Polymorphic Transformation of Drugs Induced by Glycopolymeric

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Polymorphic Transformation of Drugs Induced by Glycopolymeric Vesicles Designed for Anti-Cancer Therapy Probed by Solid-State NMR Spectroscopy Eliska Prochazkova, Cheng Cao, Aditya Rawal, Martin Dracinsky, Saroj Bhattacharyya, Ivana Cisarova, James M. Hook, and Martina H. Stenzel ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b05514 • Publication Date (Web): 10 Jul 2019 Downloaded from pubs.acs.org on July 19, 2019

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ACS Applied Materials & Interfaces

Polymorphic Transformation of Drugs Induced by Glycopolymeric Vesicles Designed for AntiCancer Therapy Probed by Solid-State NMR Spectroscopy

Eliška Procházková,*,a Cheng Cao,b Aditya Rawal,c Martin Dračínský,a Saroj Bhattacharyya,c Ivana Císařová,e James M. Hook,*,c,d and Martina H. Stenzel*,b

a Institute

of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague 166 10, Czech Republic [email protected]

b Centre

for Advanced Macromolecular Design, School of Chemistry, The University of New South Wales, Sydney 2052, NSW, Australia [email protected]

c

Mark Wainwright Analytical Centre, and d School of Chemistry, The University of New South Wales, Sydney 2052, NSW, Australia [email protected] e

Faculty of Science, Charles University, Prague 128 43, Czech Republic

Abstract Understanding the nature of the drug-polymer interactions in micellar drug delivery systems and what happens with the drug and the polymer once the complex has formed is essential for the rational design of the polymeric matrices suitable for a particular drug. In this work, glycopolymeric vesicles - a block copolymer, poly(1-O-methacryloyl-β-D-fructopyranose)-b-poly(methyl methacrylate), PFru36- PMMA160), - designed to target tumor cells loaded with two drugs, ellipticine and curcumin, were characterized. Advanced solid-state NMR spectroscopy and single-crystal/powder XRD

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combined with CASTEP calculations shed light on the nature of the drug, the polymer and their interactions. While the low drug loading (ca. 5%) ensured that the structure, size and shape of the polymeric vesicles did not change significantly, the solid-state forms of the drugs changed markedly. Upon loading into the vesicles, ellipticine favored a highly-ordered form distinctly different from the bulk drug as indicated by calculated

13C

13C

solid-state NMR spectroscopy. Detailed analysis of the CASTEP

spectra derived from crystallographic data based on the lowest mean absolute error

showed the best match with Form I. Moreover, ellipticine before loading was found as a new polymorph and was described by single-crystal XRD as a new orthorhombic Form III. Likewise, curcumin, originally present in monoclinic Form I was found to recrystallize as metastable orthorhombic Form II inside the vesicles. Intermolecular interactions between the polymeric vesicles and the drugs, ellipticine as well as curcumin, were detected using 2D 1H MAS experiments indicating that the drugs are localized in the hydrophobic layer of the vesicles.

Keywords: CASTEP Calculations, Drug Delivery, Ellipticine, Glycopolymeric Vesicles, Polymorphism, Solid-State NMR, XRD

Introduction Current research in the field of drug delivery systems involves the redeployment of known drugs as composites with macromolecular carriers such as polymeric vesicles,1-2 which may then serve as an active scaffold for the drug, thereby increasing their permeability, transport, retention, stability and targeting of specific cells (e.g. tumor cells) or tissues.3-4 Polymeric vesicles are widely favored due to their tunable size, shape, surface functionalization, high drug loading efficiency and long life-time circulating properties.5 However, it is still not fully understood, how much the polymer and the drug influence the behavior of each other, which, consequently, dictates the overall performance of the polymer-drug conjugate. In recent studies on glycopolymeric vesicles,6-7 it has been shown that the amount of drug can affect the morphology or the packing of the polymer chains, water content and mobility of the functional groups on the surface, which are exposed to solvent.8 In other studies, it was shown that the polymer matrix can provide a medium in which the drug can recrystallize in different polymorphic forms.9-11 Chemically diverse polymers are able to induce the growth of a wide range of polymorphs


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crystallizing in various space groups including metastable forms,12 which are preferred when the molecular mobility of the drug is sufficiently high.13 Therefore, the final behavior of the polymer-drug composite is critically dependent on both components and their interactions,14 such as hydrogen bonding,15 and should be considered when studying new polymer/drug system. It is known that structural changes of the drug can lead to significant modulation of solubility, stability or non-covalent interactions within the polymer matrix, and may significantly affect biological properties such as cellular uptake or drug release.16 It is therefore reasonable to believe that morphology changes of the drug will affect the interaction with the drug carrier and ultimately the behavior of the polymeric nanoparticle. However, it is quite challenging to investigate how the drug loading affects the structure and physicochemical properties of both the polymer and the drug, their interaction and where the drug is localized within the vesicles. Commonly used methods including transmission electron microscopy (TEM), dynamic light scattering (DLS), small angle X-ray scattering (SAXS) or small-angle neutron scattering (SANS) allow the average size and shape of the vesicles to be determined. SAXS and SANS can provide additional information such as the location of the drugs in a micelle.7,

17

The polymer/drug interaction can be

probed by thermal analysis (calorimetry),18 but no clear structural information is obtained. Finally, powder X-ray diffraction (PXRD) is able to provide some structural information, however, due to the poor crystallinity of the polymeric vesicles and/or low concentration of the drug inside the vesicles, it can be quite challenging to obtain high-quality PXRD patterns, even if the drug is in a crystalline form. Limitations to diffraction methods can be readily overcome using solid-state NMR spectroscopy (SS-NMR). The NMR parameters such as chemical shift or dipolar couplings are very sensitive to changes in molecular geometry and chemical environment (e.g. protonation, solvation), which makes SS-NMR an excellent tool for probing the structure, properties of both, the drug and the polymer, as well as their interaction or changes in molecular dynamics.19-23 Furthermore, the NMR measurements can be used to assess the quality of the starting homopolymers, the polymeric vesicles formed before and after the drug loading, the purity and form of the drug inside the polymeric vesicles. In this work, we report the detailed structural study of glycopolymeric vesicles used as a nanocarrier for targeting tumor cells24-25 with a payload of bioactive drugs: 1) the DNA-intercalator, ellipticine26 and 2) the potent anti-oxidant, curcumin27 (Figure 1). We provide clear spectroscopic evidence that the polymer matrix induces significant changes in the polymorphic forms of both drugs



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and there are interactions with the polymer indicating that the drugs are located in the hydrophobic PMMA layer.

Figure 1. Schematic of the self-assembly of glycopolymeric vesicles formed from the block copolymer: the hydrophobic polymethyl methacrylate (PMMA, orange) and the hydrophilic polyacryly-fructose (PFru, blue); structure of the drugs, ellipticine and curcumin located inside the PMMA block. The parameters of the shell are provided on the basis of the previous work.7

Results and Discussion Nature of the Glycopolymeric Vesicles The PMMA160-PFru36 block copolymer forms vesicles with high monodispersity and average diameter size of ~150 nm as found by TEM (Figure S1 in the SI) and confirmed by DLS (Table 1). The DLS results are typically larger than the TEM results due to the dehydrated state of the TEM sample. TEM revealed a vesicular morphology, which is not influenced by drug loading and PXRD confirmed amorphous structure of the vesicles (Figure S2 in the SI). The

13C

CP-MAS spectrum (Figure 2A)

showed a group of high-intensity aliphatic signals corresponding to the CH3 of the hydrophobic polymer backbone C ~15 ppm; the sharp signal at C 45 and 50 ppm corresponding to the PMMA, quaternary carbons and methoxy groups, respectively. The carbonyls from both blocks resonate at C ~180 ppm. Finally, the spectral window around C ~ 60–100 ppm belongs to the fructose carbons from the PFru block. While fructose exists in solution as an equilibrium of several forms such as furanose/pyranose and α/β anomers,28 crystalline fructose displays one set of sharp


13C

signals indicating a single

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dominant form crystallographically identified as β-D-fructopyranose, which has been found as the major form in aqueous solution

29

(Figure 2B). Interestingly, inside the vesicles, the minor fructose

forms can also be detected as broad signals at C ~80 ppm in the

13C

CP-MAS spectra, in addition to

the peaks in the anomeric carbon region C ~100 ppm (Figure 2B, pink frames). Successful incorporation of the fructose moiety into the polymer chain was confirmed by heteronuclear 2D correlation (HETCOR), where

13C-1H

13C-1H

spin pairs close in space can be observed through dipolar

coupling. An interaction between fructose moiety (C ~ 60–100 ppm) and the backbone protons (H ~ 1.5 ppm) is evident (Figure 2C, pink frame). Although the 1H signals of fructose and OCH3 from the PMMA unit overlap in the 1D 1H spectrum (4.2 ppm), based on 13C-1H HETCOR spectrum (Figure 2C, black frame), we can estimate that the chemical shift of OCH3 groups from PMMA moiety is lower than that in PFru part. Moreover, PMMA is expected to give arise of sharp high-intensity signal in contrast to the PFru moiety, where several forms are present, as discussed above. The local structure of the polymer was probed further with homonuclear 1H-1H doublequantum (DQ) single-quantum (SQ) MAS correlation, where the spin magnetization evolves under double-quantum coherence generated by dipole-dipole interaction between protons close in space (typical distance 2–4 Å).30 The DQ-SQ MAS spectrum of the polymer (Figure 2D) exhibits a strong autocorrelation cross-peak at H ~ 4.2 ppm (SQ) corresponding to the methoxy groups of PMMA, which interact with each other. The autocorrelation cross-peak at H ~ 1.5 ppm (SQ) indicates the interaction between backbone protons. Furthermore, there is a pair of cross-peaks at δSQ=4.2 and 1.5 ppm showing a clear interaction between methoxy group and backbone in the PMMA block.



Figure 2. (A)

13C

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CP-MAS spectrum (MAS 6.5 kHz) of the glycopolymeric vesicles with signal assignment. (B)

Fructose NMR window of the vesicles compared to the fructose in aqueous solution and in solid state (MAS 10 kHz), confirming also the presence of minor fructose forms inside the vesicles (pink frames). (C)

13C-1H

HETCOR (MAS 8 kHz) showing fructose-backbone interaction. (D) Homonuclear 1H DQ-SQ MAS spectrum (MAS 30 kHz) indicating the side chain-backbone interaction in the PMMA block.

In order to explore the domain size of these glycopolymeric vesicles, a series of 1H-1H SQ/SQ experiments was acquired with varying mixing times.19 This series enabled the estimation of the equilibration spin diffusion time in this system, which is dependent on the inter-proton distance, a function of the domain size. The equilibration time of the PMMA-PFru vesicles was found to be ~20 ms (Table 1 and Figure S3 in the SI), which fits in magnitude the previously published value (~30 ms) of a similar polymeric system, which included polymethylacrylic acid, PMAA, instead of the methyl ester, PMMA, studied in this work.8


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Table 1. Characteristics of the glycopolymeric vesicles before and after the drug loading. Drug loading

Drug loading

Vesicles

Spin diffusion Dh / nm (DLS)

T2 / ms

capacity / %

efficiency / %

equilibration / ms

PMMA-PFru

0

0

185.8  2.9

20

0.41  0.01

PMMA-PFru-ELP

6.8

27.2

182.1  9.7

30

0.59  0.01

PMMA-PFru-CCM

5.9

23.6

160.5  1.8

30

0.45  0.01

Glycopolymeric vesicles loaded with the drugs The physical size of the vesicles is readily confirmed by TEM images, ~ 150 nm (Figure S1 in the SI) and agreed well with DLS results (Table 1), which changed little after drug loading, or after additional processing by freeze-drying, and reconstitution in water. The domain size of the drug-loaded vesicles was estimated from a series of SQ-SQ experiments.19 The spin diffusion time in both types of loaded vesicles (with ellipticine denoted as PMMA-PFru-ELP as well as with curcumin denoted as PMMA-PFru-CCM) was close to 30 ms (Table 1, Figures S4 and S5 in the SI). This increase in equilibration time indicates that upon drug loading the hydrophobic domains of the vesicles increased in size. Moreover, the broad aromatic signal corresponding to drugs did not equilibrate with the micellar signal even at 500 ms, which indicates that a very small fraction of the drug molecules forms aggregates, not included into the vesicles. Molecular dynamics of the vesicles was estimated by comparing T2 relaxation time extracted from a series of spin-echo experiments varying in the number of cycles. The magnetization decay (of the signal at 4.4 ppm (of PMMA methoxy group in all cases) was fitted by mono-exponential function (Figure S6 in the SI) and the T2 value was extracted. As shown in Table 1, T2 of the parent vesicles is only slightly lower (0.4 ms) than T2 values of the drug-loaded vesicles (0.6 ms for ELP-loaded and 0.5 ms for CCM-loaded) indicating that the drugs do not alter the flexibility of the vesicles significantly. The

13C

chemical shifts of the polymeric vesicles did not change markedly after the drug

loading (Figure 3) indicating that neither drug significantly affects the chemical environment of the copolymer, which can be readily ascribed to the low drug loading (ca. 5%, Table 1). The

13C

CP-MAS

spectra of the copolymer reveal an optimal spectral window between C ~ 100–160 ppm, suitable for detecting both aromatic drugs (ellipticine and curcumin), where no signals from the glycopolymeric vesicles appear (Figure 3, in frame). Although the drug loading is low, the presence of the drugs can be easily detected by their sharp NMR signals indicating their crystalline form inside the vesicles. 7 ACS Paragon Plus Environment


Surprisingly,

13C

CP-MAS spectra of the drugs before and after the loading differed significantly,

indicating that the solid-state structure of the drugs had changed.

Figure 3.

13C

CP-MAS spectra (MAS 6.5 kHz in all cases) of the polymeric vesicles before (PMMA-PFru) and

after loading with ellipticine (PMMA-PFru-ELP) and with curcumin (PMMA-PFru-CCM). To show that the polymorphic structure of the drugs changed after loading,

13C

CP-MAS spectra of the bulk drugs are included as

separated traces.

In order to probe the drug localization in the glycopolymeric particles, we employed fluorescence lifetime measurements for both types of particles (loaded with ellipticine as well as curcumin). The fluorescence lifetime can provide the key information about the environment of the drugs due to the non-radiative decay.31 For these measurements, we used the same nanoparticles as used for the NMR studies, but re-dispersed in water for this analysis. The decay plots (Figure 4) and the obtained average lifetime AV (Table 2) revealed similarity between the drugs in the nanoparticles and the one in methyl methacrylate (MMA), which represents the polarity of the PMMA core. This indicates that the drugs are located in the PMMA layer. In contrast, the life-time of the drugs in hydrophilic environments such as water is significantly different, suggesting that the drugs have not leached into the hydrophilic shell or water.


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Figure 4. Fluorescence lifetime decay plot of (A) ellipticine loaded into nanoparticles (PMMA-PFru-ELP), dissolved in water and dissolved in MMA; (B) curcumin loaded into nanoparticles (PMMA-PFru-CCM), dissolved in water and dissolved in MMA.

Table 2. The average lifetime of the drugs loaded in the nanoparticles and MMA, sample details can be found in Table 1. Sample

1

2



AV



PMMA-PFru-CCM

0.59

1.30

0.28

0.49

1.44

CCM in MMA

1.10

0.386

-

0.679

1.05

PMMA-PFru-ELP

6.66

19.22

67.88

21.77

1.05

ELP in MMA

74.91

6.42

19.53

21.03

1.07

Ellipticine-Loaded Glycopolymeric Vesicles In order to describe the polymorphic transformation of ellipticine induced by the glycopolymeric vesicles, the polymorphic form of the drug before and after the loading had to be determined. Ellipticine is a rigid planar molecule with known two polymorphs: Form I adopts the monoclinic space group P21/c and Form II has the orthorhombic space group Pbca.32-33 However, the PXRD pattern of ellipticine used as the starting material in this study was different from the PXRD patterns simulated from the reported crystal structures (Forms I and II)32-33 as shown in Figure 4A. Unfortunately, no single-crystal (SC) XRD could be recorded on this sample due to the small crystallite size. To support the XRD data, we performed CASTEP calculations to extract NMR parameters from the two reported crystal structures, Forms I and II. The experimental

13C

CP-MAS spectrum of our sample was not in



agreement with the calculated

13C

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spectra of the reported forms (Figure 4B) supporting the discovery

of new polymorph of ellipticine, as proposed by PXRD. More details including NMR signal assignment (Figure S7 and S8) are in Section 3 in the SI. To validate our findings, a second batch of ellipticine was acquired from a different source, which displayed higher crystallinity visible to the eye. Indeed, its

13C

CP-MAS spectra were identical

with the first batch (Figure S9 in the SI), except for requiring a longer relaxation delay (d1 = 150 s instead of 15 s used in the first batch) in the CP-MAS data acquisition. The larger crystals enabled SCXRD to be collected and after careful analysis the crystal was found to possess the orthorhombic space group, Pna21, with cell unit parameters: a = 18.7736 (14) Å, b = 3.9630 (3) Å, c = 16.317 (1) Å , V = 1213.98 (15) Å3, which is a new polymorph of ellipticine, Form III (Tables S1 and S2 in the SI). Its structure is consigned to the crystallographic database (CCDC) with reference number 1902766. This crystal structure then served as an input file for simulation of its PXRD pattern and further Rietveld analysis of the PXRD data. Cell parameters calculated from the PXRD measured at room temperature closely matched those of the CIF measured at 120 K indicating this crystal structure is also present at room temperature (Figure 5A). The crystal structure was also used for prediction of the 13C

SS-NMR spectrum using CASTEP. Although at first sight the calculated

does not match perfectly with the experimental

13C

13C

spectrum of Form III

CP-MAS spectrum (Figure 5B), detailed analysis

revealed the best match of the experimental data with the calculated NMR parameters of Form III with lowest mean absolute error (MAE) of 1.08 as shown in Table S3 and Figure S10 in the SI. Similarly, experimental 1H MAS spectrum is in a good agreement with the calculated spectrum of Form III (Figure S11 in the SI). 1H

and 2D spectra such as

13C-1H

HETCOR or 1H-1H DQ-SQ MAS of ellipticine Form III

confirmed the interactions observed in the crystal structure, such as N-H…N intermolecular hydrogen bonding (Figure 5C and D) with an intermolecular N…N distance of 2.9 Å. The 1H chemical shift of NH at 12.7 ppm indicates strong intermolecular hydrogen bonding. The HETCOR cross-peaks, such as C1-NH or C7-H1 suggest geometry of two ellipticine molecules depicted in Figure 5C, which is supported by the DQ-SQ MAS cross-peak pairs (Figure 5D), such as NH-H1 or H1-H7. Furthermore, there is an autocorrelation cross-peak on the diagonal of DQ-SQ MAS indicating methyl-methyl intermolecular interaction between ellipticine layers (˂ 4 Å). Due to this well-defined hydrogen bonding and π-π stacking, the solid structure is very rigid.


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Figure 5. (A) PXRD patterns and (B)

13C

CP-MAS spectra (MAS 6.5 kHz) of bulk ellipticine used in this study (in

green) compared to the data of the reported crystal structures, Forms I and II (in grey) and extracted from the obtained crystal structure of the Form III (in black). (C) HETCOR (MAS 6.5 kHz) and (D) DQ-SQ MAS spectrum (MAS 28 kHz) of ellipticine in the new Form III indicating intermolecular N-H…N hydrogen bonding.

Ellipticine loaded inside the vesicles displayed one set of sharp 13C signals indicating a highlyordered structure, seemingly distinct from Forms I, II or III (Figure S12 in the SI). The sharp 13C signals in combination with NQS (Figure S13 in the SI) and

13C-1H

HETCOR (Figure S14 in the SI) enabled

signal assignment (Table S3 in the SI). Encouraged by the results for Form III discussed above, we calculated mean absolute errors of the correlations between the experimentally obtained chemical shifts of ellipticine inside the vesicles and the calculated values for all the three forms I–III. Surprisingly, we found the lowest error when compared with Form I (MAE = 1.05, Table S3 in the SI). Moreover, the error is quite close to the lowest error determined for Form III (MAE = 1.08) as discussed above. The similar values for error might lead to conclusion that ellipticine may be present inside the vesicles as Form I. Unfortunately, PXRD of the ellipticine-loaded vesicles did not provide any signal in the corresponding 2θ positions of ellipticine (Figure S15 in the SI), suggesting that the drug crystallites of the drug are below the limit of detection of the PXRD instrument used.



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The effect of the polymer environment on the dynamics of ellipticine, could be deduced by comparing the T2 values extracted from the NH signal of ellipticine (H ~ 13 ppm) in a series of spin echo measurements using mono-exponential fitting (Figure S16 in the SI). While T2 relaxation time of ellipticine inside the vesicles was 1.2 ms, the pure ellipticine relaxes faster at 0.8 ms. This result suggests some degree of increased dynamics in the drug molecules when they are in the polymer matrix.

Ellipticine-vesicles interaction The most straightforward NMR experiment, which can reveal polymer-drug interaction, is 1H

13C-

HETCOR. However, even at higher contact time of 1.5 ms, no polymer-drug cross-peaks were

observed. Unlike in a previous study, where a platinum-based drug was covalently bound to the PMAA polymer inside the vesicles,8 ellipticine loading relies on weaker non-covalent hydrophobic interactions, where both components are not as close in space as necessary for 13C-1H interaction. The homonuclear 1H-1H DQ-SQ MAS spectrum is potentially more sensitive for probing intermolecular polymer-drug interactions than HETCOR, which has to rely on the ~1.1% natural abundance of

13C

nuclei. On the other hand, the 1H spectra suffer from lower resolution leading to a

strong signal overlap, which can complicate the signal assignment. Nevertheless, clear experimental evidence that ellipticine interacts with the polymer was confirmed by the detection of a cross-peak pair at δSQ = 8.8 (ellipticine, H-1) and 4.4 (polymer) ppm (Figure 6A). The cross-peak pair represented as a 1D slice was compared to the 1H spectrum of ellipticine itself, to that of ellipticine-loaded vesicles and of the polymer itself (Figure 6B): H-1 of ellipticine has an intermolecular interaction with the polymer (dashed line). Although there may be some ambiguity with the fructose and methoxy 1H signals overlapping, the interaction is assigned on the basis that the methoxy groups of PMMA unit are expected to provide a sharp 1H signal unlike the fructose moiety yielding broad signals due to the presence of several fructose forms as shown in the 13C CP-MAS spectrum (Figure 2B). The interaction with the PMMA methoxy group leads to the conclusion that ellipticine is located in the hydrophobic layer.


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Figure 6. (A) 1H,1H-DQ-SQ MAS spectrum (MAS 30 kHz) of PMMA-PFru-ELP polymeric vesicles representing intermolecular polymer-drug interaction. (B) Comparison of 1D slice extracted from the DQ-SQ MAS spectrum with 1H spectra of ellipticine, the vesicles and the polymer (PMMA-PFru) itself.

To be sure that no drug decomposition during the loading procedure or in the polymer occurred, NMR spectra of the polymeric vesicles loaded with ellipticine were dissolved and measured in DMSOd6 (Figure S17 in the SI). As expected, there is no spectral difference in 1H spectra of the drugs themselves and the drug released from the polymeric vesicles.

Curcumin-Loaded Glycopolymeric Vesicles Curcumin is decidedly less rigid than ellipticine, and several polymorphic forms have been described, differing in the orientation of the phenyl rings with respect to the carbonyls, and to each other.34-40 Furthermore, as expected for a β-diketone, several keto/enol tautomeric forms have been obtained. Commercially available curcumin usually contains curcuminoids as impurities41 (Figure S18 in the SI). Therefore, it was purified chromatographically prior to the spectroscopic measurements (Figure S19 in the SI). By comparison of the PXRD pattern and

13C

CP-MAS spectrum with the

reported data,42-43 the purified curcumin can be unambiguously assigned to Form I (Figures S20 and 7A, respectively). It is the thermodynamically more stable form42 and crystallizes in a monoclinic system with a single crystallographically distinct molecule in the asymmetric unit, clearly observed as one set of sharp signals in its

13C

CP-MAS spectrum (Figure S21 in the SI). Curcumin is present in an

enol tautomer in this form, which is clearly visible in 1H spectrum (Figure 7B). The OH group is involved in intramolecular hydrogen bond with the neighboring carbonyl oxygen atom of the enol and resonates at δH 16.8 ppm, even more deshielded than in solution (δH = 16.4 ppm, Figure S22 in the SI), indicating an unusually strong intramolecular hydrogen bond classified as a resonance-assisted 13 ACS Paragon Plus Environment


hydrogen bond.44 In

13C,1H-HETCOR,

the interaction between 2’-OH and C7 (or C7’) indicating

intermolecular O-H…O hydrogen bonding was found (Figure S23 in the SI), and in 1H-1H DQ/SQ MAS spectrum, where an OH-CH3 interaction was revealed (Figure S24 in the SI), as described recently in a detailed solid-state NMR study.44 After loading into the vesicles, curcumin also recrystallized, but, interestingly, as a metastable Form II recognized by comparison of its experimental

13C

CP-MAS and 1H MAS spectra with the

published NMR data (Figures 7A and B).43 As with ellipticine, this structural change was unexpected but clearly detected by SS-NMR; there are two non-equivalent molecules in Form II leading to the two sets of NMR signals, which was corroborated by the 1H MAS spectrum. While curcumin in Form I exhibits one signal at δH 16.8 ppm, curcumin inside the vesicles provided two signals with lower chemical shifts of δH 16.2 and 14.9 ppm corresponding to two non-equivalent OH groups participating in weaker intramolecular hydrogen bonding in Form II (Figure 7B, OH region in the yellow frame). The chemical shift of the OH signals in both samples is confirmed by 1H,1H-SQ-SQ MAS spectrum, where one diagonal cross-peak belonging to Form I and two diagonal cross-peaks from the Form II can be detected (Figure S25 in the SI). The presence of Form II inside the vesicles was also deduced from PXRD pattern of the PMMA-PFru-CCM polymeric vesicles, although the PXRD pattern of the encapsulated curcumin was weak and strongly affected by scatter from the polymeric matrix. However, three peaks at diffraction angles 14°, 25.5°and 26.5° in 2θ were observed after baselinecorrection that confirmed that Form II (BINMEQ06, Figure 7C) is present.42 Curcumin most probably forms crystalline aggregates as proposed by negligible spin diffusion equilibration of curcumin signals in 1H-1H SQ-SQ correlation mentioned above (Figure S4 in the SI). To assure that the polymorphic transformation is really caused by the polymer, curcumin was dissolved in THF and then water was slowly added, as during the drug loading. However, no solid material precipitated indicating that the glycopolymeric vesicles create a unique environment inducing crystallization of curcumin in Form II.1HT2 values extracted from the mono-exponential fitting of the aromatic signal intensity decay in a series of spin-echo experiments (Figure S26 in the SI) are 1.0 ms and 1.6 ms for the neat curcumin and curcumin inside the vesicles, respectively. The reduced transverse relaxation rate for curcumin inside the vesicles indicates increased dynamics of the drug molecules when they are inside the polymeric matrix.


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Curcumin-vesicles interaction In order to investigate the curcumin-micelle interaction, the 1H DQ-SQ MAS spectrum was recorded and the cross-peak couple at δSQ = 8.4 ppm (curcumin) and 4.4 ppm (polymer) (Figure 7D) confirmed the intimate contact between both components (˂ 4 Å). Comparison of the 1D slice extracted from the DQ-SQ correlation at δDQ=12.2 ppm with the 1H MAS spectra of curcumin itself (Form I), curcumin polymeric vesicles and the polymer itself (Figure S27 in the SI) indicates the interaction with the methoxy groups of PMMA, the same as found in the case of ellipticine-loaded vesicles. The polymer-drug interactions were found as cross-peaks in the HETCOR spectrum with higher contact time (5 ms, Figure S28 in the SI). The cross-peak from curcumin to the PMMA is significantly stronger than the one to the PFru moiety, which may indicate that the drug is located in PMMA.

Figure 7. (A)

13C

CP-MAS (MAS 6.5 kHz) and (B) 1H-MAS spectra (MAS 26.5 kHz) of bulk curcumin and

curcumin inside the glycopolymeric vesicles PMMA-PFru-CCM compared to the NMR spectra of reported Forms I and II (in grey).43 (C) PXRD pattern of the curcumin-loaded vesicles (with baseline correction) compared to the simulated pattern of reported Form II (BINMEQ06). (D) 1H DQ-SQ MAS spectrum (MAS 28 kHz) of the vesicles,



where the intermolecular curcumin-polymer interaction was detected. The spectra from the literature were reproduced with permission from reference 43. Copyright 2019 The American Chemical Society.

To be sure that no drug decomposition during the loading procedure or in the polymer occurred, NMR spectra of the polymeric vesicles loaded with pure curcumin were dissolved and measured in DMSO-d6 (Figure S29 in the SI). As expected, there is no spectral difference in 1H spectra of the drugs themselves and the drug released from the polymeric vesicles.

Glycopolymeric vesicles loaded with crude curcumin In polymer chemistry/material science, crude curcumin (as received), has often been used without further purification and contains ca. 25% of structurally related impurities, curcuminoids desmethoxycurcumin and bis-desmethoxycurcumin.42, 45 These curcuminoids provide two new sets of NMR signals with chemical shifts close to those of curcumin itself (Figure S18 in the SI). The situation is even more complicated when the crude curcumin is loaded into the glycopolymeric vesicles. No signals were detected in PXRD pattern indicating an amorphous structure or an insufficient concentration of the drug due to these impurities (Figure S30 in the SI). The 13C CP-MAS signals were broadened significantly, which complicates signal assignment and determination of the polymorphic form (Figure S31 in the SI). However, in 1H MAS spectrum of these vesicles, one broader OH signal at δH 17.2 ppm could be clearly distinguished indicating Form I as distinct from the vesicles loaded by pure curcumin, where curcumin occurs as Form II with two OH signals at δH 16.2 and 14.9 ppm (Figure S32 in the SI). Even with the curcuminoid impurities, the same curcumin-vesicles interaction was found in the PMMA-PFru-CCM polymeric vesicles as those found in vesicles loaded with the pure curcumin (Figure S33 in the SI). Nevertheless, the content and nature of impurities can alter after the loading and can further complicate structural investigation. Because of this ambiguity, we decided to probe purity of curcumin inside the vesicles by NMR spectroscopy in solution. Recording 1H NMR spectra of the vesicles dissolved in DMSO-d6 and integrating diagnostic singlet signals of H-1 resonating at δH ca. 6 ppm provided clear evidence that the impurities increased significantly in concentration (>50% instead of 25% found in crude curcumin), most probably due to the higher solubility of curcuminoids (Figure S34 in the SI). This dramatic increase in impurities needs to be taken into account in biological screening or studies based on optical spectroscopy such as drug release, because all the curcuminoids have similar UV/Vis spectra (419–425 nm).46 16 ACS Paragon Plus Environment

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Conclusions Investigation of the behavior of the glycopolymeric vesicles before and after loading with two anti-cancer drugs, ellipticine and curcumin, revealed important structural aspects of polymeric vesicles as the drug delivery package. Interestingly, both drugs were found to recrystallize in polymorphic forms different from their bulk starting material, under the influence of the glycopolymeric environment. In the process, we also discovered and described a new polymorph of ellipticine (Form III), crystallizing in the orthorhombic space group (Pna21) and provided the first solid-state NMR analysis of ellipticine. NMR chemical shifts of Forms I, II and III were calculated from their crystal structures using CASTEP and were compared to the experimental data of ellipticine before the loading and inside the vesicles. CASTEP calculations suggest that ellipticine inside the vesicles may be present as the Form I polymorph. Polymorphic transformation of curcumin was easier to monitor, and led to unambiguous assignment of the polymorph before (Form I) and after (Form II) loading. Finally, given the low loadings of the drugs (~ 5 %), no significant difference in behavior of the polymeric vesicles loaded by ellipticine or curcumin was detected. There was nevertheless, clear evidence of drugpolymer interactions indicating that both drugs are localized in the hydrophobic PMMA layer as also indicated by fluorescence lifetime measurements. This study clearly demonstrates the power of solid-state NMR analysis to probe in depth the behavior of glycopolymeric vesicles as an active scaffold to induce polymorphic transformation of the loaded drugs. This transformation can, in turn, significantly affect biological properties such as cellular uptake or drug release, which need to be taken into account in the rational design of nanocarriers designed for delivering a therapeutic effect.

Experimental Section Materials Ellipticine was commercially available in purity ˃ 99% from Sapphire Bioscience, Redfern NSW 2016, Australia. The second batch was purchased from Fluka, Czech Republic. Curcumin was purchased from Sigma Aldrich with declared purity of ca. 80%. The structure and purity of the drugs was assessed before use, with NMR spectroscopy as a DMSO-d6 solution (1H,

13C,

2D experiments,

details in the SI). Ellipticine was sufficiently pure as declared by the supplier, as deduced from the NMR spectra entirely consistent with literature47-48 and, therefore, used without further purification. On



the other hand, crude curcumin, as received, contained up to ca. 25% of structurally similar curcuminoids (Figure S19 in the SI). Therefore, it was purified by column chromatography on silica gel eluting with 2–4% MeOH/CHCl3 and was obtained as a yellow solid, used for loading into the vesicles. Crude curcumin was also loaded into the vesicles and used to probe their selectivity of the drug components.

Synthesis of Poly(1-O-MAFru)36 and Poly(1-O-MAFru)36-b-PMMA160 The monomer1-O-methacryloyl-2,3:4,5-di-O-isopropylidene-β-D-fructopyranosewas prepared using a technique already reported in literature.49 The polymerization was carried out in a Schlenk tube. The monomer 1-O-methacryloyl-2,3:4,5-di-O-isopropylidene-β-D-fructopyranose (900 mg, 2.74 mmol), the initiator AIBN (2 mg, 1.12 × 10-2 mmol) and the RAFT agent CPADB (17 mg, 0.06 mmol) were mixedin 1, 4-dioxane (2 mL). The tube was sealed and the solution wasdegassed using three freeze-pump-thaw cycles. The mixture was polymerized at 70 °C for 7 hours. The polymerization was stopped by placingthe Schlenk tube in ice water (conversion: 80 %). The polymer was isolated by pouring the mixture into a large excess of diethyl ether, which led to the precipitation of the viscous polymer. The product was dried under vacuum for 24 hours. Poly(1-O-MAFru)36, was chain extended with MMA to generate the block copolymer. The macroRAFT agent Poly(1-O-MAFru)36-b-PMMA160 (100 mg, 0.825 × 10-2 mmol) was mixed with MMA (240 mg, 2.40 mmol) and the initiator AIBN (0.2717 mg, 1.66 × 10-3 mmol) and dissolved in 1,4dioxane (1.5 mL) in a Schlenk vial. The tube was sealed, followed by degassing using three freezevacuum-thaw cycles. The mixture was polymerized at 70 °C for 18 hours. After stopping the polymerization by cooling the vial in ice water, the block copolymer was isolated by precipitation in excess n-hexane. The block copolymer was dried under vacuum for 24 hours. The block copolymer was deprotected under acidic conditions to remove the isopropylidene protecting groups. The polymer (80 mg) was stirred with trifluoroacetic acid /H2O (9:1 v/v, 1.59 mL) at ambient temperature for 30 minutes. The polymer was subsequently purified by dialysis against deionized water for two days (MWCO 3500).


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Drug loading The block copolymer (4 mg) and the drug curcumin (1 mg/mL) were dissolved in THF (0.2 mL). Once a clear solution is obtained, MQ water (1.8 mL) was added slowly to the polymer drug solution at room temperature using a syringe pump, which was set to a rate of 0.2 mL/h. Free drug and organic solvent were removed by dialysis against deionized water for 8 hours while replacing the deionized water three times. The same procedure was applied for loading with ellipticine (1 mg/mL). During the self-assembly, the stirring rate was maintained at 500 r/min. Similar morphologies (vesicles) were obtained since the temperature, stirring rate, solvent, polymer concentration, and water content were kept at values similar to the previous work.7 The concentration of the drugs loaded inside the vesicles was determined by UV-Vis spectroscopy. The concentration of the encapsulated curcumin and ellipticine were determined with a Cary 100 UV−vis spectrophotometer using the wavelength maximum of curcumin and ellipticine at 421 nm and 380 nm, respectively.

The drug loading efficiency (DLE) (%) was calculated using:

DLE(wt%) =

the amount of curcumin in polymersomes the amount of curcumin in feed

(Equation 1)

× 100%

The drug loading capacity (DLC) (%) was calculated using:

DLC(wt%) =

the amount of curcumin in polymersomes the amount of polymer

(Equation 2)

× 100%

Methods NMR Spectroscopy Solution state NMR experiments such as 1H,

13C

or 2D (COSY, HSQC, HMBC, ROESY,

standard Bruker pulse sequences) were acquired on a Bruker Avance III NMR spectrometer operating at 600 MHz for 1H (14 T) fitted with a 5 mm HCN TCI-cryo-probe. The samples were measured in DMSO-d6 solution at room temperature and referenced to TMS (δ=0) using the residual solvent signal (DMSO δH 2.5 ppm for 1H and δC 39.7 ppm for 13C). NMR data were used to verify the purity of starting materials, drugs and polymers, for signal assignments and for comparison with SS NMR spectra. Solid state NMR experiments were recorded on 700 MHz (16.4 T) and on 300 MHz (7.0 T) Bruker Avance III NMR spectrometers with probes for solid-state experiments using 2.5 mm or 4 mm zirconia rotors, allowing sample spinning speeds at the magic angle (MAS) of up to 30 kHz or 15 kHz, 19 ACS Paragon Plus Environment


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respectively. Using the 2.5 mm rotor, we set the MAS at 28–30 kHz depending on the sample. Using 4 mm rotor, we set the MAS at 6–9 kHz depending on the sample. A Jeol NMR spectrometer operating at 600 MHz fitted with a probe for solid-state experiments using 3.2 mm zirconia rotors, was employed with the MAS set to 18 kHz. Solid-state pulse sequences were adapted from Bruker library in Topspin 3.2–3.7 or Jeol/Delta. Cross-polarization (CP)50 experiments with MAS (CP-MAS) and with total suppression of spinning sidebands (TOSS)51 were performed to obtain isotropic values of chemical shift, which was useful especially for the quality control and deductions about the state of the drug, whether crystalline (sharp signals) or amorphous (broad signals). To assist with signal assignment, edited

13C

spectra containing only quaternary and methyl signals were obtained using

dipolar dephasing (also known as non-quaternary suppression, NQS) during the TOSS period.52 The heteronuclear 2D experiment, HETCOR,53 was used to reveal 1H-13C contacts and to assist in signal assignment. For HETCOR measurements, lghetfq pulse sequence (Bruker library), including homonuclear decoupling, was employed. The data were acquired by collecting 96 transients in the indirect dimension with increments of 68 s, 256 scans, and relaxation delay in the range of 3–15 s depending on the material. In both 1D and 2D

13C

experiments, Hartman-Hahn 1H to

13C

cross

polarization transfer was applied via 0.1–5 ms contact time. 1H-1H DQ/SQ MAS spectra were recorded at 700 MHz using the rotor synchronized BABA (BAck-to-BAck) recoupling pulse sequence54 measured with a 2.5 mm NMR probe with MAS spinning at 30 kHz. Homonuclear NOESY-type spin diffusion 1H-1H SQ-SQ MAS correlation (2D exchange)55 were also acquired at 700 MHz with a 1H spin diffusion times of 0.1–500 ms with the data sets comprising 222 transients in the indirect domain with increments of 36 s. The 1H SQ-SQ correlation was employed in order to make deductions about domain size. 1H spin echo experiments (Bruker pulse program sronec, from Schmidt-Rohr library) were performed to estimate 1H spin-spin relaxation T2. The

13C

spectra were referenced to glycine (δC

176 ppm for its carbonyl) and the 1H spectra were referenced to adamantane (δH 1.8 ppm).

Powder X-ray Diffraction (P-XRD) Powder XRD data were measured at room temperature by mounting the sample between two Kapton foils and measuring the flat spinning disc in transmission mode to avoid the strong preferred orientation observed when measuring in a reflection geometry. The data were collected using an Empyrean XRD (supplier: PANalytical, Netherlands) fitted with a copper X-ray source (Cu Kα1, λ =


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1.540598 Å) with focusing mirror in the incident beam and measuring the diffracted beam with a PIXcel 3D 1 × 1 area detector in 1D scanning mode from 5 to 50° 2θ. The PXRD patterns from the reported structures were simulated using Mercury program.

Single-Crystal X-ray Diffraction (SC-XRD) Crystallographic data were collected on Bruker D8 VENTURE Kappa Duo PHOTON100 by IμS micro-focus sealed tube CuKα (λ= 1.54178) at a temperature of 120(2) K. The structure was solved by direct methods (XT)56 and refined by full matrix least squares based on F2 (SHELXL2018)57. The hydrogen atoms on carbon were fixed into idealized positions (riding model) and assigned temperature factors either Hiso(H) = 1.2 Ueq(pivot atom) or Hiso(H) = 1.5 Ueq (pivot atom) for methyl moiety, the hydrogen atom in –N-H moiety was found on difference Fourier maps and refined under rigid body assumption with assigned temperature factors Hiso(H) = 1.2 Ueq(N(6)). Crystal data for CCDC 1902766 C17H14N2, Mr = 246.30; Orthorhombic, Pna21, (No 33), a = 18.7736 (14) Å, b = 3.9630 (3) Å, c = 16.317 (1) Å , V = 1213.98 (15) Å3, Z = 4, Dx = 1.348 Mg m-3, Plate, yellow of dimensions 0.37 × 0.27 × 0.07 mm, multi-scan absorption correction (µ = 0.62 mm-1) Tmin = 0.69, Tmax = 0.96; a total of 6413 measured reflections (θmax= = 67.1˚), from which 1995 were unique (Rint = 0.058) and 1816 observed according to the I > 2σ(I) criterion. The refinement converged (Δ/σmax= 0.001) to R = 0.058 for observed reflections and wR(F2) = 0.141, GOF = 1.14 for 174 parameters and all 1995 reflections. The final difference map displayed no peaks of chemical significance (Δρmax = 0.25, Δρmin -0.29 e.Å-3). Absolute structure parameter: 0.5 (6)58 X-ray crystallographic data have been deposited with the Cambridge Crystallographic Data Centre (CCDC) under deposition number 1902766 and can be obtained free of charge from the Centre via its website (www.ccdc.cam.ac.uk/getstructures).

Quantum Chemical Calculations The

13C

NMR shieldings of ellipticine crystals (forms I, II and III) were calculated by the

CASTEP program,59 version 17.2. The crystal structures were optimized prior to the NMR calculation, but the unit cell dimensions were fixed. The PBE functional,60 a plane wave basis set energy cutoff of 600 eV, default ‘on the fly pseudopotentials, and a k-point spacing of 0.05 Å-1 via a Monkhorst-Pack grid61 were used. The calculation of NMR shieldings was done using the GIPAW method.62-63 The



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calculated chemical shielding (σX) of each atom was re-calculated to chemical shift (δX) using the equation δX = 171.4–σX, where the reference shielding value of 171.4 was chosen to minimize differences between experimental and calculated simulated

NMR

spectra

were

created

using

13C

chemical shifts of ellipticine form III. The

Gsim

program

written

by

Vadim

Zorin

(https://sourceforge.net/projects/gsim/) using linewidth 95 Hz and 8192 points.

Transmission Electron Microscopy (TEM) The TEM micrographs were recorded on a JEOL 1400 transmission electron microscope, which consists of a dispersive X-ray analyser and a Gatan CCD facilitating the acquisition of digital images. The TEM micrographs were obtained at an accelerating voltage of 80 kV. For the sample preparation, a polymer solution (1 mg/mL) was dropped on a copper grid and the solvent was allowed to evaporate under air. The samples were negatively stained with uranyl acetate.

Fluorescence lifetime Fluorescence lifetime was measured by Fluoromax-4 at room temperature using an excitation wavelength of 460 nm. All the data were analyzed in custom software written in Labview. The lifetimes were amplitude-weighted average lifetimes, and a multi-exponential decay law was used to determine the average lifetime (AV). The following equation describes the three-exponential decay:

τ=

∝ 1τ12 + ∝ 2τ22 + ∝ 3τ33

(Equation 3)

∝ 1τ1 + ∝ 2τ2 + ∝ 3τ3

where τ1, τ2, and τ3 are the lifetimes of the multi-exponential decay model, and τ is the average lifetimes.  is the amplitudes of the individual component.

Acknowledgments This work was funded by the Czech Academy of Sciences (E.P., Mobility grant No. MSM200551801). We acknowledge the Australian Research Council (ARC, Equipment Grants No. LE0989541 and LE120100027) for supporting the purchase NMR spectrometers at MWAC, UNSW and for financial support for ARC DP160101172. We thank Sandy Wong for the purified curcumin sample, Dr. Mohan Bhadbhade for single-crystal XRD analysis of fructose and Dr. Chris Marjo for


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discussion of our crystallographic data and Dr. Michal Šála for the “blank experiment”, curcumin precipitation in THF/water system.

Supporting Information Additional solid-state NMR spectra, NMR spectra in solution confirming quality and purity of the material before and after loading into the glycopolymeric particles, T2 plots extracted from the solidstate NMR data, crystallographic parameters from single-crystal XRD, powder XRD patterns, CASTEP-calculated NMR spectra (1H,

13C)

and a plot of experimental chemical shifts vs. calculated

chemical shieldings.

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