Analysis of Rf-PEGs By 13C(T1) and 19F(T1) Relaxation NMR

Steven Han1, Yong Ba1, Mike Mackel2 and Julie A. Kornfield2

1California State University, Los Angeles: Department of Chemistry and Biochemistry; Los Angeles, CA 900322California Institute of Technology: Department of Chemistry and Chemical Engineering; Pasadena, CA 91101

4 6 8 10 12 14 16 18

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

T1 (

s)

Weight Percent of 20KC10 Liquid Rf-PEG

-CF3

-CF2

4 6 8 10 12 14 16 18

0.65

0.70

0.75

0.80

0.85

0.90

0.95

1.00

1.05

1.10

1.15

1.20

T1 (

s)

Weight Percent of 20KC8 liquid Rf-PEG

-CF3

-CF2

ABSTRACTThe chain dynamics of Poly(ethylene glycol)s (PEGs) withterminally substituted hydrophobic fluoro-alkyl (Rf) groups (Rf-PEGs) were investigated by NMR relaxation techniques. Bystudying the intramolecular chain dynamics within the Rf

- polymerwith 19F NMR relaxation, a more-detailed understanding of thedissociation of the micelles could be gathered. Additionally, anunderstanding of the PEG backbone by 13C NMR relaxationeffects gave an in-depth analysis to the physical behavior of thecomplete Rf-PEGs for the one-phase species of 20KC8 (20g/molPEG, –(CH2)2CnF2n+1 (n=8) and 20KC10 (n=10) polymers.

INTRODUCTION•PEG is an essential polymer with respect to its physicalproperties. Rf-PEGs have been widely studied as modelassociative polymers. Some of the applications include peptideand drug conjugates, drug release systems, and surfacemodification. This is due to the fact that depending on the PEGlength and modification of the end groups an existence of thesol/gel phase characteristics within the polymer can beimplemented thus leading to a more controlled handling of PEGin an open environment.•Previously, these modified PEGs have been investigated andstudied with a combination of surface erosion, rheology, andsmall angle neutron scattering (SANS). Using surface plasmaresonance (SPR), it was discovered that single phase Rf-PEGs(20KC8 and 5K-M-C8) had a faster surface erosion rate than two-phase systems (6KC8 and 10KC8) due in large part to theamount of sol concentration in the two-phase Rf-PEG as seen inFigure 1. Using rheology, it was found that in one-phasesystems, there was a linear correlation between correlation timeand end-group relaxation time and that increasing weightconcentration increases both the plateau modulus and end-groupdissociation time. Finally, with SANS it was found that varying thetemperature of a fixed Rf-PEG system doesn’t effect thehydrophobic core of the PEG aggregation unit, varying the chainlength and aggregation of the PEG block affects the wave factor(1/A) and in the process, distance between micelles in thehydrophobic end groups has direct dependence on both thechain length of PEG and the fluoro-alkyl ends. Varying theweight concentration of the Rf-PEG also effects the latticestructure of the polymer depending on the ordering of thehydrophobic core (PEG molar mass).•To understand the local motions of the these-single phase gels,we introduce relaxation NMR. In using the relaxation data, wecan perform the dependency on weight concentration vs.relaxation time, and effects between relaxation time vs.correlation time. Thus by using these two graphs, a more, clearunderstanding to the external motions of the micelles within theRf-PEGs can be accomplished. In this study we use both 20KC8Rf-PEG and 20KC10 Rf-PEG to illustrate these differences in thebehavior of the micelles.

EXPERIMENTAL

PART 1: Synthesis of Rf-PEGs

(Note: all reactions were done under Argon gas)

• Azeotropic Distillation of PEG (20K) was dried intoluene.

• The dried PEG was mixed with a 100 molar fold of IPDI(isopropyl diisocyanate) was vacuum dried in THF for 2days.

• Anhydrous Ethyl Ether was used to remove unreactedIPDI from the IPDI-PEG-IPDI intermediate (A).

• A 10-fold excess of the 1H,1’H,2H,2’H-perfluorodecanoland 1H,1’H,2H,2’H-perfluoro dodecanol wererespectively mixed with A and continuously stirred withTHF as solvent.

• After completion of the 20KC10 Rf-PEG and 20KC8 Rf-PEG, anhydrous ethyl ether was used to remove anyunreacted IPDI and recrystallized with THF repeatedlyfor best purity of the polymer.

Part 2: Formation of Gels/Solutions

• Firstly, the purity of the 20KC8 Rf-PEGs and 20KC10Rf-PEGs were determined by single pulse 19F NMRwith an internal standard of CF3COONa (~78 ppm) andHPLC (detection ofdisubstituted/monosubstituted/PEG) with a RP-18column and a solvent mixture of 80/20water/acetonitrile ramped to 100 percent acetonitrile.

• Using D2O, a weight concentration of 5%, 12%, and17% of both 20KC8 Rf-PEG and 20KC10 Rf-PEG wereplaced in 4 ml vials thus forming 100µl of gel/sol.

• The vial was then mixed with a wrist-handed shakerand left for 1-3 days depending of the concentration.

• Similar procedures were employed for the RfPEGsdissolved in MeOD-d4.

Part 3: Relaxation NMR

• The sol or gel in both the 20KC8 Rf-PEG and 20KC10Rr-PEG were analyzed using the 200 MHz NMRSpectrometer for 13Canalysis and 500 MHz NMRSpectrometer for 19F analysis.

1. In both experiments, a delay time from 1ms-8000 mswas used.

2. Integration of both the –CF3 and –CF2 (sum) were doneat their respective times and plotted vs. their respectivedelay time for final T1 by the first order exponentialcurve fit.

• For the liquid MeOD-d4 samples, those solutions wereplaced in 5mm NMR thin wall tubes to be analyzed F-19 analysis with 400 MHz Spectrometer.

1. Samples were done much like for the D2O except thatintegration of each of the –CF2 groups were done forboth 20KC8 Rf-PEG and 20KC10 Rf-PEG

2. Again, both the individual integral of the –CF3 and–CF2’s were plotted vs. their delay time and final T1

was done on all integrals.

NOTE: Both the sol or gel samples of 20KC8 Rf-PEG and20KC10 Rf-PEG along with liquid samples of the samepolymer’s T1’s were plotted vs their respectivecorrelation time. In addition, the T1 of the respect Rf-PEG plotted vs.its corresponding weight concentrationto illustrate its correlation relationship.

CAUTION: these sol/gel samples must be refridged tokeep the moiety of the phase

Figure 1-Schematic diagram of the three different conditions in anopen environment:

a) Single-phase gel, b) lypophobic cubic-phase gel, c) sol-gel phasecoexisting gel

CONCLUSIONBy using NMR relaxation techniques a completeexplanation of the external motions of the Rf-PEGsystem for carbon and fluorine groups in thepolymer could be established. In addition, adetailed understanding of the molecular tumblingeffects residing in both atoms was explained bythe relaxation theory and mechanisms.

FUTURE WORKCurrently, we are finishing studying the one-phase Rf-PEG (20KC8) and working on two-phase sol-gel Rf-PEG (6KC6, 6KC8, 10KC8,10KC10) to give a detailed mechanism of themicelles within the Rf-PEGs by NMR relaxationstudies. Next, we will use these in NMR diffusionstudies, MRI, and REDOR experiments. With theaid of these studies, a more, clear understandingas to how Rf-PEGs are essential to a drugreleasing system in the biomedical field.

ACKNOWLEDGEMENTSSH thanks SURF Fellowship and CSEM for theirhospitality in working at CalTech. For theprelimarily studies of liquid NMR, much thanks toSusan Schafer and Dr. Scott Ross at CalTech.In addition, thanks to Ali Jabalemeli and SonjongHwang for their technical help in NMR relaxationstudies. Additional thanks to Osmundo Francoand Lucy Liu for current assistance in both oneand two phase Rf-PEGs. Finally, this project issupported by NSF PREM program.

REFERENCES1. Harris, R. K. Nuclear Magnetic Resonance

Spectroscopy; John Wiley & Sons, Inc.: NewYork, 1986

2. Levitt, M. H.; Suter, D.; Ernst, R. R. J. Chem.Phys. 1986, 8, 4233-4255

3. McBrierty, V. J.; Packer, K. J. Nuclear MagneticResonance in Solid Polymers; Cambridge: NewYork, 1993

4. Mooney, E. F. An Introduction to 19F NMRSpectroscopy; Heyden & Son Ltd.: London, 1970

5. Samosom, A.; Tuherm, T.; Past, J. J. Magn.Reson. 2001, 149, 264-267

6. Tse, G.; Kornfield, J. A.; Hubbell, J. A.;Johannsmann, D.; Hogen-Esch, T. E.Macromolecules 2001, 34, 6409-6419

7. Tse, G.; Kornfield, J. A.; Hubbell, J. A.; Lai, J.Macromolecules 2002, 35, 4448-4457

8. Tse, G. In Situ Forming Hydrogels Using Self-Assembly of Fluoroalkyl-Ended Poly(EthyleneGlycol)s (thesis); California institute ofTechnology: Pasadena, 2001

9. Utz; M. NMR Spectroscopy of Polymers,UCONN, 2001 www.giotto.ims.uconn.edu

10.Zhou, J.; Zhuang, D.; Yuan, X.; Jiang, M.; Zhang,Y. Langmuir 2000, 16, 9653-9661

4 6 8 10 12 14 16 18

0.35

0.40

0.45

0.50

0.55

0.60

0.65

0.70

0.75

T1 (

s)

Weight Percent of 20KC10 Rf-PEG

-CF3

-CF2

-15.45-15.40-15.35-15.30-15.25-15.20-15.15-15.10-15.05-15.00-14.95-14.90-14.85

-0.25

-0.20

-0.15

-0.10

-0.05

0.00

0.05

0.10

0.15

0.20

0.25

0.30

log10

T1 (

s)

log10

Tc (s)

-CF3

-CF2

20KC10 RF-PEG (Liquid)

-15.20 -15.16 -15.12 -15.08 -15.04 -15.00 -14.96

-0.18

-0.16

-0.14

-0.12

-0.10

-0.08

-0.06

-0.04

-0.02

0.00

0.02

0.04

0.06

0.08

log10

T1 (

s)

log10

Tc (s)

-CF3

-CF2

20KC8 Rf-PEG Liquid

-15.00 -14.95 -14.90 -14.85 -14.80 -14.75 -14.70

-0.40

-0.35

-0.30

-0.25

-0.20

-0.15

-0.10

log10

T1 (

s)

log10

Tc (s)

-CF3

-CF2

20KC10 Rf-PEG (Gel)

-6.88 -6.87 -6.86 -6.85 -6.84

-0.31

-0.30

-0.29

-0.28

-0.27

-0.26

log10

T1 (s

)

log10

Tc (s)

PEG

4 6 8 10 12 14 16 18

0.49

0.50

0.51

0.52

0.53

0.54

0.55

T1 (

s)

Weight Percent of 20KC10 Rf-PEG

PEG

-130-120-110-100-90-80

-130-120-110-100-90-80

-130-120-110-100-90-80

-130-120-110-100-90-80

-130-120-110-100-90-80

-130-120-110-100-90-80

-130-120-110-100-90-80

-130-120-110-100-90-80

-130-120-110-100-90-80

4000 ms

1 ms

t (ms)

-130-120-110-100-90-80

525456586062646668707274767880828486889092949698

525456586062646668707274767880828486889092949698

525456586062646668707274767880828486889092949698

525456586062646668707274767880828486889092949698

525456586062646668707274767880828486889092949698

525456586062646668707274767880828486889092949698

525456586062646668707274767880828486889092949698

525456586062646668707274767880828486889092949698

525456586062646668707274767880828486889092949698

525456586062646668707274767880828486889092949698

2000 ms

1 ms

t (ms)

525456586062646668707274767880828486889092949698

RESULTS

20KC10 Rf-PEG13C NMR

20KC10 Rf-PEG19F NMR

a b

-120-110-100-90-80

-120-110-100-90-80

-120-110-100-90-80

1 ms

4000 ms

t (ms)

-120-110-100-90-80

-120-110-100-90-80

-120-110-100-90-80

-120-110-100-90-80

-120-110-100-90-80

-120-110-100-90-80

-120-110-100-90-80

20KC10 Rf-PEG19F NMR(MeOH)

a b

DISCUSSIONAnalysis of the sol/gel one-phase of the 20KC10samples (solid) by the 13C NMR spectra indicates thepresence of the PEG midblock in the aggregation ofthe Rf-PEG as illustrated in Figure 2. In addition, the -CF3 and –CF2 overlapped in the corresponding 19FNMR spectra indicating the possible interactionbetween the –CF2 groups in the coil of the hydrophobeof the system. Even more evident is the weightdependence of the hydrophobe as seen in thecorresponding graph. Using these two NMR studies, acomplete behavior of the system having an effect duethe aggregation number of the 20K PEG has beenestablished.

As for the liquid samples in MeOD-d4, there is moredistinction in the –CF2 groups of the Rf-PEG illustratingthat the solvent has less effect on the aggregation ofthe polymer as D2O. Despite that, there is a dramaticeffect in the weight percent of the polymer even morethan in water indicating greater molecular tumbling asseen by their relaxation times. In addition to explainingthe molecular tumbling, the 13C primarily had dd-homo(inter) and fluorine-19 had primarily ue-dipolar(inter) mechanism. To further explain this, thedd-homo(inter) mechanism has contributions also fromintermolecular interactions and with that, londondispersion forces are possible to give rise to thismechanism of the PEG midblock.

Figure 3- Dependency of the weight concentration and molecular tumbling by graph of T1 vs. Tc

(correlation time) for a) 20KC10 (solid) b) 20KC8 (liquid in MeOD-d4) and c) 20KC10 (liquid inMeOD-d4)

Figure 2-Relxation Spectra of20KC10 (solid) by 13C NMRalong with its correspondingdependency in weightconcentration and moleculartumbling by T1 vs. Tc

Figure 4-Relaxation Spectra of 20KC10 (solid) by 19F NMR in a) solid-D2O and b) in MeOD-d4