Pseudopolyrotaxane Formation in the Synthesis of Cyclodextrin

Jan 24, 2017 - Pseudopolyrotaxane Formation in the Synthesis of Cyclodextrin Polymers: Effects on Drug Delivery, Mechanics, and Cell Compatibility. Th...
0 downloads 24 Views 3MB Size
Download PDF
Article pubs.acs.org/bc

Pseudopolyrotaxane Formation in the Synthesis of Cyclodextrin Polymers: Effects on Drug Delivery, Mechanics, and Cell Compatibility Thimma R. Thatiparti, Dajan Juric, and Horst A. von Recum* Department of Biomedical Engineering, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106, United States S Supporting Information *

ABSTRACT: Numerous groups have reported the use of cyclodextrin (CD)-based polymers for drug delivery applications due to their capacity to form inclusions with small molecule drugs, delaying the rate of drug release beyond that of diffusion alone (termed “affinity-based” drug delivery). Herein we demonstrate synthesis and characterization of a new family of CD-based polymers, some as pseudopolyrotaxanes, generated under mild (aqueous, room temperature) conditions. The formation of these new affinity polymers results in broad mechanical properties. Three diglycidylether cross-linkers which vary in length from 0 to 10 ethylene glycol units were examined. Pseudopolyrotaxane formation was found only with the highest-length cross-linker, noted first by a sharp change in both material properties and then confirmed by chemical signature. Materials were thoroughly evaluated by NMR, DSC, DMA, TGA, XRD, and FTIR. Cross-linker choice was also tested for impact on drug loading and delivery capacity, using antibiotics as model drugs. Chemically similar polymers without showing affinity rapidly saturated in loading experiments, while affinity materials showing high capacity for drug loading, even beyond the solubility limit of the drugs. When using the polymers with these new cross-linkers, affinity-based drug delivery is maintained: the materials are capable of antibiotic delivery, and clearance of Staphylococcus aureus, at least an order of magnitude better than diffusion-only control polymers. In cell compatibility studies, CD-based polymers were shown to have low overt cell toxicity and even resisted cell adhesion, presumably due to their highly hydrated state.

■

INTRODUCTION

hydrophilic exterior, CDs are able to form inclusion complexes with small molecules, a property which has been exploited to prolong the release of small molecule10 and complex drugs.11 In order to use this drug interaction property, many investigators have immobilized CDs onto different materials to enhance the loading and control release of drugs.12−15 On the other hand, cyclodextrins can also act as moieties capable of generating physical cross-links, by making inclusion complexes with hydrophobically modified polymers, or with amphiphilic polymers having hydrophobic chains, both of which combine to form supramolecular polymer networks. These physically cross-linked materials have also been investigated for their capacity for drug delivery;16−18 however,

Extensive efforts have been made to improve the controlled release of drugs from polymer for countless applications.1−4 Among the modifications, using a specific affinity between matrix polymer and delivered drug has been shown to be useful to control drug release profiles and total drug loading.5 Introduction of affinity modifications is usually done with compounds capable of forming supramolecular complexes with the target drug molecule. One such example compound is the family of cyclodextrins (CD).6,7 Cyclodextrins are naturally occurring cyclic oligosaccharides composed of 6, 7, and 8 Dglucopyranose units linked with α-(1,4) glucosyl bonds, and are named α-, β-, and γ-CD, respectively. These molecules are often depicted as torus-shaped, having a polar exterior and nonpolar interior, which enable cyclodextrins to form supramolecular complexes in polar solvents where they dissolve/ swell.8,9 Because of their relatively hydrophobic pocket and © 2017 American Chemical Society

Received: December 15, 2016 Revised: January 17, 2017 Published: January 24, 2017 1048

DOI: 10.1021/acs.bioconjchem.6b00721 Bioconjugate Chem. 2017, 28, 1048−1058

Article

Bioconjugate Chemistry

polysaccharide, and therefore cannot form inclusion complexes like CD. Polymer networks were formed by reacting known moles of CD with known moles of diglycidyl ethers. The polymer network had formed within 1 d at room temperature; however, the reactions were allowed to proceed for 4 d to ensure that cross-linking was complete. Here, we unexpectedly found that the CD makes an inclusion complex with PEGDGE526 to give pseudopolyrotaxanes. During the formation of the polymers for this study, initially when we were trying to make Dex-PEGDGE526 (1:0.7) polymers, the PEGDGE526 was totally unable to dissolve in the dextran solution (Figure S2(a)), whereas PEGDGE526 was easily dissolved in the CD solution (Figure S2(b)). This was the first indication of a facile inclusion formation between PEGDGE526 and CD. It has previously been reported that free poly(ethylene glycol) above 200 molecular weight can make an inclusion complex with alphaCD but not with beta-CD, because the PEG chains are easily dethreaded from beta-CD due to its higher pocket size.30 In contrast, beta-CD makes an inclusion complex with free poly(propylene glycol) due to propylene groups which block the dethreading from beta-CD.29 In these studies, similar initial threading may have occurred, with the diglycidyl groups at the end of PEGDGE526 stopping the dethreading and facilitating the cross-linking further, creating end-caps with CD to stabilize this network (Figure 1). This pseudopolyrotaxane arrangement and network formation was further confirmed through the analyses below.

these polymers end up with weak mechanical strength, as there is competition within these amphiphilic polymers where pockets have to serve a role both for physical cross-links and for drug loading.19 CD-based polymers, nevertheless, have grown in interest due to their capacity to generate slower, more sustained delivery profiles than similar polymers without affinity groups.15 In most cases these CD polymers were formed through either high temperature or harsh, nonaqueous solvents. These synthesis or conjugation conditions can damage a biomedical implant upon coating, can result in incorporation of toxic byproducts, or can leach out crucial implant material additives. Additionally, since mechanical properties were often of lowest concern, these CD polymers are often brittle and can fragment upon their high water uptake.7 In this work we show the use of digylcidyl ether cross-linkers to overcome some of the previous complications in synthesis and mechanical properties of CD polymers, while still maintaining their affinity-based drug delivery capacity. Along the way one particular polymer chemistry seems to be even further enhanced by formation of a pseudopolyrotaxane. To carry out this study we have chosen a β-CD in prepolymer form (i.e., a low-molecular-weight β-CD prepolymer) and prepared final network polymers at room temperature using, as a crosslinker, either ethylene glycol diglycidyl ether (EDGE) or one of two different poly(ethylene glycol) diglycidyl ethers (Mn = 200: PEGDGE200; or Mn = 526: PEGDGE526). Our group has previously reported the use of EDGE to cross-link CD polymers and to coat hernia repair meshes.20 These materials performed well in vivo, where the antibiotic-loaded, polymercoated meshes showed the capacity to prevent bacterial infection for up to a month. However, the mechanical properties of this particular polymer were suboptimal, due to a limited capacity to form a mechanically durable coating. To improve CD coatings, both milder reaction conditions and a cross comparison of different cross-linkers were examined herein, with an effort to improve mechanical properties while preserving drug delivery advantages. Here, unexpectedly, we found that PEGDGE526 formed inclusion complex with CD during cross-linking, to create pseudopolyrotaxanes which are stabilized when the covalent bonds form. In this article, we report synthesis and characterization of the different polymers, including the pseudopolyrotaxanes, and compare them with nonaffinity polymers made from dextran, which is chemically similar to CD but incapable of forming drug complexes. The dextran polymers were similarly prepared, cross-linked with EDGE and PEGDGE200. Lastly, the in vitro drug loading, drug release, bactericidal properties, and cell adhesion properties of these new materials were tested.

Figure 1. Schematic representation of pseudopolyrotaxane formation followed by covalent cross-linking between CD and PEGDGE526.

The main objective of the present study was to explore the possibility of altering the CD network architecture at under mild (e.g., room temperature) conditions (Figure 1) to achieve mechanically stable, flexible, and robust polymers for device coatings, with a consequence of forming pseudopolyrotaxanes. To confirm this architecture, in addition to preliminary miscibility studies, we used NMR, DSC, DMA, TGA, and XRD. NMR results (Figure S3) indicate that only the expected cross-linking reactions occur, since PEGDGE526 in the pseudopolyrotaxane was found to be identical to pure PEGDGE526. Similar observations were made previously in the case of alpha-CD and PEG pluronics,24 showing only the expected bond formations, where additionally measured properties must be explainable by noncovalent interactions (e.g., some PEGDGE526 physically associated within CD). Figure 1 shows how the partially covered PEGDGE526 chains by CD can covalently cross-link in the network. In this work, the physical associations are indicated by further analysis. Differential Scanning Calorimetric (DSC) Analysis. DSC was used to obtain data on glass transition temperatures (Tg) for both dextran and CD polymers, with expected changes

■

RESULTS AND DISCUSSION Historically, EDGE cross-linking at room temperature has been attempted with hydroxypropyl β-CD (HPCD) and cationic hydroxyethyl cellulose (CHC); however, both HPCD and CHC showed no polymer formation at room temperature.22,23 In this investigation we successfully cross-linked CD with three different cross-linkers including EDGE in room temperature conditions, in order to avoid a heating process which might be destructive to either the CD polymers or the devices they are coating. Figure S1 shows the schematic of cross-linking CD with diglycidyl ethers. Chemically similar (“diffusion only”) molecular networks were also created using dextran as a control, since dextran is a linear rather than cyclic 1049

DOI: 10.1021/acs.bioconjchem.6b00721 Bioconjugate Chem. 2017, 28, 1048−1058

Article

Bioconjugate Chemistry

otaxanes with CD. This is confirmed at least in theory by chain length and CD pocket size, using molecular modeling (http:// interactions.cyclodextrin.net/web/docking, data not shown). The disappearance of thermal transitions for CD-PEGDGE526 at all temperatures tested could therefore be further attributed to the formation of pseudopolyrotaxanes between CD and PEGDGE526. XRD Analysis. XRD was utilized as a final chemical analysis method to confirm the pseudopolyrotaxane formation between CD and the higher length cross-linker. From Figure 4 it is

occurring upon pseudopolyrotaxane formation. From Figure 2, glass transitions for both dextran and CD polymers cross-linked

Figure 2. DSC thermograms of CD polymers cross-linked with three different cross-linkers and dextran polymers cross-linked with two cross-linkers. A significant variation of Tg was observed upon change of both prepolymer type and cross-linker.

with EDGE and PEGDGE200 were not highly evident, but were approximated at the inflection points in the data. When comparing the Tg of both dextran and CD polymers crosslinked with EDGE and PEGDGE200, a lower Tg with increasing PEG chain length is observed. The differences between dextran and CD can be attributed to the bulky nature of CD compared to dextran. In contrast, no thermal transition was observed for CD-PEGDGE526, which can again be explained by the presence of rotaxane architecture.25,26 For more direct understanding of chemical interactions, we used DMA to further elucidate temperature transitions. Dynamic Mechanical Analysis (DMA). We further confirmed the thermal transitions through DMA analysis (Figure 3). Figure 3 shows the plot of storage modulus (E′)

Figure 4. XRD patterns of CD polymers formed with different crosslinker length using the same mole ratio of polymer and cross-linker. The reduced semicrystalline peak intensity and existence of small peak at 13° in the case of CD-PEGDGE526 indicates the formation of pseudopolyrotaxane with the longer cross-linker.

evident that all cross-linked polymers showed a broad diffraction peak at 20°, which corresponds to CD. However, the peak intensity gradually reduced with increasing cross-linker chain length. A decrease in intensity is expected upon the formation of CD pseudopolyrotaxanes. However, demonstrating intensity shift with pseudopolyrotaxanes has been difficult to show with CD/PEG polymers due to high incidence of PEG dethreading from either β- or γ-CD’s.27 Something capable of blocking the dethreading, namely, a group larger than β- or γCD pocket, such as another CD as shown here, is required. However, the presence of glycidyl groups on both ends of the PEG cross-linker, used in this study, possibly delayed the dethreading of PEG chains, allowing reactions with free oligomeric CDs after pseudopolyrotaxanes formation. While initial PEG threading has been demonstrated in this other work,27 the addition of bulky CD groups to either end would prevent the dethreading seen when using unmodified PEGs. It is unlikely that a simultaneous reaction to CD occurs at both ends, so therefore we suspect that the PEGDGE526 crosslinker may first form CD pseudopolyrotaxanes, and then crosslink covalently with hydroxyls of CD oligomers or pure CDs (bulky stoppers), thus locking in the rotaxane structure as shown in the Figure 1. Alternatively, a partially reacted crosslinker conjugated on only one side will have reduced dethreading capability, allowing it to be locked in place by a second CD conjugation (again confirmed by molecular modeling, as above). Similar mechanisms have been proposed recently by the Harada group in the case of β-CD28,29 where CD-PPG complexes were covalently blocked with bulky stoppers. The decreased intensity of the broad crystalline peak around 20° and appearance of small peak at lower angle (13°), similar to what they and others have observed, could be from the threading of PEGDGE526 into CD.25,30

Figure 3. DMA analysis of CD polymers cross-linked with two different length cross-linkers. A significant change in Tg with the variation of cross-linking chain length is observed.

and loss modulus (E″) versus temperature of the CD polymers. Previously it has been shown that, at low frequencies, the temperature at a maximum value of E″ could be considered a polymer’s glass transition temperature (Tg).21 Figure 3 indicated that there is E″ transition for CD-PEGDGE200 at 50 °C, which supports the transition that is observed in DSC. From other literature, it is clear that when PEG chains make pseudopolyrotaxanes with CD, the peaks at higher temperatures disappear in thermal analysis.25 However, in the case of CD-PEGDGE200, the appearance of a weak peak at 50 °C may be because PEGDGE200 is too small to form pseudopolyr1050

DOI: 10.1021/acs.bioconjchem.6b00721 Bioconjugate Chem. 2017, 28, 1048−1058

Article

Bioconjugate Chemistry Mechanical Properties. The tensile strength and elongation at break for the different cross-linked samples was measured, and due to their unique properties we report the results of different mole ratio compositions of CDPEGDGE526 in Figure 5. The tensile strength of these cross-

Figure 5. Uniaxial stress/strain curves of CD polymers formed with two different mole ratios of cross-linkers. Significant variation of tensile strength and elongation at break was observed after changing the mole ratio of cross-linker.

linked CD polymers reflects a trend observed due to the length and composition of this cross-linker. Of all of the polymers that were prepared with Dex/CD and glycidylethers, the one crosslinked with PEGDGE526 showed the highest tensile strength and elongation at break, varying with cross-linker ratio. CDEDGE and CD-PEGDGE200 cross-linked polymers are too brittle to obtain reliable measurements on the Universal Testing Machine; therefore, we do not report them. The mechanical strength of the CD-PEGDGE526 (1:0.7) and CDPEGDGE526 (1:0.35) was measured after fabricating them into a dogbone shape. The stress−strain curves for these polymers are shown in Figure 5. The mechanical properties of these polymers vary with composition. The unexpected high mechanical properties showed by the CD-PEGDGE526 could again be due to the formation of inclusion complexes followed by covalent cross-linking and generation of pseudopolyrotaxane architecture. These mechanical properties are at least an order of magnitude higher than the comparable conventional polymers, prepared similarly for drug delivery and optimized for mechanical properties.1,16,31 Viscoelastic Measurements. The mechanical properties of these polymers were further confirmed through rheological experiments. To examine the influence of cross-linker chain length on rheological properties of the CD polymers, three types of polymers, each formed with the three different chain length cross-linkers, were used to determine the storage (G′) and loss (G″) modulus. For all tested polymers, viscoelastic properties were examined by conducting both frequency and stress sweep experiments. Due to the large difference in magnitudes, CD-PEGDGE526 results are presented separately from CD-PEGDGE200 and CD-EDGE. Figure 6a shows the G′ and G″ of the CD-PEGDGE526 polymer while varying angular frequency, while Figure 6b shows that of CD-EDGE and CDPEGDGE200 polymers. From Figure 6a,b a pronounced plateau of G′ and considerably smaller G″ in the full frequency range was observed. Higher values for G′ compared to G″ indicates the formation of strong polymers. From Figure 6b it is evident that in changing cross-linker length from EDGE to PEGDGE200 that there was a decrease in G′, in this case 2fold. The decrease in modulus with increase in cross-linker chain length could be due to introduction of more flexible

Figure 6. Dynamics of elastic (G′) and viscous (G″) moduli of CD networks cross-linked with three different cross-linkers. A clear distinction between elastic and viscous moduli in frequency sweep experiment indicates strong polymer formation. A significant change in the moduli is obtainable by changing cross-linker length.

chains in the polymer network. Interestingly, this is not true when moving to even longer chains, in the case of CDPEGDGE526, which instead of showing lower G′ values, showed very high G′ values compared to other two crosslinkers, at the same mole ratio of cross-linker. The anomalous behavior of CD-PEGDGE526 could be due to the inclusion complex architecture formation between CD and PEGDGE526 by forming hydrogen bonding and hydrophobic interactions, and further reducing the mobility that would otherwise be gained from the longer chains. The higher modulus values of CD-PEGDGE526 compared to the other two polymers is therefore due to this higher perceived crosslinking density (νe) and decreased molecular weight between cross-links (Mc) (Table 1 as calculated based on our previously published work.8) From Table 1 it is also evident that the calculated interaction parameters are fairly similar for CDEDGE, and CD-PEGDGE200 and the Dex-EDGE, diffusiononly control. CD-PEGDGE526 differs for several parameters, most notably though for a decreased χ observed. This study indicates that the mechanical properties of the CD polymers can be adjusted across orders of magnitude for desired applications32 by changing the cross-linker length. Stress sweep experiments conducted on three CD polymers (CD-EDGE, CD-PEGDGE200, and CD-PEGDGE526) are shown in Figure 7 a,b. In general, the modulus of CD-EDGE and CD-PEGDGE200 decreased with increasing shear stress. More specifically though, when the shear stress exceeded 1000 Pa the CD-EDGE polymer showed a sharp decrease in modulus, while the modulus of CD-PEGDGE200 withstood up to 1500 Pa, but began to drop gradually at 2000 Pa. Interestingly, the storage modulus of CD-PEGDGE526 showed a constant value under the applied shear stress, with only a small change detected. While mechanical properties of cyclodextrin polymers are not frequently reported, the G′ 1051

DOI: 10.1021/acs.bioconjchem.6b00721 Bioconjugate Chem. 2017, 28, 1048−1058

Article

Bioconjugate Chemistry Table 1. Mechanical Properties and Network Parameters for the Cross-Linked CD and Dex Polymers sample code

φ2

E′, MPa

G′, MPa

νe (mol/m3)

Mc (kg/mol)

χ

Dex-EDGE(1:0.7) CD-EDGE(1:0.7) CD-PEGDGE200(1:0.7) CD-PEGDGE526(1:0.7)

0.203 0.222 0.183 0.171

0.3060 0.2518 0.1090 3.840

0.102 0.0839 0.0363 1.2800

69.83 55.69 25.68 924.49

46.57 35.00 118.47 4.87

0.566 0.578 0.564 0.298

higher hydrophilicity of PEGDGE200 vs EDGE. Interestingly, in the case of CD-PEGDGE526, instead of increasing the swelling ratio further, we saw a decrease: again possibly due to the shielding of PEGDGE526 chains by CD molecules due to inclusion complexation of the cross-linker. Morphology of the Polymers. SEM analysis of polymer films is shown in Figure S5. Surface morphology of the polymer films varied somewhat, depending on the type of the crosslinker used. From Figure S5 it is evident that all polymers showed a smooth surface morphology. The surface morphology of diglycidyl ether cross-linked polymers is entirely different from polymers that we have previously reported using an isocyanate cross-linker.36 The isocyanate cross-linked polymers show a more porous morphology after lyophilization. However, glycidyl ether cross-linked polymers do not appear to be porous even though they have higher swelling ratios than isocyanate cross-linked polymers.8 Therefore, the type of cross-linker used can significantly affect the surface morphology of the CD polymers. This may be attributed to the state of the water present in the polymer systems. The water present in the polymer system has different states (e.g., bound water and free water). The ratio of bound water to free water varies depending on the chemical composition of polymer. For example, isocyanate cross-linked CD polymers may have more free water compared to diglycidyl ether cross-linked CD polymers. Since free water is easily removed, an isocyanate cross-linked CD polymer will result in morphology which is more porous than that of the diglycidyl ether cross-linked polymers. The nonporous morphology of diglycidyl ether cross-linked CD polymers may be due to the high water retention capacity of these polymers (with comparatively low free water) preventing easy water loss, and preventing formation of pores. Difference in Loading between Affinity and Nonaffinity Molecules. From swelling studies, the above data showed that 1 g of polymers absorbs 3−5 g of water. Therefore, a solution absorption method was used to load different concentrations of water-soluble antibiotic drugs into the crosslinked polymers, in order to determine the comparative potential that these materials had for maximum drug loading as compared to previously studied materials. In an effort to probe maximal loading, solutions of both drugs, each at four different concentrations (0.1%, 1%, 2%, and 5% w/v) were prepared and then Dex-EDGE and CD-EDGE polymers were loaded by incubating in each of the above drug solutions. Initially we chose to examine only these two polymers, as one represents diffusion-only release and the other represents affinity-based release. Also, these two representatives are potentially the most similar since they have near-identical swelling in water and they also possess a similar cross-linking density. Therefore, the drug loading differences obtained for these two polymers will mostly be explained by the difference between having and not having a high-affinity interaction, since all other parameters are same. The percent loading is displayed in Figure 8a. From Figure 8a it is evident that the percent loading into cross-linked polymers depends on several

Figure 7. Dynamics of elastic (G′) moduli of CD networks crosslinked with three different cross-linkers. The presence of a sharp drop of elastic moduli at higher stress value in the case of lower-length cross-linkers (EDGE and PEGDGE200) as opposed to a stable modulus when cross-linked with long chain cross-linkers (PEGDGE526) indicates formation of inclusion complexes between CD and the long chain length cross-linkers.

values observed in this work are higher than those of selfassembled CD-based polymers reported elsewhere.33 Due to the capacity of these polymers to withstand the higher stresses applied in rheological experiments, we confirm the contribution of covalent cross-links as well, unlike noncovalent cross-linking CD polymers.33,34 Previously, it was shown that pseudopolyrotaxane formation was possible with PEG chains of higher molecular weight30 and lower molecular weight of polypropylene glycols (PPG400).35 In both cases, pseudopolyrotaxane dethreading was prevented by end-capping them with suitable molecules. Similarly, the polymer in this study, CDPEGDGE526, was formed by both inclusion and end-capping (in this case epoxy groups are reacting with bulky CD molecules and blocking the dethreading of PEGDGE). Swelling Studies of the Polymers. By varying the size of the cross-linker added to CD, we were able to influence significant changes to its mechanical properties; however, we still needed to examine how this would impact drug loading and release properties. Swelling is one of the most important parameters in controlling drug release. Therefore, for a more accurate comparison between diffusion-only drug release systems (in this case dextran) and affinity-based release systems (in this case CD), the swelling of these polymers should be set fairly similar to each other. We therefore optimized the polymer synthesis conditions to get the same swelling ratios for DexEDGE and CD-EDGE and applied the same conditions to prepare CD-PEGDGE200 and CD-PEGDGE526. From Figure S4 it is evident that both the Dex-EDGE and CD-EDGE swelling ratios are nearly identical; however, under the same synthesis conditions the swelling ratio increased in the synthesis of CD-PEGDGE200 which may be due to the (comparatively) 1052

DOI: 10.1021/acs.bioconjchem.6b00721 Bioconjugate Chem. 2017, 28, 1048−1058

Article

Bioconjugate Chemistry

CDs are lost to drug loading since they are used in pseudopolyrotaxane formation), reducing the ability of drug interaction with CD, and therefore the percentage loaded. This is seen in physically cross-linked CD polymers where some CD groups are consumed in formation of the physical cross-links. However, even with this loss, the drug loading of the CDPEGDGE526 materials never quite reaches that of the dextran polymers indicating that there are still some CDs available for drug complexation. Drug Release Studies. Regulation of drug release in these materials depends on several parameters including hydrophilicity of the drug, interaction of the drug and polymer (diffusion-only, or affinity interaction), swelling, and possibly degradation of the polymer networks. In the present system, the impact on swelling and the affinity interaction in CD polymers, allows substantial control over drug release. For drug release experiments we examined Dex-EDGE, CD-EDGE, and CD-PEGDGE526 polymers loaded with 5% VM and NB, again to best observe the difference between the diffusion-only release and affinity-based release. Figure 9 shows the VM and Figure 8. Effect on loading efficiency of drug concentrations during loading (0.1%, 1%, 2%, 5% w/v drug solution), with varying drug choice and cross-linker (EDGE, PEGDGE200, PEGDGE526). A and B represent loading for only EDGE cross-linked polymers across all loading concentrations. C represents loading for all cross-linkers across one drug loading concentration (5%). Experiments were done in triplicate (n = 3) and error bars represent ± standard deviation. NB = novobiocin, VM = vancomycin.

parameters such as the type of polymer, the type of drug, and the concentration of the drug solution. Although both dextran and CD polymers have the same level of swelling ratio, they differed in drug loading in all the concentrations tested. Dextran polymers in general showed lower loading in all the concentrations used in comparison to CD polymers. Interestingly, while loading in dextran polymers eventually plateaued and did not seem to continue to increase with increasing drug concentration, this was not the case for CD polymers. For CD polymers the drug loading continued to increase even to the maximum limit of drug solubility (∼5% for each drug). The continued increase of drug loading into only the CD polymers is presumed to be due to specific complexation of drug into CD molecules, in addition to the nonspecific placement of drug in and around polymer chains. It is also evident from Figure 8b that novobiocin (NB) loading is higher in both dextran and CD polymers when compared to vancomycin (VM) loading. This is presumed to be due to the more hydrophobic nature of NB compared to VM. We have previously reported that NB has a higher affinity than VM toward CD,36 which here results in higher ultimate loading. To compare the percentage loading across groups of polymers that were cross-linked with different chain length cross-linkers, only the 5% drug solution was used to load this group of polymers. Figure. 8c displays the percentage total loading of these different cross-linked polymers. From Figure 8c it is clear that drug loading is higher in CD polymers when compared to dextran polymers; however, this difference is most significant in NB loaded polymers. Within the CD polymers, the CD-PEGDGE526 polymers had lower loading when compared to the other two polymers. The poor loading of CD-PEGDGE526 can again be attributed to reduction of the number of CD available for complexation (as we presume some

Figure 9. Effect of the drug concentration and type of cross-linker and drug on drug release from dextran and CD based polymers in PBS, pH 7.4 at 37 °C. 5% VM loaded Dex-EDGE and CD polymers, crosslinked with two different cross-linkers. Dextran loaded with VM polymers showed a rapid, burst release. However, drug release from CD polymers showed a reduced burst followed by longer period of gradual release, varying depending on drug and cross-linker length.

NB release from Dex-EDGE, CD-EDGE, and CDPEGDGE526 polymers. From Figure 9, it is evident that the dextran polymers release at a faster rate than the respective drugs release from CD polymers. When comparing drug release within the CD polymers, VM is released at a faster rate than NB. This can be attributed to the more hydrophilic nature of VM leading it to make a less suitable inclusion complex with CD. From Figure 9 it is also evident that the release takes place in two phases. In the first phase there is a rapid burst release, followed by a slower release in the second phase. In the burst release phase most of the drug from dextran polymers had been released; however, in the case of CD polymers, depending on the setting, only 60−75% of the drug was released in the first phase and the remaining drug is released in a considerably more sustained manner depending on the hydrophobicity of the drug. For example, VM is released more rapidly in all the CD polymers when compared to NB probably due to a difference in hydrophobicity and corresponding complex formation. Although both dextran and CD were carefully cross-linked to the same extent, the slower rate of drug release from CD polymers observed in the two drugs studied could primarily be attributed to the formation of inclusion complex with CD. As 1053

DOI: 10.1021/acs.bioconjchem.6b00721 Bioconjugate Chem. 2017, 28, 1048−1058

Article

Bioconjugate Chemistry

polymer extracts were added to cultured cells. Specifically, NIH3T3 fibroblast cells were used in culture in the presence of the polymer extracts over 24 h at 37 °C. After 24 h the latent toxicity of the polymer extracts was determined by conducting a cell viability (MTS) assay. The MTS assay results, showing all conditions being statistically similar to controls (Figure S6), indicated that all polymer extracts capable of eluting in aqueous solution within the first 24 h are, at least indirectly, nontoxic. Direct cytotoxicity of the polymers was then explored by examining cells cultured directly on the materials. NIH3T3 cells were seeded onto the various polymers to investigate cell attachment and proliferation. As seen in Figure 11, cell adhesion and proliferation depends on the type of

expected, the drug release rate is also faster in CDPEGDGE526 than the other polymers cross-linked with EDGE, even though the swelling of these polymers (Figure S1) is higher than that of CD-PEGDGE526 cross-linked polymers. This can again be possibly explained by the presence of PEGDGE526 cross-linker in the pockets of some of the CDs which may reduce the total number of CDs available for complexation. Previously, release of antibiotics has been examined by exploiting the complexation with CD to improve the solubility, stability, and controlled release properties. However, these previously reported formulations can only be used in limited applications for coating biomedical implants/devices, as they entail a caustic approach (high temperature, solvent use, etc.).37 In contrast, the polymers that were developed herein possess good mechanical and sustained release properties that can be utilized for device coating in a mild approach, and still maintain favorable release properties. Antibacterial Activity of Drug Loaded Polymers. A zone-of-inhibition assay (Kirby-Bauer Assay) was conducted to check the difference in microbicidal activity between antibiotic loaded dextran and CD polymers. Zone of inhibition studies provide further demonstration of the difference between the dextran and CD polymers, in this case in the ability to inhibit bacterial growth. For this study, drug-loaded polymers were punched into 8 mm polymer disks initially using Dex-EDGE and CD-PEGDGE526, and were placed in agar plates spread with S. aureus, which is the most common type of bacteria to cause device failure due to infection. The number of days the polymer disks inhibited bacterial growth is represented in Figure 10. Although the release studies comparing Dex-EDGE

Figure 11. Cell adhesion property of cross-linked polymers, using NIH3T3 cells. Cells were seeded at either low density (A−F) or high density (G−I). At low cell densities, the materials used are (A) CDEDGE(1:0.7) polymer, (B) CD-PEGDGE200(1:0.7) polymer, (C) tissue culture polystyrene substrate under the CD-EDGE(1:0.7) polymer, (D) Dex-EDGE(1:0.7) polymer, (E) DexPEGDGE200(1:0.7) polymer, and (F) the tissue culture polystyrene control. At the high cell densities cells were grown on (G) DexEDGE(1:0.7) polymer, (H) Dex-EDGE200(1:0.7) polymer, (I) the tissue culture polystyrene control, and corresponding CD materials (data not shown). Cells do not attach to CD materials at either high or low densities, but remain viable as indicated by good cell attachment on the underlying tissue culture polystyrene substrate. At low cell densities on the dextran-based materials cells showed noticeable attachment, however, not as robust as the polystyrene control. At higher cell densities the cells detached from the dextran surfaces into aggregates, confirming the weak adhesion to the polymer and preference of cell−cell interactions to cell−material interactions.

Figure 10. Bactericidal activity of VM-loaded dextran or CDPEGDGE526 delivery polymers against S. aureus. The cleared zone size (mm) and the number of days cleared is plotted. The VM-loaded dextran polymers stop clearing the bacteria beyond 10 days, whereas VM-loaded CD polymers continuously showed a zone of inhibition until 20 days.

polymer and density of the cells used for culturing on the polymer films. At low cell densities (5.0 × 103 cells/well in a 48 well plate) the NIH3T3 cells attached and proliferated on DexEDGE (Figure 11d), Dex-PEGDGE200 (Figure 11e), and the control, unmodified, tissue culture polystyrene well (Figure 11f). However, when a higher cell density was used (1.0 × 104 cells/well) the cells were not as well-adhered and appeared aggregated on the dextran surfaces (Figure 11g−i). In contrast, no cell adhesion was observed on CD-EDGE (Figure 11a) and CD-PEGDGE200 (Figure 11b) polymers at either low or high cell densities (high density cultures on CD surfaces not shown). Cells were still viable, as they could still be seen around the polymer, attached to the substrate culture dish (Figure 11c). Figure 11g and h shows the aggregated cell morphology on dextran polymers, while Figure 11i shows that even at this

and CD-PEGDDE526 polymers were chosen in part because they showed the smallest difference in release rates when loaded with VM, this small difference had a pronounced effect in inhibiting bacterial growth. Dextran polymers were able to clear bacteria for only 10 days, while even the CD polymer with the fastest delivery time was able to clear bacteria for at least 20 days. Cell Adhesion on CD and Dextran Polymers. A principal feature of polymeric materials for use in drug delivery and device coatings is that they should not be toxic by themselves (direct) or release cytotoxic species (indirect). To test the indirect cytotoxicity of the polymers in these studies, 1054

DOI: 10.1021/acs.bioconjchem.6b00721 Bioconjugate Chem. 2017, 28, 1048−1058

Bioconjugate Chemistry

■

higher density, cell morphology on a control tissue-culture polystyrene dish is comparatively normal. This experiment is the first to demonstrate that cells show low to no adhesion to CD polymers when seeded with both low and high numbers of cells, but can adhere to dextran polymers when seeded at a low enough density. At higher densities the comparatively poor adhesion even to the dextran polymer gives way to cell−cell adhesions, resulting in cell detachment and aggregation. This reduced cell adhesion which we found in CD polymers may be comparable to that seen in PEG polymers,38 biological environments (such as the glycocalyx on the endothelial lumen), and mimics thereof using other polysaccharides such as maltose.39 We feel that this opens the door for cell and perhaps protein resistant coatings; however, CD coatings have an additional advantage over PEG polymers used as bioactive modification, since CD has the potential for inclusion complexation with various small and larger molecules, which PEG and linear polysaccharides do not.

■

Article

EXPERIMENTAL SECTION

Materials. Soluble β-cyclodextrin prepolymer (CD, 2−15 kDa, lightly epichlorohydrin cross-linked with an average 10 CDs per chain) was purchased from Cyclodextrin Technologies Development (CTD) Inc., (High Springs, FL); dextran (Dex, 15−20 kDa) was obtained from Polysciences, Inc. (Warrington, PA). Ethylene glycol-diglycidyl ether (EDGE), poly(ethylene glycol) diglycidyl ether (PEGDGE, Mn = 200), and poly(ethylene glycol) diglycidyl ether (PEGDGE, Mn = 526) was purchased from Laysan Bio, Arab, AL. Novobiocin sodium salt (NB) and vancomycin hydrochloride (VM) were purchased from MP Biomedicals, Inc. (Solon, OH). All other reagents were purchased from Fisher Scientific and used as received. Cross-Linking of Cyclodextrin and Dextran. CD polymers were prepared by cross-linking with three types of cross-linkers under mild conditions. Briefly, 25% w/w CD solution in 0.2 M KOH was prepared under stirring. Known amounts of EDGE, PEGDGE200, and PEGDGE526 were added to the above solution to become CD:EDGE, CD:PEGDGE200, and CD:PEGDGE526 at a molar ratio of 1:0.7. These CD solutions containing each of the three different types of cross-linkers were mixed for 2 min separately, cast into a Teflon Petri dish, and kept at room temperature for 4 days to facilitate polymer cross-linking. The cross-linked polymers were punched into different-sized discs as needed and kept in distilled water for 3 days to remove unreacted cross-linker and other impurities. The water was replaced twice per day. Crosslinking of dextran was carried out in a manner similar to that of CD, however, primarily only with EDGE. The synthesized polymers were designated as Dex-EDGE (1:0.7), CD-EDGE (1:0.7), CD-PEGDGE200 (1:0.7), and CD-PEGDGE526 (1:0.7). The 1:0.7 is indicated as the mole ratio of CD:crosslinker. In mechanical studies an additional polymer, CDPEGDGE526 (1:0.35), with a 1:0.35 mole ratio of CD:crosslinker was generated to further explore the unique mechanical properties of these unique materials, while in cell culture studies and DSC studies another additional polymer, DexPEGDGE200 (1:0.7), was generated to further explore conformation and cell adhesion on nonaffinity controls. 1H NMR analysis of CD, PEGDGE526, and CD-PEGDGE526 in DMSO-d6 was carried out using a Varian Unity-300 (300 MHz, Varian Inc., CA, USA). All the peaks were labeled on the spectra (Figure S3). Characterization of the Polymers. Differential scanning calorimetry (DSC) analyses for both Dex and CD polymers were performed using TA Instruments Q100 DSC. A sample weight of 5 mg was placed in an aluminum pan, sealed hermetically, and heated at a rate of 10°/min in a temperature range from 25 to 250 °C under a nitrogen atmosphere. The temperature dependence of the dynamic mechanical (DMA) properties was obtained using a TA Instruments Q800 under a dry nitrogen purge in tensile mode across a temperature range of −10 to 100 °C. Samples were cooled to −10 °C and data were subsequently taken at a test frequency of 1 Hz and a heating rate of 2 °C/min. The dried cross-linked polymer films were used for X-ray diffraction analysis using a Scintag X-1 XRD instrument (Cuppertino, CA, USA) at room temperature. The tube was operated at 40 kV and 40 mA. Monochromated Cu Kα radiation (λ = 0.154 nm) was directed incident to the sample in a conventional horizontal-axis configuration with a scan rate of 2°/min for 2θ = 5−35°, a divergent slit of 2 mm (1°), using a

CONCLUSIONS

In this study, we have synthesized a series of covalently crosslinked CD network polymers displaying different mechanical properties without compromising the drug loading and release rates available due to affinity interactions. Mechanical properties can be further tuned by varying the composition and length of the cross-linker. The drug release and antibacterial activity of these antibiotic loaded polymer networks can be prolonged beyond that capable of chemically similar control polymer systems relying on diffusion alone. The various polymers in this study showed low cytotoxicity in both direct and indirect observations. Cells do not adhere to the CD-based polymer platforms but remain viable. The CD polymers developed in this study may be applied to a broad range of biomedical applications, in cases where controlled cell adhesion and prolonged drug delivery are required. The low cell adhesion property makes them particularly useful for tailored adhesion, growth, and binding properties in tissue engineering applications similar to that accomplished with customized PEG polymers. Additional advantages of the materials described herein are their high mechanical properties and their capacity to incorporate both small and larger, more complex molecules through inclusion formation. More specifically, the polymers in this study can also be useful for coating devices to prevent bacterial adhesion and deliver drugs to prevent bacterial infection and biofilm formation, one of the leading causes of medical device failure. The larger-sized cross-linker examined herein favored the formation of pseudopolyrotaxanes, the formation of which is supported by miscibility, NMR, DSC, DMA, and XRD analyses. Moreover, the network formed with CD-PEGDGE526 is more stable compared to other cross-linked polymers since it can withstand very high stress values and showed high modulus throughout the studied frequency in rheological experiments. This preponderance of evidence suggests that pseudopolyrotaxane formation occurred, and that when followed by covalent cross-linking this gave a robust network with high flexibility compared to other polymers cross-linked with lower chain length cross-linkers, and which do not have enough chain length to make pseudopolyrotaxanes. 1055

DOI: 10.1021/acs.bioconjchem.6b00721 Bioconjugate Chem. 2017, 28, 1048−1058

Article

Bioconjugate Chemistry

mL phosphate buffer saline (PBS, pH 7.4) with each disk in its own 20 mL vial. The sample vials were incubated at 37 °C while shaking at 100 rpm. At periodic intervals, an aliquot of the drug release medium was withdrawn for drug concentration determination and, in all cases, replaced with the same volume of fresh PBS. The collected samples containing NB and VM were analyzed spectrophotometrically at 306 and 282 nm, respectively, using a Tecan Safire UV/vis plate reader. Similarly, absorbance from blank samples (from polymers without drug) as a function of time was systematically measured and subtracted from the absorbance values of samples from drugloaded polymers. The amount of NB and VM released from the polymers in release medium, at a given time, was calculated using standard curves of each drug in corresponding buffers and expressed as a percentage of total drug content of the investigated polymers. Each drug release experiment was repeated three times (n = 3). The antibacterial activity of Dex-EDGE(1:0.7)-VM and CDPEGDGE526(1:0.7)-VM polymer disks was determined against Staphylococcus aureus (S. aureus kindly provided by Dr. Ed Greenfield, Case Western Reserve University) using a nutrient agar method as described previously.8 Briefly, tripticase soy broth and granulated agar were added into water in a 1:2 weight ratio to make a 4.5% (w/v) solution. The solution was autoclaved, and then poured into Petri dishes and air-dried under sterile conditions. To each plate a 50 μL of freshly grown S. aureus was added and spread over the plate using a sterile glass spreader. Immediately, drug-loaded disks (8 mm in diameter) were placed on the inoculated plate and incubated overnight at 35 °C. The bactericidal activity was observed as the size of the clearance as measured by calipers across an average diameter. Drug-loaded pellets were moved daily to a new lawn using the same process described above. This process was repeated until no zone of inhibition was observed. The CD and dextran polymers were prepared in a 96 well plate by adding 150 μL of 25% of CD/dextran solution containing the required amount of EDGE or PEGDGE200. Once the solutions were cast, the plates were completely closed and allowed to cure for 4 days as described in the polymer synthesis section above. After 4 days the formed polymer disks were washed with deionized water several times, and UV sterilized. The resultant polymer disks were rinsed with DMEM solution containing 5% fetal bovine serum (FBS) and maintained in aseptic conditions. Finally, 200 μL DMEM + FBS was added to each well and kept for 24 h for extraction. NIH3T3 cells (ATCC.org, Manassas, VA) were seeded at a density of 1.0 × 105 cells/well into new wells containing 100 μL of the respective polymer extracts. Wells containing only cells and normal culture medium served as controls. The cells were incubated for 24 h at 37 °C with 5% CO2. At the end of the exposure time, cell viability was measured (n = 4) using an MTS Assay according to protocol (Invitrogen, Grand Island, NY). Absorbance was measured at 490 nm and values relative to control were reported. The cell adhesion and proliferation on both CD and dextran polymers were studied using NIH3T3 fibroblast cells. CD and dextran polymers were prepared as above, but in a 48 well format, followed by extensive washing with water and DMEM solution, and UV sterilization. Wells without polymers were used as a control. NIH3T3 cells were seeded at low (1.0 × 104 cells/well) and high (5 × 103 cells/well) densities and incubated at 37 °C with 5% CO2 for 24 h. Attached cells

scattering slit of 1 mm (1°) and receiving slit of 0.5 mm. Tensile strength of the CD-PEGDGE526 with two different mole ratios was measured using a tensile testing machine (Instron Model 1125 Universal Testing apparatus) with a crosshead speed of 10 mm/min. Before analysis, the samples were formed into a dogbone shape with a gauge length of 15 mm, and a thickness of 0.8 mm using a dogbone-shaped die. For each sample, four specimens were tested. The surface morphology of lyophilized cross-linked polymer samples was studied using a scanning electron microscope (SEM) (Hitachi S4500, Japan). Before SEM examination, all specimens were fixed on a brass stub using double-sided tape and coated with palladium for 10s using a Fine Coater. The water absorption capacity of the polymers was determined gravimetrically. The dried weight (Wd) of several 8-mm-diameter polymer disks was measured. The disks were added into distilled water. The swollen weight (Ws) of the polymer disks was determined after 24 h. The swelling ratio (Q) was calculated from the following equation Q = (Ws − Wd)/Wd. These studies were performed in triplicate (n = 3) with the average values reported. The polymer volume fraction, φ2, at equilibrium swelling at 25 °C was calculated based on our previously reported work.8,21 An AR2000(ex) Rheometer (TA Instruments) was used to generate rheometry measurements applying stainless steel, parallel plate geometry, with a 12 mm plate and 1 mm gap size. A solvent trap was used to prevent evaporation of the solvent. Two different rheological tests were conducted to determine both storage (G′) and loss (G″) modulus of the swollen polymer disks by applying a sinusoidal shear stress, τ. The stress sweep experiment was performed at a constant frequency of 1 Hz, in the stress range of 0 to 10 000 Pa and the frequency sweep study was conducted at a constant stress of 5 Pa in the frequency range of 0.1 to 160 Hz at 25 °C. All the rheological tests were conducted in triplicate and average results are presented. The polymer network parameters were determined from the G′ values as previously reported.21 Antibiotics, either novobiocin (NB) or vancomycin (VM), were loaded into cross-linked polymer disks using a common solvent/solution absorption method. Four different loading concentrations (0.1, 1, 2, and 5 wt % each) of VM and NB were prepared in water. Polymer disks were incubated in one of the concentrated drug solutions separately at room temperature for 48 h. After 48 h the drug-loaded disks from each drug solution were taken out, blotted, and air-dried first, followed by vacuum drying at room temperature. The dried drug-loaded polymer disks were used for drug release after briefly washing 3 times with pure water to remove surface-adsorbed drug. The drugloaded samples were coded as follows. For the VM samples: Dex-EDGE(1:0.7)-VM, CD-EDGE(1:0.7)-VM, CDPEGDGE200(1:0.7)-VM, and CD-PEGDGE526(1:0.7)-VM; and for the NB samples: Dex-EDGE(1:0.7)-NB, CDEDGE(1:0.7)-NB, CD-PEGDGE200(1:0.7)-NB, and CDPEGDGE526(1:0.7)-NB. The percentage loading of NB and VM was determined spectrophotometrically at 306 and 282, respectively, after extensive extraction in water. Loading percentage was calculated as percentage loading = Wdrug /(Wsample + Wdrug)× 100

where Wdrug and Wsample are weight of the drug and weight of the polymer sample, respectively. The in vitro drug release rate was measured from all the drug loaded polymer disks by placing the drug loaded disks into 10 1056

DOI: 10.1021/acs.bioconjchem.6b00721 Bioconjugate Chem. 2017, 28, 1048−1058

Article

Bioconjugate Chemistry

Tunable Affinity-Based Drug Delivery Platform. Ann. Biomed. Eng. 39 (9), 2466−2475. (11) Thatiparti, T. R., Averell, N., Overstreet, D., and von Recum, H. A. (2011) Multiplexing Interactions to Control Antibiotic Release from Cyclodextrin Polymers. Macromol. Biosci. 11 (11), 1544−1552. (12) Yamamoto, Y., and Tagawa, S. (2004) Radiolytically prepared poly(vinyl alcohol) polymer containing alpha-cyclodextrin. Radiat. Phys. Chem. 69 (4), 347−349. (13) Xu, J., Li, X., Sun, F., and Cao, P. (2010) PVA Polymers Containing beta-Cyclodextrin for Enhanced Loading and Sustained Release of Ocular Therapeutics. J. Biomater. Sci., Polym. Ed. 21 (8−9), 1023−1038. (14) Liu, Y., Fan, X., Kang, T., and Sun, L. (2004) A cyclodextrin micropolymer for controlled release driven by inclusion effects. Macromol. Rapid Commun. 25 (22), 1912−1916. (15) Zhang, J., Huang, S., Gao, F., and Zhuo, R. (2005) Novel temperature-sensitive, beta-cyclodextrin-incorporated poly(N-isopropylacrylamide) polymers for slow release of drug. Colloid Polym. Sci. 283 (4), 461−464. (16) Daoud-Mahammed, S., Ringard-Lefebvre, C., Razzouq, N., RoSilio, V., Gillet, B., Couvreur, P., Amiel, C., and Gref, R. (2007) Spontaneous association of hydrophobized dextran and poly-betacyclodextrin into nanoassemblies. Formation and interaction with a hydrophobic drug. J. Colloid Interface Sci. 307 (1), 83−93. (17) Li, J., Ni, X., and Leong, K. (2003) Injectable drug-delivery systems based on supramolecular polymers formed by poly(ethylene oxide) and alpha-cyclodextrin. J. Biomed. Mater. Res. 65A (2), 196− 202. (18) Huh, K., Ooya, T., Lee, W., Sasaki, S., Kwon, I., Jeong, S., and Yui, N. (2001) Supramolecular-structured polymers showing a reversible phase transition by inclusion complexation between poly(ethylene glycol) grafted dextran and alpha-cyclodextrin. Macromolecules 34 (25), 8657−8662. (19) Abu Hashim, I. I., Higashi, T., Anno, T., Motoyama, K., AbdElGawad, A. E., El-Shabouri, M. H., Borg, T. M., and Arima, H. (2010) Potential use of gamma-cyclodextrin polypseudorotaxane polymers as an injectable sustained release system for insulin. Int. J. Pharm. 392 (1−2), 83−91. (20) Harth, K. C., Rosen, M. J., Thatiparti, T. R., Jacobs, M. R., Halaweish, I., Bajaksouzian, S., Furlan, J., and von Recum, H. A. (2010) Antibiotic-Releasing Mesh Coating to Reduce Prosthetic Sepsis: An In Vivo Study. J. Surg. Res. 163 (2), 337−343. (21) Reddy, T. T., Kano, A., Maruyama, A., Hadano, M., and Takahara, A. (2008) Thermosensitive transparent semi-interpenetrating polymer networks for wound dressing and cell adhesion control. Biomacromolecules 9 (4), 1313−21. (22) Rodriguez-Tenreiro, C., Alvarez-Lorenzo, C., Rodriguez-Perez, A., Concheiro, A., and Torres-Labandeira, J. (2006) New cyclodextrin polymers cross-linked with diglycidylethers with a high drug loading and controlled release ability. Pharm. Res. 23 (1), 121−130. (23) Rodriguez, R., Alvarez-Lorenzo, C., and Concheiro, A. (2003) Cationic cellulose polymers: kinetics of the cross-linking process and characterization as pH-/ion-sensitive drug delivery systems. J. Controlled Release 86 (2−3), 253−265. (24) Li, J., Ni, X., Zhou, Z., and Leong, K. W. (2003) Preparation and characterization of polypseudorotaxanes based on block-selected inclusion complexation between poly(propylene oxide)-poly(ethylene oxide)-poly(propylene oxide) triblock copolymers and alpha-cyclodextrin. J. Am. Chem. Soc. 125 (7), 1788−95. (25) Abu Hashim, I., Higashi, T., Anno, T., Motoyama, K., Abd-El Gawad, A., El-Shabouri, M., Borg, T., and Arima, H. (2010) Potential use of gamma-cyclodextrin polypseudorotaxane polymers as an injectable sustained release system for insulin. Int. J. Pharm. 392 (1− 2), 83−91. (26) Farcas, A., Jarroux, N., Guegan, P., Fifere, A., Pinteala, M., and Harabagiu, V. (2008) Polyfluorene Copolymer with a Multiply Blocked Rotaxane Architecture in the Main Chain: Synthesis and Characterization. J. Appl. Polym. Sci. 110 (4), 2384−2392.

were observed by phase contrast microscope after 24 h (Nikon TE300 with a Q-Imaging Retiga camera). Statistical Analysis. All data was processed using Microsoft Excel 2003 software and the results were plotted as mean ± standard deviation of three experiments, unless otherwise stated. Statistical analyses were performed using one-way ANOVA with Minitab (Minitab Inc., State College PA, U.S.A). A p-value smaller than 0.05 was considered statistically significant.

■

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.6b00721. Chemistry of polymer synthesis, 1H NMR of CD and cross-linker formulations, difference in clarity of solutions between CD and dextran with PEGDGE526 cross-linker, swelling, morphology, and cytotoxicity of CD polymers (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Horst A. von Recum: 0000-0002-9323-8100 Notes

The authors declare no competing financial interest.

■ ■

ACKNOWLEDGMENTS The authors would like to acknowledge support from the Coulter-Case Translation and Innovation Partnership. REFERENCES

(1) Jeon, O., Song, S., Lee, K., Park, M., Lee, S., Hahn, S., Kim, S., and Kim, B. (2007) Mechanical properties and degradation behaviors of hyaluronic acid polymers cross-linked at various cross-linking densities. Carbohydr. Polym. 70 (3), 251−257. (2) Mandal, T. K., Bostanian, L. A., Graves, R. A., and Chapman, S. R. (2002) Poly(D,L-lactide-co-glycolide) encapsulated poly(vinyl alcohol) polymer as a drug delivery system. Pharm. Res. 19 (11), 1713−9. (3) Wu, L., and Brazel, C. S. (2008) Modifying the release of proxyphylline from PVA polymers using surface crosslinking. Int. J. Pharm. 349 (1−2), 144−51. (4) Ruiz, J., Mantecon, A., and Cadiz, V. (2002) Investigation of loading and release in PVA-based polymers. J. Appl. Polym. Sci. 85 (8), 1644−1651. (5) Wang, N. X., and von Recum, H. A. (2011) Affinity-based drug delivery. Macromol. Biosci. 11 (3), 321−32. (6) Bibby, D. C., Davies, N. M., and Tucker, I. G. (2000) Mechanisms by which cyclodextrins modify drug release from polymeric drug delivery systems. Int. J. Pharm. 197 (1−2), 1−11. (7) Rodriguez-Tenreiro, C., Alvarez-Lorenzo, C., Rodriguez-Perez, A., Concheiro, A., and Torres-Labandeira, J. J. (2007) Estradiol sustained release from high affinity cyclodextrin polymers. Eur. J. Pharm. Biopharm. 66 (1), 55−62. (8) Thatiparti, T., Shoffstall, A., and von Recum, H. (2010) Cyclodextrin-based device coatings for affinity-based release of antibiotics. Biomaterials 31 (8), 2335−2347. (9) Stella, V. J., and Rajewski, R. A. (1997) Cyclodextrins: their future in drug formulation and delivery. Pharm. Res. 14 (5), 556−67. (10) Fu, A. S., Thatiparti, T. R., Saidel, G. M., and von Recum, H. A. (2011) Experimental Studies and Modeling of Drug Release from a 1057

DOI: 10.1021/acs.bioconjchem.6b00721 Bioconjugate Chem. 2017, 28, 1048−1058

Article

Bioconjugate Chemistry (27) Harada, A. (1996) Preparation and structures of supramolecules between cyclodextrins and polymers. Coord. Chem. Rev. 148, 115−133. (28) Sakamoto, K., Takashima, Y., Yamaguchi, H., and Harada, A. (2007) Preparation and properties of rotaxanes formed by dimethylbeta-cyclodextrin and oligo(thiophene)s with beta-cyclodextrin stoppers. J. Org. Chem. 72 (2), 459−465. (29) Okada, M., and Harada, A. (2004) Preparation of betacyclodextrin polyrotaxane: Photodimerization of pseudo-polyrotaxane with 2-anthryl and triphenylmethyl groups at the ends of poly(propylene glycol). Org. Lett. 6 (3), 361−364. (30) Loethen, S., Ooya, T., Choi, H. S., Yui, N., and Thompson, D. H. (2006) Synthesis, characterization, and pH-triggered dethreading of alpha-cyclodextrin-poly(ethylene glycol) polyrotaxanes bearing cleavable endcaps. Biomacromolecules 7 (9), 2501−6. (31) Kloxin, C. J., Scott, T. F., Park, H. Y., and Bowman, C. N. (2011) Mechanophotopatterning on a Photoresponsive Elastomer. Adv. Mater. 23 (17), 1977−1981. (32) Vanderhooft, J. L., Alcoutlabi, M., Magda, J. J., and Prestwich, G. D. (2009) Rheological properties of cross-linked hyaluronan-polymeratin polymers for tissue engineering. Macromol. Biosci. 9 (1), 20−8. (33) Daoud-Mahammed, S., Couvreur, P., Bouchemal, K., Cheron, M., Lebas, G., Amiel, C., and Gref, R. (2009) Cyclodextrin and polysaccharide-based nanopolymers: entrapment of two hydrophobic molecules, benzophenone and tamoxifen. Biomacromolecules 10 (3), 547−54. (34) Daoud-Mahammed, S., Grossiord, J. L., Bergua, T., Amiel, C., Couvreur, P., and Gref, R. (2008) Self-assembling cyclodextrin based polymers for the sustained delivery of hydrophobic drugs. J. Biomed. Mater. Res., Part A 86 (3), 736−48. (35) Ooya, T., Inoue, D., Choi, H. S., Kobayashi, Y., Loethen, S., Thompson, D. H., Ko, Y. H., Kim, K., and Yui, N. (2006) pHresponsive movement of cucurbit[7]uril in a diblock polypseudorotaxane containing dimethyl beta-cyclodextrin and cucurbit[7]uril. Org. Lett. 8 (15), 3159−62. (36) Thatiparti, T. R., and von Recum, H. A. (2010) Cyclodextrin Complexation for Affinity-Based Antibiotic Delivery. Macromol. Biosci. 10 (1), 82−90. (37) Blanchemain, N., Haulon, S., Boschin, F., Traisnel, M., Morcellet, M., Martel, B., and Hildebrand, H. F. (2007) Vascular prostheses with controlled release of antibiotics Part 2. In vitro biological evaluation of vascular prostheses treated by cyclodextrins. Biomol. Eng. 24 (1), 143−8. (38) Lin, C. C., and Anseth, K. S. (2009) Controlling Affinity Binding with Peptide-Functionalized Poly(ethylene glycol) Polymers. Adv. Funct. Mater. 19 (14), 2325. (39) Ruegsegger, M. A., and Marchant, R. E. (2001) Reduced protein adsorption and platelet adhesion by controlled variation of oligomaltose surfactant polymer coatings. J. Biomed. Mater. Res. 56 (2), 159−167.

1058

DOI: 10.1021/acs.bioconjchem.6b00721 Bioconjugate Chem. 2017, 28, 1048−1058