Revista de Ciencias Tecnológicas (RECIT). Volumen 3 (1): 10-22.

Revista de Ciencias Tecnológicas (RECIT). Universidad Autónoma de Baja California ISSN 2594-1925

Volumen 2 (1): 20-25 Enero-Marzo 2019 https://doi.org/10.37636/recit.v212025

ISSN: 2594-1925

Diabetic foot ulcers treatment with silver nanoparticles

Tratamiento de las úlceras del pie diabético con nanopartículas de plata

Almonaci Hernández Cesar Alejandro

, Cabrera Torres Isis Marion

, López-Acevedo

Rosangela

, Juárez-Moreno Karla Oyuki

2,3

Castañeda-Juárez Martin E.

, Almanza-Reyes

Horacio

, Pestryakov Alexey

, Bogdanchikova Nina

Integral and Advance Clinic, Ensenada, Baja California, México.

Center of Nanoscience and Nanotechnology, National Autonomous University of Mexico. Km107

Carretera Tijuana-Ensenada, Ensenada, Baja California, México.

CONACYT Research Fellow at Center of Nanoscience and Nanotechnology, National Autonomous

University of Mexico. Ensenada, Baja California, México.

School of Medicine. National Autonomous University of Mexico, Ensenada, Baja California, México

School of Medicine and Psychology, Campus Tijuana. Autonomous University of Baja California, Tijuana, Baja

California, México.

Tomsk Polytechnic University, Tomsk, Russia.

Corresponding author: Nina Bogdanchikova, Center of Nanoscience and Nanotechnology, National

Autonomous University of Mexico. Km107 Carretera Tijuana-Ensenada, Baja California, México. E-mail:

nina@cnyn.unam.mx. ORCID: 0000-0003-0929-3535.

Recibido: 01 de Julio del 2018 Aceptado: 12 de Diciembre del 2018 Publicado: 20 de Enero del 2019

Resumen. - Las úlceras del pie diabético son altamente susceptibles a infecciones microbianas y son la

principal causa de hospitalización y amputación de extremidades inferiores en pacientes diabéticos. Debido a

sus propiedades antimicrobianas y anti-inflamatorias, las nanopartículas de plata (AgNPs) son el nanomaterial

más utilizado en biomedicina. En este trabajo presentamos el uso de AgNPs para el tratamiento de úlceras de

pie diabético grado II de la clasificación de Wagner. Además del tratamiento con antibióticos convencionales,

las úlceras fueron tratadas mediante la administración tópica de AgNPs a una concentración de 1.2 mg/mL de

plata metálica. Los resultados obtenidos, revelan una mejora significativa en la evolución de las úlceras, en

donde los bordes de la lesión se acercaron de manera efectiva, observándose tejido de granulación,

disminución del edema y placas de fibrina. Estos resultados constituyen la base para nuevos estudios sobre el

uso de AgNPs para el tratamiento de úlceras crónicas de diferentes orígenes.

Palabras clave: Úlceras del Pie Diabético; Diabetes Mellitus; Nanopartículas de Plata; Nanomedicina; Úlceras

Crónicas.

Abstract. - Diabetic foot ulcers are highly susceptible to microbial infections and are the leading cause of

lower limb hospitalization and amputation in diabetic patients. Due to their antimicrobial and anti-

inflammatory properties, silver nanoparticles (AgNPs) are the most widely used nanomaterial in biomedicine.

Herein, we present the usage of AgNPs for the treatment of diabetic foot ulcers with a stage II in Wagner

classification. In addition to conventional antibiotic treatment, the ulcers were treated with topical

administration of AgNPs at a concentration of 1.2 mg/ml of metallic silver. The results showed a significant

improvement in the evolution of the ulcers, where the edges of the lesion were effectively approached,

granulation tissue being observed, edema decreased and fibrin plaques. These results form the basis for further

studies on the use of AgNPs for the treatment of chronic ulcers of different origins.

Keywords: Diabetic Foot Ulcers; Diabetes Mellitus; Silver Nanoparticles; Nanomedicine; Chronic Ulcers.

Revista de Ciencias Tecnológicas (RECIT). Volumen 2 (1): 20-25

ISSN: 2594-1925

1. Introduction

Diabetes mellitus (DM) is the second major

cause of death in Mexico [1]. Twenty-five

percentage of patients with DM developed

complications of lower limbs known as

diabetic foot ulcers (DFU) [2]. These types of

ulcers are characterized by poly-microbial

infection, ulceration and destruction of the

deepest tissues of lower limbs, associated with

neurologic alterations and diverse degrees of

peripheral vascular disease [3]. Therefore,

DFU are the main cause of hospitalization and

lower limbs amputation [3][5].

The assessment of a patient with DFU starts

with wound cleaning, debridement and

antibiotic schedule treatment [7]. If no

evidence of healing is shown and

complications of the DFU such as gangrene

and osteomyelitis are observed, then a surgical

management is recommended involving the

amputation of the lower limb [8].

Infection of DFU with multi-drug resistance

(MDR) microorganisms may increase the time

of wound healing, hospitalization, treatment

costs and patient mortality [9]. Therefore, due

to its well- known antimicrobial properties,

silver has been extensively used in a wide

variety of products on the market to treat

bacterial infection and to prevent wound

sepsis [10]. However, all of these formulations

have limitations concerning the need for a

high-frequency application and the

inactivation of silver. In this sense,

nanotechnology has generating new

applications for biomedicine through the

synthesis of nanomaterials [11]. One of the

most used nanomaterial in medical products is

silver nanoparticles (AgNPs).

Owning to their antibacterial and anti-

inflammatory properties [12, 13], AgNPs have

been intensively applied for wound healing

[13] but scarcely studied in vitro and in animal

models for the treatment of diabetic ulcers,

this may be due to the high risk of limb

amputation in patients [14, 15]. However, the

use of AgNPs to heal DFU in DM patients has

never been studied before our group began to

investigate it [16]. Taking this in

consideration, the aim of this study is to

investigate the potential use of AgNPs for the

treatment of DFU in patients.

2. Methodology

2.1 Silver nanoparticle formulation

After comparison with different AgNPs

commercially available, we concluded that

only Argovit preparation resulted to have

multiple certificates for their usage in

veterinary and human applications [17].

Argovit (Scientific and Production Center

Vector-Vita, Novosibirsk, Russia) is a

preparation of highly dispersed AgNPs with

an overall concentration of 200 mg/mL (20%)

of polyvinylpyrrolidone-coated AgNPs in

water. The content of metallic silver in

Argovit preparation is 12 mg/ml, stabilized

with 188 mg/ml of polyvinylpyrrolidone

(PVP). AgNPs dilutions were calculated

according to metallic silver content in Argovit

preparation. Solutions of AgNPs were

prepared with distilled and sterile water and

were kept at 4°C in darkness.

2.2 Silver nanoparticle characterization

Size distribution and morphology of AgNPs

were determined on the basis of the results

obtained by high-resolution transmission

electron microscopy (HRTEM) using a JEOL-

JEM-2010 microscope. Hydrodynamic radius

and Zeta potential were measured by dynamic

light scattering (DLS) (Malvern Instruments

Zetasizer Nano NS model DTS 1060, UK)

Revista de Ciencias Tecnológicas (RECIT). Volumen 2 (1): 20-25

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equipped with a green laser operating at λ =

532 nm at 25 ºC. AgNPs were characterized

by UV–Vis spectroscopy in the range of 200

to 900 nm using a Cary 60 UV-Vis

spectrophotometer (Agilent Technologies,

Santa Clara, CA, USA). Further

characterization of lyophilized Argovit was

performed with Fourier transform infrared

spectroscopy-attenuated total reflectance

(FTIR-ATR) analysis in the range of 400 to

4000 cm-1 with a resolution of 2 cm-1 on a

universal diamond ATR top plate accessory

(Perkin Elmer, USA); the sample spectrum

was compared with that of standard solid PVP

(Mw 100kD).

2.3 Method of topical AgNPs application

The novel method for topical administration of

AgNPs for DFU treatment was developed by

Med. César Alejandro Almonaci Hernández.

The researching protocol for this study was

approved by the Bioethic Committee of the

General Hospital of Tijuana, Baja California

Mexico. All patients included in this study

signed an informed consent letter.

2.4 Clinical cases

Here it is presented two clinical cases of

diabetic patients with a DFU of stage II

according to Wagner classification. The first

patient is a 46-years old male with a 10 years’

evolution of controlled type-2 diabetes. He

presented a non-improved plantar ulcer in the

right forefoot at metatarsus level without

vascular involvement. DFU area is

approximately 4 cm. After surgical cleaning

protocol and treatment with conventional

antibiotics, the surface area of the wound did

not diminished and no significant

improvement was noticed. Second patient is a

40- years old male with 8 years’ evolution of

insulin- controlled type 2-diabetes. Patient

referred the wound appear after 2 weeks of

been subjected to an Achilles tendon surgery.

He presented a non- improved asymmetric

ulcer in the back face of right leg with 5 cm in

length. DFU presents a red-brown color with

yellow patches of dispersed granular tissue.

Although infection was not detected, wound

could not be cured after almost 8 months.

After surgical cleaning protocol and treatment

with conventional antibiotics, the surface area

of the wound did not diminished and no

significant improvement was noticed.

Therefore patient was included in this study.

2.5 AgNPs treatment of foot ulcer in diabetic

patients

The treatment of DFU with AgNPs, started

after the patient signed an informed consent

letter given his approval to be included in the

protocol.

Afterwards, a complete exploration of the

wound area, debridement when necessary and

cleaning with surgical soap solution. Was

performed. Then topical administration of

AgNPs solution (with a metallic silver

concentration of 1.2 mg/ml) was done firstly,

and this treatment was repeated every 24 h for

12 days. The amount of metallic silver to be

applied in the injury was calculated on the

basis of the content of metallic silver present

in the AgNPs solution Argovit, as mentioned

in section 2.1.

To record the evolution of the wound healing

process, photographs were taken before and

after the treatment with AgNPs.

3. Results

3.1. Silver nanoparticle characterization

Our group has recently reported

physicochemical characteristic of AgNps [18].

In summary, AgNPs are spheroidal in

morphology, the size distribution goes from 1

to 90 nm with an average size of 35 nm, and

Revista de Ciencias Tecnológicas (RECIT). Volumen 2 (1): 20-25

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their hydrodynamic radio considering both the

PVP covering and the metallic silver

nanoparticle is 70 nm. Zeta potential of

AgNPs is -15 mV and their plasmon

absorbance is found at 420 nm wavelength. A

TEM micrograph of the AgNPs used in this

study is shown in figure 1.

Figure 1. TEM image of AgNPs showing their

spheroidal morphology and size.

3.2 Evolution of diabetic foot ulcer healing

The evolution of the wound healing process of

DFU treated with AgNPs was documented by

photography. In figure 2 it is observed the

chronological evolution of DFU from two the

patients. In figure 2A it is shown the initial

appearance of a Wagner II ulcer after the

debridement process. The DFU is located in

the forefoot and imposed on the metatarsal

region of the right foot. The extended area of

the DFU is approximately of 3 cm x 4 cm and

0.4 cm deep, with irregular contours and

hyperkeratosis active edges. After 8 days of

treatment with AgNPs solution, there was an

improvement of the ulcer with reduction of

edema. It exhibits a red coloration without

signs of infection. The outer contour of the

ulcer shows a progressive decrease of granular

tissue and fibrin plaques at the center of the

ulcer. The evolution of the healing process

after 12 days of topical administration of

AgNPs solution, in this picture, a progressive

diminishment of the lesion extent is

noticeable. An evident improvement of the

coping is observed along the edges of the DFU

while granulation tissue is depicted at the

center of the ulcer. Indeed, the central zone of

the ulcer shows a diminishment of its diameter

with an evident improvement in the

pigmentation that confirms the imminent re-

epithelialization process with active edges that

favoring closure of the injury.

Figure 2. Chronological evolution of the lesion of

diabetic foot ulcers classified as Wagner grade II,

treated with topical administration of AgNPs. DFU

from (A) 46 years-old male patient with type-2 diabetes

and (B) 40 years-old male patient with type-2 diabetes.

In both figures (A and B) initial appearance of the ulcer

is shown and the evolution of the DFU after several

days of treatment with AgNPs solution.

Figure 2B depicts the initial Wagner II ulcer

located in the back of the right leg forefoot.

DFU has an extension of 5 cm approximately

with asymmetric edges. After 18 days of

treatment with AgNPs solution, there was

50% of improvement in healing process of the

ulcer. DFU exhibits coping of active edges

through re-epithelialization and no signs of

infection were observed. The contours of DFU

Revista de Ciencias Tecnológicas (RECIT). Volumen 2 (1): 20-25

ISSN: 2594-1925

show a progressive closure. The presence of

granular tissue, red color and well-moisturized

skin was observed. On day 26, the edges of the

wound were active and allowed the coping of

the borders of the lesion reducing its area.

Granular tissue and red color in the middle of

the ulcer were observed. Finally, after 45 days

of treatment with AgNPs, DFU has attempted

the closure in 95%. Granular tissue is observed

along 5 cm. No edema, infection or flush was

detected. Coping of wound edges was

successful and reached the closure of the ulcer.

4. Conclusions

Thus, we present herein two clinical cases of

successful treatment of diabetic foot ulcers of

II degree of Wagner classification with AgNPs

topically administered into the wound. Daily

administration of AgNPs solution with

metallic silver concentrarion of 1.2 mg/mL

causes an improvement of the wound healing

in less than 2 months. To the best of our

knowledge, successful treatment of diabetic

ulcers with AgNPs, discovered by our group,

for the first time was described in our previous

work [16]. This work is the second work of

series of publications of our group dedicated

to clinical cases of rapid wound healing of

diabetic foot ulcers treated with AgNPs. It has

been extensively reported the use of AgNPs

for healing a wide spectrum of wounds

including different types of ulcers. However,

its study for the successful treatment of

diabetic foot ulcers in patients has never been

explored before our two publications (our

previous article [16] and this work).

Therefore, the results of our series of works

will permit to include diabetic ulcers type,

characterized by high extremity amputation

risk, to this spectrum.

5. Acknowledgements

The authors wish to thank the funding through

the International Network of

Bionanotechnology with impact in

Biomedicine, Food and Biosafety

(CONACYT project No. 279889) and to the

CONACYT project No. 269071. This work

was also supported by DGAPA PAPIIT-

UNAM IT200114 project. Authors wish to

thank Dr. Oxana Martyniuk and Sr. Francisco

Ruiz Medina for their technical support.

Authors acknowledge the medical support

given by Carmen Enriques Enriques, Willfred

Cárdenas Acosta, Michel Natay Peraza

Perales, Alejandra Rembao Hernández and

Nora Inés Carricury Chequer. We are grateful

to Dr. Leonel Cota (CNyN-UNAM) for the

studentships given through the CONACYT-

SNI 3 researcher fund. Argovit AgNPs were

kindly donated by Professor Dr. Vasily

Burmistrov from the Scientific and Production

Center Vector-Vita (Russia).

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