Reversible self-assembly nano vesicle of UCST response prepared

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Reversible self-assembly nano vesicle of UCST response prepared with multi-L-arginyl-poly-L-aspartate conjugated with polyethylene glycol Wen-Chi Tseng, Tsuei-Yun Fang, Yu-Chih Lin, Shing-Jong Huang, and Yi-Hao Huang Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01274 • Publication Date (Web): 05 Nov 2018 Downloaded from http://pubs.acs.org on November 10, 2018

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Reversible self-assembly nano vesicle of UCST response prepared with multi-L-arginyl-poly-L-aspartate conjugated with polyethylene glycol Wen-Chi Tseng,∗,† Tsuei-Yun Fang,‡ Yu-Chih Lin,† Shing-Jong Huang,¶ and Yi-Hao Huang† †Chemical Engineering Department, National Taiwan University of Science and Technology, Taipei, Taiwan ‡Department of Food Science, National Taiwan Ocean University, Keelung, Taiwan ¶Instrumentation Center, National Taiwan University, Taipei, Taiwan E-mail: [email protected] Abstract Multi-arginyl-poly-aspartate (MAPA), also known as cyanophycin, containing a backbone of poly-aspartate with arginine and lysine as side chains was prepared with recombinant Escherichia coli. The insoluble part (iMAPA) was conjugated with polyethylene glycol (PEG) at two different levels, high (iMAPA(PEG)h) and low (iMAPA(PEG)l). Both levels of conjugation exhibited UCST-type responses in the pH range of 3 to 10 at a concentration of 2 mg/mL. The cloud-point temperature of each conjugate also showed a positive correlation with concentration in PBS, falling between 20 to 58 ◦C at a concentration from 0.1 to 3 mg/mL. Hysteresis was observed to follow approximate paths under the same condition during repeated heating and cooling. Notably,

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reversible formation of core-shell vesicles appeared at room temperature in PBS with a size of around 25 to 60 nm as measured by DLS and observed under TEM. The reversibility was further employed to encapsulate doxorubicin (Dox) at different weight ratios of Dox to iMAPA(PEG)h. An encapsulation efficiency could reach as high as 70% with an equivalent of loading capacity of 1.5 mg Dox/mg iMAPA(PEG)h. The Dox-loaded vesicles stayed as stable at 4 ◦C up to 4 weeks with a minimal leakage below 2% and a slightly dilated morphology. Temperature-triggered release of Dox from the vesicles could be achieved by a step change of 5 ◦C successively from 37 to 62 ◦C in an effort to induce an initial 10% release at 37 ◦C gradually to complete release at 62 ◦C.

Introduction Stimuli-responsive materials undergo conformational changes after receiving external stimuli, such as heat, acidity or certain chemical molecules. 1 The conformational changes generally occur with the alterations in physiochemical properties that can be applied to a variety of fields such as analytical assays, drug delivery systems, and biomaterials. 2,3 The developments of those responsive polymers have been attracted lots of attentions, specifically the thermal responsive ones. There exist two major types of thermal responses, depending on the miscibility gap of a polymer solution. 4 One is the LCST (low critical solution temperature) type of which the change in miscibility accompanies the turbidity of solution from transparent to turbid upon raising temperature. Among the LCST type polymers, poly(Nisopropylacrylamide) (polyNIPAM) and its derivatives are the most studied. 5 The transition temperature of polyNIPAM at 32 ◦C can be altered through adjusting the inter- and intramolecular forces by different pendant groups. 4 On the other hand, the solution turns from turbid to transparent upon raising the temperature for the UCST (upper critical solution temperature) type polymers. 6 Unlike its counterpart of LCST type, the UCST property has been seldom identified in aqueous solution, especially under the physiological condition. 6 A couple of polymers have recently been shown to display UCST behavior in aqueous 2


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solution. Depending on the interactions within the supramolecular structures before phase transition occurs, UCST polymers can be further classified as non-ionic and Zwitterionic types. The non-ionic polymers are held intermolecularly mainly by hydrogen bond, and the phase transition becomes less dependent on the aqueous pH condition. Several such polymers have been synthesized such as poly(N-acryloyl glycinamide) (PNAGA) based polymers, 6–8 ureido-derivatized polymers, 9–11 and poly(N-acryloylaspargineamide). 12 The other type of UCST polymers belongs to polyelectrolytes, and the polysulfobetaine based polymers have been the focus of study. 13,14 Due to the charge interactions among molecules, many environmental factors, such as ionic strength and pH, can determine the ionization of those charged groups, and subsequently affect the phase change. 15 Similar to the studies of LCST type polymers, the attachment of a pendant group or the modification of the side chain has been shown to alter the UCST response of either a non-ionic type or a Zwitterionic type polymer. A reversible assembly possessing UCST was observed for poly(phosphonate)s bearing carboxylic acids. 16 The phase change of polyacrylamide copolymers bearing bile acid or β-cyclodextrin could occur around 37 ◦C. 17 The transition temperature was decreased when the incorporation of N,N’-dimethyl(methacryloylethyl)ammonium propane changed the linear structure of poly(sulfobetaine). 18 Post-polymerization modification with hydrophobic group resulted in enhancing the UCST of poly(sulfobetaine) derivatives. 19 In spite of the attentions to the LCST type polymer and the emerging development of UCST type polymer, only a comparably few polypeptides are known to be thermal responsive, and most of those polypeptides exhibit LCST responses, such as the elastin like peptides, 20 poly(N-substituted α/β asparagine), 21 and (polyethylene glycol) modified polyL-glutamates. 22 However, the polypetides undergoing UCST type responses are rarely reported especially under the physiological condition, and protein in nature is even less known as a thermoresponsive material. One of the few proteins is resilin, a natural polypeptide of elastomeric property found in many insects, which can undergo both LCST and UCST 3



phase changes in aqueous solution. 23 In some recent studies, multi-L-arginyl-poly-L-aspartic acid (MAPA), a non-ribosomal polypeptide also known as cyanophycin, has been shown to possess dual pH and thermal responsiveness. 24,25 MAPA occurs as a nutrient reservoir in most cyanobacteria. 26,27 Unlike most polypeptides, its synthesis is directed by a single enzyme cyanophycin synthetase. 28 Therefore, in addition to the synthesis by cyanobacteria, MAPA can be produced heterologously by the bacterial transformed with the cphA gene that codes for cyanophycin synthetase. 28–30 The recombinant MAPA is composed of poly-aspartate as a backbone with either arginine or lysine as the side chain linking to the carboxylic group of each aspartate. 31 The contents of lysine and arginine have been shown to affect the solubility of MAPA under the physiological condition. MAPA exhibits insoluble at a low lysine content and becomes soluble when the lysine content increases. 32,33 The dependence of solubility on temperature and acidity of solution have been shown to attribute to the dual thermal and pH responses of MAPA. However, at the physiological pH, the UCST of the insoluble fraction (iMAPA) hardly exists due to its limited solubility. 24,25 Despite of being biocompatible, 34 the undesirable temperatures of phase change could limit its further biomedical applications which generally favor the physiological condition. Previous studies have demonstrated that the pendant groups can successfully alter the responsive temperature of phase transition, and the UCST can be modulated by hydrogen bonds. 16–18 The gaunido group of arginine provides hydrogen bond for the intermolecular interactions which bound the molecules into agglomerates under a neutral condition and become dismantled in an acidic solution. In this study, conjugation of polyethylene glycol onto the primary amine of lysine was attempted to moderate the interactions contributed from the guanido of arginine through widening the molecular distances between two iMAPA molecules, and the decreased interactions was expected to enhance the solubility of iMAPA at the neutral pH while maintaining a similar characteristic of UCST response as that under an acidic condition. 4


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Experimental Section Materials Poly(ethylene glycol) monomethyl of molecular weight 2000, phosphotungstic acid, potassium bromide, 2,4,6-trinitrobenzene sulfonic acid, chloroacetaldehyde diethyl acetal, and sodium cyanoborohydride were from Sigma-Aldrich (St. Louis, MO). Sodium bicarbonate, sodium chloride, sodium hydroxide, phosphoric acid, hydrogen chloride, ether were obtained from J.T. Baker (Phillipsburg, NJ). Reagents of bacteria culture medium were purchased from Becton-Dickinson (Franklin Lakes, NJ). Doxorubicin hydrochloride was from LC Laboratories (Woburn, MA). Dialysis membrane of MWCO 5000 was obtained from Spectrum Laboratories (Rancho Dominguez, CA). All chemicals were used as received unless otherwise stated. Water was deionized by a Milli-Q water purification system (Bedford, MA).

Preparation of insoluble multi-arginyl-poly-aspartate Insoluble multi-arginyl-poly-aspartate (iMAPA) was obtained from the culture of recombinant Escherichia coli BL21-CodonPlus(DE3)-RIL carrying an expression vector pET21b with the cyanophycin synthetase gene cphA from Synechocystis sp. PCC 6803 as previously described. 32 Purification proceeded with a total volume of 18 L broth harvested from different batches of culture. The purity was checked on SDS-PAGE, and the amino acid composition was determined by HPLC with a post-derivation method using PITC as previously described. 32 After lyophilization, the product was stored at room temperature for further reaction.

Conjugation of polyethylene glycol Polyethylene glycol monomethyl of molecular weight 2000 (PEG) was used to conjugate iMAPA. An acetaldehyde diethyl acetal derivative was first prepared by reacting PEG with chloroacetaldehyde diethyl acetal and NaOH in dioxane, and then the dried intermediate was 5



reacted in phosphoric acid solution to obtain PEG acetaldehyde as previously described. 35 Two different levels of conjugation, iMPAP(PEG)l and iMAPA(PEG)h with a low and a high level of conjugated PEG, respectively, were prepared by a reductive reaction between the aldehyde group of the activated PEG and the amine group of lysine of iMAPA in the presence of sodium cyanoborohydride. The reaction was carried out in 0.1 M citrate phosphate buffer (pH 3.0) at 45 ◦C for 68 h. At the conclusion of reaction, the product was dialyzed with membrane of MWCO 5000 against deionized water at a volume ratio of 1/100 for 6 times followed by lyophilization.

Characterization of conjugate The chemical structures of conjugate and iMAPA were investigated by FTIR and NMR spectroscopy. A weight of around 2 mg sample was mixed with potassium bromide at a weight ratio of 1:20. The mixture was finely pulverized, and formed a transparent pellet which was subsequently subjected to FTIR analysis (FTS-3500, Bio-Rad, Hercules, CA). The iMAPA(PEG)h conjugate and unmodified iMAPA were dissolved in a solvent containing D2O, HCl, and H2O (1:1:8), receptively, and the 1H-NMR spectra were recorded on a Bruker AVIII-500MHz FT-NMR spectrometer or a AVIII-800MHz one with a water suppression pulse. The extent of PEG conjugation was quantitated by TNBSA (2,4,6-trinitrobenzene sulfonic acid) assay which measured the differences in the amounts of primary amine on the lysine before and after conjugation.

Measurement of phase change temperature For the measurement of phase change, a desired concentration of the conjugate was prepared by adding the conjugate into phosphate buffered saline (PBS). When the pH effect was examined, 0.1 M of the following buffers containing 0.5 mg/mL of either iMPAP(PEG)l or iMAPA(PEG)h was used: citrate-phosphate for pH 3 and 5.5 and borate for pH 9 and 11. Before the measurement of phase change, the solution was under sonication at room 6


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temperature for 15 min in an ultrasonic bath (Branson, Danbury, CT). The changes in turbidity was monitored by a spectrophotometer (UV-750, Jasco, Tokyo, Japan) equipped with a stirring unit, a piezo heating unit, and a cooling water bath. Temperature change was monitored between 2 to 72 ◦C with a temperature gradient of 1 ◦C/min for either heating or cooling. The corresponding temperature of the midpoint between the lowest and highest transmittances during heating was designated as the cloud-point temperature (Tcp ) of the phase change after a pretreatment of one cycle of heating and cooling.

Encapsulation of doxorubicin The UCST property of iMAPA(PEG)h was employed to encapsulate doxorubicin (Dox), a commonly used chemotherapeutic drug for various cancers. A desired amount of Dox was mixed with iMAPA(PEG)h at different weight ratios of 1:0.5, 1:1, 1:1.5, and 1:2, respectively. Each mixture containing a final concentration of 2 mg/mL iMAPA(PEG)h was under constant stirring at 55 ◦C for 30 min, was cooled down stepwise to 40, 30, and 20 ◦C with an incubation time of 5 min at each temperature, and then was transferred to a cup of a Slide-A-Lyzer MINI Dialysis of MWCO 10 KDa (ThermoFisher Scientific, Waltham, MA) containing 40 mL PBS outside the cup. The unencapsulated Dox was removed by incubating the whole conical tube at 20 ◦C water bath under reciprocating shaking with a 10 cm displacement at 150 rpm. Every 24 h the dialysis buffer was refreshed, and the amount of Dox within the dialysis buffer was quantitated by a fluorimeter with an excitation wavelength of 493 nm and emission wavelength of 591 nm. The dialysis proceeded for 72 h till the encapsulation efficiency stayed almost unchanged. The encapsulation efficiency was calculated by the sum of Dox quantity in each replaced buffer divided by the amount of initially added Dox.

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Drug release triggered by temperature After the removal of unencapsulated Dox, the whole conical tube with fresh PBS buffer was further incubated at 37 ◦C water bath under the same reciprocating shaking condition. Examination of the effect of temperature on triggering the release was carried out by raising the incubation temperature from 37 to 62 ◦C with a step raise of 5 ◦C every 24 h. At the specified time intervals of 1, 2, 4, 6, and 24 h after each temperature raise, an aliquot of buffer was taken for the measurement of the released Dox, and the same amount of buffer was replenished immediately. The percentage of cumulative release was calculated based on the amount of encapsulated Dox.

Measurement of particle size by dynamic light scattering The hydrodynamic sizes at different concentrations in PBS were measured by a ZetaPALS system (Brookhaven, Holtsville, NY). Each sample was measured 10 times for 120 s, and the distribution was analyzed by automatic mode.

Examination of particle size under transmission electron microscopy The conjugate was dissolved in PBS at a concentration of 2 mg/mL. The staining solution containing 1% phosphotungstic acid was adjusted with 0.5 N NaOH to pH 7 followed by filtration through a 0.22 µm membrane filter. An aliquot of 10 µL conjugate was applied onto a copper mesh and allowed to stand for 5 min. The excessive liquid was wiped off followed by an addition of 10 µL staining solution. After 30 sec, the excessive solution was wiped off and then waited further for 10 to 15 min prior to observation under a transmission electron microscope (H-7100, Hitachi, Chiyoda-ku, Tokyo, Japan)

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Results and Discussion Characterization of conjugate Two levels of PEG conjugation were prepared via reaction between the aldehyde of activated PEG and the primary amine of iMAPA. The unmodified iMAPA contains a molar ratio of Asp/Arg/Lys around 5/4/1 by HPLC analysis. About 70% and 25% of the primary amine of lysine reacted with the activated PEG to form a high level (iMAPA(PEG)h) and a low level of conjugation (iMAPA(PEG)l), respectively, as assayed by the TNBSA method. Both iMAPA and its PEG conjugates become readily dissolved in an acidic aqueous environment, but are comparably insoluble in a number of organic solvents including chloroform, dichloromethane, dimethyl sulfoxide, and dimethylformamide. Thus an NMR measurement might preclude the use of those deuterated solvents. Similar to measure the NMR spectra of hydrophilic peptides in H2O/D2O (9/1), a solvent consisting of H2O/HCl/D2O (8/1/1) was used for detecting the NMR spectra of iMAPA and the PEG conjugate. On the 1H

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H-COSY-DQF spectra of

iMAPA and the iMAPA(PEG)h (Figure 1B, S1), most of the peaks are congruent with data of the reported chemical shifts of aspartic acid, arginine, and lysine. 36,37 The cross peaks of the HN with Hα suggested the existence of peptide bond linkages along the aspartic acid backbone and the side-chain linkages of β-carboxylic group of aspartic acid to the α-amine of arginine as well as to that of lysine. Under the acidic condition, the η-NH of arginine and the newly formed secondary amine between the lysine and PEG of the conjugate were undetected in the measurement presumably due to the deuterium-hydrogen exchange. Additionally, the deuterium-hydrogen exchange also limited a precise determination of the unreacted amine of lysine after conjugation by NMR spectroscopy. The IR analysis depicted the spectra of iMAPA(PEG) conjugates and iMAPA in Figure 1C. After the conjugation, the C H stretching vibration from PEG became noticeable in the region of 2840 to 3000 cm−1 , and N H bending vibrations of the primary amine of iMAPA was found to decrease in the region of 1580 to 1650 cm−1 . The diminishing from primary 9



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Figure 1: NMR and IR spectra of iMAPA(PEG)h (A) the constituents of iMAPA(PEG)h: β-arginyl aspartate, β-lysyl aspartate, β-lysyl aspartate conjugated with PEG (from left to right). (B) 1H 1H-COSY-DQF spectrum of iMAPA(PEG)h associated with 1H spectra. (C) the IR spectra of unmodified iMAPA, the low degree of PEG conjugation iMAPA(PEG)l, and the high degree of PEG conjugation iMAPA(PEG)h with the shaded area from left to right indicating the following bonding: 3300 to 3350 cm−1 secondary amine N–H stretch, 2840 to 3000 cm−1 C C stretch, 1590 to 1650 cm−1 primary amine N–H2 deformation.

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amine indicated the formation of linkage between iMAPA and PEG, and a high extent of reduction associated with a high degree of conjugation further suggested the attachment of more PEG molecules. The formation of secondary amine could also be detected in the region 3300 to 3350 cm−1 .

Phase change 60 A

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65 ◦C

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Figure 2: Concentration dependence of cloud-point temperature of iMAPA(PEG) iMAPA bearing a low level (A) and a high level (B) of PEG conjugation at various concentrations ( : 0.1 mg/mL, : 0.5 mg/mL, : 1 mg/mL, : 1.5 mg/mL, : 2 mg/mL, : 2.5 mg/mL, : 3 mg/mL) in PBS being heated from 2 to 72 ◦C with a gradient of 1 ◦C/min. (C) the relationships of concentration with the Tcp of the low level ( ) and Tcp of the high level ( ) of PEG conjugation. (D) the corresponding observed transmittances at different concentrations at 15 and 65 ◦C, respectively. Conjugation of PEG increased the solubility of iMAPA under the physiological condition. When the conjugate was dissolved in PBS, a suspension of small submicron aggregates formed below the cloud-point temperature (Tcp ) in contrast to the unmodified polymer which exits as visible flakes subsided at the tube bottom under the same condition. As the temperature was raised from 2 to 72 ◦C, the transmittance of solution began to increase, turning from opaque to transparent at temperature around 50 - 60 ◦C (Figure 2A, 2B). The phase change 11



marked a Tcp ranging from 28 to 48 ◦C, depending on both concentration and conjugation level (Figure 2C). No sharp phase change was observed. Instead, the phase change occurred gradually over the entire temperature range from 2 to 72 ◦C. However, the transmittance of iMAPA(PEG)l solution stayed between 50% and 90% after phase change, and a lower transmittance was observed for a higher concentration (Figure 2A), indicating that incomplete dissolution existed presumably due to a saturated solubility. When iMAPA was conjugated with more PEG molecules as in iMAPA(PEG)h, the solution below 2 mg/mL became complete transparence after phase change, but at a higher concentration, for example 2.5 mg/mL and 3 mg/mL, still remained 80% transmittance as shown by the translucence of solution (Figure 2B, 2D). After phase change, the higher transmittance of iMAPA(PEG)h indicated that iMAPA(PEG)h had a higher solubility than iMAPA(PEG)l at the same concentration. Overall, the thermal response behaved as a function of concentration, and the phase change temperature was positively correlated with concentration. The concentration dependence of both levels of conjugation seemed to be parallel, and the low level conjugation had a higher Tcp at the same concentration (Figure 2C). The lysine and arginine of unmodified iMAPA exhibit tight interactions of hydrogen bond which hold the unmodified iMAPA molecules together. The unmodified iMAPA was insoluble at neutral pH and in a number of organic solvents. In an acidic condition of pH 4 and 5 but not at neutral pH, the unmodified iMAPA shows UCST responses. In addition to provide a solvation effect in aqueous solution, the conjugation of PEG onto iMAPA might enlarge the intermolecular distances between the lysine and some of the arginine side chains and weaken the hydrogen-bond interactions, therefore enhancing the solubility of iMAPA(PEG) and leading to the UCST responses under the physiological condition. More molecules of attached PEG as in iMAPA(PEG)h had a higher solubility and resulted in a higher transmittance and a lower Tcp as compared with iMAPA(PEG)l, suggesting that the Tcp could be altered according to the level of conjugated PEG by balancing the strength of hydrogen bond and the solvation effect. 12


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Figure 3: pH dependence of cloud-point temperature of iMAPA(PEG) iMAPA bearing a low level (A) and a high level (B) of PEG conjugation in various pH buffer solutions ( : pH=3, : pH=4, : pH=5.5, : pH=7.4, : pH=9, : pH=10) being heated from 2 to 72 ◦C with a gradient of 1 ◦C/min. (C) the Tcp as a function of pH for the low level ( ) and the high level ( ) of PEG conjugation (D) the corresponding transmittances at different pHs at 15 and 65 ◦C, respectively. The guanidino group and primary amine at the side chains of iMAPA become ionizable at different pHs, resulting in pH-dependent solubility of iMAPA. For the conjugates, pH could still affect the ionization of remaining guanidino group as well as the solubility of conjugates after some of the primary amine of lysine reacted with PEG. At pH 3, the solution exhibited partially soluble with a transmittance around 40 – 50% before phase change, resulting in a low Tcp around 15 ◦C (Figure 3A, 3B). When pH was raised to 4, some large particles were found to settle at the bottom of tube, and a high Tcp peaked around 55 – 55 ◦C. A low transmittance also reflected the suspension of insoluble particulates after phase change (Figure 3A, 3D). Then the Tcp gradually decreased as the pH increased for iMAPA(PEG)l. For iMAPA(PEG)h, the Tcp dropped at pH 5.5, slightly increased between pH 7.4 to 9 and slightly decreased again at pH 10 (Figure 3C). After phase change, complete transparence of nearly 100% transmittance was observed for iMAPA(PEG)h in the pH range of 3 to 10 13



but the transmittance of iMAPA(PEG)l was between 35 to 90 at the same condition except at an acidic condition of pH 3. Before conjugation the unmodified iMAPA was soluble at an acidic condition, and the conjugate still tended to become soluble at an acidic condition of pH 3 with an enhanced solubility. It was observed that the pH of forming precipitates shifted from pH 7 to a lower pH of 4, presumably caused by the decrease in primary amines of the conjugate. The lower isoelectric point was presumably due to the exclusion of the primary amines of lysine which became less ionizable after conjugation. On the side chains of unmodified iMAPA, arginine has one carboxylic group pairing with one guanidino group, and lysine has one carboxylic group pairing with one amine group. The diminishing of each amine group resulted in one more carboxylate to bring down the isoelectric point, resulting a higher Tcp at pH 4.

Hysteresis and reversible-assembly vesicles Similar to other UCST-type polymers, the PEG-conjugated iMAPA exhibited hysteresis during phase change. Such hysteresis was observed for both iMAPA(PEG)l and iMAPA(PEG)h in PBS in the concentration range from 0.1 to 3 mg/mL and at different pH values from 3 to 10 at a concentration of 2 mg/mL. Under some specific conditions, the conjugate failed to become completely dissolved with less than 100% transmittance after heating (Figure 4A), or could hardly exhibited null transmittance after cooling (Figure 4B). Irrespective of the terminal states either after heating or after cooling, the changes in transmittance with temperature followed almost the same paths during repeated heating and cooling under the same condition, indicating that the Tcp of the same condition stayed invariant (Figure 4, S1-S4). Some variations among those routes were noticed but minimal, and shifting of the hysteresis patterns were hardly found. These results suggest that the intermolecular hydrogen bonds of the PEG-conjugated iMAPA could renature during cooling in a robust and responsive way. The unmodified iMAPA showed hysteresis during phase change only at an acidic condition due to the limited solubility under the physiological condition. After the 14


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transmittance (%)

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Figure 4: Hysteresis of thermal response of iMAPA(PEG) Typical hysteresis of iMAPA bearing a low level (A) and a high level (B) of PEG conjugation at a concentration 2 mg/mL during three consecutive cycles of heating and cooling ( : the first cycle, : the second cycle, : the third cycle; solid: heating, hollow: cooling) in the temperature range of 2 and 70 ◦C with a gradient of 1 ◦C/min. PEG conjugation, similar patterns of hysteresis were retained, and could be observed not only under the physiological condition but also under various pH buffers when PEG provided high solvation capability. The extension of PEG from the lysine of iMAPA enhanced the solubility but might affect the hydrogen-bond interactions very minimally because the polymers followed almost identical routes without too much hindrance from the conjugated PEG molecules on the renaturation of hydrogen bonds . Furthermore, the conjugation of PEG facilitated the formation of a self-assembled hollow structure (Figure 5), presumably due to a balance between the intrinsic hydrogen bonds of iMAPA and the solvation effect of PEG. After a pretreatment of one cycle of heating and 15


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102 103 101 102 103 101 102 103 size (nm) size (nm) size (nm)

Figure 5: Reversible formation of iMAPA(PEG)h vesicles during repeated thermal cycles (A) Size distribution of iMAPA(PEG)h of 2 mg/mL after repeated thermal treatments of heating and cooling in the temperature range of 2 ◦C and 70 ◦C with a gradient of 1 ◦C/min : one cycle, : three cycles, : five cycles. (B) The corresponding TEM images after different cycles with the indicated average hydrodynamic diameters (one, three, five cycles from left to right). cooling which enabled complete dissolution, the iMAPA(PEG)h was found to spontaneously form such vesicles after being subject to each subsequent cycle of heating and cooling treatment. A reversibility of the vesicle formation was noticed when up to five times of repeated processes were employed to heat a suspension full of submicron assemblies to completely transparent followed by the returning to assemblies of the approximate sizes below Tcp . The structure sizes seemed a heterogeneous of distribution around 30 to 70 nm randomly assembled by iMAPA(PEG)h (Figure 5). The sizes slightly depended on the concentration, and a larger size was found at a higher concentration, presumably due to an aggregation of more molecules (Figure S5).

Encapsulation of doxorubincin In an attempt to encapsulate Dox, iMAPA(PEG)h of 2 mg/mL was used because the polymer exhibited as complete dissolution with 100% transmittance after phase change at this concentration. After two rounds of heating and cooling, the conjugate was employed to encapsulate Dox. At an elevated temperature, 55 ◦C, that is higher than the Tcp , the hy-

16


60

A

100 80 60

1.6 1.2

40 20 0

loading capacity (mg dox/mg polymer)

80

encapsulation efficiency (%)

100

0

10

20

30 40 50 dialysis time (h)

60

70

0.8

40

0.4

20

cumulative leakage (%)

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

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encapsulation efficiency (%)

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0

0.5 1.0 1.5 2.0 dox/polymer weight ratio (mg/mg) after encapsulation

4

4 weeks later

B

3 2 1 0

5

10

15 20 time (day)

25

30

Figure 6: Encapsulation of Dox with iMAPA(PEG)h (A) Encapsulation efficiency ( ) and loading capacity ( ) of Dox with iMAPA(PEG)h at different weight ratios and the inset showing the changes of encapsulation efficiency at each weight ratio (Dox/polymer) with time during dialysis ( : 2, : 1.5, : 1, : 0.5). (B) Leakage of the Dox-loaded vesicles at the Dox/polymer weight ratio of 2 during the storage at 4 ◦C and the morphologies of the vesicles before storage and storage after 4 weeks at 4 ◦C, respectively. drogen bond was dismantled, and the vesicles were unfolded. Dox was encapsulated during the subsequent cooling process, and the unencapsulated Dox was removed by dialysis. After 24h the amount of Dox in the dialysis buffer fell almost below the detection limit. However, the dialysis was carried out for additional 48 h in an effort to completely remove the unencapsulated dox (Figure 6A inset). The relationship of the encapsulation efficiency and the Dox/polymer ratio exhibited similar to the rectangular hyperbola of an enzymatic reaction. At a Dox/iMAPA(PEG)h ratio of 0.5 mg/mg, around 37% of Dox was included into the vesicles during cooling. Then the proportion increased to 53% at the ratio of 1 mg/mg, and began to level off to around 70% at 1.5 mg/mg and 2 mg/mL (Figure 6A). On the other hand,

17



the loading capacity per unit weight of polymer increased linearly from 0.2, 0.5, 1.0 to 1.4 mg dox/mg iMAPA(PEG)h, presumably due to more amounts of iMAPA(PEG)h interacting with Dox. Compared with Doxil, a currently used delivery vehicle of Dox in cancer treatments, which formulation generally contains 2 mg/mL Dox in 16 mg/mL lipid of liposomes with a loading capacity of 0.125 mg Dox/mg lipid, the loading capacity of iMAPA(PEG)h could reach more than ten folds. The high encapsulation efficiency might also suggest that some unexpected interactions between Dox and iMAPA(PEG) enahnced the inclusion of Dox into the hollow structure. The Dox-loaded vesicles of 2 mg Dox/mg iMAPA(PEG)h were further kept at 4 ◦C to examine the stability. The leakage of Dox was increased to around 1% of the total loaded amount after the first week, and then slowly to 1.7% over the next three weeks (Figure 6B). The morphology of the vesicles still remained intact with a slight dilation after 4 weeks of storage. Most of the sizes were found to increase less than one fold in diameter under TEM observation, and Dox precipitates at the low temperature seemed to form a shade area inside the hollow structure. The observations indicated the Dox-loaded vesicles can be stored at 4 ◦C as stable for least 4 weeks.

Release of doxorubincin Release of Dox was performed by a series of stepwise increases in temperature for the vantage of iMAPA(PEG)h which showed gradual increases in transmittance with temperature and was completely soluble at an elevated temperature. Upon each temperature change, a burst release of Dox was observed immediately, most of the release finished within 6 h, and then the release continued increasing slowly with time before the next temperature raise was applied to induce another quick release of Dox (Figure 7). The release followed a ladder pattern escalating from 0% to complete release at 62 ◦C accompanying the stepwise increases in temperature. Disproportional releases at each temperature raise were observed with some larger releases at 52 ◦C and 57 ◦C of which the latter temperature could cause almost more 18


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120

37 ◦ C

42 ◦ C

47 ◦ C

52 ◦ C

57 ◦ C

62 ◦ C

100 cumulative release (%)

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

Biomacromolecules

80

62𝑜 C

60 40 37𝑜 C

20 0

47𝑜 C

0

20

40

60 80 100 120 140 time (h)

Figure 7: Temperature triggered release of Dox Profile of Dox released from the Dox-loaded vesicles at the Dox/polymer weight ratio of 2 when temperature being heated from 37 to 62 ◦C with a step raise of 5 ◦C and a duration of 24 h for each step with different symbols representing three independent experiments and the solid line passing through the averages; the photos of Dox release at 37, 47, and 62 ◦C as indicated, respectively, by the conical tubes showing different Dox/polymer weight ratios of 0.25, 0.5, 1, and 2 from left to right in each photo. than 95% release of loaded Dox. From the relationship of transmittance and temperature, half of the vesicles was assumed to disintegrate at the Tcp of around 38 ◦C at 2 mg/mL of iMAPA(PEG)h from the earlier pattern of thermal response (Figure 4B). However, the release of Dox at 42 ◦C was about 25%, far less than the estimation from the Tcp (Figure 7). A release of 60% loaded Dox was observed at an elevated temperature of 52 ◦C. The inconsistency between transmittance and Dox release might be due to the lack of proportionality between the percentage of disintegrated vesicles and the transmittance. Another reason of the inconsistency might come from some interactions between iMAPA(PEG)h and Dox which strengthened the original mutual interactions of iMAPA(PEG)h to become disintegrated at a higher temperature than the Tcp . Different weight ratios of Dox to iMAPA(PEG)h had similar profiles of the temperature triggered release in terms of the loaded Dox, resulting in a predictable release rate by a fixed temperature irrespective of the drug/polymer ratios

19



(Figure S6). As the temperature increased, more of the released Dox diffused out of the dialysis membrane into the dialysis buffer which color was observed to turn from almost transparent to pale tint orange, and then to dark orange. Although a complete release occurred at a temperature higher than the normal body temperature of 37 ◦C, a gradual and continuing release near the body temperature could be expected when the Tcp decreases with the decreasing concentration after the excretion of iMAPA(PEG)h.

Conclusion In conclusion, iMAPA originally possesses an UCST response at an acidic condition, and such thermal properties could be observed under the physiological condition of neutral pH after conjugation of PEG. The conjugate exhibited almost the same pattern of thermal responses during several cycles of repeated heating and cooling at the same concentration and pH environment, providing a predictable state for the inclusion of other chemicals. After complete dissolution, a subsequent cooling process was observed to prompt a reversible formation of core-shell nano-structure which can be further employed for drug encapsulation. Both a high encapsulation efficiency of 70% and a loading capacity of 1.5 mg Dox/mg polymer could be achieved, and the Dox-loaded structure could maintain stable up to 4 weeks of storage at 4 ◦C. Additionally, the vantage of gradual Tcp changes of the conjugate allows the regulation of Dox release rates through elevating the temperature to different levels. The conjugate iMAPA(PEG)h presents a potential as a drug delivery system with a capability of temperature regulating release.

acknowledgements The research was supported by the grant MOST 107-2221-E-011-063 from the Ministry of Science and Technology at Taiwan. The authors wish to thank Miss Ching-Yen Lin and Miss Ya-Yun Yang for the assistance of performing TEM at the Instrumentation Center of 20


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National Taiwan University.

Supporting Information Available The Supporting Information contains the NMR spectrum of iMAPA, the hysteresis of iMAPA(PEG) during repeated heating and cooling, the morphology and size distribution of iMAPA(PEG)h observed under TEM and detected by DLS.

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Biomacromolecules

Graphical TOC Entry Asp

Asp

Asp

Arg

Lys

Lys

1

monomers cin ← in ←←←←←→ ub ←←←← or←←←←←←←←←ng x do ←←←← oli + ←←←←←←←← co ←←←

heating ←↽ ←←←←←←←←←←←←←←←←←←←←←←⇀ ←←←←← cooling < UCST

120

37 ◦ C

42 ◦ C

47 ◦ C

52 ◦ C

57 ◦ C

62 ◦ C

100 cumulative release (%)

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

Page 24 of 24

PEG

heating ←←←←←←←←←←←←←←←←←→ ←

80 60 40 20 0

> UCST

24

0

20

40

60 80 100 120 140 time (h)


Reversible self-assembly nano vesicle of UCST response prepared

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