A Peptide-Based Mechano-sensitive, Proteolytically Stable Hydrogel

Subscriber access provided by GAZI UNIV

Article

A Peptide based Mechano-sensitive, Proteolytically stable Hydrogel with Remarkable Antibacterial Properties Abhishek Baral, Subhasish Roy, Srabanti Ghosh, Daniel Hermida Merino, Ian W Hamley, and Arindam Banerjee Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b03789 • Publication Date (Web): 27 Jan 2016 Downloaded from http://pubs.acs.org on January 31, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Langmuir is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 29

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

Langmuir

A Peptide based Mechano-sensitive, Proteolytically stable Hydrogel with Remarkable Antibacterial Properties Abhishek Baral,† Subhasish Roy,† Srabanti Ghosh,§ Daniel Hermida-Merino,# Ian W. Hamley,‡ and Arindam Banerjee*† †

A. Baral, Dr. S. Roy, and Prof. A. Banerjee Department of Biological Chemistry

Indian Association for the Cultivation of Science Jadavpur, Kolkata-700032, India Fax: (+ 91) 33-2473-2805 E-mail: [email protected] §

Dr. S. Ghosh

Dept. of Chemical, Biological and Macromolecular Sciences, S. N. Bose National Centre for Basic Sciences, Block-JD, Sector-III, Salt Lake, Kolkata-700098, India #

Dr. D. Hermida-Merino

DUBBLE @ ESRF – The European Synchrotron CS40220, 38043 Grenoble Cedex 9, France ‡

Prof. I. W. Hamley

Department of Chemistry University of Reading, Whiteknights Reading, RG6, 6AD, UK

ACS Paragon Plus Environment

Langmuir

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

Abstract A long chain amino acid containing dipeptide has been found to form a hydrogel in phosphate buffer whose pH ranges from 6.0-8.8. The hydrogel formed at pH 7.46 has been characterized by small angle X-ray scattering (SAXS), wide angle powder X-ray diffraction (PXRD), FT-IR, field emission scanning electron microscopic (FE-SEM), high-resolution transmission electron microscopic (HR-TEM) imaging and rheological analyses. The microscopic imaging studies suggest the formation of a nanofibrillar 3D network for the hydrogel. As observed visually and confirmed rheologically, the hydrogel at pH 7.46 exhibits thixotropy. This thixotropic property can be exploited to inject the peptide. Furthermore, the hydrogel exhibits remarkable antibacterial activity against Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa which are responsible for many common diseases. The hydrogel has potential applications due to biocompatibility with human red blood cells and human fibroblast cells. Interestingly, this hydrogel shows high resistance towards proteolytic enzymes making it a new potential antimicrobial agent for future applications. It has also been observed that a small change in molecular structure of the gelator peptide not only turns the gelator into a non-gelator molecule under similar conditions but also has a significant negative impact on its bactericidal character.

Introduction Low molecular weight gels (LMWGs) hold a key position in current research due to their versatility and usefulness as smart soft materials.1-20 These supramolecular gels offer extensive applications in various fields including drug delivery,21-24 wound healing,25 oil spill recovery7,26 designing of template for making nanohybrid systems27 and others.28-29 Biocompatibility and biodegradability of peptide and amino acidbased supramolecular gels make them very attractive candidates to prepare biomaterials for various potential applications.30-33 Apart from thermo-responsiveness, only a few supramolecular gels exhibit mechano-responsive behaviour. In these cases, they can be broken by mechanical shaking by applying a shear strain/stress and upon the withdrawal of the mechanical force or shear strain/stress, the gel phase is


Page 2 of 29

Page 3 of 29

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

Langmuir

reformed. This behaviour, known as thixotropy, has been exploited in recent years to prepare injectable hydrogels for the application in drug delivery system.33-38 Bacterial infections are one of the most common problems encountered by medical practitioners in recent years. Although there are several antibiotics available for treatment of bacterial infections, they have their own limitations. Growing bacterial resistance towards existing antibiotics39 leads to the challenge to discover and design of new antibiotics. Antibiotics that can be delivered locally have two vital advantages over the oral delivery. It reduces the amount of antibiotic needed to cure infection, thus making it cost-effective and as only the infected site gets the drugs, it also minimizes the side-effects. One of the ways to deliver a drug locally is by surgical implantation, but the patient has to face the pain of surgery. In this regard, injectable hydrogels are wonderful candidates for local delivery.34,40 Their shear thinning property helps them to be administered by using a simple syringe. After injection the material can recover its gel form and it remains localized in the infected area. Several studies were carried out to design hydrogel based delivery systems for sustained release of drugs.21,22,37 Apart from being injectable, it is also essential that the drug carrier must be biocompatible and can be easily extracted from the body. Thus, exploration of injectable antibacterial hydrogels is very important. Several antibacterial hydrogels are reported in the literature so far.41-52 Most of these gels exhibit their antibacterial properties through the release of encapsulated silver nanoparticles44-45,51 or silver ions.4647

However, silver containing antibacterial agents has several side effects like pigmentation of skin or

eyes, oxidative DNA damage or inflammation.53 Liposome containing hydrogel system stabilized with gold nanoparticle has also shown its bactericidal activity by fusing into the bacterial membrane.41 Vancomycin is a known antibiotic; however, when loaded with biomaterials, the release of the drug is much faster than the biomaterials degradation, which requires further surgery to remove the biomaterial. This problem is addressed by covalent linking of vancomycin with a hydrogel backbone, so that the drug release rate and hydrogel degradation rate are similar.42 Antibacterial agents based on polymeric and supramolecular hydrogels containing positively charged ammonium or pyridinium moieties are also known.48-49 An interesting and efficient way of bacterial inhibition has been achieved by inserting a


Langmuir

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

hydrogel precursor into a bacterial cell. Then the enzymatic cleavage inside the cell resulted in the gel formation and bacterial cell death.50 A polycationic gel has also been reported that attract the anionic membrane of the microbes into the pores of the hydrogel like ‘anion sponge’ leading to the microbial death.52 Although there are several antibacterial gels, most of them contain either silver ions/particles or net positive charge on gelator molecules. This drives our curiosity to examine the bactericidal activity of a hydrogel with neither silver ions/particles nor a net positive charge. Thus, keeping in mind the rising demand about peptide-based antibacterial agents, we choose a simple dipeptide and have studied its gelation and antibacterial activity. Important properties crucial to design antibacterial gelator molecules are good biocompatibility, injectability and proteolytic stability. Furthermore, injection of the hydrogel into the human body by a simple syringe can be easily carried out only if the hydrogel is compatible with human blood and healthy human cell. Thus, it will be remarkable to produce a hydrogel that not only shows injectability but also exhibits very little or no toxicity towards human blood cells. In this study, we report the hydrogelation of an N-Boc protected dipeptide P1 (Figure 1) in phosphate buffer of physiological pH 7.46. The Phe (L-phenylalanine) residue present in the gelator also triggers self-association and gelation using - stacking and hydrophobic interactions.23,37,54 The hydrogel P1 exhibits antibacterial activity against Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa. The hydrogel also shows negligible toxicity to human erythrocytes at its minimum inhibitory concentration (MIC) for E. coli and only small toxicity at MIC for P. aeruginosa. The human fibroblast cells also exhibits very low cytotoxicity in the presence of the hydrogel. Furthermore, the dipeptide P1 shows stability towards proteolytic enzymes present inside the cells, thus enhancing the hydrogel’s applicability for practical purposes. The hydrogel P1 can be exploited for the treatment of localized bacterial infections through subcutaneous injection.38,55 Bacteria like Pseudomonas aeruginosa frequently infect persons having burns, wounds and other skin infections.56,57 In such cases, subcutaneous injection can be given in the fatty layer of tissue just under the skin for antibacterial treatment. Moreover, localised bacterial infections arising from orthopaedic surgeries can also be treated with the P1 hydrogel. Bacterial biofilm formation (inflicted by several pathogens including Pseudomonas aeruginosa and Escherichia


Page 4 of 29

Page 5 of 29

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

Langmuir

coli) over orthopaedic implants during joint replacements is a major worry for orthopaedic surgeons across the world. The hydrogel P1 can be applied for dealing with these prosthetic-related infections that are often highly resistant to antibiotics as well as to patients’ immune responses.58,59 We are also interested to know whether a structurally similar peptide P2 forms a hydrogel or not and whether it shows any significant amount of antibacterial activity comparable to the gelator peptide P1 at similar conditions. However, it has been found that a dipeptide P2 that contains Phg (L-phenylglycine) residue instead of Phe residue fails to produce hydrogel under similar conditions. Interestingly, the P2 solution also exhibits very low activity against Gram-negative bacteria. In short, we have successfully designed a peptide based injectable and human blood-compatible hydrogel for antibacterial treatment and observed its superiority over a structurally similar non-gelator peptide.

Experimental section Materials and methods 11-aminoundecanoic acid, L-phenylalanine and L-phenylglycine were purchased from Aldrich. HOBt, sodium dihydrogen phosphate, and disodium hydrogen phosphate were purchased from Merck. DCC, NaOH, MeOH, silica gel (100−200 mesh), Et2O, petroleum ether, EtOAc, and DMF were purchased from SRL (India). The water used in all experiments was of Millipore MilliQ grade. For the antibacterial study, Escherichia coli (MTCC 1687), Pseudomonas aeruginosa (MTCC 424), Staphylococcus aureus (MTCC 7405) and Bacillus subtilis (MTCC 441) were obtained from the Institute of Microbial Technology, Chandigarh, India. The bacterial strains were maintained on nutrient agar slants. Details of the synthetic procedures of gelator peptide and instrumentation details are given in the Supporting Information.

Results and Discussion Gelation study


Langmuir

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

Two structurally similar N-terminally protected long chain amino acid-containing aromatic dipeptides Boc-AUDA-Phe-COOH (P1) and Boc-AUDA-Phg-COOH (P2) were synthesized, purified, characterised and studied for gelation (Figure 1 and Figures S1-S6) (AUDA: 11-aminoundecanoic acid). P1 and P2 were separately placed in glass vial and each of these materials was dissolved in phosphate buffer of physiological pH 7.46 by heating on a hot plate. A translucent hydrogel was obtained for P1 on standing after four hours. On the other hand, a clear solution was obtained for P2 even after 48 hours of resting. The detailed characterization and properties of P2 solution are given in the Supporting Information. The stability of the P1 hydrogel was checked by vial inversion (Figure 1). The dipeptide can gelate water molecules in a wide pH range between 6.0 and 8.8. The minimum gelation concentration (MGC) of the hydrogel P1 in phosphate buffer at pH 7.46 is 0.48 % (w/v) or 4.8 mg / mL. Figure S7 shows the increase of sol–gel transition temperature with an increase in concentration (% w/v) of the gelator.

Figure 1. Chemical structures of the peptides and their respective gelation behaviour.

Morphological study To obtain information on the morphological features of the dipeptide-based hydrogel P1, field emission scanning electron microscopic (FE-SEM) and high resolution transmission electron microscopic (HRTEM) experiments were carried out (Figure 2). FE-SEM and HR-TEM images of the xerogel (prepared from hydrogel at pH 7.46) suggest a nanofibrilar network structure for the hydrogel P1. The width of these nanofibers varies from 120 nm to 240 nm and they extend to several micrometers in length. The


Page 6 of 29

Page 7 of 29

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

Langmuir

gelator molecules which are self-assembled by using different non-covalent interactions like hydrogenbonding and - interactions results in the formation of nanofibrillar network structure for the xerogel of P1 when viewed microscopically. This nanofibrillar network structure provides a lot of space that is responsible for entrapping large number of water molecules to form a self-supporting gel.

Figure 2. (a) Field emission scanning electron microscopic and (b) High resolution transmission electron microscopic images of the P1 xerogel.

FT-IR analysis A Fourier transform infrared (FT-IR) spectroscopic study of the P1 xerogel was performed to get structural insight into gelators in the gel state. Significant peaks were obtained at 3368 cm-1, 1688 cm-1, 1650 cm-1, 1627 cm-1 and 1525 cm-1 in the FT-IR spectra of the xerogel (Figure 3). The peak at 3368 cm-1 [marked 1 (red) in Figure 3] arise from hydrogen bonded N-H stretching, while the peak at 1627 cm-1 [marked 4 (orange) in Figure 3] is the characteristic peak for >C=O stretching coming from extended backbone ( -sheet-like) structures.19 The N-H bending peak appears at 1525 cm-1 [marked 5 (purple) in Figure 3] and the IR absorption peak at 1688 cm-1 [marked 2 (blue) in Figure 3] indicates the >C=O stretching of the hydrogen-bonded urethane group (Boc >C=O).60 On the other hand, the presence of another peak at 1650 cm-1 [marked 3 (black) in Figure 3] suggests the presence of some random coils within the supramolecular network formed by the gel.61


Langmuir

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

Figure 3. FT-IR analysis of the P1 xerogel. The peaks are assigned with 1(red), 2(blue), 3(black), 4(orange) and 5(purple).

SAXS and powder Powder X-ray diffraction (PXRD) studies Small angle X-ray scattering (SAXS) of the wet gel and wide angle powder X-ray diffraction (PXRD) of the P1 dried gel (xerogel) were carried out to provide information on the interactions between gelator molecules. The SAXS intensity profile shows a peak at 26.8 Å (D) (Figure 4a) which matches with the molecular length of P1. Taking the molecular length into consideration, periodic peaks at d values of 14.64 Å (2 = 6.03°), 7.32 Å (2 = 12.07°), 6.49 Å (2 = 13.62°), 5.73 Å (2 = 17.7°) and 4.22 Å (2 = 21.01°) in the wide angle diffraction pattern (Figure 4b) can be assigned as D/2, D/3, D/4, D/5 and D/6 respectively. These data indicate a lamellar one-dimensional ordered structure of the gelator molecule in its self-assembled state.26,37 The peak at d-spacing of 4.66 Å (2 = 19.01°) along with another at 9.97 Å (2 = 8.86°) give a strong hint towards an extended backbone (antiparallel -sheet-like) alignment of these peptide molecules in their gel state. The spacing between the peptide chains within a -sheet-like backbone structure is characterized by d= 4.66 Å, while the peak at 9.97 Å can be attributed to the distance between -sheet-like layers.22 Further, the - interactions between the aromatic rings of the phenylalanine residue within P1 xerogel is expressed in the PXRD spectra as a peak at 3.81 Å (2 = 23.25°).22,26


Page 8 of 29

Page 9 of 29

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

Langmuir

Figure 4. (a) SAXS of the hydrogel (1 % (w/v)) and (b) wide angle X-ray diffraction pattern of the P1 xerogel.

Thixotropic property The gelator P1 contains a non-proteinaceous undecanoic amino acid residue which is known to promote thixotropy in addition to gelation in some previous reports.14,37 This leads us to test the mechanoresponsive property for the hydrogel P1. So, to test the thixotropic nature of the hydrogel P1, a glass vial containing P1 hydrogel of concentration 1.0 % (w/v) in phosphate buffer of pH 7.46 was shaken vigorously by hand to get a viscous solution. The solution on standing for about 6 hours without disturbance, regained its self-supported gel form (Figure S11) indicating the self-healing ability of the supramolecular network structure responsible for gelation. When gel reformation is noted at elevated temperatures of 32 °C, 37 °C (physiological temperature) and 45 °C, it was found that at higher temperature the hydrogel needs longer time (6 h 40 mins, 7 h 5 mins and 24 hours respectively) to restore its self-supporting nature than the gel re-appearing time required at 25 °C.

Rheological studies Rheological experiments were performed to study the mechanical strength and stability of the hydrogel P1. We were also interested to examine whether the self-repairing nature can be explained in the light of the rheological data. Performing dynamic shear measurements, the storage modulus (G ) and loss


Langmuir

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 10 of 29

modulus (G ) were measured as a function of different parameters including angular frequency and time. In this study, gel samples with 1 % (w/v) gelator concentration were used for all rheological studies. For the frequency sweep experiment G and G values were measured as a function of angular frequency, at a fixed strain of 0.1 % (Figure 5a). In the low frequency region (8 – 100 rad / sec), G and G are almost independent of angular frequency which indicates the formation of a stable gel. The hydrogel P1 was found to exhibit a thixotropic property. This can be best explained though a step-strain rheological experiment. Thus, a step-strain experiment (Figure 5b) was performed to observe the recovery of the gel as it gives the most convincing view of the gel-strength recovery process in real situations.62 The stepstrain experiment was carried out in three intervals. At first, a low strain of 0.1 % was applied for the first 200 seconds to measure the storage and loss moduli values. The G value was greater than G value in this interval indicating the gel nature of this sample. The strain was then suddenly increased to 30 % for the next 200 seconds and G was found to be higher than G in this regime. This proves that the gel was transformed to its sol form as a consequence of the high strain. This observation is analogous to what is observed when the gel was rigorously shaken by hand mechanically to get the gel – sol transition. Then in the third interval, the gel recovery kinetics was examined for 400 seconds maintaining a low strain of 0.1 %. It was found that the gel regained its original strength within 80 seconds after the withdrawal of the large strain. The time-dependent step-strain experiment shows that the recovery time is only about 80 seconds, but in our thixotropic experiment it is found that it takes several hours (about 6 hours) to regain the self-supporting nature of the gel (within a glass vial). This reveals a disparity between the microscopic and macroscopic behavior implying only a few minutes are required for the gel reforming process to complete at a microscopic level, but several hours of dynamic process are needed to heal the supramolecular network sufficiently for the gel to become self-supporting at the macroscopic level. Thus, the rheological study gives a significant explanation regarding the mechanical properties associated with the thixotropy of the hydrogel P1. In most cases, the thixotropic nature bestows an injectable property onto a hydrogel. Thus to test this, the gel was pulled by a syringe and then injected back into an empty vial. This viscous solution on standing for 6 hours regains its gel form (Figure 6).


Page 11 of 29

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

Langmuir

Figure 5. (a) Frequency sweep analysis of the P1 hydrogel (1 % (w/v)) at a constant strain of 0.1 %. (b) Step-strain strain rheological analysis of the P1 hydrogel of concentration 1 % (w/v) at a fixed angular frequency of 1 rad/s.

Figure 6. Illustration of the injectable nature of the P1 hydrogel.

Aggregation study with pyrene The aggregation nature within a gelator system can be nicely studied by using a fluorescent fluor probe like pyrene.19 The different structural features of the non non-planer planer excited and the planar ground state of pyrene


Langmuir

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 12 of 29

results in increasing I3/I1 (I3, I1 = Intensity of the vibronic bands of pyrene) values with the enhancement of hydrophobicity (which is closely related to aggregation) in its environment.19 Thus, it will be interesting to investigate the difference in the I3/I1 ratio between P1 and P2 which will also help us to compare the critical aggregation concentration (CAC) for both these peptides (gelator and non-gelator). To determine the CAC, solutions with ten different concentrations (0.0008-0.4 % (w/v)) each of P1 and P2 were prepared. Then, in the solutions (1 mL each) having different concentrations of peptides 50 L of methanol solution of pyrene (0.5 mg/mL) were added. Fluorescence of the solution was recorded and I3/I1 ratio was calculated. The plot of I3/I1 vs. concentration gives critical aggregation concentration (CAC) values of 0.04 % (w/v) and 0.1 % (w/v) for P1 and P2 respectively (Figure S12). This was noted by a sudden and almost linear increase of the I3/I1 ratio beyond these CAC points with an increase in peptide concentration. Here, we are curious to know that whether the non-gelator peptide P2 aggregates or not under conditions similar to the gelator peptide P1. It has been found (from Figure S13) that the critical aggregation concentration of the non-gelator P2 is significantly higher than that of the gelator peptide P1 (at same pH of phosphate buffer solution and temperature). So, it can be said that the gelator peptide P1 is more prone to aggregate in phosphate buffer at pH 7.46 (which ultimately leads to gelation as discussed above) than that of the non-gelator peptide P2 and a subtle change in the molecular structure (only one CH2- less in the side chain) causes a change in the self-association and gelation property of a peptide based amphiphile in aqueous medium. However, the exact reason for this change is yet to be explored. There are some previous reports from our group and also from others that suggests variation in the molecular structure can turn a gelator into a non-gelator under specific conditions.7,26,54

Antibacterial study To explore the possibility whether the peptide based P1 hydrogel can be used as an antibacterial agent, antibacterial activity in vitro against a range of pathogenic micro-organisms was tested. The antimicrobial activity of hydrogel was investigated against Gram-negative and Gram-positive bacteria using the wellplate diffusion method. In an agar plate, gel sample is used as test material and phosphate buffer as


Page 13 of 29

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

Langmuir

negative control. The inhibition zone was formed in the screening test indicating the antimicrobial activity of hydrogel against Gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa (as shown in Figure 7 and Table 1). For comparison, the solution P2 was also screened for antibacterial activity. The microdilution technique was used to determine minimum inhibitory and minimum bactericidal concentration (MIC and MBC) for the P1 hydrogel (details in the SI). The hydrogel P1 was found to be quite effective against Gram-negative bacteria Escherichia coli (causing diseases like urinary tract infections, gastroenteritis) and Pseudomonas aeruginosa (responsible for pneumonia, septic shock, urinary tract infections etc.). Minimum inhibitory concentration (MIC), the lowest hydrogel concentration at which no viable bacterial cell is present, against E. coli is around 50 g/mL (5 L of 1 % (w/v) of P1 gel) whereas the MIC value is around 100 g/mL (10 L of 1 % (w/v) of P1 gel) for P. aeruginosa. Minimum bactericidal concentrations ranged from 50 to 100 µg/mL (5-10 L of 1 % (w/v) of P1 gel). These MIC values of the dipeptide based P1 hydrogel are higher than some of the silver containing hydrogels reported previously in the literature.46,47 This can arise from the difference in the way this peptide shows its antibacterial property in comparison to its Ag-containing counterparts. The Agcontaining hydrogels exhibit antibacterial activity through the release of Ag nanoparticles/ions from their polymer or supramolecular matrix, while in this study small dipeptide based hydrogel itself holds antibacterial activity. Thus, this hydrogel also has some advantages. This type of hydrogel is devoid of any silver induced side–effects that can frequently be associated with silver containing antibacterial agents. These side effects include irreversible pigmentation of the skin and eyes or oxidative DNA damage and inflammation by increasing reactive oxygen species (ROS) in biological systems.53 The hydrogel P1, on the other hand, was ineffective against both Gram-positive bacteria Staphylococcus aureus and Bacilus subtilis even upto a concentration of 500 g/mL (50 L of 1 % (w/v) P1 gel). This may be due to the variation in the composition of the outer wall of the Gram-negative and Gram-positive bacteria which can give rise to a permeability difference in the outer walls.54,55 The thick cell wall of Gram-positive bacteria mainly consists of peptidoglycan, while the outer wall of the Gram-negative


Langmuir

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 14 of 29

bacteria contains lipopolysaccharide, lipoprotein, and very little peptidoglycan.63,64 Some of the previously reported antimicrobial agents suggests discrimination towards Gram-negative bacteria over Gram-positive bacteria64 or selectivity towards Gram-positive bacteria at the expense of Gram-negative bacteria.65

Biocompatibility Studies with the Hydrogel Hydrogel with human blood Furthermore, our study aims to unravel the cellular events that occur upon exposure to hydrogel with human blood cells. For practical use as an antimicrobial agent, the hydrogel must exhibit low hemolysis. Figure 7 (e, f) illustrates a dose–response profile of hemolysis of the red blood cells in presence of hydrogel. The hemolytic activity increased with increasing hydrogel concentration, the hydrogel showed negligible hemolysis (less than 1%) at MIC for E. coli and only 13 % at MIC for P. aeruginosa. Though 13 % hemolysis is more than the observed value for E. coli, it is not considered to be significant for invitro analysis.66 As shown in Figure 7 (e, f), 40 % hemolysis was observed only at hydrogel concentration of 500 g / mL (50 L of 1 % (w/v) P1 gel) which is much higher than the MIC values. Thus, it is interesting to observe that the antibacterial activity of the non-gelator peptide P2 is much less than that of the peptide gel P1. This suggests that the gelator peptide P1 not only aggregates more than that of the non-gelator peptide P2 but also display much more efficient antibacterial activity compared to that of the non-gelator peptide P2 under similar experimental conditions.


Page 15 of 29

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

Langmuir

Figure 7. (a-d) Determination of the effect of hydrogel on Gram-negative and Gram-positive bacteria by agar-diffusion assay method. (a) Escherichia coli, (b) Pseudomonas aeruginosa, (c) Staphylococcus aureus and (d) Bacilus subtilis were spread on an Agar plate. In each case, two different amounts of 1 % (w/v) P1 hydrogel, g1 (10 µL/well) and g2 (20 µL/well) were added to the wells where C represents 50 L of phosphate buffer. (e) (i-v) Hemolysis assay for the hydrogel using Triton-X as a positive control and PBS as a negative control, where Red blood cells are treated with (i) 5 L, (ii) 7.5 L, (iii) 10 L, (iv) 20 L and (v) 50 L of 1 % (w/v) hydrogel respectively. (f) A dose–response plot for the hemolysis of human red blood cells by hydrogel. Each value represents the mean ± SD of six measurements.

Table 1. Antibacterial activity of hydrogel P1 determined by agar-diffusion method

! " #$

" #%

#

" #%

" #&

#

' " #(

#

#

#

#

#


Langmuir

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 16 of 29

Interaction of the hydrogel with hemoglobin protein: The hemolytic data suggests that the P1 hydrogel has affinity towards hemoglobin protein. Understanding the interaction of the hydrogel with protein is crucial to address the engineering of hydrogel materials towards biocompatibility. Figure S14 displays the UV-Vis absorption spectra of Hemoglobin (Hb) in the absence and presence of the hydrogel P1. The absorption spectra of pure Hb show electronic bands located at 278 nm due to the presence of the phenyl group of Trp and Tyr residues of the protein, while the band at 406 nm can be designated as the heme or Soret band.67 The change in intensity of the Trp/Tyr band as well as heme band, suggests that the hydrogel can interact with both the heme and the Trp residues. However, in the presence of hydrogel, the band at 278 nm is blue-shifted to 259 nm, whereas the heme band remains at same position. The fluorescence (FL) of the Hb molecule was also found to be quite sensitive to hydrogel P1 in micromolar range and caused a FL enhancement of Hb (Figure S15). As displayed in Figure S16, there is a consistent rise in FL intensity with blue shifting upto 16 L of 1 % (w/v) P1 hydrogel which is equivalent to 160 g of the gelator. This enhancement could be attributed to the electrostatic interaction between carboxylic groups of P1 in the hydrogel with the amino acid residues of Hb.68 This indicates that the hydrogel is directly involved in binding with Trp/Tyr in the protein and did not affect the overall protein structure.69 The circular dichroism spectroscopic data of Hb was also obtained in the presence of hydrogel (Figure S16). The 209 nm band corresponds to the - * transition of the -helix, whereas the 222 nm band is assigned to a - * transition for either the -helix or the random coil.59 The intensity change of these absorption peaks shows a negligible change of secondary structure. Thus, the CD data suggests that at a suitable concentration of hydrogel the molecular structure of Hb molecule is maintained properly without any significant toxicity. Thus UV-Vis, fluorescence (FL) and circular dichroism (CD) experiments show significant interaction between the hydrogel P1 and the Hb molecules without perturbing the structure of Hb molecules at the minimum inhibitory concentration of the hydrogel which kills Gram-negative bacteria. The preservation of protein structure despite strong interaction between the hydrogel and the amino acid residues of Hb is consistent with previous literature.68,70


Page 17 of 29

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

Langmuir

Hydrogel with normal human cell line Human fibroblast cell line has been used to determine the cytotoxicity of the as prepared hydrogel P1. The number of surviving cells was expressed as Percent viability = The absorbance of the sample (treated cells)-background/the absorbance of the control (untreated cells)-background) × 100. Cellular toxicity of extracted contents from scaffolds was rated as follows: severe (90%) of MTT activity, compared to the control cells cultured in extract-free medium. Cytotoxic effects of gel against normal human fibroblast cells (W138) was examined by MTT cell

proliferation

assay

(MTT,

3-(4,

5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazoilum bromide).

Importantly, the hydrogel was practically non-toxic for W138 cells upto gelator concentration of 500 g/mL (50 L of 1 % (w/v) P1 hydrogel), since cell viability was higher than 88% (Figure X) after 24 and 48 hours incubation periods. This suggests that the hydrogel is biocompatible to a normal human cell line.

Figure 8. Cell viability of normal human fibroblast cells (W138) after 24 (black line bar) and 48 (red line bar) hours treatment with different concentrations of gel as calculated from the MTT assay.

Proteolytic Stability


Langmuir

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 18 of 29

The dipeptide P1 contains a naturally occurring alpha amino acid (Phe) at the carboxylic end and this is covalently attached to a non-proteinaceous long chain containing 11-amino undecanoic acid residue. Due to the absence of any peptide linkage between two alpha proteinaceous amino acid residues (one is alpha amino acid but the other is not), the dipeptide P1 and the non-gelator peptide P2 show resistance towards proteolysis upon the treatment with proteolytic enzymes, proteinase k and chymotrypsin (Figures 9, S18, S19 and S20). To examine the proteolytic stability, the gelator peptide P1 and the non-gelator P2 were incubated with proteolytic enzymes proteinase K and chymotrypsin in HEPES buffer at physiological temperature (37 °C) and pH (7.46). Then, at different time intervals mass spectra were taken to monitor if there is any change in the mass spectrum of the HEPES buffer solution of the dipeptides in presence of both enzymes (proteinase K and chymotrypsin). However, no change of the mass spectrum was observed during various intervals upto 36 hours (Figures 9a, b, S18, S19 and S20). As high performance liquid chromatography (HPLC) can be a better tool than mass spectrometry to study enzymatic degradation, the digestion experiment of the incubated HEPES buffer (pH 7.46) solution of peptide P1 was also carried out using HPLC in presence of proteinase K and chymotrypsin. Peak area under the curve between 6 to 8 minutes of elusion time from the chromatograms (Figure S21) suggests about 20 % and 25 % degradation after 24 hours and 36 hours respectively of incubation of the P1 gelator (Figure 9c, d). The amount of the compound remained after 24 and 36 hours show only a small amount of degradation compared to the results obtained for peptide hydrogels containing only L- -amino acids reported by other groups.71 Thus, only a small amount of degradation of the gelator peptide P1 occurred during its interaction with proteolytic enzymes (proteinase K and chymotrypsin) even after 24 and 36 hours. Thus it can be concluded that the dipeptide P1 is proteolytically stable at physiological pH and temperature and the injectable hydrogel P1 can potentially be applied in human body as an antibacterial agent.


Page 19 of 29

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

Langmuir

Figure 9. Digestion curve of the hydrogelator P1 upon treatment of (a), (c) proteinase K and (b), (d) chymotrypsin measured by using (a-b) mass spectrometry and (c-d) digestion profile obtained from high performance liquid chromatography (HPLC) traces at different time interval indicating that there is little cleavage or degradation of the gelator peptide in presence of the enzymes.

Conclusion This study convincingly demonstrates the formation of a proteolytically stable, mechano-sensitive (thus injectable) hydrogel exhibiting remarkable and selective antimicrobial activity, for Gram-negative bacteria. Moreover, this gel-based material also shows good compatibility with human red blood cells as well as human fibroblast cells under concentrations where antimicrobial behavior is observed. So, we have successfully designed a peptide based proteolytically stable biocompatible and injectable soft material with exceptional promise in the development of new antibacterial agents. We have also noticed that a dipeptide P2 which is structurally similar to the gelator peptide does not produce a hydrogel under


Langmuir

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 20 of 29

similar conditions. Moreover, antibacterial activity of P2 solution is considerably lower than the P1 hydrogel. Thus, it can be concluded that a small structural modification leads to a completely different scenario at the supramolecular level and a solution instead of gel is obtained which in turn also disturbs its antibacterial potency to a great extent.

Acknowledgement A. Baral gratefully acknowledges CSIR, New Delhi (India) for financial assistance. We also acknowledge Dr. P. Jaisankar and Rahul Gajbhiye of Chemistry Division, CSIR-Indian Institute of Chemical Biology, Kolkata for performing the HPLC experiments.

Associated Content *Supporting Information Instrumentation, antibacterial and hemolytic study details, CD, UV-Vis and fluorescence spectroscopic studies, synthetic procedures, NMR, mass, Tgel, mass spectra from proteolytic studies, HPLC traces.

References (1)

Du, X.; Zhou, J.; Xu, B. Supramolecular Hydrogels Made of Basic Biological Building Blocks. Chem. Asian J. 2014, 9, 1446-1472.

(2)

Raeburn, J.; Cardoso, A. Z.; Adams, D. J. The importance of the self-assembly process to control mechanical properties of low molecular weight hydrogels. Chem. Soc. Rev. 2013, 42, 5143-5156.


Page 21 of 29

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

Langmuir

(3)

Lan, Y.; Corradini, M. G.; Weiss, R. G.; Raghavan, S. R.; Rogers, M. A. To gel or not to gel: correlating molecular gelation with solvent parameters. Chem. Soc. Rev. 2015, 44, 60356058.

(4)

Du, X.; Zhou, J.; Shi, J.; Xu, B. Supramolecular Hydrogelators and Hydrogels: From Soft Matter to Molecular Biomaterials. Chem. Rev. 2015, 115, 13165−13307.

(5)

Lan, Y.; Corradini, M. G.; Liu, X.; May, T. E.; Borondics, F.; Weiss, R. G.; Rogers, M. A. Comparing and Correlating Solubility Parameters Governing the Self-Assembly of Molecular Gels Using 1,3:2,4-Dibenzylidene Sorbitol as the Gelator. Langmuir 2014, 30, 14128−14142.

(6)

Okesola, B. O.; Vieira, V. M. P.; Cornwell, D. J.; Whitelaw, N. K.; Smith, D. K. 1,3:2,4Dibenzylidene-D-sorbitol (DBS) and its derivatives – efficient, versatile and industrially relevant low-molecular-weight gelators with over 100 years of history and a bright future. Soft Matter 2015, 11, 4768-4787.

(7)

Jadhav, S. R.; Vemula, P. K.; Kumar, R.; Raghavan, S. R.; John, G. Sugar-Derived PhaseSelective Molecular Gelators as Model Solidifiers for Oil Spills. Angew. Chem. Int. Ed. 2010, 49, 7695-7698.

(8)

Banerjee, S.; Das, R. K.; Terech, P.; Geyer, A. d.; Aymonier, C.; Loppinet-Serani, A.; Raffy, G.; Maitra, U.; Guerzo, A. D.; Desvergne, J. –P. Hybrid organogels and aerogels from coassembly of structurally different low molecular weight gelators. J. Mater. Chem. C 2013, 1, 3305-3316.

(9)

Lalitha, K.; Prasad, Y. S.; Maheswari, C. U.; Sridharan, V.; John, G.; Nagarajan, S. Stimuli responsive hydrogels derived from a renewable resource: synthesis, self-assembly in water and application in drug delivery. J. Mater. Chem. B 2015, 3, 5560-5568.


Langmuir

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

(10) Datta S.; Bhattacharya, S. Multifarious facets of sugar-derived molecular gels: molecular features, mechanisms of self-assembly and emerging applications. Chem. Soc. Rev. 2015, 44, 5596-5637. (11) Rohner, S. S.; Ruiz-Olles, J.; Smith, D. K. Speed versus stability–structure–activity effects on the assembly of two-component gels. RSC Adv. 2015, 5, 27190-27196. (12) Díaz-Oltra, S.; Berdugo, C.; Miravet J. F.; Escuder, B. Study of the effect of polymorphism on the self-assembly and catalytic performance of an L-proline based molecular hydrogelator. New J. Chem. 2015, 39, 3785–3791. (13) Tian, Y.; Wang, H.; Liu, Y.; Mao, L.; Chen, W.; Zhu, Z.; Liu, W.; Zheng, W.; Zhao, Y.; Kong, D.; Yang, Z.; Zhang, W.; Shao, Y.; Jiang, X. A Peptide-Based Nanofibrous Hydrogel as a Promising DNA Nanovector for Optimizing the Efficacy of HIV Vaccine. Nano Lett. 2014, 14, 1439-1445. (14) Roy, S.; Baral, A.; Banerjee, A. An Amino-Acid-Based Self-Healing Hydrogel: Modulation of the Self Healing Properties by Incorporating Carbon-Based Nanomaterials. Chem. Eur. J. 2013, 19, 14950-14957. (15) Liu, K.; Steed, J. W. Triggered formation of thixotropic hydrogels by balancing competitive supramolecular synthons. Soft Matter 2013, 9, 11699-11705. (16) Sajisha, V. S.; Maitra, U. Remarkable isomer-selective gelation of aromatic solvents by a polymorph of a urea-linked bile acid–amino acid conjugate. RSC Adv. 2014, 4, 43167-43171. (17) Kumar, D. K.; Steed, J. W. Supramolecular gel phase crystallization: orthogonal selfassembly under non-equilibrium conditions. Chem. Soc. Rev. 2014, 43, 2080-2088. (18) Li, T.; Kalloudis, M.; Cardoso, A. Z.; Adams, D. J.; Clegg, P. S. Drop-Casting Hydrogels at a Liquid Interface: The Case of Hydrophobic Dipeptides. Langmuir 2014, 30, 13854−13860. (19) Baral, A.; Basak, S.; Basu, K.; Dehsorkhi, A.; Hamley, I. W.; Banerjee, A. Time-dependent gel to gel transformation of a peptide based supramolecular gelator. Soft Matter 2015, 11, 4944-4951.


Page 22 of 29

Page 23 of 29

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

Langmuir

(20) Babu, S. S.; Praveen, V. K.; Ajayaghosh, A. Functional

Gelators and Their Applications.

Chem. Rev. 2014, 114, 1973−2129. (21) Hoffman, A. S. Hydrogels for biomedical applications. Adv. Drug Delivery Rev. 2012, 64, 18-23. (22) Naskar, J.; Palui G.; Banerjee, A. Tetrapeptide-Based Hydrogels: for Encapsulation and Slow Release of an Anticancer Drug at Physiological pH. J. Phys. Chem. B 2009, 113, 11787-11792. (23) Nanda, J.; Banerjee, A.

-Amino acid containing proteolitically stable dipeptide based

hydrogels: encapsulation and sustained release of some important biomolecules at physiological pH and temperature. Soft Matter 2012, 8, 3380-3386. (24) Castelletto, V.; Hamley, I. W.; Stain, C.; Connon C. Slow-Release RGD-Peptide Hydrogel Monoliths. Langmuir 2012, 28, 12575−12580. (25) Yang, Z.; Liang, G.; Ma, M.; Abbah, A. S.; Lu, W. W.; Xu, B. D-Glucosamine-based supramolecular hydrogels to improve wound healing. Chem. Commun. 2007, 843-845. (26) Basak, S.; Nanda, J.; Banerjee, A. A new aromatic amino acid based organogel for oil spill recovery. J. Mater. Chem. 2012, 22, 11658-11664. (27) Nanda, J.; Biswas, A.; Adhikari, B.; Banerjee, A. A Gel-Based Trihybrid System Containing Nanofibers, Nanosheets, and Nanoparticles: Modulation of the Rheological Property and Catalysis. Angew. Chem. Int. Ed. 2013, 52, 5041-5045. (28) Wada, A.; Tamaru, S.; Ikeda, M.; Hamachi, I. MCM-Enzyme-Supramolecular Hydrogel Hybrid as a Fluorescence Sensing Material for Polyanions of Biological Significance. J. Am. Chem. Soc. 2009, 131, 5321-5330. (29) Galler, K. M.; Aulisa, L.; Regan, K. R.; D’Souza, R. N.; Hartgerink, J. D. Self-Assembling Multidomain Peptide Hydrogels: Designed Susceptibility to Enzymatic Cleavage Allows Enhanced Cell Migration and Spreading. J. Am. Chem. Soc. 2010, 132, 3217-3223.


Langmuir

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

(30) Scott, G.; Roy, S.; Abul-Haija, Y. M.; Fleming, S.; Bai, S. Ulijn; R. V. Pickering Stabilized Peptide Gel Particles as Tunable Microenvironments for Biocatalysis. Langmuir 2013, 29, 14321−14327. (31) Fleming, S.; Debnath, S.; Frederix, P. W. J. M.; Hunt N. T.; Ulijn, R. V. Insights into the Coassembly of Hydrogelators and Surfactants Based on Aromatic Peptide Amphiphiles. Biomacromolecules 2014, 15, 1171-1184. (32) Krysmann, M. J.; Castelletto, V.; Kelarakis, A.; Hamley, I. W.; Hule R. A.; Pochan, D. J. Self-Assembly and Hydrogelation of an Amyloid Peptide Fragment. Biochemistry 2008, 47, 4597-4605. (33) Yan, C.; Mackay, M. E.; Czymmek, K.; Nagarkar, R. P.; Schneider, J. P.; Pochan; D. J. Injectable Solid Peptide Hydrogel as a Cell Carrier: Effects of Shear Flow on Hydrogels and Cell Payload. Langmuir 2012, 28, 6076−6087. (34) Yu, L.; Ding, J. Injectable hydrogels as unique biomedical materials. Chem. Soc. Rev. 2008, 37, 1473-1481. (35) Huang, H.; Shi, J.; Laskin, J.; Liu, Z.; McVey, D. S.; Sun, X. S. Design of a shear-thinning recoverable peptide hydrogel from native sequences and application for influenza H1N1 vaccine adjuvant. Soft Matter 2011, 7, 8905-8912. (36) Foo, C. T. S. W. P.; Lee, J. S.; Mulyasasmita, W.; Parisi-Amon, A.; Heilshorn, S. C. Twocomponent protein-engineered physical hydrogels for cell encapsulation. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 22067-22072. (37) Baral, A.; Roy, S.; Dehsorkhi, A.; Hamley, I. W.; Mohapatra, S.; Ghosh, S.; Banerjee, A. Assembly of an Injectable Noncytotoxic Peptide-Based Hydrogelator for Sustained Release of Drugs. Langmuir 2014, 30, 929-936. (38) Latxague, L.; Ramin, M. A.; Appavoo, A.; Berto, P.; Maisani, M.; Ehret, C.; Chassande, O.; Barthélémy, P. Control of Stem-Cell Behavior by Fine Tuning the Supramolecular


Page 24 of 29

Page 25 of 29

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

Langmuir

Assemblies of Low-Molecular -Weight Gelators. Angew. Chem. Int. Ed. 2015, 54, 45174521. (39) Campoccia, D.; Montanaro, L.; Speziale, P.; Arciola, C. R. Antibiotic-loaded biomaterials and the risks for the spread of antibiotic resistance following their prophylactic and therapeutic clinical use. Biomaterials 2010, 31, 6363-6377. (40) Guvendiren, M.; Lu, H. D.; Burdick, J. A. Shear-thinning hydrogels for biomedical applications. Soft Matter 2012, 8, 260-272. (41) Gao, W.; Vecchio, D.; Li, J.; Zhu, J.; Zhang, Q.; Fu, V.; Li, J.; Thamphiwatana, S.; Lu, D.; Zhang,

L.

Hydrogel

Containing

Nanoparticle

Stabilized

Liposomes

for

Topical

Antimicrobial Delivery. ACS Nano 2014, 8, 2900-2907. (42) Lakes, A. L.; Peyyala, R.; Ebersole, J. L.; Puleo, D. A.; Hilt; J. Z.; Dziubla, T. D. Synthesis and Characterization of an Antibacterial Hydrogel Containing Covalently Bound Vancomycin. Biomacromolecules 2014, 15, 3009-3018. (43) Li, W. –R.; Xie, X. –B.; Shi, Q. –S.; Zeng, H. –Y.; Yang, Y. –S. O.; Chen, Y. –B. Antibacterial activity and mechanism of silver nanoparticles on Escherichia coli. Appl. Microbiol. Biotechnol. 2010, 85, 1115-1122. (44) Fullenkamp, D. E.; Rivera, J. G.; Gong, Y.; Lau, K. H. A.; He, L.; Varshney, R.; Messersmith, P. B. Mussel-inspired silver-releasing antibacterial hydrogels. Biomaterials 2012, 33, 3783-3791. (45) Valle, H.; Rivas, B. L.; Fernández, M.; Mondaca, M. A.; Aguilar, M. R.; Román, J. S. Antibacterial Activity and Cytotoxicity of Hydrogel–Nanosilver Composites Based on Copolymers from 2-Acrylamido-2-methylpropanesulfonate Sodium. J. Appl. Polym. Sci. 2014, 131, 39644 (1-11). (46) Liu, Y.; Ma, W.; Liu, W.; Li, C.; Liu, Y.; Jiang; X.; Tang, Z. Silver(I)–glutathione biocoordination

polymer

hydrogel:

effective

antibacterial

cytocompatibility. J. Mater. Chem. 2011, 21, 19214-19218.


activity

and

improved

Langmuir

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 26 of 29

(47) Xu, F.; Padhy, H.; Al-Dossary, M.; Zhang, G.; Behzad, A. R.; Stingl, U.; Rothenberger, A. Synthesis and properties of the metallosupramolecular polymer hydrogel poly[methyl vinyl ether-alt-mono-sodium maleate].AgNO3:Ag+/Cu2+ ion exchange and effective antibacterial activity. J. Mater. Chem. B 2014, 2, 6406-6411. (48) Wang, H.; Zha, G.; Du, H.; Gao, L.; Li, X.; Shen, Z.; Zhu, W. Facile fabrication of ultrathin antibacterial hydrogel films via layer-by-layer “click” chemistry. Polym. Chem. 2014, 5, 6489-6494. (49) Brahmachari, S.; Debnath, S.; Dutta, S.; Das, P. K. Pyridinium based amphiphilic hydrogelators as potential antibacterial agents. Beilstein J. Org. Chem. 2010, 6, 859-868. (50) Yang, Z.; Liang, G.; Guo, Z.; Guo, Z.; Xu, B. Intracellular Hydrogelation of Small Molecules Inhibits Bacterial Growth. Angew. Chem. Int. Ed. 2007, 46, 8216-8219. (51) Hu, Y.; Xu, W.; Li, G.; Xu, L.; Song, A.; Hao, J. Self-Assembled Peptide Nanofibers Encapsulated with Superfine Silver Nanoparticles via Ag+ Coordination. Langmuir 2015, 31, 8599−8605. (52) Li, P.; Poon, Y. F.; Li, W.; Zhu, H. –Y.; Yeap, S. H.; Cao, Y.; Qi, X.; Zhou, C.; Lamrani, M.; Beuerman, R. W.; Kang, E. –T.; Mu, Y.; Li, C. M.; Chang, M. W.; Leong, S. S. J.; Chan-Park, M. B. A polycationic antimicrobial and biocompatible hydrogel with microbe membrane suctioning ability. Nat. Mater. 2011, 10, 149-156. (53) Yeo, E. D.; Yoon, S. A.; Oh, S. R.; Choi, Y. S.; Lee, Y. K. Degree of the Hazards of Silver-Containing Dressings

on MRSA-Infected Wounds

in Sprague-Dawley and

Streptozotocin-Induced Diabetic Rats. Wounds 2015, 27, 95-102. (54) Banerjee, A.; Palui, G.; Banerjee, A. Pentapeptide based organogels: the role of adjacently located phenylalanine residues in gel formation. Soft Matter 2008, 4, 1430–1437. (55) Lee, A. L. Z.; Ng, V. W. L.; Gao, S.; Hedrick, J. L.; Yang, Y. Y. Injectable Hydrogels from Triblock Copolymers of Vitamin E-Functionalized Polycarbonate and Poly(ethylene glycol)


Page 27 of 29

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

Langmuir

for Subcutaneous Delivery of Antibodies for Cancer Therapy. Adv. Funct. Mater. 2014, 24, 1538-1550. (56) Bodey, G. P.; Bolivar, R.; Fainstein, V.; Jadeja, L. Infections Caused by Pseudomonas aeruginosa. Rev. Clin. Infect Dis. 1983, 5, 279-313. (57) Foca, M.; Jakob, K.; Whittier, S.; Latta, P. D.; Factor, S.; Rubenstein, D.; Saiman, L. Endemic Pseudomonas aeruginosa Infection in a Neonatal Intensive Care Unit. N. Engl. J. Med. 2000, 343, 695-700. (58) Song, Z.; Borgwardt, L.; Høiby, N.; Wu, H.; Sørensen, T. S.; Borgwardt, A. Prosthesis infections after orthopedic joint replacement: the possible role of bacterial biofilms. Orthopedic Reviews 2013, 5, 65-71. (59) Hengzhuang, W.; Wu, H.; Ciofu, O.; Song, Z.; Høiby, N. In Vivo Pharmacokinetics/ Pharmacodynamics of Colistin and Imipenem in Pseudomonas aeruginosa Biofilm Infection. Antimicrobial Agents and Chemotherapy 2012, 56, 2683–2690. (60) Moretto, V.; Crisma, M.; Bonora, G. M.; Toniolo, C.; Balaram, H.; Balaram, P. Comparison of the Effect of Five Guest Residues on the

-Sheet Conformation of Host (L-Val)n

Oligopeptides. Macromolecules 1989, 22, 2939-2944. (61) Morris, K. L.; Chen, L.; Rodger, A.; Adams, D. J.; Serpell, L. C. Structural determinants in a library of low molecular weight gelators. Soft Matter, 2015, 11, 1174–1181. (62) Qazvini, N. T.; Bolisetty, S.; Adamcik, J.; Mezzenga, R. Self-Healing Fish Gelatin/Sodium Montmorillonite Biohybrid Coacervates: Structural and Rheological Characterization. Biomacromolecules 2012, 13, 2136-2147. (63) Schleifer, K. H.; Kandler, O. Peptidoglycan Types of Bacterial Cell Walls and their Taxonomic Implications. Bacteriol. Rev. 1972, 36, 407-477. (64) Ghosh, S.; Ghosh, D.; Bag, P. K.; Bhattacharya, S. C.; Saha, A. Aqueous synthesis of ZnTe/dendrimer

nanocomposites

and

their

antimicrobial

therapeutics. Nanoscale 2011, 3, 1139-1148.


activity:

implications

in

Langmuir

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 28 of 29

(65) Brahmachari, S.; Mandal, S. K.; Das, P. K. Fabrication of SWCNT-Ag Nanoparticle Hybrid Included Self-Assemblies for Antibacterial Applications. PLOS One 2014, 9, e106775. (66) Amin, K.; Dannenfelser, R. –M. In Vitro Hemolysis: Guidance for the Pharmaceutical Scientist. J. Pharm. Sci 2006, 95, 1173-1176. (67) Boys, B. L.; Kuprowski, M. C.; Konermann, L. Symmetric Behavior of Hemoglobin - and -

Subunits

during

Acid-Induced

Denaturation

Observed

by

Electrospray

Mass

Spectrometry. Biochemistry 2007, 46, 10675-10684. (68) Mahato, M.; Pal, P.; Kamilya, T.; Sarkar, R.; Chaudhuri A.; Talapatra, G. B. Hemoglobinsilver interaction and bioconjugate formation: a spectroscopic study. J. Phys. Chem. B 2010, 114, 7062-7070. (69) Adler, A. J. Methods in Enzymology; Academic Press, New York, 1973, 27. (70) Ghosh, S.; Ray, M.; Das, M. R.; Chakrabarti, A.; Khan, A. H.; Sarma, D. D.; Acharya, S. Modulation of glyceraldehyde-3-phosphate dehydrogenase activity by surface functionalized quantum dots. Phys. Chem. Chem. Phys. 2014, 16, 5276-5283. (71) Liang, G.; Yang, Z.; Zhang, R.; Li, L.; Fan, Y.; Kuang, Y.; Gao, Y.; Wang, T.; Lu, W. W.; Xu, B. Supramolecular Hydrogel of a D-Amino Acid Dipeptide for Controlled Drug Release in Vivo Langmuir 2009, 25, 8419–8422.


Page 29 of 29

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

Langmuir

Table of Contents (TOC)


A Peptide-Based Mechano-sensitive, Proteolytically Stable Hydrogel

Recommend Documents