Broad-Spectrum Antibacterial Activity of Proteolytically Stable Self

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Broad-spectrum antibacterial activity of proteolytically stable self-assembled ##-hybrid peptide gels Kamal Malhotra, Sudha Shankar, Rajkishor Rai, and Yashveer Singh Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01582 • Publication Date (Web): 31 Jan 2018 Downloaded from http://pubs.acs.org on February 2, 2018

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Broad-spectrum antibacterial activity of proteolytically stable self-assembled αγ-hybrid peptide gels Kamal Malhotraa, Sudha Shankarb,c, Rajkishor Raib,c, and Yashveer Singha* a

Department of Chemistry, Indian Institute of Technology Ropar, Rupnagar-140001, Punjab, India

b

Medicinal Chemistry Division, CSIR- Indian Institute of Integrative Medicine, Canal Road, Jammu Tawi-180001, J & K, India c

Academy of Scientific and Innovative Research, New Delhi, India

KEYWORDS: Self-assembled, α/γ hybrid peptide, peptide gels, broad spectrum, antibacterial activity, proteolytic stability

ABSTRACT: Bacterial infections pose a serious threat to mankind and there is immense interest in the design and development of self-assembled peptide gels using ultra short peptides for antibacterial applications. The peptide gels containing natural amino acids suffer from poor stability against proteolytic enzymes. Therefore, there is a need to design and develop peptide gels with improved stability against proteolytic enzymes. In the present work, we report the synthesis and characterization of / hybrid peptides, Boc-D-Phe-4-L-Phe-PEA (NH007) and Boc-L-Phe-4-L-Phe-PEA (NH009) to improve the proteolytic stability. Both the dipeptides were

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found to self-assemble into gels in aqueous DMSO (3-5% w/v) and the self-assembly process was studied using FTIR and CD, which indicated antiparallel β-sheet formation with random coils in NH007 gels, and random or unordered conformation in NH009. The rheological studies indicated viscoelastic characteristics for both gels- the storage modulus (G’) for NH007 and NH009 gels (3% w/v) were estimated as 0.2 and 0.5 MPa, higher than the loss modulus (G’’). Also, both gels demonstrated self-healing characteristics for six consecutive cycles when subjected to varying strains of 0.1 and 30% (200 sec each). The peptide gels were incubated with a mocktail of proteolytic enzymes, proteinase K, pepsin, and chymotrypsin and stability was monitored using RP HPLC. Up to 23 and 40% degradation was observed for NH007 (3%, w/v) in 24 and 36 hours, and 77 and 94% degradation for NH009 (3%, w/v), within the same period. Thus, / hybrid peptide gels containing D-Phe exhibited higher stability than gels fabricated using L-Phe peptide. The use of D-residue in / hybrid peptide significantly enhanced the stability of peptides against proteolytic enzymes, as the stability data reported in this work are possibly the best in class. Both peptide gels exhibited broad-spectrum antibacterial activity against gram negative and gram positive bacteria, such as Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus. The Pseudomonas aeruginosa and Staphylococcus aureus, in particular, are known to develop resistance. The NH007 (3%, w/v) demonstrated 65% inhibition, whereas NH009 (3%, w/v) showed 78% inhibition, with potent activity against Pseudomonas aeruginosa. Mechanistic studies, using SEM, HR-TEM, and bacterial live-dead assay, indicated entrapment of bacteria in gel networks, followed by interaction with cell membrane components, and lysis. Cell viability (MTT assay) and toxicity (LDH assay) studies showed that both gels are not toxic to NIH 3T3 mouse embryonic fibroblast cells (mammalian). MTT assay showed more than 85% cell viability and LDH assay exhibited


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not more than 15% cytotoxicity, even at higher concentrations (5%, w/v) and prolonged exposures (48 hours). Overall, studies indicate the potential application of gels developed from the  hybrid peptides in preventing biomaterial-related infections.

1. INTRODUCTION Bacterial infections pose significant threat because of increasing prevalence of antibiotic resistance. Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa are responsible for nosocomial infections1. Pseudomonas aeruginosa, a multi-drug resistance pathogen, is the major culprit and alone accounts for 11% of all hospital-acquired infections2. Antibacterial strategies involving the use of silver ions3, silver nanoparticles4, gold nanoparticles5, carbon nanotubes6, and graphene oxide7 have been proposed to prevent the bacterial infections but, unfortunately, these are invariably associated with limitations like cytotoxicity8,9,10 irreversible discoloration of skin11, inflammation12, and oxidative DNA damage12. Consequently, there is an increasing interest in exploring antibacterial surfaces, which not only prevent infection but also helps in circumventing antibacterial resistance. Biomaterials like gels and hydrogels, with unique capability to hold water, have immense potential in such applications13,14,15,16,17. In particular, peptide-based self-assembled gels have generated significant interest for antibacterial applications and as extracellular matrix (ECM)-mimicking scaffold for cell growth and differentiation18. For antibacterial applications, gels are usually fabricated from peptides with inherent antibacterial activity or small molecule antibiotics are passively entrapped within the gel network19. These peptide gels have been found to be biocompatible and biodegradable, and give by-products, which are non-immunogenic20. Poly-cationic gels, fabricated from peptides, have been explored for antimicrobial activities19. For instance, a long 20 residue poly-


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cationic21 and arginine-rich19 self-assembled peptide gels were developed and evaluated for antibacterial activity against both gram positive (Staphylococcus epidermis and Staphylococcus aureus) and gram negative strains19,21 (Klebsiella pneumonia, Escherichia coli, and multidrug resistance Pseudomonas aeruginosa). Similarly, N-terminally protected long chain amino acid containing aromatic dipeptide was also synthesized to develop proteolytically stable, antibacterial gel against gram negative strains, Escherichia coli and Pseudomonas aeruginosa22. In all instances, long peptides were used, which are difficult to synthesize and often associated with high costs. Therefore, the focus of current research has shifted towards gels fabricated from ultra-short self-assembled peptides because of their ease of synthesis on large scale and applications in areas, like drug delivery, tissue engineering, and regenerative medicine20. Several antibacterial gels, synthesized using ultra-short self-assembled peptides and containing antibiotics23, Ag ions/particles24 and positive charge, have been reported in literature. Ultra-short peptide gels fabricated using diphenylalanine, the core recognition motif of Alzheimer’s β amyloid polypeptide owing to its potential to form molecular self-assemblies25, has generated immense interest for applications, like 3D cell culture, drug delivery, bioimaging, and biosensor26. Mostly, fluorenylmethyloxycarbonyl (Fmoc)-protected diphenylalanine have been used to fabricate gels, where Fmoc acts as an enhancer of gelation due to aromatic π-π stacking27. For instance, Laverty and coworkers28 fabricated gels using peptides, such as FmocFF, Fmoc-FFKK, Fmoc-FFFKK, and Fmoc-FFOO, and explored for antibacterial applications and it was found that Fmoc-FF gel was more active than other peptide gels in inhibiting biofilm formation by Staphylococcus aureus, Staphylococcus epidermis, Escherichia coli, and Pseudomonas aeruginosa but the gels showed higher cytotoxicity and hemolysis. It was contrary to the earlier report by Paladini et al.29, who showed that the Fmoc-FF gel is an inert material and


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doesn’t possess anti-bacterial activity against Staphylococcus aureus treated flax. Despite the promise shown as antibacterial materials, Fmoc-protected gels exhibit lower stability at physiological pH, which seriously limits their use in biological applications13 like drug delivery and tissue engineering30. This has led to a strong emphasis on developing diphenylalanine derivatives with improved proteolytic stability. Peptides containing -amino are susceptible to cleavage by proteolytic enzymes. The stability of peptides towards proteolytic enzymes can be improved by incorporating non-protein amino acids into peptide sequences. Earlier studies have demonstrated the proteolytic stability of polypeptides containing α/β31, amphipathic peptide hydrogelators

(H-FEβ3hFFQβ3hFFK-OH,

H-FEβ3hFFQβ3hFFK-NH2,

and

H-

FEβ3hFYQβ3hFYK-NH2) containing mixed α/β32 but the stability of α/γ-hybrid peptides has not been explored. Wani et al.33 have shown that the peptides containing β- and γ-amino acids exhibit antibacterial activity against Pseudomonas aeruginosa and Staphylococcus aureus. It has been also shown that the incorporation of un-natural D-amino acid improves the proteolytic stability of peptides, in vivo34,35. Similarly, change in chirality from L- to D- at N-terminal amino acid aided in the formation of self-assembled gels, in case of a tripeptide36,37 but it has not been investigated for diphenylalanines. In the present work, we report the synthesis and characterization of / hybrid peptides, BocD-Phe-4-L-Phe-PEA (NH007) and Boc-L-Phe-4-L-Phe-PEA (NH009), to improve the proteolytic stability. Both the peptides were self-assembled into gels and their viscoelastic, selfhealing, swelling, and degradation characteristics investigated. The stability of peptide gels against a mocktail of proteolytic enzymes, proteinase K, pepsin, and chymotrypsin, was monitored using RP HPLC and antibacterial activities assessed against gram negative and gram positive bacteria, such as Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and


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Staphylococcus aureus. Finally, SEM, HR-TEM and in-vitro bacterial live-dead assay studies were also carried out to shed light on the mechanism of antibacterial activity. Cell viability and toxicity studies using MTT and LDH assays showed that both peptide gels are not toxic to mammalian cells (NIH 3T3). This is possibly the first report on gelation behavior and antibacterial properties of gels developed from short  hybrid peptides. 2. EXPERIMENTAL SECTION 2.1. Materials and Methods. All reagents used were procured from commercial sources and used without further purification. Deionized water (18.2 MΩ•cm) was used in all experiments (Bio-Rad Milli-Q). Bacterial cultures, Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, and Staphylococcus aureus, with accession # MTCC 1687, MTCC 441, MTCC 424, and MTCC 7443, were procured from CSIR- IMTECH, Chandigarh. NIH 3T3 mouse embryonic fibroblast cells were a generous gift from Dr. Javed N. Agrewala, Chief Scientist, CSIRIMTECH, Chandigarh. MTT dye and Lactate dehydrogenase assay kit (cell cytotoxicity assay) were procured from Himedia and absorbance was measured using Tecan Infinite M Plex plate reader. Peptides were purified by RP HPLC on a C18 column (10 × 250 mm) using methanol/water gradient and characterized by HRMS (Agilent Technologies 6540). The 1H & experiments were carried out in CDCl3 on a Bruker 400 MHz (100 MHz for

13

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C NMR

C NMR), using

TMS as an internal standard. Fourier transform infrared (FTIR) spectra of peptide gels (dried) were recorded on a SHIMADZU IR spectrometer in 4000–400 cm−1 region. Scanning electron microscopy (SEM) studies were carried out using JEOL JSM-6610LV microscope. The gels were dried on metal stubs with vacuum drying and coated with platinum by sputtering at an accelerating voltage of 10-15kV. RP HPLC analysis (stability against proteolytic enzymes) was


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performed on a Waters system equipped with XBridge BEH 300 RP C18 column (250 × 4.6 mm, 5 µm), pump, degasser, injector with 100 µl loop, PDA and UV/VIS detectors, and data were analyzed using Empower 3 software. An isocratic mobile phase of acetonitrile:water::50:50 (v/v) was applied for 30 min at a flow rate of 1 ml/min. Samples were filtered through a 0.2 µm membrane filter, prior to injection (50 µl) and peaks were monitored at 220 nm. Unless mentioned otherwise, all experiments were done at least in triplicate and data reported are mean ± standard error (SE). 2.2. Synthesis and Characterization Studies 2.2.1. Boc-4-L-Phe–OH (5). 4-(tert-butoxycarbonyl)amino-5-phenylpentanoic acid (5) was synthesized using a procedure reported in the literature38. Briefly, Boc-L-Phe-OH was coupled with Meldrum’s acid at 0 oC in DCM using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl) and 4-dimethylamino pyridine (DMAP) to yield the intermediate 2, which was reduced using sodium borohydride in glacial acetic acid to yield 3. The intermediate 3 was refluxed in toluene for 3 h to yield 4, which on saponification using acetone/methanol and NaOH (1 N) for 30 minutes at 30 oC, yielded the desired product 5 (1.51 g, yield: 75.4%). HRMS(ESI)m/z: Mcal = 293.1435; Mobs = 294.1508 [M+H]+. 1H NMR (400 MHz, CDCl3) δ 7.2 (m, 2H), 7.13 (m, 3H), 5.84 (s, 1H), 4.37 (d, J = 8 Hz, 1H), 3.73 (d, J=17.3 Hz, 1H), 2.79- 2.62 (m, 2H), 2.34 (s, 2H), 1.86-1.53 (m, 2H), 1.30 (s, 9H).

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C NMR (101 MHz, CDCl3) δ 178.11,

155.78, 137.66, 129.41, 128.44, 126.47, 79.55, 51.26, 41.57, 31.02, 29.51, 28.34. 2.2.2. Boc-4-L-Phe-PEA. Boc-4-L-Phe-OH (2.9 g, 10 mmol) was dissolved in dry DCM (10 ml) and cooled in an ice bath, followed by the addition of NMM (10 ml, 20 mmol), EDC.HCl (2.2 g, 10 mmol), phenethylamine (PEA, 1.6 ml, 10 mmol) to the reaction mixture, and it was stirred for 12 h. The progress of the reaction was monitored using TLC at regular intervals. The


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solvent was evaporated completely and crude product was extracted with ethyl acetate (3 × 15 ml). The organic layer was washed successively with HCl (2N, 1 × 15 ml), NaHCO3 (1M, 1 × 15 ml), and brine solution. It was passed over anhydrous sodium sulfate and evaporated under vacuum to yield white crystalline solid Boc-4-L-Phe-PEA (3.5 g, yield = 89.7%), which was used for further synthesis without purification. 2.2.3. Boc-D-Phe-4-L-Phe-PEA (NH007). Boc-4-L-Phe-PEA (1.9 g, 5.0 mmol) was deprotected with TFA (30%) in DCM (10 ml) and reaction was monitored using TLC. After 2 h, TFA was evaporated completely and the free base was added to the pre-cooled solution of BocD-Phe-OH (1.3 g, 5.0 mmol) in dry DCM (5 ml), followed by addition of NMM (1.5 ml, 5.0 mmol) and EDC.HCl (1.1 g, 5.0 mmol). The reaction mixture was allowed to attain room temperature and stirred for 12 h. The progress of the reaction was monitored using TLC at regular intervals. After completion of the reaction, DCM was evaporated under reduced pressure and reaction was worked up, as described above. The crude peptide was purified by column chromatography over silica gel (60-120 mesh) to yield Boc-D-Phe-4-L-Phe-PEA (2 g, yield = 76.5%). 1H NMR (400 MHz, CDCl3) δ 7.14 (m, 13H), 6.89 (d, J = 2H), 6.31 (s, 1H), 5.96 (s, 1H), 4.98 (d, J = 7.4 Hz, 1H), 4.11 (q, J = 14.4, 7.0 Hz, 1H), 3.97 (q, 1H), 3.46 (dd, J = 13.1, 6.3 Hz, 1H), 3.35 (dd, J = 13.0, 6.1 Hz, 1H), 2.85 (d, 2H), 2.74 (t, J = 7.0 Hz, 2H), 2.59 (dd, J = 8.2 Hz, 1H), 2.46 (dd, J = 13.4, 6.5 Hz, 1H), 1.96 (t, J = 6.4 Hz, 2H), 1.74 (d, J = 3.7 Hz, 2H), 1.30 (s, 9H). ESI-MS: Mcal = 543.7; Mobs = 544.7 [M+H]+.

13

C NMR (126 MHz, CDCl3) δ 172.93,

171.48, 155.46, 139.01, 137.20, 136.64, 129.42, 128.80, 128.66, 128.56, 128.52, 127.02, 126.66, 126.43, 56.35, 49.78, 41.18, 40.68, 38.49, 35.62, 33.04, 30.19, 28.28. 2.2.4. Boc-L-Phe-4-L-Phe-PEA (NH009). The dipeptide NH009 was obtained using a procedure similar to one described above for NH007 but Boc-L-Phe-OH (1.3 g, 5.0 mmol) was


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used instead of Boc-D-Phe-OH (1.8 g, yield = 70.2%). 1H NMR (400 MHz, CDCl3) δ 7.35 – 6.98 (m, 15H), 6.20 (s, 1H), 5.82 (s, 1H), 4.87 (s, 1H), 4.19 (q, J = 4 Hz, 1H), 3.97 (t,J=12,1H), 3.55 (dd, 1H), 3.50 (dd, 1H),2.91 (m, 2H), 2.82 (t, J = 8 Hz, 2H), 2.70 (dd,2H), 1.86 (m, 4H), 1.36 (s, 9H). ESI-MS: Mcal = 543.7; Mobs = 544.7 [M+H]+. 13C NMR (126 MHz, CDCl3) δ 172.54, 171.12, 155.41, 139.04, 137.29, 136.55, 129.40, 129.31, 128.78, 128.76, 128.57, 128.49, 127.03, 126.62, 126.44, 56.19, 49.70, 41.16, 40.53, 38.02, 35.55, 32.85, 30.12, 28.24. 2.3. Fabrication of Self-assembled Peptide Gels. The gel from peptide NH007 was fabricated by dissolving the peptide (3 and 5 mg) in DMSO (50 µl), which was then transferred to deionized water (50 µl), with continuous tapping, whereas to fabricate gel from peptide NH009, deionized water (50 µl) was added drop wise to the peptide solution in DMSO (50 µl), with tapping. Self-assembled gels (3 and 5%, w/v) were immediately formed and were kept unperturbed for 40 min, prior to use. 2.4. Rheology Studies: Oscillatory rheology experiments were performed on a MCR 102 rheometer (Anton Paar) using a 25 mm stainless steel parallel plate geometry (80 mm diameter, 0.2 mm gap). Peptide gels NH007 and NH009 (3 and 5% w/v) were prepared in a mold and kept unperturbed for 40 min, prior to experimentation. To prevent evaporation, the free surface was covered with low viscosity silicon oil. Gelation kinetics was monitored by time sweep measurements at 37 °C, keeping the angular frequency constant at 10 rad/sec. Dynamic amplitude sweep test was performed by varying the strain from 0.01-100%, keeping the angular frequency constant at 10 rad/sec and temperature 37 °C, whereas dynamic frequency sweep test (100-0.1 rad/sec) was performed at 37 °C by keeping the strain constant at 0.1%. 2.5. Self-healing Properties. Self-healing capability of gels were determined using timedependent recovery measurements by applying alternating strains of 0.1 and 30%, replicating


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conditions that provide stable gel and complete deformation. The strains were applied in a cycle of 200 sec each (each cycle included 200 seconds of 0.1%, 200 seconds of 30%, and 200 seconds of 0.1%) and repeated for six such cycles. The sixth cycle of 0.1% strain was applied for 400 sec. Self-healing studies were carried out at macroscopic level too. The gel fabricated from peptide NH009 (3%, w/v) was stained with rhodamine dye, whereas the gel fabricated from peptide NH007 was stained with methylene blue. Both gels were stacked together and observed for selfhealing capabilities after 6 and 36 h. 2.6. Gel Stability in Presence of Proteolytic Enzymes. A mocktail of proteolytic enzymes, proteinase K (3 units/ml), chymotrypsin (5.48 units/ml), and pepsin (5.5 units/ml), were prepared in phosphate buffer (10 mM). The mocktail (1 ml) was transferred to peptide gels (3%, w/v), which were incubated at 37 °C for 36 h. Aliquots (100 µl) were withdrawn at predetermined time intervals, filtered through 0.2 µm membrane filter, and analyzed by RP HPLC. 2.7. Antibacterial Studies. Experiments were done according to a reported protocol21, with slight modifications. Briefly, gels (3 and 5%, w/v) were formed in separate wells of a 96-well polystyrene plate (non-treated), using aqueous DMSO, as described above. The plate was subjected to vacuum drying for 2-3 days. The dried gels were sterilized in UV and equilibrated with Luria broth (100 µl) at 37 °C for overnight. Finally, media was removed from top of gels. Stock solution of bacterial cultures were prepared by suspending the bacterial powder in Luria broth (10 ml) and quadrant streaked on Luria plates, followed by incubation at 37 °C, overnight on an orbital shaker (100 rpm). Sub-culturing was done by inoculating bacteria (from fourth quadrant) into Luria broth (100 ml) and incubating it at 37 °C, overnight on an orbital shaker (100 rpm). The optical density of the suspension was adjusted to OD600nm = 0.1 AU by adding Luria broth, resulting in 107-108 cfu /ml bacterial solutions. Each bacterial culture (100 µl) was


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poured on to gels, which were incubated at 37 °C for 48 h. OD600nm was monitored at 6 h intervals, for up to 48 h using a plate reader. Gentamicin sulfate was taken as a positive control at concentrations of 10, 50, 100, and 150 µg/ml and pure buffer was taken as a negative control. Peptides alone (3 and 5 mg) were also assessed for their antibacterial activities by dissolving them in DMSO (100 µl), followed by vacuum drying and addition of media on top of it. Nonviable counts (%) of bacterial cultures were determined at an interval of 6 h, for up to 48 h after incubation. 2.8. Mechanistic Studies. 2.8.1. SEM. The peptide gel NH007 and NH009 (0.1%, w/v) were taken on a metal stub each and dried by vacuum drying. Bacterial cultures of Pseudomonas aeruginosa and Staphylococcus aureus were grown till mid log phase and 10 µl of each culture was inoculated on the stubPseudomonas aeruginosa was incubated with NH007 gel, whereas Staphylococcus aureus with NH009 gel at 37 °C for 12 hours in an incubator and coated with platinum by sputtering at an accelerating voltage of 10-15 kV. 2.8.2. HR TEM. The bacterial samples in presence and absence of NH007 and NH009 gels (3%, w/v) were prepared as follows: for a given well, peptide gels (100 µl, 3%, w/v) were prepared in aqueous DMSO and dried under vacuum for 2-3 days. The dried gels were sterilized in UV and equilibrated with Luria broth (100 µl) at 37 °C overnight. Prior to start of assay, Luria broth was removed from the top of gels. Bacterial cultures (Staphylococcus aureus and Pseudomonas aeruginosa) were grown till mid log phase and each bacterial culture (100 µl) was poured on the gel and incubated at 37 °C for 12 hours. Multiple wells for each bacterial strain were prepared to have enough bacteria. Control samples were prepared without peptide gel. After 12 hours of incubation, bacterial cultures were centrifuged (5500 rpm) at 4 °C for 15


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minutes and suspended in PBS (400 µl, pH 7.4) after discarding the supernatant. Process was repeated two more times. Fixation was carried out by adding glutaraldehyde into each bacterial culture, followed by incubation at 4 °C for overnight. Samples were centrifuged again and dehydrated, using graded ethanol (50 and 90%). Finally, bacterial samples were centrifuged and resuspended in 100% ethanol (100 µl). These bacterial samples were shaken well and used for preparation of samples for TEM studies. Microscopy was performed using a FEI Tecnai G2 F20, Netherlands at 120 kV electron energy. 2.9. Bacterial Live-dead Assay. Peptide gels (NH007 and NH009, 3%, w/v) were prepared in a 96-well plate, as described earlier. The gels were dried under vacuum, sterilized using UV, washed with DI water, and equilibrated with Luria broth overnight. The broth in each well was removed from the top of gels. Bacterial cultures (Staphylococcus aureus and Pseudomonas aeruginosa) were grown till mid log phase (OD600nm = 0.1, 107 cfu/ml) separately in eppendorf tubes. About 100 µl of each culture was poured over gels and incubated at 37 °C for 12 hours. Multiple wells for each strain were prepared so as to have enough bacteria. Control samples were prepared without gels. After incubation, cultures were collected and poured into a separate 96well plate. Finally, cultures were stained with 100 µl of Syto 9 (6 µM) and propidium iodide (30 µM) solutions for 30 minutes. The dye-stained bacterial cells (10 µl) were poured onto a slide and imaged using Leica microscope (63x). 3.0. Cell Viability and Toxicity Assay. 3.0.1. MTT Assay. Cell viabilities of NIH 3T3 cells against NH007 and NH009 gels were assessed using MTT assay39,40, with slight modifications. Briefly, gels (3% and 5% w/v) were prepared in a 96-well tissue culture treated plate (Nunclon Delta Surface, Thermofisher). DMSO, DPBS (negative), and 70% ethanol (positive) were taken as controls. The gels were dried under


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vacuum, and sterilized using UV, washed with DI water, and equilibrated with DMEM (100 µl) at 37º C for overnight. Media was removed from the top of gels. Simultaneously, cells were grown in DMEM supplemented with 10% FBS and 1% antibiotic antimycotic solution on a T25 Nunc cell culture treated flask, at 37 ºC in a humidified atmosphere containing 5% CO2 and subcultured at 80-90% confluency. Subconfluent cells were trypsined using trypsin-EDTA solution, harvested and counted using hemocytometer. Cells were diluted using DMEM complete media and seeded at a concentration of 4 x 105 cells per well in a 96 well plate containing gels and incubated for 24 and 48 hours. MTT (5 mg/ml, 20 µl) reagent was added after 24 and 48 hours and gels were further incubated for 4 hours. DMSO (100 µl) was added to dissolve the formazan salt. The dissolved solutions were transferred to a new plate and absorbance was measured at 570 nm (n=3) using a plate reader. The percentage cell viability was estimated as given below: % Cell Viability = 100 – [(Abs570nm peptide – Abs570nm PBS)/ (Abs570 70% ethanol – Abs570nm PBS) x 100] 3.0.1. Lactate Dehydrogenase (LDH) Assay. Cell cytotoxicity was assessed using LDH assay kit. Gels (3 and 5%) were prepared as described earlier and incubated for 24 and 48 hours with NIH 3T3 cells seeded with concentration of 4 x 105 cells per well. Following the incubation, LDH test reagent (10 µl) was added to gels and gels were incubated for 5 minutes. Finally, 50 µl of stop solution was added. Lysis buffer (5 µl) was added to wells without peptide gels, containing only seeded cells, 15 minutes prior to addition of LDH test reagent. Absorbance was measured at 490 nm using a plate reader. Cell toxicity was calculated using the equation given below: % toxicity = (Abs490nm peptide – Abs490nm PBS)/ (Abs490 Lysis buffer – Abs490nm PBS) x 100


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3. RESULTS AND DISCUSSION Polypeptide-based cationic gels have been explored earlier for broad spectrum antibacterial activities19,21 but these are associated with tedious and costly synthetic procedures and, therefore, there is an increasing interest in the field to explore the ultra-short peptide gels for antibacterial, drug delivery, and tissue engineering20 applications. The ultra-short peptides are more interesting because these are potential extracellular matrix mimic and associated with the ease of synthesis. Unfortunately, both polypeptides and ultra-short peptides are prone to cleavage by proteolytic enzymes30. The development of proteolytically stable ultra-short peptide gels is of significant research interest to the field.

Scheme 1. Synthesis of  hybrid dipeptide: (A) Boc-D-Phe-4-L-Phe-PEA (NH007); and (B) Boc-L-Phe-4-L-Phe-PEA (NH009). The present work is focused on developing antibacterial ultra-short self-assembled dipeptide gels with proteolytic stability. Earlier studies have shown that proteolytic stability can be achieved by incorporating non-natural amino acid analogs, D-amino acid41, terminal


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fluorination30, and non-protein analogs of α- and β-linkages42,43. In order to explore the use of α/γ hybrid peptides in the development of gel and their applications as well as proteolytic stability, we synthesized and characterized the dipeptides incorporating γ4-L-Phe as a non-protein amino acid, Boc-D-Phe-4-L-Phe-PEA (NH007) and Boc-L-Phe-4-L-Phe-PEA (NH009). The incorporation of γ-amino acid residue into peptide sequences is expected to provide increased stability against cleavage by proteolytic enzymes32. In addition, the presence of tertbutyloxycarbonyl (Boc) group at N-terminus and phenyl ethyl (PEA) group at C-terminus of dipeptide provides increased hydrophobicity44,45,46,47.

Figure 1. Images of gel fabricated from dipeptides in aqueous DMSO (50%, v/v): (A) NH007 (3%); (B) NH009 (3%); (C) NH007 (5%); and (D) NH009 (5%). SEM images of gels: (E) NH007 (0.2%) and (F) NH009 (0.2%).


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The dipeptides were synthesized using conventional solution-phase peptide synthesis (Scheme 1). Briefly, the Boc and methyl ester groups protecting N- and C- termini were removed with 30% TFA in DCM, followed by a saponification reaction. The coupling was carried out using EDC.HCl in presence of NMM as a base. Final peptides were purified using RP HPLC on a C18 column (Figure S1-S2) and characterized by HRMS (Figure S3-S4), 1H and

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C NMR data

(Figure S5-S10). The dipeptides, NH007 and NH009, were self-assembled into gels using aqueous DMSO (50:50, v/v). Other solvents / solvent systems were tried but self-assembly was observed only in aqueous DMSO, with minimum gelation concentration (MGC) being 3% (w/v). Both peptides formed opaque gels and beyond 5% (w/v), the peptides became insoluble (Figure 1). Earlier studies have reported no molecular self-assemblies with uncapped homo-chiral tripeptides36,48,49 but in our case, Boc- protected homo-chiral dipeptides could be self-assembled into gels. The FTIR spectra of dried gels, fabricated from NH007, gave peaks at 3327, 3026, 1649.06, 1633, 1527, 1492 and 1170 cm-1 (Figure S11). The peaks at 3327 and 3026cm-1 were attributed to N-H (stretching) and =C-H (stretching), indicating H-bonding interactions. The peaks at 1649, 1633, 1527, and 1170 cm-1 were attributed to amide I (>C=O stretching), amide carbonyl (>C=O stretching), amide II (N-H bending), and ester (C–O asymmetric stretching). Finally, the peaks at 1649 and 1494 cm-1 indicated β-sheet like conformation50 and random coils22 within the supramolecular structure. The spectra for gel, fabricated from NH009, gave peaks at 3328.99, 3290, 1683.78, 1650.99, 1539.12, 1519.84, 1166.81, 1043, and 696.27 cm-1, which were assigned as discussed earlier. Of particular interest was amide I peak at 1681 cm-1, which indicated that peptide adopts turn like structure51,52. The circular dichroism (CD) studies were carried out to confirm the results obtained from FTIR (Figure S12). Both dipeptides exhibited a minimum in


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Biomacromolecules

210-220 nm region, indicating antiparallel beta sheets23. The NH009 peptide showed a band at ~222 nm, which was assigned to π-π* transition for either α-helix or random coil22. In addition, it also gave a negative band of small amplitude at ~235-238 nm, corresponding to unordered structure. Thus, CD studies confirmed the presence of antiparallel β-sheets along with random coil in NH007, as indicated by FTIR. However, CD studies for NH009 indicated random coil or unordered conformation53 contrary to FTIR data. Both dipeptide gels self-assembled due to non-covalent interactions, like hydrogen bonding and π-π interactions. As indicated by the FTIR and CD spectra, both dipeptides adopt different conformations in hybrid dipeptides due to change in chirality at single amino acid. The schematic packing model of both dipeptides, as proposed in Figure S13, indicated the possibility of both intermolecular and intramolecular Phe-Phe interactions in the formation of NH007 gels but only intermolecular Phe-Phe interactions in NH009 gels. The SEM images of dried gels exhibited porous morphology, with NH007 gels exhibiting interwoven morphology, which bear prominent pores, whereas NH009 gels exhibited flake-like network with comparatively smaller porous structures (Figure 1). The viscoelastic characteristics of NH007 and NH009 gels were investigated using rheology, and both storage modulus (G’) and loss modulus (G’’) were determined as a function of frequency and strain (Figure 2). Time kinetics of both gels were determined at 3 and 5% and it indicated that gelation kinetics increased with time, attaining a plateau after 30 min (Figure 2AB). Thus, both gels are formed instantly and get stiffer with time. Earlier studies have shown that L-amino acid in the N-terminus of an uncapped peptide does not form gel but gels are formed when D-amino acid is present48. Similar to earlier studies, the D-amino acid containing peptide


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formed gels with slightly higher storage modulus but contrary to earlier studies, both chiral capped amino acid containing dipeptides formed stable gels. The amplitude sweep was performed to assess the linear viscoelastic (LVE) range and found to be 0.1%. Beyond LVE, storage modulus decreased abruptly and crossover (structure breakdown) points were observed at 9.89 and 10.17% for NH007 and NH009 (3% each), and 8.62 and 5% for 5% gels. Frequency sweep studies showed that the average G’, depicting elastic characteristics, were around 0.2 and 0.5 MPa for NH007 and NH009 gels (3%), which was higher than the storage modulus reported for other peptide gels22,43,48 (Figure 2E-F). The G’ values were slightly higher for 5% gels, suggesting more rigidity at higher concentrations. The G’ values were always one factor higher than G’’, indicating that both gels are viscoelastic in nature54. Moreover, the G’ values did not change with increasing angular frequency (100 rad/sec), indicating that gels maintain their viscoelastic characteristics even at higher angular frequency, and no cross over points were observed in the frequency range of 0.1-100 rad/sec. The NH007 gels, containing a D-amino acid, showed slightly higher storage modulus than NH009 gels.

Figure 2. Rheology studies. Gelation kinetics of: (A) NH007 and NH009 (3%) and (B) NH007 and NH009 (5%); Amplitude sweep at a constant strain of 0.1%: (C) NH007 and NH009 (3%)


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Biomacromolecules

and (D) NH007 and NH009 (5%); Frequency sweep at a constant strain of 0.1%: (E) NH007 and NH009 (3%) and (F) NH007 and NH009 (5%); Self-healing characteristics at periodic strains of 0.1 and 30% (200 seconds) for six consecutive cycles and constant angular frequency of 10 rad/sec: (G) NH007 (3%) and (H) NH009 (3%). The self-healing characteristics were investigated using rheology by subjecting gels to varying strains of 0.1 to 30%, for a cycle of 200 sec each (Figure 2G-H). Storage modulus (G’) was found to be constant for six consecutive cycles, with little deformation after the first cycle. G’ values were always higher than G’’, suggesting that gels were regaining their structure after getting deformed at higher strain. Gel-to-sol transition was obtained at higher strain of 30%, where G’ decreased to G’’, indicating network destruction55. At this point, G’ decreased to 1572 Pa, suggesting a collapsed state. Both gels quickly recovered their structure, without significant decrease in repeatable cycle, upon decreasing the strain from 30 to 0.1%. Self-healing characteristics of gels were also investigated at macroscopic level by staining NH007 and NH009 gels (3%) with rhodamine and methylene blue dyes (Figure S15). Both gels were stacked together and complete diffusion of dyes was observed from one gel to other after 6 h, leading to the formation of intact gels in about 36 h. These results demonstrated that both gels have the capability to self-heal, as they regain their original structure quickly after being sheared. Thermoresponsive, swelling, and degradation properties of gels were also studied and are included in the supporting information (Figure S14 and S16). The stability of NH007 and NH009 gels, against proteolytic enzymes, were estimated qualitatively using RP HPLC (Figure 3). Both gels were incubated in a mocktail of proteolytic enzymes containing proteinase K22, pepsin30, and chymotrypsin22,30 to mimic in vivo conditions. Proteinase K cleaves the alpha amino acid at the carboxyl end of aliphatic and aromatic amino


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acids; pepsin cleaves the peptide bond preferentially at N-terminal of aromatic amino acid; and chymotrypsin cleaves at the amide bond of C-terminal aromatic amino acids56. Aliquots (100 µl) were withdrawn at 0, 12, 24, and 36 h and analyzed using RP HPLC (Figure 3). Both peptides were found to elute between 16-18 min, and amount of degradation was assessed by estimating the area under the curve. The NH007 peptide exhibited ~23 and 40% degradation in 24 and 36 h (Figure 3 A-D), whereas NH009 exhibited 77 and 94% degradation in the same time (Figure 3 E-H). Thus, NH007 peptide exhibited higher stability against proteolytic enzymes when compared to NH009 peptide. Even though the NH009 peptide underwent 77% degradation in 24 h, the results are still better when compared to L-derived peptides reported in literature, which degraded in 1 h of incubation49. The improved stability of our peptides against proteolytic enzymes was resulting from the presence of D-amino acid and γ- linkage at C-terminal instead of α, which is enhancing the biostability57.

Figure 3. RP HPLC profiles of dipeptide gels (3%) incubated with a mocktail of proteolytic enzymes, containing proteinase K, pepsin, and chymotrypsin (37 oC, pH 7.4): (A) 0 h, NH007; (B) 12 h, NH007; (C) 24 h, NH007; (D) 36 h, NH007; (E) 0 h, NH009; (F) 12 h, NH009; (G) 24 h, NH009 and (H) 36 h, NH009.


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Biomacromolecules

The antibacterial activities of gels, fabricated from NH007 and NH009 (3 and 5%, w/v), were evaluated against gram negative (Escherichia coli and Pseudomonas aeruginosa) and positive (Bacillus subtilis and Staphylococcus aureus) strains, associated with biomaterial-related infections and compared with standard antibiotic, gentamicin sulfate (positive control), used for biomaterial-related infection (Figure 4). Pseudomonas aeruginosa is quiet common in nosocomial infections58, pneumonia, septic shock and urinary tract infections22, whereas Staphylococcus aureus is responsible for hospital-acquired infections19, sepsis, and biomaterialrelated infections59. Escherichia coli cause urinary tract, gastroenteritis and nosocomial infections19. All strains, with the exception of Bacillus subtilis, are known for acquiring resistance over time with repeated usage of antibiotics60.

Figure 4. Antibacterial activities of dipeptide gels, NH007 and NH009 (3%): (A) E. coli; (B) B. subtilis; (C) P. aeruginosa; (D) S. aureus; and (E-H) A-D repeated with 5% gels. Antibacterial bacterial activities of gentamicin sulfate (positive control) are given in the supporting information. Data reported are mean ± SE (n = 3). The NH007 and NH009 gels (3%) exhibited 59 and 66% inhibition against Escherichia coli; 65 and 67% against Bacillus subtilis; 51 and 71% against Pseudomonas aeruginosa; and 40 and


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60% against Staphylococcus aureus, after 18 hours of incubation at 37 oC (Figure 4A-D). The values are comparable with 100 g/ml of gentamicin sulfate for gram negative strains and 10-50 g/ml for gram positive strains (Figure S17). Also, the NH009 gels (3%, w/v) were most active against multi-drug resistant pathogen, Pseudomonas aeruginosa, where it gave 77% inhibition after 36 hours of incubation. As such both gels can be explored against biofilm inhibition and nosocomial-related infections61,62,63. The NH007 and NH009 (5%) gels showed either similar or slightly higher inhibition compared to 3% gels (Figure 4E-H). Overall, the antibacterial data revealed that NH007 (3%) exhibit up to 60% inhibition against gram negative and 65% against gram positive strains, whereas NH009 exhibit up to 78% inhibition against gram negative and 67% inhibition against gram positive strains, under same conditions. Thus, NH009 gels, containing L-amino acid, were slightly more active (3 and 5%) than NH007 gels, containing D-amino acid. A possible reason could be that L-form (natural form) is able to interact more efficiently with cell membrane components of bacteria, compared to D-form. Another reason could be the different secondary conformations of NH007 and NH009, which allows it to interact differently with bacterial cell membrane components, as discussed earlier. Dipeptides (3 and 5 mg) were also evaluated and these exhibited 45-55% inhibition but it is difficult to predict whether the activity is coming from peptide solutions or gels because peptides were first dissolved in DMSO and media was added over it (50:50 aqueous DMSO). Some sort of aggregation cannot be ruled out completely. To the best of our knowledge, gelation behavior and antibacterial activities of diphenylalanine derivatives containing / linkage has not been explored earlier.


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Figure 5. Mechanistic studies using SEM and HR TEM: (A-B) SEM images of P. aeruginosa entrapped in NH007 (0.1%) gel networks; (C-H) HR TEM images of P. aeruginosa: (C-E) control; and (F-H) treated with NH007 gel (3%), showing ruptured outer membrane, shrunken cells and cytoplasmic contents. To gain insight into the mechanism, further studies were carried out using SEM and HR TEM (Figure 5). NH007 (0.1%) xerogel was incubated with Pseudomonas aeruginosa and observed under SEM, which revealed entrapment of bacteria in porous networks of gel (Figure 5A-B). HR-TEM images of untreated group revealed rod shaped cells with intact membrane integrity


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and uniform distribution of cytoplasm (Figure 5C-E), whereas the images for the treated group revealed shrunken cells with collapsed and/or ruptured cell membrane and uneven distribution of cytoplasm (Figure 5F-H). Antibacterial activity of peptide gels could possibly be due to the phenyl group/hydrophobic core of the peptide piercing the bacterial membrane integrity44 or the entrapment of bacteria into the gel network is altering the permeability of bacteria via membrane integration mechanism64.

Figure 6. Fluorescence microscopy images of Pseudomonas aeruginosa incubated with NH007 gel (3%) for 12 hours: (A-C) Control bacterial/non-treated culture; and (D-F) Bacterial culture after treatment. The bacterial solutions were subjected to live-dead cell staining with Syto 9 and propidium iodide, prior to imaging. The live-dead staining of Pseudomonas aeruginosa incubated with NH007 and Staphylococcus aureus incubated with NH009 gels (3%, w/v) were carried out using Syto 9 and propidium iodide. The gels were inoculated with high inoculum of 107 cfu/ml, which was quiet high when compared to infections associated with biomedical devices (

Broad-Spectrum Antibacterial Activity of Proteolytically Stable Self

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