Evaluation of Biocompatibility of the AC8 Peptide ... - ACS Publications

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Evaluation of Biocompatibility of the AC8 Peptide and Its Potential Use as a Drug Carrier Sheva Naahidi,†,‡ Yujie Wang,§ Man Zhang,§ Rong Wang,§ Mousa Jafari,†,‡ Yongfang Yuan,*,§ Brian Dixon,∥ and P. Chen*,†,‡ †

Department of Chemical Engineering, University of Waterloo, 200 University Avenue West, Waterloo, Ontario N2L 3G1, Canada Waterloo Institute for Nanotechnology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario N2L 3G1, Canada § Department of Pharmacy, Shanghai Third People’s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 201999, China ∥ Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario N2L 3G1, Canada ‡

ABSTRACT: Peptide-based nanoparticles have emerged as promising drug delivery systems for targeted cancer therapy. Yet, the biocompatibility of these nanoparticles has not been elucidated. Here, the in vitro biocompatibility and toxicity and in vivo immunocompatibility and bioactivity of the self/coassembling peptide AC8 in its nanoparticle form are evaluated. AC8 showed minimal hemolytic activity (5%) and did not cause aggregation of red blood cells. The in vitro assay revealed that AC8 did not activate the complement system via the classical or alternative pathway but did activate the lectin pathway to a small extent. However, AC8 showed no C3a and C5a anaphylotoxin activation suggesting that complement activation did not proceed to the later, inflammatory, stages. The in vivo immune response assay showed that administration of AC8 to BALB/c mice had no effect on the weight of immune organs or body weight of mice at doses less than 0.1 mg/kg. This peptide also did not have any effect on the expression of CD3+ T-cells and natural killer (NK) cells, the ratio of CD4+/CD8+ T-cell, and the proliferation of B-cells. These results suggest that AC8 can be a potential carrier candidate for drug delivery. KEYWORDS: biocompatibility, cytotoxicity, complement system activation, self-assembling peptides drug delivery system

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studies of nanoparticles.14 Additionally, recent ongoing research, for instance work by Liangfang Zhang’s group, has focused on using or mimicking erythrocytes as drug delivery system.15 A number of mechanisms for drug-mediated hemolysis have been recommended, yet the true mechanism is yet to be identified. It is now well-known that surface properties (especially surface charge) play an important role for nanoparticles and can directly damage erythrocyte membranes. For instance, in the presence of certain concentrations of unprotected primary amines (positive charge), red blood cell damage was observed on the surface of poly amidoamine, carbosilane, polypropyleneimine, and poly lysine.16−21 Another important aspect that greatly affects the delivery of drugs is their uptake by immune cells and clearance from intended target sites (immunocompatibility). It is now wellknown that the complement system, which plays a crucial role in this process, consists of more than 20 different plasma proteins and protein fragments as inactive zymogens in the

INTRODUCTION Since the first application of biomaterials as conventional therapeutics, there has been tremendous interest in materials capable of sustained macromolecular delivery.1,2 As research has progressed, modern pharmacotechnology has evolved such that the main goal now is to improve the therapeutic efficacy and controlled targeted release of drugs by using a variety of nanoparticles as carrier systems.3−9 Recent years have witnessed a tremendous expansion of the use of nanoparticles for drug delivery applications.10−13 Given their advantages and potential, the necessity of understanding biocompatibility of nanoparticles is undeniable, as bioincompatibility is the one drawback these systems can face. Hence, the study of nanoparticle interaction with blood components and immune cells, as well as its toxic effects, should be seriously considered in order to translate the use of nanoparticles from research to preclinical or clinical studies. Hemolysis, immune system responses such as complement activation, and cytotoxicity are the main concepts considered for nanoparticle biocompatibility. We know that erythrocytes occupy a larger volume fraction of the blood than leukocytes and lysing of excessive number of red blood cells can lead to anemia; therefore, in vitro examination of hemolysis is an instrumental part of preclinical © XXXX American Chemical Society

Received: February 9, 2014 Revised: June 18, 2014 Accepted: July 23, 2014

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dx.doi.org/10.1021/mp5001185 | Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Molecular Pharmaceutics

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blood.22,23 Complement system activation results in the activation of the cell-killing membrane attack complex (MAC).24 There are three major complement pathways that may become activated: the classical, alternative, and lectin pathways. One pathway that was recently discovered but not fully understood is the ficolin innate immune recognition.25 Ficolins are oligomeric defense proteins that bind to acetylated molecules as well as neutral carbohydrates that trigger the lectin pathway.26,27 In the classical pathway, the C1q protein recognizes and attaches to antibody bound to the pathogen, C1r and C1s, through electrostatic and hydrophobic interactions. The lectin pathway is activated via mannanbinding lectin (MBL) binding to neutral sugar residues, while the alternative pathway is started by C3b protein binding to the hydroxyl or amino groups on a pathogen’s surface.28−30 Recent studies have recognized the importance of nanoparticle surface charge in activation of the complement system. For instance, polypropylene sulfide nanoparticles, lipid nanocapsules, cyclodextrin-containing polycation-based nanoparticles and polystyrene nanospheres have shown that charged nanoparticles activate the complement system more efficiently than neutral nanoparticles.31−33 In addition to possessing immunocompatibility, peptide carriers such as AC8 should show low bioactivity in other areas, such as no or low hemolysis. Indeed the ability of a nanoparticle to evade immune recognition represents an area of interest in the field of drug delivery. One of the promising approaches toward this goal has been through the development of the self-assembled peptide nanomaterials due to their perceived biocompatibility and structural diversity. A new, promising class of self-assembling peptides is the self-assembling ionic-complementary peptides. As such, the self-assembling peptide AC8 nanoparticle is a short (eight amino acids long) peptide with the sequence AcFEFQFNFK-NH2. The capability of the AC8 as the selfassembling ionic-complementary peptides to stabilize the hydrophobic anticancer drug ellipticine in aqueous solution has been already investigated.34 The schematic of the molecular structure of this peptide is shown in Figure 1.

delivery applications. All of this together makes AC8 peptide a candidate to be evaluated as a carrier for drug delivery. Here, we report the systematic investigation of the in vitro biocompatibility and in vivo immunocompatibility and bioactivity properties of self-assembling ionic-complementary peptide AC8. We also show the potential ability of this peptide to be utilized as a carrier for the delivery of the hydrophobic anticancer drug pirarubicin. Nuclear uptake of AC8−pirarubicin complexes by A549 cells was shown by confocal microscopy. Additionally, the toxicity of AC8 was evaluated on A549 lung cancer cell line due to their availability to our lab. However, toxicity was also examined to breast cancer cell line MC7 with similar results (less than 3−5% differences), which will be presented in another journal publication. Finally, we demonstrated that AC8 peptides had favorable biocompatibility and poor bioactivity in vivo.

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EXPERIMENTAL SECTION In Vitro. Peptide Preparation. The peptides AC8 (purity >98%) were purchased from CanPeptide Inc. (Montreal, Canada) and used without further purification. The primary structure of AC8 is FEFQFNFK and its secondary structure is beta-strand. The N-terminus and C-terminus of the peptides were protected by acetyl and amino groups, respectively. The peptide AC8 has been patented by World Intellectual Property Organization (Patent Number: 2009026729). Cytotoxicity Assay. Nonsmall cell lung carcinoma cells A549 were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). The MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) cell proliferation and viability assay kit (TOX1 from Sigma-Aldrich, Oakville, ON, Canada) was used to perform cytotoxicity assays. A549 cells were seeded onto 96-well plates at a density of 104 cells per well and treated 24 h later. Twenty microliters of the AC8 peptide was diluted in 180 μL of growth media (DMEM containing 10% FBS) and added to each well. DMEM served as the blank, and untreated cells served as the control. The plates were incubated for 24 and 48 h prior to performing the cell viability assay. Then, 100 μL of MTT substrate was added to each well and incubated for an additional 4 h at 37 °C in the dark before the addition of 100 μL solubilization. Absorbance was measured at a wavelength of 570 nm using a microplate reader (model 550; Bio-Red, CA, USA). Cell survival was expressed as a percentage of the absorbance value determined for control cultures using the following calculation: viability = (sample absorbance − blank absorbance) /(negative absorbance − blank absorbance)

Figure 1. Schematic of the chemical structure of AC8. Abbreviations: AC8, Ac-FEFQFNFK-NH2.

Hemolysis Studies. Hemolysis experiments were carried out by using approximately 5 mL of fresh human anticoagulated blood. The blood was centrifuged at 1500 rpm for 10 min and washed three times by adding Dulbecco’s Phosphate Buffered Saline (DPBS). After washing, red blood cells (RBCs) were isolated, suspended in DPBS to the original volume, and then diluted further with DPBS to make a 5% hematocrit solution. Varying concentrations of the peptide in 800 μL of DPBS were added to the 200 μL of RBCs. DPBS and water served as negative and positive controls, respectively. After incubation for 2 h, all of the samples were then centrifuged after which the supernatant was removed and placed into a clean cuvette. Subsequently, the absorbance was measured at 541 nm. The hemolysis in the water solution was considered to be 100%,

It is worth mentioning that AC8 peptides comprise all complementary amino acid pairs in the sequence and thus the name, amino acid pairing peptide (AAP). This peptide selfassembles into β-sheet-rich nanofibers. As discussed in Fung et al.,34 a model formulation of AAP peptide that has been proposed for AC8 via amino acid pairing has a stronger nanostructure, lower critical aggregation concentration (0.1 mg/mL), and less charged residues than most ionic complementary self-assembling peptides. However, the biocompatibility properties of AC8 have not yet been explored, which is a necessary first step in an investigation of drug B



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Fetal Bovine Serum (FBS) was from Gibco-Invitrogen Corporation (NY USA). MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-CD3 PerCP, anti-CD4 FITC, anti-CD8 FITC, antipan NK PE, and isotype-specific antibodies were purchased from eBioscience incorporation (San Diego, CA, USA). Animals. Healthy male BALB/c mice, 4 weeks-old and weighing 10−12 g, were obtained from B&K Universal Group Limited (Shanghai, China). Mice were maintained under a 12 h light/dark cycle at 25 °C and a humidity of 60 ± 10%. Experiments were performed in accordance with institutional and governmental regulations on the use of experimental animals. Animals in Immune Activity Experiments. BALB/c mice were separated into five groups: normal group (N group); model group (M group); thymalfasin group (Th group); AC8 low group treated with low AC8 dose (AC8 L); and AC8 high group treated with high AC8 dose (AC8H) as shown in Figure 2. N group mice were injected intraperitoneally with sterile

while the hemolysis in the DPBS solution was taken as 0%. The results were thus expressed as percentage hemolysis using the following calculation: %hemolysis = 100 × (absorbance of sample − absorbance of negative control) /(absorbance of positive control − absorbance of negative control)

Complement System Activation Studies. In order to determine the amount of complement activation caused by the AC8 peptides in vitro, the active forms of four complement products (C4d, SC5-b9, Bb, and MBL) were analyzed using enzyme-linked immunosorbent assay kits from Quidel Corp, (San Diego, CA, USA) and ALPCO Diagnostics kit (Salem, MA, USA). Peptide samples were incubated with human serum at a volume ratio of 1:5 in a shaking incubator (100 rpm) for 60 min at 37 °C. It is worth to mention that three time points were measured (1−3 h). However, there was no increase at later time points (data are not shown).The samples were then diluted further with Sample Diluent according to the manufacturer’s instructions to determine the amount of C4d, Bb, SC5b-9, or mannan-binding protein that was formed by the complement system during the incubation. The C4d immunoassay measures the amount of active C4 fragments present in human serum, which indicates the activation of the classical pathway. The Bb Plus immunoassay assesses the amount of Factor B cleavage that occurs, providing a measure of the activation of the alternative pathway. SC5b-9 measures the terminal complement complex, which is formed by the classical, alternative, or lectin pathways. MBL is the protein indicator that the lectin pathway has been activated. Zymosan (a known complement activator) was used as a positive control, and untreated human serum was used as a negative control: both were assayed in the same way as all the other samples. The results were then read at 450 nm (FLUOstar OPTIMA, BMG, NC). Standard curves were made for quantification of complement activation products by using the assigned concentration of each individual standard supplied by the manufacturer and validated. The slope, intercept, and correlation coefficient of the derived best fit line for SC5b-9, Bb, C4d, and MBL standard curves were within the manufacturer’s specified range. The efficacy of the treatments was determined by comparison with baseline levels using paired t-test. Anaphylatoxin Studies. In order to assess the amount of C3a and C5a, samples were incubated with human serum in a 1:5 ratio in a shaking incubator (100 rpm) for 60 min at 37 °C. The samples were then tested according to manufacturer’s instructions for the enzyme-linked immunosorbent assay kits from Quidel Corp. (San Diego, CA, USA). The results were then read at 450 nm (FLUOstar OPTIMA, BMG, NC). Since C3a is a factor produced by the activation of the classical, alternative, or lectin complement pathways, Zymosan was used as the positive control and human serum was used as the negative control. In Vivo. Reagent and Chemicals. Cyclophosphamide (CY) was purchased from Shanxi Pude Pharmaceutical Co., Ltd. (Shanxi, China). Thymalfasin (Th) was purchased from Hainan Shuangcheng Pharmaceuticals Co., Ltd. (Hainan, China). Dulbecco’s modified eagle medium (DMEM) was purchased from Gibco-Invitrogen Corporation (Carlsbad, CA, USA).

Figure 2. Method of separating Blab/c mice into five groups. Normal group (N group); model group (M group); thymalfasin group (Th group); AC8 low group treated with low AC8 dose (AC8 L); and AC8 high group treated with high AC8 dose (AC8H).

physiological saline. M group, Th group, AC8L group, and AC8H group mice were injected intraperitoneally with a single dose of cyclophosphamide, CY (100 mg/kg), in the morning of the first 2 days. These four mice groups received the following material once daily afternoon for 7 consecutive days: M group, i.p. sterile physiological saline; Th group, i.h. 10 mg/kg Th as positive controls; AC8L group, i.p. 2.5 mg/kg AC8; AC8H group, i.p. 10 mg/kg AC8. After 8 days, all the mice were humanely sacrificed according to institutional protocols. Body Weight and Index of Immune Organs. During the experiment, the body weights of BALB/c mice were measured every day, and the change of body weight was detected as relative growth rate. After 8 days, all the animals were sacrificed and the thymus and spleen were promptly removed and weighed. The indexes of thymus and spleen were expressed as the percentage (%) of thymus and spleen wet weight versus body weight, respectively. relative growth rate = C

(maximum body weight − basic body weight) basic body weight dx.doi.org/10.1021/mp5001185 | Mol. Pharmaceutics XXXX, XXX, XXX−XXX


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Flow Cytometry. After mice were anesthetized by i.p. pentobarbital injection (50 mg/kg), blood was taken from orbital sinus, collected in heparinized tubes and analyzed within 2 h after blood sampling. Whole blood was labeled directly with conjugated monoclonal antibodies, including anti-CD3 PerCP, anti-CD4 FITC, anti-CD8 FITC, antipan NK PE, and isotypespecific antibody controls according to the manufacturer’s instructions. Cells were fixed in 2% para-formaldehyde and stored in the dark before analysis using Beckman Coulter FACS flow cytometer. Flow cytometry data were processed by Cell Quest software (Beckman Coulter). LPS-Induced Splenocyte Proliferation Assay. Mice were sacrificed and spleens were removed aseptically. Cell suspension was prepared by squeezing and flushing the spleens through a gauze filter. After centrifugation at 1000 rpm, 4 °C for 5 min, erythrocytes were lysed by hypotonic solution and the cell pellets were washed twice with DMEM. The cells were resuspended, and the cell concentration was adjusted with DMEM to 5 × 105 cell/mL. The splenocyte proliferation assay was measured according to the MTT method. Two hundred microliters of splenocytes suspensions (5 × 105 cells/mL) was added into a 96-well plate. LPS (5 μg/mL) was used as a mitogen, and 0.1% dimethyl sulfoxide (DMSO) in DMEM was used as control. After incubation at 37 °C in humidified 5% CO2 for 48 h, 20 μL/well of MTT (5 mg/mL) and 40 μL of DMEM were added into each well. Cells were further incubated with MTT for 4 h. The insoluble formazan was dissolved in DMSO (100 μL/well) solution. The plate was further incubated for 5 min at room temperature. The absorbance was measured on a microplate reader (model 550; Bio-Red, CA, USA) at a wavelength of 570 nm using a 96-plated reader (Bio-Red, CA, USA). The percentage of proliferation was calculated by the following equation: stimulation index =

Figure 3. Cytotoxicity of AC8 was measured against the lung cancer cell line A549. Three independent experiments were performed for each data point. AC8H represents the group treated with high AC8 dose (0.1 mg/mL) and AC8L represents the group treated with low AC8 dose (0.025 mg/mL). Error bars represent standard error of mean (SEM) of three independent experiments. *p < 0.05 vs nontreated group in 24 h; #p < 0.05 vs nontreated group in 48 h.

0.03, respectively). In addition, the cellular viability of AC8 in the low dose group (0.025 mg/mL) at 48 h was also significantly lower than the nontreated cell group (0.76 ± 0.07). However, the therapeutic dose of AC8 at 24 h did not show any impact on cellular viability. These results suggested that AC8 at therapeutic concentration was not toxic, whereas at high concentrations it significantly affected cell viability of A549 cells compared with nontreated cells. Although with increasing incubation time and dose, the antiproliferative effect of AC8 became more significant (Figure 3), it is safe in low dose as a drug carrier. Cytotoxicity studies in a range of concentration of this peptide in other cell lines have been already reported.34 Although, it is preferable for carriers in a drug delivery system to have inert biological activity, it would be of interest if AC8 would contain antitumor activity similar to the drug for pharmacological research. In this work, it was concluded that in addition to its safety at therapeutic concentration within 24 h, as a drug carrier, when overdosed, AC8 demonstrated antitumor activity at high doses. Hemolysis. Hemolysis is the breakage of the RBC’s membrane, causing the release of hemoglobin and other internal components into the surrounding fluid; therefore, it is a crucial factor in evaluating the biocompatibility of biomaterials. It is also a common occurrence seen in serum samples and may compromise the laboratory’s test parameters. When determining the hemolytic activity, a ratio of 5% or less hemolytic activity is considered biocompatible.35 In the present study the hemolytic potential of treated human blood with the self-assembling peptide AC8 was investigated to see the possible hemolytic effect on RBC. As shown in Figure 4A, the results of the in vitro hemolytic assay showed that the self-assembling peptide AC8 has no hemolytic activity within the therapeutic range of concentrations. However, at concentration as high as 0.1 mg/mL, AC8 showed some hemolytic activity (data is not shown). Nevertheless, in terms of aggregation, the result was the same as the normal saline, a negative control (Figure 4C). There was no RBC aggregation with the AC8 peptides, at neither high nor

OD(sample) − OD(control) × 100 OD(control)

Nuclear Localization Track of Pirarubicin by Confocal Microscopy. Subcellular localization of peptide was analyzed by confocal microscopy (Zeiss LSM 510, Canada) using a 63× objective and a slice thickness of 300 nm. A549 cells were seeded in 35 mm glass bottom dishes (MatTek, MA, USA) at a density of 1 × 105 cells per mL and allowed to attach for 24 h. The cells were treated with peptide and controls for 20 min and washed three times with warm PBS and fixed with 4% PFA for 20 min, followed by washing with PBS and adding DAPI (Sigma-Aldrich, Oakville, Canada) to stain the cell nuclei. DAPI was excited at 405 nm and was visualized by a 420−480 nm band path filter. Statistical Analysis. Statistical analysis was carried out using ANOVA or Student’s t test where applicable. Relative growth rate was analyzed by nonparametric tests (Mann−Whitney U). In vitro experiments were repeated at least three times. Differences were considered significant when the p value was less than 0.05.

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RESULTS AND DISCUSSION In Vitro Results. Cytotoxicity of Peptide. The cellular toxicity of AC8 was determined by MTT assay as shown in Figure 3. The cellular viability in the high AC8 dose group (0.1 mg/mL) at both 24 and 48 h time intervals were significantly lower than the nontreated cell group (0.88 ± 0.03 and 0.62 ± D



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Figure 4. (A) Hemolytic activity of AC8. AC8H represents the group treated with high AC8 dose (0.1 mg/mL) and AC8L represents the group treated with low AC8 dose (0.025 mg/mL). Error bars represent SEM of three independent experiments. (B) Detection of RBC aggregation by light microscope (400×). (C) RBC in normal saline as a negative control (400×).

Complement System Activation. The complement system, as one of the key effector mechanisms of humoral and innate immunity, consists of several, generally inactive, serum and cell surface proteins, which are activated only under particular conditions to generate products that mediate a variety of effector functions. The classical pathway uses a C1 plasma protein to detect IgM, IgG1, or IgG3 antibodies attached to the surface of a microbe or other structures. The alternative pathway is elicited by the spontaneous cleavage of the thioester

low concentration (Figure 4B). Therefore, while both monomers and polymers are present in samples, aggregates are not. This is important because aggregates are probably more likely to induce immune responses if they get inside the cell; HSPs will bind them and induce responses. These observations confirm that AC8 is not hemolytic within the therapeutic range of concentrations and do not cause erythrocyte aggregation within the low and high range of concentrations. E



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bond in C3 (“C3-tickover”) or when the internal thioester bond in the α-chain of nascent C3b go through nucleophilic attack by the direct detection of a microbial surface structure rich in nucleophilic groups (particularly hydroxyl- and aminorich surfaces).36,37 The lectin pathway is activated by an MBL plasma protein, which identifies terminal mannose residues on microbial glycoproteins and glycolipids. Therefore, not surprisingly, the majority of the consequences of complement activation results from the pharmacological effect of some of the activated components. Accordingly, clarification of the potential to activate complement can be used as one decisive factor in testing the biocompatibility of a variety of nanoparticles. Different types of nanoparticles, especially polymers, have been used in medicine for targeted or controlled release of various drugs and diagnostic agents, of which many have been assessed for complement activation.38−41 Here, we report the effect of AC8 peptide on the three major complement pathways. After incubation with human serum, Bb and C4d concentrations were measured to assess the complement system activation via alternative and classical pathways. Zymosan (a known complement activator) was used as a positive control, and untreated human serum was used as a negative control: both were assayed in the same way as all the other samples. As shown in Figure 5A, complement activation via the classical pathway increased in small amount compared with serum, as negative control; but enhancement was not significant for AC8 peptides as determined by two-sample t test (p < 0.05). Therefore, there was no classical activation upon exposure to AC8 peptide. Classical activation can be due to binding of C1q (main recognition unit of classical pathway) to the peptide via electrostatic or hydrophobic interactions.42 The MBL pathway was assessed using a kit that measures the amount of functional MBL oligomer remaining in the solution. When the pathway is activated, the oligomer will bind to the surface of the invading particle rendering it dysfunctional. After incubating the samples with human serum for an hour to allow time for the oligomer to bind to the surface of the peptides and peptide complexes, the amount of remaining functional MBL oligomer was measured. Figure 5C shows that for the positive control, there is very little functional MBL oligomer remaining, indicating that Zymosan activated the lectin pathway. This means that in the graph there is a shorter bar for the positive control (Zymosan) in comparison with serum (negative control) in which all the functional MBL oligomer remains. For our samples, there is activation for AC8 peptides, although it is less than the positive control (Figure 5C). While there was lectin pathway activation, nevertheless it did not reach the “threshold” level of activation required for the next step of complement activation because there was no significant Bb activation (Figure 5B). Anaphylotoxin Activation. Upon classical, lectin, or alternative pathway activation, the C3 protein is split into two fragments, C3b and a potent anaphylotoxin C3a. Therefore, the presence of C3a in a test sample proves that peptide nanoparticles can activate complement by one or both pathways. However, C3b can participate in the formation of a new enzyme, the C5 convertase, which cleaves C5 to C5b, which drives the rest of the common terminal pathway, and C5a, also a very potent anaphylotoxin. One outcome of C5b production is cell killing by forming a MAC. However, a large amount of the C5b generated with in vitro samples is diverted to the fluid phase by reacting to S protein to form soluble SC5b-9, which can be measured as proof of terminal pathway

Figure 5. Complement activation of classical (A), alternative (B), and mannan binding lectin (C) pathways of the AC8s (60 μg/mL). Error bars represent SEM of three independent experiments. * denotes statistically significant difference compared to the negative control (serum), as determined by two-sample t test (p < 0.05).

activation. An important point is that for each mole of detected SC5b-9, an equal number of moles of C5a are generated. C5a is a small, sticky and exclusive molecule with a short half-life. In contrast, SC5b-9, as a marker for C5 cleavage, is an extremely stable soluble macromolecular complex and can be used as a F



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Figure 6. (A) Complement activation of the terminal pathway by the AC8 (60 μg/mL). Bars represent the mean concentration of SC5b-9, a biomarker of C5a through both the classical and alternative pathways. (B) Anaphylatoxin C3a assay comparing the concentration of C3a in AC8 treated samples, and positive (Zymosan) and negative control (Human serum). Error bars represent SEM of three independent experiments. * denotes statistical significance compared to the negative control (serum), as determined by two-sample t test (p < 0.05).

Figure 7. Confocal microscopy of A549 cells treated with AC8−pirarubicin complexes confirming the presence of pirarubicin inside the nucleus. Violet nucleus (C) implies the colocalization of pirarubicin (red) (B) and DAPI (blue) (A) at the same confocal layer.

marker for C5a anaphylotoxin generation. Accordingly, to further assess the in vitro extent of NP-induced activation of complement, SC5b-9, C3a, and C5a were evaluated. SC5b-9 concentration was measured after incubation with human serum. As shown in Figure 6A, it was found that AC8 formulations did not activate the terminal pathway of complement system with respect to human serum (negative control) and were significantly less than Zymosan (positive control). Similar results were observed, as expected, in regards to C3a and C5a anaphylotoxin (Figure 6B). Since the C5a anaphylotoxin results were similar to C3a, data are not shown. In summary, the complementary amino acid pairing peptide AC8 (AAP8) did not activate the complement system in vitro, highlighting the potential of this peptide to be used as a drug delivery system. Nuclear Localization Track of Pirarubicin by Confocal Microscopy. Confocal fluorescent microscopy confirmed the nuclear uptake of anticancer drug pirarubicin (tetrahydropyranyladriamycin) when complexed with the peptides as shown by its colocalization with the nucleus (Figure 7A−C). Pirarubicin’s hydrophobicity and fluorescence characteristics make it a good candidate to be used for our drug delivery

system using AC8 as its carrier. The peptide mediated the nuclear uptake of pirarubicin, represented as violet areas (merged), as the result of localization of pirarubicin (red) in stained nuclei (blue). In addition, confocal microscopy of A549 cells treated with just pirarubicin lacking AC8 peptide (control) has been also investigated. Indeed, there were just a few red dots in nucleolus indicating significantly reduced uptake of pirarubicin to the nucleus in comparison with AC8−pirarubicin complex. This highlights the necessity of choosing an efficient compatible carrier for drug delivery. It is worth mentioning that the range of concentrations used in all conducted experiments highly exceeds the dose considered for therapeutic purposes (i.e., 0.05). However, the thaymalfasin group significantly increased the percentage of CD3+ cells (p < 0.05) in comparison with the model group. It is worth mentioning that thymalfasin (thymosin-alpha 1) is a 28-amino acid peptide that is an immunomodulating agent able to stimulate stem cells and also increases production of NK, CD4, and CD8 cells. It has beneficial effects on numerous immune system parameters, including increasing T-cell differentiation and maturation. As shown in Figure 8B, compared with the normal group, the model group had no significant effect on the ratio of CD4+/ CD8+ cells (p > 0.05). Similarly, there was no difference among the high and low dose AC8 peptide groups (p > 0.05). As shown in Figure 8C, compared with the normal group, the model group had no significant effect on the percentage of NK cells in peripheral blood (p > 0.05). Similarly, there was no difference among the high and low dose AC8 peptide groups (p > 0.05). Therefore, in this study, AC8 peptide in high or low dosage could not stimulate the percentage of CD3+ cells, which can be suppressed by CY, did not disrupt the balance of CD4+/CD8+ cells in peripheral blood, and had no effect on NK cells. All of these results together would suggest that the complementary, ionic self-assembling peptide AC8 could potentially be used as a drug carrier. In Vivo Effects of The AC8 Peptide on Proliferation of Splenocytes. B-cells produce antibodies and mediate many other functions essential for immune homeostasis.61 They can release immunomodulatory cytokines that influence a variety of immune cell functions and regulate lymphoid tissue organization.62−66 However, the absence of B-cells leads to abnormalities in the immune system.67 Some studies indicate that in pemphigus vulgaris and systemic lupus erythematosus, B-cells are highly proliferative.68−70 Moreover, B-cells have a crucial role in the initial phase of these diseases.71 In some research studies, several peptides could be combined with B-cell epitopes to regulate B-cell proliferation. For instance, the peptide pA20-−36 could bind to the B-cell receptor (BCR) of A20 cells,72 and the calcitonin gene-related peptide could inhibit early B-cell development.73 In this study, CY highly depressed LPS-induced splenocytes proliferation, as shown in Figure 9. AC8 peptide could not increase the splenocytes proliferation induced by LPS in the mice treated by CY. Compared with the normal group, the model group significantly decreased the proliferation of splenocytes (p < 0.05). However, the AC8 peptide, in either high or low doses, had no obvious effect on the proliferation of splenocytes compared to the model group (p > 0.05). In this

be observed in immunosuppressive organism but not in a healthy individual.46,47 In this study, the immune response of AC8 peptide was detected in an immunosuppressive model, which was induced by high-dose cyclophosphamide (CY). Cyclophosphamide was biotransformed principally in the liver to active alkylating metabolites. These metabolites leaded to myelosuppression and immunosuppression by interfering crosslinking of cell DNA. It could induce high immunosuppressive effects. However, in vivo, normal immune system is complex and could be modulated by itself. Immunostimulatory effects would be evaluated by immunosuppressed murine model.48,49 Accordingly, cyclophosphamide was used to find the immunosuppressed model and to learn the possible immunostimulatory effect of AC8. In Vivo Effects of AC8 Peptide on Body Weight and Index of Immune Organs. As shown in Table 1, compared with the normal group (N group), the relative growth rate of BALB/c mice was lower in the model group (M group) (p < 0.05). Treatment with thymalfasin (Th) and high dose AC8 (AC8H group) significantly increased the body weight compared with the M group (p < 0.05); but no difference was found in the body weight between M group and low dose AC8 (AC8L group). Thymus index and spleen index were significantly decreased in the M group compared with that in the N group (p < 0.05); but there was no difference among the M, Th, AC8L, and AC8H groups. Overall, the results of thymus index and spleen index showed that the AC8 peptide in high concentration had no effect on the weight of immune organs. The peptides did not exacerbate the immunocompromised state. In Vivo Effects of The AC8 Peptide on the Percentage of CD3+ (T-Cells) and Ratio of CD4+/CD8+ and CD49+ (NK Cell) in the Peripheral Blood. The CD3 complex is a multichain T-cell receptor (TCR), which can activate T-cells and trigger an immune response.50 While TCR/CD3 binds a pMHC, CD3 heterodimers transform the structure of TCR.51 Therefore, CD3+ cells represent T-cells, and the subsets mainly include CD4+ T-cells and CD8+ T-cells. The ratio of CD4/ CD8 indicates the function of immune system.52,53 Antimouse CD49b was used to detect the expression of NK cells of BALB/c mice.54 NK cells not only attack cells infected by some pathogens but also attack transformed cells.55,56 In addition, NK cells regulate adaptive immune responses.57 Some clinical studies showed that NK cells can mediate antibodies against tumors and viruses.58,59 Subtle changes of peptides on the NK receptors can regulate the expression of NK cells.60,61 Therefore, T-cells, subsets of T-cells, and NK cells were elevated in the study. As shown in Figure 8A, compared with the normal group, the model group significantly reduced the percentage of CD3+ cells in peripheral blood (p < 0.05). The H



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Figure 8. continued (n = 10). (C) Effects of peptides and Th on the NK cells in vivo. Peptides and Th were injected intraperitoneally into the immune suppressed mice, which was induced by CY. The mice were killed on day 8, and flow cytometry were used to detect the percentage of panNK+ cells in peripheral blood. The values were presented as mean percentage ± SE (n = 10).

Figure 9. Effects of peptides and Th on LPS-induced proliferation of splenocytes in vivo. Cellular proliferation was measured by the MTT method. The values are presented as means ± SE (n = 10). Significant differences compared to the N group are designated as *p < 0.05.

study, results indicated that the AC8 peptides had no effects on spleen index or proliferation of splenic B-cells.

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CONCLUSIONS The in vitro and in vivo biocompatibility of the complementary, ionic self-assembling AAP peptide AC8 was systematically investigated. It was found that the AC8 peptide showed potential in vitro compatibility in terms of cytotoxicity, hemolytic activity, RBC aggregation, and anaphylotoxin as an end result of complement activation. In vivo studies suggested that the AC8 peptide in low dose produced no immune response in immunosuppressed mice, indicating that it has favorable biocompatibility. In addition, the AC8 peptide itself could significantly inhibit the proliferation of A549 cells at a high dose and raise the body weight of immunosuppressive mice. Hence, AC8 could be considered not only as a drug carrier at therapeutic concentrations but also as a potential antitumor drug when used at higher doses (0.1 mg/mL), in the future. It is worth mentioning that nuclear uptake of pirarubicin was also demonstrated using AC8 as a carrier. The in vitro data presented here is helpful in predicting the effects of AC8 peptide in vivo. Indeed, as more mechanistic studies take place in this regard, a more complete picture can be proposed for the use of AC8 in drug delivery. On the basis of all of the in vitro and in vivo data presented here, the AC8 peptide would be a favorable drug delivery vehicle, if treatment adheres to the recommended therapeutic concentration, for future studies.

Figure 8. (A) Effects of peptides and Th on the CD3+ T-cells in vivo. Peptides and Th were injected intraperitoneally into the immune suppressed mice, which was induced by CY. The mice were sacrificed on day 8, and flow cytometry were used to detect the percentage of PE-CD3+ in peripheral blood. The values were presented as mean percentage ± SE (n = 10). Significant differences compared to the M group were designated as *p < 0.05. (B) Effects of peptides and Th on the ratio of CD4+/CD8+ T-cells in vivo. Peptides and Th were injected intraperitoneally into the immune suppressed mice, which was induced by CY. The mice were killed on day 8, and flow cytometry were used to detect the FITC-CD4+ and PE-CD8+ T-cells in peripheral blood. The values were presented as mean percentage ± SE

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AUTHOR INFORMATION

Corresponding Authors

*(Y.Y.) E-mail: [email protected]. Tel: +8621-56786907. Fax: +8621-56786907. I



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*(P.C.) E-mail: [email protected]. Tel: 1-519-888-4567, ext. 35586. Fax: 1-519-888-4347.

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Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank Dale Weber, TEM/SEM/ Confocal Specialist, Department of Biology, University of Waterloo for technical assistance with confocal microscopy. The authors also would like to thank Katherine Taylor and Terence Tang for their technical assistance with the in vitro part as well as Faramarz Edalat for the proofreading of the initial draft. The authors are grateful for the financial support from the Natural Sciences and Engineering Research Council of Canada (NSERC), the Canadian Foundation for Innovation (CFI), Waterloo Institute of Nanotechnology (WIN), and the Canadian Research Chairs (CRC) program. The authors are also grateful for the financial support from Shanghai Committee of Science and Technology, China (Grant No. 10410711300) and Excellent Young Teachers Training Fund of Shanghai Municipal Education Commission (Grant No. jdy11003).

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