Effect of encapsulating arginine containing molecules on PLGA: a solid-state NMR study
Jean-Baptiste Guilbaud(a), Helen Baker(b), Brian C. Clark(c), Elisabeth Meehan(c) and YaroslavZ.Khimyak(a,*)
(a) Department of Chemistry, The University of Liverpool, Crown St, Liverpool, L69 7ZD, UK
(b) School of Chemical Engineering and Analytical Science, The University of Manchester, PO Box 88, Manchester, M60 1QD, UK
(c) AstraZeneca, Macclesfield SK10 2NA, Cheshire, UK
(*) to whom correspondence should be addressed, e-mail address:
Electronic Supporting Information
Fig. ESI-1. Formula of the amorphous pseudo-decapeptide AZD.
Fig. ESI-2. PXRD patterns of L-arginine and its PLGA composites.
Fig. ESI-3. Glass transition temperature of Arginine -PLGA formulations.
Fig. ESI-4. Experimental and simulated powder XRD patterns of L-arginine.
The assignment of the 1H-13C CP/MAS spectrum of arginine is supported by the 1H-13C heteronuclear correlation (HETCOR) spectrum recorded with a contact time of 200ms (Fig. ESI-5). A strong cross-peak between the broad NH resonance and the CH2d (43.1ppm) is noted. A weaker contribution of the NH protons is also found for the CHa resonance at 56.3 ppm. This confirms that the 13C peaks at 43.1 and 56.3 ppm correspond to the CH2d of the guanidine tail and CHa respectively. Based on previously reported results, the two higher field doublets (ca. 24.6 and 32.2 ppm) were assigned to the CH2g and CH2b of the guanidine tail (ca. 26.3 and 31.0 ppm for the CH2g and CH2b respectively).(1-5)
Fig. ESI-5. 1H-13C HETCOR NMR spectrum of L-arginine recorded with 200 ms contact time and 10 kHz MAS rate.
15N solid-state NMR spectra were measured on a Bruker Avance 400 DSX spectrometer operating at 40.54MHz for 15N and 400.13MHz for 1H. The values 15N chemical shift is referred to liquid ammonia. 1H®15N Cross Polarisation Magic Angle Spinning (CP/MAS) NMR experiment was carried out at MAS rate of 5.0kHz using zirconia rotors of 4mm in diameter. The 1H p/2 pulse was 3.0µs. The TPPM decoupling was used during the acquisition. The Hartmann-Hahn condition was set using glycine. RAMP-amplitude CP was implemented to recover a broad Hartmann-Hahn matching condition. The recycle delay was 5.0and the contact time 1.5ms.
Fig. ESI-6. 1H-15N CP/MAS NMR spectrum of ARARAF recorded with 1.5 ms contact time and 5 kHz MAS rate.
Fig. ESI-7. CP kinetics curves for the crystalline L-arginine.
Fig. ESI-8. CP kinetics curves for the amorphous ARARAF hexapeptide.
Fig. ESI-9. 1H-13C CP/MAS NMR spectra of the PLGA 5050_1 film and its arginine composites, * denotes the spinning sidebands, the FWHH in Hz derived from the Gaussian deconvoluted spectra are quoted along the peak for each resonance.
Fig. ESI-10. 1H-13C CP/MAS NMR spectra of the PLGA 5050_1 film and its ARARAF composites, * denotes the spinning sidebands, the FWHH in Hz derived from the Gaussian deconvoluted spectra are quoted along the peak for each resonance.
Fig. ESI-11. Variable temperature 1H-13C CP/MAS NMR spectra of F-Arg5 (Tg=319 K), the FWHH in Hz and the integral I derived from the Gaussian deconvoluted spectra are quoted along the peak for each resonance.
Fig. ESI-12. Variable temperature 1H-13C CP/MAS NMR spectra of F-ARARAF13.5 composite (Tg= 315 K), the FWHH in Hz and the integral I derived from the Gaussian deconvoluted spectra are quoted along the peak for each resonance.
Fig. ESI-13. (A) 1H-13C WISE spectra of (a) PLGA 5050_1 film, (b) 550_1/Arg2.5% and (c) 5050_1/Arg5%, MAS=7kHz, contact time=200ms. (B) 1H projections of the 1H-13C WISE spectra of 5050_1 and its Arginine composites.
Fig. ESI-14. (A) 1H-13C WISE spectra of (a) PLGA 5050_1 film, (b) 550_1/ARARAF6.8% and (c) 5050_1/ARARAF13.5%, MAS=7kHz, contact time=200ms. (B) 1H projections of the 1H-13C WISE spectra of 5050_1 and its ARARAF composites.
Table ESI-1. T1rC-VSL times of L-Arginine.
ppm / I .10-8 / a.u. / T1rC / ms / R2C=O / 180.7 / - / ∞ / -
179.5 / - / ∞ / -
C guan. / 158.7 / 1.37 / ∞ / 0.817
CHa / 56.3 / 3.47 / 34.7 / 0.979
CH2d / 43.1 / 3.93 / 21.3 / 0.988
CH2b / 32.5 / 2.72 / 17.5 / 0.987
31.8 / 2.51 / 21.0 / 0.979
CH2g / 24.8 / 2.61 / 31.3 / 0.970
24.4 / 2.36 / 21.1 / 0.968
Table ESI-2. TrC-VSL times of ARARAF.
ppm / I .10-8 / a.u. / TrC / ms / R2C=O / 174.6 / - / ∞ / -
C guan.- / 162.3 / 1.13 / 36.5 ± 3.6 / 0.953
C guan. / 157.3 / 2.22 / 22.2 ± 2.0 / 0.937
C aromatic / 134.1 / 0.75 / 36.6 ± 7.2 / 0.767
CH aromatic / 129.1 / 2.14 / 9.2 ± 1.1 / 0.919
CH / 54.0 / 3.80 / 20.4 ± 1.3 / 0.967
CH / 50.2 / 4.32 / 22.8 ± 1.6 / 0.956
CH2 / 41.9 / 2.19 / 7.0 ± 0.7 / 0.952
CH2 / 38.2 / 1.58 / 10.2 ± 0.8 / .0.972
CH2 / 27.4 / 2.00 / 7.2 ± 0.8 / 0.942
CH3 / 17.2 / 3.28 / 32.4 ± 2.2 / 0.955
Theory of Cross-Polarisation
The transfer of magnetisation from an abundant spin (I), to a dilute one (S), by cross-polarisation (CP) is widely used in numerous pulse sequences in solid-state NMR. The CP experiments must be carefully optimised to record high-quality spectra, derive kinetic parameters describing molecular mobility and make the measurements quantitative. The knowledge of CP kinetics (dependence of the peak intensity vs. contact time) is therefore crucial for the correct interpretation of the CP spectra.
Different models of CP kinetics have been developed.(6-10) The simplest model of CP dynamics (I-S) was derived for homogeneous solids where the I-S heteronuclear interactions are relatively weak and the I-I homonuclear dipolar interactions are strong sufficiently to provide efficient spin diffusion. For a system of abundant and dilute ½ spin nuclei, providing that the transfer of magnetisation occurs at a faster rate that the relaxation in the rotating frame of the S spins (), the I-S model leads to the kinetic equation:(6)
(1)
where I0 is the absolute amplitude; T1ρ is the relaxation time in the rotating frame; TIS is the CP time constant.
Although the I-S model is simple to understand and is widely applied, it is not sufficient to describe the CP kinetics for solids with heterogeneous populations of the source spins. The I-I*-S model(6,10) takes into account the efficiency of spin diffusion, which relies on homonuclear dipolar interactions and proceeds through flip-flop spin transitions. Within this model, it is assume that the I-S heteronuclear dipolar interactions are strong enough in comparison to the I-I homonuclear dipolar interactions. Thus, the I-I*-S model relies on the existence of different I populations, denoted I* for the I spins directly bound to an S spin under study and I for the rest of the I network. The CP proceeds in two steps. A fast rise of the intensity is observed initially due to the transfer of the magnetisation to a dilute spin (I*-S) by the abundant spins in close proximity followed by a slow rise of the intensity or damped oscillation. For long contact times, a decay of magnetisation or a plateau is observed.
Several equations have been proposed to describe the CP kinetics, the simplest is shown below (equation 2).(6)
(2)
where T1ρI is the I spin lattice relaxation time in the rotating frame; Tdf is the 1H spin-diffusion time constant describing the strength of the homonuclear dipolar interactions and the homogeneity of the I spin pool; λ is defined by the number n of I spins attached to the S spin under study (); T2 is the spin-spin relaxation time. Although the parameter T2 needs to be fitted, due to its low value it has very little impact on the quality of the fitting.(6)
Ultimately, the applicability of the two models is subject to the homogeneity of the I spin population and depends on whether (I-S model) or not (I-I*-S model) the spin-diffusion is faster than the polarisation transfer from directly bonded I spins.
Table ESI-3. CP kinetics parameters and T1rH-VSL times of PLGA 5050_1 in F-Arg2.5.
Assignment / CP kinetics / VSL-T1rHI .107 (a.u) / l=1/(n+1) / T1rH (ms) / TIS (ms) / T2 (ms) / Tdf (ms) / R² / T1rH (ms) / R²
C=O Lac. / 2.94 / - / 5.55 / 0.95 / - / - / 0.997 / 8.8 / 1.00
C=O Gly. / 2.55 / - / 5.19 / 0.81 / - / - / 0.998 / 8.5 / 0.999
CH / 3.97 / 0.39 / 4.46 / - / 0.015 / 1.10 / 0.993 / 8.2 / 0.999
CH2a / 2.03 / - / 4.03 / 0.02 / - / 0.994 / 8.2 / 0.998
2.04 / 0.33 / 4.03 / - / 0.028 / 0.03 / 0.994
CH3a / 6.99 / - / 8.83 / 0.16 / - / - / 0.992 / 8.2 / 1.00
7.41 / 0.65 / 8.28 / - / 0.059 / 0.30 / 0.997
a for these groups, both models lead to a satisfactory fitting of the data set. l was fixed to its expected value for the I-I*-S fitting of the CH2 glycolide in both formulations
Table ESI-4. CP kinetics parameters and T1rH-VSL times of PLGA 5050_1 in F-Arg5.
Assignment / CP kinetics / VSL-T1rHI .108 (a.u) / l=1/(n+1) / T1rH (ms) / TIS (ms) / T2 (ms) / Tdf (ms) / R² / T1rH (ms) / R²
C=O Lac. / 3.30 / - / 9.97 / 0.88 / - / - / 0.998 / 14.9 / 0.998
C=O Gly. / 2.96 / - / 9.24 / 0.79 / - / - / 0.998 / 14.5 / 0.999
CH / 3.59 / 0.28 / 8.28 / - / 0.014 / 0.86 / 0.993 / 13.6 / 0.998
CH2a / 2.45 / - / 7.25 / 0.02 / - / - / 0.997 / 14.1 / 0.997
2.45 / 0.33 / 7.24 / - / 0.028 / 0.03 / 0.997
CH3 / 6.60 / 0.52 / 8.60 / - / 0.070 / 0.33 / 0.996 / 13.4 / 1.00
a for these groups, both models lead to a satisfactory fitting of the data set. l was fixed to its expected value for the I-I*-S fitting of the CH2 glycolide in both formulations
The difference in T1rH times measured using VSL the CP-VCT experiments has been reported for PVC, PMMA and PVA.(11) In CP-VCT experiments, the transfer of magnetisation occurs directly after the 1H p/2 pulse, the 1H magnetisation does not equilibrate before CP. In the case of independent T1rH measurements during the spin-lock field the 1H magnetisation evolves and equilibrates over a larger distance leading to a more homogeneous transfer of magnetisation during CP.
Table ESI-5. T1rH times of the pure L-arginine and of arginine in its PLGA formulations.
Ppm / T1rH (ms) / R² / T1rH (ms) / R² / T1rH (ms) / R²
158.7 / 19.7 / 0.979 / - / - / 6.81 / 0.959
56.3 / 22.3 / 0.977 / 2.68 / 0.960 / 8.79 / 0.889
43.1 / 22.2 / 0.980 / - / - / 10.2 / 0.940
24.8 / 22.6 / 0.984 / - / - / 8.01 / 0.734
Table ESI-6. Variable temperature T1rH times (in ms) of the PLGA 5050_1 film.
303 K / 313 K / 323 K / 333 KC=O Lac. / 26.9 ± 1.5 / 17.8 ± 0.7 / 13.1 ± 0.5 / 5.5 ± 0.9
C=O Gly. / 27.5 ± 2.4 / 18.9 ± 1.3 / 11.2 ± 0.4 / 5.2 ± 0.5
CH / 27.2 ± 1.2 / 16.8 ± 0.5 / 11.4 ± 0.3 / 5.0 ± 0.3
CH2 / 22.7 ± 1.1 / 18.5 ± 0.9 / 10.6 ± 0.4 / 4.2 ± 0.4
CH3 / 25.9 ± 1.4 / 18.1 ± 0.3 / 11.0 ± 0.2 / 5.8 ± 0.1
Table ESI-7. Variable temperature T1rH times (in ms) of PLGA in F-Arg2.5.
303 K / 313 K / 323 K / 333 KC=O Lac. / 4.4 ± 0.2 / 2.1 ± 0.3 / 1.2 ± 0.5 / 1.4 ± 0.3
C=O Gly. / 4.3 ± 0.1 / 2.1 ± 0.3 / 1.0 ± 0.2 / 2.4 ± 0.8
CH / 3.8 ± 0.1 / 1.6 ± 0.1 / 2.8 ± 0.6 / 1.1 ± 0.2
CH2 / 3.7 ± 0.2 / 1.4 ± 0.3 / - / -
CH3 / 4.2 ± 0.1 / 2.3 ± 0.0 / 1.9 ± 0.0 / 2.1 ± 0.1
Table ESI-8. Variable temperature T1rH times (in ms) of PLGA in F-Arg5.
303 K / 313 K / 323 K / 333 KC=O Lac. / 14.9 ± 0.2 / 15.8 ± 1.2 / 11.6 ± 0.9 / 6.8 ± 0.5
C=O Gly. / 14.5 ± 0.2 / 15.5 ± 0.6 / 10.9 ± 0.7 / 6.9 ± 0.4
CH / 13.6 ± 0.2 / 16.8 ± 1.5 / 10.2 ± 0.9 / 6.9 ± 0.3
CH2 / 14.1 ± 0.3 / 14.7 ± 0.9 / 10.6 ± 1.0 / 6.4 ± 0.4
CH3 / 13.4 ± 0.1 / 14.0 ± 0.4 / 10.4 ± 0.3 / 6.5 ± 0.1
Table ESI-9. Variable temperature T1rH times (in ms) of PLGA in F-ARARAF6.8.
303 K / 313 K / 323 K / 333 KC=O Lac. / 6.0 ± 0.3 / 3.2 ± 0.3 / 0.9 ± 0.1 / -
C=O Gly. / 6.3 ± 0.2 / 2.7 ± 0.3 / 1.4 ± 0.1 / -
CH / 6.2 ± 0.1 / 2.6 ± 0.3 / - / -
CH2 / 6.1 ± 0.4 / - / - / -
CH3 / 5.9 ± 0.0 / 2.8 ± 0.0 / 1.8 ± 0.1 / 1.3 ± 0.1
Table ESI-10. Variable temperature T1rH times (in ms) of PLGA in F-ARARAF13.5.
303 K / 313 K / 323 K / 333 KC=O Lac. / 12.8 ± 0.7 / 5.6 ± 0.1 / 2.9 ± 0.3 / -
C=O Gly. / 13.2 ± 0.6 / 5.7 ± 0.3 / 2.7 ± 0.2 / 2.4 ± 0.5
CH / 13.4 ± 0.5 / 5.4 ± 0.2 / 2.3 ± 0.2 / -
CH2 / 12.8 ± 0.4 / 5.2 ± 0.4 / 2.7 ± 0.9 / -
CH3 / 12.7 ± 0.2 / 5.7 ± 0.0 / 2.7 ± 0.0 / 1.8 ± 0.1
References
1. Siemer AB, Ritter C, Steinmetz MO, Ernst M, Riek R, Meier BH 2006. C-13, N-15 resonance assignment of parts of the HET-s prion protein in its amyloid form. J Biomol NMR 34(2):75-87.
2. Igumenova TI, McDermott AE, Zilm KW, Martin RW, Paulson EK, Wand AJ 2004. Assignments of carbon NMR resonances for microcrystalline ubiquitin. J Am Chem Soc 126(21):6720-6727.
3. Pauli J, Baldus M, van Rossum B, de Groot H, Oschkinat H 2001. Backbone and side-chain C-13 and N-15 signal assignments of the alpha-spectrin SH3 domain by magic angle spinning solid-state NMR at 17.6 tesla. Chembiochem 2(4):272-281.