Effect of Catalytic Nanomedicine on Amputation-Stage Chronic Venous Ulcers: Four Case Studies

Chronic ulcers are the main complication in patients with microcirculation problems, such as type 2 diabetes and venous insufficiency.^1,2 It is estimated that about 15% to 25% of patients with diabetes present with chronic ulcers³ and face limb amputation rates that are 10 to 20 times higher than those of individuals without diabetes.⁴ Normal wound healing is a multiple and dynamic process that requires the response of several cellular agents to achieve the phases of coagulation, inflammation, epithelial cell proliferation, and tissue maturation.⁵ Nonetheless, defects in reepithelialization and angiogenesis, growth factor deficits, and inefficient mechanisms to remove recurrent infections are major impediments to adequate healing.^6-8 Currently, although there are numerous therapeutic strategies based on the pathophysiology of the chronic ulcer—such as reducing edema, promoting reepithelialization, and addressing underlying diseases and complications (such as multi-strain bacterial infections)^9-11—there is insufficient information to choosing one therapy over another in a generalized manner, so treatment has to be determined on an individual basis.¹² 

New approaches have opted for the design of nanostructured composites capable of disinfection and optimization of the healing response in chronic wounds.¹³ Catalytic nanomedicine has excelled in the development of catalytic nanostructures based on a mixture of oxides with selective antibacterial properties.¹⁴ These compounds, called bionanocatalysts, are capable of crossing the cell wall of a wide range of pathogenic microorganisms, destabilizing key processes of pathogenic microorganisms.¹⁵ The 3-way catalytic functioning of these nanostructures results in the formation of molecular oxygen as a byproduct. While it is not yet fully tested, reoxygenation of the affected area appears to facilitate capillary angiogenesis,¹⁶ as seen in previous studies.^17,18 The result is an optimization of the normal process of tissue regeneration through the improvement of local blood flow conditions and the elimination of the chronic inflammatory response that results from infection. 

Furthermore, the integration of antibacterial agents into nanostructured hydrogels, carefully regulated for viscosity and pH, has demonstrated an enhanced ability to promote both antibacterial efficacy and the spreading capacity of the gel to fully cover the wound.¹⁹ Importantly, this first-line approach also preserves the essential microenvironment conditions of humidity and temperature conducive to optimal wound healing.²⁰

The intent of this study was to evaluate the use of antibacterial and tissue regeneration bionanocatalysts made of copper coated in a titanium dioxide-silicate matrix deployed in a gel in the treatment of 4 patients with amputation-stage chronic ulcers. The impact of their application on wound progression was also studied. 

Treatment

Bionanocatalysts in gel matrix. The mixed titania and silicate matrix was obtained through the hydrolysis and alkoxylation of precursors for both oxides. Synthesis parameters were adjusted according to the previous studies²¹ to optimize the surface-to-volume ratio and particle coating, resulting in a coated powder (bionanocatalyst). Complementary physicochemical characterization was carried out as described previously.²² The bionanocatalysts were embedded in a generic mixed-polymeric gel matrix developed by the authors’ team, given its results in cutaneous application.¹⁹ 

Debridement, gel matrix application, and dressing. The treatment protocol is depicted in Figure 1. Regular, local sharp debridement using scalpels, scissors, or forceps was performed by the same surgeon once a month, following current standard-of-care guidelines.²³ Debridement was performed to achieve the complete removal of nonviable tissue. Other methods to perform ulcer debridement, such as larval, autolytic hydrosurgery, and ultrasonic therapy, were not used. Every second day, enough gel with embedded bionanocatalysts was applied to cover the entire surface of the wound. No further medications or therapies were applied. 

After gel application, wounds were covered with standard-of-care secondary dressings. Treatment was carried out until wounds were fully closed. 

Ethics approval. The research protocol was approved by the Direction of Teaching and Research of the Specialized Center for the Care of Diabetic Patients, Dr. Manuel Gonzalez Rivera, under the Ministry of Health of the Federal District with registration number 101/100/014/13.

Patient 1: A 53-year-old male with a significant clinical history of diabetes, hypertension, and grade 2 obesity presented with a chronic ulcer of 6 months’ duration in the upper third of the outside of his left leg. The recommendation of amputation, as per standard of care, made the patient seek out alternative treatments. On physical examination, the ulcer volume measured 2.10 cm³ (6.2 cm × 3.4 cm × 0.1 cm); the ulcer exhibited moderate, non-fetid exudate and had irregular edges, adherence with edge effect, and hyperkeratotic perilesional skin (Figure 2A). The patient complained of mild pain (5/10 on a visual analog scale [VAS]).

Edema was observed to decrease after 2 weeks of treatment, with full revascularization, elimination of infected tissue, and improvement in the closure of the wound due to fibroblast production. One month from the first application, the wound bed was clean and presented granulation tissue as well as optimal epithelialization. By the time of discharge at the 8-week mark, the wound had closed, showing a normal, fine-line scar without inflammatory signs. Slight hyperpigmentation was still present in the adjacent tissue (Figure 3).

Patient 2: A 79-year-old female with a clinical history of chronic obstructive pulmonary disease due to exposure to wood smoke, which was treated with supplemental oxygen for more than 15 hours a day. She also had systemic arterial hypertension of 12 years’ duration that was managed with losartan (50 mg/day), chronic venous insufficiency (CVI) without treatment, and a saphenectomy in the right leg at the age of 54 years. The patient presented with two chronic ulcers on the left leg, one located in the internal malleolus (10 years’ duration) and the second in the forefoot (6 months’ duration). The patient was experiencing pain of 8/10 per VAS and indicated that she was tired of performing daily wound treatments that caused burning and intense pain that required nonsteroidal anti-inflammatory drugs. The patient had been told that if no improvement was observed in the following month, amputation would be required. 

On physical examination, the first ulcer had a volume of 4.18 cm³ (5.5 cm × 3.8 cm × 0.2 cm), with 30% granulation tissue, 70% sphaceli tissue, scarce exudate, irregular edges adhered without edge effect, and indurated, hyperkeratotic perilesional skin (Figure 1B). Pedial and tibial pulses were present with an ankle brachial index of 1.1. The second ulcer had a volume of 0.39 cm³ (2.2 cm × 1.8 cm × 0.1 cm), with 10% granulation tissue, 90% sphaceli tissue, moderate exudate, irregular edges adhered without edge effect, and erythematous perilesional skin (Figure 2B). 

After 1 week of treatment, the first ulcer had dried and showed sharper edges in the perilesional tissue, and the second ulcer exhibited a reduction in erythema and edema. At 10 weeks, edema had disappeared in the first ulcer, wound size and depth had decreased substantially (4.0 cm × 0.8 cm × 0.1 cm), and granulation tissue formation was present. At 14 weeks, the second ulcer was almost completely closed. The first ulcer was completely epithelialized after 3 months, and the second wound was healed after 6 months of treatment (Figure 4). To avoid recurrence, compression therapy was recommended.

Patient 3: A 68-year-old male with a history of Parkinson’s disease of 6 years’ duration (treated with pramipexol), hypothyroidism of 6 years’ duration (treated with levothyroxine), and arterial hypertension of 8 years’ duration (treated with enalapril). The patient sought consultation for a chronic ulcer of 9 months’ duration in the middle third of the outside of the left leg. He mentioned having received several unsuccessful treatments, including 2 rejected grafts, and had been informed that the next step was an amputation. On physical examination, the ulcer measured 11.25 cm² (2.5 cm × 4.5 cm), and the wound bed exhibited moderate exudate, serous regular edges adhered with edge effect, and healthy perilesional skin (Figure 2C). 

After 1 month of treatment, the ulcer exhibited no sign of suppuration, with sharp edges and edema reduction. After 2 months, the wound had significantly decreased in size and depth (2.0 × 2.8 cm) and presented an erythematous clean bed and granulation tissue. After 3 months of treatment, the ulcer had completely reepithelialized and showed no edema nor hyperpigmentation. Healthy skin had formed in the surroundings of the atrophic scar (Figure 5).

Patient 4: An 86-year-old female with systemic arterial hypertension of 15 years’ duration (treated with candesartan [16 mg/twice daily] and felodipine [5 mg/day]), and sequelae of left hemiplegia secondary to an ischemic cerebral vascular event 10 years previous, had a consultation for a chronic ulcer on the internal malleolus of the right leg of 15 months’ duration. On physical examination, the ulcer had an area of 5.4 cm² (3.0 cm × 1.8 cm) and the wound bed exhibited 20% fibrin tissue and 30% dehydrated necrotic tissue with scarce exudate, irregular edges adhered without edge effect, and a hyperkeratotic perilesional skin with ocher dermatitis (Figure 2D).

The ulcer dried after the first week of treatment, with the appearance of a clean wound bed, granulation tissue, and no necrotic tissue. After 5 months of application, the wound significantly decreased in size (from a total area of 5.4 cm² to a total area of 0.96 cm²). At the 8-month mark, the wound had completely reepithelialized, and no scar was observed (Figure 6). 

The chronic nature of venous ulcers of the lower extremities, the high incidence rate in the general population (1-2%),^24,25 and the disastrous consequences for patients and their families (loss of working days, early retirement, prolonged therapeutic expenses, restriction of daily activities, among others)²⁶ make this condition a problem of crucial importance. Given the diverse etiologies of chronic ulcers— primarily venous (70-80%) and arterial (5-10%), as well as arteriovenous, neuropathic, diabetic, and pressure, among others²⁷—the correct identification of the underlying causes of the condition is crucial for the determination of the ulcer management protocol, especially in cases where an interdisciplinary approach is required not only for the treatment of the wound, per se, but also of the concomitant diseases involved, as is the case with CVI or type 2 diabetes. 

In any scenario, as described in the 4 case studies, the first step in the healing of a nonhealing wound involves debridement and cleansing of the ulcer to remove any biofilm of microorganisms that may have formed on the exposed tissue bed.²⁸ Surgical debridement has been widely used for the removal of nonviable tissue that, if not removed, can lead to necrosis, gangrene, or, as determined for these 4 patients, preemptive limb amputation.²⁹ However, debridement alone is often insufficient to enable reepithelialization of the wound edges. In this regard, Thomas et al³⁰ conducted a comprehensive systematic review of 318 case studies in which they analyzed the role of debridement in wound bed preparation, studying the types of debridement (autolytic, enzymatic, biological, mechanical, and surgical). After analysis, the authors concluded that, although promising, both new and traditional debridement therapies offer inconclusive results, especially considering that patient safety is not always clearly defined. Moreover, in several forms of debridement, the risk for reinfection is increased due to tissue exposure and further morbidity. While debridement remains the first approach to managing a chronic wound, it is not the only one; if no improvement is observed within 2 weeks, additional treatments are necessary.³¹

Along these lines, catalytic nanomedicine and bionanocatalysts with antibacterial and wound support properties can help mitigate the reinfection risk in chronic wounds. As previously described, the holistic functioning of bionanocatalysts originates from the intrinsic antibacterial capacity of coated elements in the nanostructure that constitutes these nanoparticles.³² As evidenced in Figure 7, the evaluation of the nanostructures by transmission electron microscopy (TEM) demonstrates a crystallinity pattern indicative of the presence of the titanium (TiO₂) and silicone (SiO₂) mixture,³³ in addition to evidencing the selected size (<5 nm), characteristic of the individual unit of the bionanocatalysts.³⁴

This is one of the main advantages of catalytic nanomedicine, since the miniaturization of these materials enables the development of high surface areas within the material (Table 1), which translates into larger contact zones and, therefore, a more effective reaction.³⁵ This coating phenomenon potentiates the intrinsic activity of the metal, so the addition of a lower concentration is necessary to acquire similar effects to those from bulk metal.³⁶ In a recent review of the application of catalytic nanomedicine in the treatment of chronic wounds, the mechanism of action of bionanocatalysts was proposed.¹⁴ Normally, the organism has intrinsic mechanisms for tissue regeneration in the face of wounds. Upon tissue discontinuity, the coagulation cascade is activated.³⁷ 

The mediators released during this process complement activation and platelet adhesion/aggregation to constitute a potent stimulus for the influx of inflammatory cells to the wound site.³⁸ The polymorphonuclear neutrophil is the first cell to arrive on the scene: its basic function is to protect from infection, phagocytizing microorganisms and cellular debris from the affected tissue.³⁹ This antibacterial action depends largely on the adequate supply of oxygen,⁴⁰ which forms unstable radicals with polyunsaturated fatty acids that destabilize the biological membranes of microorganisms.⁴¹ However, it is precisely this crucial first step that is impaired in patients with microcirculatory problems. Given the impaired neutrophil response, the wound becomes susceptible to contamination by agents as varied as Pseudomonas aeruginosa, Klebsiella pneumoniae, Serratia marcencens, Proteus vulgaris, Enterophathogenic Escherichia coli (EPEC), Salmonella typhimurium, Shigella dysenteriae, Staphylococcus aureus (ATCC 25923), and methicillin-resistant Staphylococcus aureus (MRSA).⁴² The diversity and antibiotic susceptibility pattern of these microorganisms make their widespread treatment extremely difficult. In addition, this polymicrobial aggregation can lead to the appearance of biopolymers that allow the adhesion of microorganisms to surfaces, their cohesion, and their resistance to biocidal agents.⁴³ These biofilms stimulate a chronic inflammatory response that results in the constant recruitment of macrophages and neutrophils,⁴⁴ which come to the wound site at the levels allowed by the patient’s affected microcirculation. The main problem at this point is that the action of neutrophils is ephemeral, so they die quickly following the phagocytosis of microbes.⁴⁵ Normally, such action is necessary because the apoptosis of these neutrophils results in the release of their intracellular content, which becomes part of the inflammatory exudate necessary to promote moist wound healing.⁴⁶ However, with chronic inflammation and inefficient clearance of the infection, the exudate becomes a source of nutrients for the perpetuation of the biofilm, as hypothesized by Lawrence et al.⁴⁷

Bionanocatalysts exhibit a broad-spectrum antibacterial capacity, as previous studies have shown.^48-49 In addition, a previous prospective study demonstrated their ability to eliminate infections despite the presence of biofilms.¹⁸ This property seems to be linked both to their size (which allows greater penetration through the polymeric structures of the biofilm) and to their organic surface coating, which endows them with biocompatibility and selectivity for damaged cells without apparent effect on the surrounding tissues.⁵⁰ In addition, the elimination of these organisms results in the appearance of molecular oxygen that seems to favor angiogenesis in the area, as evidenced by the appearance of reinnervated tissue in these and other case studies.^18,51

Thus, once the main impediment to the proper action of the coagulation processes has been eliminated, the wound can heal following the normal mechanisms of tissue regeneration and granulation which, although diminished in patients with microcirculation disorders, can act in the absence of biofilm. 

Notably, although the mechanism of action of bionanocatalysts is known and their effectiveness against bacteria has been demonstrated, the way biofilm remotion is catalyzed by these nanostructures remains to be elucidated. Future research should focus on the catalytic processes that occur between bionanocatalysts and biomolecules in biofilms to determine the complete mechanism of action.

Case series can be prone to bias, which limits their generalizability to larger populations of patients. Nonetheless, the information extracted from the healing processes of the 4 patients evaluated in the current study allows researchers to hypothesize on the efficacy and mechanism of action of the bionanocatalysts in chronic wound healing. In addition, it is important to highlight that a complementary surgical debridement treatment was also applied, which standard-of-care practices note is important for the effectiveness of the treatment. In the present study, no direct comparison was made against treatment with debridement alone, other than a comparison with the literature. This was done for the benefit of the patients because of the insufficiency of debridement as the only treatment for chronic wounds. However, such a comparison will be made in future work.

The use of bionanocatalysts incorporated into a gel, in conjunction with monthly surgical debridement, efficiently enabled tissue generation in 4 patients with chronic venous ulcers destined for amputation. In all cases, complete wound closure was observed in terms of tissue reepithelialization. The patients reported no side effects from the application of the treatment, which supports previous extensive research into the biocompatibility of these nanostructured agents. This work offers a reinterpretation of the working hypothesis on the mechanism of action of bionanocatalysts in the tissue regeneration of chronic wounds; however, further research is needed, especially on their action on biofilms. The authors recommend future study of the impact of bionanocatalysts on the normal processes of the signaling cascade to determine whether there is an interaction between bionanocatalysts and these bioprocesses.