Preclinical safety of negatively charged microspheres (NCMs): Optimization of radiolabeling for in vivo and ex vivo biodistribution studies after topical administration on full-thickness wounds in a rat model.

Negatively charged microspheres (NCMs) are postulated as a new form of treatment for chronic wounds. Despite the efficacy shown at clinical level, more studies are required to demonstrate their safety and local effect. The objective of the work was to confirm the lack of NCM systemic absorption performing a biodistribution study of the NCMs in an open wound rat animal model. To this end, radiolabeling of NCMs with technetium-99 m was optimized and biodistribution studies were performed by in vivo SPEC/CT imaging and ex vivo counting during 24 h after topical administration. The studies were performed on animals treated with a single or repeated dose to study the effect of macrophages during a prolonged treatment. NCM radiolabeling was achieved in a simple, efficient and stable manner with high yield. SPECT/CT images showed that almost all NCMs (about 85 %) remained on the wound for 24 h either after single or multiple administrations. Ex vivo biodistribution studies confirmed that there was no accumulation of NCMs in any organ or tissue except in the wound area, suggesting a lack of absorption. In conclusion, NCMs can be considered safe as local wound treatment since they remain at the administration area.

Generation of a novel human dermal substitute functionalized with antibiotic-loaded nanostructured lipid carriers (NLCs) with antimicrobial properties for tissue engineering.

BACKGROUND: Treatment of patients affected by severe burns is challenging, especially due to the high risk of Pseudomonas infection. In the present work, we have generated a novel model of bioartificial human dermis substitute by tissue engineering to treat infected wounds using fibrin-agarose biomaterials functionalized with nanostructured lipid carriers (NLCs) loaded with two anti-Pseudomonas antibiotics: sodium colistimethate (SCM) and amikacin (AMK). RESULTS: Results show that the novel tissue-like substitutes have strong antibacterial effect on Pseudomonas cultures, directly proportional to the NLC concentration. Free DNA quantification, WST-1 and Caspase 7 immunohistochemical assays in the functionalized dermis substitute demonstrated that neither cell viability nor cell proliferation were affected by functionalization in most study groups. Furthermore, immunohistochemistry for PCNA and KI67 and histochemistry for collagen and proteoglycans revealed that cells proliferated and were metabolically active in the functionalized tissue with no differences with controls. When functionalized tissues were biomechanically characterized, we found that NLCs were able to improve some of the major biomechanical properties of these artificial tissues, although this strongly depended on the type and concentration of NLCs. CONCLUSIONS: These results suggest that functionalization of fibrin-agarose human dermal substitutes with antibiotic-loaded NLCs is able to improve the antibacterial and biomechanical properties of these substitutes with no detectable side effects. This opens the door to future clinical use of functionalized tissues.

Overcoming the Inflammatory Stage of Non-Healing Wounds: In Vitro Mechanism of Action of Negatively Charged Microspheres (NCMs).

Negatively charged microspheres (NCMs) represent a new therapeutic approach for wound healing since recent clinical trials have shown NCM efficacy in the recovery of hard-to-heal wounds that tend to stay in the inflammatory phase, unlocking the healing process. The aim of this study was to elucidate the NCM mechanism of action. NCMs were extracted from a commercial microsphere formulation (PolyHeal® Micro) and cytotoxicity, attachment, proliferation and viability assays were performed in keratinocytes and dermal fibroblasts, while macrophages were used for the phagocytosis and polarization assays. We demonstrated that cells tend to attach to the microsphere surface, and that NCMs are biocompatible and promote cell proliferation at specific concentrations (50 and 10 NCM/cell) by a minimum of 3 fold compared to the control group. Furthermore, NCM internalization by macrophages seemed to drive these cells to a noninflammatory condition, as demonstrated by the over-expression of CD206 and the under-expression of CD64, M2 and M1 markers, respectively. NCMs are an effective approach for reverting the chronic inflammatory state of stagnant wounds (such as diabetic wounds) and thus for improving wound healing.

Safety and effectiveness of sodium colistimethate-loaded nanostructured lipid carriers (SCM-NLC) against P. aeruginosa: in vitro and in vivo studies following pulmonary and intramuscular administration.

The usefulness of nanotechnology to increase the bioavailability of drugs and decrease their toxicity may be a tool to deal with multiresistant P. aeruginosa (Mr-Pa) respiratory infections. We describe the preparation and the in vivo efficacy and safety of sodium colistimethate-loaded nanostructured lipid carriers (SCM-NLC) by the pulmonary and intramuscular routes. Nanoparticles showed 1-2 mg/L minimum inhibitory concentration against eight extensively drug-resistant P. aeruginosa strains. In vivo, SCM-NLC displayed significantly lower CFU/g lung than the saline and similar to that of the free SCM, even the dose in SCM-NLC group was lower than free SCM. There was no tissue damage related to the treatments. Biodistribution assessments showed a mild systemic absorption after nebulization and a notorious absorption after IM route. Altogether, it could be concluded that SCM-NLC were effective against P. aeruginosa in vivo, not toxic and distribute efficiently to the lung and liver after pulmonary or intramuscular administrations.

Composite nanofibrous membranes of PLGA/Aloe vera containing lipid nanoparticles for wound dressing applications.

Electrospun nanofibrous dressings present suitable characteristics to be used in wound healing, such as high porosity and high surface area-to-volume ratio. In this study, a wound dressing based on PLGA and Aloe vera containing lipid nanoparticles (NLCs) was developed. NLCs were added in order to add a lipid component that could avoid the adhesion of the dressing to the wound and improve its handling. Membranes with and without NLCs were composed of uniform fibers of about 1 µm in diameter. Their porosity was above 80% and their thickness was about 160 µm. Both dressings showed similar water vapour transmission rate 1100 g/m2day. The formulation containing NLCs presented a higher ultimate tensile strength (2.61 ±â€¯0.46 MPa) and a higher water uptake. Both formulations were biocompatible in vitro. Furthermore, the cell adhesion assay demonstrated that both membranes had a low adherence profile, although it was lower with the dressing containing NLCs. Finally, their efficacy was evaluated in a full thickness wound healing assay conducted in db/db mice, where both enhanced healing similarly. Accordingly, the PLGA-AV-NLC membrane might be a promising strategy for the treatment of chronic wounds, since it improved handling in comparison to the formulation without NLCs.

Immuno-modulatory effect of local rhEGF treatment during tissue repair in diabetic ulcers.

Wound healing is a complex process that can be severely impaired due to pathological situations such as diabetes mellitus. Diabetic foot ulcers are a common complication of this pathology and are characterized by an excessive inflammatory response. In this work, the effects of local treatment with recombinant human epidermal growth factor (rhEGF) were studied using a full-thickness wound healing model in streptozotocin-induced diabetic rats. Wound healing process was assessed with different concentrations of rhEGF (0.1, 0.5, 2.0 and 8.0 µg/mL), placebo and both diabetic and non-diabetic controls (n = 53). The macroscopic healing observed in treated diabetic rats was affected by rhEGF concentration. Histologically, we also observed an improvement in the epithelialization, granulation tissue formation and maturation in treated groups, finding again the best response at doses of 0.5 and 2.0 µg/mL. Afterwards, the tissue immune response over time was assessed in diabetic rats using the most effective concentrations of rhEGF (0.5 and 2.0 µg/mL), compared to controls. The presence of macrophages, CD4+ T lymphocytes and CD8+ T lymphocytes, in the reparative tissue was quantified, and cytokine expression was measured by quantitative real-time PCR. rhEGF treatment caused a reduction in the number of infiltrating macrophages in the healing tissue of diabetic, as well as diminished activation of these leukocytes. These findings show that local administration of rhEGF improves the healing process of excisional wounds and the quality of the neoformed tissue in a dose-dependent manner. Besides, this treatment reduces the local inflammation associated with diabetic healing, indicating immuno-modulatory properties.

Novel nanofibrous dressings containing rhEGF and Aloe vera for wound healing applications.

Nanofibrous membranes produced by electrospinning possess a large surface area-to-volume ratio, which mimics the three-dimensional structure of the extracellular matrix. Thus, nanofibrous dressings are a promising alternative for chronic wound healing, since they can replace the natural ECM until it is repaired. Therefore, in this study we have developed a PLGA nanofibrous membrane that contains recombinant human Epidermal Growth Factor (rhEGF) and Aloe vera (AV) extract. Both of them promote wound healing, as EGF is a wound healing mediator and AV stimulates the proliferation and activity of fibroblast. The obtained membranes were composed of uniform and randomly oriented fibers with an average diameter of 356.03±112.05nm, they presented a porosity of 87.92±11.96% and the amount of rhEGF was 9.76±1.75µg/mg. The in vitro viability assay demonstrated that the membranes containing rhEGF and AV improved fibroblast proliferation, revealing the beneficial effect of the combination. Furthermore, these membranes accelerated significantly wound closure and reepithelisation in an in vivo full thickness wound healing assay carried out in db/db mice. Overall, these findings demonstrated the potential of PLGA nanofibers containing rhEGF and AV for the treatment of chronic wounds.

LL37 loaded nanostructured lipid carriers (NLC): A new strategy for the topical treatment of chronic wounds.

The LL37 is a human antimicrobial peptide which not only has a broad spectrum of antimicrobial activity, but it has also been proved to modulate wound healing by participating in angiogenesis, epithelial cell migration and proliferation, and immune response. In this work, LL37 has been encapsulated in nanostructured lipid carriers (NLCs), produced by the melt-emulsification method, in order to improve its effectiveness. The characterisation of the NLC-LL37 showed a mean size of 270nm, a zeta potential of -26mV and an encapsulation efficiency of 96.4%. The cytotoxicity assay performed in Human Foreskin Fibroblasts demonstrated that the NLC-LL37 did not affect cell viability. Moreover, the in vitro bioactivityassay evidenced that the peptide remained active after the encapsulation, since the NLC-LL37 reversed the activation of the macrophages induced by LPS in the same way as the LL37 in solution. In addition, the in vitro antimicrobial assay revealed the NLC-LL37 activity against Escherichia coli. The effectiveness of the nanoparticles was assessed in a full thickness wound model indb/dbmice. The data demonstrated that NLC-LL37 significantly improved healing compared to the same concentration of the LL37 solution in terms of wound closure, reepithelisation grade and restoration of the inflammatory process. Overall, these findings suggest a promising potential of the NLC-LL37 formulation for chronic wound healing.

Advances in drug delivery systems (DDSs) to release growth factors for wound healing and skin regeneration.

Current advances in novel drug delivery systems (DDSs) to release growth factors (GFs) represent a great opportunity to develop new therapies or enhance the effectiveness of available medical treatments. These advances are particularly relevant to the field of regenerative medicine, challenging healthcare issues such as wound healing and skin repair. To this end, biocompatible biomaterials have been extensively studied to improve in vivo integration of DDSs, to enhance the bioactivity of the released drugs and to deliver bioactive molecules in a localised and controlled manner. Thus, this review presents an overview of DDSs to release GFs for skin regeneration, particularly emphasising on (i) polymeric micro and nanospheres, (ii) lipid nanoparticles, (iii) nanofibrous structures, (iv) hydrogels and (v) scaffolds. In addition, this review summarises the current animal models available for studying wound healing and the clinical trials and marketed medications based on GF administration indicated for chronic wound treatment. FROM THE CLINICAL EDITOR: Chronic wounds currently pose a significant burden worldwide. With advances in science, novel drug delivery systems have been developed for growth factors delivery. In this comprehensive review, the authors highlighted current drug delivery systems for the enhancement of wound healing and their use in clinical settings.

The topical administration of rhEGF-loaded nanostructured lipid carriers (rhEGF-NLC) improves healing in a porcine full-thickness excisional wound model.

The development of an effective treatment able to reduce the healing time of chronic wounds is a major health care need. In this regard, our research group has recently demonstrated the in vivo effectiveness of the topical administration of rhEGF-loaded lipid nanoparticles in healing-impaired db/db mice. Here we report the effectiveness of rhEGF-NLC (rhEGF loaded nanostructured lipid carriers) in a more relevant preclinical model of wound healing, the porcine full-thickness excisional wound model. The rhEGF-NLC showed a particle size of around 335nm, negative surface charge (-27mV) and a high encapsulation efficiency of 94%. rhEGF plasma levels were almost undetectable, suggesting that no systemic absorption occurred, which may minimise potential side effects and improve treatment safety. In vivo healing experiments carried out in large white pigs demonstrated that 20µg of rhEGF-NLC topically administered twice a week increased the wound closure and percentage of healed wounds by day 25, compared with the same number of intralesional administrations of 75µg free rhEGF and empty NLC. Moreover, rhEGF-NLC improved the wound healing quality expressed in terms of number of arranged microvasculature, fibroblast migration and proliferation, collagen deposition and evolution of the inflammatory response. Overall, these findings demonstrated that topically administered rhEGF-NLC may generate de novo intact skin after full thickness injury in a porcine model, thereby confirming their potential clinical application for the treatment of chronic wounds.

A novel strategy for the treatment of chronic wounds based on the topical administration of rhEGF-loaded lipid nanoparticles: In vitro bioactivity and in vivo effectiveness in healing-impaired db/db mice.

Lipid nanoparticles are currently receiving increasing interest because they permit the topical administration of proteins, such as recombinant human epidermal growth factor (rhEGF), in a sustained and effective manner. Because chronic wounds have become a major healthcare burden, the topical administration of rhEGF-loaded lipid nanoparticles, namely solid lipid nanoparticles (SLN) and nanostructured lipid carries (NLC), appears to be an interesting and suitable strategy for the treatment of chronic wounds. Both rhEGF-loaded lipid nanoparticles were prepared through the emulsification-ultrasonication method; however, the NLC-rhEGF preparation did not require the use of any organic solvents. The characterisation of the nanoparticles (NP) revealed that the encapsulation efficiency (EE) of NLC-rhEGF was significantly greater than obtained with SLN-rhEGF. The in vitro experiments demonstrated that gamma sterilisation is a suitable process for the final sterilisation because no loss in activity was observed after the sterilisation process. In addition, the proliferation assays revealed that the bioactivity of the nanoformulations was even higher than that of free rhEGF. Finally, the effectiveness of the rhEGF-loaded lipid nanoparticles was assayed in a full-thickness wound model in db/db mice. The data demonstrated that four topical administrations of SLN-rhEGF and NLC-rhEGF significantly improved healing in terms of wound closure, restoration of the inflammatory process, and re-epithelisation grade. In addition, the data did not reveal any differences in the in vivo effectiveness between the different rhEGF-loaded lipid nanoparticles. Overall, these findings demonstrate the promising potential of rhEGF-loaded lipid nanoparticles, particularly NLC-rhEGF, for the promotion of faster and more effective healing and suggest their future application for the treatment of chronic wounds.

rhEGF-loaded PLGA-Alginate microspheres enhance the healing of full-thickness excisional wounds in diabetised Wistar rats.

Diabetic foot ulcers (DFUs) represent a major clinical challenge in the ageing population. To address this problem, rhEGF-loaded Poly-Lactic-co-Glycolic-Acid (PLGA)-Alginate microspheres (MS) were prepared by a modified w/o/w-double-emulsion/solvent evaporation method. Different formulations were evaluated with the aim of optimising MSs properties by adding NaCl to the surfactant solution and/or the solvent removal phase and adding alginate as a second polymer. The characterisation of the developed MS showed that alginate incorporation increased the encapsulation efficiency (EE) and NaCl besides increasing the EE also became the particle surface smooth and regular. Once the MS were optimised, the target loading of rhEGF was increased to 1% (PLGA-Alginate MS), and particles were sterilised by gamma radiation to provide the correct dosage for in vivo studies. In vitro cell culture assays demonstrated that neither the microencapsulation nor the sterilisation process affected rhEGF bioactivity or rhEGF wound contraction. Finally, the MS were evaluated in vivo for treatment of the full-thickness wound model in diabetised Wistar rats. rhEGF MS treated animals showed a statistically significant decrease of the wound area by days 7 and 11, a complete re-epithelisation by day 11 and an earlier resolution of the inflammatory process. Overall, these findings demonstrate the promising potential of rhEGF-loaded MS (PLGA-Alginate MS) to promote faster and more effective wound healing, and suggest its possible application in DFU treatment.