Bioinspired gelatin/bioceramic composites loaded with bone morphogenetic protein-2 (BMP-2) promote osteoporotic bone repair.

There are currently several commercialized products approved by the Food and Drug Administration and the European Medicines Agency based on the use of recombinant human BMP-2 for the treatment of non-unions long fractures and spinal fusion. However, the adverse effects recorded with the use of BMPs suggest the need for drug delivery carriers that allow reducing the required doses and improve their cost-effectiveness. Herein, we have developed a new osteoconductive scaffold that reduces the required doses of BMP-2 for promoting bone regeneration in an osteoporotic defect model. The composite is, in brief, a gelatin-based 3D scaffold reinforced with either calcium sulfate or hydroxyapatite as an inorganic osteoconductive biomaterial. To this end, the organic/inorganic composite systems showed high hydration capacity and good in vitro degradability. The incorporation of 7.5% (m/v) ceramic compounds resulted in scaffolds with stiffer Young modulus (179 and 75 kPa for CaSO4_7 and HA_7, respectively) than bare gelatin hydrogels (48 kPa). Studies with human bone-marrow derived mesenchymal stem cells (hBM-MSCs) revealed that the 3D scaffolds promote cell adhesion and proliferation along with osteogenic differentiation capabilities. Specifically, downregulation of stemness (Nanog, Oct4) genes and upregulation of osteogenic markers (ALP, Col1a1, Fmod) by two fold were observed over 10 days under basal culture conditions. Promisingly, the sustained in vitro release of BMP-2 observed from the porous reinforced scaffolds allowed us to address the critical-sized osteoporotic mice calvarial defects with a relatively low growth factor doses (600 ng BMP-2/scaffold) compared to conventional doses at 2-15 micrograms. Overall, this study demonstrates the promising potential of osteoconductive gelatin/calcium bioceramics composites as osteogenic growth factors delivery carriers for bone-regeneration via ultra-low growth factor doses.

Development, characterization and sterilisation of Nanocellulose-alginate-(hyaluronic acid)- bioinks and 3D bioprinted scaffolds for tissue engineering.

3D-bioprinting is an emerging technology of high potential in tissue engineering (TE), since it shows effective control over scaffold fabrication and cell distribution. Biopolymers such as alginate (Alg), nanofibrillated cellulose (NC) and hyaluronic acid (HA) offer excellent characteristics for use as bioinks due to their excellent biocompatibility and rheological properties. Cell incorporation into the bioink requires sterilisation assurance, and autoclave, ß-radiation and γ-radiation are widely used sterilisation techniques in biomedicine; however, their use in 3D-bioprinting for bioinks sterilisation is still in their early stages. In this study, different sterilisation procedures were applied on NC-Alg and NC-Alg-HA bioinks and their effect on several parameters was evaluated. Results demonstrated that NC-Alg and NC-Alg-HA bioinks suffered relevant rheological and physicochemical modifications after sterilisation; yet, it can be concluded that the short cycle autoclave is the best option to sterilise both NC-Alg based cell-free bioinks, and that the incorporation of HA to the NC-Alg bioink improves its characteristics. Additionally, 3D scaffolds were bioprinted and specifically characterized as well as the D1 mesenchymal stromal cells (D1-MSCs) embedded for cell viability analysis. Notably, the addition of HA demonstrates better scaffold properties, together with higher biocompatibility and cell viability in comparison with the NC-Alg scaffolds. Thus, the use of MSCs containing NC-Alg based scaffolds may become a feasible tissue engineering approach for regenerative medicine.

Type 1 Diabetes Mellitus reversal via implantation of magnetically purified microencapsulated pseudoislets.

Microencapsulation of pancreatic islets for the treatment of Type I Diabetes Mellitus (T1DM) generates a high quantity of empty microcapsules, resulting in high therapeutic graft volumes that can enhance the host's immune response. We report a 3D printed microfluidic magnetic sorting device for microcapsules purification with the objective to reduce the number of empty microcapsules prior transplantation. In this study, INS1E pseudoislets were microencapsulated within alginate (A) and alginate-poly-L-lysine-alginate (APA) microcapsules and purified through the microfluidic device. APA microcapsules demonstrated higher mechanical integrity and stability than A microcapsules, showing better pseudoislets viability and biological function. Importantly, we obtained a reduction of the graft volume of 77.5% for A microcapsules and 78.6% for APA microcapsules. After subcutaneous implantation of induced diabetic Wistar rats with magnetically purified APA microencapsulated pseudoislets, blood glucose levels were restored into normoglycemia (<200 mg/dL) for almost 17 weeks. In conclusion, our described microfluidic magnetic sorting device represents a great alternative approach for the graft volume reduction of microencapsulated pseudoislets and its application in T1DM disease.

Low molecular-weight hyaluronan as a cryoprotectant for the storage of microencapsulated cells.

The low-temperature storage of therapeutic cell-based products plays a crucial role in their clinical translation for the treatment of diverse diseases. Although dimethylsulfoxide (DMSO) is the most successful cryoprotectant in slow freezing of microencapsulated cells, it has shown adverse effects after cryopreserved cell-based products implantation. Therefore, the search of alternative non-toxic cryoprotectants for encapsulated cells is continuously investigated to move from bench to the clinic. In this work, we investigated the low molecular-weight hyaluronan (low MW-HA), a natural non-toxic and non-sulfated glycosaminoglycan, as an alternative non-permeant cryoprotectant for the slow freezing cryopreservation of encapsulated cells. Cryopreservation with low MW-HA provided similar metabolic activity, cell dead and early apoptotic cell percentage and membrane integrity after thawing, than encapsulated cells stored with either DMSO 10% or Cryostor 10. However, the beneficial outcomes with low MW-HA were not comparable to DMSO with some encapsulated cell types, such as the human insulin secreting cell line, 1.1B4, maybe explained by the different expression of the CD44 surface receptor. Altogether, we can conclude that low MW-HA represents a non-toxic natural alternative cryoprotectant to DMSO for the cryopreservation of encapsulated cells.

Advances in the slow freezing cryopreservation of microencapsulated cells.

Over the past few decades, the use of cell microencapsulation technology has been promoted for a wide range of applications as sustained drug delivery systems or as cells containing biosystems for regenerative medicine. However, difficulty in their preservation and storage has limited their availability to healthcare centers. Because the preservation in cryogenic temperatures poses many biological and biophysical challenges and that the technology has not been well understood, the slow cooling cryopreservation, which is the most used technique worldwide, has not given full measure of its full potential application yet. This review will discuss the different steps that should be understood and taken into account to preserve microencapsulated cells by slow freezing in a successful and simple manner. Moreover, it will review the slow freezing preservation of alginate-based microencapsulated cells and discuss some recommendations that the research community may pursue to optimize the preservation of microencapsulated cells, enabling the therapy translate from bench to the clinic.

Engineering a Clinically Translatable Bioartificial Pancreas to Treat Type I Diabetes.

Encapsulating, or immunoisolating, insulin-secreting cells within implantable, semipermeable membranes is an emerging treatment for type 1 diabetes. This approach can eliminate the need for immunosuppressive drug treatments to prevent transplant rejection and overcome the shortage of donor tissues by utilizing cells derived from allogeneic or xenogeneic sources. Encapsulation device designs are being optimized alongside the development of clinically viable, replenishable, insulin-producing stem cells, for the first time creating the possibility of widespread therapeutic use of this technology. Here, we highlight the status of the most advanced and widely explored implementations of cell encapsulation with an eye toward translating the potential of this technological approach to medical reality.

Biologically active and biomimetic dual gelatin scaffolds for tissue engineering.

We have designed, developed and optimized Genipin cross-linked 3D gelatin scaffolds that were biologically active and biomimetic, show a dual activity both for growth factor and cell delivery. Type B gelatin powder was dissolved in DI water. 100mg of genipin was dissolved in 10ml of DI water. Three genipin concentrations were prepared: 0.1%, 0.2% and 0.3% (w/v). Solutions were mixed at 40°C and under stirring and then left crosslinking for 72h. Scaffolds were obtained by punching 8 mm-cylinders into ethanol 70% solution for 10min and then freeze-drying. Scaffolds were biologically, biomechanically and morphologically evaluated. Cell adhesion and morphology of D1-Mesenchymal stem cells (MSCs) and L-929 fibroblast was studied. Vascular endothelial grwoth factor (VEGF) and Sonic hedgehog (SHH) were used as model proteins. Swelling ratio increased and younǵs module decreased along with the concentration of genipin. All scaffolds were biocompatible according to the toxicity test. MSC and L-929 cell adhesion improved in 0.2% of genipin, obtaining better results with MSCs. VEGF and SHH were released from the gels. This preliminary study suggest that the biologically active and dual gelatin scaffolds may be used for tissue engineering approaches like bone regeneration.

Pharmacokinetics of Benznidazole in Healthy Volunteers and Implications in Future Clinical Trials.

Despite its toxicity and low efficacy in the chronic phase, benznidazole is the drug of choice in Chagas disease. Scarce information about pharmacokinetics and pharmacodynamics of benznidazole has been published. We performed a phase I, open-label, nonrandomized pharmacokinetic study of benznidazole (Abarax) conducted with 8 healthy adult volunteers at the Infectious Diseases Department of the Vall d'Hebron University Hospital (Barcelona, Spain). The separation and detection of benznidazole were performed on a Waters Acquity ultraperformance liquid chromatography system (UPLC) coupled with a Waters Xevo TQ MS triple quadrupole mass spectrometer. The pharmacokinetic parameters were calculated based on a noncompartmental body model using Phoenix WinNonlin version 6.3 software. Furthermore, computational simulations were calculated for the multiple-dose administration at two dose regimens: 100 mg of benznidazole administered every 8 h and 150 mg of benznidazole administered every 12 h. After benznidazole administration, the median area under the concentration-time curve from time zero to time t (AUC0-t ) and extrapolated to infinity (AUC0-∞) were about 46.4 µg · h/ml and 48.4 µg · h/ml, respectively. Plasma benznidazole concentrations peaked at 3.5 h, with maximal concentrations of 2.2 µg/ml, and benznidazole exhibited a terminal half-life of 12.1 h. The median maximum concentration (Cmax) of benznidazole was lower in men than in women (1.6 versus 2.9 µg/ml), and median volume of distribution (V) as a function of bioavailability (F) was higher in men than in women (125.9 versus 88.6 liters). In conclusion, dose regimens (150 mg/12 h or 100 mg/8 h) reached a steady-state range concentration above of the minimum experimental therapeutic dose. Sex differences in the benznidazole pharmacokinetics were observed; mainly, men had lower Cmax and higher V/F than women.

Use of Flow Focusing Technique for Microencapsulation of Myoblasts.

Alginate cell microencapsulation implies the immobilization of cells within a polymeric membrane that allows the bidirectional diffusion of nutrients and oxygen inside the microcapsules and the release of waste and therapeutic molecules outside them. This technology has been applied to several cell types and it has been extensively described with pancreatic islets. However, other cells such as myoblasts are being currently studied and showing high interest. Moreover, different systems and approaches have been developed for cell encapsulation such as electrostatic extrusion and Flow focusing technology. When Flow focusing technology is applied for myoblast encapsulation, several factors should be considered, such as the pressure, the flow of the system, or the diameter size of the nebulizer, which will determine the final diameter size and shape of the microcapsules containing the myoblasts. Finally, viability of encapsulated myoblasts needs to be assessed before further studies are performed.

Microencapsulated Cells for Cancer Therapy.

The microencapsulation of different types of cells that are able to produce therapeutic factors is being investigated for the treatment of several human diseases. Most efforts are focused on chronic and degenerative diseases as this strategy could become an alternative to some commonly used parenteral treatments that need to be repeatedly administered. But, this approach has also been investigated in the field of oncology with the aim of providing immunomodulatory antibodies that are able to enhance the patient's inherent immune response against the tumor. These kind of treatments would provide the patient with the therapeutic drug produced in situ, de novo, and in a sustained way, making the therapy more comfortable.Although different devices are nowadays available to produce cell-enclosing alginate-microcapsules, here, we describe the most important steps and advices in order to fabricate alginate-poly-L-lysine-alginate microcapsules containing hybridoma cells for cancer management using an electrostatic bead generator, and how to evaluate the viability of those cells over the time.

Morphological Changes in a Severe Model of Parkinson's Disease and Its Suitability to Test the Therapeutic Effects of Microencapsulated Neurotrophic Factors.

The unilateral 6-hydroxydopamine (6-OHDA) lesion of medial forebrain bundle (MFB) in rats affords us to study the advanced stages of Parkinson's disease (PD). Numerous evidences suggest synergic effects when various neurotrophic factors are administered in experimental models of PD. The aim of the present work was to assess the morphological changes along the rostro-caudal axis of caudo-putamen complex and substantia nigra (SN) in the referred model in order to test the suitability of a severe model to evaluate new neurorestorative therapies. Administration of 6-OHDA into MFB in addition to a remarkable depletion of dopamine in the nigrostriatal system induced an increase of glial fibrillary acidic protein (GFAP)-positive cells in SN and an intense immunoreactivity for OX-42, vascular endothelial growth factor (VEGF), and Lycopersycum esculentum agglutinin (LEA) in striatum and SN. Tyrosine hydroxylase (TH) immunostaining revealed a significant decrease of the TH-immunopositive striatal volume in 6-OHDA group from rostral to caudal one. The loss of TH-immunoreactive (TH-ir) neurons and axodendritic network (ADN) was higher in caudal sections. Morphological recovery after the implantation of microspheres loaded with VEGF and glial cell line-derived neurotrophic factor (GDNF) in parkinsonized rats was related to the preservation of the TH-ir cell number and ADN in the caudal region of the SN. In addition, these findings support the neurorestorative role of VEGF+GDNF in the dopaminergic system and the synergistic effect between both factors. On the other hand, a topological distribution of the dopaminergic system was noticeable in the severe model, showing a selective vulnerability to 6-OHDA and recovering after treatment.

Stability study of sodium colistimethate-loaded lipid nanoparticles.

In the last decades, the encapsulation of antibiotics into nanoparticulate carriers has gained increasing attention for the treatment of infectious diseases. Sodium colistimethate-loaded solid lipid nanoparticles (Colist-SLNs) and nanostructured lipid carriers (Colist-NLCs) were designed aiming to treat the pulmonary infection associated to cystic fibrosis patients. The nanoparticles were freeze-dried using trehalose as cryoprotectant. The stability of both nanoparticles was analysed over one year according to the International Conference of Harmonisation (ICH) guidelines by determining the minimum inhibitory concentration (MIC) against clinically isolated Pseudomonas aeruginosa strains and by studying their physico-chemical characteristics. The results showed that Colist-SLNs lost their antimicrobial activity at the third month; on the contrary, the antibacterial activity of Colist-NLCs was maintained throughout the study within an adequate range (MIC ≤16 µg/mL). In addition, Colist-NLCs exhibited suitable physico-chemical properties at 5 °C and 25 °C/60% relative humidity over one year. Altogether, Colist-NLCs proved to have better stability than Colist-SLNs.

The influence of the polar head-group of synthetic cationic lipids on the transfection efficiency mediated by niosomes in rat retina and brain.

The development of novel non-viral delivery vehicles is essential in the search of more efficient strategies for retina and brain diseases. Herein, optimized niosome formulations prepared by oil-in water (o/w) and film-hydration techniques were characterized in terms of size, PDI, zeta potential, morphology and stability. Three ionizable glycerol-based cationic lipids containing a primary amine group (lipid 1), a triglycine group (lipid 2) and a dimethylamino ethyl pendent group (lipid 3) as polar head-groups were part of such niosomes. Upon the addition of pCMS-EGFP plasmid, nioplexes were obtained at different cationic lipid/DNA ratios (w/w). The resultant nioplexes were further physicochemically characterized and evaluated to condense, release and protect the DNA against enzymatic digestion. In vitro experiments were performed to evaluate transfection efficiency and cell viability in HEK-293, ARPE-19 and PECC cells. Interestingly, niosome formulations based on lipid 3 showed better transfection efficiencies in ARPE-19 and PECC cells than the rest of cationic lipids showed in this study. In vivo experiments in rat retina after intravitreal and subretinal injections together with in rat brain after cerebral cortex administration showed promising transfection efficiencies when niosome formulations based on lipid 3 were used. These results provide new insights for the development of non-viral vectors based on cationic lipids and their applications for efficient delivery of genetic material to the retina and brain.

Killing effect of nanoencapsulated colistin sulfate on Pseudomonas aeruginosa from cystic fibrosis patients.

Pseudomonas aeruginosa frequently infects the respiratory tract of cystic fibrosis (CF) patients. Multidrug-resistant phenotypes and high capacity to form stable biofilms are common. Recent studies have described the emergence of colistin-resistant isolates in CF patients treated with long-term inhaled colistin. The use of nanoparticles containing antimicrobials can contribute to overcome drug resistance mechanisms. The aim of this study was to explore antimicrobial activity of nanoencapsulated colistin (SLN-NLC) versus free colistin against P. aeruginosa clinical isolates from CF patients and to investigate their efficacy in biofilm eradication. Susceptibility of planktonic bacteria to antimicrobials was examined by using the broth microdilution method and growth curve assay. Minimal biofilm eradication concentration (MBEC) and biofilm prevention concentration (BPC) were determined to assess antimicrobial susceptibility of sessile bacteria. We used atomic force microscopy (AFM) to visualize treated and untreated biofilms and to determine surface roughness and other relevant parameters. Colistin nanoparticles had the same antimicrobial activity as free drug against planktonic bacteria. However, nanoencapsulated colistin was much more efficient in the eradication of biofilms than free colistin. Thus, these formulations have to be considered as a good alternative therapeutic option to treat P. aeruginosa infections.

Intranasal Administration of chitosan-Coated Nanostructured Lipid Carriers Loaded with GDNF Improves Behavioral and Histological Recovery in a Partial Lesion Model of Parkinson's Disease.

Parkinson's disease (PD) is the second most frequent neurodegenerative disorder, but current therapies are only symptomatic. A promising alternative to address the neurodegenerative process is the use of neurotrophic factors, such as the glial cell-derived neurotrophic factor (GDNF). However, its clinical use has been limited due to its short half-life and rapid degradation after in vivo administration, in addition to difficulties in crossing the blood-brain barrier (BBB). This barrier is a limiting factor in brain drug development, making the future progression of neurotherapeutics difficult. In the past few years, intranasal drug delivery has appeared as an alternative non-invasive administration route to bypass the BBB and target drugs directly to the CNS. Thus, the aim of this work was to study the in vivo neuroprotective effect of intranasally administered GDNF, encapsulated in chitosan-coated nanostructured lipid carrier (CS-NLC-GDNF), in a 6-OHDA partially lesioned rat model. The developed CS-NLC-GDNF showed a particle size of approximately 130 nm and high encapsulation efficiency. The in vitro study in PC-12 cells demonstrated the ability of the encapsulated GDNF to protect these cells against 6-OHDA toxin. After two weeks of daily intranasal administration of treatments, the administration of CS-NLC-GDNF achieved a behavioral improvement in rats, as well as a significant improvement in both the density of TH+ fibres in the striatum and the TH+ neuronal density in the SN. Thus, it can be concluded that the nose-to-brain delivery of CS-NLC-GDNF could be a promising therapy for the treatment of PD.

Enhanced Hippocampal Neurogenesis in APP/Ps1 Mouse Model of Alzheimer's Disease After Implantation of VEGF-loaded PLGA Nanospheres.

During adult life, hippocampus is an important brain region involved in neurogenesis. The generation and cell death of newly generated neuronal cells in this region have critical roles in brain maintenance and alterations in these processes are seen in Alzheimer's disease (AD). For the purpose of carrying out a neuroregenerative strategy, we propose a novel approach based on the encapsulation of vascular endothelial growth factor (VEGF) in poly (lactic co-glycolic acid) (PLGA) biodegradable nanospheres (NS) administered by craniotomy to stimulate the proliferation of neuronal precursors in a transgenic mouse model of AD. VEGF loaded nanospheres were prepared by double emulsion solvent evaporation technique, obtaining 200 nm nanospheres with a biphasic release profile. After demonstrating their efficacy in the proliferation and differentiation of neuronal cell cultures, in vivo studies were carried out. 3 months after VEGF-NS were implanted directly into the cerebral cortex of APP/Ps1 mice, the determination of BrdU(+) cells in the whole hippocampal region and specifically in the dentate gyrus, demonstrated a significantly enhanced cellular proliferation in VEGF-NS treated group. These results were also confirmed showing an increased number of DCX(+) and NeuN(+) cells. Hence, PLGA-VEGF nanospheres may be a potential strategy to modulate proliferative neuronal progenitors in the hippocampal region, and therefore, provide new insight for future therapeutic approaches in AD.

Protamine/DNA/Niosome Ternary Nonviral Vectors for Gene Delivery to the Retina: The Role of Protamine.

The present study aimed to evaluate the incorporation of protamine into niosome/DNA vectors to analyze the potential application of this novel ternary formulation to deliver the pCMS-EGFP plasmid into the rat retina. Binary vectors based on niosome/DNA and ternary vectors based on protamine/DNA/niosomes were prepared and physicochemically characterized. In vitro experiments were performed in ARPE-19 cells. At 1:1:5 protamine/DNA/niosome mass ratio, the resulted ternary vectors had 150 nm size, positive charge, spherical morphology, and condensed, released, and protected the DNA against enzymatic digestion. The presence of protamine in the ternary vectors improved transfection efficiency, cell viability, and DNA condensation. After ocular administration, the EGFP expression was detected in different cell layers of the retina depending on the administration route without any sign of toxicity associated with the formulations. While subretinal administration transfected mainly photoreceptors and retinal pigment epithelial cells at the site of injection, intravitreal administration produced a more uniform distribution of the protein expression through the inner layers of the retina. The protein expression in the retina persisted for at least one month after both administrations. Our study highlights the flattering properties of protamine/DNA/niosome ternary vectors for efficient and safe gene delivery to the rat retina.

Pulmonary drug delivery: a review on nanocarriers for antibacterial chemotherapy.

As the WHO stated, lower respiratory infections are the third leading cause of death. In addition, it is remarkable that antimicrobial resistance represents a huge threat. Thus, new therapeutic weapons are required. Among the possible alternatives, antibiotic encapsulation in nanoparticles has gained much attention in terms of improved tolerability, activity and ability to combat the resistance mechanisms of bacteria. In this regard, this review article focuses on the latest nanocarrier approaches for inhalatory therapy of antibiotics. First, the technology related to lung disposition will be reviewed. Then, nanocarrier systems will be introduced and the challenges required to perform adequate pulmonary deposition analysed. In the following part, drug delivery systems (DDSs) on the market or in clinical trials are described and, finally, new approaches of nanoparticles that have reached pre-clinical stage are enumerated. Altogether, this review aims at gathering together the novel nanosystems for anti-infectious therapy, underlining the potential of DDSs to improve and optimize currently available antibiotic therapies in the context of lung infections.

Graphene oxide increases the viability of C2C12 myoblasts microencapsulated in alginate.

Cell microencapsulation represents a great promise for long-term drug delivery, but still several challenges need to be overcome before its translation into the clinic, such as the long term cell survival inside the capsules. On this regard, graphene oxide has shown to promote proliferation of different cell types either in two or three dimensions. Therefore, we planned to combine graphene oxide with the cell microencapsulation technology. We first studied the effect of this material on the stability of the capsules and next we analyzed the biocompatibility of this chemical compound with erythropoietin secreting C2C12 myoblasts within the microcapsule matrix. We produced 160 µm-diameter alginate microcapsules with increasing concentrations of graphene oxide and did not find modifications on the physicochemical parameters of traditional alginate microcapsules. Moreover, we observed that the viability of encapsulated cells within alginate microcapsules containing specific graphene oxide concentrations was enhanced. These results provide a relevant step for the future clinical application of graphene oxide on cell microencapsulation.

Development and in vitro evaluation of lipid nanoparticle-based dressings for topical treatment of chronic wounds.

This research addresses the development and in vitro evaluation of lipid nanoparticle (NP)-based dressings to optimize the delivery of human recombinant epidermal growth factor (rhEGF) for the topical treatment of chronic wounds. The systems investigated were rhEGF-loaded solid lipid nanoparticles (rhEGF-SLN) and rhEGF-loaded nanostructured lipid carriers (rhEGF-NLC) formulated in wound dressings comprising either semi-solid hydrogels or fibrin-based solid scaffolds. Following detailed characterisation of the NP, in vitro diffusion cell experiments (coupled with dermatopharmacokinetic measurements), together with confocal microscopic imaging, conducted on both intact skin samples, and those from which the barrier (the stratum corneum) had been removed, revealed that (a) the particles remained essentially superficially located for at least up to 48h post-application, (b) rhEGF released on the surface of intact skin was unable to penetrate to the deeper, viable layers, and (c) sustained release of growth factor from the NP "drug reservoirs" into barrier-compromised skin was observed. There were no significant differences between the in vitro performance of rhEGF-SLN and rhEGF-NLC, irrespective of the formulation employed. It is concluded that, because of their potentially longer-term stability, the fibrin-based scaffolds may be the most suitable approach to formulate rhEGF-loaded lipid nanoparticles.