Efficient Protein Encapsulation within Thermoresponsive Coacervate

Mar 27, 2018 - Presented here is a novel method for encapsulating proteins into biodegradable, thermoresponsive coacervate-type polyesters. Bovine ser...
0 downloads 9 Views 2MB Size
Letter Cite This: ACS Macro Lett. 2018, 7, 477−481

pubs.acs.org/macroletters

Efficient Protein Encapsulation within Thermoresponsive Coacervate-Forming Biodegradable Polyesters Megan A. Cruz,† Daniel L. Morris,‡ John P. Swanson,† Mangaldeep Kundu,† Steven G. Mankoci,† Thomas C. Leeper,§ and Abraham Joy*,† †

Department of Polymer Science, The University of Akron, Akron, Ohio 44325, United States Department of Chemistry and Biochemistry, The University of Akron, Akron, Ohio 44325, United States § College of Science and Mathematics, Kennesaw State University, Kennesaw, Georgia 30144, United States ‡

S Supporting Information *

ABSTRACT: Presented here is a novel method for encapsulating proteins into biodegradable, thermoresponsive coacervate-type polyesters. Bovine serum albumin (BSA) was efficiently incorporated into coacervate droplets via a simple thermoresponsive encapsulation mechanism. Tunable modular systems for encapsulation such as the one presented here may be useful in a range of protein delivery applications.

P

whose biological function is dependent on maintaining their native structure.17,18 Despite these benefits, far fewer examples of thermoresponsive coacervate-type polymers exist in literature as compared to coil−globule types. Even rarer are reports of thermoresponsive coacervate-type polymers capable of biodegradation. Traditional encapsulation delivery systems have thus far been limited to small molecule drugs. It is much easier to encapsulate and release small, hydrophobic drugs compared to large, sensitive biomolecules such as proteins. Proteins are much more complex, as they can contain hydrophobic, hydrophilic, and charged surfaces, which must be balanced by complementary functional groups on the desired delivery system. Additionally, the native conformation of the protein must not be affected by encapsulation for it to maintain its therapeutic potential after delivery and release. As such, there is a need for polymers capable of capture and release of active biomolecules. A selection of recent reports on thermoresponsive encapsulation of proteins exist. For example, Shea and co-workers were able to synthesize cross-linked poly(NIPAM-co-acrylic acid-co-tbutylacrylamide) nanoparticles that exhibited “catch-andrelease” of lysozyme due to nanoparticle domains capable of protein interaction.19 Tirrell and co-workers have shown that complex coacervates formed by the sequential addition of oppositely charged polypeptides are capable of encapsulating

rotein-based therapeutics are becoming a major fraction of the world’s approved drug portfolio and are now used to treat a variety of diseases such as diabetes1−3 and cancer.4−6 Although protein-based therapeutics have high efficacy, selectivity, and minimal side effects, they suffer from premature degradation in vivo, which in turn increases cost of treatment. As a result, numerous studies have been devoted to the development of polymer carriers to provide assisted delivery of protein therapeutics.7−14 Of the proposed systems, thermoresponsive materials are well-suited for protein encapsulation: polymer and protein are mixed together in an aqueous solution, and by simply increasing the temperature, the protein can be safely sequestered within the polymer. One class of thermoresponsive materials is coacervate-forming polymers. The term coacervate refers to a phase-separated solution in which a dense polymer-rich phase (coacervate) is separated from the polymer-depleted solution (aqueous phase).15,16 Coacervation-type thermoresponsive polymers are ideal for encapsulation of biomolecules. They exhibit minimal conformational change when brought above their Lower Critical Solution Temperature (LCST) as compared to the drastic conformational changes observed in polymers that undergo efficient dehydration (and subsequent coil−globule transition) above the LCST. This conformational stability is primarily due to significant amounts of water present in the coacervate phase, which enables a network of stabilizing hydrogen bonds. Coacervates can form with pockets of hydrophilicity, which makes these polymers ideal systems for the segregation of sensitive biomolecules, for example, nucleic acids and proteins, © XXXX American Chemical Society

Received: February 12, 2018 Accepted: March 19, 2018

477

DOI: 10.1021/acsmacrolett.8b00118 ACS Macro Lett. 2018, 7, 477−481

Letter

ACS Macro Letters Scheme 1. Synthetic Route for the Preparation of TR-PEsa

Reagents and conditions: (i) Et3N, DCM, 0 °C to room-temperature, 1 h; (ii) diethanol amine (DEA), neat, 80 °C, vacuum, 16 h; (iii) SA, DIC, DPTS, BocGlu, DCM, 0 °C to room-temperature, 72 h; (iv) 4 N HCl/1,4-dioxane, anhydrous DCM, 45 min.

a

charged proteins.20 However, to the best of our knowledge, there exist no synthetic biodegradable materials that can encapsulate and release proteins by a simple thermoresponsive mechanism. Ideally, such a system would be based on a modular, coacervate-type polymer to allow for nondamaging encapsulation and release of proteins within a wide range of biodiversity. Recently, our lab has reported a new system of high molecular weight, biodegradable, thermoresponsive polyesters (TR-PEs) exhibiting LCST behavior.21−23 TR-PEs underwent inefficient temperature-induced phase separation leading to coacervation above their cloud point temperatures (Tcp, commonly used to approximate LCST). TR-PEs were shown to undergo relatively quick degradation and studies indicated the materials to be noncytotoxic, even at high concentrations. Herein, we describe a new biodegradable, coacervate forming TR-PE for the efficient encapsulation of proteins. Fluorescently tagged bovine serum albumin (FITC-BSA) was chosen as a model protein as it is widely used, readily accessible, and allows for simple observation via fluorescence microscopy. A number of factors were explored for the successful design of ionic TRPEs such as the identity of the pendant group and the amount of charge. It was hypothesized that positively charged pendant groups would facilitate interactions with the anionic charge of BSA. However, incorporation of charged groups would increase the Tcp and would need to be countered by copolymerization with pendant groups that can bring down the Tcp. Based on our previous studies,23 nonionic TR-PEs, which showed low LCSTs, were chosen, as they would be useful in counterbalancing the increase in Tcp brought about by incorporating amine groups in this new class of TR-PEs. A library of ionic TR-PEs was created in order to optimize the pendant groups, amount of charge, and solvent necessary for this application (Table S1). The information gained from this data led us to design TR-PEs shown in Scheme 1 with cationic groups along the polyester backbone. By varying the ratio of succinic acid to glutamic acid, we were able to control the relative cationic charge of the polyester backbone. Two TR-PEs based on succinic acid/glutamic acid and N-substituted diols were studied: TR-PE12.5 and TR-PE15, bearing a positive charge on 12.5% and 15% of their repeat units, respectively. The polyesters were characterized using nuclear magnetic resonance spectroscopy (NMR) and size exclusion chromatography (SEC). Encapsulation efficiency of FITC-BSA was studied via fluorescence microscopy and total protein assays. Polymer degradation and the effect of biologically relevant pH levels on ionic TR-PE thermoresponsivity were examined by measuring Tcp on a UV−vis spectrophotometer. BSA secondary structure

preservation was determined by circular dichroism (CD). Finally, viability of NIH 3T3 mouse fibroblast cells were measured in the presence of polymer to examine the potential toxicity of this delivery system. The typical synthetic route for preparation of TR-PEs is illustrated in Scheme 1. Synthesis of the monomer (cyclopropyl diethanolamine, cPrDEA) followed the same general procedure described in previous studies.21,23 To synthesize the cationic TR-PE, a DIC (N,N′-diisopropylcarbodiimide) coupling reaction was carried out using cPrDEA as the diol and a mixture of succinic acid (SA) and boc-L-glutamic acid (BocGlu) as the diacids.23 TR-PEs were purified via dialysis against MeOH. 1H NMR was used to confirm the purity of the polymers and to quantify the ratio of BocGlu:SA. Molecular weights were analyzed via SEC (TR-PE12.5 = 29.0 kDa, PDI = 1.4 and TR-PE15 = 29.7 kDa, PDI = 1.3). The Boc group was deprotected using 4 N HCl in dioxane. After removal of HCl/ dioxane under reduced pressure, the deprotection was confirmed via 1H NMR in DMSO-d6 (Figure S1). Coacervate-induced interactions between BSA and TR-PE12.5 were probed using saturation transfer difference (STD) NMR spectroscopy.24−27 STD NMR is a technique used to study binding interactions between small molecules and protein receptors by saturating protein resonances and observing saturation transfer in the form of signal attenuation in ligand peaks. Ligand peak attenuation is visualized by obtaining a difference spectrum where the saturating experiment is subtracted from a nonsaturating experiment. Peaks appearing in the difference spectrum are engaging in a binding and release event with the receptor during the experiment. Although STD NMR is most frequently applied to systems containing small molecules and proteins, binding mechanisms of TR-PE12.5 with BSA can also be observed by this method. However, because the polymer is a large macromolecule with a substantial hydration network, one must allow for a degree of spurious saturation during interpretation of control experiments containing only the polymer. This is due to intentional saturation of the water signal, which is required to obtain a clear spectrum in these samples. Normally a nonissue in standard STD experiments with small-molecule ligands, saturation of water molecules subsequently gets transferred into the polymer even in the absence of protein due to the extensive hydration networks within TR-PEs.28 In the same vein, since the polymer and coacervate droplets are much larger than small molecule ligands, the significantly decreased molecular tumbling rate enhances STD signal attenuation by accelerating the saturation transfer efficiency. However, despite these limitations, an enhancement of the STD effect can be 478

DOI: 10.1021/acsmacrolett.8b00118 ACS Macro Lett. 2018, 7, 477−481

Letter

ACS Macro Letters

In order to determine the Tcp of TR-PEs, absorbance measurements were taken at 500 nm for 10 mg mL−1 solutions in 100 mM phosphate buffer (Figure 2A). Solid circles

observed for protein-loaded coacervate samples relative to polymer-only controls. Background saturation transfer for the polymer-only samples is relatively constant throughout temperature changes (Figure 1, black). When BSA is present, increasing protein-enhanced

Figure 1. STD 1H NMR control spectra of TR-PE12.5 (black, bottom) and TR-PE12.5 with BSA (red, top) at temperatures varying from 5 to 37 °C.

Figure 2. UV−vis spectra (500 nm, 1 °C min−1 heat) of TR-PE12.5 and TR-PE15 solutions (10 mg mL−1 in 100 mM PB) at varying pH (A) and hydrolytic degradation of TR-PE12.5 incubated at 37 °C in 100 mM pH 6 PB (B).

saturation transfer is evident until around the Tcp (17.1 °C), after which the sample difference spectra reconverges with the control as a function of increasing temperature (Figure 1, red). This temperature-dependent saturation pattern suggests that below the Tcp a relatively low affinity binding and release event occurs in which BSA interacts transiently with the polymer. At the Tcp saturation transfer efficiency is increased, displaying a stronger affinity binding event, because the coacervated complex is in equilibrium capturing and releasing BSA rapidly. Above the Tcp, BSA is locked inside the stable coacervate, and thus, saturation cannot be enhanced since the polymer does not release from its complex with BSA. Interactions observed by STD NMR indicate that the Kd between polymer and protein is between 10−3 and 10−8 M, suggesting that the interaction is neither extremely weak nor extremely strong. The polymer protons appear to saturate uniformly in the presence of BSA, which excludes the probability of forming one distinct and unique protein-bound form. Rather, the polymer−protein complexes probably exchange between a number of positionally varied configurations. This saturation pattern is also observed in reverse as a function of cooling temperatures implying the stable release of BSA. Absence of precipitate in the cooled sample is also a good indicator of intact protein globular structure. Admittedly, BSA is a stable blood protein, and this stability may not hold up when applied to other systems. Further biophysical measurements will be required to define the affinity and specificity more precisely and to determine if encapsulation is unique to BSA or can be applied to more medically significant protein therapeutics.

represent TR-PE12.5 and open circles represent TR-PE15. An increase in pH is correlated with a reduction in cloud point temperature. At pH 6, the Tcp of TR-PE12.5 and TR-PE15 is 17.1 and 27.5 °C, respectively. When the pH is increased to 7, the Tcp decreases to 10.0 and 14.1 °C, respectively. The Tcp drops even further to 4.9 and 9.3 °C, respectively, when the pH is raised to 8. In previous work, we have shown that polyesters based on succinic acid and N-substituted diols degrade relatively quickly.21,23 Since the TR-PEs are charged, their Mn cannot be measured under the same conditions as the protected polymers. However, it has been shown that lower molecular weight TR-PEs have higher T cp, and hence, polymer degradation can be qualitatively monitored by a change in Tcp.21,29 A solution of TR-PE12.5 in phosphate buffer was incubated at 37 °C to explore degradation. Over a period of 7 days, a clear shift in Tcp from 23.4 to 45.7 °C was observed (Figure 2B). As expected, hydrolytic degradation occurs over the tested time period, with the maximum difference in Tcp corresponding to the largest change in molar mass between days 0 and 1 after 24 h. STD NMR results show compelling evidence that the TRPEs are interacting with BSA; however, additional data to support our claim that the protein is being encapsulated would be beneficial. As an initial method to characterize the potential for protein encapsulation, fluorescence images (Figure 3) were taken to quantify encapsulation efficiency at 1 wt %/vol FITC479

DOI: 10.1021/acsmacrolett.8b00118 ACS Macro Lett. 2018, 7, 477−481

Letter

ACS Macro Letters

Figure 3. Fluorescence microscopy images (20× magnification, FITC filter) of FITC-BSA (1 mg mL−1 in 100 mM PB) and TR-PE coacervates (10 mg mL−1 in 100 mM PB) at pH 6. FITC-BSA is encapsulated in TR-PE12.5 (left, 50 μm scale bar) and TR-PE15 (middle, 50 μm scale bar), but remains outside the nonionic control polymer (TR9, right, 20 μm scale bar). Figure 4. In vitro encapsulation of FITC-BSA (1 mg mL−1 in 100 mM PB) into TR-PE12.5, TR-PE15, and TR9 coacervates (10 mg mL−1 in 100 mM PB) at pH 6. Error bars denote standard deviation; n = 4.

BSA loading. Phosphate buffer (PB) was selected as the model solution, as it is a common biological medium that contains less salt than traditional PBS. Highly salinated solutions are undesirable as they cause polyester Tcp to decrease so the amount of additional charge required to counterbalance this decrease would likely accelerate TR-PE degradation. Additionally, highly salinated solutions may result in reduced BSApolymer interactions due to a charge screening effect. As shown in Figure 3, at pH 6, both TR-PE12.5 and TR-PE15 were observed encapsulating FITC-BSA via the formation of fluorescent coacervates. Increasing the pH decreases the cationic charge of the TR-PE, resulting in reduced encapsulation efficiency. The reduction in encapsulation efficiency, particularly at pH 8, can be observed as an increase in comparative background fluorescence, as more FITC-BSA remains outside the coacervates (Figure S2). At pH 6, TRPE12.5 and TR-PE15 displayed an encapsulation efficiency greater than 75% (the maximum value for this fluorescence assay). At pH 7, this value dropped to 23 ± 1% and 14 ± 1%, respectively. Protein encapsulation efficiency drops to less than 2% for both TR-PEs at pH 8, indicating the pH sensitivity of this system. A nonionic control sample with a Tcp similar to that of TR-PE12.5 (TR9, Tcp = 16 °C)22 was also tested to determine if the cationic group is necessary for BSA encapsulation. As shown in Figure 3 (right), encapsulation of FITC-BSA was unsuccessful with a nonionic TR-PE. To better quantify encapsulation efficiency, a bicinchoninic acid (BCA) assay was performed at a 10:1 polymer to protein ratio. This assay relies on the reduction of Cu2+ from CuSO4 to Cu1+ caused by peptide bonds in the protein. Two molecules of BCA then form a purple chelate complex with Cu1+ that has strong absorbance at 562 nm. The amount of Cu2+ reduced is directly proportional to the amount of protein in solution, and a concentration can be calculated when compared to a standard curve. As shown in Figure 4, TR-PE 12.5 and TR-PE 15 encapsulated FITC-BSA at pH 6.0 with 78.3% and 85.3% efficiency, respectively. In contrast, the nonionic control polymer (TR9, Tcp = 16 °C) was only able to encapsulate FITC-BSA with 8.3% efficiency at pH 6. At pH 7, BSA encapsulation dropped to less than 1% for TR-PE12.5 and 7.9% for TR-PE15. For both TR-PE12.5 and TR-PE15, protein encapsulation was less than 1% at pH 8 (Figure S3). As pH is increased, encapsulation efficiency is reduced due to the reduction in cationic charge of the TR-PEs. Circular dichroism experiments were conducted in order to examine the secondary structure of BSA after encapsulation and release by TR-PE coacervates (Figure S4). BSA (5 mg mL−1) was encapsulated in the polyester TR-PE12.5 (10 mg mL−1).

The resulting polymer−protein coacervate was diluted 100-fold (to prevent saturation of the detector), cooled below Tcp and the CD spectrum was collected. Figure S4 shows two characteristic negative bands for α-helix structure at 208 and 222 nm, showing the preservation of protein conformation. The possible toxicity of the TR-PEs was investigated by examining NIH 3T3 mouse embryonic fibroblast proliferation in medium supplemented with varying concentrations of the TR-PEs. After 1 day of growth in the presence of the cationic polyesters, cell viability was probed using a CellTiter Blue assay kit. As shown in Figure 5, TR-PE15 at high concentrations (1

Figure 5. Cell viability of TR-PE12.5 and TR-PE15 against NIH 3T3 cells, 1 day; n = 4, p < 0.05. Error bars denote standard deviation.

mg mL−1) showed slight toxicity, which was statistically significant compared to an untreated control sample (ANOVA, p < 0.05). This result is not surprising as TR-PE15 has a greater amount of charge, and thus has a stronger impact on cell viability compared to TR-PE12.5. At high concentrations (1 mg mL−1) of TR-PE12.5, there was only a 10% decrease in cell viability compared to solutions containing an untreated control sample (ANOVA, p < 0.05). The implied low cytotoxicity of cationic TR-PEs is promising for their potential as therapeutic delivery agents. In this work, we have described an efficient method for encapsulating proteins by simple mixing with thermoresponsive polyesters that form coacervates above their Tcp. To the best of our knowledge, this represents the first example of a biodegradable polymer capable of thermoresponsive encapsulation of sensitive biomolecules. The described experiments 480

DOI: 10.1021/acsmacrolett.8b00118 ACS Macro Lett. 2018, 7, 477−481

Letter

ACS Macro Letters

(11) Vermonden, T.; Censi, R.; Hennink, W. E. Hydrogels for protein delivery. Chem. Rev. 2012, 112 (5), 2853−2888. (12) Lee, Y.; Ishii, T.; Cabral, H.; Kim, H. J.; Seo, J. H.; Nishiyama, N.; Oshima, H.; Osada, K.; Kataoka, K. Charge-conversional polyionic complex micelles-efficient nanocarriers for protein delivery into cytoplasm. Angew. Chem., Int. Ed. 2009, 48 (29), 5309−5312. (13) Li, F. Controlled Release of Bevacizumab Through Nanospheres for Extended Treatment of Age-Related Macular Degeneration. Open Ophthalmol. J. 2012, 6 (1), 54−58. (14) Degim, I. T.; Ç elebi, N. Controlled delivery of peptides and proteins. Curr. Pharm. Des. 2007, 13 (1), 99−117. (15) Khokhlov, A. R.; Nyrkova, I. A. Compatibility enhancement and microdomain structuring in weakly charged polyelectrolyte mixtures. Macromolecules 1992, 25 (5), 1493−1502. (16) Hwang, D. S.; Zeng, H.; Srivastava, A.; Krogstad, D. V.; Tirrell, M.; Israelachvili, J. N.; Waite, J. H. Viscosity and interfacial properties in a mussel-inspired adhesive coacervate. Soft Matter 2010, 6 (14), 3232. (17) Maeda, T.; Takenouchi, M.; Yamamoto, K.; Aoyagi, T. CoilGlobule Transition and/or Coacervation of Temperature and pH Dual-Responsive Carboxylated Poly(N-isopropylacrylamide). Polym. J. 2009, 41 (3), 181−188. (18) Maeda, T.; Takenouchi, M.; Yamamoto, K.; Aoyagi, T. Analysis of the formation mechanism for thermoresponsive-type coacervate with functional copolymers consisting of N-isopropylacrylamide and 2hydroxyisopropylacrylamide. Biomacromolecules 2006, 7 (7), 2230− 2236. (19) Yoshimatsu, K.; Lesel, B. K.; Yonamine, Y.; Beierle, J. M.; Hoshino, Y.; Shea, K. J. Temperature-responsive ‘catch and release’ of proteins by using multifunctional polymer-based nanoparticles. Angew. Chem., Int. Ed. 2012, 51 (10), 2405−2408. (20) Black, K. A.; Priftis, D.; Perry, S. L.; Yip, J.; Byun, W. Y.; Tirrell, M. Protein Encapsulation via Polypeptide Complex Coacervation. ACS Macro Lett. 2014, 3 (10), 1088−1091. (21) Swanson, J. P.; Martinez, M. R.; Cruz, M. A.; Mankoci, S. G.; Costanzo, P. J.; Joy, A. A coacervate-forming biodegradable polyester with elevated LCST based on bis-(2-methoxyethyl)amine. Polym. Chem. 2016, 7 (28), 4693−4702. (22) Swanson, J. P.; Cruz, M. A.; Monteleone, L. R.; Martinez, M. R.; Costanzo, P. J.; Joy, A. The effect of pendant group structure on the thermoresponsive properties of N -substituted polyesters. Polym. Chem. 2017, 8, 7195−7206. (23) Swansson, J. P.; Monteleone, L. R.; Haso, F.; Costanzo, P. J.; Liu, T.; Joy, A. A Library of Thermoresponsive, Coacervate-Forming Biodegradable Polyesters. Macromolecules 2015, 48 (12), 3834−3842. (24) Viegas, A.; Manso, J.; Nobrega, F. L.; Cabrita, E. J. SaturationTransfer Difference (STD) NMR: A Simple and Fast Method for Ligand Screening and Characterization of Protein Binding. J. Chem. Educ. 2011, 88 (7), 990−994. (25) Venkitakrishnan, R. P.; Benard, O.; Max, M.; Markley, J. L.; Assadi-Porter, F. M. Use of NMR saturation transfer difference spectroscopy to study ligand binding to membrane proteins. Methods Mol. Biol. 2012, 914, 47−63. (26) Bhunia, A.; Bhattacharjya, S.; Chatterjee, S. Applications of saturation transfer difference NMR in biological systems. Drug Discovery Today 2012, 17 (9), 505−513. (27) Mayer, M.; Meyer, B. Group epitope mapping by saturation transfer difference NMR to identify segments of a ligand in direct contact with a protein receptor. J. Am. Chem. Soc. 2001, 123 (25), 6108−6117. (28) Dalvit, C.; Fogliatto, G.; Stewart, A.; Veronesi, M.; Stockman, B. WaterLOGSY as a method for primary NMR screening: Practical aspects and range of applicability. J. Biomol. NMR 2001, 21 (4), 349− 359. (29) Shimokuri, T.; Kaneko, T.; Akashi, M. Effects of thermoresponsive coacervation on the hydrolytic degradation of amphipathic poly(gamma-glutamate)s. Macromol. Biosci. 2006, 6 (11), 942−951.

show interactions between the protein and polymer that appear to be responsible for the efficient encapsulation of FITC-BSA. The TR-PEs described in this work appear to be noncytotoxic, have no negative effect on the native fold of the protein after encapsulation and release, and their hydrolytic degradation would be advantageous for timed released applications in vivo. A system such as the one presented here could be the starting point for the synthesis of other thermoresponsive polyesters designed to encapsulate specific proteins with precisely tuned interactions and for their subsequent release in a controlled manner.



ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.8b00118. Details on experimental methods, Figures S1−S4, and Table S1 (PDF).



AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Abraham Joy: 0000-0001-7781-3817 Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS The work described here was funded in part by an NSF CAREER Grant to A.J. (NSF DMR #1352485).



REFERENCES

(1) Dewitt, D. E.; Hirsch, I. B.; Care, R.; Pro, P. Clician’s Corner Outpatient Insulin Therapy in Type 1 and Type 2 Diabetes Mellitus. J. Sci. Rev. Clin. Appl. 2003, 289 (17), 2254−2264. (2) Gilroy, C. A.; Luginbuhl, K. M.; Chilkoti, A. Controlled release of biologics for the treatment of type 2 diabetes. J. Controlled Release 2016, 240, 151−164. (3) Valla, V. Therapeutics of diabetes mellitus: focus on insulin analogues and insulin pumps. Exp. Diabetes Res. 2010, 2010, 178372. (4) Satchi-Fainaro, R.; Duncan, R.; Barnes, C. M. Polymer therapeutics for cancer: Current status and future challenges. Adv. Polym. Sci. 2006, 193, 1−65. (5) Matsumura, Y.; Maeda, H. A new concept for macromolecular therapies in cancer chemotherapy: mechanisms of tumortropic accumulation of proteins and the antitumor agents SMANCS. Cancer Res. 1986, 46, 6387−6397. (6) Scott, A. M.; Wolchok, J. D.; Old, L. J. Antibody therapy of cancer. Nat. Rev. Cancer 2012, 12 (4), 278−287. (7) Schweizer, D.; Serno, T.; Goepferich, A. Controlled release of therapeutic antibody formats. Eur. J. Pharm. Biopharm. 2014, 88 (2), 291−309. (8) Schaefer, E.; Abbaraju, S.; Walsh, M.; Newman, D.; Salmon, J.; Amin, R.; Weiss, S.; Grau, U.; Velagaleti, P.; Gilger, B. Sustained Release of Protein Therapeutics from Subcutaneous Thermosensitive Biocompatible and Biodegradable Pentablock Copolymers (PTS gels). J. Drug Delivery 2016, 2016, 1−15. (9) Bhuchar, N.; Sunasee, R.; Ishihara, K.; Thundat, T.; Narain, R. Degradable thermoresponsive nanogels for protein encapsulation and controlled release. Bioconjugate Chem. 2012, 23 (1), 75−83. (10) Lu, Y.; Sun, W.; Gu, Z. Stimuli-responsive nanomaterials for therapeutic protein delivery. J. Controlled Release 2014, 194, 1−19. 481

DOI: 10.1021/acsmacrolett.8b00118 ACS Macro Lett. 2018, 7, 477−481