Role of Oligoethylene Glycol Side Chain Length in Responsive

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Article Cite This: Langmuir 2019, 35, 7929−7936

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Role of Oligoethylene Glycol Side Chain Length in Responsive Polymeric Nanoassemblies Kishore Raghupathi,†,⊥ Vikash Kumar,†,⊥ Uma Sridhar,† Alexander E. Ribbe,‡,∥ Huan He,† and S. Thayumanavan*,†,§,∥ †

Department of Chemistry, ‡Department of Polymer Science and Engineering, §Molecular and Cellular Biology Program, and Center for Bioactive Delivery, Institute for Applied Life Sciences, University of Massachusetts, Amherst, Massachusetts 01003, United States

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S Supporting Information *

ABSTRACT: An oft-desired feature of a responsive nanomaterial is that it should undergo disassembly or morphological change upon application of a specific stimulus. The extent of response has been found to depend on factors such as the nature and the number of responsive functionalities incorporated into these particles. In this work, the length of oligoethylene glycol (OEG) side chains associated with the polymers has been shown to greatly influence the responsive behavior of polymeric nanoparticles. The integrity of these OEG-based polymeric assemblies was found to depend not only on the chemical cross-links but also on the physical cross-links in these aggregates in cases where the polymer chains bear long OEG side chains. The physical cross-linking in longer OEG side chain containing polymeric nanogels is present in the form of crystalline domains. Our results here highlight that these ethylene glycol-based hydrophilic units are not to be ignored as spectator units with water-solubilization characteristics but must be analyzed in the context of assembly stabilization and triggerability with the targeted stimulus.

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agent.24−30 Similarly, acetal- and ketal-based systems are responsive to pH in that they lose their integrity under acidic conditions.31−37 In this work, we aim to explore the role played by the OEG chains in the redox responsive behavior of polymeric nanogels. A series of redox responsive acrylate polymer nanogels is studied here with OEG units of different side chain lengths, viz., OEG1, OEG4, OEG5, OEG6, and OEG9, where the number indicates the number of ethylene glycol repeat units in the side chain. The nanogels in these systems were obtained using an inverse emulsion method. The resultant nanogels show remarkably different responsive behaviors in the presence of a reducing agent, dithiothreitol (DTT), as the stimulus. The role played by OEG chains in imparting stability to these assemblies is studied using dynamic light scattering (DLS) and guest release experiments. To further understand the reason behind this unusual responsive behavior, high-resolution transmission electron microscopy (TEM) was used to probe the OEG chains in these nanogels, which reveals a crystalline ordering in the case of longer OEG chains. The impact of such ordering in the stability of the assemblies is studied (Scheme 1).

INTRODUCTION Polymeric nanoparticles have emerged to be of great importance as stimuli-responsive materials for the targeted delivery of cargos such as anticancer drugs1−3 and therapeutic proteins.4−6 An attractive feature of the polymer-based system is that it offers the opportunity to incorporate functional groups that cause them to be triggered in response to specific microenvironments.7 Although there have been several structure−property relationship studies that elucidate the role of responsive functional groups in polymers,8−10 the role of hydrophilic surface functional groups is largely ignored, beyond their primary function of endowing the nanoassembly with requisite solubility in the aqueous milieu. One of the most commonly used side chain functional groups in polymeric nanoparticles involves oligoethylene glycol (OEG) or poly(ethylene glycol) (PEG) chains.11 Apart from providing aqueous solubility, these functionalities impart many other important properties in the context of drug delivery, such as shielding the nanocarriers from uptake by immune cells,12 improved stealth and circulation properties, and thus an overall better stabilization of the nanoparticles.13−15 These features have led to their use in other scaffolds, beyond polymers as well.16−20 Although OEG units have been extensively used as side chain functional groups, the integrity of a triggerable polymeric nanoparticle is mostly determined by the type of responsive unit incorporated in the polymer.21−23 For example, disulfide cross-linked polymeric nanogels are redox responsive where they disassemble in the presence of a reducing © 2019 American Chemical Society

Received: March 6, 2019 Revised: April 25, 2019 Published: May 16, 2019 7929

DOI: 10.1021/acs.langmuir.9b00676 Langmuir 2019, 35, 7929−7936

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Scheme 1. Cartoon Representation of the Stimuli-Responsive Behavior of Nanogels Formulated using (A) Larger OEG Chains and (B) Shorter OEG Chains

Table 1. Amount of Reagents Used for the Synthesis of Pentaethylene Glycol Acrylate and Hexaethylene Glycol Acrylate pentaethylene glycol (mmol) pentaethylene glycol acrylate hexaethylene glycol acrylate

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hexaethylene glycol (mmol)

triethylamine (mmol)

acryloyl chloride (mmol)

dichloromethane (mL)

14.2

0.018 0.013

10 6.97

50 50

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solvent was evaporated, and the crude product was purified by column chromatography using 1:1 mixture of ethyl acetate and hexane as the mobile phase. The product was analyzed by 1H NMR (400 MHz, CDCl3) δ 6.39 (dd, J = 17.3, 1.5 Hz, 1H), 6.12 (dd, J = 17.3, 10.4 Hz, 1H), 5.78 (dd, J = 10.4, 1.5 Hz, 1H), 4.32−4.25 (m, 2H), 3.74−3.61 (m, 14H) (Supporting Information S2). 13 C NMR (100 MHz, CDCl3) δ 61.43, 63.62, 68.98, 70.03, 70.40, 70.46, 72.61, 128.07, 131.17, 166.10 (Supporting Information S3). ESI-MS: (M + Na)+ = 271.12 (Supporting Information S4). Synthesis of Pentaethylene Glycol Acrylate and Hexaethyleneglycol Acrylate. A procedure similar to that for tetraethyleneglycol acrylate was followed for the synthesis and purification. The amount of each reagent used for the synthesis is given in Table 1. Characterization of Pentaethylene Glycol Acrylate. 1H NMR (400 MHz, CDCl3) δ 6.35 (dd, J = 17.3, 1.5 Hz, 1H), 6.08 (dd, J = 17.3, 10.4 Hz, 1H), 5.76 (dd, J = 10.4, 1.5 Hz, 1H), 4.29−4.16 (m, 2H), 3.72−3.47 (m, 18H) (Supporting Information S5). 13 C NMR (100 MHz, CDCl3) δ 68.97, 70.08, 70.37, 70.39, 70.42, 70.45, 72.54, 128.19, 131.31, 166.10 (Supporting Information S6). (M + Na)+ = 315.15 (Supporting Information S7). Characterization of Hexaethylene Glycol Acrylate. 1H NMR (400 MHz, CDCl3) δ 6.40 (dd, J = 1.4 Hz, 1H), 6.15 (dd, J = 17.3, 10.4 Hz, 1H), 5.83 (dd, J = 10.4, 1.4 Hz, 1H), 4.38−4.21 (m, 2H), 3.78−3.56 (m, 22H) (Supporting Information S8). 13 C NMR (100 MHz, CDCl3) δ 61.34, 63.71, 69.00, 69.88, 70.02, 70.32, 70.35, 70.41, 70.43, 72.83, 128.02, 131.01, 165.81 (Supporting Information S9). (M + Na)+ = 359.17 (Supporting Information S10). Synthesis of Nanogels. In a 7 mL glass vial, 0.3 g of Brij L4 was dissolved in 2.5 g of heptane, and the mixture was vortexed to obtain a clear pre-emulsion mixture. In a separate Eppendorf tube, 0.408 mmol of the acrylate monomer, 0.005 mmol of cystine diacrylamide, and 0.0438 mmol of the ammonium persulfate initiator were dissolved in 200 μL of distilled water. The hydrophilic guest molecule was introduced at this stage. In our study, for calcein encapsulation, 0.5 mg of calcein (dissolved in 50 μL of pH 8 buffer) was added. All of the polymer precursors along with the hydrophilic guest constitute the nanogel precursor solution. The nanogel precursor solution was then added to the pre-emulsion mixture to make an inverse nanoemulsion.

EXPERIMENTAL SECTION 1

Measurements. H NMR spectra were recorded in a 400 MHz Bruker NMR spectrometer with the residual proton of the solvent as the internal standard where chemical shifts are reported in parts per million (ppm). 13C NMR spectra were recorded in a 100 MHz Bruker NMR spectrometer using the carbon signal of the deuterated solvent as the internal standard. Electrospray ionization-mass spectra (ESIMS) were recorded using a Bruker MicroTOF ESI-TOF Mass Spectrometer. Dynamic light scattering (DLS) was performed using a Malvern nanozetasizer with a 637 nm laser source with noninvasive backscattering detected at 173°. Sonication was performed in a Branson 3510 bath sonicator. Absorbance measurements for the calcein release study were performed on a SpectraMax M5 microplate reader at 495 nm. Reagents. L-Cystine (>98%, Aldrich), NaOH pellets (>97%, Fischer Scientific), NaHCO3 (99.7−100%, Fischer Scientific), concentrated HCl (36.5−38.0 wt % in water, Fischer Scientific), hydroxyethyl acrylate (96% Acros Organics), poly(ethylene glycol) acrylate (Mn ∼ 480, Aldrich), ammonium persulfate (>98%, Aldrich), tetramethylethylenediamine (>99.5%, Aldrich), acryloyl chloride (97%, Aldrich), tetraethylene glycol (99%, Aldrich), pentaethylene glycol (98%, Acros Organics), hexaethylene glycol (97%, Aldrich), calcein disodium salt (Fluka Chemical Corp), and dithiothreitol (DTT) (99%, Aldrich) were obtained from the mentioned commercial sources and were used as received, unless otherwise mentioned. Synthesis of Redox-Responsive Cross-Linker. This product was synthesized according to a previously reported procedure.38 1H NMR (400 MHz, acetone-d6) δ 7.78 (d, J = 7.7 Hz, 2H), 6.41 (dd, J = 17.0, 10.2 Hz, 2H), 6.25 (dd, J = 17.0, 1.6 Hz, 2H), 5.65 (dd, J = 10.1, 1.6 Hz, 2H), 4.99−4.80 (m, 2H), 3.41−3.27 (m, 2H), 3.26−3.07 (m, 2H) (Supporting Information S1). Synthesis of Tetraethylene Glycol Acrylate. Tetraethyleneglycol (3.0 g, 15 mmol) and 2.0 mL of triethylamine (14 μmol) were dissolved in 50 mL of dichloromethane in a 250 mL round-bottom flask. To this mixture at 0 °C, 0.7 g of acryloyl chloride (7.7 mmol) was added dropwise for about 10 min. The reaction was allowed to warm up and stir at ambient temperature for about 4 h. The excess 7930

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Langmuir This solution was then purged with argon for 5 min with gentle stirring to remove any dissolved oxygen, and 25 μL of tetramethylethylenediamine was added. The mixture was allowed to stir for an additional 10 min in an inert atmosphere to form nanogels. Extraction of Nanogels. The nanogel solution was added to 8 mL of PBS pH 7.4 buffer along with 2 mL of n-butanol in a 20 mL vial and stirred for about 5 min. This solution was taken into a 15 mL centrifuge tube and centrifuged for 5 min at 3000 rpm which resulted in the phase separation. The organic phase containing heptane and surfactant was discarded, and the water layer containing nanogels was recovered. The aqueous solution containing the nanogel was then dialyzed using a 3000 MWCO snakeskin dialysis membrane for 24 h with two water changes to remove any unreacted monomer. The final volume of the nanogel solution was adjusted to 8 mL by using a 3000 MWCO Amicon centrifuge dialysis tube, and this solution was used as the nanogel stock. Nanogel Treatment with Reducing Agent and DLS Measurements. Dithiothreitol (DTT) was used as a reducing agent to test the redox responsive behavior of nanogels. To check the redox response of the nanogels, 100 μL of nanogel stock was mixed with 900 μL of 0.1 M DTT solution and incubated for 24 h. This solution was diluted 10× with distilled water before analyzing the size by DLS. The final concentration of DTT in the solution to be analyzed by DLS was 9 mM. As a control, the same amount of distilled water was used instead of 0.1 M DTT. Calcein Release Study. In a 0.5 mL 3000 MWCO Amicon centrifuge dialysis tube, 400 μL of the nanogel stock and 100 μL of 0.5 M DTT stock solution in distilled water were added, and followed by incubation for 24 h. Calcein, released from nanogels, was dialyzed by centrifugation for 30 min at 15 000 rpm. The final volume was made up to 500 μL using 0.1 M DTT stock solution, incubated for 24 h, and then the released calcein was removed by centrifugation. These centrifugation and DTT incubation steps were performed for 7 days. The absorption of calcein at 495 nm present in the nanogel solution (200 μL) was monitored every time before the centrifugation step to analyze the dye release. Ultrasound Exposure to Nanogels. The nanogel stock (100 μL) was dissolved in 900 μL of distilled water in a 7 mL glass vial and placed in the sonication bath for 30 min. The frequency and power of the sonicator used were 42 kHz and 100 W, respectively. The water bath temperature was maintained at 4 °C using ice, and the solution was transferred from the glass vial to the DLS cuvette for size measurements. Transmission Electron Microscopy. Transmission electron microscopy (TEM) was carried out on a JEOL 2200FS at cryogenic temperatures at an acceleration voltage of 200 kV. The samples were drop-cast on an ultrathin lacey carbon-supported carbon film and were frozen to −175 °C after transfer to the TEM using a GATAN 626 cryo double tilt holder. Imaging and diffraction experiments were performed under equal low dose conditions for all samples.

Figure 1. Structure of NG1 and the DLS size characterization of NG1 and NG1 upon adding DTT.

synthesized as nanoaggregates using the same inverse emulsion methodology used for synthesizing the nanogels. The investigation into the influence of the seemingly innocuous hydrophilic OEG side chains started with the comparison of the redox sensitivity of nanogels NG1 and NG9. In nanogels, the covalent cross-links provide significant structural integrity. In the case of the NG series nanogels, the disulfide functionalities in the cross-linkers make them susceptible to cleavage under reducing conditions, which in turn compromises their structural integrity to cause partial or complete disassembly of the nanogels. To test this possibility, we first monitored the effect of treating NG1 in the presence of a reducing agent, dithiothreitol (DTT). In this case, the size of aggregates reduced from 58 ± 1 to 35 ± 1 nm (Figure 1 and Supporting Information S11), which suggests a significant change in the NG1 structure under reducing conditions due to the cleavage of its redox-labile cross-linker. The polydispersity index for the nanogel solution increased from 0.087 to 0.347, which further confirmed the disassembly of nanogels in the presence of DTT. Interestingly, however, when NG9 was subjected to a similar DTT treatment, the size of NG9 slightly increased, which could be due to the swelling of nanogels upon the cleavage of cross-links that leads to loose aggregates (Figure 2 and Supporting Information S12). The polydispersity index for this nanogel solution also changed only slightly from 0.186 to 0.205, which implies that the integrity of nanogels is preserved even in the presence of DTT. This difference offered the first clue to suggest that the OEG side chains might play a significant role in the redox sensitivity of these polymeric nanogels. To further probe the difference in behavior, we systematically evaluated the effect of DTT on NG4, NG5, and NG6. Interestingly, there was a sharp change in the behavior between tetra- and pentaethylene glycol side chains. NG4 exhibited a significant difference in size change as well as the polydispersity index from 0.292 to 0.575, whereas the sizes of NG5 and NG6 were unchanged in the presence of DTT (similar to that of NG9) (Figure 3 and Supporting Information S13−S15). We hypothesized that the observed variations in redox sensitivity could be due to the accessibility of the reducing agent into the nanogel core, aided by the steric crowding offered by the longer OEG side chains. To test this, we encapsulated a hydrophilic dye molecule (calcein) inside the nanogel and monitored its release upon exposure to the redox trigger, since the reducing agent should uncrosslink the nanogel and cause the dye molecules to leak. Thus, we envisaged that the molecular leakage will test the penetrability of the reducing agent into the nanogel core. Interestingly,

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RESULTS AND DISCUSSION Nanogels were synthesized using an inverse emulsion protocol, which uses heptane as the continuous phase and Brij L4 as the surfactant.38 2-Hydroxyethyl acrylate, tertaethyleneglycol acrylate, pentaethylene glycol acrylate, hexaethylene glycol acrylate, and poly(ethylene glycol) acrylate (Mn = 480 containing ∼9 OEG units) were used as monomers to synthesize the respective nanogels, cross-linked using the cystine bis(acrylamide) cross-linker to synthesize nanogels NG1, NG4, NG5, NG6, and NG9 respectively. The disulfide bond present in the cystine bis(acrylamide) cross-linker imparts redox sensitivity to the nanogels, whereas methylenebis(acrylamide) was used as a cross-linker to synthesize control nanogels that do not exhibit sensitivity to redox conditions, CNG1, C-NG4, C-NG5, C-NG6, and C-NG9, respectively. Polymers synthesized in the absence of any cross-linker are labeled as P1, P4, P5, P6, and P9. Polymers P1 to P9 were 7931

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Figure 2. Structure of NG9 and the DLS size characterization of NG9 and NG9 upon adding DTT.

DTT to the nanogels. There was no effect of temperature on the size of NG1 and NG9, which was attributed to the crosslinking of assemblies that lock the nanogel (Supporting Information S17). We then reasoned that the lack of size change in the nanogels based on longer OEG side chains could be due to side chain entanglements or physical cross-linking of these nanogels. To test this possibility, we performed inverse emulsion polymerization to synthesize P1 and P9 without any cross-linker and control nanogels C-NG1 and C-NG9 using methylene-bisacrylamide, which lacks redox sensitivity. P1 forms aggregates of size around 30 nm, by itself, which did not change upon adding the reducing agent (Figure 5B). We also found that cleaving cross-links in NG1 resulted in a hydrodynamic size comparable to that of P1 (Figure 5A). However, the redox insensitive control nanogel, C-NG1, did not show this stimulus-responsive behavior (Figure 5C). From these results, it can be inferred that the integrities of NG1 and C-NG1 assembly are due to the chemical cross-links in the polymer network. Then, if our hypothesis that the physical cross-links are aided by OEG chain entanglements in nanogels containing longer OEG chains were correct, then P9 should be able to form aggregates similar to those of NG9 and C-NG9. Indeed, we found the hydrodynamic size of the polymer to be in the same range as that of nanogels and similar to the nanogels, these aggregates also did not show any response to the redox trigger (Figure 6).

Figure 3. (A) Structure of NG4, NG5, and NG6 with the variation in the OEG chain length. (B) Size of nanogels upon treatment with the reducing agent (DTT) obtained from DLS.

however, the guest release behavior was found to be similar in all of the nanogels (Figure 4 and Supporting Information S16). We also tested the effect of temperature on the stability of nanogels to make sure that the nanogels are in a swollen state at room temperature, because a change in temperature may bring about a change in size of nanogels due to the ethylene glycol chain hydration. This could affect the accessibility of

Figure 4. Calcein release study of (A) NG1 and (B) NG9 with and without the redox trigger at 495 nm. 7932

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Figure 5. DLS size analysis upon application of redox stimulus in (A) redox-responsive NG1, (B) P1, and (C) redox-insensitive control NG1.

Figure 6. DLS size analysis upon application of redox stimulus in (A) redox-responsive NG9, (B) P9, and (C) redox-insensitive C-NG9.

Figure 8. DLS size analysis of redox-insensitive control nanogels before and after the application of mechanical force.

Figure 7. DLS size analysis of polymer aggregates before and after the application of mechanical force through sonication.

Although the polymers containing longer OEG side chains have the propensity to form aggregates aided by the physical cross-links through polymer entanglements, they should lack the stability provided by the chemical cross-links present in their nanogel counterparts. If this were to be true, we could potentially remove these cross-links by the application of mechanical force. Ultrasonication is one such method through which mechanical force can be applied to the polymer aggregates in the solution. The solvodynamic shear produced

by ultrasound is also known to even induce the polymer chain scission.39,40 Thus, we hypothesized that ultrasonication should be able to disentangle polymer aggregates, which would manifest in a decrease in the hydrodynamic size. However, nanogels with covalent cross-links are less likely to disintegrate upon the application of mechanical force. To test this possibility, we sonicated the OEG polymers (without any cross-linker) aggregates for 30 min, and we observed that the 7933

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NG9 along the direction shown in Figure 10B, but such a structural order was absent in NG1 (Figure 11B). The ordered structure in NG9 was further supported by the diffraction pattern of the sample (Figure 10B inset), which is indicative of the presence of crystalline domains offered by the longer OEG chains, which manifest themselves into physical cross-links. No such diffraction patterns were observed (Figure 11B inset) for NG1, however, which is attributed to the shorter OEG side chains that are not capable of providing those crystalline domains.

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Figure 9. DLS size analysis of redox-responsive nanogels before and after the application of mechanical force through sonication.

CONCLUSIONS

In summary, through a systematic study of polymers and two different series of nanogels containing varying lengths of OEG side chains, we find that these side chains play a significant role in the fidelity of these assemblies. We show here that (a) longer OEG chains provide physical cross-linking that keeps the nanogels intact even when the chemical cross-links are broken. On the other hand, the integrity of responsive nanogels containing shorter OEG chains is mainly determined by the chemical cross-links. Therefore, we show here that one has to exercise caution in designing responsive nanogels based on aggregations of long OEG side chains. (b) The physical cross-links, caused by these long OEG side chains, are weak enough to be broken in the presence of a mechanical force provided in the form of ultrasonication. (c) The chain entanglements manifest themselves as a long-range ordered structure and crystalline packing, as revealed by the diffraction patterns. The findings from the systematic study of the nanogel series provide valuable design guidelines for responsive polymeric nanogels potentially used in drug delivery vehicles, while using oligoethylene glycol units as side chains. Specifically, we show that ethylene glycol-functional groups must not be ignored as simple charge-neutral spectator units for imparting water solubility to a polymer or a nanoassembly but must be analyzed in the context of assembly stabilization due to potential crystallization of these functionalities.

size of these polymer aggregates indeed decreased significantly (Figure 7). However, sonication of the control nanogels containing a redox insensitive cross-linker only resulted in a very small change in its size (Figure 8). Although we expected no change in its size because of its stable covalent cross-links, we attribute this slight change to the polymer chain scission of nanogels to the ultrasound.41−43 On the contrary, although redoxresponsive nanogels contain covalent disulfide cross-links, we observed that these nanogels upon sonication resulted in a drastic decrease in size (Figure 9). We attribute this behavior of these nanogels to the mechanosensitive properties of the disulfide bond.44 These results support the fact that longer OEG side chains provide physical cross-links in the polymer aggregates thereby providing structural integrity to the polymeric nanogels. We then were interested in understanding how these cross-links present themselves in the aggregate. It is well known that polymers containing polyethylene glycol chains tend to show a crystalline behavior, and the extent of crystallization is dependent on the molecular weight of ethylene glycol moieties.45−47 Based on these reports, we hypothesized that longer OEG-chain-containing nanogels can show crystalline behavior due to the physical cross-linking of the side chains. To test this, we analyzed NG9 and NG1 using high-resolution TEM at cryogenic temperature. Interestingly, we observed highly ordered structures with a lattice spacing of about 2 Å in

Figure 10. (A) Low-magnification Cryo TEM image of NG9 at −175 °C. (B) High-resolution TEM image of NG9 under the same conditions. The double-headed arrow indicates the orientation of the lattice plane. The top right inset is the corresponding electron diffraction pattern. 7934

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Figure 11. (A) Low-magnification TEM image of NG1 at −175 °C. (B) High-magnification TEM image of NG1 under the same conditions. Note the absence of electron diffraction pattern (inset) due to the lack of anisotropy in the nanoparticles.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.9b00676. 1

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H NMR of the redox-responsive cross-linker; 1H NMR for tetraethylene glycol acrylate; 13C NMR of tetraethylene glycol acrylate; ESI-MS of tetraethylene glycol acrylate; DLS plot and correlation function; dye release study; and the effect of temperature on the size of NG1 and NG9 (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Alexander E. Ribbe: 0000-0002-9924-3429 S. Thayumanavan: 0000-0002-6475-6726 Author Contributions ⊥

K.R. and V.K. contributed equally to this manuscript.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Army Research Office (W911NF-15-1-0568) for support. REFERENCES

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DOI: 10.1021/acs.langmuir.9b00676 Langmuir 2019, 35, 7929−7936

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DOI: 10.1021/acs.langmuir.9b00676 Langmuir 2019, 35, 7929−7936