The use of nanomaterials in medicine involves the applications of nanoparticles and manufactured nanosystems to provide regeneration at the cellular and tissue levels [
Biomaterials used as tissue engineering scaffolds have specific physical properties and might form fibrous networks similar to collagenous extracellular matrix. They also can be programmed to carry chemical and physical cues to provide bioactivity for cell-materials interactions. In the search for more improved bioactive materials for tissue engineering purposes, peptide amphiphile (PA) molecules are good candidates to bring scaffold properties and bioactivity together [
The use of medicinal plants as remedies for numerous disorders has formed the basis of current medicinal approach. Various plants are used ethnomedicinally for prevention of excessive bleeding and as wound dressing to staunch blood flow [
ABS provides vital erythroid aggregation covering the entire physiological hemostatic process via a unique protein network depending primarily on the interactions between ABS and blood proteins, particularly with fibrinogen gamma and prothrombin [
In this work, we present a chimeric hemostatic agent, ABS Nanohemostat, via combining a self-assembling PA molecule with the traditional Ankaferd hemostat. The first step of our research was the synthesis of the specific self-assembling peptide molecules capable of being a part of the combined ABS Nanohemostat compound. The second step was the assembly of the peptide nanofibers and ABS to generate the ABS Nanohemostat. The third step of our study was testing the
9-Fluorenylmethoxycarbonyl (Fmoc), ter. Butoxycarbonyl (Boc) protected amino acids, Rink Amide MBHA resin, and 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) were purchased from NovaBiochem or ABCR. The other chemicals were purchased from Fisher, Merck, Alfa Aesar, or Aldrich and used as provided.
Peptides were constructed on Rink Amide MBHA resin. Amino acid couplings were done with 2 equivalents of Fmoc protected amino acid, 1.95 equivalents HBTU, and 3 equivalents of N,N-diisopropylethylamine (DIEA) for 2 hours. Fmoc removal was performed with 20% Piperidine/Dimethylformamide (DMF) solution for 20 min. Cleavage of the peptides from the resin was carried out with a mixture of TFA : TIS : H2O in ratio of 95 : 2.5 : 2.5 for 2 h. Excess TFA was removed by rotary evaporation. The remaining viscous peptide solution was triturated with ice-cold ether, and the resulting white product was dried under vacuum. PA molecules were characterized by liquid chromatography-mass spectrometry (LC-MS) (Figure
Liquid chromatography (a) and mass spectrometry (b) characterization of the peptide amphiphile (PA) molecule.
The PA was synthesized by Fmoc Solid Phase Peptide Synthesis (SPPS) method. It is composed of a lauryl (C12) group, hydrophobic region of the PA, and a peptide sequence. VVAG peptide sequence is used as
Schematic representation of ABS Nanohemostat formation by self-assembly of peptide amphiphile molecules into nanofibers upon addition of Ankaferd solution.
JASCO J815 CD spectropolarimeter was used at room temperature. 1 × 10−4 M peptide solutions were measured from 300 nm to 190 nm, data interval and data pitch being 0.1 nm and scanning speed being 100 nm/min, all measurements with three accumulations. Digital integration time (DIT) was selected as 1 sec, band width as 1 nm, and the sensitivity was standard.
Oscillatory rheology measurements were performed with Anton Paar Physica RM301 Rheometer operating with a 25 mm parallel plate configuration at 25°C. Each sample of 100
SEM experiments were performed with FEI Nova NanoSEM 230, using the ETD detector at low vacuum mode with 30 keV beam energy. Small amounts of gels with a final peptide concentration of 1% were put on a metal mesh, dried at critical point (1072 Psi, 31°C) with Tousimis Autosamdri-815 B Series C critical point dryer and coated with 6 nm Au-Pd. Figure
Scanning Electron Microscopy images of peptide amphiphile (PA) gel with Ankaferd (a) or alone (b) at pH 10.
An amount of 250
All animal experimentations described in this paper were carried out in accordance with national guidelines for the use and care of laboratory animals and were approved by the local animal review and ethics committee. All procedures were in full compliance with Turkish Law 6343/2, Veterinary Medicine Deontology Regulation 6.7.26, and with the Helsinki Declaration of World Medical Association recommendations on animal studies. The animals were obtained from the center of medical experimental research of Ankara Training and Research Hospital. The rats were housed in stainless steel cages in an animal room maintained at a temperature of 22–24°C with 12-hour light/dark periods. All were fed with the same amount of laboratory pellet diet and with water supplied
A total of 24 Wistar rats weighing 200 to 300 g were divided into 4 groups of 6 each and underwent PN. One surgeon with an assistant performed all the surgical procedures. All operations were performed under total anesthesia with injection of 50 mg/kg intramuscular ketamine hydrochloride. After sterile preparation and draping, a midline incision was made on the abdomen. For each rat, renal artery and vein were revealed by hilar vascular dissection. Subsequently, renal artery and vein were clamped with Rommel vascular clamp. The lower third of the left kidney was resected in guillotine fashion with a single stroke of an amputating knife. Four different hemostatic techniques were applied to the groups. (i) Group 1 (G1) is the left PN with hilar vascular control including intracorporeal suturing of the renal parenchyma and collecting duct (control group). (ii) Group 2 (G2) is the conventional PN with only 0.5 mL traditional Ankaferd hemostat (ABS) application without suturing. (iii) Group 3 (G3) is the conventional PN with ABS (0.25 mL) + peptide (0.25 mL) gel (ABS Nanohemostat) mixture application with no suturing. (iv) Group 4 (G4) is the conventional PN with only 0.5 mL peptide solution application.
Two objective parameters were recorded during the surgical procedure: warm ischemia time (WIT) and amount of bleeding (AOB). The unit of WIT was the “second,” while the AOB was measured with the bleeding area (cm2) onto the sponges. The abdominal incision was afterwards closed with surgical sutures. All the rats were allowed to feed and drink water for the following 4 weeks. After that, each rat was sacrificed, and total nephrectomy was performed for histopathological examination.
Each hemostatic method was used during the period of warm ischemia (WI). WI started with clamping the renal artery and vein and finished with taking the clamp out. In G1, traditional hemostasis method was used as compression onto the renal excised area and suturing the renal vessels and collecting duct with absorbable sutures (Figures
(a) and (b) Traditional partial nephrectomy model with suture on resected area dissection of kidney. (c) and (d) Transection of kidney and application of ABS.
Light microscopic sections were reviewed by a pathologist blinded to the treatment groups. Coronal 1 to 2 mm thick slices of the kidney were fixed in 4% formaldehyde and embedded in paraffin, and 3 to 4
Data analysis was performed using IBM Statistical Package for the Social Sciences (SPSS) statistics software. Continuous variables were tested for normality by the Kolmogorov-Smirnov test. Values were presented as median and range because all numeric data were nonnormally distributed. Comparisons of numerical variables among four groups were initially performed using the Kruskal-Wallis test and Mann-Whitney
In this work, we synthesized a positively charged PA molecule as shown in Figure
Comparison of mechanical character of PA-Ankaferd gel with PA gel formed at pH 10 by using oscillatory rheology.
Left lower pole PN was surgically performed successfully to each rat in all groups. The kidney size and shapes were similar. Likewise, the size of the resected area was standardized for groups. The same surgical equipments were used, and Rommel clamps succeeded the warm ischemia during all surgical procedures.
Mean ± SD WITs were
Comparison of warm ischemia time (WIT) and amount of bleeding (AOB) in study groups.
WIT (sec) | AOB (cm2) | |
---|---|---|
Group 1 ( |
232.8 ± 56.3 | 7.3 ± 3.3 |
Group 2 ( |
65.6 ± 11.4 | 5.7 ± 2.3 |
Group 3 ( |
75.5 ± 17.2 | 5.2 ± 3.2 |
Group 4 ( |
58.1 ± 17.6 | 16.4 ± 7.7 |
|
0.003* | 0.035** |
*G1 versus G2
**G1 versus G2
(a) and (b) Application of the ABS Nanohemostat on the resected area.
All rats were sacrificed at the first month following the surgery. Each specimen was protected in formalin solution. Fibrosis was not different among the groups (
Histopathological features of study groups.
Parameters | Group 1 (%) | Group 2 (%) |
Group 3 (%) |
Group 4 (%) |
|
---|---|---|---|---|---|
Presence | |||||
Fibrosis | 0 | 1 (16.6) | 3 (50) | 2 (33.2) | NS |
Inflammation | 0 | 1 (16.6) | 4 (66.7) | 4 (66.7) | 0.048* |
Erythroid aggregation | 2 (33.2) | 1 (16.6) | 5 (83.3) | 5 (83.3) | 0.036* |
Hemosiderin | 2 (33.2) | 2 (33.2) | 5 (83.3) | 4 (66.7) | NS |
Calcification | 0 | 0 | 6 (100) | 5 (83.3) | <0.001* |
Overall ( |
6 (100) | 6 (100) | 6 (100) | 6 (100) |
NS: not significant.
*Significant difference was found between G1 and G2 versus G3 versus G4.
Erythrocyte aggregation in glomerular field and interstitium in ABS Nanohemostat group (20–40 xHE).
Bleeding and congestion without erythrocyte aggregation with significant calcification in Nanopeptide group (10 xHE).
In this study, we revealed that PA-Ankaferd gel mixture (ABS Nanohemostat) is effective as traditional Ankaferd hemostat. Moreover, this unique nanomedicine proves itself as an effective and easily applicable alternative hemostatic method which can successfully be used even in complicated hemorrhagic situations such as aggressive surgical tissue bleeding due to PN.
Several hemostatic agents are preferred to control external and internal bleedings; yet, commercially available products are not sufficiently effective or fast acting to achieve hemostasis in extreme occasions. Ankaferd hemostat is a topical hemostatic agent of plant origin, including molecules with high density of negative and positive charges, and is proven to work as an efficient hemostatic agent [
Sustaining hemostasis in clinical hemorrhages is a challenging task and requires extensive effort to stabilize traumatic and surgical injuries. Our experimental animal model is therefore clinically relevant since bleeding is one of the most important major complications leading to the morbidity and even mortality during PN for renal masses. Providing hemostasis following the renal mass excision is the most important step of PN [
The hemostatic effect of local ABS application in a patient who underwent an open PN has already been shown [
Controlled clinical studiesconducted to evaluate the effectiveness of ABS in distinct states of bleeding disorders documented the safety and efficacy of traditional Ankaferd hemostat in comparison to conventional antihemorrhagic medications. The first randomized controlled clinical study was reported by Teker et al. [
The ability of ABS to induce formation of a protein network not only makes it an effective hemostatic agent, but also confers anti-infective, antineoplastic, and healing modulator properties to the extract [
In summary, ABS Nanohemostat has comparable hemostatic efficacy to the traditional Ankaferd hemostat in the PN experimental model. ABS Nanohemostat-induced erythroid aggregation is prominent at the kidney tissue level. These observations prompt the design of future experimental and clinical studies focusing on the antibleeding and vascular repair effects of this novel hemostatic nanomedicine. The production of ABS Nanohemostat in gel formation is also important for using the hemostatic material during the laparoscopic urologic surgery. The steady scaffold onto the resected area with ABS Nanohemostat predicts the intracorporeal use of ABS hemostat as a promising agent. Likewise, future controlled studies are needed to shed further light on the expanding spectrum of the effects of ABS compounds in hemostasis and related areas.
The authors declare no conflict of interests related to this paper.
The authors would like to thank Busra Mammadov for helping them to illustrate Figure