Formation of an interpolymer complex in supercritical carbon dioxide and its application in the encapsulation of probiotics

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The Formation of an Interpolymer Complex in Supercritical Carbon Dioxide and its Application in the Encapsulation of Probiotics

Presented at the CSIR R&I Conference Materials Science and Manufacturing Dr. Sean Moolman

Polymers, Ceramics and Composites 27 February 2006

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Structure of talk

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Supercritical fluids

What are they & what makes them special?

•

^Probiotics

What is it & why do we want to encapsulate it?

•

Polymers and scCO₂

Love/hate relationship!

•

Interpolymer complexes

A potential solution!

•

The CSIR technology

So how do we do it?

•

^Results

(Does it actually work?!)

•

Conclusions

Where to from here?

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Supercritical fluids

What are they & what makes them special?

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Slide 5 © CSIR 2006 www.csir.co.za

Comparison of typical values of physico-chemical properties for a liquid, gas and supercritical fluid

1 10^-5

10^-1 Gas

300 10^-5

10^-3 Supercritical fluid

1000 10^-3

10^-5 Liquid

Density [kg/m³] Viscosity [Pa.s]

Diffusivity [cm²/s]

Material state

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Supercritical carbon dioxide

Most commonly used supercritical fluid, because:

•

Non-toxic & non-flammable

•

Inert – cannot be oxidised

•

Relatively mild supercritical conditions (31.0 ºC, 73.8 bar)

•

Inexpensive

•

Easy separation by depressurization & no residual solvent

•

Environmentally benign (zero net effect – already produced by many industries, e.g. beer production) But:

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SCF processing relatively expensive, therefore higher value add applications

(Commercially used in e.g. decaffeination, hops extraction)

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CO₂ Total Solubility Parameter - Experimental vs. Calculated

0 2 4 6 8 10 12 14 16 18

1 10 100 1000

Pressure (bar)

Hildebrand parameter (MPa0.5)

Exp. 31 ºC LW 31 ºC Hansen 31 ºC Adjusted molar vol.

Calculation base values:

LW: d_d = 16.6 d_p = 5.3 d_h = 5.7 MPa^0.5 Hansen: d_d = 15.3 d_p = 6.9 d_h = 4.1 MPa^0.5

T_ref = 25 ºC, P_ref = 1655 bar, V_ref = 3.61E-5 m³/mol Adjusted V_ref = 4.15E-5 m³/mol, P_ref = 606 bar

Temperature = 31 ºC

Hansen LW Adjusted

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Probiotics

What is it & why do we want to encapsulate it?

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Bacteria & the human body

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No. of bacteria 20 times more than no. of cells!

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Large intestine – 10¹⁰ – 10¹¹ bacteria / g intestinal contents;

400 – 500 species

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Up to 1.5 kg of bacteria!

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Friendly bacteria – help promote digestion, aid in absorption of nutrients

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Pathogens – responsible for digestive problems (diarrhoea, etc.)

(From Holzapfel et al. 1998. Overview of gut flora and Probiotics. Int. Jnl. Food Microb. 41:85-101)

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Probiotics - definitions

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Live microbial cultures fed by mouth and surviving transit through the large intestine where they colonise the system (Frost and Sullivan, 2000; Saarela et al., 2000)

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A preparation of or a product containing viable, defined microorganisms in sufficient numbers, which alter the microflora by implantation or colonization, in a

compartment of the host and by that, exert beneficial

effects on host health (Schrezenmeir and de Vrese, 2001)

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Live microorganisms which when administered in

adequate amounts confer a health benefit on the host (FAO/WHO, 2001).

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Most commonly Lactobacillus and Bifidobacterium

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Potential benefits of probiotics as supplement or in functional foods

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Prevention and treatment of diarrhoea caused by rotavirus, especially in children

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Immune system enhancement

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Reducing some allergic reactions\

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Treating and preventing respiratory infections, especially in children

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Decreased faecal mutagenicity

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Decrease in the level of pathogenic bacteria

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Decreased faecal bacterial enzyme activity

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Prevention of the recurrence of superficial bladder cancer

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The restoration of the correct balance of natural microflora after stress, antibiotic treatment, alcohol use and

chemotherapy

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Problems with putting probiotics in food products…

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Lactobacillus – microaerophilic & Bifidobacterium – anaerobic, thus sensitive to OXYGEN…

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Short shelf-life…→ encapsulation?

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Encapsulation difficult because the bacteria also sensitive to HEAT and SOLVENTS

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Also sensitive to the aggressive GASTRIC environment

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Thus a ‘soft’ encapsulation method required – perhaps supercritical CO₂-based? (no solvent, low temp., no oxygen)

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Enteric release would be advantageous (i.e. protection in stomach, release in intestines)

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Polymers and scCO

₂

Love/hate relationship!

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Polymers and scCO

₂

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High-pressure CO₂ can depress polymer T_g and/or melting point of polymers and improve processing…

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However, many polymers have insufficient solubility in or compatibility with scCO₂ to enable processing at mild

pressures and temperatures

Graphs from: Kirby CF, McHugh MA. 1999. Phase Behaviour of Polymers in Supercritical Fluid Solvents. Chem. Rev. 99:565-602.

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Approaches to overcome low affinity between polymers and scCO

₂

Limited flexibility with regards to properties

Fats, waxes and oils are generally soluble in scCO₂

Use fats / waxes for encapsulation

These polymers generally have low mechanical integrity and/or barrier properties

These polymers are more amenable to scCO₂processing.

Use low molar mass and low polarity polymers

Reintroduces requirement for use of a solvent - many actives are

sensitive to solvents Use scCO₂as an anti-solvent to extract

the solvent from a sprayed polymer solution and thus precipitate the polymer

Gas anti-solvent (GAS)

technique

No obvious second supercritical fluid with desired combination of properties (low/no toxicity, low critical temperature & pressure, low cost, etc.)

The use of a second supercritical fluid to enhance polymer processability Mixtures of SCFs

Reintroduces requirement for use of a solvent - many actives are

sensitive to solvents The addition of a cosolvent such as

methanol or ethanol to increase the solvation power of scCO₂

Cosolvents

Need FDA approval for surfactants The addition of CO₂-soluble surfactants

Surfactants

Need FDA approval for new polymers Incorporation of "CO₂-philic" functional

groups in new polymers Polymer design

Limitations Elaboration

Approach

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Interpolymer complexes

A potential solution!

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What are interpolymer complexes?

“Non-covalent interaction between two or more polymers through forces such as ionic forces, hydrophobic interactions, hydrogen bonding and Van der Waals forces”

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Proven, proprietary CSIR technologies based on interpolymer complexes…

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Controlled release drug delivery systems

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Barrier coating for packaging applications

0 20 40 60 80 100

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

PMVE-MA as wt fraction of (PVOH + PMVE-MA)

Normalized viscosity

0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 0.05

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

Gantrez content OTR (cm3@stp/bottle/day)

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Desired properties of polymers for encapsulation system

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^scCO₂-processable (soluble / plasticizable)

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pH-dependent release in intestinal tract

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Complementary (form interpolymer complex)

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FDA approved for food or pharma

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The CSIR technology

So how do we do it?

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Polymer system

Vinyl pyrrolidone repeat unit Vinyl acetate repeat unit

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SCF processing unit

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SCF processing unit

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Important parameters

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Temperature, pressure

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Back pressure in product chamber

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Nozzle configuration (e.g. capillary vs. orifice)

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Processing aids

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Stirrer design

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Results

(Does it actually work?!)

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Plasticization of PVP and PVAc-CA

PVP

PVAc-CA

2000 – 3000 g/mol

45 000 g/mol

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Formation of interpolymer complex in scCO

₂

PVAc-CA Complex PVP

Carbonyl absorption band

acetate absorption band overlapping with two carbonyl stretching modes of the free and self- associated carboxylic acid groups

1664 cm^-1

1681 cm^-1

0 1 2 3 4 5 6 7 8 9

Moisture absorption (%)

PVP:PVAc-CA PVP-VAc:PVAc-CA PEO-PPO-PEO:PVAc-CA Moisture absorption, 72 hours, 60% RH, 30 ºC

Dry blend scCO2-processed

24 h, 30 ºC, 60% RH

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Product particles containing B. lactis

Particle size (µm)

0.1 1 10 100 1000 10000

Normalized volume distribution

0 20 40 60 80 100

PVP:PVAc-CA 0.25:0.75 GMS:PVP:PVAc-CA 0.75:0.0625:0.1875

Sauter mean diameter 168 µm Sauter mean

diameter 6.9 µm

3.0E+12 3.0E+12

9.3E+10 0.0E+00

5.0E+11 1.0E+12 1.5E+12 2.0E+12 2.5E+12 3.0E+12

Viable cell count (cfu/g)

Control SCF-encapsulated Spray-dried Survival of B. longum through encapsulation process

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0 5 10 15 20 25

% of encapsulated drug released

0 20 40 60 80 100

pH 6.8 buffer pH 1.2 buffer

pH-dependent release of indomethacin

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Dissolution/swelling of encapsulated B. longum

Freeze-dried control

SCF encapsulated

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Shelf-life improvement

*Cui et al. 2000. Survival and stability of bifidobacteria loaded in alginate poly-l-lysine microparticles. Internat. J. of Pharmaceutics 210:51-59

Bb12 (B. lactis ) survival after 7 weeks

1.0E+06 1.0E+07 1.0E+08 1.0E+09 1.0E+10 1.0E+11 1.0E+12 1.0E+13 1.0E+14

Bb12 - RT PL3/129 - RT Bb12 - 4 ºC PL3/129 - 4 ºC Bb12 - RH PL3/129 - RH B. bifidum in alginate- polylysine, 4ºC*

Sample / conditions

Bacteria survival (cfu/g)

RH = Humidity cabinet, 30 ºC, 60% RH RT = Room temperature, dessicated 4 ºC = 4 ºC, dessicated

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SGJ & SIF results – vs time

1.E+08 1.E+09 1.E+10 1.E+11 1.E+12 1.E+13 1.E+14

-1 1 3 5 7

Time (h)

Viable cells (cfu/g)

Bb-46 control

Encapsulated - pellet

Encapsulated - supernatant Encapsulated - total

Before experiment

Simulated gastric juice (HCl, pepsin, saline, pH 2)

Simulated intestinal fluid (KH₂PO₄, NaOH, pancreatin, pH 6.8)

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Summary of B. longum results - final counts

1.E+06 1.E+07 1.E+08 1.E+09 1.E+10 1.E+11 1.E+12 1.E+13

Viable bacteria (cfu/g)

Controls (cfu/g) Encapsulated (cfu/g)

Controls (cfu/g) 2.25E+08 1.16E+07 1.16E+07 1.21E+08 1.07E+10 2.05E+08 Encapsulated (cfu/g) 3.29E+09 1.35E+08 1.52E+09 9.45E+08 1.52E+12 1.20E+09

Normal system

Normal system +

Pluronic

Normal system

Normal system - low er press.

Normal system +

GMS

Normal + GMS +

gelatin

Counts after exposure to simulated gastric juice and simulated intestinal fluid

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Conclusions

Where to from here?

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Conclusions

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Can form interpolymer complexes in scCO₂

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Can process these interpolymer

complexes in scCO₂, without complexation inhibitor

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Can successfully encapsulate drugs and bacteria using PVP:PVAc-CA and the PGSS system, with minimal

damage to bacteria

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PVP:PVAc-CA interpolymer complex insoluble in acidic environments, but swells & releases in alkaline

environments

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SCF-encapsulated B. longum has improved survival through gastric environment compared to controls

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Further work

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Investigate other applications (e.g. oral vaccines)

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Improve shelf-life performance / testing methodology

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Shelf-life trials with subsequent SGJ-SIF testing

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Trials on SHIME system (Gent University – Belgium)

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Atomistic simulation to investigate optimum stoichiometric ratios

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Culture B. bifidum and determine effect of SCF-based encapsulation

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Acknowledgements

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Project team:

• Philip Labuschagne, Dr. Thilo van der Merwe (CSIR)

• Mapitsi Thantsha, Prof. Eugene Cloete (UP)

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IDC (joint investment)

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DST (funded first supercritical fluid reactor system)