up and low environmental impact
‰ Main drawback for their industrial
implementation:
Lack of the required facilities in
pharmaceutical production plants
100 nm
2010 - Nanomol Technologies S.A.
Prof. Jaume Veciana,
Dr. Santi Sala,
Evelyn Moreno,
Preparation,
processing
andDr.characterization
Dr. Elisa Elizondo, Ingrid Cabrera (Cu), Alba Córdoba, Lidia
of functional
molecular
materials
Ferrer, Mildrey
Sanchez (Cu)
Institut de Ciència de Materials de Barcelona (ICMAB) of CSIC
Universitat Autònoma de Barcelona
Financial and institutional support
Thank you for your attention!
Equipment
for industrial scale
IMPULSO
A LA TRANSFERENCIA
cGMP pilot plant
0,5-2 tons/year
TECNOLOGIAS NANOMOL
Small batches (5-10Kg)
for test clinical phases
Source: Natex
Equipment for industrial scale
DIAM - Plant for cork treatment (Extremadura, Spain)