Injectable Hydrogel: Amplifying the pH Sensitivity of a Triblock

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Injectable Hydrogel: Amplifying the pH Sensitivity of a Triblock Copolypeptide by Conjugating the N-termini via Dynamic Covalent Bonding Maria-Teodora Popescu, George Liontos, Apostolos Avgeropoulos, Efstathia Voulgari, Konstantinos Avgoustakis, and Constantinos Tsitsilianis ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b03977 • Publication Date (Web): 24 Jun 2016 Downloaded from http://pubs.acs.org on June 29, 2016

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ACS Applied Materials & Interfaces

Injectable Hydrogel: Amplifying the pH Sensitivity of a Triblock Copolypeptide by Conjugating the N-termini via Dynamic Covalent Bonding Maria-Teodora Popescu, a George Liontos, b Apostolos Avgeropoulosb Efstathia Voulgari,c Konstantinos Avgoustakisc and Constantinos Tsitsilianis a* a

Department of Chemical Engineering, University of Patras, 26504, Patras, Greece. Department of Materials Science and Engineering, University of Ioannina, University Campus, 45110 Ioannina, Greece. c Department of Pharmacy, University of Patras, 26504, Patras, Greece. b

Keywords: copolypeptide, pH-responsive, injectable hydrogel/polymersome formulation, sustained drug delivery.

hydrogel,

Abstract: We explore the self-assembly behavior of aqueous solutions of

an

amphiphilic, pH-sensitive poly(L-alanine)-b-poly(L-glutamic acid)-b-poly(L-alanine), (A5E11A5) triblock copolypeptide, end-capped by benzaldehyde through Schiff base reaction. At elevated concentrations and under physiological pH (7.4) and ionic strength (0.15M), the bare copolypeptide aqueous solutions underwent a sol-gel transition after heating and slow cooling thermal treatment, forming opaque stiff gels due to a hierarchical self-assembly that led to the formation of β-sheet-based twisted super fibers (Popescu et al. Soft Matter, 2015, 11, 331-342.). The conjugation of the Ntermini with benzaldehyde (Bz) through a Schiff base reaction, amplifies the copolypeptide pH-sensitivity within a narrow pH window relevant for in vivo applications. Specifically, the dynamic character of the imine bond allowed coupling/decoupling of the Bz upon switching pH. The presence of Bz conjugates to the N-termini of the copolypeptide, resulted in enhanced packing of the elementary superfibers into thick and short piles, that inhibited the ability of the system for 1 ACS Paragon Plus Environment


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gelation. However, partial cleavage of Bz upon lowering pH to 6.5 prompted recovery of the hydrogel. The sol-gel transition triggered by pH was reversible, due to the coupling/decoupling of the benzoic-imine dynamic covalent bonding, endowing thus the gelling system with injectability. Undesirably, the gelation temperature window was significantly reduced, which however can be regulated at physiological temperatures by using a suitable mixture of the bare and the Bz-conjugated coplypeptide. This triblock copolypeptide gelator was investigated as a scaffold for the encapsulation of polymersome nanocarriers, loaded with a hydrophilic model drug, calcein. The polymersome/polypeptide complex system showed prolonged probe release in pH 6.5, which is relevant to extracellular tumor environment, rendering the system potentially useful for sustained delivery of anticancer drugs locally in the tumor.

1. Introduction Artificial block copolypeptides, incorporating hydrophilic and hydrophobic building blocks, that adopt specific secondary structural motifs, like α-helix, β-sheet or random coil, have attracted considerable attention as self-assembly biomaterials which can find biomedical applications as drug nanocarriers (in the form of micellar nanoassemblies) or scaffolds (hydrogels) for tissue engineering.1,2,3,4,5,6 Concerning hydrogels, in most of the cases they hierarchically self-assemble in aqueous media forming nanofibrous 3 dimensional structures. In this case, fibrillogenesis is based on the formation of β-sheet or α-helix secondary conformations of the polypeptide building blocks, mainly driven by hydrophobic interactions and stabilized by hydrogen bonding, developing fibrillar nanostructures of high aspect ratio.7 Block copolypeptides can be designed through bottom-up strategies using the chain growth polymerization method, specifically by ring opening polymerization of amino acid N-carboxyanhydrites, initiated by an amine group which leads to welldefined macromolecules with high fidelity N-termini.3,8,9,10 For instance, amphiphilic block copolypeptides, constituted of poly(L-lysine) and poly(L-leucine), have been previously designed by Deming et al.11,12 These block copolymers, irrespective of their macromolecular topology (diblock, triblock or pentablock), associate into

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hydrogels at very low polymer concentrations, through the formation of hydrophobic α-helices, associated perpendicularly to fibril long dimensions. Stimuli responsive copolypeptides are a very interesting class of biomacromolecules which form physical hydrogels through noncovalent interactions, triggered by an external stimulus.7,13,14,15 In this case, we are talking about the socalled injectable hydrogels, the formulations of which are in the sol state under the preparation conditions, enabling “in situ” gelling after injection in the body. The appearance of the sol–gel transition can be triggered by various stimuli including salt,16,17,18, temperature,19,20,21,22 , enzymes23,24,25,26 and pH.7,27,28,29,30 Alternatively to conventional stimuli responsive systems, a novel class of amphiphiles constructed on the basis of dynamic covalent bonds (DCB) has emerged.31,32,33 The imine bond is the most widely investigated DCB, since it has demonstrated the formation of nanoparticles with strong pH sensitivity, suitable for biomedical applications. An exciting work based on coupling of methoxypoly(ethylene

glycol)-b-poly(L-lysine

hydrochloride)

(PEG-b-PLKC)

to

4-

(decyloxy)benzaldehyde (DBA) has been reported by Zhang et al.34 The system formed micellar assemblies at pH 7.4, which disintegrated at pH 6.5, by cleavage of the imine bond, which could further be reformed for several pH cycles. This concept has been expanded to conjugation of hydrophobic drugs onto double hydrophilic polymers that self-assemble into micelles at physiological pH and dissociate at acidic conditions, triggering drug release.35 Similarly, many pH responsive gelling systems have been also constructed through this Schiff’s base reaction, either by coupling modified block copolymers

36,37,

or by incorporating of DCB as cross-links.38,39 The

dynamic covalent bonds endow the hydrogels with sharper stimuli-responsive sol-gel transition, enhancing considerably their potential in delivery applications. While the literature covering the use of dynamic bonds on polymer chains or on random coil polypeptide chain is quite abundant,31-33 to the best of our knowledge, there is no report on the influence of such functionality on more ordered peptide structures such as α-helix or β-sheet. Several reports have demonstrated that end-capping of short polypeptide endblocks of a (DL/L-PA)-PLX-(DL/L-PA) thermoresponsive hydrogelator, results in increased β-sheet and in a reduction of the sol-gel transition temperature.40,41 In addition, fabricated

nucleobase-functionalized from

self-assembly

supramolecular of

nanofibers

adenine-terminated

were

recently

oligopeptide

and 3

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complementary nucleobase interaction was employed as a functional factor for controlling the peptide self-assembly.42 The general trend that derives from these studies is that the hydrophobic/hydrophilic balance alters drastically the structural transition of the peptide chain. Thus, it is of interest to impart reversibility to such transition by the incorporation of dynamic bonds onto peptidic hydrogels. To this end, we explore herein the gelation ability of a triblock copolypeptide, endcapped with benzaldehyde through benzoic-imine dynamic covalent bond. As we have shown recently, the bare poly(L-alanine)-b-poly(L-glutamic acid)-b-poly(Lalanine) (A5-E11-A5) triblock copolypeptide, self-assembles hierarchically at elevated concentrations and physiological conditions, forming a β-sheet-based fibrilar hydrogel.43 The initial motivation was to explore the effect of increasing hydrophobicity of the end-blocks, by conjugating the amino end groups with benzaldehyde (Bz) through Schiff’s base reaction, as to improve the gel properties in terms of critical gelation concentration, pH responsiveness and injectability. Although not expected, the ability of the system for gelation was inhibited at physiological conditions. Importantly, upon switching pH to 6.5 the hydrogel was recovered. It is demonstrated that the benzaldehyde end-capped triblock copolypeptide undergoes reversible sol-gel transition in response to pH, in a narrow window which is relevant for biomedical applications. The Bz-conjugated triblock copolypeptide was used as a scaffold for encapsulation of polymersome nanocarriers, loaded with calcein as hydrophilic model drug. This complex drug delivery system exhibited much slower drug release by switching pH from 7.4 to 6.5, which is relevant to extracellular tumor environment, and might be considered for prolonged drug delivery to tumors in anticancer chemotherapy.

2. Experimental Section 2.1 Materials. All solvents used were analytical or HPLC grade and were purchased from Merck (Germany). Calcein and other materials (buffer preparation stuff) were of analytical grade and were purchased from Sigma-Aldrich Hellas. 2.2 Synthesis of the triblock copolypeptide.


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The NCAs of γ-benzyl-L-glutamate (BLG-NCA) and L-alanine (Ala-NCA) were synthesized from the corresponding α-amino acids and triphosgene in ethyl acetate at 70 0C under inert atmosphere according to the literature.44,45 The A5E11A5 triblock copolypeptide was synthesized by sequential addition of the NCAs of the corresponding α-amino acids in THF/DMF, using 1,6-hexamethylenediamine as initiator, followed by alkali-hydrolysis deprotection of the central PBLG block. Characterization of the copolypeptide was performed prior to hydrolysis by GPC and 1

H NMR and its characteristics are presented in Table 1. Details of synthesis and

characterization are reported elsewhere. 43 Table 1. Molecular characteristics of the triblock copolypeptide.

Poly(L-alanine)-b-(γ-benzyl-L-glutamate)-b-(Lalanine) (PAla-PγBG-Pala) Poly(L-alanine)-b-(L-glutamic acid)-b-(Lalanine) (A5E11A5) By a 1H-NMR and b GPC

Ib

E/BGa

Aa

E/BGa

Mn PGA

Mn PAla

(g/mol)a

(g/mol)a

2 400

760

1.11

11

10

0.76

1 420

760

1.11

11

10

0.65

%wt

2.3 Synthesis of benzaldehyde-capped A5E11A5. Benzaldehyde (Bz) and PAla-PGA-PAla triblock copolypeptide were dissolved in 20 mL of DMSO at the molar ratio PAla-PGA-PAla: Bz of 1:4 using twofold excess of the benzaldehyde groups to ensure quantitative end-capping. The mixture was stirred and heated to 40°C for 24 h before the solvent was evaporated by a rotary evaporator. The rough product was washed with diethyl ether and methanol, and dried under reduced pressure at 40°C to gain a pale yellow powder. The conjugated polypeptide was denoted as Bz-A5E11A5-Bz. 2.4 Methods. NMR spectroscopy.

1

H-NMR spectra were obtained on a Brucker AC-200

spectrometer, at room temperature, in D2O at different values of pD, adjusted by addition of appropriate amounts of NaOD or DCl. Scanning electron microscopy. SEM imaging of freeze dried samples was performed using a LEO SUPRA 35 VP scanning electron microscope. All specimens were sputtered with gold before imaging. Rheology. Rheological measurements were carried out using a stress-controlled rheometer AR-2000ex (TA Instruments) equipped with a plate and plate geometry (diameter = 20 mm, truncation = 500 µm). After sample loading, a delay of 5 min was



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applied prior to any measurement to erase any shear history. The linear viscoelastic regime was established by oscillatory strain sweeps using a frequency of 1 Hz. Dynamic oscillatory sweeps (in linear regime at 25 0C) were performed for samples viscous enough to provide meaningful results. The rheometer was thermally regulated (±0.10C) by the Peltier controlling system. To prevent changes in concentrations from water evaporation during the experiments, a solvent trap was used. 2.5 Sample Preparation. Hydrogel preparation. The copolypeptide was dissolved in PBS with 0.15M NaCl at room temperature and stirred for 24 h to ensure complete dissolution. The pH was adjusted by adding appropriate amounts of 1N HCl or 1N NaOH. For hydrogel preparations, the as-obtained milky concentrated solutions were then heated by increasing temperature at a rate of 1 0C/min under stirring (400 rpm) in a water bath until clear solutions were obtained. After equilibration for 30 min, the solutions were slowly cooled down, with a rate of 10C/min, until free standing opaque hydrogels were obtained.43 The as-prepared hydrogels were kept overnight at 40C before use. Calcein encapsulation in polymersomes and release. Calcein was encapsulated in the polymer vesicles by the film hydration method. The pentablock terpolymers were first solubilized in CHCl3 (P5b) or in CHCl3/MeOH (Q5b). A thin film was obtained by solvent evaporation, that was subsequently hydrated with a calcein solution 100 mM in PBS 0.15M NaCl at pH 7.4. The resulting polymeric dispersions were placed in a bath-type sonicator (Branson 1200) for 3 cycles of 15 min. To prepare the composite formulations, the triblock copolypeptide was dissolved in the polymersome dispersion, followed by stirring for 24 h. Then, the solution was heated at 370C in a water bath at a slow heating rate as described above. It should be noted here that this temperature treatment was monitored by DLS and did not affect the vesicular integrity. Further on, the warm composite solution was split in two, placed in dialysis tubes (SpectraPOR MWCO 2 000) and dialyzed against PBS (0.15M NaCl) at pH 7.4 and 6.5, respectively for 8 hours at 40C to ensure the physical stability of the incorporated vesicles. The pH of the medium was periodically checked to determine when the equilibrium was attained. This was also confirmed by visual inspections of the tubes, with a free flowing opaque solution at pH 7.4 and an immobile gel at pH 6.5. In both cases, the polymersome concentration was 10 mg/g polypeptide (1 wt%). The release of calcein was then monitored at room temperature by fluorescence spectroscopy (EX 470 nm, EM 520 nm). The amount of calcein released was 6 ACS Paragon Plus Environment

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calculated as: cumulative calcein release (%) = (Mt /M0) x 100, where Mt is the total amount of drug released at time t, and M0 is the amount of calcein loaded in the polymer vesicles. 2.6 Cytotoxicity study. A549 cells (ATCC) were cultured in RPMI supplemented with 10% (v/v) fetal bovine serum and a cocktail of antibiotic agents with 100 µg/ml penicillin- streptomycin at 37oC, in a humidified atmosphere with 5% (v/v) CO2. The culture medium was changed every 48 h and cells were harvested with 0.25% (v/v) trypsin in PBS. The cytotoxicity of the benzaldehyde conjugated polypeptide (Bz-A5E11A5-B) was evaluated against the A549 cell line, using the propidium iodide (PI) fluorescence method.46 Data analysis was performed with the WinMDIcytometry analysis software. The cells were seeded in the presence of medium into 24-well plates at a density 5x104 cells/well and allowed to attach and proliferate for 24 hours under standard cell culture conditions. Then, the supernatant in each well was replaced with fresh medium (pH 7.4) and dilute HCl (pH 6.5) at 37°C, containing various concentrations (1, 5, 10, 20, 50 and 100 µg/ml) of Bz-A5E11A5-Bz.

After 24 hours

the supernatant was removed and the cells were washed with phosphate buffered saline (PBS). The cells were harvested with 0.25% (w/v) trypsin, transferred to FACS tubes and then centrifuged (1600 rpm for 5 min).The pellet was washed with PBS and the cells from the pellet were incubated with 5 µl PI solution (Propidium Iodide stock 1mg/ml) for 1 min. The PI fluorescence (cell death) was determined with flow cytometry (excitation λ= 488 nm, emission λ= 620 nm), in a FACS Calibur, Coulter Epics XL-MCL apparatus. To calculate the background fluorescence of the cells, unlabeled cells without any addition of studied conjugates or drug, were used as a negative control in every measurement.

3. Results and Discussion

The A5E11A5 triblock copolypeptide under investigation exhibits pH/thermoresponsive properties thanks to the ionic nature of the central block and its general protein-like character (e.g. denaturation upon heating). As we have shown previously,43 at elevated concentrations and in aqueous media of physiological conditions, pH and ionic strength (pH 7.4, 0.15M salt), the copolypeptide aqueous 7 ACS Paragon Plus Environment


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solutions underwent a sol-gel transition after heating and slow cooling thermal treatment, forming opaque stiff gels. Structural investigation by electron microscopy (TEM and SEM) revealed that the copolypeptide chains underwent hierarchical growth of β-sheet-based fibrils, into giant bundles of superfibers that were disrupted and rearranged upon thermal treatment into a supermolecular network of twisted rigid superfibers (Figure 1). The so-formed hydrogel was characterized by pH sensitivity and fast self-healing after shear induced gel disruption. Moreover, a critical copolypeptide concentration was determined at 4.5 wt%, to form a hydrogel whereas below this value, clear solutions were obtained at all temperatures. Thus, upon tuning the copolypeptide concentration the gelation can be adjusted to physiological temperature. 43

Figure 1. SEM images of A5E11A5 freeze dried from a 10wt% solution of pH 7.4 (A) before and (B) after thermal treatment. Inset: schematic representation of the A5E11A5 forming βsheet motif.

3.1 Synthesis and characterization of Bz-A5E11A5-Bz In thermogeling systems based on triblock copolymers, bearing β-sheet forming peptide end-blocks, it was demonstrated that the increase of the hydrophobicity of the end-blocks by capping the N-termini with alkyl groups affect the sol-gel transition. More importantly, as the alkyl group length increased from acetyl, to butyryl, the b-sheet content of PAla increased and the self-assembled morphology of the polymer changed from spherical micelles to fibrous nanostructures. 41,47 Inspired from these findings, the N-termini of the copolypeptide were slightly modified to enhance the hydrophobic character of the associative PAla


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end-blocks

by

end-capping

reaction

using

benzaldehyde

(Bz-A5E11A5-Bz).

Additionally, the preference of this reaction was dictated from the pH responsive dynamic character (reversible) of the formed imine bonds (Scheme 1). It has been established that the imine bond has a dynamic reversible character upon switching pH values.31-33 Consequently, we have inspected by 1H NMR (Figure S1) this dynamic bonding by decreasing pH to 6.5, relevant to extracellular tumor environment, having in mind potential biomedical applications. At pH 7.4 a large peak at 8 ppm is observed, indicative of the imine bond between the Bz and end-chain amino groups of the triblock copolypeptide. Some residues of free benzaldehyde are observed (at 10 ppm), probably caged into the system during purification.

Scheme 1. Illustrative end-conjugation of the A5E11A5 with benzaldehyde and its pH responsiveness.

Decreasing the pH to mildly acidic conditions (pH 6.5), results in a reduction of the imine bond content and an increase of free benzaldehyde in the solution corresponding to ~14 molar %. This result demonstrates that acid treatment at pH 6.5 caused partial hydrolysis of the imine linkages and, thus, the position of the imine equilibrium for this end-capped triblock copolypeptide system might be shifted towards lower pH values. It should be noted that the 1H NMR spectra have been recorded for samples prepared before any thermal treatment. In addition to pH, the imine bond is also sensitive to changes in temperature particularly in the pH region where the disassembly is initialized.48,49 Since the morphology of the samples was significantly different, it is possible that increasing temperature probably push the 9 ACS Paragon Plus Environment


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equilibrium further in the direction of hydrolysis. Nonetheless, even though the hydrolysis of the imine bond is not complete, a significant difference in the mechanism of self-assembly was observed, emphasizing the importance of the endchain functionality for the association of these triblock copolypeptides.

3.2 Hydrogel preparation and pH responsiveness The gelation ability of the Benzaldehyde end-capped block copolypeptide was investigated for a 10 wt% solution in PBS (0.15 M NaCl), at different values of pH. At pH 7.4, an opaque suspension was obtained from Bz-A5E11A5-Bz with no hydrogel formation upon temperature treatment. Specifically, by heating this suspension up to 700C, a clear solution was obtained; however, by cooling back to room temperature the opaque suspension was recovered, in contrast to gelation observed in the bare copolypeptide. However, upon decreasing pH at 6.5 (tumor environment), a free standing opaque hydrogel was formed by heating/cooling cycle as depicted in the digital micrographs of Figure 2 (top). More importantly, the temperature of the sol-gel

Figure 2. Digital photographs showing the thermal procedure for the gelation of Bz-

A5E11A5-Bz 10 wt% in PBS, pH 6.5 (top) and the pH responsive sol-gel transition for two cycles of pH switch at room temperature.

transition was significantly reduced at 27 oC with respect to 52 oC observed in the unmodified A5E11A5 hydrogel. In addition, the pH responsiveness of the Bz-A5E11A5Bz gel was explored by switching the pH between 7.4 and 6.5. As can be observed in Figure 2, the pH induced sol-gel transition is reversible for two cycles of pH variation.


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This amplification of pH sensitivity in such a narrow pH window, could be proved important for drug delivery biomedical applications, as it will be discussed below. In order to get insight into the mechanism of the pronounced pH sensitivity of the Bz-A5E11A5-Bz hydrogel, the 10wt% formulations obtained after thermal treatment were freeze dried and visualized by SEM. As depicting in Figure 3, at pH 7.4 the Bz conjugated triblock copolypeptide self-assembles forming thick tapes of about 200 nm in width, which probably consists of a great number of much thinner elementary ribbons. These structures were too short, with lengths varying from 0.5 to 2 µm, to entangle with each other, thus no three-dimensional network enabling solvent trapping could be formed.50 It has been previously reported that by increasing the attraction energy, infinite sheets of stacked ribbons are formed, instead of individual fibers.51 The same effect seems to occur in the present system as well, since at pH 7.4, the hydrophobic attraction of the elementary tapes formed by the Pala endblocks is enhanced by π-π stacking of the benzene end-groups. On the contrary, at pH 6.5 the presence of an extended entangled network, composed of thin fibers similar with those formed by the bare triblock copolypeptide (Figure 1B) and bundles of stacked fibers with an average width of ~ 160 nm, was revealed. As mentioned before, due to the increased attractive interactions, the elementary twisted fibers stack together resulting in a mixture of thin supermolecular fibers and bundles of such fibers, with sufficient flexibility to entangle and form a free standing hydrogel. In fact, it has been previously reported that the changes in total hydrophobicity of artificial and amphiphilic peptides by guest additives, such as cyclodextrin52 and azobenzene derivatives53 affects the self-assembling process of the amphiphilic peptides. In particular, it was reported that the hydrophobicity enhancement of the peptides promote the structural transition into β-sheet drastically. Therefore, it can be assumed that the hydrophobic benzene ends triggers the formation of very thick βsheet stacked tapes. On the contrary, cleavage of the hydrophobic group increases the flexibility of the polypeptide chain that can be further rearranged by thermal



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Figure 3. SEM images Bz-A5E11A5-Bz aqueous suspensions freeze dried from a 10 wt% formulation at pH 7.4 and 6.5.

treatment, favoring controlled growth into matured superfibers that further entangle into a three dimensional supramolecular network. Another important issue is that the gelation temperature window for the BzA5E11A5-Bz hydrogel was undesirable shifted below 27 oC at pH 6.5, which prevents potential biomedical applications. As demonstrated above (Fig. 3), SEM imaging revealed a structure constituted of a mixture of fine superfibers and thick bundles of stacked fibers at pH 6.5, arising from the partial hydrolysis of the imine bonds. The fact that the Bz-A5E11A5-Bz chains form stacked ribbons, which prevents hydrogel

Figure 4. Sol-Gel transition for A5E11A5 (A), Bz-A5E11A5-Bz (B) and mixtures of the two polymers (50/50 w/w) (C) of 10wt% in PBS, pH 6.5 and 0.15M NaCl, upon thermal treatment.

formation at pH 7.4, implies that the gelation depends on the fraction of the nonstacked superfibers, which arise from the partial cleavage of the benzene from the conjugated triblock copolypeptide upon decreasing pH to 6.5. This result is in line 12 ACS Paragon Plus Environment

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with the concentration dependence of the gelation temperature.43 Hence, we have considered increasing the fraction of the thin superfibers by mixing conjugated and bare copolypeptides in order to attempt tuning the gelation window. For this purpose, a mixture of triblock copolypeptide with its Bz conjugate at 50/50 weight ratio, was investigated in 10 wt% overall polymer concentration aqueous solution of pH 6.5. Figure 4 demonstrates the phase behavior of the three systems (blend and pure constituents) induced by heating and slow cooling treatment. As observed, the gelation temperature window for the A5E11A5/Bz-A5E11A5-Bz 50/50 blend was broadening up to 35 oC, approaching the targeting physiological temperature window confirming our expectations. The above findings therefore indicate that the gelation temperature can be regulated to physiological conditions just by tuning the mass ratio of the Bz-conjugated and the bare copolypeptide precursor (see ESI, Fig. S2). Figure 5 shows the viscoelastic behavior of the Bz-A5E11A5-Bz hydrogel at pH 6.5. The storage modulus G’ was always higher than loss modulus G’’ and both moduli were nearly frequency independent, illustrating the formation of an elastic hydrogel with high storage modulus of the order of 105 Pa. Interestingly, G’ was almost one order of magnitude lower than that of the hydrogel formed by the bare copolypeptide at pH 7.4. Obviously, the origin of this difference should be attributed to the structural differences between the involved copolypeptides. Again, the fraction

G', G'' (Pa)

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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7

10

6

10

5

10

4

10

3

10

2

10

-2

10

-1

10

0

10

1

Frequency (Hz)

Figure 5. Storage modulus (G’) (filled symbols) and loss modulus (G’’) (open symbols) as a function of frequency for the bare hydrogel A5E11A5 (●, magenta), and Bz-A5E11A5-Bz at pH 6.5 (■, blue) and pH 4 (■, purple), of 10wt% copolypeptide in PBS 0.15M NaCl.

of the isolated superfibers (versus stacked), seems to be the determining factor affecting the overall elasticity of the hydrogel. Although speculative, a possible 13 ACS Paragon Plus Environment


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correlation between the mechanical rigidity of the gels and the local morphology of their respective assemblies can be made. The thinner fibers formed by the bare triblock copolypeptide, as opposed to the mixtures of higher order stacked fibers obtained by the end-capped Bz-A5E11A5-Bz at pH 6.5, afford the most rigid hydrogel. It may be that the smaller width and more flexible fibers can entangle more efficiently in the three-dimensional network, resulting in more rigid hydrogels. To confirm this hypothesis, the recovery of the initial A5E11A5 hydrogel by full decoupling of benzaldehyde from the Bz-A5E11A5-Bz conjugate at even stronger acidic conditions (pH 4) was attempted. As depicted in Figure 5, the network is significantly strengthened with the storage modulus approaching the G’ determined from the A5E11A5 hydrogel at pH 7.4, suggesting a complete recovery of the hydrogel structure upon complete cleavage of the Bz groups. This result demonstrates that the strong pH responsiveness of the imine dynamic covalent bond can be used in order to modulate both gelation temperature window and mechanical rigidity of the copolypeptide hydrogel.

3.3 Hydrogel/polymersome drug delivery nanocomposite system As was stated in the Introduction, the motivation behind the present work is to investigate the ability of the injectable triblock copolypeptide hydrogel, in which drug-loaded polymersomes have been entrapped, to release in a controlled fashion the encapsulated drug. Environmentally sensitive hydrogels have been widely used for controlled drug release applications via a volume phase transition or a gel-sol process in response to various external stimuli.7,13-15 Despite many advantageous properties, hydrogels also have several limitations mainly related to the high water content and large pore sizes that often result in relatively rapid drug release. As a result, growing interest has focused on overcoming the inherent pharmaceutical limitations of hydrogels by co-formulating particulate systems into the hydrogel matrix, to form composite hydrogel networks. To date, various classes of composite hydrogels have been reported which were mainly based on liposomal formulations, embedded within physically or chemically cross-linked three dimensional networks.54,55,56 In view of biomedical applications, peptidic fibrillar hydrogels have been mainly developed as substrates for cell growth and delivery of cells for tissue regenerative therapy.

4,57,58

Therefore, it was of interest to study this novel pH sensitive hydrogel as a scaffold for 14 ACS Paragon Plus Environment

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drug release from vesicular nanocarriers, aiming at potential applications as antitumor sustained-release injectable preparations. 59,60 Hence, the Bz-A5E11A5-Bz triblock copolypeptide injectable gelator was further investigated as a 3D matrix for encapsulation of polymersomes charged with a model hydrophilic model drug (e.g. calcein). Having in mind the benefits of the PCLPEO-P2VP-PEO-PCL pentablock terpolymers vesicles (see ESI),61,62 and the modulation potential of their membrane permeability (i.e. through pH or quaternization), we have chosen them as nanocarriers embedded within the polypeptide hydrogel. As illustrated schematically in Fig. 6 (top), the membrane of the polymersome is formed by the hydrophobic association of the P2VP central block and the PCL outer blocks of the terpolymer, while the internal and external soluble shell is constituted of PEO chain loops, as have been described in details in previous reports. 61-62 Two types of vesicles were used, differing in the degree of ionization of the P2VP membrane forming block, i.e non-ionized and 19 mol% ionized (bearing positively charged quaternized groups) denoted as P5b and Q5b vesicles respectively. The morphology of the composite system was visualized by SEM. Figure 6 shows the SEM micrograph of the fibrilar hydrogel network containing pentablock terpolymer spherical polymersomes, dispersed into the fibrillar network, without affecting the structural integrity of the polypeptide scaffold.

Figure 6. TEM image (scale bar 25 nm) and schematic representation of the polymersomes resulted from the self-assembly of PCL-PEO-P2VP-PEO-PCL pentablock terpolymers (top) and SEM image of freeze dried Bz-A5E11A5-Bz 10 wt% at pH 6.5 enclosing 1 wt% polymersomes (bottom).



In the following, we explored possible effects of the presence of the pentablock terpolymer vesicles on the rheological features of the composite hydrogel. Figure 7 shows dynamic oscillatory shear data for a Bz-A5E11A5-Bz gelator concentration of 10 wt % at pH 6.5 entrapping the P5b and Q5b vesicles. It is observed that all the samples behave as strong physical elastic hydrogels since both the elastic (G’) and viscous (G’’) modulus, remain independent on frequency. However, the composite hydrogel containing the neutral P5b vesicles form more rigid gels, since the elastic modulus increased about three times. This might be attributed to the better rearrangement of the fibrils in the presence of polymersomes. A similar effect was observed in a previous reported liposome/hydrogel nanocomposite system.59 On the contrary, for the charged Q5b vesicles embedded into the polypeptide hydrogel, the elastic modulus is identical to the blank hydrogel despite the electrostatic interactions that could be developed between the positively charged polymersomes and the negatively charged copolypeptide hydrogel.

G', G'' (Pa)

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10

7

10

6

10

5

10

4

10

3

10

2

0.01

0.1

1

10

Frequency (Hz)

Figure 7. Storage modulus (closed symbols) and loss modulus (open symbols) as a function of frequency for 10 wt% Bz-A5E11A5-Bz hydrogel prepared at pH 6.5 (blue) and loaded with 1 wt% P5b (green) or Q5b (red) vesicles.

In Vitro Drug Release. A hydrophilic model drug, calcein, was selected to investigate drug release from the polymersome/polypeptide composite formulations at physiological pH (sol) and at extracellular tumor pH 6.5 (gel). To ensure comparable data, the triblock copolypeptide/polymersome formulation, was heated at 370C and after equilibration the solution was split into two similar parts, placed in dialysis tubes and dialyzed against PBS 0.15M NaCl at pH 7.4 and 6.5 respectively. The release profiles were monitored at room temperature. 16 ACS Paragon Plus Environment

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We note here that the formulation at pH 6.5 was turned to gel implying rapid proton exchange. As has been reported recently, pH responsive hydrogels, based on imine dynamic bonds, are formed rapidly after subcutaneous injection within short time.39 Thus, the in vitro confirmation of the in situ gelation capability of the copolypeptide under investigation, render it a promising injectable hydrogel for in vivo potential applications. As it can be observed from Figure 8, sustained release has been obtained from the compex formulations relatively to the blank polymersomes in all pH conditions. More importantly, the Bz-A5E11A5-Bz polypeptide matrix presents a significantly different release profiles at different pH conditions depending on the state of the formulation, i.e. sol vs. gel. As plotted in Figure 8A, after 144 h the cumulative release of calcein is 32% from the polypeptide sol (pH 7.4) encapsulating uncharged pentablock terpolymer P5b, whereas only 16% of the drug is released under extracellular tumor conditions (pH 6.5), where the scaffold is an entangled fibrillar network. Furthermore, increasing the degree of ionization of P2VP membrane forming block by partial quaternization (Q5b) (Figure 8B), has led to an accelerated release of calcein from the control vesicles, whereas the peptidic formulations were able to significantly increase drug retention. At 144 h, 28% of calcein had escaped from the composite Q5b/Bz-A5E11A5-Bz formulation at pH 7.4, while at pH 6.5 significantly slower release was observed accounted for 8%. These results indicate that the solution pH, which affects the imine linkages, plays an important role in the controlled release of the model drug. At lower pH values, the hydrolysis of the imine linkages allows the formation of a free standing hydrogel, and such viscous scaffold compensates the swelling of the vesicular membrane as a result of P2VP ionization at pH 6.5.61 For the vesicular assemblies bearing quaternized moieties (Q5b), a faster release profile at physiological pH relatively to the nonquaternized counterpart would have been expected as a result of decreased hydrophobicity of the membrane as observed for the bare vesicles presented in Figure 8B. However, for the composites presented herein, slower release was observed from Q5b polymersomes at both pH values investigated. In this case, the PGA chains are deprotonated (pKa 5.2) resulting in electrostatic attractive interactions with the quaternized moieties at the outer surface of the vesicles, despite the screening effect arising from the presence of salt (150 mM NaCl). Indeed, for the specific case of Q5b vesicles, it was measured that



polypeptides incorporating Q5b vesicles exhibit increased zeta-potential (−19.5 mV) compared to the composite loading P5b vesicles (−22.0 mV).

50

A Calcein release (%)

40 30 20 10 0 0

20

40

60

80

100

120

140

100

120

140

Time (h) 50

B 40 Calcein release (%)

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30 20 10 0 0

20

40

60

80

Time (h)

Figure 8. (A) Cumulative amount of calcein released per time (hours) from the control (P5b) polymersomes (○), composite polymersome-in-polypeptide formulation for a Bz-A5E11A5-

Bz concentration of 10 wt% loaded with 1 wt% polymersomes at pH 7.4 (■) and from the composite hydrogel at pH 6.5 (▲). Each point is the mean of three independent measurements and bars represent standard deviation of means. (B) As in A but with the partially quaternized (Q5b) polymersomes.

Yet, the drug release profile from the neutral vesicles (P5b) and the polypeptide formulation at physiological pH seem to follow the same path within the first 40 h, from where it deviates with a suppression of drug release from the Bz-A5E11A5-Bz formulation, suggesting that calcein diffusion from the vesicles is the dominant mechanism. On the contrary, the cationic Q5b vesicles/polypeptide formulations have shown an inhibition of drug release relatively to the blank polymersome at all pH conditions, probably as a result of electrostatic attractive interactions with the peptide scaffold. The suppression of drug escape from the Q5b/Bz-A5E11A5-Bz system is even more pronounced at pH 6.5, with less than 10% calcein released within 142 h, where 18 ACS Paragon Plus Environment

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there is an additional electrostatic attraction between hydrogel and the polymersomes for the drug to overcome. Therefore, it is rationalized that the presence of the polypeptide hydrogel matrix creates an additional barrier to diffusion of drug from the vesicular formulation into the bulk media, resulting in a lower initial release. Sustained drug release from peptide hydrogels has been observed previously for ProAsp-(Phe-Asp)5-Pro peptide hydrogels which released Dox slowly, ~30% release within 120 h, as a result of strong interactions with the matrix through electrostatic, ππ stacking and hydrophobic interactions.63 This comparison clearly demonstrates the importance of the composite formulation for sustained drug release, even in the absence of interactions as for the case of P5b/Bz-A5E11A5-Bz hydrogel, where less than 20% calcein leakage was observed within 144 h. Previous studies concerning the release of hydrophilic drugs from liposomal hydrogels formulations have reported on the importance of liposome-membrane rigidity, that determines the release rate of the drug from liposomal gels.64 In our case, electrostatic attractive interactions between the cationic polymersomes Q5b and the negatively charged hydrogel segments, overcomes the membrane leaky nature of the blank vesicles, enhancing drug retention. Indeed, when calcein was encapsulated in Q5b vesicles and dispersed in gels, it was released at a significantly lower rate compared to its release from P5b vesicles with a more robust membrane. This observation implies that both processes, the release of calcein from the polymersomes, as well as its diffusion through the gel, are important determining factors for calcein release from such composite polypeptidic gels. 3.4 Cytotoxicity study

The cytotoxicity of the benzaldehyde-polypeptide derivative was tested at the physiological pH of 7.4 and at the lower pH of 6.5, which may be encountered at solid tumors.65 The benzaldehyde-polypeptide derivative exhibited very low cytotoxicity at both pHs (Fig. 9). Although several studies have suggested that benzaldehyde can have carcinostatic or antitumor properties,66 the obtained results indicate that the benzaldehyde molecules, liberated through the partial hydrolysis of imine bonds at the lower pH of 6.5, did not increase cellular toxicity.



100

pH 7.4 pH 6,5 80

% cytotoxicity

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60

40

20

0 0

20

40

60

80

100

ppm

Figure 9. Cytotoxicity of Bz-A5E11A5-Bz against the A549 cell line at two different pHs.

Conclusions We have studied the aqueous self-assembly behavior of a benzaldehyde endcapped

poly(L-alanine)-b-poly(L-glutamic

acid)-b-poly(L-alanine),

copolypeptide (Bz-A5E11A5-Bz), As previously reported,

43

triblock

at physiological pH and

ionic strength (7.4, 0.15M NaCl) and elevated concentrations, the bare triblock copolypeptide underwent hierarchical growth of fibrils into giant supermolecular tapes that could be disrupted and rearranged into a supermolecular network of twisted fibers upon temperature treatment. In the following, the N-termini of the triblock copolypeptide, were end-capped by benzaldehyde (Bz) through dynamic imine bond. The presence of Bz, conjugated to the end-chains of the A5E11A5 copolypeptide, resulted in enhanced packing of the hydrophobic groups into piles of stacked supermolecular tapes, that inhibited gelation. However, partial cleavage of Bz upon lowering pH to 6.5, resulted in recovery of the ability of hydrophobic PAla endgroups to self-assemble into supermolecular fibers, either isolated or stacked in bundles, leading to hydrogel formation. The sol-gel transition was reversible by coupling/decoupling of the benzoic-imine linkage, triggered by a slight change of pH, in addition to an undesirable reduction of the gelation temperature. However, the gelation threshold could be tuned by mixing the conjugated copolypeptide with the unmodified counterpart. Therefore, the Bz conjugation endows the gelling system with pronounced pH-sensitivity and in turns with injectability. Such novel dually responsive triblock copolypeptide formulation, based on imine dynamic covalent 20 ACS Paragon Plus Environment

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bond, was investigated as a scaffold for encapsulation of polymer pentablock vesicular nanocarriers, loaded with a hydrophilic probe, calcein, in their aqueous lumen. The release of calcein from the composite was examined at pH 7.4 (sol) and 6.5 (gel) relevant for in vivo applications. For instance, the formulation exists as sol at physiological conditions, thus being injectable, while it can be transformed to hydrogel at pH 6.5, related to the extracellular tumor environment, capable to be immobilized therein. As expected, the polymersome/polypeptide sol, prepared at physiological pH, showed significantly faster release profile relatively to the composite hydrogel at pH 6.5. Additionally, both formulations (i.e., sol and hydrogel), showed a significantly improved drug retention as compared to the bare polymersome nanocarriers.

Such

nanocomposite

polymersome/hydrogel

formulations,

encompassing the benefits of biocompatibility, dual pH/thermo-responsiveness of the hydrogel matrix (injectability), together with the robustness and tunability of the polymersome nanocarrier, might find potential applications as controlled drug delivery systems for anticancer therapies. Thus, intratumoral injection of an anticancer drug-loaded polymersome/copolypeptide sol formulation would result to the formation of a hydrogel depot system (sol-gel transition at the mildly acidic tumor environment) which would release its drug content in a sustained fashion, leading to prolonged drug availability in the tumor.

ASSOCIATED CONTENT

Supporting information Additional data on the pH responsiveness by 1H NMR, polymersome preparation and gelation temperature window regulation are included. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (C.T.).

ACKNOWLEDGEMENTS



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M-T. P and C. T. would like to thank the University of Patras for the financial support through the C. Karatheodoris Program (C897).

References

1

Nowak, A. P.; Breedveld, V.; Pakstis, L.; Ozbas, B.; Pine, D.J.; Pochan, D. Deming, T. J.

Rapidly Recovering Hydrogel Scaffolds From Self-Assembling Diblock Copolypeptide Amphiphiles. Nature 2002, 417, 424-428. 2

Dasgupta, A.; Mondal, J. H.; Das, D. Peptide hydrogels. RSC Adv. 2013, 3, 9117-9149.

3

Deming T. J. Synthetic Polypeptides for Biomedical Application. Prog. Polym. Sci., 2007,

32, 858-875. 4

Schlaad H.; Antoniety, M. Block Copolymers with Amino Acid Sequences: Molecular

Chimeras of Polypeptides and Synthetic Polymers. Eur. Phys. J. E. 2003, 10, 17-23. 5

Checot, F.; Rodriguez-Hernandez, J.; Gnanou, Y.; Lecommandoux, S. pH-responsive

Micelles and Vesicles Nanocapsules Based on Polypeptide Diblock Copolymers. Biomol.

Eng. 2007, 24, 81-85. 6

Koga, T.; Matsuoka, M.; Higashi, N. Structural Control of Self-Assembled Nanofibers by

Artificial β-Sheet Peptides Composed of d- or l-Isomer. J. Am. Chem. Soc. 2005, 127, 1759617597. 7

Chassenieux, C.; Tsitsilianis, C. Recent Trends on Ph/Thermo-Responsive Self-Assembling

Hydrogels: From Polyions to Peptide-Based Polymeric Gelators. Soft Matter, 2016, 12, 13441359. 8

Zhao, W.; GnanouY.; Hadjichristidis N. Fast and Living Ring-Opening Polymerization of

α-Amino Acid N-Carboxyanhydrides Triggered by an “Alliance” of Primary and Secondary Amines at Room Temperature. Biomacromolecules, 2015, 16, 1352–1357. 9

Hadjichristidis, N.; Iatrou, H.; Pitsikalis, M.; Sakelariou, G. Synthesis of Well-Defined

Polypeptide-Based Materials via the Ring-Opening Polymerization of α-Amino Acid NCarboxyanhydrides. Chem. Rev., 2009, 109, 5528-5578. 10

Lu, H.; Wang, J.; Song, Z.; Yin, L.; Zhang, Y.; Tang, H.; Tu, C.; Lin Y.; Cheng, J. Recent

Advances in Amino Acid N-Carboxyanhydrides and Synthetic Polypeptides: Chemistry, SelfAssembly and Biological Applications. Chem. Commun. 2014, 50, 139-155. 11

Deming, T. J. Polypeptide Hydrogels via a Unique Assembly Mechanism. Soft Matter

2005, 1, 28-35. 22 ACS Paragon Plus Environment

Page 23 of 28

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


12

Nowak, A. P.; Sato, J.; Breedveld, V.; Deming, T.J. Hydrogel Formation in Amphiphilic

Triblock Copolypeptides. Supramol Chem 2006, 18, 423-427. 13

Jeong, B.; Bae, Y. H.; Lee, D. S.; Kim, S. W. Biodegradable Block Copolymers as

Injectable Drug Delivery Systems. Nature 1997, 388, 860-862. 14

Qiu, Y.; Park, K. Environment-Sensitive Hydrogels for Drug Delivery. Adv. Drug Delivery

Rev. 2001, 53, 321-339. 15

Hoffman, A. S. Hydrogels for Biomedical Applications. Adv. Drug Delivery Rev. 2002, 53,

3-12. 16

Collier, J. H.; Messersmith, P. B. Self-Assembling Polymer-Peptide Conjugates:

Nanostructural Tailoring. Adv. Mater. 2004, 16, 907-910. 17

Capito, R. M.; Azevedo, H. S.; Velichko, Y. S.; Mata, A.; Stupp, S. I. Self-Assembly of

Large and Small Molecules into Hierarchically Ordered Sacs and Membranes. Science 2008,

319, 1812-1816. 18

Gillette, B. M.; Jensen, J. A.; Tang, B. X.; Yang, G. J.; Bazargan-Lari, A.; Zhong, M.; Sia

S. K. In situ Collagen Assembly for Integrating Microfabricated Three-Dimensional CellSeeded Matrices. Nat. Mater. 2008, 7, 636-640. 19

Cohn, D.; Sosnik, A.; Garty S. Smart Hydrogels for In Situ Generated Implants.

Biomacromolecules 2005, 6, 116-1175. 20

Rajagopal, K.; Ozbas, B.; Pochan, D. J.; Schneider, J. P. Probing the Importance of Lateral

Hydrophobic Association in Self-Assembling Peptide Hydrogelators. Eur. Biophys. J. 2006,

35, 162-169. 21

Hacker, M. C.; Klouda, L.; Ma, B. B.; Kretlow, J. D.; Mikos A. G. Synthesis and

Characterization of Injectable, Thermally and Chemically Gelable, Amphiphilic Poly(Nisopropylacrylamide)-Based Macromers. Biomacromolecules 2008, 9, 1558-1570. 22

Moon, H. J.; Ko, D. Y.; Park, M. H.; Joo M. K.; B. Jeong B. Temperature-responsive

Compounds as In Situ Gelling Biomedical Materials. Chem. Soc. Rev. 2012, 41, 4860-4883. 23

Jun, H. W.; Yuwono, V.; Paramonov, S. E.; Hartgerink J. D. Enzyme-Mediated

Degradation of Peptide-Amphiphile Nanofiber Networks Adv. Mater. 2005, 17, 2612-2617. 24

Collier, J. H.; Messersmith, P. B. Enzymatic Modification of Self-Assembled Peptide

Structures with Tissue Transglutaminase. Bioconjugate Chem. 2003, 14, 748-755. 25

Toledano, S.; Williams, R. J.; Jayawarna, V.; Ulijn, R. V. Enzyme-Triggered Self-

Assembly of Peptide Hydrogels via Reversed Hydrolysis. J. Am. Chem. Soc. 2006, 128, 10701071. 26

Yang, Z.; Ma, M.; Xu, B. L. Using Matrix Metalloprotease-9 (MMP-9) to Trigger

Supramolecular Hydrogelation. Soft Matter 2009, 5, 2546-2448.



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

27

Page 24 of 28

Shim, W. S.; Yoo, J. S.; Bae, Y. H.; Lee D. S. Novel Injectable pH and Temperature

Sensitive Block Copolymer Hydrogel. Biomacromolecules 2005, 6, 2930-2934. 28

Yang, J.; Xu, C.; Wang, C.; Kopecek, J. Refolding Hydrogels Self-Assembled from N-(2-

Hydroxypropyl)Methacrylamide Graft Copolymers by Antiparallel Coiled-Coil Formation.

Biomacromolecules 2006, 7, 1187-1195, 29

Shim, W. S.; Kim, S. W.; Lee D. S. Synthesis of Multiresponsive and Dynamic Chitosan-

Based Hydrogels for Controlled Release of Bioactive Molecules. Biomacromolecules 2006, 7, 1935-1941 30

Shen, W.; Kornfield, J. A.; Tirell, D. A. Dynamic Properties of Artificial Protein Hydrogels

Assembled Through Aggregation of Leucine Zipper Peptide Domains. Macromolecules 2007,

40, 689-692. 31

Zhang, X.; Wang C. Supramolecular Amphiphiles. Chem. Soc. Rev. 2011, 40, 94-101.

32

Jackson, A. W.; Fulton D. A. Making Polymeric Nanoparticles Stimuli-Responsive with

Dynamic Covalent Bonds. Polym. Chem. 2013, 4, 31-45. 33

Xin, Y.; Yuan J, Schiff's Base as a Stimuli-Responsive Linker in Polymer Chemistry.

Polym. Chem. 2012, 3, 3045-3055. 34

Wang, C.; Wang, G.; Wang, Z.; Zhang X. A pH‐Responsive Superamphiphile Based on

Dynamic Covalent Bonds. Chem. Eur. J. 2011, 17, 3322-3325. 35

Bae, Y.; Fukushima, S.; Harada, A.; Kataoka K. Design of Environment-Sensitive

Supramolecular Assemblies for Intracellular Drug Delivery: Polymeric Micelles That Are Responsive to Intracellular pH Change. Angew. Chem., Int. Ed. 2003, 42, 4640-4643. 36

He, L.; Jiang, Y.; Tu, C.; Li, G.; Zhu, B.; Jin, C.; Zhu, Q.; Yan. D.; Zhu X. Self-Assembled

Encapsulation Systems with pH Tunable Release Property Based on Reversible Covalent Bond. Chem. Commun. 2010, 46, 7569-7571. 37

Jackson, A.; Stakes, C.; Fulton D. The Formation of Core Cross-Linked Star Polymer and

Nanogel Assemblies Facilitated by the Formation of Dynamic Covalent Imine Bonds. Polym.

Chem. 2011, 2, 2500-2511. 38

Zhang, Y.; Tao, L.; Li, S.; Wei Y. Synthesis of Multiresponsive and Dynamic Chitosan-

Based Hydrogels for Controlled Release of Bioactive Molecules. Biomacromolecules 2011,

12, 2894-2901. 39

Li, L.;

Gu, J.; Zhang, J.;

Xie,Z.; Lu,Y.; Shen, L.;

Dong,Q, Y. Injectable and

Biodegradable pH-Responsive Hydrogels for Localized and Sustained Treatment of Human Fibrosarcoma. ACS Appl. Mater. Interfaces, 2015, 7, 8033–8040.


Page 25 of 28

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


40

Kim, J.Y.; Park, M. H.; Joo, M. K.; Lee, S. Y.; Jeong B. End Groups Adjusting the

Molecular Nano-Assembly Pattern and Thermal Gelation of Polypeptide Block Copolymer Aqueous Solution. Macromolecules 2009, 42, 3147-3151. 41

Yu, L.; Zhang, H.; Ding J. A. A Subtle End-Group Effect on Macroscopic Physical

Gelation of Triblock Copolymer Aqueous Solutions. Angew. Chem., Int. Ed. 2006, 45, 22322235. 42

Koga, T. Watanabe, T.; Higashi N. Fabrication of Nucleobase-Functionalized

Supramolecular Nanofiber Through Peptide Self-Assembly. J. Nanosci. Nanotechnol. 2009,

9, 584-590. 43

Popescu, M.-T.; Liontos, G.; Avgeropoulos, QA.; Tsitsilianis C. Stimuli Responsive

Fibrous Hydrogels from Hierarchical Self-Assembly of a Triblock Copolypeptide.

Soft

Matter, 2015, 11, 331-342. 44

Pickel, D. L.; Politakos, N.; Avgeropoulos, A.; Messman, J. M. A Mechanistic Study of a-

(Amono Acid)-N-Carbonxyanhydance Polymerization: Comparing Initiation and Termination Events in High-Vacuum and Traditional Polymerization Techniques. Macromolecules 2009,

42, 7781-7788. 45

Mondeshki, M.; Spiess, H. W.; Aliferis, T.; Iatrou, H.; Hadjichristidis, N.; Floudas G.

Hierarchical Self-Assembly in Diblock Copolypeptides of Poly(Γ-Benzyl-L-Glutamate) with Poly(L-Leucine) and Poly(O-Benzyl-L-Tyrosine). Eur. Polym. J. 2011, 47, 668-674. 46

W.A Dengler, J. Schulte, D.P. Berger, R. Mertelsmann, H.H. Fiebig, Development of a

Propidium Iodide Fluorescence Assay for Proliferation and Cytotoxicity Assays. Anti-Cancer

Drugs 1995, 6, 522-532. 47

Park, M. H.; Joo, M. K.; Choi B. G.; jeong, B. Biodegredable Hydrogels. Acc. Chem. Res.

2012, 45, 424-433. 48

Minkenberg, C. B.; Florusse, L.; Eelkema, R.; Koper, G. J. M.; van Esch J. H. J. Triggered

Self-Assembly of Simple Dynamic Covalent Surfactants. J. Am. Chem. Soc. 2009, 131, 11274-11275. 49

Belowich, M. E.; Stoddart J. F. Dynamic Imine Chemistry. Chem. Soc. Rev. 2012, 41,

2003-2024. 50

Stupp, S. I.; Zha, R. H.; Palmer, L. C.; Cui H.; Bitton, R. Self-assembly of Biomolecular

Soft Matter. Faraday Discuss. 2013, 166, 9-30.



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

51

Page 26 of 28

Nyrkova, I. A.; Semenov, A.N.; Aggelli, A.; Boden, N. Fibril Stability in Solutions of

Twisted Β-Sheet Peptides: A New Kind of Micellization in Chiral Systems. Eur. Phys. J. B 2000, 17, 481-497. 52

Takahashi, Y.; Ueno, A.; Mihara, H. Design of a peptide undergoing α-β structural

transition and amyloid fibrillogenesis by the introduction of a hydrophobic defect. Chem. Eur.

J. 1998, 4, 2475-2484. 53

Koga, T.; Ushirogochi, M.; Higashi, N. Regulation of self-assembling process of a cationic-

sheet peptide by photoisomerization of an anionic azobenzene derivative Polymer J. 2007, 39, 16-17. 54

Mourtas, S.; Fotopoulou, S.; Duraj, S.; Sfika, V.; Tsakiroglou, C.; Antimisiaris, S. G.

Liposomal Drugs Dispersed in Hydrogels. Effect of Liposome, Drug and Gel Properties on Drug Release Kinetics. Colloids Surf., B : Biointerfaces 2007, 55, 212-221. 55

Pavelic, Z.; Skalkobasnet, N.; Jalsenjak, I. Characterisation and In Vitro Evaluation of

Bioadhesive Liposome Gels for Local Therapy of Vaginitis. Int. J. Pharm. 2005, 301, 140148. 56

Popescu, M.-T.; Mourtas, S.; Pampalakis, G.; Antimisiaris, S. G.; Tsitsilianis C. pH-

Responsive

Hydrogel/Liposome

Soft

Nanocomposites

for

Tuning

Drug

Release.

Biomacromolecules 2011, 12, 3023-3030. 57

Petka, W.A.; Harden, J. L.; McGrath, K. P.; Wirtz, D.; Tirrell, D. A. Reversible Hydrogels

from Self-Assembling Artificial Proteins. Science 1998, 281, 389-392. 58

Silva, G. A.; Czeisler, C.; Niece, K. L.; Beniash, E.; Harrington, D. A.; Kessler, J. A.;

Stupp, S. I. Selective Differentiation of Neural Progenitor Cells by High-Epitope Density Nanofibers. Science 2004, 303, 1352-1355. 59

Xu, L.; Zhang, H.; Song, W. Current Advances in Sustained-Realease Injectable

Preparations. Int. J. Pharm. Sci. & Res.2012, 3, 2888-2896. 60

Kang, Y. M.; Kim, G. H.; Kim, J. I. Kim, D. Y.; Lee, B. N.; Yoon, S. M.; Kim, J. H.; Kim

M. S. In Vivo Efficacy of an Intratumorally injected In Situ-Forming Doxorubicin /Poly(Ethylene Glycol)-B-Polycaprolactone Diblock Copolymer. Biomaterials, 2011, 32, 4556-4564. 61

Popescu, M.-T.; Tsitsilianis, C. Controlled Delivery of Functionalized Gold Nanoparticles

by pH Sensitive Polymersomes. ACS Macro Lett. 2013, 2, 222-225.


Page 27 of 28

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


62

Popescu, M.-T.; Korogianaki, M. ; Marikou, K. ; Tsitsilianis, C. CBABC Terpolymer-

Based Nanostructured Vesicles with Tunable Membrane Permeability as Potential Hydrophilic Drug Nanocarriers. Polymer, 2014, 55, 2943-2951. 63

Zarzhitsky, S.; Rapaport, H. The Interactions Between Doxorubicin and Amphiphilic and

Acidic Β-Sheet Peptides Towards Drug Delivery Hydrogels. J. Colloid and Interface Sci. 2011, 360, 525. 64

Mourtas, S.; Haikou, M.; Theodoropoulou, M.; Tsakiroglou, C.; Antimisiaris S. G. The

Effect of Added Liposomes on The Rheological Properties of a Hydrogel: A Systematic Study. J. Colloid Interface Sci. 2008, 317, 611-619. 65

Tannock I. F.; Rotin, D. Acid pH in Tumors and its Potential for Therapeutic Exploitation.

Cancer Res., 1989, 49, 4373-4384. 66

Andersen, A. Final Report on the Safety Assessment of Benzaldehyde. International

Journal of Toxicology 2006, 25, Suppl 1, 11-27.



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Injectable Hydrogel: Amplifying the pH Sensitivity of a Triblock

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