Evaluation of the hemocompatibility of RADA 16-I peptide

Abstract

RADA 16-I is an ionic self-assembling peptide that can form macroscopic scaffolds through β-sheet structures which are used in favor of cell growth and tissue engineering. This peptide has also the ability to stop bleeding effectively and quickly (∼20 seconds) when applied directly to the injuries. This study is focused on coagulation process, platelet aggregation, C3 and C4 concentrations, CBC counting, hemolysis, and white blood cell morphology tests to analyze hemocompatibility of RADA 16-I at different concentrations – 0.1, 0.2, 0.3 and 0.5%. According to the results, RADA 16-I hydrogel decreased the number of blood cells, slightly increased clot formation time and platelet aggregation, and yielded negligible hemolysis and only small changes in C3 and C4 concentrations and white blood cell morphology. All by all, the in vitro tests of hemocompatibility showed no perturbation in the blood composition when the peptides were in contact with the blood. The observed rapid hemostasis might be a result of increasing local concentrations of molecules involved in the formation of clot near the peptide hydrogel, thereby making a barrier which ended up with complete hemostasis. In conclusion, our experiments strongly supported further development of biomaterials based on RADA 16-I peptide.

Keywords

Aggregation blood–material interaction hemocompatibility hemostasis self-assembly

Introduction

Self-assembling peptides have found various tissue-engineering applications such as drug delivery, membrane protein stabilization and hemostasis.¹ RADA 16-I is a peptide that consists of 16 amino acid residues belonging to the family of self-complementary peptides. It consists of repeated segments of hydrophobic (Ala) and hydrophilic (Arg and Asp) amino acids.² These self-assembling peptides can be spontaneously assembled to form not only stable nano-scale fibers, but also three-dimensional macroscopic scaffolds with extremely high water content, called hydrogels, by changing pH or adding salt solutions (i.e. physiological saline, blood or cerebrospinal fluid).^3,4 The hydrogels have been observed to achieve complete hemostasis in ∼20 seconds, without employing any of traditional hemostatic therapies. Application of many protein-based biomaterials (such as, collagen and fibrin) for achieving hemostasis has been extensively studied; however, the rate of success with such biomaterials has been limited due to the lack of adequate hemorrhage control.^5,6

Hemocompatibility is a very important factor to consider when deciding on the application of biomaterials. The study of host's response to biomaterials in a blood-contacting environment is the key to understand hemocompatibility with respect to toxicity. As most of functional biomaterials come into contact with blood, red blood cells may undergo hemolysis to release hemoglobin (Hb), leading to anemia or kidney failure.⁷ While other plasma components like platelets, white blood cells, and complements in blood come into contact with biomaterial, there are chances that those are activated to cause coagulation and inflammation.^8,9 Hence, evaluation of hemocompatibility of these biomaterials through material–host interactions should be first considered.

There are reports on the investigation of RADA 16-I as an extracellular matrix to facilitate neuronal or tissue regeneration. To the best of our knowledge, as of current, no study investigating hemocompatibility of and the mechanism through which this peptide treats wounds is reported. Interestingly, the mechanism was not tissue-specific, as complete hemostasis was observed in various organs and tissues.^10–12 Elucidation of the mechanisms underlying this somewhat surprising hemostatic ability may serve as a key to facilitate a revolutionary approach to the design of blood-contacting biomaterials.

In this work, in vitro evaluation of hemocompatibility of the RADA 16-I is reported. This investigation involves complement activation, coagulation assays for estimation of peptide-induced abnormalities to the intrinsic and extrinsic coagulation pathways, platelet and whole blood cell attachment to peptide, morphology tests on white blood cells, and hemolysis. This work provides an in-depth hemocompatibility analysis of RADA 16-I hydrogel for the first time.

Materials and methods

Materials

Briefly, whole blood was collected from healthy donors. The blood collected in this manner was used for all subsequent plasma-related assays, as described below.

RADA 16-I (Ac-R-A-D-A-R-A-D-A-R-A-D-A-R-A-D-A-NHCOCH₃) peptide was from Innovagen and supplied as trifluoroacetic salt in the form of a lyophilized powder. The material was used as received without further purification. Purity of the peptide was ∼98%, with a calculated MW of 1713.2.

Peptide solution was prepared by dissolving RADA 16-I peptide powder in MilliQ water. The dissolution was done by sonicating the peptide solution. Aqueous peptide solutions were diluted with phosphate buffer saline, such that the final working peptide concentrations were 0.1, 0.2, 0.3, and 0.5% (w/v). Peptides were stored at 4℃ until use. At these concentrations, the peptide had gel structure which corresponded to storage modulus of 4000 Pa for 0.5% solution.¹³

Activated partial thromboplastin time (APTT)

APTT is a simple yet highly reliable method through which the capacity of blood to coagulate through intrinsic coagulation mechanism can be measured, with possible effect of peptide on the process evaluated.¹⁴ Briefly, the blood was collected from human vein into test tubes. After incubation at 37℃ for 30 min, the platelet-poor plasma (PPP) was obtained by centrifugation at 5000 r/min for 10 min. Various concentrations of the RADA 16-I hydrogels (0.1%, 0.2%, 0.3%, and 0.5% (w/v)) were thoroughly mixed and incubated with equal volume of PPP plus APTT reagent (Fisher Scientific) at 37℃ for 30 min. The clotting reaction was initiated by adding an equal volume of 0.025 M CaCl₂ into the samples and the optical density at 405 nm was measured with a BioTek ELx808 plate reader, at 5 seconds intervals over a period of 120 seconds. BSA was used as negative control. All experiments were repeated three times.

Prothrombin time (PT)

Prothrombin time was measured to evaluate peptide-induced abnormalities of the extrinsic coagulation pathway.¹⁵ Platelet-poor plasma plus PT reagents (Fisher Scientific) were incubated at 37℃ with various concentrations of the RADA 16-I hydrogels (0.1%, 0.2%, 0.3%, and 0.5% (w/v)). The time to the onset of clot formation was recorded as PT.

Analysis of hemolytic properties

Free Hb was used as an indicator for hemolysis, i.e. red blood cell damage. Percentage of hemolysis was determined by comparisons versus a 100% hemolysis sample. Peptide samples at concentrations of 0.1, 0.2, 0.3 and 0.5% were placed in the wells of a 24-well plate. PBS and H₂O were used as negative and positive controls, respectively. 0.2 ml of whole heparinized rabbit blood was added to the samples in triplicate. The plates were then agitated on a shaker incubator at 37℃ for 3 h. Aliquots of the initial and post-agitation blood samples were removed from the plate and transferred to Eppendorf tubes and treated with Drabkin reagent for 15 min. The tubes were then centrifuged at 1000 r/min for 15 min. Supernatant absorption was then measured against PBS at a wavelength of 540 nm. The percentage of hemolysis (% H) was calculated according to the following formula:

(% H) = (D_{t} - D_{nc}) / (D_{pc} - D_{nc}) \times 100

where D_t is the average absorbance of the test sample, and D_nc and D_pc are absorbances of the negative and positive controls, respectively.

Assessment of white blood cell morphology after contact with hydrogels

Buffered Wright/Giemsa staining is useful to differentiate and study blood cell types, their morphology and platelet aggregation in a sample. The RADA 16-I was treated with human blood at different concentrations, namely 0.1, 0.2, 0.3, and 0.5%, for 30 min, and the smear was prepared and stained according to Wright/Giemsa staining protocol (Sigma). The slides were studied by optical microscopy at 100 × magnification and on the images captured by a high-definition color camera head Nikon DS-Fi2. All images were recorded and saved in JPG format. For comparison, all images were also manually segmented into nucleus and cell (or cytoplasm) areas and classified into normal or abnormal leukocytes, namely lymphocyte, monocyte, and neutrophil, by a hematologist. Water and latex were used as negative and positive controls, respectively.

Blood cell adsorption on to peptide

Whole blood cell counting was carried out to assess blood cell adsorption on to peptide. CBC analysis was done on 2 ml of anti-coagulated blood drawn just prior to the experiment; the samples were already incubated with various concentrations of RADA 16-I hydrogels (0.1%, 0.2%, 0.3%, and 0.5% (w/v)) using an Automatic Biochemistry Analyzer (Benaka Healthcare) according to the manufacturer's instructions. Whole blood (without peptide) was considered as control. Adsorbed quantities of erythrocytes, leukocytes, and platelets by the biomaterial were determined by subtracting the number of the cells in the solution phase of the peptide containing samples from the corresponding value in control.

Complement C3 and C4 measurement

Complementary C3 and C4 studies were performed using platelet-poor human plasma. Briefly, plasma was incubated at 37℃ with equal volume of peptide at different concentrations (0.1, 0.2, 0.3 and 0.5%) for 30 min. Subsequently, EDTA was added to a final concentration of 15 µM for quenching the reaction. The final reaction volumes were handled using direct immunoturbidimetry assays (Pars Azmun, Tehran, Iran) as per the manufacturer's protocol. Serum C3 and C4 absorptions were measured at 340 nm and their concentrations were obtained by putting the results on a calibration curve. Limits of detection of the method for C3 and C4 were found to be 0.04 and 0.02 g/L, respectively. Normal ranges were 0.9–1.8 g/L and 0.1–0.4 g/L for C3 and C4, respectively.

Statistical analysis

Data are presented as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used to analyze significant differences within the data. A P-value smaller than 0.05 was considered as an indication of statistical significance. Calculations were performed using Excel 2013.

Results

Our previous work has confirmed successful hemostasis function of RADA 16-I-based hydrogels at various concentrations. Investigation of secondary structures along with the results of molecular dynamic studies have revealed that, as a monomer, peptide has a typical α-helical structure.¹⁶ It was demonstrated that RADA-16 was not only able to retain its characteristic α-helical structure, but also exhibited an increase in relative β-sheet content with increasing peptide concentration from 0.1% to 1% (w/v) due to the increase in the peptide interactions at higher concentrations (not published). Even though the net charge of RADA 16-I peptide is zero at pH ∼7.4, the presence of electrostatic charges on the argenine and aspartatic acid residues seems to play a significant role in the assembly of the peptide to three-dimensional structures.¹⁷

Many interactions are possible when surface of these peptide hydrogels is exposed to blood, with these interactions being expected to interfere many processes at interface. Taking this into account, we analyzed the blood clotting kinetics, the hemolysis process, whole blood cell adsorption, morphology of white blood cells and C3 and C4 concentrations, when the peptide hydrogel surface was in contact with blood.

Plasma activated partial thromboplastin time and prothrombin time

Coagulation pathway involves a series of proteolytic reactions (including intrinsic and extrinsic pathways) which result in the formation of a fibrin clot.¹² Evaluation of anticoagulant activity based on plasma coagulation has been recognized as a standard test to estimate blood compatibility of a biomaterial.

The results of PT and APTT analysis are shown in Table 1. It can be concluded from Table 1 that the PT and APTT of the samples were seen to be longer than those of the control groups, but these differences were not significant and converged to a stable value at peptide concentrations beyond 0.3%.

Table 1.

Prothrombin time (PT) and activated partial thromboplastin time (APTT) analysis. For in vitro PT (a) and APTT (c) experiments, the platelet-poor plasma (PPP) was treated with various concentrations of the RADA 16-I hydrogels (0.1%, 0.2%, 0.3%, and 0.5% (w/v)) and incubated with equal volume of APTT or PT reagent at 37℃ for 30 min. ***p < 0.0005 compared to control PT and APTT clotting times.

	Control	0.1%	0.2%	0.3%	0.5%
APTT (second)	51 ± 1.1	51 ± 1.1	53 ± 1.2	55 ± 0.9	55 ± 1.2
PT (second)	12.5 ± 0.7	14.6 ± 1.1	15.59 ± 0.9	16 ± 0.9	16 ± 0.6

In order to analyze kinetics of APTT, turbidimetric assay was conducted to monitor the changes in optical density, where an increase in turbidity served as an indication of the formation of fibrin clots. Figure 1 shows the absorbance profile (at 405 nm) of APTT for a range of hydrogel concentrations. Following the addition of CaCl₂ to the plasma-rich hydrogel systems, an initial lag phase was observed, followed by a rise and then plateau in optical density that coincided with clot development. All of the peptide-containing samples and control showed a brief lag period for the initiation of the clot formation (∼20 s), reaching a plateau within ∼60 s upon adding CaCl₂ (Figure 1). In Figure 1, the turbidity (absorbance) exhibits an evident dose-dependency which might be due to increase in turbidity with increasing peptide concentration. Furthermore, an increase in turbidity was not only dose-dependent with various hydrogel systems, but also statistically significant (p < 0.05, one-way ANOVA).

Upon studying PT and APTT clot formation kinetics using platelet-poor human plasma, it could be concluded that the blood-stopping property of RADA 16-I was not because of accelerating the coagulation process. Because blood flow stopped within ∼15–20 seconds using these peptide hydrogels, whereas blood clots formation took as long as 1 minute (Figure 1). It is less possible that the hydrogel serves as a simple barrier for injuries or wounds because this hydrogel is not enough stiff.¹⁸ There are possibilities that the hydrogel makes a surface that the activated platelets adhere to fibrinogen and cause the release of thrombin and form a fibrin matrix on the hydrogel surface.

Hemolytic properties in the presence of peptide

Erythrocytes are perfect osmometers which can undergo lysis by disturbances in osmotic and physical changes in blood. In general, erythrocyte destruction can be caused by several factors such as bacterial infection, intrinsic erythrocytes membrane defects, mechanical shear due to blood flow, osmotic and pH changes in blood, drug-induced hemolysis, excessive complement, and platelet activation.^19,20 Exposure of the erythrocytes to biomaterials can result in hemolysis. Because hemolysis results in the release of red-colored Hb, colorimetric assays can be used to estimate the extent of the damage. Accordingly, the effect of peptide on erythrocytes hemolysis was analyzed. Absorbance results of hemolysis tests on the samples exposed to peptide, positive control, and negative control are reported in Table 2. According to the results, the degree of hemolysis in the presence of RADA 16-I was 1.

Table 2.

Percentage of hemolysis at different concentration of peptide. Water and PBS were used as positive and negative control, respectively.

Samples	Optical density at 540 nm	Hemolysis percentage
H₂O	0.0408 ± 0.0020	1.1 ± 0.1
PBS	0.017 ± 0.0040	0.048 ± 0.1
0.1% peptide	0.286 ± 0.0042	0.78 ± 0.1
0.2% peptide	0.357 ± 0.0031	1 ± 0.1
0.3% peptide	0.370 ± 0.0041	1.07 ± 0.1
0.5% peptide	0.380 ± 0.0021	1.1 ± 0.1

Microscopic examination of white blood cells morphology after contact with hydrogels

Peptide samples at various concentrations (0.1, 0.2, 0.3 and 0.5%) and controls (negative and positive) were exposed to human blood (EDTA anticoagulant) under predefined conditions, and their effect on blood cell morphology was determined by Wright/Giemsa staining. Water and latex were used as negative and positive controls. When they come in contact with blood, materials may damage white blood cells, resulting in changes in function of these cells or the blood properties. About 100 smear slides were prepared and all of the areas in smears were observed. The cells were counted and recorded on a sheet as intact or destroyed. The results are shown in Table 3. Morphological analysis of human white blood cells showed a slight deformation or decomposition of the cells in the presence of peptide (Figure 2), with no obvious abnormality seen in the cells. Most cells exhibited round or oval geometries, with chromatin being loosely and non-uniformly distributed in their nuclei; the nuclei were large and clear, with cytoplasm being stained with blue.²¹

Table 3.

White blood cell morphology changes upon incubation with RADA 16-I of 0.1–0.5% (w/v) stained with Wright/Giemsa.

	H₂O	Latex	0.1% peptide	0.2% peptide	0.3% peptide	0.5% peptide
Total	1000	1000	1000	1000	1000	1000
Damaged	20	300	40	50	50	80

Blood cell adsorption on to peptide

When surface of a biomaterial is exposed to blood, many interactions may occur. Plasma proteins adsorption is one of the first events that may occur in such a case, followed by cells adherence, affecting the blood composition and functions. Numerical reduction of erythrocytes and leukocytes remaining in the solution phase relative to control was used to estimate the degree of adsorption of blood cells onto scaffold materials during incubation with whole blood samples.

Analysis of the blood formula presented in Table 4 indicates relative adsorption of RBC, WBC and PLT onto peptide hydrogels. In contrast, the numbers of adsorbed cells were not significantly different between different concentrations of peptide (p > 0.05) (Table 4). The Wright/Giemsa analysis of cells showed that most of the hemocytes adsorbed onto the surface were neutrophilic granulocytes and erythrocytes. Deposition of erythrocytes and neutrophilic granulocytes on the hydrogel surface might be because of the chemistry of the peptide hydrogels.

Table 4.

Blood formula (blood cell counts) in the absence and presence of various concentrations of peptide (0.1–0.5% (w/v)).

Sample	WBC (10³)	RBC (10³)	PLT (10³)
Control	6.8	4.58	312
0.1% peptide	4.5	2.44	114
0.2% peptide	4.1	2.42	105
0.3% peptide	4.4	2.57	111
0.5% peptide	5.4	2.59	130

Quantity of platelets was also reduced in peptide-containing samples in comparison to control. The result of staining by Wright/Giemsa showed that these cells become aggregated dose-dependently. Platelets in contact with foreign materials become distorted and form thick reticulate structures with the fibrinogen deposited on the surfaces.

Complement C3 and C4 measurement

Biomaterials are known to be potential agonists for complement activation, where the complement system has a primary function in host defense and removes foreign cells, microorganisms, and cell debris from body.^22,23 Moreover, the complement system is interlinked with the coagulation cascade. Various serine proteases belonging to the coagulation system are able to activate the complement cascade independent of the so far established pathways. The complement system can be activated through three pathways,²⁴ namely “classical” activation pathway, alternative pathway, and lectin pathway. It is believed that some biomaterials tend to activate alternative pathway, while others go for classical pathway, depending on the nature of the considered biomaterial. The initial step in complement activation is the adsorption of C3 and C4 on the biomaterial surface.²³ Therefore, the material–complement interaction is considered a very important aspect of hemocompatibility of the material. Activation of the complement system, both in vivo and in vitro, leads to the generation of activation products such as anaphylatoxins C3a and C5a and fragments of C3 and C4 produced by sequential proteolysis.²⁵ Interestingly, a previous study has revealed a significant increase in complement activation by materials containing several functional groups such as COOH, NH₂, and OH, because of the covalent bonds established between these functional groups and C3 and C4. Thus RADA 16-I is expected to significantly activate complement due to the abundance of aspartic acid and arginine residues.²⁶ It is suggested that surfaces with repeating unit or nucleophilic groups tend to activate alternative pathways. The charged groups on the biomaterials have been discussed as an important factor in determining interactions between biomaterials and neutrophils. In the literature, much effort has been made to attenuate the adsorption of plasma proteins onto the artificial surfaces to improve the hemocompatibility. The purpose of this section was to investigate effects of C3 and C4 adsorption on the hydrogel, a key step in assessment of complement activation pathway. So, the concentrations of these two factors were measured in serum after hydrogel treatment.

As shown in Table 5, concentration of the serum C3 in contact with peptide hydrogel at different concentrations (0.1–0.5% (w/v)) was lower than that of control (Table 5), but the difference was not significant. Although the C3 and C4 concentration ranges for RADA 16-I varied at different peptide concentrations, no clear dose-dependent trend was seen in the generation of the fragments. The obtained data suggested that C3 and C4 adsorptions are less dependent on the peptide concentration. Remarkably, reduction in C3 and C4 concentrations mediated by these peptides was less than that of the polymers widely used as biocompatible material (such as dextran, PEG and PHB). It is thought that C3 and C4 bind to biomaterials quickly after exposure. In some instances, this rapid binding disappears, in which cases the quantities of C3 and C4 are comparable to those in normal serum.^27–29 We suggest that the extent of covalent C3 and C4 surface binding (which occurs in complement activation) is minor, and the deposition of C3 and C4 most likely occurs via electrostatic and hydrophobic interactions rather than covalent binding.

Table 5.

Concentration of C3 and C4 in human platelet-poor plasma (PPP) exposed to different concentrations of RADA 16-I hydrogel systems. Human PPP incubated with PBS (pH 7.4) is used as a control. The experiments were repeated two times for each system using the same-pooled plasma.

	C3 (mg/dl)	C4 (mg/dl)
Control	70 ± 5	7 ± 1
0.1% peptide	69 ± 4	6.2 ± 1
0.2% peptide	70 ± 6	6.1 ± 1
0.3% peptide	71 ± 5	4.1 ± 1
0.5% peptide	71 ± 4	2.9 ± 1

Discussion

In this study, RADA 16-I peptide hydrogels were successfully produced in an effort to gain further insight into the hemocompatibility of these self-assembling peptides.

One of the tests which was evaluated was coagulation process compatibility. In clot formation process, prothrombin is converted to thrombin by activated Xa enzyme, followed by the conversion of fibrinogen to fibrin.³⁰ Any loss in any of the enzymes or substrates involved in coagulation process can lengthen the clotting times. The interaction between RADA 16-I hydrogel and plasma proteins can reduce fibrinogen concentration due to adsorption, as can be observed as longer PT and APTT. Most of the studied biomaterial surfaces showed increased PT and APTT. These increases in PT and APTT were probably due to simple adsorption of coagulation proteins, like fibrinogen and thrombin, which could directly alter plasma fibrinogen, making it abnormal in its formation of fibrin when catalyzed via activated thrombin.^31,32 Dysfibrinogen or abnormal fibrinogen has been shown to prolong both clotting times as fibrinogen is an integral part of the common coagulation pathway for PT (extrinsic coagulation) and APTT (intrinsic coagulation).

Generally, a usable biomaterial should yield less than 2% hemolysis. A higher degree of hemolysis indicates poor hemocompatibility of the biomaterial. All materials currently used in medical applications, including PVC, meet this standard.²³ The data suggested that RADA 16-I provides an acceptable level of hemolysis.

In the case of the adherence of cells to peptide, RADA 16-I appeared to merely induce the adhesion of erythrocytes without serious damage to the cell membrane, as judged by the low hemolysis rate (∼1%). Leukocytes, especially neutrophilic granulocytes, have strong chemotactic stimulus properties, encouraging their emigration to and retention by the surfaces of exogenous substances. An activated neutrophilic granulocyte can release many enzymes and inflammatory factors that induce an inflammatory response. This inflammatory response is sometimes severe, causing damage to organs or even systemic reactions such as fever, arrhythmia, acute lung injury, and chronic inflammation.^33,34 The neutrophil adsorption onto hydrogels suggested that peptide might promote an inflammatory response. However, examination of neutrophil activation and release of inflammatory cytokines are beyond the scope of the current study.

Here, few platelets adhered and aggregated, and when studied under microscope, the adherent platelets exhibited no significant deformation. Consequently, RADA 16-I has compatibility with platelets. According to reports in literature, positively charged particles interact more strongly with platelets.^35,36 Therefore, platelets can interact with positive charge of arginine, thereby increasing the aggregation.

Conclusion

In vitro biocompatibility assays are vital for the design of any blood-contacting biomaterial. Based upon the obtained results, it seems that peptide tends to prolong clotting time without accelerating coagulation process as an efficient hemostatic agent. The complement activation studies indicated that the presence of RADA 16-I hydrogels resulted in insignificant complement activation, when compared to the controls. This conclusion was further supported by the fact that; no red blood cell or platelet abnormalities were found after achieving complete hemostasis with RADA 16-I hydrogel.

The observed hemostasis cannot be adequately explained by mechanical inhibition of blood flow; as upon testing a stiffer gel than RADA 16-I, the modified gel failed to arrest bleeding. It is likely that as an early interaction between blood and a material, plasma proteins (and fibrinogen, in particular) are adsorbed on the material surface. The adsorbed fibrinogen becomes denatured of its native conformation, leading to exposure of the fibrinogen receptor binding sites and subsequent adhesion of activated platelets. Accordingly, the activated platelets that bind to the surface of the fibrinogen release thrombin and form a fibrin matrix on the material surface. Further studies need to be conducted to reveal the exact mechanism which drives such rapid hemostasis. This system is almost biocompatible and can serve as safe material in wound healing and blood stopping.

Footnotes

Acknowledgements

The authors would like to thank the research council of Malek-Ashtar University of Technology.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The authors would like to thank the research council of Malek-Ashtar University of Technology for the financial support of this investigation.

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