Polymeric nanocarriers for parenteral drug administration

Dr. Oana G. Schramm Aachen, February 07.2012

Outline
Introduction to polymer-based drug delivery systems
micells dendrimers star-shaped polymers

4- and 6-arm starPCL-pPEGMAs

synthesis characterization core degradation encapsulation properties

Conclusions

Acknowledgements

Introduction drug-delivery systems

Most of clinically used drugs are small hydrophobic molecules with molecular weights < 500 g/ mol.
rapid diffusion into healthy tissues small amounts of drugs reach the target sites therapeutics is associated with side effects

Major goal: improving the therapeutic index!

(defined as ratio of the toxic dose to the therapeutic dose)

Polymer therapeutics

term coined by Helmut Ringsdorf and Ruth Duncan

protein-polymer conjugates drug-polymer conjugates supramolecular drug-delivery systems

Angew. Chem. Int. Ed. 1981, 20, 305-325, Nat. Rev.Drug Discovery 2003, 2, 347-360.

Drug-delivery systems (DdS)

drug-polymer conjugates - specific (active) targeting
drug targeting moiety polymer unit linker tether

targeted polymer-drug conjugate

solubilizers etc.

drug carriers - passive targeting

enhanced permeability and retention (EPR) effect

amphiphiles hydrophilic drug hydrophobic drug vesicle micelles nanoparticles

high molecular weights (> 20 kDa) are required for passive targeting renal threshold ca. 30 kDa
H. Maeda et al., J. Control. Release 2000, 65, 271-284 R. Haag, F. Kratz, Angew. Chem. Int. Ed. 2006, 45, 1198-1215

DdS: Properties (I)

Ideal drug-delivery candidate:

able to solubilize the hydrophobic drugs

to protect sensitive drugs from hydrolitic and oxidative conditions high loading capacity able to release the drug in a controlled way

for passive targeting molecular weights > 20 kDa

biocompatible biodegradable or easy to eliminate from organism

DdS: Properties (II)

Single polymer chain micelle

Micelles
Drawbacks: unstable under different enviromental conditions: high dilution pH temperauture pressure sterilization

hydrophilic chain

Hydrophobic chain cmc

weakly water-soluble drug

Dendrimers
Drawbacks: low loading capacity high costs (multisteps synthesis) not suitable for large scale applications
weakly water-soluble drug

Star-shaped polymers: General concept

Star-shaped polymers can effciently combine the properties of selfassembled micelles and dendrimers
core-shell structure unimolecular micellar behavior high loading capacity low stability high synthesis costs
degradable biocompatible hydrophobic core

.. and they circumvent the main drawbacks of these systems:

guest molecules

(bio)degradation

6-hydroxycaproic acid

biocompatible hydrophilic shell

pPEGMA

Star-shaped Polymers: Synthesis

R1
HO RO OH
R = H or O HO OH OH O Br Br

O OH + O
130 C, 4 h Tin(II)-octoate

R O RO OR

O O
1

R = R1 or
1

O RO

OR OR

R3
O RO RO OR
3 3

NEt3, THF 24-72 h, RT

R2
CuBr/PMDETA toluene, 90 C

O RO RO
2

O O
hydrophobic

nO

Br

O O OR
2

nO

Br

O
hydrophilic

O PEG
O O PEG

R = R3 or
3

O RO

OR OR

R = R2 or
2

OR OR

RO

Quantitative fuctionalization of star PCL

RO RO OR
1 1 1

O O

NEt3, THF H O n 24-72 h, RT O Br Br

O RO RO OR
2 2 2

O O

O n

Br

Polymer 1 2 3 4
4-armStar-PCL

Conversion ~ 100% ~ 100% ~ 8000 9000 100% ~ 100%

4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2

Mw 3200
4armStar-PCL

Mw 5600
4-armStar-PCL
3000 4000 5000 Mw 7200 6000 7000

2000

10000

m/z

6-armStar-PCL

Mw 9800

d, ppm

ATRP of PEGMA
O RO RO OR
2 2 2

O O

O n

Br

CuBr/PMDETA toluene, 90 C

O RO RO OR
3 3 3

O O

O n O

Br p O PEG

O O PEG
normalized RI response, a.u

0.40
o

Temp. 90 C, [M]/[I] = 200:1

1.2
o

Temp. 70 C, [M]/[I] = 200:1

0.32 Temp. 90 C, [M]/[I] = 20:1

Temp. 70 C, [M]/[I] = 20:1

10 min 30 min 1h 2 10 min h 32 h h 44 h h Star-PCL StarPCL-Br

1.0

0.8

normalized RI response, a.u.

RI response

1.0 0.8 0.6

0.24 0.16 0.08 0.00

normalized RI response, a.u

0.8

0.4

10 min 30 min 1 h min 10 2 h min 20 3 h min 40 4h 70 min 5h 2h StarPCL

0.6

3h 4h

0.0

4-arms

StarPCL-Br

0.4 0.2 0.0 14

ln([M0]/[M])

14

15

16

17

18

19

20

0.4

14

16

18

20

elution volume, mL

elution volume, mL

1.2

0.2

1.2

0.8

15

16

17

18

19

20

8.5

ln([M0]/[M])

0.0

0.8

9.0
0.4

9.5

10.0

10.5

11.0

11.5

elution volume, mL
0.4

elution volume, mL

0.0 0 50 100 150 200 250

time, minutes

0.0 0 50 100 150 200 250 300

time, minutes

ATRP of PEGMA 1H NMR

O RO RO OR
2 2

O Br
CuBr/PMDETA toluene, 90 C

O O
R= R2 or O RO
2

RO RO

O O OR
3

Br

O
R = R3 or
3

O PEG

OR OR

O O PEG

O RO

OR OR

star-initiator 10 minutes 30 minutes

60 minutes

ppm

All arms initiated the polymerization of PEGMA

Star PCLp PEGMAs

polymer M/I no. of arms PEGMA Mw (Da) Mn (Da) (GPC) Mn (Da) (1H NMR) Conversion no. of PEGMA/arm PDI (GPC) Dh (nm) (DLS, H2O) P1 40:1 4 475 12,600 21,400 80% 8 1.3 6 nd P2 40:1 4 1100 24,900 48,000 85% 8.5 1.24 7.6 6.4 P3 200:1 4 475 25,000 51,800 48% 24 1.16 9.2 nd P4 200:1 4 475 26,600 59,400 56% 28 1.13 9.8 8.2 P5 60:1 6 475 20,700 32,100 80% 8 1.29 7.2 5.8 P6 60:1 6 1100 39,600 65,400 85% 9 1.3 11.8 9.2 P7 240:1 6 475 35,000 83,400 70% 26 1.18 13.6 11.8 P8 240:1 6 1,100 41,000 170,000 60% 24 1.15 17.4 13.4

Dh (nm) (DLS, CH3Cl)

All starPCL-pPEGMA present unimolecular micellar behavior

Upscaling of polymer P4, after precipitation: 17 g, PDI = 1.13

ATRP of PEGMA
ATRP of acrylates using star macroinitiators is still a challenge due to star-star coupling reactions not all of the bromine end-capped sites act as initiators for ATRP Consequences: broad polydispersity indices and unsymmetrical GPC profiles

Our optimized procedure allows the synthesis of well-defined 4- and 6-arm starPCL-pPEGMA (PDI <1.3) at moderate to high conversion (48-85%)

Mn (kg/mol), PDI

Mn (kg/mol), PDI

(P4) 59.4, 1.13 (P3) 52.8, 1.16 (P2) 48.0, 1.24 (P1) 21.4, 1.30 4-arm starPCL-Br 4.6, 1.17 8 12 16 20

(P8) 170, 1.15 (P7) 83.4, 1.18 (P6) 65.4, 1.3 (P5) 32.1, 1.29 6-arm starPCL-Br 7.0, 1.17 8 12 16 20
elution volume/mL

elution volume/mL

Selective PCL degradation

acid or base catalyzed degradation enzymatic degradation
O PEG O Br O PEG O O O O
nO

Br

PEG O O O

O Br
p

O PEG OH
6n HO

O
O

aq. HCl, dioxane, RT

O
6 HO

O Br
p

nO

O
O
n

+
HO HO OH OH

O O

O
OH

O O
n

PEG O O
p

HO

O O O O O Br
p

O
n

PEG O

Br

PEG

Br

For enzymatic degradation of PCL see i.e. Eur. J. Sci. 2007, 31, 119-128.

Acid catalyzed degradation

PEG
P8 after hydrolysis at 60 C P8 after hydrolysis at R.T. P8
o

PDI 1.17 PDI 1.15

room temperature selective hydrolysis of the PCL-core

6arm starPCL-Br

12

16

20

24

Elution volume, mL

H2O

(a)
5.0
1H

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

NMR spectra of (a) p(PEGMA) from the hydrolysis of 6-arm starPCL-pPEGMA, (b) 6-arm star-PCL-pPEGMA, (c) 6-arm starPCL-Br.

(b)
5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5

(c)
5.0

d, ppm

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

Enzymatic degradation of PCL core

Lipase from Rhizopus arrhizus
Conditions: lipase/polymer 1:5, in phosphate buffer solution (pH=7), at 37 C

1.2 1.0

RI response, a.u.

0.8 0.6 0.4 0.2 0.0 14 16

P9 - t0 P9 - 1 day P9 - 2 days P9 - 4 days

biodegradable core

in vitro

18

20

Elution volume, mL

Dye encapsulation disperse orange 3

[dye]/[P4], [P4] = 25M

1.2

20:1 16:1

absorption/ a.u.

polymer

0.8

12:1 10:1 8:1

0.4

4:1 1:1

0.0

disperse orange 3
300 400 500 600 700
wavelength/ nm

20:1 18:1 16:1 14:1 12:1 10:1 9:1 8:1 7:1 6:1 5:1 4:1 3:1 2:1 1:1 0.5 mM dye, 0 mM P4

800

O N O N N NH2

water insoluble dye

Dye encapsulation (II)

4-arm starPCL-pPEGMA, P4
[dye]/[polymer]
20:1 18:1 16:1 14:1 12:1 10:1 9:1 8:1 7:1 6:1 5:1 4:1 3:1 2:1 1:1

6-arm starPCL-pPEGMA, P7
1.2
[dye]/[polymer]

1.2

Absorption/ a.u.

0.8

0.8

0.4

0.4

26:1 24:1 22:1 20:1 18:1 16:1 14:1 12:1

absorption/ a.u.

0.0 300 400 500 600

0.0 300
1.2

400

500

600

700

wavelength/ nm
1.2
absorption at 442 nm/ a.u.

wavelength/ nm

absorption at 443 nm/ a.u.

1.0

0.8

0.8

0.4

0.6

10

15

20

12

16

20

24

28

no. of encapsulted molecules of guest 1 per molecule of P4

no. of encapsulated molecules of guest 1 per molecule of P7

Dye encapsulation (III)

in the literature: encapsulation by polymer aggregation

R. Haag et al., Angew.Chem. Int. Ed. 2007, 46, 1265. R. Haag et al., Macromol. Rapid Commun. 2008, 29, 172. Simple PEGylation is not sufficient to build up stable unimolecular core-shell systems! our systems: encapsulation inside the polymer

Unloaded polymer (P4) Dh (DLS, H2O)

Polymer (P4) loaded with disperse orange 3

9.8 nm

11.6 nm

DLS measurements strongly support the formation of unimolecular host-guest architectures.

Drug encapsulation: Furosemide

O a O H2N S O Cl
H2O DMF a f b d+e c
P4 + guest 2 P3 + guest 2 P2 + guest 2 P1 + guest 2
P5 + guest 2

OH H N c b

e f O

antihypertensive and antidiuretic activity for the treatment of excessive fluid accumulation and swelling (edema) of the body caused by heart failure, cirrhosis, chronic kidney failure, and nephrotic syndrome

DMF

H2O

DMF

DMF a f b d+e c
P8 + guest 2 P7 + guest 2 P6 + guest 2

guest 2 + DMF in D2O

guest 2 + DMF in D2O

4
d / ppm

d / ppm

polymer
guest 2

P1
4

P2
5

P3
7

P4
7

P5
6

P6
5

P7
10

P8
10

Drug encapsulation: Hydrochlorothiazide

O H2N S O Cl
DMF a b
P4 + guest 3

O S N H

O NH

antihypertensive and antidiuretic activity for the treatment of heart failure, cirrhosis, chronic kidney failure, also effective for nephrogenic diabetic insipidus and hypercalciuria

H2O

DMF
DMF a b
P8 + guest 3

H2O

DMF

P3 + guest 3
P7 + guest 3

P2 + guest 3

P6 + guest 3

P1 + guest 3

P5 + guest 3

guest 3 +DMF in D2O

guest 3 + DMF in HDO in 2 O

2

4
d / ppm

d / ppm

polymer

P1

P2

P3

P4

P5

P6

P7

P8

guest 3

22

18

24

24

34

18

36

36

Conclusions
New water-soluble, well-defined 4- and 6-arm star-shaped polymers as
unimolecular micelles have been successfully synthesized;
High loading encapsulation capacities of different hydrophobic drugs and other hydrophobic guests;

Room temperature selective degradation of the PCL core has proven the
homogeneous distribution of the PEGMA units over the arms; Enzymatic degradation experiments using lipase from Rhizopus arrhizus have demonstrated that the PCL core of the starPCLpPEGMAs is in vitro biodegradabile; Potential nanocarriers for parenteral drug administration.

Acknowledgement

Infos under http://www.schubert-group.com

Dr. Richard Hoogenboom, Dr. Michael Meier, Prof. Jean-Franois Gohy (CMAT, Belgium), Prof. Dr. Ulrich S. Schubert

3rd Generation: Higher hydrophlicity

Synthesis of homo-branching agents
O Br O Cl O O Br O PCl5 O O HO O O Br O Br Br O Br O HO OH OH

O RO RO OR
1 1 1

O O Br O O Br

O O

O n

R2

Property screening
Methylorange: pH-Indicator
N N N O S O
+

+H+

O Na basic form; yellow color acidic form; red color

N N H

O S O O Na
+

[methylorange] /(mol/L)

0,04

0,03
4 6

0,02

0,01

Maximal loading: 6.6 7.2 MO/micelle INI: approx. 0.5 MO/micelle

0,00 0,000

0,005

0,010

0,015

J. Am. Chem. Soc. 2004, 126, 11517.

[polymer] /(mol/L)

Transport of guest-molecules: AUC

1.0

2.00 3500

unloaded micelles: P1
0.8

Non UV

Normalized c(M)

0.6

Absorbance Meff (g/mol)

P2 absorbingP3 P4 P5 P6

1.75 3000 1.50

2500 1.25 2000 1.00 1500 0.75

loaded micelles unloaded micelles

0.4

0.2

0.50 1000

loaded micelles: Guest is UV absorbing

0 1000 2000 3000 4000 5000 6000

0.25 500 0.00

0 0

0.0

300

400

500 9

Meff (g/mol)

DP PCL block

(nm)

600 12

700 15

18