Scientific resources: patents and publications

Researchers: Gainza, E., Villullas, S., Ibarrola, O., Gainza, G., Herrán, E., Aguirre, J.J., Del Pozo, A., Pedraz, J.L., Esquisabel, A., Moreno, M., Pastor, M., Viñas, M., Vinuesa, T., Bachiller, D.

Title: Tobramycin lipid nanoparticles (P201431894)

Priority country: Spain

Priority date: 19/12/2014

Countries to which it has spread: Europe, Brazil, Colombia, USA

Summary: The present invention relates to a lipid nanoparticle understood as a matrix comprising a core of lipid nature and / or lipophilic in mixtures of solid lipids and liquids that conveys at least one antibiotic tobramycin for use in the treatment and / or prevention of infections in the respiratory tree. In this sense, the studies carried out by the inventors have demonstrated the ability of these lipid nanoparticles, as well as the pharmaceutical compositions and / or drugs that comprise these nanoparticles, to obtain a stable lipid nanoparticle with sustained and / or regulated release effect of the antibiotic, protect the antibiotic from premature degradation, have the ability to penetrate the biofilm generated by the bacteria themselves, and obtain better values of minimum inhibitory concentration than the free antibiotic.

Researchers: Gainza, E., Gainza, G., Ibarrola, O., Villullas, S., Pastor, M., Del Pozo, A., Pedráz, J.L., Hernández, R.M. and Igartua, M

Title: Lipid nanoparticles for wound healing (EP13382275.9)

Priority country: Spain

Priority date: 04/07/2013

Countries to which it has spread: Europe, Brazil, Colombia, USA, Russia, China and Japan.

Summary: The invention relates to lipid nanoparticles comprising a growth factor and / or an antimicrobial lipid and its method of preparation. It also refers to pharmaceutical compositions comprising such lipid nanoparticles and a pharmaceutically acceptable vehicle. Finally, it refers to that pharmaceutical composition for use as pharmaceutical compositions for wound healing, in particular as a suspension of nanoparticles, dressings or gels for topical application.

Researchers: Gainza, E., Del Pozo, A., Gainza, G., Ibarrola, O., Villullas, S., Fernández, R., Bachiller, D., Pedraz, J.L., Esquisabel, A., Pastor, M., Fuste, E., Sans, E. and Gil, I.

Title: Lipid nanoparticles of polymyxin (13382268)

Priority country: Spain

Priority date: 04/07/2013

Countries to which it has spread: Europe (Spain, Italy, France, Germany, United Kingdom) and the USA. Under evaluation in Brazil, Colombia.

Summary: The present invention relates to a lipid nanoparticle comprising at least one antibiotic of the family of polymyxins, a lipid fraction and one or more surfactants, for use in the prevention and / or treatment of infections of the respiratory tree. These nanoparticles are able to adhere to or interact with the mucous layer of the respiratory tract or the biofilm generated by the bacteria themselves, favoring that at a lower therapeutic dose of antibiotic optimal minimum inhibitory concentration results are obtained. The nanoparticles of the invention are protected against premature degradation and also have a sustained release of the antibiotic in the alveolar and bronchial epithelium.

Researchers: Gainza, E., Gainza, G., Ibarrola, O., Villullas, S., Del Pozo, A., Pedraz, J.L., Hernández, R.M. and Igartua, M.

Title: Microparticles with EGF, method of preparation and use (EP12382476.5)

Priority country: Spain

Priority date: 30/11/2012

Countries to which it has spread: Spain

Summary: The present invention relates to microparticles comprising biocompatible polymers and epidermal growth factor. It also refers to the method of preparation of these microparticles and their use to promote wound healing. With the microparticles of the present invention, high efficiencies of encapsulation of EGF (up to 88%) and a sustained release of EGF for at least 30 days are achieved, which allows to obtain excellent healing results, even with a single administration. In addition, the sustained release rate of EGF is of the order of nanograms, which is also an advantage since prolonged exposures to higher concentrations of EGF can favor cell proliferation, which can be aggressive for the body.

Hameed Z, Garcia-Zapirain B, Aguirre JJ, Isaza-Ruget MA. Multiclass classification of breast cancer histopathology images using multilevel features of deep convolutional neural network. Sci Rep. 2022;12(1):15600.

Breast cancer is a common malignancy and a leading cause of cancer-related deaths in women worldwide. Its early diagnosis can significantly reduce the morbidity and mortality rates in women. To this end, histopathological diagnosis is usually followed as the gold standard approach. However, this process is tedious, labor-intensive, and may be subject to inter-reader variability. Accordingly, an automatic diagnostic system can assist to improve the quality of diagnosis. This paper presents a deep learning approach to automatically classify hematoxylin-eosin-stained breast cancer microscopy images into normal tissue, benign lesion, in situ carcinoma, and invasive carcinoma using our collected dataset. Our proposed model exploited six intermediate layers of the Xception (Extreme Inception) network to retrieve robust and abstract features from input images. First, we optimized the proposed model on the original (unnormalized) dataset using 5-fold cross-validation. Then, we investigated its performance on four normalized datasets resulting from Reinhard, Ruifrok, Macenko, and Vahadane stain normalization. For original images, our proposed framework yielded an accuracy of 98% along with a kappa score of 0.969. Also, it achieved an average AUC-ROC score of 0.998 as well as a mean AUC-PR value of 0.995. Specifically, for in situ carcinoma and invasive carcinoma, it offered sensitivity of 96% and 99%, respectively. For normalized images, the proposed architecture performed better for Makenko normalization compared to the other three techniques. In this case, the proposed model achieved an accuracy of 97.79% together with a kappa score of 0.965. Also, it attained an average AUC-ROC score of 0.997 and a mean AUC-PR value of 0.991. Especially, for in situ carcinoma and invasive carcinoma, it offered sensitivity of 96% and 99%, respectively. These results demonstrate that our proposed model outperformed the baseline AlexNet as well as state-of-the-art VGG16, VGG19, Inception-v3, and Xception models with their default settings. Furthermore, it can be inferred that although stain normalization techniques offered competitive performance, they could not surpass the results of the original dataset.

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Garcia-Orue I, Santos-Vizcaino E, Sanchez P, Gutierrez FB Aguirre JJ, Hernandez RM, Igartua M). Bioactive and degradable hydrogel based on human platelet-rich plasma fibrin matrix combined with oxidized alginate in a diabetic mice wound healing model. 1. Mater Sci Eng C Mater Biol 2022;135:112695

In the present study we developed an injectable, bioactive and degradable hydrogel composed of alginate at 2.5% oxidation degree and calcium-activated platelet rich plasma (PRP) for wound healing applications (PRP-HG-2.5%). The alginate gives mechanical support to the hydrogel while the activated PRP provides growth factors that enhance wound healing and fibrin which creates an adequate microenvironment for cell migration and proliferation. The rheological and mechanical properties of the hydrogel were characterized. Further characterization revealed that PRP-HG-2.5% showed a faster 4ydrolytic degradation rate than unmodified alginate and a similar platelet derived growth factor (PDGF-BB) release profile. In vitro efficacy studies, carried out in human fibroblasts and keratinocytes, showed that PRP-HG-2.5% was not cytotoxic and that it was able to promote cell adhesion and proliferation. Thereafter, in an in vivo full thickness wound healing study conducted in diabetic mice, no differences were found among PRP-HG-2.5% and its counterpart without PRP, likely due to the xenogeneic origin of the PRP. This hypothesis was validated in vitro, since a cytotoxic effect was observed after human PRP application to mouse fibroblasts. Therefore, PRP-HG-2.5% might be a promising strategy for chronic woundstreatment, although its effectiveness should be evaluated in a more reliable preclinical model.

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Collantes M, Vairo C, Erhard Á, Navas C, Villullas S, Ecay M, Pareja F, Quincoces G, Gainza G, Peñuelas I. 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. Eur J Pharm Biopharm. 2022 Aug;177:61-67.

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.

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Luengo, Y.; Díaz-Riascos, Z.V.; García-Soriano, D.; Teran, F.J.; Artés-Ibáñez, E.J.; Ibarrola, O.; Somoza, Á.; Miranda, R.; Schwartz, S., Jr.; Abasolo, I.; et al. Fine Control of In Vivo Magnetic Hyperthermia Using Iron Oxide Nanoparticles with Different Coatings and Degree of Aggregation. Pharmaceutics 2022, 14, 1526.

The clinical implementation of magnetic hyperthermia has experienced little progress since the first clinical trial was completed in 2005. Some of the hurdles to overcome are the reliable production of magnetic nanoparticles with controlled properties and the control of the temperature at the target tissue in vivo. Here, forty samples of iron oxide superparamagnetic nanoparticles were prepared by similar methods and thoroughly characterized in terms of size, aggregation degree, and heating response. Selected samples were intratumorally administered in animals with subcutaneous xenografts of human pancreatic cancer. In vivo experiments showed that it is possible to control the rise in temperature by modulating the field intensity during in vivo magnetic hyperthermia protocols. The procedure does not require sophisticated materials and it can be easily implemented by researchers or practitioners working in magnetic hyperthermia therapies.

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Alex Zabeo, Fabio Rosad,Lisa Pizzol, Fanny Caputo, Sven Even Borgos, Jeremie Parot, Robert E. Geertsma, Joost Jacob Pouw, Rob J. Vandebriel, Oihane Ibarrola Moreno, Danail Hristozov. A Decision Support System for preclinical assessment of nanomaterials in medical products: the REFINE DSS. Drug Delivery and Translational Research (2022) 12:2101–2113.

The application of nanomaterials in medicine has led to novel pharmaceuticals and medical devices that have demonstrated a strong potential for increasing the efficacy/performance and safety of therapeutic and diagnostic procedures to address a wide range of diseases. However, the successful translation of these technologies from their inception (proof-of-concept) to clinical practice has been challenged by substantial gaps in the scientific and technical capacity of R&D companies, especially SMEs, to keep up with the ever-evolving regulatory expectations in the emerging area of nanomedicine. To address these challenges, the EU Horizon 2020 project REFINE has developed a Decision Support System (DSS) to support developers of nanotechnology-enabled health products in bringing their products to the clinic. The REFINE DSS has been developed to support experts, innovators, and regulators in the implementation of intelligent testing strategies (ITS) for efficient preclinical assessment of nanotechnology-enabled health products. The DSS applies logical rules provided by REFINE experts which generate prioritized lists of assays to be performed (i.e. ITSs) for physicochemical characterisation and for immunotoxicological endpoints. The DSS has been tested against several case studies and was validated by internal project experts as well as external ones.

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Jesús Chato-Astrain, David Sánchez-Porras, Óscar Darío García-García, Claudia Vairo, María Villar-Vidal, Silvia Villullas, Indalecio Sánchez-Montesinos Fernando Campos, Ingrid Garzón, Miguel Alaminos. “Improvement of cell culture methods for the successful generation of human keratinocyte primary cell cultures using EGF-loaded nanostructured lipid carriers”. Accepted 4.nov Biomedicines. 2021;(9):1634.

Human skin keratinocyte primary cultures can be established from skin biopsies with culture media containing epithelial growth factor (EGF). Although current methods are efficient, optimization is required to accelerate the procedure and obtain these cultures in less time. In the present study, we evaluated the effect of novel formulations based on EGF-loaded nanostructured lipid carriers (NLC). First, biosafety of NLC containing recombinant human EGF (NLC-rhEGF) was verified in immortalized skin keratinocytes and cornea epithelial cells, and in two epithelial cancer cell lines, by quantifying free DNA released to the culture medium. Then we established primary cell cultures of human skin keratinocytes with basal culture media (BM) and BM supplemented with NLC-rhEGF, liquid EGF (L-rhEGF), or NLC alone (NLC-blank). The results showed that cells isolated by enzymatic digestion and cultured with or without a feeder layer had a similar growth rate regardless of the medium used. However, the explant technique showed higher efficiency when NLC-rhEGF culture medium was used, compared to BM, L-rhEGF, or NLC-blank. Gene expression analysis showed that NLC-rhEGF was able to increase EGFR gene expression, along with that of other genes related to cytokeratins, cell–cell junctions, and keratinocyte maturation and differentiation. In summary, these results support the use of NLC-rhEGF to improve the efficiency of explant-based methods in the efficient generation of human keratinocyte primary cell cultures for tissue engineering use

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Hameed Z, Zahia S, Garcia-Zapirain B, Aguirre JJ, Vanegas AM. “Breast Cancer Histopathology Image Classification Using an Ensemble of Deep Learning Models”. Sensors 2020; 20(16):4373.

Breast cancer is one of the major public health issues and is considered a leading cause of cancer-related deaths among women worldwide. Its early diagnosis can effectively help in increasing the chances of survival rate. To this end, biopsy is usually followed as a gold standard approach in which tissues are collected for microscopic analysis. However, the histopathological analysis of breast cancer is non-trivial, labor-intensive, and may lead to a high degree of disagreement among pathologists. Therefore, an automatic diagnostic system could assist pathologists to improve the effectiveness of diagnostic processes. This paper presents an ensemble deep learning approach for the definite classification of non-carcinoma and carcinoma breast cancer histopathology images using our collected dataset. We trained four different models based on pre-trained VGG16 and VGG19 architectures. Initially, we followed 5-fold cross-validation operations on all the individual models, namely, fully-trained VGG16, fine-tuned VGG16, fully-trained VGG19, and fine-tuned VGG19 models. Then, we followed an ensemble strategy by taking the average of predicted probabilities and found that the ensemble of fine-tuned VGG16 and fine-tuned VGG19 performed competitive classification performance, especially on the carcinoma class. The ensemble of fine-tuned VGG16 and VGG19 models offered sensitivity of 97.73% for carcinoma class and overall accuracy of 95.29%. Also, it offered an F1 score of 95.29%. These experimental results demonstrated that our proposed deep learning approach is effective for the automatic classification of complex-natured histopathology images of breast cancer, more specifically for carcinoma images.

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Santos-Vizcaino E, Salvador A, Vairo C, Igartua M, Hernandez RM, Correa L, Villullas S, Gainza G. “Overcoming the Inflammatory Stage of Non-Healing Wounds: In Vitro Mechanism of Action of Negatively Charged Microspheres (NCMs)”. Nanomaterials. 2020; (10):1108.

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.

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Las Heras K, Santos-Vizcaino E, Garrido T, Gutierrez FB, Aguirre JJ, De la Caba K, Guerrero P, Igartua M, Hernandez RM. “Soy protein and chitin sponge-like scaffolds: from natural by-products to cell delivery systems for biomedical applications”. Green Chem. 2020 (22): 3445-3460.

The increasing necessity of developing new devices for biomedical applications has added a growing social need of being environmentally respectful. In this work, we have shown that natural by-products from the food industry (soy protein and β-chitin) can be an excellent source of biomaterials to produce 3D scaffolds through simpler and cleaner processes. With the mixture of these two polymers, we have developed sponge-like scaffolds (SLS) with great physicochemical properties. Furthermore, a dialysis pre-conditioning step was enough to obtain negligible cytotoxicity in vitro. The predominant M2 macrophage profile, an elevated deposition of collagen fibres and the enhanced neovascularization capacity suggested excellent biocompatibility also in vivo. Moreover, these SLS were able to promote cell adhesion, proliferation and high loading capacity. Finally, h-MSCs 3D-cultured in these SLS released four times higher VEGF than h-MSCs seeded onto 2D plates. The green thinking strategy, properties and biocompatibility of this SLS highlight its potential as a cell delivery system for biomedical applications.

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Vairo C, Basas J, Pastor M, et al. In vitro and in vivo antimicrobial activity of sodium colistimethate and amikacin-loaded nanostructured lipid carriers (NLC) [published online ahead of print, 2020 Jul 1]. Nanomedicine. 2020;29:102259.

Sodium colistimethate (SCM) and amikacin (AMK) are among the few antibiotics effective against resistant P. aeruginosa, K. pneumoniae and A. baumannii; however, their toxicity severely limits their use. Enclosing antibiotics into nanostructured lipid carriers (NLC) might decrease drug toxicity and improve antibiotic disposition. In this work, SCM or AMK was loaded into different NLC formulations, through high pressure homogenization, and their in vitro and in vivo effectiveness was analyzed. The encapsulation process did not reduce drug effectiveness since in vitro SCM-NLC and AMK-NLC drug activity was equal to that of the free drugs. As cryoprotectant, trehalose showed better properties than dextran. Instead, positive chitosan coating was discarded due to its limited cost-efficiency. Finally, the in vivo study in acute pneumonia model revealed that intraperitoneal administration was superior to the intramuscular route and confirmed that (-) SCM-NLC with trehalose, was the most suitable formulation against an extensively drug-resistant A. baumannii strain

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Vairo C, Collantes M, Quincoces G, Villullas S, Peñuelas I, Pastor M, Gil AG, Gainza E, Hernandez RM, Igartua M, Gainza G. “Preclinical safety of topically administered nanostructured lipid carriers (NLC) for wound healing application: biodistribution and toxicity studies”. Int J Pharm. 2019 (569):118484

Re-activation of the healing process is a major challenge in the field of chronic wound treatment. For that purpose, lipid-nanoparticles, especially nanostructured lipid carriers (NLC), possess extremely useful characteristics such as biodegradability, biocompatibility and long-term stability, besides being suitable for drug delivery. Moreover, they maintain wound moisture due to their occlusive properties, which have been associated with increased healing rates. In the light of above, NLC have been extensively used topically for wound healing; but to date, there are no safety-preclinical studies concerning such type of application. Thus, in this work, biodistribution studies were performed in rats with the NLC previously developed by our research group, using technetium-99 m (99mTc-NLC) as radiomarker, topically administered on a wound. 99mTc-NLC remained on the wound for 24 h and systemic absorption was not observed after administration. In addition, toxicological studies were performed to assess NLC safety after topical administration. The results obtained demonstrated that NLC were non-cytotoxic, non-sensitizing and non-irritant/corrosive. Overall, it might be concluded that developed NLC remained at the administration area, potentially exerting a local effect, and were safe after topical administration on wounds.

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Hernando S, Requejo C, Herran E, Ruiz-Ortega JA, Morera-Herreras T, Lafuente JV, Ugedo L, Gainza E, Pedraz JL, Igartua M, Hernandez RM. “Beneficial effects of n-3 polyunsaturated fatty acids administration in a partial lesion model of Parkinson’s disease: The role of glia and NRf2 regulation”. Neurobiol Dis. 2019; (121):252-262.

Omega-3 polyunsaturated fatty acids (n-3 PUFAs) have been widely associated to beneficial effect over different neurodegenerative diseases. In the present study, we tested the potential therapeutic effect of docohexanoic acid (DHA) and its hydroxylated derivate, DHAH, in a partial lesion model of Parkinson’s disease (PD). One month before and four months after the striatal lesion with 6-OHDA was made, the animals were daily treated with DHA (50 mg/kg), DHAH (50 mg/kg), vehicle or saline, by intragastric administration. Animal groups under n-3 PUFA treatments exhibited a trend to improve in amphetamine-induced rotations and cylinder test. The beneficial effect seen in behavioral studies were confirmed with TH immunostaining. TH+ fibers and TH+ neurons increased in the experimental groups treated with both n-3 PUFAs, DHA and DHAH. Moreover, the n-3 PUFAs administration decreased the astrogliosis and microgliosis, in both the striatum and substantia nigra (SN), with a higher decrease of GFAP+ and Iba-1+ cells for the DHAH treated group. This experimental group also revealed a positive effect on Nrf2 pathway regulation, decreasing the positive Nrf2 immmunostaining in the striatum and SN, which revealed a potential antioxidant effect of this compound. Taking together, these data suggest a positive effect of n-3 PUFAs administration, and more concretely of DHAH, for PD treatment as it exhibited positive results on dopaminergic system, neuroinflammation and oxidative stress.

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Vallejo-Diez S, Fleischer A, Martín-Fernández JM, Sánchez-Gilabert A, Castresana M, Aguillón D, Villegas A, Mastronardi CA, Espinosa LG, Arcos-Burgos M, Del Pozo Á, Herrán E, Gainza E, Isaza-Ruget M, Lopera F, Bachiller D. “Generation of one iPSC line (IMEDEAi006-A) from an early-onset familial Alzheimer’s Disease (fAD) patient carrying the E280A mutation in the PSEN1 gene”. Stem Cell Res. 2019; (37):101440

The mutation E280A in PSEN1 (presenilin-1) is the most common cause of early-onset familial Alzheimer’s Disease (fAD). It presents autosomal dominant inheritance and frequently leads to the manifestation of the disease in relatively young individuals. Here we report the generation of one PSEN1 E280A iPSC line derived from an early-onset patient. OriP/EBNA1-based episomal plasmids containing OCT3/4, SOX2, KLF4, L-MYC, LIN28, BCL-xL and shp53 were used to reprogram oral mucosa fibroblasts. The iPSC line generated has normal karyotype, carry the E280A mutation, is free of plasmid integration, express high levels of pluripotency markers and can differentiate into all three germ layers.

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Carreira-Barral I, Rumbo C, Mielczarek M, Alonso-Carrillo D, Herran E, Pastor M, Del Pozo A, García-Valverde M, Quesada R. “Small molecule anion transporters display in vitro antimicrobial activity against clinically relevant bacterial strains” 2019; 55(68):10080-10083.

Highly active transmembrane anion transporters have demonstrated their activity against antibiotic-resistant and clinically relevant bacterial strains. This type of compound offers promise as a strategy to develop novel antibacterial agents.

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Garcia-Orue I, Gainza G, Garcia-Garcia P, Gutierrez FB, Aguirre JJ, Hernandez RM, Delgado A, Igartua M. “Composite nanofibrous membranes of PLGA/Aloe vera containing lipid nanoparticles for wound dressing applications”. Int J Pharm. 2019 (556):320-329

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 ± 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.

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Pastor M, Basas J, Vairo C, Gainza G, Moreno-Sastre M, Gomis X, Fleischer A, Palomino E, Bachiller D, Gutiérrez FB, Aguirre JJ, Esquisabel A, Igartua M, Gainza E, Hernandez RM, Gavaldà J, Pedraz JL. “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”. Nanomedicine. 2019 (18):101-111.

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.

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García-Honduvilla N, Cifuentes A, Ortega MA, Pastor M, Gainza G, Gainza E, Buján J, Álvarez-Mon M. “Immuno-modulatory effect of local rhEGF treatment during tissue repair in diabetic ulcers.” Endocr Connect. 2018 (4):584-594.

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.

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Garcia-Orue, I, Gainza G, Villullas S, Pedraz JL, Hernandez RM, Igartua M. “Nanotechnology approaches for skin wound regeneration using drug-delivery systems.” Nanobiomaterials in soft tissue engineering. 2016;(5):31-55

The application of nanotechnology in medicine represents a great opportunity to enhance the effectiveness of currently available medical treatments, especially focused on challenging healthcare issues, such as skin wound regeneration. Hence, in the last few decades, nanobiomaterials have been extensively studied and optimized for the development of nanoscale drug-delivery systems releasing drugs, such as growth factors, cytokines, or antimicrobials, for skin wound repair. In this regard, several natural or synthetic materials have been studied due to their similarities to the skin and biocompatible properties, or due to their antibacterial or antiseptic effect. In addition, materials can also be engineered as scaffolds for the development of novel wound dressings. Thus, this chapter presents an overview of the current nanotechnological approaches used for the controlled release of drugs in the field of skin wound regeneration, particularly emphasizing polymeric and lipid nanoparticles (NPs), silver NPs, nanofibrous structures, nanosheets, and nanohybrids.

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Garcia-Orue I, Gainza G, Girbau C, Alonso R, Aguirre JJ, Pedraz JL, Igartua M, Hernandez RM. LL37 loaded nanostructured lipid carriers (NLC): A new strategy for the topical treatment of chronic wounds. Eur J Pharm Biopharm. 2016;108:310-316.

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 270 nm, a zeta potential of −26 mV 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 bioactivity assay 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 in db/db mice. 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.

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Gainza G, Villullas S, Pedraz JL, Igartua M, Hernandez RM. “Advances in drug delivery systems (DDSs) to release growth factors for wound healing and skin regeneration” Nanomedicine 2015;(11):1551-1573

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.

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Gainza G, Celdran Bonafonte D, Moreno B, Aguirre JJ, Gutierrez FB, Villullas S, Pedraz JL, Igartua M, Hernandez RM. “The topical administration of rhEGF-loaded nanostructured lipid carriers (rhEGF-NLC) improves healing in a porcine full-thickness excisional wound model” Journal of Control Release. 2015; (197):41-47

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.

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Gainza G, Pastor M, Aguirre JJ, Villullas S, Pedraz JL, Hernandez RM, Igartua M. “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” Journal of Control Release. 2014; (185):51-61

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.

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