Prevalence of azole-resistant Aspergillus fumigatus and other aspergilli in the environment from Argentina.

Azole resistance has emerged as a new therapeutic challenge in patients with aspergillosis. Various resistance mutations are attributed to the widespread use of triazole-based fungicides in agriculture. This study explored the prevalence of azole-resistant Aspergillus fumigatus (ARAF) and other aspergilli in the Argentine environment. A collection of A. fumigatus and other aspergilli strains isolated from soil of growing crops, compost, corn, different animal feedstuffs, and soybean and chickpea seeds were screened for azole resistance. No ARAF was detected in any of the environmental samples studied. However, five A. flavus, one A. ostianus, one A. niger and one A. tamarii recovered from soybean and chickpea seeds showed reduced susceptibility to medical azole antifungals (MAA). The susceptibility profiles of five A. flavus isolates, showing reduced susceptibility to demethylase inhibitors (DMIs), were compared with those of 10 isolates that exhibited susceptibility to MAA. Aspergillus flavus isolates that showed reduced MAA susceptibility exhibited different susceptibility profiles to DMIs. Prothioconazole and tebuconazole were the only DMIs significantly less active against isolates with reduced susceptibility to MAA. Although no ARAF isolates were found in the samples analysed, other aspergilli with reduced susceptibility profile to MAA being also important human pathogens causing allergic, chronic and invasive aspergillosis, are present in the environment in Argentina. Although a definitive link between triazole-based fungicide use and isolation of azole-resistant human pathogenic aspergilli from agricultural fields in Argentina remains elusive, this study unequivocally highlights the magnitude of the environmental spread of azole resistance among other Aspergillus species.

This study intended to inform about the prevalence of Aspergillus species showing triazole resistance in the Argentinian environment. Since azole fungicides are used for crop protection, it was expected that azole resistance in this species with cross-resistance to medical azoles could occur.

Isolation of Microascus sp. and Cephalotheca sp. from Bats in Argentina.

Bats worldwide play significant roles in ecosystem functions, encompassing pollination, seed dispersal, and pest control while concurrently serving as diseases reservoirs. As part of a comprehensive wildlife health surveillance effort, bats were systematically sampled within two national protected areas in Argentina. During this study 67 bats were examined and samples were collected from eight Molossus spp. individuals exhibiting conspicuous yellowish or white lesions on their noses. All samples were cultured on Sabouraud dextrose agar and lactrimel agar for fungal growth evaluation. Fungal isolates were identified using morphologic and molecular taxonomic techniques, leading to the detection of Microascus sp. in three Molossus rufus from Ibera National Park and Cephalotheca sp. in five Molossus molossus from Marsh Deer National Park. No fungal growth was identified in samples collected from the healthy hairs of the bats displaying lesions on their noses. The two fungi, which have not previously been isolated from bats, should be considered potentially pathogenic, evidenced by diseased hairs in the affected individuals.

Histoplasma antigens as novel players for the development of new enzyme immunoassays for the serodiagnosis of histoplasmosis: A comparative study of their analytical performance.

Definitive diagnosis of histoplasmosis relies on culture and/or cytology/histopathology; however, these procedures have limited sensitivity and cultures are time-consuming. Antibodies detection by immunodiffusion has low sensitivity in immunocompromised individuals and uses histoplasmin (HMN), a crude antigenic extract, as reagent. Novel protein antigen candidates have been recently identified and produced by DNA-recombinant techniques to obtain standardized and specific reagents for diagnosing histoplasmosis. To compare the analytical performance of novel enzyme-linked immunosorbent assays (ELISAs) for antibodies testing for diagnosing histoplasmosis using different Histoplasma capsulatum antigens as reagents. The H. capsulatum 100 kDa protein (Hcp100), the M antigen and its immunoreactive fragment F1 were produced by DNA-recombinant techniques. Galactomannan was purified from both the yeast and mycelial cell walls (yGM and mGM, respectively). The analytical performance of the ELISA tests for the serological detection of antibodies against these antigens was evaluated and compared with those obtained using HMN as reagent. Antibodies detection by the Hcp100 ELISA demonstrated 90.0% sensitivity and 92.0% specificity, versus 43.3% sensitivity and 95.0% specificity of the M ELISA, 33.3% sensitivity and 84.0% specificity of the F1 ELISA, 96.7% sensitivity and 94.0% specificity of the yGM ELISA, 83.3% sensitivity and 88.0% specificity of the mGM ELISA, and 70.0% sensitivity and 86.0% specificity for the HMN ELISA. In summary, Hcp100 is proposed as the most promising candidate for the serodiagnosis of histoplasmosis. The primary immunoreactive element in HMN proved to be GM rather than the M antigen. Nevertheless, a higher incidence of cross-reactions was noted with GM compared to M.

Hcp100 is a promising serodiagnostic candidate for histoplasmosis, boasting high sensitivity and specificity. Notably, GM, rather than M antigen, emerged as the primary immunoreactive element in HMN, despite a higher incidence of cross-reactions with GM compared to M.

Evaluation of the Histoplasma capsulatum 100-kilodalton antigen dot blot for the rapid diagnosis of progressive histoplasmosis in HIV/AIDS patients.

Among people living with HIV (PLHIV), progressive disseminated histoplasmosis (PDH) represents an important cause of mortality. Since antigen detection allows a rapid diagnosis and the instauration of a specific treatment this study aimed to evaluate the analytical performance of the Hcp100 dot blot, an in-house assay that detects the Histoplasma capsulatum 100-kilodalton antigen in urine and compare it with 2 commercially available assays the Histoplasma Urine Antigen Lateral Flow Assay (MVD-LFA) (MiraVista® Diagnostics) and the Clarus Histoplasma Galactomannan EIA (Clarus HGM) (IMMY). Urine specimens from 23 PLHIV with PDH, 13 patients with other infectious diseases, and 20 healthy individuals were tested. The Hcp100 dot blot showed higher sensitivity (87.0%), specificity (97.0%) and accuracy (92.9%) than the MVD-LFA (73.9%, 78.8%, and 76.8%, respectively) and the Clarus HGM (78.3%, 90.9%, and 85.7%, respectively). The Hcp100 dot blot had high analytical performance and would be a valuable screening tool for diagnosing PDH among PLHIV.

Antifungal susceptibility patterns for Aspergillus, Scedosporium, and Exophiala isolates recovered from cystic fibrosis patients against amphotericin B, and three triazoles and their impact after long-term therapies.

In cystic fibrosis (CF) patients, fungal colonization of the respiratory tract is frequently found. Aspergillus fumigatus, Scedosporium genus, and Exophiala dermatitidis are the most commonly isolated moulds from the respiratory tract secretions of CF patients. The aim of this 5-year surveillance study was to identify trends in species distribution and susceptibility patterns of 212 mould strains identified as Aspergillus spp., Scedosporium spp., and Exophiala spp., isolated from sputum of 63 CF patients who received long-term therapy with itraconazole (ITR) and/or voriconazole (VRC). The Aspergillus isolates were identified as members of the sections Fumigati (n = 130), Flavi (n = 22), Terrei (n = 20), Nigri (n = 8), Nidulantes (n = 1), and Usti (n = 1). Among the 16 species of the genus Scedosporium, 9 were S. apiospermum, 3 S. aurantiacum, and 4 S. boydii. Among the 14 Exophiala species, all were molecularly identified as E. dermatitidis. Overall, 94% (15/16) of Scedosporium spp., 50% (7/14) of E. dermatitidis, and 7.7% (14/182) of Aspergillus spp. strains showed high MIC values (≥8 µg/ml) for at least one antifungal. Particularly, 8.9% (19/212) of isolates showed high MIC values for amphotericin B, 11.7% (25/212) for ITR, 4.2% (9/212) for VRC, and 3.3% (7/212) for posaconazole. In some cases, such as some A. fumigatus and E. dermatitidis isolates recovered from the same patient, susceptibility to antifungal azoles decreased over time. We show that the use of azoles for a long time in CF patients causes the selection/isolation of mould strains with higher MIC values.

The use of azoles for a long time in cystic fibrosis patients causes the selection/isolation of Aspergillus, Scedosporium, and Exophiala species with higher MIC values.

Evidence of anti-Hcp100 antibodies in COVID-19 critically ill patients with detectable anti-histoplasmin antibodies in a highly endemic area for histoplasmosis.

Patients with severe COVID-19 are at increased risk for invasive fungal infections, which are underestimated. Histoplasmosis reactivation in endemic areas should not be overlooked in this population. In a previous study, seroconversion to anti-histoplasmin antibodies by ELISA was detected in 6/39 (15.4%) patients with severe COVID-19. In this work, samples were further investigated to detect seroconversion to antibodies against the Histoplasma capsulatum 100-kDa antigen (Hcp100) by ELISA. Seroconversion to anti-Hcp100 antibodies was detected in 7/39 patients, of whom 6 also seroconverted anti-histoplasmin antibodies. These results reinforce previous findings that show histoplasmosis as an underdiagnosed fungal entity complicating COVID-19.

This study verifies that patients with severe COVID-19 at intensive care units are at risk for histoplasmosis reactivation in endemic areas. Accurate diagnosis of this deadly fungal disease among critically ill patients with COVID-19 living in endemic areas for histoplasmosis is needed.

Seroprevalence of sporotrichosis infection in cats living in the Buenos Aires epizootic area.

This study aimed to perform a seroepidemiological survey on the prevalence of sporotrichosis among cats living in the Northern area of Buenos Aires, where a four-fold increase of Sporothrix brasiliensis infections were diagnosed during the last decade. For this purpose, an in-house indirect ELISA test sensitized with S. brasiliensis crude antigens was used. The ELISA test showed 100.0% sensitivity and 95.0% specificity. Antibodies against S. brasiliensis antigens were detected in 3.7% (9/241) of healthy cats evaluated, suggesting likely exposure or infection to this fungus. This ELISA test would be a valuable screening tool for diagnosing sporotrichosis and for seroepidemiological surveys.

S. brasiliensis is the primary cause of feline sporotrichosis in Argentina. The seroprevalence of sporotrichosis infection in urban localities of Buenos Aires province is reported for the first time. An ELISA test using S. brasiliensis crude antigens is also described.

Role of recombinant Histoplasma capsulatum 100-kilodalton antigen in the diagnosis of histoplasmosis among HIV/AIDS patients: Antigenuria and antibodies detection.

BACKGROUND: Diagnosing progressive disseminated histoplasmosis (PDH) is still challenging in many countries where this disease is highly endemic. Definitive diagnosis is established by culture and/or by cytology/histopathology but both procedures have limited sensitivity and cultures are time-consuming. Antibodies detection by immunodiffusion has a low sensitivity in immunocompromised individuals. Commercially available antigen detection assays have high sensitivity in PDH cases; however, they are expensive and only performed in few laboratories. AIMS: To describe the potential use of a novel ELISA for antibodies testing and a dot blot assay for antigen testing for diagnosing PDH using the recombinant 100 kDa protein of Histoplasma capsulatum (Hcp100) and their polyclonal antibodies as novel reagents, respectively. METHODS: Serum and urine samples from a cohort of patients with HIV/AIDS and proven PDH were studied for the detection of anti-Hcp100 antibodies by ELISA and Hcp100 antigen by dot blot, respectively. Sensitivity, specificity and cross-reactions with other diseases were estimated for each assay and compared with those obtained using histoplasmin (HMN) as a reagent for antibodies detection by ELISA and immunodiffusion, and using a commercial antigenuria test. RESULTS: Antibodies detection by the Hcp100 ELISA demonstrated 78.6% sensitivity and 88.4% specificity, versus 85.7% sensitivity and 81.0% specificity for the HMN ELISA and 26.1% sensitivity and 100% specificity for the immunodiffusion assay. Antigen detection by the Hcp100 dot blot demonstrated 89.3% sensitivity and 97.0% specificity versus 82.1% sensitivity and 90.9% specificity for the commercial test. CONCLUSION: The immunoassays described herein based on Hcp100 would be a valuable screening tool for diagnosing PDH.

Detection of anti-Histoplasma capsulatum antibodies and seroconversion patterns in critically ill patients with COVID-19: An underdiagnosed fungal entity complicating COVID-19?

The patients with severe COVID-19 are at increased risk for invasive fungal infections, such as invasive pulmonary aspergillosis and candidiasis, which increase morbidity and mortality. However, clinicians should also consider the possibility of reactivating latent Histoplasma capsulatum in patients with severe COVID-19 living within areas of endemicity who have worsening respiratory function or sepsis, even if they do not have classical risk factors for histoplasmosis (e.g., HIV/AIDS). Bearing in mind this scenario, serum samples of 39 non-HIV/AIDS patients from Buenos Aires hospitalized due to severe COVID-19 pneumonia were analyzed for anti-H. capsulatum-specific IgG antibodies by an in-house ELISA. Antibodies against H. capsulatum were detected in the sera of 8/39 patients (20.51%). To exclude the possibility that these antibodies arose from past exposure of these patients to the fungus, paired serum samples obtained after an interval of at least 10 days were evaluated. Of them, five patients (62.5%) with negative anti-H. capsulatum antibodies at baseline became seropositive 7-10 days later. Three patients (37.5%) had positive anti-H. capsulatum antibodies at baseline, but at time point 2, one of them became seronegative and the other one diminished the antibody titers (4000 vs. 16000 at baseline). The remaining patients displayed higher antibody titers at time point 2 (4000 vs. 1000 at baseline) and died immediately thereafter. In conclusion, awareness of the possibility of fungal co-infections is essential to reduce delays in diagnosis and treatment in order to help prevent severe illness and death from these infections. LAY SUMMARY: This study verifies that patients with severe COVID-19 at ICU are at risk for histoplasmosis reactivation in endemic areas. Accurate diagnosis of this deadly fungal disease among critically ill patients with COVID-19 living in endemic areas for histoplasmosis is needed.

Invasive fusariosis in a critically ill patient with severe COVID-19 pneumonia: A case report.

Invasive fungal infections as aspergillosis and candidiasis are well-documented complications in critically ill patients with acute respiratory distress syndrome due to COVID-19. However, invasive infections by other molds are rarely reported. We describe a case of invasive fusariosis in a patient with severe COVID-19 with a fatal outcome.

Sporothrix Brasiliensis: A Review of an Emerging South American Fungal Pathogen, Its Related Disease, Presentation and Spread in Argentina.

Sporotrichosis, caused by Sporothrix schenckii and related species, is the most frequent implantation mycosis in Latin America. In Argentina, over the last 8 years, there have been 0.16 new cases per month of feline sporotrichosis in 2011, increasing to 0.75 cases per month in 2019 and involving zoonotic transmission to humans. Molecular identification by polymerase chain reaction (PCR) detected Sporothrix brasiliensis in these feline and zoonotic outbreaks. This study will focus on different feline and human sporotrichosis outbreaks caused by S. brasiliensis in Argentina during 2011-2019. We will address the sources of infection and environmental hotspots, as well as the application of several treatment strategies for improving the pharmacotherapy of the different clinical forms of the disease. Finally, we will provide a detailed summary of the clinical aspects and new advances in host-pathogen interactions, virulence factors and immune response, focusing on state-of-the-art diagnostic tools and potential vaccine candidates.

Antifungal susceptibility profile and molecular identification of Cyp51C mutations in clinical and environmental isolates of Aspergillus flavus from Argentina.

BACKGROUND: The emergence of azole resistance in non-fumigatus Aspergillus strains is on the raise. OBJECTIVES: To study the susceptibility profiles and the molecular mechanisms of azole resistance of environmental and clinical strains of Aspergillus flavus from Argentina. METHODS: Thirty-five A flavus isolates (18 from soybean seeds and chickpea seeds and 17 from the clinic) were analysed for amphotericin B and azole resistance using the standard microbroth dilution method according to CLSI M38-A2 guidelines. Sequencing analysis of the cyp51 genes was conducted in those isolates displaying high MICs values to itraconazole, voriconazole and/or posaconazole. RESULTS: Among the environmental isolates, 33.3% of them showed high MIC values for at least one triazole whereas 23.5% of the clinical isolates displayed high MIC values for amphotericin B. Point mutations in the Cyp51C gene were recorded in most environmental isolates with non-wild-type MIC values. CONCLUSIONS: Susceptibility differences among environmental A flavus isolates might suggest the possibility of native resistance to certain triazole antifungals used in the clinic. To the best of our knowledge, this is the first report of antifungal screening of environmental strains of A flavus in soybean seeds and chickpea seeds from Argentina that showed increased resistance to voriconazole and itraconazole in comparison to clinical strains.

Innovative Therapeutic and Delivery Approaches Using Nanotechnology to Correct Splicing Defects Underlying Disease.

Alternative splicing of pre-mRNA contributes strongly to the diversity of cell- and tissue-specific protein expression patterns. Global transcriptome analyses have suggested that >90% of human multiexon genes are alternatively spliced. Alterations in the splicing process cause missplicing events that lead to genetic diseases and pathologies, including various neurological disorders, cancers, and muscular dystrophies. In recent decades, research has helped to elucidate the mechanisms regulating alternative splicing and, in some cases, to reveal how dysregulation of these mechanisms leads to disease. The resulting knowledge has enabled the design of novel therapeutic strategies for correction of splicing-derived pathologies. In this review, we focus primarily on therapeutic approaches targeting splicing, and we highlight nanotechnology-based gene delivery applications that address the challenges and barriers facing nucleic acid-based therapeutics.

High prevalence of triazole-resistant Aspergillus fumigatus sensu stricto in an Argentinean cohort of patients with cystic fibrosis.

BACKGROUND: Triazole resistance in Aspergillus fumigatus sensu stricto due to mutations in the cyp51A gene has been widely reported. Data from Argentina, and particularly from cystic fibrosis (CF) patients, are limited. OBJECTIVES: To investigate the prevalence and molecular mechanisms of azole resistance in A. fumigatus sensu stricto recovered from this population. METHODS: Ninety-three A. fumigatus isolates from 50 CF patients were retrospectively analysed for azole resistance using the standard microbroth dilution method according to CLSI M38-A2 guidelines. Sequencing analysis of the cyp51A gene and its promoter region was conducted in those isolates displaying high MIC values to itraconazole, voriconazole and/or posaconazole. RESULTS: Overall, 14% of isolates displayed high MIC values to at least one azole. Of them, 30.7% had the mutation TR34-L98H. No mutations in the cyp51A gene or its promoter were found in the remaining non-wild-type strains. Therefore, other mechanisms associated with azole resistance can be highly prevalent in these isolates. CONCLUSIONS: To the best of our knowledge, this is the first study in Latin America reporting azole-resistant A. fumigatus strains recovered from respiratory secretions of CF patients. Noteworthy, the prevalence of azole resistance in A. fumigatus sensu stricto in the studied Argentinean CF population is alarmingly high.

A Novel Combination of CYP51A Mutations Confers Pan-Azole Resistance in Aspergillus fumigatus.

The treatment of invasive and chronic aspergillosis involves triazole drugs. Its intensive use has resulted in the selection of resistant isolates, and at present, azole resistance in Aspergillus fumigatus is considered an emerging threat to public health worldwide. The aim of this work is to uncover the molecular mechanism implicated in the azole resistance phenotype of three Aspergillus fumigatus clinical strains isolated from an Argentinian cystic fibrosis patient under long-term triazole treatment. Strain susceptibilities were assessed, and CYP51A gene sequences were analyzed. Two of the studied Aspergillus fumigatus strains harbored the TR34-L98H allele. These strains showed high MIC values for all tested triazoles (>16.00 µg/ml, 1.00 µg/ml, 1.00 µg/ml, and 2.00 µg/ml for itraconazole, isavuconazole, posaconazole, and voriconazole, respectively). The third strain had a novel amino acid change (R65K) combined with the TR34-L98H mutations. This new mutation combination induces a pan-azole MIC augment compared with TR34-L98H mutants (>16 µg/ml, 4.00 µg/ml, 4.00 µg/ml, and 8.00 µg/ml for itraconazole, isavuconazole, posaconazole, and voriconazole, respectively). The strain harboring the TR34-R65K-L98H allele showed no inhibition halo when voriconazole susceptibility was evaluated by disk diffusion. The effect of these mutations in the azole-resistant phenotype was confirmed by gene replacement experiments. Transformants harboring the TR34-L98H and TR34-R65K-L98H alleles mimicked the azole-resistant phenotype of the clinical isolates, while the incorporation of the TR34-R65K and R65K alleles did not significantly increase azole MIC values. This is the first report of the TR34-L98H allele in Argentina. Moreover, a novel CYP51A allele (TR34-R65K-L98H) that induces a pan-azole MIC augment is described.

Histoplasma capsulatum 100-kilodalton antigen: recombinant production, characterization, and evaluation of its possible application in the diagnosis of histoplasmosis.

The goal of the present work was to develop a novel reagent with potential for histoplasmosis diagnosis. For this purpose, the genetic sequence of the 100 kDa protein of Histoplasma capsulatum (Hcp100) was cloned and expressed as a secretory protein in Pichia pastoris. After optimizing the culture conditions and purifying by immobilized metal ion affinity chromatography, the highest yield of Hcp100 reached approximately 1.3 mg/l with > 90% purity in shake flasks using basal salt medium supplemented with casamino acids after 72 h of methanol induction. To investigate its potential for diagnosis, its detection in urine samples using specific polyclonal antibodies as reagent was evaluated by dot blot in 6 patients with progressive disseminated histoplasmosis (PDH), of whom all had AIDS. Antigen was detected in urine from all 6 (100%) PDH patients. Urine samples from a pool of 20 healthy individuals did not react with the anti-Hcp100 antibodies. The dot blot assay performed in this study provides preliminary data of a simple technology that can be performed in medical institutions with limited resources to facilitate the rapid diagnosis of histoplasmosis, particularly the disseminated forms. Hence, use of these assays may provide a rapid diagnostic tool of PDH in endemic areas for histoplasmosis where PDH-related mortality is high, hastening treatment and improving patient survival. Finally, this novel antigen and its specific antibodies may provide an alternative diagnostic reagent to the largely unknown and poorly characterized polysaccharide antigens (HPA, galactomannan, histoplasmin) frequently used in the diagnostic tests. KEY POINTS: Few antigens are used as laboratory tools for the immunodiagnosis of histoplasmosis. P. pastoris was an excellent system for recombinant Hcp100 expression. Maximum expression levels of rHcp100 were achieved in BSM with 1% casamino acids. Dot blot assays with anti-rHcp100 antisera can be successfully used for diagnosing PHD.

Molecular epidemiology of Aspergillus species and other moulds in respiratory samples from Argentinean patients with cystic fibrosis.

In cystic fibrosis (CF) patients, fungal colonization of the respiratory tract is frequently found. Aspergillus fumigatus is the most frequently recorded and is associated with loss of pulmonary function and allergic disease (ABPA). The knowledge on prevalence rates of filamentous fungi in CF patients in Latin America is scarce. One hundred and seventy-six fungal isolates recovered from the upper respiratory tract of CF patients from Argentina were identified to species by morphology and DNA sequencing. In total, 90% of CF patients were colonized by Aspergillus sp., followed by Exophiala sp. (14%) and Scedosporium sp. (10%). Among Aspergillus, six species complexes (Fumigati, Flavi, Terrei, Nigri, Usti, and Nidulante) and different cryptospecies were found. Among Scedosporium, three species were observed (Scedosporium apiospermum, Scedosporium aurantiacum and Scedosporium boydii). All Exophiala isolates were identified as Exophiala dermatitidis. Rare filamentous fungi were also found. All cases of ABPA were associated to the presence of A. fumigatus. Mixed colonization with other mould or rare fungi was observed in half of them. To our knowledge, this is the first prospective study of mould species in CF using molecular methods in Latin America. This study shows that Aspergillus sp., E. dermatitidis and Scedosporium sp. have a high frequency in CF patients from Argentina, and by far, A. fumigatus was the most commonly cultured species. Continuous clinical surveillance is required to detect the emergence of new fungal pathogens and to detect resistant or difficult-to-treat species capable of chronic colonizing the airways and of hematogenous dissemination in case of lung transplantation.

Development and characterization of an improved formulation of cholesteryl oleate-loaded cationic solid-lipid nanoparticles as an efficient non-viral gene delivery system.

Nanoparticle-mediated plasmid delivery is considered a useful tool to introduce foreign DNA into the cells for the purpose of DNA vaccination and/or gene therapy. Cationic solid-lipid nanoparticles (cSLNs) are considered one of the most promising non-viral vectors for nucleic acid delivery. Based on the idea that the optimization of the components is required to improve transfection efficiency, the present study aimed to formulate and characterize cholesteryl oleate-containing solid-lipid nanoparticles (CO-SLNs) incorporating protamine (P) to condense DNA to produce P:DNA:CO-SLN complexes as non-viral vectors for gene delivery with reduced cytotoxicity and high cellular uptake efficiency. For this purpose, CO-SLNs were used to prepare DNA complexes with and without protamine as DNA condenser and nuclear transfer enhancer. The main physicochemical characteristics, binding capabilities, cytotoxicity and cellular uptake of these novel CO-SLNs were analyzed. Positively charged spherical P:DNA:CO-SLN complexes with a particle size ranging from 330.1 ± 14.8 nm to 347.0 ± 18.5 nm were obtained. Positive results were obtained in the DNase I protection assay with a protective effect of the genetic material and 100% loading efficiency was achieved at a P:DNA:CO-SLN ratio of 2:1:7. Transfection studies in human embryonic kidney (HEK293T) cells showed the versatility of adding protamine to efficiently transfect cells, widening the potential applications of CO-SLN-based vectors, since the incorporation of protamine induced almost a 200-fold increase in the transfection capacity of CO-SLNs without toxicity. These results indicate that CO-SLNs with protamine are a safe and effective platform for non-viral nucleic acid delivery.