Manufacturing of Polymeric Nanomaterials for Biomedical - - PowerPoint PPT Presentation

Manufacturing of Polymeric Nanomaterials for Biomedical applications

Yvon Durant Advanced Polymer Laboratory Nanostructured Polymer Research Center

Presented at the International Congress of Nanotechnology- October 31-November 3, 2005 San Francisco

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Architecture - Size @ 100KD

linear chain

24nm

Random coil

700nm

G5 dendrimer

7nm 10nm

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Block copolymer architecture

diblock-copolymers Tri block-copolymers gradient-copolymers Block-gradient -copolymers Star block copolymer

Block pendant copolymer

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Why are polymer well suited for nanoscale manufacturing ?

• Assume a block copolymer PEG-PGLA 55K-b-45K

– Random coil size = Rg= l(na)0.5 with l=0.2nm – Density of PGLA = 1.1 g/cm3

• PGLA assembled in a 10nm “dry” core
• Number of chains/particle, n= πD3/6 *ρ /m.A
• N= 3.14*(10E-7) 3/6*1.1/45000*6.02E23=8 chains
• Rg =l(na)0.5 =0.2*((55000)/44) 0.5=0.2*(1250)0.5=7nm
• D=10+7*2=24nm

O O

n

O OH O O

m x y

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• Polymeric Nanoparticles synthesis processes

– Mini-emulsion Polymerization – Self assembly – Directed assembly

• Application to biotechnologies

– liposomes for transmembrane delivery – biosensors by molecularly imprinted polymers – Drug delivery

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Emulsion Polymerization : soap opera

Micelle:5nm Stabilized Monomer droplet:5-50m Stabilized Polymeric Particle: 50-500nm

ygd1

Slide 6 ygd1

Yvon Durant, 1/28/2002

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• Create a meta-stable emulsion of the monomer(s).
• Use 2 key elements :

– High shear source to break large droplets

• Sonicator
• Microfluidizer
• Homogeneizer

– Use a water insoluble molecule to stabilize the particle

• Sometimes called cosurfactant (missleading)
• Hexadecane, Eicosane, polymer, macromonomer, macroinitiator,

CTA, ...

Miniemulsion Polymerization

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Monomer(s) Stabilizer Water Surfactant(s)

No stabilizer With stabilizer

Miniemulsion stability

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Particle size control

• K. Landfester, N. Bechthold, F. Tiarks, and M. Antonietti, Miniemulsion Polymerization with Cationic and Nonionic Surfactants: A Very

Efficient Use of Surfactants for Heterophase Polymerization. Macromolecules 1999, 32, 2679.

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Mini to micro emulsion

• K. Landfester, Recent Developments in Miniemulsions - Formation and

Stability Mechanisms. Macromol. Symp. 2000, 150, 171.

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Encapsulation of magnetite in polymer particles by miniemulsion

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Magnetite encapsulation

SEM TEM

Magnetite PS-PMAA PEG shell cNRG targeting peptide 50nm

Magnetic nanoparticles functionalized with cNGR for atherosclerotic plaque diagnostic.

cNRG targets CD13 – tracer of engiogenesis

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• Polymeric Nanoparticles synthesis processes

– Emulsion Polymerization – Mini-emulsion Polymerization – Self assembly – Directed assembly

• Application to biotechnologies

– biosensors by molecularly imprinted polymers – liposomes for transmembrane delivery – Bypassing the BBB

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1. Selection of template molecule and functional monomers 2. Self-assembly of template molecule and functional monomers 3. Polymerization 4. Analyte Extraction

Molecularly Imprinted Polymers

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SINP : Surface Imprinted NanoParticle

P(MMA-EGDMA) Core Extraction by dialysis P(MAA-EGDMA) shell Caffeine

P(MMA-EGDMA) Core P(MMA-EGDMA) Core

MAA EGDMA Caffeine

1st stage Miniemulsion Polymerization 2nd stage Emulsion Polymerization

MJB-20: miniemulsion seed Organic phase = 23% : MMA 85.5%, EGDMA 9.5%, Hexadecane 5%, Water phase = 77% : Water 99%, SDS 0.6%, KPS 0.025%, NP-50 0.39% Prepare the two phases, mix them together, magnetically stir them for 15 minutes, then, sonicate the resulting emulsion for 2 minutes (90%, 9) in ice. SCexp = 22.25%, Conversion = 98.96%, Size = Malvern Nanosizer: Dz = 107.1 nm, Dv = 111.9 nm MJB-21: 2nd stage imprinting Water 57.74% MJB20 (wet) 33.44% NaHCO3 0.042% KPS 0.047% Caffeine 5.78% EGDMA 2.63% MAA 0.31% Water, MJB-21, NaHCO3, were mixed and heated at 80C. When at temperature, add caffeine and start degassing. After 15 minutes, add KPS and start feeding with egdma+maa. Dilute with 250g of hot water (336%) while stirring. SCexp = 2.635% (dilution) Conversion = 57.86% Size = Malvern nanosizer Dz= 108.4 nm, Dv = 114.2nm Brookhaven 90+: Dz = 104.9 nm, Effective Dv = 105.2 nm

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SEM+DLS of SNIP

MJB21

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Adsorption studies by HPLC

Caffeine adsorption isotherm 0.00E+00 2.00E-03 4.00E-03 6.00E-03 8.00E-03 1.00E-02 1.20E-02 1.40E-02 1.60E-02 1.80E-02 0.00E+00 5.00E-03 1.00E-02 1.50E-02 2.00E-02 2.50E-02 3.00E-02 3.50E-02 4.00E-02

caffeine free-gm. caffeine bound-g

EGDMA-MA -caf imprint in ACN (bulk-1) Binding constant specific site 1027 l/mol Binding constant non-specific site 47 l/mol Nanoparticles EDGMA-MA in H2) caf(MJB40) Binding constant specific site 888 l/mol Binding constant non-specific site 51 l/mol

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Biomimetic electrochemical sensors based on molecular imprinting

• A chemical sensor selectively recognizes a target analyte molecule in a complex matrix and

gives an output signal which correlates with the concentration of the analyte. The transducer: When the analyte interacts with the recognition element of a sensor, there is a change in one or more physicochemical parameters associated with the interaction. Transducer convert these parameters into an electrical output signal than can be amplified, processed and displayed in a suitable form.  Molecular imprinting use as sensing materials Advantage: cheap, stable and robust under a wide range of conditions including pH, humidity and temperature Problem: Signal transduction is so low that it seem to be environmental artifacts. Due to the insulating nature of the polymer constituting the MIP

Biomimetic electrochemical sensors based on molecular imprinting / Chap.18 MIP – D. Kriz, R. J. Ansell- Vol 23 -Elsevier

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QCM

• A QCM consists of a thin quartz disc sandwiched between a

pair of electrodes. Due to the piezoelectric properties of quartz, it is possible to excite the crystal to oscillation by applying an AC voltage across its electrodes.

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Q-Sense D300

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Coated QCM sensor Fracture SEM

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Raw data

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QCM results

Adsorption of caffeine at different caffeine solution concentrations

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 5 10 15 20 25

time in minutes F1/F1max

caffeine 0.05g/L caffeine 0.0005g/L caffeine 0.005 g/L

With the Langmuir equation the quantity adsorbed can be calculated for the caffeine MIP at a concentration of 0.0005g/L. This value is found to be equal to 7.3×10-6g of caffeine per gram of MIP. The mass of MIP on the crystal is equal to 4×10-

• 5g. With these two values, the minimum amount detected in

this experiment was equal to 0.3nanogram.

150Hz 12Hz 1.6Hz

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Guanosine Recognition

• Perfect complement to imprint

guanosine is cytidine

• Modified cytidine monomer

N O O N O O O NH2 N N N O N H O N H2 O O O

Guanosine Cytidine

N N O O O NH2 O O H OH O OH N N O O H NH2 O O H OH

+

H3PO4 1.3eq EDIC 1.5eq DMAP 2.5eq in water RT 12 hrs

EDCI: 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride DAMP: 4-dimethylaminopyridine

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RT: 0.05 - 29.98 2 4 6 8 10 12 14 16 18 20 22 24 26 28 Time (min) 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative Abundance 13.10 13.06 24.94 25.06 25.13 24.89 12.61 1.93 1.87 1.81 11.14 10.64 20.72 20.64 20.80 2.12 28.64 28.04 1.65 3.74 9.66 5.04 5.51 24.53 23.13 6.22 20.43 19.78 15.72 17.72 13.68 7.02 NL: 1.57E6 Base Peak F: MS marine_sampl e_05042011 4728

N N O O NH2 OH HO HO

Na+ 266

N N O O NH2 OH HO O O

Na+

334.1

Two different Isomers apparently m/z 226, 174, etc m/z 112, 266

LC/MS Base peak chromatogram

m/z 334

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SINP : Guanosine detection

P(MMA-EGDMA) Core Extraction by dialysis P(MAA-EGDMA) shell Guanosine

P(MMA-EGDMA) Core P(MMA-EGDMA) Core

Cytidine-MA EGDMA Guanosine

1st stage Precipitation Polymerization 2nd stage Emulsion Polymerization

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Precipitation Polymerization in ACN

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Precipitation polymerization

• Smaller…
• 20nm
• Higher sensitivity

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Low cost QCM

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QCM200

• 650
• 600
• 550
• 500
• 450
• 400
• 350
• 300

0.0 1.0 2.0 3.0 4.0 5.0 6.0

Time (Hours) Frequency (Hz) 0.1 0.2 0.3 0.4 0.5 0.6 Caffeine (g/L) LAN28-a-6-6th Event

• 30
• 20
• 10

10 20 30 40 50

0.0 0.5 1.0 1.5 2.0 2.5 3.0

Time (Hours) Frequency (Hz) 0.1 0.2 0.3 0.4 0.5 0.6 Caffeine (g/L)

MJB18-a-2 Event

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• Polymeric Nanoparticles synthesis

processes

– Emulsion Polymerization – Mini-emulsion Polymerization – Self assembly – Directed assembly

• Application to biotechnologies

– biosensors by molecularly imprinted polymers – liposomes for transmembrane delivery – Drug delivery

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Self assembly (Claverie)

PGlu PLA PEG

insulin

pH = 7.4 Spontaneous self association

O O O Me n O m O NH O HN H HN NH2 O O k CO2H CO2H j j + k + 1 = l

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Parental delivery of insulin

Enteric Coating Small Intestine Nanoparticle dispersion protease Digestion of the PGlu hairy layer hydrophobic particle is adsorbed epithelial cell Insulin delivery endocytosis Endosome (pH = 5) Acidic degradation of PLA microvilii

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Properties of the vesicles

• White / translucent liquid (nanosize)
• Does not contain any solid in suspension
• Has the viscosity of water

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Electronic Microscopy

FMC 146

160nm

FMC 179

100nm

FMC 66

300nm 140nm

FMC 150

130nm

Linear Triblock Branched Triblock

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• Polymeric Nanoparticles synthesis processes

– Emulsion Polymerization – Mini-emulsion Polymerization – Micro-emulsion Polymerization – Self assembly – Directed assembly

• Application to biotechnologies

– biosensors by molecularly imprinted polymers – liposomes for transmembrane delivery – Drug delivery

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Trigger strategy (in vitro)

PEPTIDE PEPTIDE TRIGGER

PEPTIDE

PEPTIDE

RELEASE STUDY

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Liposomes

Lipid bilayer Liposome

http://www.avantilipids.com/PreparationOfLiposomes.html

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Self assembly of Liposome

Multi Lamellar Vesicles

Photo courtesy of FEI Company Japan Ltd.)

Small Unilamellar Vesicles Large Unilamellar Vesicles : LUV

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Vent Sample injection Sample reservoir loop Membrane filter holder Water bath Collect liposome

Directed assembly : extrusion

High pressure N2 tank

• Operates above Tc
• Membrane pore size control vesicle size
• Multiple extrusion (typically 5 passes)
• Good reproducibility
• Can operate at up to 10 bar (typically 4)
• “Wide” range of LUV

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DPPC liposome size distribution after extrusion through a 400 nm polycarbonate membrane filter. Negatively-stained TEM

400 nm

Can be VERY monodispersed

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Release study strategy

37 oC buffer

• 1. Take 0.5 ml sample out

periodically

• 2. Centrifugal extraction
• 3. filtration (MWCO 10 K/50K)

FLD

"blank' release

20 40 60 80 100 120 140 160 5 10 15 20 25 30 35 40 Time (hr) Adjusted Fluorescence

37C

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Transmembrane transport mechanism

• f insulin with excipient triggering

Phase I Phase II Phase III

CPE-215 molecules Liposome

T=0

Low insulin leak rate High insulin leak rate Medium insulin leak rate Low insulin leak rate

Defect

Release at 37C with cholesterol 0.001 0.002 0.003 0.004 0.005 0.006 5 10 15 20 25 30 35 40 45 50 55 Time (hour) C

• n

cen t ratio n(m g /m l)

Blank 1XCPE&CSO 1XCPE&CSO+B-

Phase I Phase II Phase III

flux

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Acknowledgements

• Julien Ogier, Marine Barasc, Romuald Couronne
• Dr. Zhengmao Li
• Pr. Jerome Claverie, Floraine Collette, Sayantan Roy
• Funding : NOAA, Bentley Pharmaceuticals, NSF, DOT

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Microemulsion

Recipe MJB-10: microemulsion (seed) Water 82.84% NaHCO3 0.043% Na2O5S2 0.011% SDS 8.27% KPS 0.17% Styrene 8.67% Water, Salts, SDS, stirred, degassed. Add 20% of styrene. Heat. When at 80C, add KPS. Let react for 20

• minutes. Start feeding with styrene,
• ver 2 hours. 30 minutes of Post

polymerization. SCexp = 15.1% Conversion = 77.47% Size = CHDF: Dv = 35.5 nm, Dn = 33.2 nm Nanotrac: Dv = 36.8 nm, Dn = 25.13 nm

MJB10 MJB21 108nm 33nm