Amphiphilic Peptide Nanorods Based on Oligo ... - ACS Publications

Subscriber access provided by GRIFFITH UNIVERSITY

Article

Amphiphilic peptide nanorods based on oligophenylalanine as a biocompatible drug carrier Su Jeong Song, seulgi Lee, Kyoung-Seok Ryu, and Joon Sig Choi Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00247 • Publication Date (Web): 25 Jul 2017 Downloaded from http://pubs.acs.org on July 26, 2017

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.

Bioconjugate Chemistry 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 36

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

Bioconjugate Chemistry

Amphiphilic Peptide Nanorods Based on Oligo-Phenylalanine as a Biocompatible Drug Carrier

Su Jeong Song†, Seulgi Lee†, Kyoung-Seok Ryu*‡, and Joon Sig Choi*†

†

‡

Department of Biochemistry, College of Natural Sciences, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon 305-764, Republic of Korea

Protein Structure Group, Korea Basic Science Institute, 162 Yeongudanji-ro, Ochang-eup, Cheongwon-gu, Cheongju, Chungcheongbuk-Do 281-19, Republic of Korea

* Authors to whom correspondence should be addressed. Email addresses: [email protected], [email protected] Phone: +82-(42)-821-5489 Fax: +82-(42)-822-7548

ACS Paragon Plus Environment


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 2 of 36

Abstract Peptide nanostructure has been widely explored for drug delivery systems in recent studies. Peptides possess comparatively lower cytotoxicity and are more efficient than polymeric carriers. Here, we propose a peptide nanorod system, composed of an amphiphilic oligopeptide RH3F8 (Arg-His3-Phe8) as a drug delivery carrier. Arginine is an essential amino acid in typical cell penetration peptides, and histidine induces endo/lysosomal escape because of its proton sponge effect. Phenylalanine is introduced to provide rich hydrophobicity for stable self-assembly and drug encapsulation. The self-assembled structure of RH3F8 showed nanorod-shaped morphology, positive surface charge, and retained formation in water for 35 days. RH3F8, labeled with Nile Red, showed high cellar uptake and accumulation in both cytoplasm and nucleus. The RH3F8 nanorods demonstrated negligible cytotoxicity, as shown by

the

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide

(MTT),

lactate

dehydrogenase (LDH), and hemolysis assays. To confirm the efficiency of drug delivery, curcumin was encapsulated in the RH3F8 nanorod system (RH3F8-Cur). RH3F8-Cur showed high encapsulation efficiency (24.63 %) under the conditions of 200 µM curcumin. The RH3F8-Cur retained nano-scale size, and positive surface charge, similar to those of the empty RH3F8 nanorods. RH3F8-Cur displayed a robust anticancer effect in HeLa and A549 cells, and inhibitied the proliferation of cancer cells in a zebrafish model. These results indicate that the RH3F8 nanorods may be a promising candidate for safe and effective drug delivery system.


Page 3 of 36

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


Introduction Peptides are extensively studied as biomolecular carriers for various biomedical applications.1 Peptides and proteins are involved in important biological functions. Any alteration in functional proteins leads to various diseases such as cancer, Alzheimer’s disease, Parkinson’s disease, and sickle cell anemia.2, 3 Therefore, peptides, or oligo-peptides are a promising choice for numerous drug delivery systems.4, 5 Because of their biocompatibility and biodegradability, peptides can be combined with different nanoparticles to form drug delivery systems (DDSs). The cell-penetrating peptides (CPPs) represent such a system.6 Among the 20 amino acids, positively charged lysine and arginine facilitate cell membrane interaction and subsequent internalization of nano-carriers.7 Nevertheless, over the past decade, amphiphilic peptides (APs) have gained increased attention because of their ability to self-assemble into nanostructures, depending on their amino acid sequences.8 Recent studies with APs report several mechanisms for formulation and bio-application.9 APs containing arginine and valine have been evaluated as siRNA delivery carriers.10 Selfassembled APs-based nanostructures have been designed to function as self-adjuvants.11 Assembled structures based on cross-beta APs have shown biodegradability achieved via modification of ester linkages.12 Combining tetra-peptides and carbon chains confirmed that the formulation mechanism is dependent upon amino acid compositions and peptide sequences. Thus, APs have attracted considerable attention for biomedical applications.13, 14 Drug delivery systems (DDSs) based on amphiphilic peptide nanostructures can potentially be applied for safe and effective therapy.15, 16 Amphiphilic peptide nanostructures, consisting of a hydrophobic core and hydrophilic surface, can encapsulate drugs that have poor solubility in water. The encapsulated drug is protected from the aqueous environment and stabilized during the delivery pathway. Thus, encapsulation of labile drugs increases their



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

therapeutic efficacy.17,

18

Peptides have excellent biocompatibility and biodegradability,

which are important factors for developing successful delivery systems.19 Several recently developed polymer-based drug delivery systems are reported to have significant cytotoxicity caused by non-degradability and excessive intracellular accumulation.20 Therefore, numerous studies focused on the development of biodegradable drug carriers.21 Peptides can be degraded via proteolysis by various enzymes in intra-molecular digestion. Thus, peptide nanostructures demonstrate appropriate clearance from the body and reduced cytotoxicity in vitro and in vivo.22 Although amphiphilic peptide nanostructure is highly biocompatible, it has not yet achieved efficient drug delivery compared with that of polymer-based drug delivery systems. To improve the efficacy of amphiphilic peptide nanostructures, their stability, as well as efficiency of drug loading and delivery, needs to be optimized. In this study, we propose a new self-assembled peptide nanorod system, consisting of the amphiphilic peptides, Arg-His3-Phe8 (RH3F8). (Figure 1)

Figure 1. Schematic illustration of RH3F8 nanorods as a drug carrier.


Page 4 of 36

Page 5 of 36

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


The amphiphilic peptide is composed of arginine, histidine and phenylalanine residues. Because of its positive charge, arginine, a common component of cell-penetrating peptides (CPPs), increases cellular uptake.23 Histidine has an imidazole ring that could induce endo/lysosomal escape caused by its proton buffering capacity.24 These two amino acids form a hydrophilic part of the peptide. Because of its hydrophobic characteristics, originating from the side chain containing phenyl groups, phenylalanine constitutes the hydrophobic part. The aromatic carbon ring imparts increased loading efficiency due to its interaction with hydrophobic drugs.25 Moreover, the aromatic carbon ring allows for the formation of a stable peptide nanostructure. In the preliminary study, we designed two types of peptides (RH3F4 and RH3F8) to optimize the number of phenylalanines for the formation of nanostructure. The self-assembled RH3F8 showed nanorod structures, whereas RH3F4 did not form any nanostructure because of its weak self-assembling capacity. Multiple phenylalanines have been applied to form beta-sheet structure.26 The nanorod structure of RH3F8 is based on the formation of beta-sheets and clustering by peptide monomers.27 We then employed numerous assays to validate the ability of the RH3F8 nanostructure to function as a drug carrier. The physical characterization of RH3F8 was analyzed using dynamic light scattering (DLS), field emission-scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), and critical aggregation concentration (CAC). Cellular uptake was examined by confocal microscopy.

Cytotoxicity

was

assessed

using

the

3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH), and hemolysis assays. To evaluate drug delivering ability, the RH3F8 system was challenged with curcumin. The anticancer effect of curcumin, encapsulated by the RH3F8, was analyzed using MTT, detection of apoptosis, as well as reactive oxygen species (ROS) and cancer cell proliferation assays in zebrafish.



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

Results and discussion Preparation and characterization of peptide nanorods Peptide nanorods were prepared using two types of peptides, namely RH3F4 and RH3F8 (Table 1).

Table 1. Physical characterization of the RH3F8 nanorods. Each value presents the average and standard deviation (n=3). To enable the formation of a self-assembled nanorod system, these peptides were designed to possess a hydrophilic head (Arg and His) and a hydrophobic tail (Phe). The Peptides were composed of 1 arginine, 3 histidine and 4 or 8 phenylalanine residues. The Peptides were synthesized using solid-phase peptide synthesis, purified by high performance liquid chromatograph (HPLC), and confirmed using matrix assisted laser mass spectroscopy (MALDI-MS). (Supporting information, Figure S1 A and B) HPLC showed an elution peak at 7 min of retention time. Mass spectrum showed a sharp peak at 1763 m/z, it is similar to expected mass of 1761. These results indicate that the RH3F8 peptide was synthesized successfully. As mentioned previously, arginine, which can enhance cellular uptake, is a representative residue of cell-penetrating peptides,6 while histidine possesses a ‘proton sponge’ effect that can induce endo/lysosomal escape after endocytosis.24 Phenylalanine provides rich hydrophobicity stemming from its phenyl group, which helps generate the formation of a


Page 6 of 36

Page 7 of 36

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


stable nanostructure, and imparts high loading efficiency with respect to hydrophobic drugs. The diameter of the RH3F4 peptide-based formulation showed too small to be measured and possessed a slightly negative zeta potential value. These results suggest that it is difficult to form a nanostructure, using RH3F4, because of the relatively lower hydrophobic property of the four phenylalanine residues. However, the RH3F8 peptide has a nano-scale size of 96.2 nm and shows a positive zeta potential value of 25.9 mV. These results indicate that RH3F8 can self-assemble into a nanostructure with a high positive surface charge density. Because of these stable nanostructures formation, RH3F8 was chosen for further studies to evaluate the characteristics of the peptide as a drug delivery system.



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 2. Physical characterization of the RH3F8 nanorods (A) Size distribution by number. (B) Size change as a function of storage time. (C) Field emission-scanning electron microscopy image. (D) Transmission electron microscopy image. (E) Circular dichroism (CD) spectrum.


Page 8 of 36

Page 9 of 36

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


The size distribution of the RH3F8 nanostructure showed a relatively uniform histogram with an average size of about 100 nm (Figure 2A). The morphology of the RH3F8 nanostructure was confirmed by FE-SEM and TEM (Figure 2C and D). The results of FESEM and TEM indicated that the nanorods possessed long shapes. The results of TEM (Figure 2D) indicated that the RH3F8 nanorod structures had a diameter of 5-10 nm and a length of 10-200 nm. Additional TEM images are presented in the supporting information (Figure S2). The self-assembled structure of the RH3F8 nanorods was analyzed by circular dichroism (CD) spectroscopy (Figure 2E). The RH3F8 nanorods showed characteristics of high beta-sheet (strong negative band at 216 nm, positive band at 199 nm) and low alphahelix organization (weak negative band at 223 nm). The beta-sheet structure is generated by multiple phenylalanines,26 and the alpha-helix structure is based on arginine and histidine residues with high alpha-helix forming preference28. So, RH3F8 nanorods could be organized by the combinational effects of the peptide’s beta-sheet and alpha-helix formation tendency. DLS was used to examine the mean diameter of the RH3F8 rods or aggregates of small rods. The stability of the RH3F8 nanorods was also confirmed using DLS to monitor the diameter of the nanorods after storage for 35 days at room temperature (RT). The RH3F8 nanorods maintained their nanoscale-size, and diameter of less than 200nm, for up to 35 days. (Figure 2B) These results indicate that the RH3F8 nanorods have good stability because of the rich hydrophobic core consisting of phenylalanine. Overall, the RH3F8 nanorods can potentially be applied as drug carriers because of their stable formation of nanoparticle.

Quantification of critical aggregation concentration (CAC) The critical aggregation concentration (CAC) of the RH3F8 nanorods was determined using the Nile Red hydrophobic fluorescence probe. (Figure 3) This method is based on the spectral shift and increases the fluorescence intensity of Nile Red in a hydrophobic environment.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

Below 0.05 mg/mL of the RH3F8 nanorods, Nile Red showed a rapid increase in fluorescence intensity and a reduced maximum emission wavelength. This result indicates that below this value of CAC, the nanorods were fractured, and the exposed hydrophobic portions of the nanorods provided Nile Red with a hydrophobic environment, resulting in increased fluorescence intensity and a shift in emission wavelength. These results indicate that the CAC value of RH3F8 was 0.05 mg/mL. Moreover, CAC value of RH3F8 means that nanostructure is based on self-assemble by amphiphilic peptide monomer.

Figure 3. Critical aggregation concentration of the RH3F8 nanorods. (A) Fluorescence spectra. (B) Calculation of CAC using florescence intensity and maximum emission wavelength with respect to the concentration of RH3F8.

Cellular uptake assay Cellular permeability is related to the arginine-modified nanostructure, shape of the nanorods, and stability imparted by phenylalanine. The Nile Red-tagged RH3F8 nanorods were used to examine the cellular uptake in HeLa cells. After 6 h incubation time, cells, treated with Nile Red-loaded RH3F8 nanorods, were observed under florescence and confocal microscopy. As shown in Figure 4A and B, most of the cells show fluorescence,


Page 10 of 36

Page 11 of 36

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


demonstrating the high cellular uptake of the RH3F8 nanorods. To understand the spatial details of the uptake pattern, the cells were analyzed using confocal microscopy. (Figure 4C) The confocal micrographs showed robust florescence signaling in the cytoplasm and nucleus. This indicates that RH3F8 was readily taken up by the cells, which is an important quality in a drug delivery carrier.

Figure 4. Cellular uptake assay of the Nile Red-tagged RH3F8 nanorods. (A) and (B) Fluorescence microscopy images. (C) Confocal microscopy images.

Cytotoxicity of the RH3F8 nanorods



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

The cytocompatibility of RH3F8 was confirmed using MTT and LDH assays in HeLa cells. Additionally, RH3F8 was compared with b-PEI, a representative polymeric cationic carrier. The MTT assay indicated that b-PEI showed high toxicity below 0.025 mg/mL, whereas RH3F8 demonstrated negligible cytotoxicity at 0.1 mg/mL. (Figure 5A) The LDH assay was used to examine membrane damage caused by RH3F8. (Figure 5B). At concentrations of up to 0.1 mg/mL, b-PEI induced an approximately 60 % release of LDH, indicating considerable membrane damage. However, the RH3F8 nanorods induced negligible LDH release at all the tested concentrations. These results indicate that the RH3F8 nanorods possess excellent biocompatibility in vitro despite their positive surface charge, and show that by tailoring the material specificity of peptides, nanorod systems can minimize cellular damage during the delivery pathway.

Hemolysis assay Hemolysis refers to the damage caused to red blood cells (RBCs) membranes upon interaction with certain molecules. Negligible hemolysis is advantageous in biomedical applications. Therefore, it is crucial to evaluate the hemolytic activity biomaterials intended for drug delivery applications. We assessed the biocompatibility of the RH3F8 nanorods by examining hemolysis in human RBCs. (Figure 5C) While b-PEI showed an approximately 10% hemolytic activity, the RH3F8 nanorods dispalyed no hemolytic activity compared with that in the untreated control.


Page 12 of 36

Page 13 of 36

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 5. Cytotoxicity of the RH3F8 nanorods. (A) MTT assay. (B) LDH assay. (C) Hemolysis assay. Each value presents average and standard deviation (n=3).

Characterization of curcumin-loaded RH3F8 nanorods Curcumin, an anticancer drug, was loaded into the RH3F8 nanorods to determine their suitability as a drug carrier. For optimizing encapsulation conditions, curcumin was prepared at various concentrations (50-250 µM) and encapsulated in RH3F8 using dialysis. The curcumin-loaded RH3F8 nanorods displayed a larger diameter compared with that of the empty RH3F8; this was due to the curcumin encapsulated inside the hydrophobic core of the nanorods. The zeta potential of curcumin-loaded RH3F8 had high cationic charge of approximately 30 mV, which was similar to that of the empty RH3F8. Increases in the concentration of added curcumin altered the loading efficiency (LE) and encapsulation



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

efficiency (EE). (Table 2) Using 50 and 100 µM of added curcumin, LE and EE could not be determined because the curcumin was not detected by HPLC; this was caused by the overly low concentration of curcumin. At condition of concentration above 200 µM, LE was maintained, while EE decreased at the concentration of 250 µM. Based on these results, 200 µM was selected as the optimum concentration for encapsulating curcumin in the RH3F8 nanorods. Thereafter, curcumin-loaded RH3F8 under condition of 200 µM curcumin nanorods (RH3F8-Cur) were used for assaying the anticancer effects of this system.

Table 2. Physical characterization of the RH3F8-Cur nanorods with respect to the concentration of curcumin. Each value represents average and standard deviation (n=3).

The physical properties of RH3F8-Cur were characterized by measurements of absorbance and FE-SEM (Figure 6). As shown in Figure 6A, we analyzed the absorbance spectra of empty RH3F8 and RH3F8-Cur. Both samples showed peptide absorbance at 220 nm and phenylalanine absorbance at 280 nm. However, only RH3F8-Cur showed curcumin absorbance at 450 nm. These results indicate that curcumin was encapsulated into the peptide nanostructure. The FE-SEM and TEM image of RH3F8-Cur showed a morphology similar to that of the empty RH3F8. (Figure 6B and C).


Page 14 of 36

Page 15 of 36

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 6. Physical characterization of the RH3F8-Cur nanorods. (A) Absorbance spectrum of RH3F8-Cur and empty RH3F8 (B) Field emission-scanning electron micrograph (C) Transmission electron micrograph of RH3F8-Cur.

In vitro drug release Drug release by RH3F8 was investigated by HPLC using buffers with different pH at various time points. As shown in Figure 7, the pH of 5.5 and 7.4 induced a continuous release of curcumin over 168 h. During the first 12 h, up to 15 % curcumin was rapidly released. After 12 h, the cumulative released drug increased by 3-5% per 24 h. This sustained release pattern



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

was maintained for 168 h. The stable structure, imparted by phenylalanine, may have induced sustained release. Such sustained release maintains a constant level of a drug in cells and tissues, and is, therefore, desirable in effective DDSs.30 Furthermore, the RH3F8 nanorods increased the release rate under acidic conditions (pH 5.5). The peptide monomers in the nanostructure were fractured by hydrolysis under acidic conditions, thereby facilitating drug release. Because tumor tissue usually has an acidic pH, caused by lactic acid from anaerobic glycolysis31, this rapid release rate under acidic conditions can be highly advantageous in a drug carrier intended for cancer therapy. Although the maximum released drug percentage of the drug was around 40 %, the RH3F8 nanorods are likely to show a high release rate after intracellular uptake because of enzymatic degradation.

Figure 7. Drug release from RH3F8-Cur at pH 5.5 and 7.4.


Page 16 of 36

Page 17 of 36

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


Anticancer activity of RH3F8-Cur To confirm anticancer effect of RH3F8-Cur, we performed cell viability, ROS, and apoptosis assays in HeLa and A549 cell lines. Curcumin from Curcuma longa exhibits anticancer and antiseptic activity with low cytotoxicity.32, 33 Therefore, curcumin is model anticancer drug and has been investigated for clinical application. A combination of peptides and curcumin may be a promising therapy system consisting of natural substances. To examine the anticancer effect at various concentrations of curcumin, cells were treated with a curcumin control, RH3F8-Cur, and empty RH3F8. (Figure 8) On day 1, 5 µM of RH3F8Cur showed scant activity in HeLa and A549 cell lines. However, on day 2, RH3F8-Cur treatment in HeLa cells presented higher anticancer effect at all concentrations of 1-5 µM. At the concentration of 5 µM RH3F8-Cur, significant differences in cell viability were observed in RH3F8-Cur-treated HeLa cells compared with that of HeLa cells treated with the curcumin control (p

Amphiphilic Peptide Nanorods Based on Oligo ... - ACS Publications

Recommend Documents