Production and application of biopolymers in drug release

Maria Filomena de Andrade Rodrigues

(filomena@ipt.br)

Laboratory of Industrial Biotechnology – Center for Technology of Processes and Products

2

Variety of available degradable polymers - Biocompatibility- free from degradation related toxic

products (e.g. monomers, stabilizers, polymerization initiators, emulsifiers)

Few approved by FDA

Biodegradable polymers for drug delivery systems

Polylactic acid (PLA)

Polyglycolic acid (PGA)

Poly(lactic-co-glycolic acid) (PLGA)

Polycaprolactone (PCL)

PHA (polyhydroxyalkanoate), specially PHB (polyhydroxybutyrate),

Biodegradable polymers for drug delivery systems

• Development of technology for production of P(3HB) and P(3HB-co-3HV) from sugar cane.– Instituto de Pesquisa Tecnológicas do Estado de São Paulo (IPT);– Universidade de São Paulo (USP);– Copersucar (private company);– FINEP – Transference to the company PHBISA.

• Other developments– Application of raw materials studied for PHA production

(Xylose+glucose from lignocellulose materials, Glycerol/fat acids, vegetable oils and agroindustrial wastes)

– Development of processes for PHA application for medical application (encapsulation of drugs).

PHA in IPT

Polyesters synthesized and used by microorganisms for intracellular energy storage

Produced from renewable materials

Biodegradable

Rate of degradation controlled by varying copolymer composition

in vivo PHB degrades to hydroxybutyric acid which is a normal constituent of human blood biocompatible

Polyhydroxybutyrate (PHB) and copolymers

n = 1R = -CH3 poly(3-hydroxybutyrate) R = -CH2 - CH3 poly(3-hydroxyvalerate)R = -CH2 = CH2 poly(3-hydroxypentenoate)R ≥ -CH2 - CH2-CH3 medium-chain-PHA

Polyhydroxyalkanoate chemical structure

Different monomeric compositions

•Alcaligenes sp.• Cupriavidus sp. •Burkholderia sp.•Pseudomonas sp.•Streptomyces sp.••GGenetic MModified OOrganism

Biodiversity for PHA production

Metabolic routes for PHA biosynthesis

Objective StrategyReduction of Costs Isolation and screening

Genetic studiesLow cost substratesHigh cell density cultivation

Control of molecular weight Substrate fluxMutants deficient in depolymarase

Downstream process Use of enzymes

New polyesters New susbtratesIsolation of new bacteriaGenetic modificationsChemical modifications

PHA - Strategies for new developments

Copersucar Technology Center

SugarFactory

CaneFields

PowerPlant

Bagasse

SteamElectricity

Cane

Compost

Sugar

Mol

asse

s

Ethanol

Distillery

Vinasse Steam

Electric

ity

Ethanol

YeastSolvents

PHBFactory

Ste

amE

lect

ricity

PHB

SugarC

ompo

st

Solar

Energy

CO2

Integrated PHB, Sugar and Ethanol Mill

PHA production in Brazil

•Commodities

•Microcapsules for controlled drug releasePHA granules in bacteria

PHA applications

Scaffolds for tissue engineering

Different biopolymers

(PHB, PHB/HV, PHPE,

PHAMCL) and applications

• Thermoplastic properties• Packaging industry• Medicine - Functionalised

nanoparticles• Pharmacy - Drug delivery• Agriculture• Food industry• Raw material for

enantiomerically pure chemicals

0

20

40

60

80

100

0 5 10 15 20 25 30

Tempo (dias)

Biod

egra

daçã

o (%

CO

2 tot

al)

Padrão (Glicose) Amostra A Inibição

GlicosePHB

Degradation of PHA

Polymer t 10 (days)

Vicryl ® Vabsorbable suture (PLLA 8%-co-

PGA 92%)

18,8

Dexon® suture (PGA) 22,9

Suture PDS® (poly(p-dioxanona) 50,0

PHBV (20% HV) Mw 300.000 Da 229,1

PHBV (12% HV) Mw 350.000 Da 229,1

PHB (0% HV) Mw 800.000 Da 104,2

Reduction of 10% in weight under phisiologic condictions (37oC, pH 7,4) (HOLLAND, 1986; AMASS et al., 1998).

Degradation of PHA

PHA for drug delivery systems

Spray drying

Coacervation

Emulsion/solvent extraction

Gelification  

Polymerisation

Electrospinning

Microfluidics

Technology

Micro‐nanoencapsulation at IPT

PHA for drug delivery systems

Büchi-B190 spray dryer

Burkholderia cepacia Burkholderia cepacia

Soil bacteriaAccumulate a blend of two polyestersP(3H4PE) is produced from sucrose

P(3HB)

P(3H4PE)

0

2

4

6

8

10

12

14

16

18

20

0 10 20 30 40 50Time (h)

X, P

HA

e PH

B (g

/L)

0,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

NH

4 e

PHPE

(g/L

)

PHB

PHA

X

NH4

PHPE

Growth and polymer accumulation by B. cepacia IPT 64 at pH 7.0 and 30oC.

Production of PHA

Purification of PHA by enzymatic lysis of cells

Burkholderia IPT 64

Fermentation Enzymatic lysis

Spray drier

PHBHPE granules after cell extraction and purification steps -Granules 0.3-1.0 microns , Mw - 860 kDa , Ip - 10

Production of cells with 50-60% of PHB-HPE PHB-HPE

PHBHPE PHBHPE microspheres after spray drying already loaded with the drug NzPC

Micro e nanoparticles with biodegradable biopolymers for drug delivery from Burkholderia cepacia as effective biodegradable matrices for drug carriers

Absorption measurement (680 nm) of NzPC/PHBPHPE in PBS buffer (∆); NzPC/PHBPHPE in PBS buffer+10% BSA)( ).Release from the NzPC-loaded microspheres during the first 2 hours, followed by a continuous and slowly release until 36 hours in both medium.

Fluorescence emission of NzPC/ PHBPHPE 0.84mM in PBS+10%BSA medium at: 2h(__); 6h(- - -); 12h( __ - __); 24h( --- ); 36h(__ - __). Insert: Normalized absorption spectra of NzPC/PHBPHPE (0.84mM in PBS).

Spectroscopic measurements

35x106

30

25

20

15

10

5

Flu

orec

ence

,cps

800780760740720700W avelength, nm

1.0

0.8

0.6

0.4

0.2

0.0

O.D

. No

rma

lized

80 07 5 07 0065 060 0Wavelen gth,n m

00,5

11,5

22,5

33,5

44,5

5

0 2 6 10 12 24 36Time (h)

O.D

.

Biodegradation

pBurkholderia cepacia as effective biodegradable

matrices for drug carriers

Granules of B. cepacia before (a) and after spray drying (b).

Granules of B. cepacia before (a) and after spray drying (b).

Process for production of biodegradable microparticles from granules in aqueous medium.

Spray-dried PHA particles loaded with Acetaminophen (drug : polymer of 1:1)

0

20

40

60

80

100

0 300 600 900 1200 1500Dissolution time (min)

Dru

g re

leas

ed (

% w

/w)

Acetaminophen release profiles from spray-dried PHA microparticles loaded with mass ratio of drug : polymer of ( ) 1:1 and ( ) 4:1. The dissolution profile of the drug is presented as reference ( ).

PHBHPE aqueous suspensions are suitable to entrap drugs using the spray drying method to produce a biodegradable drug carrier with a relatively small size and a narrow size distribution and with potential use of PHBPHPE microspheres as an efficient drug delivery system.

PHBHPE and other PHA represent new biodegradable materials for application in drug release

Diversity of blends and monomer combinations open the possibility for new developments in this field

New bio-nanotechnology structure at IPT for new developments

Final Remarks

Research Financial SupportIPTFapespFinepCNPq

Workshop organization

Acknowledgments

CTPP TeamLaboratory of Industrial

Biotechnology (LBI)

Laboratory of Chemical Processes and Particle Technology (LBI)

USP – Ribeirão Preto

Obrigada!

Vielen Danke!