Drug-induced morphology transition of self-assembled glycopolymers

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Drug-induced morphology transition of self-assembled glycopolymers: Insight into the drug-polymer interaction Cheng Cao, Jiacheng Zhao, Fan Chen, Mingxia Lu, Yee Yee Khine, Alexander Macmillan, Christopher J. Garvey, and Martina H. Stenzel Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.8b01882 • Publication Date (Web): 16 Jul 2018 Downloaded from http://pubs.acs.org on July 17, 2018

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Chemistry of Materials

Drug-induced morphology transition of self-assembled glycopolymers: Insight into the drug-polymer interaction Cheng Cao,a,b Jiacheng Zhao,a Fan Chen, a Mingxia Lu, a Yee Yee Khine, a Alexander Macmillan,c Christopher J. Garvey, *,b and Martina H. Stenzel*,a a

Centre for Advanced Macromolecular Design, School of Chemistry, The University of New South Wales, Sydney, Australia. b

Australia Nuclear Science and Technology Organisation, Lucas Heights, Australia. c

Biomedical Imaging Facility, University of New South Wales, Sydney, Australia [email protected] [email protected]

Abstract It is often assumed that a hydrophobic drug will be entrapped in the hydrophobic environment of a micelle. Little attention is usually drawn to the actual location of the drug and the effect of the drug on properties. In this publication, we show how the chosen drug curcumin is not only unexpectedly located in the shell of the micelle, but that the accumulation in the hydrophilic block can lead to changes in morphology during self-assembly. A block copolymer poly(1-O-methacryloyl -β-Dfructopyranose)-b-poly(methyl methacrylate), Poly(1-O-MAFru)36-b-PMMA192, was loaded with different amounts of curcumin. The resulting self-assembled nanoparticles were analyzed using TEM, SAXS, and SANS. Initial microscopy evidence revealed that the presence of the drug induces morphology changes from cylindrical micelles (no drug) to polymersomes, which decreased in size with increasing amount of drug. SAXS and SANS analysis, supported by fluorescence studies, revealed that the drug is interacting with the glycopolymer block. The drug did not only influence the shape of the drug carrier, but also the level of hydration of the shell. Increasing the amount of drug dehydrated the nanoparticle shell, which coincided with a lower nanoparticle uptake by MCF-7 breast cancer cells and non-cancerous Raw-264.7 cells. As a result, we showed that the drug can influence the behaviour of the nanoparticle in terms of shape and shell hydration, which could influence the performance in a biological setting. Although the depicted scenario may not apply to every drug carrier, it is worth evaluation if the drug will interfere in unexpected ways.

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Introduction Polymeric micelles have been applied widely as drug delivery system due to the high drug loading efficiency, long-circulating properties1 and the possibility to decorate the surface with a high density of targeting ligands.2 The properties of micelles can be easily adjusted by fine-tuning the block lengths and ratios, which influences size and stability of the micelle. These defining properties – size, stability and surface chemistry - influence the fate of the micelles in vitro and in vivo.

3-7

Although

micelles are well studied in regards to the relationship between physico-chemical properties and behaviour in biological system, little attention has been drawn to effect of amount and type of drug on the activity. There is currently a surprisingly limited amount of reports available that investigate the effect of drug loading on the activity. The common wisdom is that high amounts of loading is preferred, which is understandable considering that the drug carrier is only the packaging material that needs to be disposed of after usage. Moreover, low drug loading content can have serious implication on the amount of nanoparticles required to treat cancer in humans or animals as the required injection volume during administration will be exceed feasible amounts.8 Therefore, researchers usually target the highest possible drug loading efficiency to limit the amount of drug carrier required. This can be achieved by reducing the crystallinity of the core-forming block to enhance the drug loading capacity.9 Other approaches to increase loading focused on increasing the compatibility between polymer and drug10-11 or on adjusting the glass transition temperature.12 To push the amount of drug in a micelle even further, stronger polymer drug interaction are introduced by attaching functional groups such as boronic acid that can display strong donor–acceptor coordination13 or, ultimately, by covalently conjugating the drug to the polymer.14-15 The mantra that the higher the drug loading the better is however not always valid as it has been shown in a study using docetaxel-loaded nanoparticles. The lower weight loading (9%-NP vs. 20%-NP) was observed to have a better pharmacokinetic profile and was more efficient in the fight against murine cancer models.16 At this point, it is crucial to understand the physico-chemical parameters to be able to relate the properties of micelles to their performance in media. The question such as the location of the drug in the micelle (shell or core) and how this may affect the mobility of the polymer chains is instrumental to understand why the amount of drug in a carrier can have such large influences. The location of the drugs can be targeted by conjugating drugs either to the shell or the core. Doxorubicin in the shell was more readily cleaved and therefore the nanoparticle was more toxic.17 However, even drugs bound to the core forming block can affect the cellular uptake as their location and the conjugated amount can influence the shell properties such as the zeta potential18 or the


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hydrophilicity and mobility of the polymer.19 Moreover, different drugs, despite being bound to the micelle in a similar manner, can influence the physico-chemical behaviour of the drug carrier and influence the outcomes in vivo.20 Binding the drug onto the polymer allows some control over the location, but a physical encapsulation of the drug into the micelle can lead to surprising results. Although it is assumed that hydrophobic drugs will prefer the hydrophobic environment, the drug can be accumulated within the shell as long as the drug can interact with the respective polymer.21-22 In this example, the size of the core influenced the crowding of the shell, which, in turn, affected the strength of binding between drug and shell-forming block.21 The effect of the drug on the properties of the micelles should therefore not be simplified as the drug – type and amount - may affect micelle stability23 and the behaviour of the shell forming block.18-20 The drug may also display strong interactions with the shell-forming block.21 However, little is known about how the drug can affect the morphology itself, which ultimately will influence the biological activity. Although we are confident it has been frequently observed in the lab that the presence of drugs can affect the self-assembly process, there are no in-depth studies to understand the underpinning principle that influence the morphology transition. In this paper, we show that the drug can have strong tendency to interact with the shell despite the disparate polarities between drug and polymer shell. We believe that this may be common when polymers such as glycopolymers are employed. Glycopolymers are attractive materials as they display bioactivity, but they can also form strong interactions with other compounds using, for example, hydrogen bonding, which would be absent in more common polymers such as PEO. This interaction can have far-reaching effects as demonstrated here using a block copolymer based on PMMA and a glycopolymer (Figure 1). Curcumin was used as the model drug as it displays antioxidant, anti-inflammatory and antitumoral properties.24-25 With the help of synchrotron small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS), we could identify the location of curcumin in the system. The drug position was found to affect the self-assembly process resulting in morphology transitions. Moreover, understanding the location of the drug and its interaction with the polymer is crucial not only to appreciate the morphology changes, but also the effect the drug location can have on the biological activity.



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Figure 1. Synthesis and self-assembly of Poly(1-O-MAFru)36-b-PMMA192 and various morphologies formed by self-assembly of Poly(1-O-MAFru)36-b-PMMA192 (2 mg/mL) in existence of different amount of curcumin: 0 mg/mL, 0.37 mg/mL and 0.75 mg/mL by TEM. Scale bar = 500 nm.

Results and discussion Self-assembly in the presence of curcumin and biological evaluation The poor bioavailability of curcumin can be addressed by the use of a suitable nanocarrier to enhance the therapeutic efficacy of this promising natural product.26 It was reported that worm-like micelles are promising nanocarriers as they lengthen the circulation time of drugs in the blood.27 We have therefore investigated the formation of spherical and cylindrical micelles of different aspect ratios using poly(1-O-MAFru)-b-PMMA. Fructose-containing polymers were observed to display an enhanced uptake by breast cancer cells and can therefore be used to selectively deliver drugs.28-29 While these glycopolymers show a high affinity to breast cancer cells, it is not clear how drug loading will affect the activity of these carriers. A block copolymer, Poly(1-O-MAFru)36-b-PMMA192, was synthesized as the chosen block lengths can be processed into cylindrical micelles in order to create potentially long-circulating drug carriers.29 Typically, block copolymer and curcumin were dissolved in THF, followed by adding of MQ water into the solution (Figure 1). After dialysis, the amount of curcumin loaded into the nanoparticle was measured using UV-Vis analysis. From this, the drug loading efficiency (DLE) and the drug loading capacity (DLC) were measured (Table S1). As a control, 4 ACS Paragon Plus Environment

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the block copolymer without added drugs was subjected to the same process. Although the drug free system led to the expected cylindrical micelles according to TEM analysis, polymersomes instead of cylindrical micelles were obtained in the presence of curcumin (Figure 1). The morphologies were stable over an extended period of time. The stabilities of the nanoparticles were confirmed by TEM analysis after the liquid samples were stored for 6 months and reanalysed (Figure S1). Moreover, DLS data showed that the size of curcumin loaded polymersomes is affected by the concentration of curcumin in the solution. The diameter of the polymersome decreased from 272 to 263 nm when the concentration of curcumin in the solution during assembly increased from 0.37 mg/mL to 0.75 mg/mL (Table 1). The diameter distributions measured by TEM for sample B and C in Table 1 are shown in Figure S2. The mean diameter is 179 nm and 134 nm, respectively, which is smaller than the sizes measured by DLS, most likely due to the dehydrated state. The result suggests that the curcumin alters with the self-assembly process in a concentration of the drug fashion. The decline in polymersome size coincides with the increase in surface curvature, which is triggered by increased area demands of the hydrophilic polymer or a decreased volume of the hydrophobic core. Our observations may suggest that curcumin is in the fructose shell, which could have implications on the properties of the shell such as hydration and swelling as well as surface charge. This would ultimately influence the biological activity. It is evident that the presence of curcumin led to a morphology transition, but at this stage, it is unknown if the aggregate changes are permanent or if the morphology can revert back to cylindrical micelles. Initially, the drug release of both drug loaded carriers (Table 1) was investigated over 72 h in PBS solution (pH=7.4). The drug loaded nanoparticles were placed in a dialysis bag and the sample was dialyzed against water. At regular time intervals, a sample was taken from inside the bag and the amount of curcumin was measured by UV-Vis spectroscopy. Although typical set-up measure the drug on the outside of the dialysis bag, this was not possible here as curcumin accumulated on the surface of the bag. Both carriers displayed similar release rates (Figure S3), albeit the drug carrier with the higher high drug loading capacity had a slightly faster rate of release (78% vs 72% of loaded drug released after 72 hours). The nanoparticles after the release of curcumin were studied using TEM analysis (Figure S4). It is interesting to note that despite the release, the morphology was retained and does not revert to the cylindrical micelles found when self-assembling the block copolymer without the drug. Once formed, the polymersomes are kinetically stable against rearrangements with changes of drug content as the glass transition temperature is well above ambient temperature.



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Table 1. Size of self-assembled drug loaded Poly(1-O-MAFru)36-b-PMMA192 nanoparticles using the different amount of curcumin. Nanoparticles

[Curcumin]/ Drug loading Drug loading mg/mL efficiency/ % capacity/ %

Dh / nm (DLS)

PDI

Zeta Potential / mV

Sample A (cylinder)

0

0

0

167 ± 5

0.062 ± 0.012

-25.7 ± 1.5

Sample B (polymersomes)

0.37

36.5

6.8

272 ± 3

0.118 ± 0.015

-22.6 ± 1.2

Sample C (polymersomes)

0.75

20.8

7.8

263 ± 2

0.124 ± 0.020

-19.5 ± 0.8

Subsequently, the effect of drug loading on the biological activity such as uptake of nanoparticles by cells was evaluated. As cylindrical micelles will naturally have different cellular uptake patterns independent of drug loading, Poly(1-O-MAFru)36-b-PMMA192 was therefore processed into drug-free polymersomes by applying a stirring rate of 300 r/min. This trapped the block copolymer in the desired spherical shape with a hydrodynamic diameter of 271 nm instead of the usual cylindrical micelle this polymer likes to form. Drug-free polymersomes and drug-loaded polymersomes can then be used for direct comparison in subsequent studies without having to take shape factors into account.

At this point, it was expected that the cellular uptake of polymersomes with higher drug loading would be more efficient as the smaller size should display a more efficient cell uptake.5, 30 The cell uptake using empty and drug-loaded polymersomes was investigated using flow cytometry measurements using the concentrations listed in Table S2. The polymers were labelled with conjugated cyanine 5 (Cy5< 1wt%), which does not interfere with the fluorescence emission spectrum of curcumin. The mean fluorescence intensity (MFI) of polymersomes was normalized after subtraction of the cellular autofluorescence (Table S2 and 3). Surprisingly, polymersomes loaded with a higher amount of drugs showed a lower uptake by MCF-7 and Raw 264.7 cell lines (Figure 2a and b) despite its smaller size. Moreover, the surface charge does not change significantly. If anything, the lower particles with the lower drug loading and the more negative charged surfaces should show preferred uptake, which contrasts actual uptake measurements. The polymersomes with the highest amount of drugs must obviously present cues that may not only lower the cellular uptake but also lower cytotoxicity (Figure 2c, d, and Table S4). Therefore, spectroscopic and scattering analyses were carried out to obtain more details on the internal structure of drug-loaded nanoparticles.


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Figure 2. Flow cytometry on MCF-7 cell line (a) and Raw-264.7 cell line (b). The polymer concentrations was set to 0.2 mg/mL, and the concentration of loaded drug ranges from 0 to 0.0375 mg mL-1 (see Table S2). (c), (d) IC50 curves of curcumin-loaded polymersomes in 2D MCF-7 and Raw246.7 cell culture models. The DLC in figure (c) and (d) can be found in Table S1

Physico-chemical characterization of curcumin loaded nanoparticles The local physicochemical environments in self-assembled nanoparticles can be probed by spectroscopic techniques using the solvatochromic shift of the UV-Vis absorbance.31 To understand the effect of the local dielectric environment sensed by the probe, the UV-Vis absorption maxima λmax of curcumin in various solvent as well as 1,4-dioxane-water mixtures were measured (Table S5 and S6). The measured λmax of curcumin in the NP equated to a dielectric constant of 39.18 (Figure S5) suggesting that curcumin is not located in the dielectric media of the PMMA shell but rather in a dielectric environment like a mixture of water and fructose.32 While the measured UV-Vis absorption maxima λmax= 421 nm is independent from the drug loading content (Figure S6), the fluorescence of curcumin is responsive to the environment,33 thus, to the changes in the polymer with increasing drug content (Figure 3a). The emission maximum of 7 ACS Paragon Plus Environment


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curcumin undergoes a 10 nm Stokes shift when the curcumin concentration in the carrier increases, indicative of an environment of decreasing polarity. In addition, increasing curcumin concentrations leads to fluorescent self-quenching, which is frequently observed when the dye undergoes aggregation or stacking. The fluorescence lifetime of the drug (fluorophore) is sensitive to the drug’s environment due to the strong effect on the non-radiative decay.34 Figure 3b depicts the lifetime τ decrease with decreasing amount of curcumin loaded in the NPs while the lifetime τ of curcumin in water is less dependent on the concentration (Figure 3c). The fluorescence lifetime of curcumin loaded into nanoparticles leads to a longer decay time, which is increasing with increasing curcumin concentration (Figure 3b and d). Direct comparison of the curcumin fluorescent lifetime in water and in the nanoparticle (Figure 3d, S7 and Table S7) reveals restricted motion of curcumin in the nanoparticle. The lifetime of curcumin in MMA is rather short contrasting the long lifetime in water (Figure 3c). Compared to the actual lifetime of curcumin in the polymersomes, the long lifetime suggests that curcumin is more likely located in the glycopolymer shell.

Figure 3. (a) The fluorescence spectra of various concentration of curcumin loaded in the polymersomes and λex = 421 nm. (b) The lifetime plots versus different amounts of curcumin loaded in NPs. (c) The lifetime plots versus different amount of curcumin in the water. (d) The average lifetime versus different amounts of curcumin loaded in the nanoparticles and different amount of curcumin in the water.


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Subsequently, we gained more insight into the location of curcumin in the aggregate by scattering studies. The analysis of small-angle scattering data has been carried out by model-dependent fitting.35 The scattering curves were fitted with a mathematical model containing both spatial and compositional information. The length scales were initially constrained by TEM (shape) and DLS (size and polydispersity) measurements (Figure 1 and Table 1). The scattering length density (SLD) is a description of the atoms in a representative volume: =

∑

(Equation 1) where is the bound coherent scattering density of all i atoms over the molecular or monomer volume, . For SAXS and SANS, the coherent scattering cross-sections are from the electronic and nuclear environments respectively.36 Each sample with different concentrations for SANS and SAXS were measured and normalized by intensity, and they do not depend on the concentrations which can ignore the structure factor. For the fitting of the SAXS and SANS data, a core-shell cylinder and a 3-shell around a water core models37 were used. The important parameters used in the models are illustrated in Figures 4b and 5a respectively. The shell of the cylinder and inner and outer shells of the polymersome structures are composed of Poly(1-O-MAFru), water and in some cases curcumin. The middle shell is composed of PMMA with the physical properties of the amorphous material used as a starting point.38 We modelled the SLD and thickness of shells as well as the radius of the nanoparticles. The SLDs of the inner and outer shells allow us to determine the chemical composition since: = ∅ ∙ + ∅ ∙ + ∅ ! ∙ ! (Equation 2) where the subscripts refer to the SLD and ∅ (volume fraction) of the shell, water, curcumin, and fructose. If ∅ ! is negligible then, ∅ + ∅ = 1, and the ratio of water to polymer is easily calculated. Initially, the SAXS of NPs without curcumin was fitted with a core-shell cylinder and with curcumin with core-shell, Model 1. There is no difference between NPs with low drug loading and high drug loading for SAXS in the high-q regime (Figure 4a). All the parameters we used are summarised in Table S8. The agreement between the SAXS data for the sample without curcumin with rod fit is



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obvious. Thus, it can be concluded that the SAXS is sensitive to the shape of the NPs, but not the structure of the shell. The modelling of the SAXS data provided some useful information, it gives an excellent indication of the average shape of the NPs. Due to the poor contrast between the Poly(1-O-MAFru), solvent and curcumin (Table S9), the amount of information about the inner and outer shells that can be extracted from the high-q regime is limited. SANS measurements have the possibility of enhancing the contrast between the inner and outer shells and the solvent by using D2O as the solvent.

Figure 4. (a) SAXS data for the sample A, B and C and the corresponding fit on the curve of sample A; (b) Core cylinder model and fitting results of sample A. The samples are listed in Table 1

Typical SANS curves from NP polymersomes in D2O for optimal contrast are shown in Figure 5b. We have used two different models to extract structure information from the scattering curves. In the low-q regime, the model can be used to extract the mean core radius of NPs, # . The high-q regime is sensitive to the thickness, $ , and the SLD of each shell of the NPs. Initially, we kept the SLD of the inner and outer poly-fructose/water/curcumin shells equal (Model 1). Although the low-q fit, corresponding to the overall size and shape of the NP converged well, the high-q regime corresponding to the internal structure of the shells was a poor fit (Figure S8 – Model 1). A much better fit can be obtained by including slight differences in the SLD’s and thicknesses between inner and outer shells (Figure 5a – Model 2). The results of the fitting are shown in the Table S10.


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Figure 5. (a) Scattering length density(SLD) file for the SANS polymersome Model 2. (b) SANS data for sample [curcumin]=0 mg/mL (No drug); 0.37 mg/mL (Low drug); 0.75 mg/mL (High drug) and their respective curves fits.

160 1200 140 1000

Tshell(Å)

120

inner shell PMMA shell outer shell

100 80

800

600

Rcore(Å)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


400

60

200

40 20

0 No Drug

Low Drug

High Drug

Figure 6. The variation of core radius and the thickness of inner shell, PMMA shell and outer shell with drug loading. The lines are only to guide the eye.



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SANS data of NPs with different amount of curcumin is modelled to obtain the SLD, thickness of the shell. Figure 5b shows the overview of the fitting of SANS curves from samples with three different loadings of curcumin: ([No drug] = 0 mg/mL; [Low drug] = 0.37 mg/mL; and [High drug] = 0.75 mg/mL). The fitting parameters are given in Table S10. The # is insensitive to the drug loading. A parameter which is very sensitive to drug loading is the thickness of PMMA shell, however, the inner and outer shells are poorly sensitive to the drug loading and show opposite trends, the inner shell getting slightly thicker and outer shell getting marginally thinner (Figure 6). The SLD of the PMMA shell 3.55 × 10*+ Å*- remains the same, close to the initial value for amorphous PMMA, for the three samples. With an increasing amount of curcumin, the SLD of the outer fructose shell decreases from 5.20 × 10*+ Å*- to 4.80 × 10*+ Å*- , by contrast, the SLD of the inner shell, increases slightly (Table S10). While the SLD should allow us to calculate the chemical composition of the shells (see equation 2) we have only measured at one contrast point (100% D2O), and because of the effects of curvature of a thickening PMMA layer on particles which have similar radius (Figure 6), we shall concentrate on the outer shell where the curvature remains almost constant. An additional assumption is that the density of Poly(1-O-MAFru) chains remains constant in the outer shell. While clearly the outer shell is composed of fructose, D2O and curcumin, from the perspective of SANS the contribution of curcumin to the overall SLD is negligible since even when it partitions completely in the inner and outer shells its perturbation to the SLD is very small (Table S11). According to the variation of the SLD in the outer shell with drug loading (Table S10), we use the equation 2 to calculate the ∅ in the outer shell (Figure 7a and b). This result shows that the water content of the outer shell decreases with the increasing amount of curcumin. In Figure 2, increasing drug content slows the cellular uptake of NP’s. The SANS results indicate that this slower cellular uptake is associated with an outer layer of the NP’s which has a lower water content and the more compact layer of Poly(1-O-MAFru) and curcumin.


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(a)

(b)

Figure 7. (a) Volume fraction of water in the outer fructose shell with respect to the different amount of curcumin according to the equation 6; (b) Possible morphologies of fructose chains in the outer shell with the different amount of curcumin.

SANS and SAXS measurements confirm that the drug is indeed located in the shell of the polymersome, which agrees on the spectroscopic studies. The block copolymer itself has a tendency to form cylindrical micelles, however, with the addition of curcumin, the morphology changed to polymersomes. These polymersomes became smaller when more curcumin was added. Association of hydrophobic curcumin with the hydrophilic glycopolymer instead of the hydrophobic PMMA led to these influences in the self-assembly process. Important is here that the presence of curcumin in the shell does have far-reaching effects on the hydration. The declining levels of hydration may be responsible for the lower cellular uptake when the nanoparticles are loaded with drugs as the fructose polymer is less mobile and less likely recognized by the receptors on MCF-7 cells.19 It needs to be noted here that the morphological transitions were specific to this block length. Polymers with the smaller hydrophobic block, poly(1-O-MAFru)35-b-PMMA160, were found to form micelle (Figure S9) independent from the amount of curcumin added although curcumin will be located in the shell 13 ACS Paragon Plus Environment


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as well. In this scenario, the influence of the surface curvature by the drug is less dramatic. We also have observed similar results using a related polymer and Paclitaxel as drug suggestion that drugs in general can potentially affect the activity of nanoparticles. This observation will be discussed in a forthcoming paper.

Conclusion We showed here that the morphologies of Poly(1-O-MAFru)36-b-PMMa192 self-assembled nanoparticles are affected by the concentration of curcumin resulting in a transition from cylindrical micelles to polymersomes. Coincidently, the cellular uptake decreased with increasing amount of curcumin drug suggesting that the drug introduced changes in the surface properties that may have been recognized by cancer cells. Increasing amount of curcumin did not only lead to lower uptake, but also to lower toxicity. We, therefore, used spectroscopic technique and SANS/SAXS studies to learn more about the polymersomes. We found that the drug is indeed located in the shell, which helped to explain the observed morphology changes. More importantly, the location of the drug led to the dehydration of the shell, which may have been the cause for the cellular uptake. In this study, we have shown that the interaction between drug and polymer can influence the selfassembly process in unexpected ways. Although it is commonly assumed that hydrophobic drugs will reside in the hydrophobic parts of self-assembled block copolymer aggregate, we showed here that the drug can a) change the morphology of the nanoparticle and b) enrich itself in the shell of the self-assembled morphology. The consequence of this behaviour is not only the changed morphology but the changes in the physico-chemical properties of the shell, which is now filled with drugs. We studied the encapsulation of curcumin as the drug and a block copolymer based on glycopolymers, but we believe that similar observation can be found when drug and polymer form strong interactions that can overcome disparate polarities. Our observation may well be specific to the glycopolymer system that we have used, but the researcher needs to consider that even if the drug is located in the core as expected, the presence of the drug may cause alterations to the property. The take-home message of this manuscript is that the drug cannot be seen as an independent variable, but the drug will interact with the polymer in various ways, which will result in physiochemical changes of the drug carrier. In some cases, this may lead to change in behaviour in a biological environment.

Experimental 14 ACS Paragon Plus Environment

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Materials D-fructose (99%, Aldrich), curcumin (>80%, Aldrich), dichloromethane (DCM; anhydrous, >99.8%, Aldrich), tetrahydrofuran (THF; 99%, Ajax Finechem), fluorescein O-methacrylate (97%, Aldrich) and N,N-dimethylformamide (DMF; 99%, Ajax Finechem) were used as received. 1,4-dioxane (99%, Ajax Finechem) and pyridine (99%, Ajax Finechem) were purified by reduced-pressure distillation. Methyl methacrylate (MMA, >99%, Aldrich) was passed over basic aluminium oxide to remove the inhibitors. 2,2-azobis(isobutyronitrile) (AIBN; 98%, Fluka) was recrystallized from methanol for purification. The RAFT agent 4-cyanopentanoic acid dithiobenzoate (CPADB) was synthesized according to a literature procedure.39

Synthesis and procedures Synthesis of Poly(1-O-MAFru)36 and Poly(1-O-MAFru)36-b-PMMA192 The synthesis of 1-O-methacryloyl-2,3:4,5-di-O-isopropylidene-β-D-fructopyranose followed the similar procedure as reported previously.40 The polymerization of the glycomonomer was described as follows: in a Schlenk tube, 1-O-methacryloyl-2,3:4,5-di-O-isopropylidene-β-D-fructopyranose (4 g, 12.2 mmol), AIBN (2.6 mg, 1.62 × 10-2 mmol) and CPADB (35 mg, 0.12 mmol) were dissolved in 1, 4dioxane (8.5 mL). Then the tube was degassed by three freeze-pump-thaw cycles. The polymerization was carried out at 70 ℃ and stopped at 7 h by cooling the solution in ice water (conversion: 62%). The polymer solution was poured into a large excess of diethyl ether for precipitation. The viscous polymer was dried under vacuum for 24 h. The macro-RAFT agent, Poly(1-O-MAFru)36, was subsequently used for chain extension with MMA. The procedure for the synthesis of Poly(1-O-MAFru)36-b-PMMA192 was described as follows: macroRAFT agent (200 mg, 2 × 10-2 mmol), MMA (516 mg, 5.16 mmol), AIBN (0.64 mg, 4 × 10-3 mmol) and were dissolved in 1,4-dioxane (3 mL). The tube was degassed by three freeze-vacuum-thaw cycles. The polymerization was carried out at 70 ℃ and stopped after 18 h by cooling the solution in ice water. The polymer solution was poured into a large excess of n-hexane for precipitation. The viscous polymer was dried under vacuum for 24 h. The deprotection of the block copolymers was carried out under acidic conditions. Specifically, the polymer (80 mg) was added into 1.59 mL of TFA/H2O (9:1 v/v) in a vial with stirring at room temperature for 30 minutes. After reaction, the polymer solution was dialyzed against deionized water for two days (MWCO 3500).



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Self-assembly of Curcumin Encapsulated Nanoparticles A typical procedure for the encapsulation of curcumin was described as follows: The block copolymer (4 mg) and curcumin (1 mg) were dissolved in THF (0.2 mL). Then, 1.8 mL MQ water was added to the polymer solution using a syringe pump with a rate of 0.2 mL/h. The micelle solution was dialysed against deionized water to remove THF and excess curcumin. The obtained micellar solutions were used for structural investigations by DLS, TEM, SAXS and SANS. According to the literature,29 the stirring rate of the solution during the self-assembly process can change the morphologies of NPs, thus polymersomes composed of Poly(1-O-MAFru)36-b-PMMA192 and different concentrations of curcumin (0 mg/mL, 0.1725 mg/mL and 0.2025 mg/mL) were prepared at a stirring rate of 380 cycles/min) in D2O for SANS measurements. The similarity in size was confirmed by DLS.

Drug release study The release study was carried out at 37 oC in the pH 7.4 phosphate buffer (20mM). Two polymeric nanoparticles (2mg/mL) carrying different concentrations of curcumin 0.115mg/mL and 0.135mg/mL were transferred into 10mL two different dialysis bags (MWCO 3500 kDa) in 1L MQ water at 37 oC. The polymer solution inside the bag was sampled every 2 h. The drug concentration in the nanoparticle was determined using the UV-Vis absorbance at 421 nm. After all curcumin was released, the empty nanoparticles were collected and analysed using TEM (JEOL1400).

Cellular uptake Cellular uptake was carried out using flow cytometry. After culturing sufficient amount of cells using medium Dulbecco’s modified Eagle’s medium (DMEM) including fetal bovine serum (FBS, Bovogen Biologicals) and plasmocin at 37 °C in a humidified atmosphere including 5% CO2, MCF-7 human breast cancer cells were seeded in the 6-well plates at a density of 3 × 102 cells per well with DMEM cell culture medium at 37 °C with 5% CO2 for 2 days prior to nanoparticle treatment. For the treatment, micelles (0.6 mg in 3 mL DMEM) were incubated with cells for 1 day. The cells were initially washed 4 times with cold PBS to remove excess nanoparticles before they were detached from the plates using tryspin/EDTA. The cell suspensions were then centrifuged and resuspended in cold Hank’s buffer. The fluorescence intensity of the cells from the suspensions (20000 cell events)


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was measured indicative of cellular uptake on a BD FACSCanto TM II Analyser using an excitation laser wavelength of 640 nm and a band-pass filter of 660/20 nm for emission spectra. Raw data was analysed using FlowJo® software and the results are shown using median fluorescence intensity (MFI) averaged from 3 individual wells for each sample.

Cytotoxicity test The cytotoxic effects of free curcumin, as well as blank and curcumin-loaded nanoparticles(NPs) were examined on MCF-7 cell lines by a standard sulforhodamine B colorimetric proliferation assay (SRB assay). After culturing sufficient amount of cells using medium Dulbecco’s modified Eagle’s medium (DMEM) including fetal bovine serum (FBS, Bovogen Biologicals) and plasmocin at 37 °C in a humidified atmosphere including 5% CO2, cells were seeded in 96-well cell culture plates at a density of 4000 cells per well for 24 h. All the sample solutions were sterilized by UV irradiation for 15 mins in the bio-safety cabinet and then the medium was replaced with 100 μL of fresh 2 × concentrated medium. Subsequently, the cells were treated with serially diluted (2 × dilution) sample solutions: blank nanoparticles, nanoparticles with less curcumin concentration and nanoparticles with high curcumin concentration, followed by incubation at 37 °C for 3 days.

After that, the culture medium was eliminated, followed by the addition of 100 µL of 10% (w/v) trichloroacetic acid (TCA) to each well and incubation of the plates at 4°C for 30 min. Then the plates were washed five times with MQ water. Next, the cells were stained with 100 μL of 0.4% (w/v) SRB dissolved in 1% acetic acid, and then the plates were covered with aluminum foil and incubated for 20 min at room temperature. After removal of SRB solution, the plates were washed five times using 1% acetic acid and the plates were air-dried for 2 h at room temperature. Finally, the dye (SRB) was solubilized using 200 μL of 10 mM Tris buffer. The absorbance of each well was read at 490 nm using a Bio-Rad BenchMark Microplate reader and the data were analyzed using GraphPad Prism 6.0. In this experiment, non-treated cells were used as controls and the cell viability was calculated using the following equation (where OD means optical density): ?@A,CD*?@A,C!E

4566 789 868:; = ?@A, !*?@A,C!E × 100 (Equation 3)

Analysis Size exclusion chromatography (SEC)



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The molecular weight and polydispersity of synthesized polymers were analyzed via size exclusion chromatography (SEC). A Shimadzu modular system comprising a SIL-10AD auto-injector, DGU-12A degasser, LC-10AT pump, CTO-10A column oven and a RID-10A refractive index detector was used. A 5.0-µm bead-size guard column (50 × 7.8 mm) followed by four 300 × 7.8 mm linear columns (500, 103, 104, and 105 Å pore size, 5 μm particle size) were employed for analysis. N,N-Dimethylacetamide [DMAc; HPLC grade, 0.05% w/v 2,6-di-butyl-4-methylphenol (BHT) and 0.03% w/v LiBr] with a flow rate of 1 mL/min at 50 ℃ was used as mobile phase. 50 μL of polymer solution with a concentration of 2 mg/mL in DMAc was used for every injection. The calibration was performed using commercially available narrow-polydispersity PMMA standards (0.5-1000 kDa, Polymer Laboratories).

Dynamic Light Scattering (DLS) The hydrodynamic diameter Dh was determined using a Malvern Zetaplus particle size analyser (laser, angle = 173°) at a copolymer concentration of 1 mg/mL. Samples were prepared in deionized water and sonicated for 30 min prior to the measurements. The polydispersity index (PDI) as a fitting parameter in the cumulants analysis of autocorrelation function shows the DLS data’s quality. Zeta potential is measured to indicate the surface charge of the nanoparticles.

Transmission Electron Microscopy (TEM) The TEM micrographs were obtained using transmission electron microscope (JEOL1400) comprising of a dispersive X-ray analyser and a Gatan CCD facilitating the acquisition of digital images. The measurement was conducted at an accelerating voltage of 80 kV. The samples were prepared by casting the copolymer micellar solution (1 mg/mL) onto a copper grid. The grids were dried by air and then negatively stained with uranyl acetate.

UV-Vis spectroscopy UV-visible spectra were measured between 250 nm- 700 nm on the Cary 100 UV−Vis spectrophotometer. The encapsulated curcumin was measured with Cary 100 UV−vis spectrophotometer. The maximum wavelength of curcumin is 421 nm, the drug loading efficiency (DLE) (%) were calculated by:


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DLE =

KLM NOPQRK PS TQUTQOVR VR WPXYOMUZPOMZ × KLM NOPQRK PS TQUTQOVR VR SMM[

100% (Equation 4)

The drug loading capacity (DLC) (%) were calculated by:

DLC =

KLM NOPQRK PS TQUTQOVR VR WPXYOMUZPOMZ × KLM NOPQRK PS WPXYOMU

100% (Equation 5)

UV-vis absorbance of the polymer samples regarding the different concentration of curcumin is used to describe the change of absorbance during the drug release.

Fluorescence Spectroscopy Fluorescence measurements were carried on a CARY Eclipse fluorescence spectrophotometer at room temperature. The excitation of measurements was 421 nm for the experiments. The fluorescent wavelength was collect at 430 – 750 nm and excitation slit is 10 and 20.

Fluorescence lifetime Fluorescence lifetime was measured by Fluoromax-4 at room temperature. The excitation was 460 nm. All the data was analysed 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 (τ). For a two-exponential decay is described by:

]=

∝_ `_ a ∝ ` ∝_ `_ a ∝ `

(Equation 6)

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

Small-angle X-ray scattering and small-angle neutron scattering SAXS measurements were made at the Australian Synchrotron’s SAXS/WAXS beamline41 from samples in 1.5 mm quartz capillaries (Charles Supper, Natick, USA). Isotropic scattering patterns 19 ACS Paragon Plus Environment


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were collected on a Pilatus 1 M detector (Dectris, Baden-Daettwil, Switerland) using a wavelength of 1.033 Å (20 KeV) and a sample to detector distance of 7.2 m. The measurement geometry was used to convert the counts per detector pixel into the radially averaged intensity versus the scattering vector on an absolute intensity scale after subtraction of background due to solvent filled capillary and normalising to the scattering of water using the beamline software Scatterbrain. The scattering vector, q, is defined by b=

?c h sin d

(Equation 7)

and Θ/2 is the scattering angle. Small angle neutron scattering (SANS) measurements were made on the fixed wavelength reactor based pin-hole instrument Quokka (ANSTO, Lucas Heights, Australia) .42

For each sample

measurements were made at three different samples to detector distances: 1.3 m, 10 m and 20 m, where the counting times were 30 min, 1 h and 4 h respectively. The first two configurations were measured using 5 Å neutrons and the final at 8 Å. In both cases the ∆λ/λ was 10%. The isotropic counts per pixel on the area detector was converted a continuous scattering curve using the by radial averaged 2-dimensional data after first correcting for the detector sensitivity, subtracting the dark field counts and the background signal due to the solvent filled empty cell and finally normalising to the incident flux of neutrons on the sample.37 Small angle scattering curves were obtained at several different concentrations (SAXS: 1 mg/mL, 2 mg/mL, 3 mg/mL; SANS: 1 mg/mL, 2 mg/mL, 3 mg/mL). When normalised to concentration of particles both SANS and SAXS curves were independent of concentration. We can therefore assume that all scattering curves were obtained under dilute conditions, and that the scattering curve is dominated by the shape and internal structure of the particles.43 The structure model for the NPs described was fitted separately to the reduced SAXS and SANS data by using the program SASview35 and specific macros written for IgorPro.37

Acknowledgements MHS and CJG like to thank the Australian Research Council ARC for funding DP160101172. We also like to thank the Electron Microscopy Unit of the UNSW Mark Wainwright Analytical Centre for their help. We acknowledge the support of the Australian Centre for Neutron Scattering, Australian NuclearScience and Technology Organization, in providing the neutron research used in this work.


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This research was also undertaken on the (SAXS/WAXS) beamline at the Australian Synchrotron, part of ANSTO

Electronic Supplementary Information (ESI) available: [curcumin release, TEM after curcumin release, dielectric constants, UV-Vis spectra, lifetime plots, SAXS and SANS fitting parameter].

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Drug-induced morphology transition of self-assembled glycopolymers: Insight into the drug-polymer interaction Cheng Cao, Jiacheng Zhao, Fan Chen, Mingxia Lu, Yee Yee Khine, Alexander Macmillan, Christopher J. Garvey, and Martina H. Stenzel


Drug-induced morphology transition of self-assembled glycopolymers

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