Alginate-Amphothericin B Nanocomplexes Covered By Nanocrystals From Bacterial Cellulose: Physico-Chemical Characterization And In Vitro Toxicity

Alginate-Amphothericin B Nanocomplexes Covered By Nanocrystals From Bacterial Cellulose: Physico-Chemical Characterization And In Vitro Toxicity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Alginate-Amphothericin B Nanocomplexes Covered By Nanocrystals From Bacterial Cellulose: Physico-Chemical Characterization And In Vitro Toxicity Victória Soares Soeiro, Ricardo Silva-Carvalho, Daniela Martins, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-105741/v2 This work is licensed under a CC BY 4.0 License Status: Under Review Version 2 posted 11 You are reading this latest preprint version Show more versions Abstract Amphotericin B (AmB) is a drug with anti-leishmanial and anti-fungal ability, but it has low water solubility and permeability, limiting its therapeutic use. Therefore, the incorporation of AmB into nanostructured systems could be profit. Nanostructured systems without surfactants have pharmacotechnical advantages such as amplify the water solubility and decrease the toxicity. For this reason, the present study aimed to produce a nanocomplex of alginate (Alg) with AmB covered by NCC in order to decrease the toxicity of AmB. This straightforward process allowed to obtain water soluble particles Furthermore, the ionic adsorption of the NCC into the Alg-AmB nanocomplex surface was confirmed by an increase in the particle size and a small surface charge decrease. The amorphous inclusion of AmB complex into the polysaccharide chain network in both formulations. AmB in the nanocomplexes was in supper-aggregated form and showed good biocompatibility, being significantly less cytotoxic in vitro against kidney cells and significantly less hemolytic compared to the free-drug. The results indicated the Alg-AmB nanocomplex can be considered a non-toxic alternative to improve the AmB therapeutic effect. Furthermore, NCC coating of the nanocomplexes brought additional protection to the system without compromised the advantages attributed to the developed formulation. Biological sciences/Biotechnology/Biomaterials Nanoscience Bacterial cellulose Nanocrystals Amphotericin B. Alginate Nanocomplexes Figures Figure 1 Figure 2 Background Amphotericin B (AmB) is a polyene antibiotic used either as a gold standard therapy for fungal infections, once it does not cause microbial resistance 1 , or as a second-line treatment for visceral leishmaniasis 2 . Despite being a drug widely used for more than half a century, AmB presents several limitations such as low water solubility and low permeability, in addition to high toxicity, especially nephrotoxicity. 3 . Different formulations containing AmB are marketed, such as AmBisome® and Abelcet®, which are based on lipids and awfully expensive formulations. Therefore, drug delivery systems have been used nowadays in order to overcome AmB limitations and improve its therapeutic efficacy. Encapsulating the drug in a nanocomplex system was an alternative found to solve the product problems. 4 . Nanocomplexes are self-organized structures composed of a polymer (in many cases natural polysaccharides) and the drug. This system has advantages such as nano scale, high stability, good dispersion in water, low toxicity, with excellent cost-benefit and does not use solvents. In the literature, there are several works using different types of polymers, such as dextrin 5 , gum arabic 6 , albumin 7 and alginate 8 . Alginate (Alg) is a polymer extracted from natural sources, widely used in the pharmaceutical area and in tissue engineering. This material has several advantages, such as immune system activation, nontoxicity, biocompatibility and biodegradability 9 – 11 . In our research, in order to reinforce and cover the system Alg-AmB, bacterial cellulose nanocrystals (NCC) were employed. NCC can be used as a reinforcement material to drug delivery systems, protecting them and enabling the administration of the drug by different routes 12 , 13 . NCC are produced by the action of cellulase in contact with bacterial cellulose. This material has a renewable and sustainable origin, with good cost-benefit, biocompatibility and biodegradability, besides presenting a good mechanical resistance, large surface area, low toxicity 10 , 14 , 15 . Since there is no delivery system to AmB using NCC, we aimed to develop nanocomplexes of Alg-AmB, evaluating the NCC as a cover system in order to protect the system, analysing physic-chemical characterization and the toxicity of AmB, assess by in vitro assays. Results Physico-chemical characterization of nanocomplexes Size, index of polydispersity (PDI) and zeta potential of the Alg-AmB nanocomplexes and the nanocomplexes covered by NCC (Alg-AmB + NCC) are shown in table I. Table I - Analysis of the size, PDI and zeta potential of Alg-AmB Alg-Amb+NCC) NCC Alg-AmB Alg-AmB + NCC Size (nm) 41.67 ± 5.55 258.87 ± 10.41 466.3 ± 17.57 Index of polydispersity 0.127 ± 0.005 0.523 ± 0.073 0.420 ± 0.05 Zeta potential (mV) 7.57 ± 2,26 mV -62.93 ± 2.02 -55.75 ± 1.23 Both nanocomplexes maintained their size on the nanometer scale. However, there was a significant increase in the size of the Alg-AmB + NCC nanocomplex, possibly due to coating by the NCC. On the other hand, when NCC was added NCCin nanocomplexes, the polydispersity index decreased. Zeta potential of the nanocomplexes increased in the sample containing NCC. The increase was indicated the possibility of nanocomplexes coating. Although the NCC have a positive characteristic, the value is very low; so the coating value has not changed significantly. Figure 1 shows Differential Scanning Calorimetry (DSC) data for AmB, Alg, NCC, Alginate + AmB (physical mixture), Alg-AmB and Alg- AmB + NCC. Note A – DSC analysis of AmB, Alg, NCC, alginate + AmB (mixture), Alg-AmB and Alg-AmB + NCC. B – FTIR of pure alginate, Alg and Alg-AmB. D – UV-Vis spectrometry of the AmB, Alg-AmB and Alg-AmB + NCC. In Amphotericin B sample, we have found a peak in 167°C degrees. For NCC sample, the peak was at 151 ° C. The physical formation of AmB-alginate mixture showed peaks at 144°C, referring to alginate, and at 161°C referring to AmB. On the other hand, when Alg-AmB nanocomplexes were formed, DSC showed only one peak, at 141°C, for alginate. The DSC value for Alg-AmB + NCC sample showed a similar peak to Alg-AmB, but with a slightly lower temperature, at 123°C. This indicates when NCC was added in the formulation, there was a decrease in the temperature. Figure 1 .B shows the FTIR data of the samples of pure alginate, Alg and Alg-AmB. The wavelength band 3424 cm − 1 presented in all samples refers to the O-H stretching vibrations of the alginate. The weak band at 2930 cm − 1 represents the C-H stretching vibrations; the bands 1630 and 1416 cm − 1 are asymmetrical and symmetrical stretching vibrations of the C ≡ O and the COO- group. Furthermore, the band around 1024 cm − 1 refers to the vibrations of the ring elongation C-O and C-O, with deformations of C-C-H and C-O-H 8 , 16 . In the Alg-AmB sample, the AmB wavelength bands were compared to data from the literature, showing the bands: OH stretching vibration (3434 cm − 1 ), CH flexion vibrations and CH 3 oscillation (1024 cm − 1 ) 17 . The hypothesis is the other bands are overlapped by the alginate bands. The Fig. 1 .D shows the UV-vis spectra of AmB, Alg-AmB nanocomplex and Alg-AmB + NCC. In this study, AmB was firstly dissolved in 0.1 M borate buffer pH 11 and then diluted in water, in a monomeric state. The ratio Abs 349 /Abs 410 was 0.36. Considering the Alg-AmB nanocomplex, the1st peak (327 nm) and the ratio between the peaks I and IV (Abs 327 /Abs 410 ) was 4.38, showing AmB is in a supper-aggregated state in the formulation, which, as referred above, is associated to a low toxicity. Despite promoting a decrease in the absorbance intensity, NCC addition to the nanocomplex surface did not affect the supper-aggregated state of AmB since a ratio of 4.45 (Abs 327 /Abs 409 ) was found. In vitro toxicity The hemolysis test was performed for AmB, Alg, Alg-AmB, Alg-AmB + NCC and AmBisome, in concentrations of 1, 2, 4, 8, 16 and 32 µM (Fig. 2 .A). Note A – AmB (*) and Alg-AmB (**) were compared to the other groups by the Duncan test (p < 0,05). B – AmB (*) was compared to the other groups by the Duncan test (p < 0.05). The standard sample of Alg showed irrelevant values of hemolysis (lower than 2%), in all concentration. AmB induced hemolysis percentage between 1.97 ± 1.66% and 81.18 ± 12.50%. When alginate was added to the formulation (Alg-AmB), there was a decrease in toxicity of 90% compared to standard AmB, with hemolysis values from − 0.08% to 30.25 ± 0.50%. When Alg-Amb was coated with NCC, the values were similar (-0.3 ± 0.19% and 44.7 ± 0.50%). Even at high concentrations, nanocomplexes reduced the rate of hemolysis in comparison to standard AmB. Regarding hemolytic porcetange of AmBisome®, data ranged from 0.34 ± 0.33% to 2.13 ± 1%. Although AmBisome's hemolysis values are lower than those found in the Alg-AmB formulation, Alg-AmB nanocomplexes had excellent results compared with free AmB. Hemolysis diminish in the sample due to the incorporation of AmB in Alg nanocomplexes shows the nanocomplexes are safe. In Fig. 2 .B is presented the cytotoxicity data for AmB, Alg, Alg-AmB, Alg-AmB + NCC and AmBisome®. Alg showed cell viability between 93.01 ± 3.83% and 108.66 ± 7.04%. AmB data indicated concentration-dependent toxicity. On the other hand, the Alg-AmB nanocomplexes demonstrated significant less toxicity than free AmB. Cell viability was high, ranging between 97.16 ± 5.61% and 109.86 ± 7.06%. For the Alg-AmB + NCC nanocomplex, the cell viability values were similar to Alg-AmB NCC, ranging from 98.55 ± 5.06% to 106.40 ± 5.35%, demonstrating safety use of coating with NCC. Cell viability for AmBisome® was around 101.40 ± 6.23% to 111.29 ± 6.92%. When comparing the results of the commercial sample, AmBisome®, with Alg-AmB and Alg-AmB + NCC nanocomplexes, there was no significant difference among them, indicating both nanocomplexes, with or without NCC, are as safe as the commercial one. Discussion In this work, alginate-amphotericin nanocomplexes (Alg-Amb) were developed aiming to improve the AmB water solubility with low toxicity. It was also evaluated the influence of NCC as a cover system to protect the system. Therefore, to achieve this Aim, it was realized physico-chemical characterization and in vitro toxicity. Taheri and Mohammadi (2015) 18 utilized cellulose nanocrystals to cover a hydroquinone system and they demonstrated an increase in particle size. The way to obtain smaller particles is important, because expanding the routes of administration. Systems with polymers nanoparticles of size around 200 nm are absorbed by the spleen, lung and liver 4 . The nanocomplexes method is capable of producing smaller particles than ionotropic gelification method. Senna et al. (2018) 19 produced particles by ionotropic gelation and observed the production of particles of 1.2 ± 0.34 mm. Compared to our method; these particles were about 3 times larger than the nanocomplex coated by NCC. One study with alginate particles containing miltefosine obtained by ionotropic gelation and emulsification method showed a polydispersity index of 0.43 ± 0.14 20 , similar to the data found in our study. Comparatively in another study, the zeta potential of alginate particles obtained by ionotropic gelation showed negative values in the range of -15.7 ± 1.7 and -9.8 ± 1.2 mV. However, when adding chitosan in the formulation, there was a change in zeta potential due to the coating 21 . AmB is characterized by two endothermic peaks in the crystalline form 22 . In our study, AmB standard showed peaks at 167 °C (melting point) and 198 °C, similar to the temperatures found by those authors. Alginate showed an endothermic peak at 149 °C, which demonstrates the degradation of the biopolymer 23,24 . Vasconcelos et al. 2017 25 . reported peaks to NCC obtained by acid hydrolysis ranging from 140 to 195 °C 25, similar to our finding (peak at 151 ° C). Regarding Alg-AmB nanocomplexes, we have described only one peak. The absence of AmB peak indicates there was a change from crystalline to amorphous state, demonstrating the formation and improvement of alginate and AmB nanocomplexes 26 . In a study with alginate associated to the polymer poly [N-(2-hydroxypropyl) methacrylamide] and the drug camptothecin, the characteristic peak of camptothecin was absent due to the drug being compatible with the polymers and its high dispersion in the formulation 27 . About alginate sample, similar results were found by Silva-Carvalho et al. (2020), indicating the importance of using safe polysaccharides for the formation of the nanocomplex. Another examples can be observed in a study whose objective was to use NCC as a reinforcement for a film containing polylactic acid, concluding there was an increase in crystallinity 28 . George et al., (2011 reported the utilization of NCC associated with polymers and showed there was an increase in the parameters of glass transition, melting temperature, enthalpy and crystalline behaviour. The degree of AmB aggregation can be determined by the ratio from the first to the fourth peak in the UV-Vis spectra, being a value of 2 related to a supper-aggregated form 4,6,8,29 . In solution, AmB has three different states that affect its activity and pharmacokinetical characteristics, such as monomers, in which AmB is water soluble normally associates with ergosterol in fungal and protozoan cell membranes; oligomers, a state in which small water aggregates are toxic towards host cells and present very low solubility; and poly-aggregates, that also referred as supper-aggregates present low in vitro and in vivo toxicity 6,8 . Fungizone® and AmBisome® are drugs with low toxicity found in the pharmaceutical market. Due to the super-aggregated state of AmB, specified by ratio values of 2.9 and 4.8 (Alg-AmB and Alg-AmB+NCC, respectively), our nanocomplexes have similar data in relation to commercial formulations 7,30 . The hemolysis test is important in studies with nanocomplexes or nanoparticles containing AmB once there is evidence of hemolytic anemia caused by the use of free AmB 31,32 . AmB has a high hemotoxicity even at low concentrations, due to the aggregated conformation 31 . Conjugates and ionic cross-linked polymeric nanoparticles of AmB with alginate show less hemotoxicity and greater hemocompatibility than free AmB, possibly by the protective effect from polysaccharide and by the change in conformation of the super-aggregated state 4,8 . The importance of cytotoxicity test with this cell strain is due to the nephrotoxicity associated with free AmB 7 . Therefore, it is important to understand the behaviour of nanocomplexes in this type of cells. The non-toxicity and the capacity for cell proliferation observed to alginate experimentation have already been described 4 . In a study, AmB solution at 15.6 µg/mL was able to induce death in renal cells 17 . On the other hand, Ravichandran and Jayakrishnan, 2018 tested AmB and alginate conjugates to verify cytotoxicity and observed a decrease in toxicity compared to free AmB. Although there are studies reporting reduction in hemo and cytotoxicity of amphotericin, our work proposes a simplified methodology, with a cost-benefit and achieves the same objectives by complex and high cost methodologies. Therefore, in addition to our material being as safe as the standard medicine (AmBisome®). As,Silva-Carvalho, et al, 2020 the findings, our work, overall, demonstrates the suitability of alginate as an AmB carrier, covered or not by NCC. Conclusion In our study, the development of an alginate nanocomplexes with AmB decreased the hemolytic and cytotoxic effect of the drug. In addition, coating the nanocomplexes with the NCC, produced by enzymatic way, brought additional protection to the system without compromised the advantages attributed to the developed formulation, which can be considered a cost benefit, non-toxic and safe as the commercial one. These findings have an important relevance once when developing a nanocomplex, the great advantage is to direct the formulation to the drug's action sites nd with the extra cover of NCC can be applied in other administration route. Methodology Reagents Amphotericin B (AmB, molecular weight of 924.08 g/mol) powder from Streptomyces sp., resazurin Sodium salt, sodium alginate, triton X-100, sodium tetraborate decahydrate were purchased from Sigma-Aldrich (Missouri, USA). Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS) and penicillin-streptomycin were obtained from Merck Millipore (Massachusetts, USA). Roswell Park Memorial Institute (RPMI) 1640. Glutamax supplemented medium and L-glutamine (GlutaMAX™-I) was purchased from Gibco (Massachusetts, USA). Dimethylsulfoxide (DMSO ATCC® 4-X™) solution for cell culture was acquired from American Type Culture Collection (ATCC, Virginia, USA). Dialysis tubing with a molecular weight cut-off of 1000 Da was obtained from Orange Scientific (Braine-l'Alleud, Belgium). AmBisome® was kindly provided by Gilead Sciences. Obtaining bacterial cellulose nanocrystals (NCC) NCC production was carried out following the methodology developed by our research group ( In press- supplementary material ). The bacterial cellulose was obtained from G. xylinus , according to Jozala et al. (2014) 33 .The process uses a mechanical treatment of bacterial cellulose with ultra-turrax and high-pressure homogenizer and a enzymatic treatment with cellulase for 72 hours. After, the NCC were separated with centrifugation and filtration. Preparation of alginate-amphotericin B (Alg-AmB) nanocomplexes The Alg-AmB nanocomplexes were prepared according to Silva-Carvalho et al. (2020) 5 . In the formulation, borate buffer (pH 11.0), sodium alginate and amphotericin B were used, resulting in a final concentration of 2.5 mg/mL of AmB in the solution. The nanocomplexes formulation were described in table II. Table II – Formulation of alginate (Alg) and alginate-amphotericin B (Alg-AmB). Formulations Borate buffer pH 11 (mL) Sodium alginate (mg) Amphotericin B (mg) Alg 9.6 120 - Alg-AmB 9.6 96 24 The samples were kept under agitation at 4 °C for 48 hours without interference from light. In the next step, the dialysis process was run on a membrane from 12000 to 14000 KDa. The membrane with the sample was immersed in 5 L of distilled water under agitation at 4 °C, without light interference, for 30 hours. During the process, the water was changed three times to remove salts, until reaching pH value in the range of 5.5-5.7. Then, the samples were collected, frozen at -80 °C for 24 hours, and lyophilized. The final yield of the process was 73.72%. Nanocomplex coating Alg and Alg-AmB nanocomplexes were covered with NCC. For that, the lyophilized nanocomplexes were dispersed in water in the proportion of 1 mg/mL. After dispersion, 1 ml of the nanocomplexes was added into 1 ml of 0.01% NCC suspension. This sample was kept on rotatory shaker (20 rpm) at 25 °C for 24 hours. Physico-chemical characterization For the physico-chemical characterization the following parameters were used: size, index of polydispersity, zeta potential, differential scanning calorimetry, infrared spectrometry by Fourier transform (FTIR) and UV-Vis spectrometry. For the evaluation of the size, index of polydispersity and surface potential of the particles (zeta potential), the Zetasizer equipment (ZEN3600) was used, at an angle of 173º, at 25 °C for dynamic light scattering (DLS), Index of Polydispersity and zeta potential. The samples Alg-AmB and Alg-AmB + NCC were analysed in six times. To characterize the stability of the nanocomplexes, Differential Scanning Calorimetry (DSC) was used. The characterization was performed by the DSC 6000 equipment (PERKIN ELMER | STEC INSTRUMENTS) in a nitrogen atmosphere with a flow of 20 mL/min, in the heating range of 25 – 250 °C with a heating rate of 10 °C/min. The test was re-performed with the samples AmB, Alg nanocomplexes, NCC, alginate + AmB (physical mixture without nanocomplexes process), Alg-AmB nanocomplex and Alg-AmB + NCC. The characterization of the chemical groups was carried out by spectrometry in FTIR. The samples were analysed by the technique of KBr tablets, with 2 mg of sample and 200 mg of KBr. The infrared spectra were obtained in the range of 4000–400 cm -1 in the Bruker Alpha II equipment, with a resolution of 4 cm -1 and 12 scans. The samples analysed were: pure alginate, Alg nanocomplexes and Alg-AmB. In addition, the samples were analysed by UV-Vis spectroscopy in the 300-450 nm range using a UV-Visible spectrophotometer (JASCO V-560) with 5 nm resolution and scanning speed of 400 nm/min. The nanocomplexes Alg and Alg-AmB were dispersed in distilled water (1 mg of sample/mL). The material was diluted again to 10 µM in distilled water in order to avoid saturation of the test. AmB control was performed using 1082 µM diluted in borate buffer (0.1 M, pH 11). The solution was also diluted to 10 µM with distilled water. To perform the measurement, 200 µL of the solutions were analyzed in a quartz cuvette. The absorbance ratio of peak I (315-350) to peak IV (408-410) was used to monitor the aggregation status of AmB and the results were evaluated for the quantification of AmB in the sample. In vitro toxicity For the in vitro toxicity tests, a commercial product (AmBisome®) was taken as a control. This drug is for injectable use and it has a liposomal formulation containing AmB. AmBisome® prescription is mentioned in several articles due to its low toxicity 6,7,34 . Hemolysis and cytotoxicity were the testes chosen to perform the safety of the new nanocomplexes. Hemolysis test Hemolysis test assesses the percentage of erythrocyte rupture. Both the protocol and the animal's blood were derived from Silva-Carvalho et al. (2020) 5 . We used ordinary blood samples (dog) from a blood bank. The whole blood was centrifuged for 10 min (4 °C, 1200 g) and the supernatant was discarded. Red blood cells were resuspended in phosphate buffer solution (pH 7.4). Resuspended red blood cells were counted in a Neubauer chamber. In a 48-well plate, 450 µL of red blood cells at a concentration of 1 x 10 8 cells / mL were placed in contact with 50 µL of the samples, at concentrations of 1, 2, 4, 8, 16 and 32 µM AmB, Alg, Alg -AmB, Alg-AmB + NCC and AmBisome®. The plate was incubated with shaking for 30 minutes (37 °C, 120 rpm). After the period, the solutions were collected and centrifuged for 10 min (4 ° C, 1200 G). The supernatants were collected and analyzed by UV-Vis spectrophotometry, with absorbance at 540 nm (referring to hemoglobin). Complete lysis (100%) was admitted by hemoglobin released with 1% triton X-100 (positive control). Cytotoxicity Human Embryonic Kidney (HEK) cell line was selected to perfume citotoxity due to the toxicity of AmB in renal cells. The HEK 293T monolayer (1 × 10 4 cell/well) was incubated for 24 h (37 °C in a 5% CO 2 atmosphere) with AmB, Alg, Alg-AmB, Alg-AmB + NCC and AmBisome® at concentrations of 0.78, 1, 17, 1.76, 2.63, 3.95, 5.93, 8.89, 13.33, 20, 30, 45 and 67.5 μM. After the incubation period, 10% (v / v) of a 2.5 mM resazurin solution was added to each well and the plates were incubated again under the same conditions as above for 4 hours. Fluorescence was measured (λ 560 / λ 590) in a SpectraMAX GeminiXS microplate reader (Molecular Devices LLC, California, USA). The results were expressed as mean percentage ± SD of viable cells in relation to the positive control (condition considered to be 100% viable cells). Statistical analysis Data from toxicity tests were expressed as mean ± standard deviation. Analysis of Variance (ANOVA) followed by the Duncan test were used to verify differences among treatment protocols, and p values <0.05 were considered significant. Results were analyzed using Statistica® v. 8.0 (Dell, Round Rock, TX, USA) and GraphPad Prism® v. 6.0 (San Diego, CA, USA). Declarations Acknowledgements The authors acknowledge financial support from Coordination for Higher Level Graduate Improvements (CAPES/Brazil, finance code 001), National Council for Scientific and Technological Development (CNPq/Brazil Process #428751/2016-4), and the State of São Paulo Research Foundation (FAPESP/Brazil, processes #2019/22626-5 and #2018/10508-5). Author contributions VSS: Investigation, Methodology, Formal analysis, Writing - original draft; RSC: Investigation, Methodology, Formal analysis, Writing - review & editing; DM: Methodology develop; PP: Methodology develop; DG: Validation, Writing - review & editing; MVC: Writing - review & editing; FMG: Conceptualization, Writing - review & editing, Supervision; AFJ: Conceptualization, Writing - review & editing, Supervision. Additional Information Competing Interests Statement: the authors declare that they have no competing interests References Gray, K. C. et al. Amphotericin primarily kills yeast by simply binding ergosterol. Proc. Natl. Acad. Sci. U. S. A. 109 , 2234–2239 (2012). TORRADO, J. J., ESPADA, R., BALLESTEROS, M. P. & TORRADO-SANTIAGO, S. Amphotericin B Formulations and Drug Targeting. J. Pharm. Sci. 97 , 2405–2425 (2008). Richter, A. R., Feitosa, J. P. A., Paula, H. C. B., Goycoolea, F. M. & de Paula, R. C. M. 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Biomater. 66 , 781–790 (2017). Wardhono, E. Y., Kanani, N. & Rahmayetty, A. &. Development of polylactic acid ( PLA ) bio- composite films reinforced with bacterial cellulose nanocrystals ( BNCC ) without any surface modification. J. Dispers. Sci. Technol. 0 , 1–8 (2019). Barwicz, J., Christian, S. & Gruda, I. Effects of the aggregation state of amphotericin B on its toxicity to mice. Antimicrob. Agents Chemother. 36 , 2310–2315 (1992). Mullen, A. B. & Carter, K. C. Comparison of the Efficacies of Various Formulations of Amphotericin B against Murine Visceral Leishmaniasis. Antimicrob. Agents Chemother. 41 , 2089–2092 (1997). Vásquez Marcano, R. G. del J., Tominaga, T. T., Khalil, N. M., Pedroso, L. S. & Mainardes, R. M. Chitosan functionalized poly (ε-caprolactone) nanoparticles for amphotericin B delivery. Carbohydr. Polym. 202 , 345–354 (2018). Jain, J. P. & Kumar, N. Development of amphotericin B loaded polymersomes based on (PEG)3-PLA co-polymers: Factors affecting size and in vitro evaluation. Eur. J. Pharm. Sci. 40 , 456–465 (2010). Jozala, A. F. et al. Bacterial cellulose production by Gluconacetobacter xylinus by employing alternative culture media. Appl. Microbiol. Biotechnol. 99 , 1181–1190 (2014). Serrano, D. R. et al. Hemolytic and pharmacokinetic studies of liposomal and particulate amphotericin B formulations. Int. J. Pharm. 447 , 38–46 (2013). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 2 posted Editorial decision: Major revision 29 Sep, 2021 Reviews received at journal 08 Sep, 2021 Reviews received at journal 03 Jul, 2021 Reviewers agreed at journal 22 Jun, 2021 Reviewers agreed at journal 11 May, 2021 Reviewers agreed at journal 30 Apr, 2021 Reviewers invited by journal 19 Feb, 2021 Editor assigned by journal 18 Feb, 2021 Editor invited by journal 08 Feb, 2021 Submission checks completed at journal 08 Feb, 2021 First submitted to journal 27 Jan, 2021 You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-105741","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[{"code":1,"date":"2020-11-17 19:11:54","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":11703348,"identity":"79430254-4354-417b-a96e-e93f48204737","order_by":0,"name":"Victória Soares Soeiro","email":"","orcid":"","institution":"Universidade de Sorocaba","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Victória","middleName":"Soares","lastName":"Soeiro","suffix":""},{"id":11703349,"identity":"b422a34a-6e22-4216-bb0d-1aa8db1a98c6","order_by":1,"name":"Ricardo Silva-Carvalho","email":"","orcid":"","institution":"University of Minho","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ricardo","middleName":"","lastName":"Silva-Carvalho","suffix":""},{"id":11703350,"identity":"f65d5d82-7d43-467f-8f21-0794d9b0e7f2","order_by":2,"name":"Daniela Martins","email":"","orcid":"","institution":"University of Minho","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Daniela","middleName":"","lastName":"Martins","suffix":""},{"id":11703351,"identity":"19267ab8-2702-45c0-9082-2594c9cd9783","order_by":3,"name":"Pier Parpot","email":"","orcid":"","institution":"University of Minho","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Pier","middleName":"","lastName":"Parpot","suffix":""},{"id":11703352,"identity":"5c5a6d7b-cc1b-42c6-b3a0-85fa8b46a4ca","order_by":4,"name":"Denise Grotto","email":"","orcid":"","institution":"Universidade de Sorocaba","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Denise","middleName":"","lastName":"Grotto","suffix":""},{"id":11703353,"identity":"dc5e0539-dbbb-4b06-abc6-bba659badb9a","order_by":5,"name":"Marco Vinicius Chaud","email":"","orcid":"","institution":"Universidade de Sorocaba","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Marco","middleName":"Vinicius","lastName":"Chaud","suffix":""},{"id":11703354,"identity":"8797718c-b65c-424f-becd-7c0a74194a1a","order_by":6,"name":"Francisco Miguel Portela Gama","email":"","orcid":"","institution":"University of Minho","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Francisco","middleName":"Miguel Portela","lastName":"Gama","suffix":""},{"id":11703355,"identity":"a663a650-d47c-4b1a-8490-8daf8b6c8c32","order_by":7,"name":"Angela Jozala","email":"data:image/png;base64,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","orcid":"","institution":"Universidade de Sorocaba","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Angela","middleName":"","lastName":"Jozala","suffix":""}],"badges":[],"createdAt":"2020-11-10 13:00:40","currentVersionCode":2,"declarations":"","doi":"10.21203/rs.3.rs-105741/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-105741/v2","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":5961929,"identity":"e5a8d6f0-a4a9-4e54-8f52-4ebbe01ffb43","added_by":"auto","created_at":"2021-02-15 12:54:56","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":156584,"visible":true,"origin":"","legend":"A – Differential Scanning Calorimetry (DSC). B – Infrared spectrum by Fourier transform (FTIR). C – Design Production of Alg-AmB and Alg-AmB + NCC. D – UV-Vis spectrometry. \nNote: A – DSC analysis of AmB, Alg, NCC, alginate + AmB (mixture), Alg-AmB and Alg-AmB + NCC. B – FTIR of pure alginate, Alg and Alg-AmB. D – UV-Vis spectrometry of the AmB, Alg-AmB and Alg-AmB + NCC.","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-105741/v2/e90507602e02833261d10f28.jpg"},{"id":5962130,"identity":"9966fe6d-b564-48c8-9863-a5aac60182b5","added_by":"auto","created_at":"2021-02-15 12:57:56","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":85615,"visible":true,"origin":"","legend":"Percentage of hemolysis after treatment of red blood cells (A) Cytotoxicity of the renal cell line HEK293T (B) with Alg, Alg-AmB, Alg-AmB + NCC, AmBisome® and AmB.\nNote: A ¬– AmB (*) and Alg-AmB (**) were compared to the other groups by the Duncan test (p\u003c0,05). B – AmB (*) was compared to the other groups by the Duncan test (p \u003c0.05).","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-105741/v2/1b1679da23c5f07dd57efdf8.jpg"},{"id":15670751,"identity":"7debe42d-73d1-498f-9adf-83d729ab4ec7","added_by":"auto","created_at":"2021-11-18 14:01:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":554128,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-105741/v2/769e5bdb-9c9f-4b81-a758-2d8bae1d71f5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eAlginate-Amphothericin B Nanocomplexes Covered By Nanocrystals From Bacterial Cellulose: Physico-Chemical Characterization And \u003cem\u003eIn Vitro\u003c/em\u003e Toxicity\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eAmphotericin B (AmB) is a polyene antibiotic used either as a gold standard therapy for fungal infections, once it does not cause microbial resistance \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, or as a second-line treatment for visceral leishmaniasis \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Despite being a drug widely used for more than half a century, AmB presents several limitations such as low water solubility and low permeability, in addition to high toxicity, especially nephrotoxicity. \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eDifferent formulations containing AmB are marketed, such as AmBisome\u0026reg; and Abelcet\u0026reg;, which are based on lipids and awfully expensive formulations. Therefore, drug delivery systems have been used nowadays in order to overcome AmB limitations and improve its therapeutic efficacy. Encapsulating the drug in a nanocomplex system was an alternative found to solve the product problems. \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eNanocomplexes are self-organized structures composed of a polymer (in many cases natural polysaccharides) and the drug. This system has advantages such as nano scale, high stability, good dispersion in water, low toxicity, with excellent cost-benefit and does not use solvents. In the literature, there are several works using different types of polymers, such as dextrin \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, gum arabic \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, albumin \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e and alginate \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Alginate (Alg) is a polymer extracted from natural sources, widely used in the pharmaceutical area and in tissue engineering. This material has several advantages, such as immune system activation, nontoxicity, biocompatibility and biodegradability \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. In our research, in order to reinforce and cover the system Alg-AmB, bacterial cellulose nanocrystals (NCC) were employed.\u003c/p\u003e\n\u003cp\u003eNCC can be used as a reinforcement material to drug delivery systems, protecting them and enabling the administration of the drug by different routes \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. NCC are produced by the action of cellulase in contact with bacterial cellulose. This material has a renewable and sustainable origin, with good cost-benefit, biocompatibility and biodegradability, besides presenting a good mechanical resistance, large surface area, low toxicity \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eSince there is no delivery system to AmB using NCC, we aimed to develop nanocomplexes of Alg-AmB, evaluating the NCC as a cover system in order to protect the system, analysing physic-chemical characterization and the toxicity of AmB, assess by \u003cem\u003ein vitro\u003c/em\u003e assays.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003cp\u003e\u003cstrong\u003ePhysico-chemical characterization of nanocomplexes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSize, index of polydispersity (PDI) and zeta potential of the Alg-AmB nanocomplexes and the nanocomplexes covered by NCC (Alg-AmB\u0026thinsp;+\u0026thinsp;NCC) are shown in table I.\u003c/p\u003e\n\u003cp style='margin-top:.25in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:left;font-size:13px;font-family:\"Arial\",sans-serif;font-weight:bold;line-height:150%;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif; font-size: 15px;\"\u003eTable I - Analysis of the size, PDI and zeta potential of Alg-AmB Alg-Amb+NCC)\u003c/span\u003e\u003c/p\u003e\u003ctable style=\"width:100.0%;border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:30.54%;border-top:solid #7F7F7F 1.0pt;border-left: none;border-bottom:solid #7F7F7F 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:13.05pt;\"\u003e\n \u003cp style='margin-top:.25in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:left;text-indent:0in;line-height:150%;font-size:16px;font-family:\"Arial\",sans-serif;'\u003e\u003cstrong\u003e\u003cspan style=\"font-family: Calibri, sans-serif; font-size: 15px;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:23.52%;border-top:solid #7F7F7F 1.0pt;border-left: none;border-bottom:solid #7F7F7F 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:13.05pt;\"\u003e\n \u003cp style='margin:0in;text-align:left;text-indent:0in;line-height:150%;font-size:16px;font-family:\"Arial\",sans-serif;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cstrong\u003eNCC\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:23.52%;border-top:solid #7F7F7F 1.0pt;border-left: none;border-bottom:solid #7F7F7F 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:13.05pt;\"\u003e\n \u003cp style='margin:0in;text-align:left;text-indent:0in;line-height:150%;font-size:16px;font-family:\"Arial\",sans-serif;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cstrong\u003eAlg-AmB\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:22.42%;border-top:solid #7F7F7F 1.0pt;border-left: none;border-bottom:solid #7F7F7F 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:13.05pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:left;text-indent:0in;line-height:150%;font-size:16px;font-family:\"Arial\",sans-serif;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cstrong\u003eAlg-AmB + NCC\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:30.54%;border:none;border-bottom:solid #7F7F7F 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:34.35pt;\"\u003e\n \u003cp style='margin-top:.25in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:left;text-indent:0in;line-height:150%;font-size:16px;font-family:\"Arial\",sans-serif;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cstrong\u003eSize (nm)\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:23.52%;border:none;border-bottom:solid #7F7F7F 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:34.35pt;\"\u003e\n \u003cp style='margin:0in;text-align:left;text-indent:0in;line-height:150%;font-size:16px;font-family:\"Arial\",sans-serif;margin-bottom:0in;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e41.67 \u0026plusmn; 5.55\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:23.52%;border:none;border-bottom:solid #7F7F7F 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:34.35pt;\"\u003e\n \u003cp style='margin:0in;text-align:left;text-indent:0in;line-height:150%;font-size:16px;font-family:\"Arial\",sans-serif;margin-bottom:0in;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e258.87 \u0026plusmn; 10.41\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:22.42%;border:none;border-bottom:solid #7F7F7F 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:34.35pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:left;text-indent:0in;line-height:150%;font-size:16px;font-family:\"Arial\",sans-serif;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e466.3 \u0026plusmn; 17.57\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:30.54%;border:none;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-top:.25in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:left;text-indent:0in;line-height:150%;font-size:16px;font-family:\"Arial\",sans-serif;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cstrong\u003eIndex of polydispersity\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:23.52%;border:none;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:left;text-indent: 0in;line-height:150%;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e0.127 \u0026plusmn; 0.005\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:23.52%;border:none;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-top:.25in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:left;text-indent:0in;line-height:150%;font-size:16px;font-family:\"Arial\",sans-serif;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e0.523 \u0026plusmn; 0.073\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:22.42%;border:none;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:left;text-indent:0in;line-height:150%;font-size:16px;font-family:\"Arial\",sans-serif;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e0.420 \u0026plusmn; 0.05\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:30.54%;border-top:solid #7F7F7F 1.0pt;border-left: none;border-bottom:solid #7F7F7F 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:19.85pt;\"\u003e\n \u003cp style='margin-top:.25in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:left;text-indent:0in;line-height:150%;font-size:16px;font-family:\"Arial\",sans-serif;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cstrong\u003eZeta potential (mV)\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:23.52%;border-top:solid #7F7F7F 1.0pt;border-left: none;border-bottom:solid #7F7F7F 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:19.85pt;\"\u003e\n \u003cp style='margin:0in;text-align:left;text-indent:0in;line-height:150%;font-size:16px;font-family:\"Arial\",sans-serif;margin-bottom:0in;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e7.57\u0026nbsp;\u0026plusmn;\u0026nbsp;2,26 mV\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:23.52%;border-top:solid #7F7F7F 1.0pt;border-left: none;border-bottom:solid #7F7F7F 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:19.85pt;\"\u003e\n \u003cp style='margin:0in;text-align:left;text-indent:0in;line-height:150%;font-size:16px;font-family:\"Arial\",sans-serif;margin-bottom:0in;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e-62.93 \u0026plusmn; 2.02\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:22.42%;border-top:solid #7F7F7F 1.0pt;border-left: none;border-bottom:solid #7F7F7F 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:19.85pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:left;text-indent:0in;line-height:150%;font-size:16px;font-family:\"Arial\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif; font-size: 15px;\"\u003e-55.75 \u0026plusmn; 1.23\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\u003cBR\u003e\n\u003cp\u003eBoth nanocomplexes maintained their size on the nanometer scale. However, there was a significant increase in the size of the Alg-AmB\u0026thinsp;+\u0026thinsp;NCC nanocomplex, possibly due to coating by the NCC. On the other hand, when NCC was added NCCin nanocomplexes, the polydispersity index decreased.\u003c/p\u003e\n\u003cp\u003eZeta potential of the nanocomplexes increased in the sample containing NCC. The increase was indicated the possibility of nanocomplexes coating. Although the NCC have a positive characteristic, the value is very low; so the coating value has not changed significantly.\u003c/p\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e shows Differential Scanning Calorimetry (DSC) data for AmB, Alg, NCC, Alginate\u0026thinsp;+\u0026thinsp;AmB (physical mixture), Alg-AmB and Alg- AmB\u0026thinsp;+\u0026thinsp;NCC.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eNote\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA \u0026ndash; DSC analysis of AmB, Alg, NCC, alginate\u0026thinsp;+\u0026thinsp;AmB (mixture), Alg-AmB and Alg-AmB\u0026thinsp;+\u0026thinsp;NCC. B \u0026ndash; FTIR of pure alginate, Alg and Alg-AmB. D \u0026ndash; UV-Vis spectrometry of the AmB, Alg-AmB and Alg-AmB\u0026thinsp;+\u0026thinsp;NCC.\u003c/p\u003e\n\u003cp\u003eIn Amphotericin B sample, we have found a peak in 167\u0026deg;C degrees. For NCC sample, the peak was at 151 \u0026deg; C. The physical formation of AmB-alginate mixture showed peaks at 144\u0026deg;C, referring to alginate, and at 161\u0026deg;C referring to AmB. On the other hand, when Alg-AmB nanocomplexes were formed, DSC showed only one peak, at 141\u0026deg;C, for alginate.\u003c/p\u003e\n\u003cp\u003eThe DSC value for Alg-AmB\u0026thinsp;+\u0026thinsp;NCC sample showed a similar peak to Alg-AmB, but with a slightly lower temperature, at 123\u0026deg;C. This indicates when NCC was added in the formulation, there was a decrease in the temperature.\u003c/p\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.B shows the FTIR data of the samples of pure alginate, Alg and Alg-AmB. The wavelength band 3424 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e presented in all samples refers to the O-H stretching vibrations of the alginate. The weak band at 2930 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e represents the C-H stretching vibrations; the bands 1630 and 1416 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are asymmetrical and symmetrical stretching vibrations of the C\u0026thinsp;\u0026equiv;\u0026thinsp;O and the COO- group. Furthermore, the band around 1024 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e refers to the vibrations of the ring elongation C-O and C-O, with deformations of C-C-H and C-O-H \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In the Alg-AmB sample, the AmB wavelength bands were compared to data from the literature, showing the bands: OH stretching vibration (3434 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), CH flexion vibrations and CH\u003csub\u003e3\u003c/sub\u003e oscillation (1024 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The hypothesis is the other bands are overlapped by the alginate bands.\u003c/p\u003e\n\u003cp\u003eThe Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.D shows the UV-vis spectra of AmB, Alg-AmB nanocomplex and Alg-AmB\u0026thinsp;+\u0026thinsp;NCC.\u003c/p\u003e\n\u003cp\u003eIn this study, AmB was firstly dissolved in 0.1 M borate buffer pH 11 and then diluted in water, in a monomeric state. The ratio Abs\u003csub\u003e349\u003c/sub\u003e/Abs\u003csub\u003e410\u003c/sub\u003e was 0.36. Considering the Alg-AmB nanocomplex, the1st peak (327 nm) and the ratio between the peaks I and IV (Abs\u003csub\u003e327\u003c/sub\u003e/Abs\u003csub\u003e410\u003c/sub\u003e) was 4.38, showing AmB is in a supper-aggregated state in the formulation, which, as referred above, is associated to a low toxicity. Despite promoting a decrease in the absorbance intensity, NCC addition to the nanocomplex surface did not affect the supper-aggregated state of AmB since a ratio of 4.45 (Abs\u003csub\u003e327\u003c/sub\u003e/Abs\u003csub\u003e409\u003c/sub\u003e) was found.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u003cspan class=\"BoldItalic\"\u003eIn vitro\u003c/span\u003e\u003c/em\u003e toxicity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe hemolysis test was performed for AmB, Alg, Alg-AmB, Alg-AmB\u0026thinsp;+\u0026thinsp;NCC and AmBisome, in concentrations of 1, 2, 4, 8, 16 and 32 \u0026micro;M (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.A).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNote\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA \u0026ndash; AmB (*) and Alg-AmB (**) were compared to the other groups by the Duncan test (p\u0026thinsp;\u0026lt;\u0026thinsp;0,05). B \u0026ndash; AmB (*) was compared to the other groups by the Duncan test (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003cp\u003eThe standard sample of Alg showed irrelevant values of hemolysis (lower than 2%), in all concentration. AmB induced hemolysis percentage between 1.97\u0026thinsp;\u0026plusmn;\u0026thinsp;1.66% and 81.18\u0026thinsp;\u0026plusmn;\u0026thinsp;12.50%. When alginate was added to the formulation (Alg-AmB), there was a decrease in toxicity of 90% compared to standard AmB, with hemolysis values from \u0026minus;\u0026thinsp;0.08% to 30.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50%. When Alg-Amb was coated with NCC, the values were similar (-0.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19% and 44.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50%). Even at high concentrations, nanocomplexes reduced the rate of hemolysis in comparison to standard AmB.\u003c/p\u003e\n\u003cp\u003eRegarding hemolytic porcetange of AmBisome\u0026reg;, data ranged from 0.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33% to 2.13\u0026thinsp;\u0026plusmn;\u0026thinsp;1%. Although AmBisome's hemolysis values are lower than those found in the Alg-AmB formulation, Alg-AmB nanocomplexes had excellent results compared with free AmB. Hemolysis diminish in the sample due to the incorporation of AmB in Alg nanocomplexes shows the nanocomplexes are safe.\u003c/p\u003e\n\u003cp\u003eIn Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.B is presented the cytotoxicity data for AmB, Alg, Alg-AmB, Alg-AmB\u0026thinsp;+\u0026thinsp;NCC and AmBisome\u0026reg;. Alg showed cell viability between 93.01\u0026thinsp;\u0026plusmn;\u0026thinsp;3.83% and 108.66\u0026thinsp;\u0026plusmn;\u0026thinsp;7.04%. AmB data indicated concentration-dependent toxicity. On the other hand, the Alg-AmB nanocomplexes demonstrated significant less toxicity than free AmB. Cell viability was high, ranging between 97.16\u0026thinsp;\u0026plusmn;\u0026thinsp;5.61% and 109.86\u0026thinsp;\u0026plusmn;\u0026thinsp;7.06%.\u003c/p\u003e\n\u003cp\u003eFor the Alg-AmB\u0026thinsp;+\u0026thinsp;NCC nanocomplex, the cell viability values were similar to Alg-AmB NCC, ranging from 98.55\u0026thinsp;\u0026plusmn;\u0026thinsp;5.06% to 106.40\u0026thinsp;\u0026plusmn;\u0026thinsp;5.35%, demonstrating safety use of coating with NCC. Cell viability for AmBisome\u0026reg; was around 101.40\u0026thinsp;\u0026plusmn;\u0026thinsp;6.23% to 111.29\u0026thinsp;\u0026plusmn;\u0026thinsp;6.92%. When comparing the results of the commercial sample, AmBisome\u0026reg;, with Alg-AmB and Alg-AmB\u0026thinsp;+\u0026thinsp;NCC nanocomplexes, there was no significant difference among them, indicating both nanocomplexes, with or without NCC, are as safe as the commercial one.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this work, alginate-amphotericin nanocomplexes (Alg-Amb) were developed aiming to improve the AmB water solubility with low toxicity. It was also evaluated the influence of NCC as a cover system to protect the system.\u0026nbsp; \u0026nbsp;Therefore, to achieve this Aim, it was realized physico-chemical characterization and \u003cem\u003ein vitro\u003c/em\u003e toxicity.\u003c/p\u003e\n\u003cp\u003eTaheri and Mohammadi (2015) \u003csup\u003e18\u003c/sup\u003e utilized cellulose nanocrystals to cover a hydroquinone system and they demonstrated an increase in particle size. The way to obtain smaller particles is important, because expanding the routes of administration. Systems with polymers nanoparticles of size around 200 nm are absorbed by the spleen, lung and liver \u003csup\u003e4\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe nanocomplexes method is capable of producing smaller particles than ionotropic gelification method. Senna et al. (2018) \u003csup\u003e19\u003c/sup\u003e produced particles by ionotropic gelation and observed the production of particles of 1.2 \u0026plusmn; 0.34 mm. Compared to our method; these particles were about 3 times larger than the nanocomplex coated by NCC. One study with alginate particles containing miltefosine obtained by ionotropic gelation and emulsification method showed a polydispersity index of 0.43 \u0026plusmn; 0.14 \u003csup\u003e20\u003c/sup\u003e, similar to the data found in our study.\u003c/p\u003e\n\u003cp\u003eComparatively in another study, the zeta potential of alginate particles obtained by ionotropic gelation showed negative values in the range of -15.7 \u0026plusmn; 1.7 and -9.8 \u0026plusmn; 1.2 mV. However, when adding chitosan in the formulation, there was a change in zeta potential due to the coating \u003csup\u003e21\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAmB is characterized by two endothermic peaks in the crystalline form \u003csup\u003e22\u003c/sup\u003e. In our study, AmB standard showed peaks at 167 \u0026deg;C (melting point) and 198 \u0026deg;C, similar to the temperatures found by those authors. Alginate showed an endothermic peak at 149 \u0026deg;C, which demonstrates the degradation of the biopolymer \u003csup\u003e23,24\u003c/sup\u003e. Vasconcelos et al. 2017 \u003csup\u003e25\u003c/sup\u003e. reported peaks to NCC obtained by acid hydrolysis ranging from 140 to 195 \u0026deg;C 25, similar to our finding (peak at 151 \u0026deg; C). Regarding Alg-AmB nanocomplexes, we have described only one peak. The absence of AmB peak indicates there was a change from crystalline to amorphous state, demonstrating the formation and improvement of alginate and AmB nanocomplexes \u003csup\u003e26\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;In a study with alginate associated to the polymer poly [N-(2-hydroxypropyl) methacrylamide] and the drug camptothecin, the characteristic peak of camptothecin was absent due to the drug being compatible with the polymers and its high dispersion in the formulation \u003csup\u003e27\u003c/sup\u003e. About alginate sample, similar results were found by Silva-Carvalho et al. (2020), indicating the importance of using safe polysaccharides for the formation of the nanocomplex. \u0026nbsp;Another examples can be observed in a study whose objective was to use NCC as a reinforcement for a film containing polylactic acid, concluding there was an increase in crystallinity \u003csup\u003e28\u003c/sup\u003e. George et al., (2011 reported the utilization of NCC associated with polymers and showed there was an increase in the parameters of glass transition, melting temperature, enthalpy and crystalline behaviour.\u003c/p\u003e\n\u003cp\u003eThe degree of AmB aggregation\u0026nbsp; can be determined\u0026nbsp; by the ratio from the first to the fourth peak in the UV-Vis spectra, being a value of\u0026nbsp; \u0026lt; 1 related to the monomeric form and a value of \u0026gt; 2 related to a supper-aggregated form \u003csup\u003e4,6,8,29\u003c/sup\u003e. In solution, AmB has three different states that affect its activity and pharmacokinetical characteristics, such as monomers, in which AmB is water soluble normally associates with ergosterol in fungal and protozoan cell membranes; oligomers, a state in which small water aggregates are toxic towards host cells and present very low solubility; and poly-aggregates, that also referred as supper-aggregates present low \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e toxicity \u003csup\u003e6,8\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eFungizone\u0026reg; and AmBisome\u0026reg; are drugs with low toxicity found in the pharmaceutical market. Due to the super-aggregated state of AmB, specified by ratio values of 2.9 and 4.8 (Alg-AmB and Alg-AmB+NCC, respectively), our nanocomplexes have similar data in relation to commercial formulations \u003csup\u003e7,30\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe hemolysis test is important in studies with nanocomplexes or nanoparticles containing AmB once there is evidence of hemolytic anemia caused by the use of free AmB \u003csup\u003e31,32\u003c/sup\u003e. AmB has a high hemotoxicity even at low concentrations, due to the aggregated conformation \u003csup\u003e31\u003c/sup\u003e. Conjugates and ionic cross-linked polymeric nanoparticles of AmB with alginate show less hemotoxicity and greater hemocompatibility than free AmB, possibly by the protective effect from polysaccharide and by the change in conformation of the super-aggregated state \u003csup\u003e4,8\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe importance of cytotoxicity test with this cell strain is due to the nephrotoxicity associated with free AmB \u003csup\u003e7\u003c/sup\u003e. Therefore, it is important to understand the behaviour of nanocomplexes in this type of cells. The non-toxicity and the capacity for cell proliferation observed to alginate experimentation have already been described \u003csup\u003e4\u003c/sup\u003e. In a study, AmB solution at 15.6 \u0026micro;g/mL was able to induce death in renal cells \u003csup\u003e17\u003c/sup\u003e. On the other hand, Ravichandran and Jayakrishnan, 2018 tested AmB and alginate conjugates to verify cytotoxicity and observed a decrease in toxicity compared to free AmB.\u003c/p\u003e\n\u003cp\u003eAlthough there are studies reporting reduction in hemo and cytotoxicity of amphotericin, our work proposes a simplified methodology, with a cost-benefit and achieves the same objectives by complex and high cost methodologies. Therefore, in addition to our material being as safe as the standard medicine (AmBisome\u0026reg;). As,Silva-Carvalho, et al, 2020 the findings, our work, overall, demonstrates the suitability of alginate as an AmB carrier, covered or not by NCC.\u003c/p\u003e"},{"header":"Conclusion","content":" \u003cp\u003eIn our study, the development of an alginate nanocomplexes with AmB decreased the hemolytic and cytotoxic effect of the drug. In addition, coating the nanocomplexes with the NCC, produced by enzymatic way, brought additional protection to the system without compromised the advantages attributed to the developed formulation, which can be considered a cost benefit, non-toxic and safe as the commercial one. These findings have an important relevance once when developing a nanocomplex, the great advantage is to direct the formulation to the drug's action sites nd with the extra cover of NCC can be applied in other administration route.\u003c/p\u003e "},{"header":"Methodology","content":"\u003cp\u003e\u003cstrong\u003eReagents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmphotericin B (AmB, molecular weight of 924.08 g/mol) powder from \u003cem\u003eStreptomyces\u003c/em\u003e sp., resazurin Sodium salt, sodium alginate, triton X-100, sodium tetraborate decahydrate were purchased from Sigma-Aldrich (Missouri, USA). Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS) and penicillin-streptomycin were obtained from Merck Millipore (Massachusetts, USA). Roswell Park Memorial Institute (RPMI) 1640. Glutamax supplemented medium and L-glutamine (GlutaMAX\u0026trade;-I) was purchased from Gibco (Massachusetts, USA). Dimethylsulfoxide (DMSO ATCC\u0026reg; 4-X\u0026trade;) solution for cell culture was acquired from American Type Culture Collection (ATCC, Virginia, USA). Dialysis tubing with a molecular weight cut-off of 1000 Da was obtained from Orange Scientific (Braine-l'Alleud, Belgium). AmBisome\u0026reg; was kindly provided by Gilead Sciences.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eObtaining bacterial cellulose nanocrystals (NCC)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNCC production was carried out following the methodology developed by our research group (\u003cem\u003eIn press- supplementary material\u003c/em\u003e). \u0026nbsp;The bacterial cellulose was obtained from \u003cem\u003eG. xylinus\u003c/em\u003e, according to Jozala et al. (2014) \u003csup\u003e33\u003c/sup\u003e.The process uses a mechanical treatment of bacterial cellulose with ultra-turrax and high-pressure homogenizer and a enzymatic treatment with cellulase for 72 hours. After, the NCC were separated with centrifugation and filtration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of alginate-amphotericin B (Alg-AmB) nanocomplexes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Alg-AmB nanocomplexes were prepared according to Silva-Carvalho et al. (2020) \u003csup\u003e5\u003c/sup\u003e. In the formulation, borate buffer (pH 11.0), sodium alginate and amphotericin B were used, resulting in a final concentration of 2.5 mg/mL of AmB in the solution. The nanocomplexes formulation were described in table II.\u003c/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size: 15px; font-family: Calibri, sans-serif;\"\u003e\u003cstrong\u003eTable II\u0026nbsp;\u003c/strong\u003e\u003c/span\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u0026ndash;\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003cspan style=\"font-size: 15px; font-family: Calibri, sans-serif;\"\u003e\u003cstrong\u003eFormulation of alginate (Alg) and alginate-amphotericin B (Alg-AmB).\u003c/strong\u003e\u003c/span\u003e\u003c/p\u003e\u003ctable style=\"float: left;border: none;border-collapse:collapse;margin-left:6.75pt;margin-right:6.75pt;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"border-top:solid windowtext 1.0pt;border-left:none;border-bottom: solid #7F7F7F 1.0pt;border-right:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:left;text-indent: 0in;line-height:150%;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cstrong\u003e\u003cspan style=\"font-family: Calibri, sans-serif; font-size: 15px;\"\u003eFormulations\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid rgb(127, 127, 127);border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:left;text-indent: 0in;line-height:150%;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cstrong\u003e\u003cem\u003eBorate buffer pH 11 (mL)\u003c/em\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid rgb(127, 127, 127);border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:left;text-indent: 0in;line-height:150%;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cstrong\u003e\u003cem\u003eSodium alginate (mg)\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid rgb(127, 127, 127);border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:left;text-indent: 0in;line-height:150%;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cstrong\u003e\u003cem\u003eAmphotericin B (mg)\u003c/em\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"border:none;border-right:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:16.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:left;text-indent: 0in;line-height:150%;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cem\u003eAlg\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: none;padding: 0in 5.4pt;height: 16.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:left;text-indent: 0in;line-height:150%;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e9.6\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"padding: 0in 5.4pt;height: 16.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:left;text-indent: 0in;line-height:150%;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e120\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"padding: 0in 5.4pt;height: 16.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:left;text-indent: 0in;line-height:150%;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e-\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:left;text-indent: 0in;line-height:150%;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cem\u003eAlg-AmB\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:left;text-indent: 0in;line-height:150%;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e9.6\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:left;text-indent: 0in;line-height:150%;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e96\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:left;text-indent: 0in;line-height:150%;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif; font-size: 15px;\"\u003e24\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\u003cBR\u003e\u003cBR\u003e\u003cBR\u003e\u003cBR\u003e\u003cBR\u003e\u003cBR\u003e\u003cBR\u003e\u003cBR\u003e\n\u003cp\u003eThe samples were kept under agitation at 4 \u0026deg;C for 48 hours without interference from light. In the next step, the dialysis process was run on a membrane from 12000 to 14000 KDa. The membrane with the sample was immersed in 5 L of distilled water under agitation at 4 \u0026deg;C, without light interference, for 30 hours. During the process, the water was changed three times to remove salts, until reaching pH value in the range of 5.5-5.7. Then, the samples were collected, frozen at -80 \u0026deg;C for 24 hours, and lyophilized. The final yield of the process was 73.72%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNanocomplex coating\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAlg and Alg-AmB nanocomplexes were covered with NCC. For that, the lyophilized nanocomplexes were dispersed in water in the proportion of 1 mg/mL. After dispersion, 1 ml of the nanocomplexes was added into 1 ml of 0.01% NCC suspension. This sample was kept on rotatory shaker (20 rpm) at 25 \u0026deg;C for 24 hours.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhysico-chemical characterization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the physico-chemical characterization the following parameters were used: size, index of polydispersity, zeta potential, differential scanning calorimetry, infrared spectrometry by Fourier transform (FTIR) and UV-Vis spectrometry.\u003c/p\u003e\n\u003cp\u003eFor the evaluation of the size, index of polydispersity and surface potential of the particles (zeta potential), the Zetasizer equipment (ZEN3600) was used, at an angle of 173\u0026ordm;, at 25 \u0026deg;C for dynamic light scattering (DLS), Index of Polydispersity and zeta potential. The samples Alg-AmB and Alg-AmB + NCC were analysed in six times.\u003c/p\u003e\n\u003cp\u003eTo characterize the stability of the nanocomplexes, Differential Scanning Calorimetry (DSC) was used. The characterization was performed by the DSC 6000 equipment (PERKIN ELMER | STEC INSTRUMENTS) in a nitrogen atmosphere with a flow of 20 mL/min, in the heating range of 25 \u0026ndash; 250 \u0026deg;C with a heating rate of 10 \u0026deg;C/min. The test was re-performed with the samples AmB, Alg nanocomplexes, NCC, alginate + AmB (physical mixture without nanocomplexes process), Alg-AmB nanocomplex and Alg-AmB + NCC.\u003c/p\u003e\n\u003cp\u003eThe characterization of the chemical groups was carried out by spectrometry in FTIR. The samples were analysed by the technique of KBr tablets, with 2 mg of sample and 200 mg of KBr. The infrared spectra were obtained in the range of 4000\u0026ndash;400 cm\u003csup\u003e-1\u003c/sup\u003e in the Bruker Alpha II equipment, with a resolution of 4 cm\u003csup\u003e-1\u003c/sup\u003e and 12 scans. The samples analysed were: pure alginate, Alg nanocomplexes and Alg-AmB.\u003c/p\u003e\n\u003cp\u003eIn addition, the samples were analysed by UV-Vis spectroscopy in the 300-450 nm range using a UV-Visible spectrophotometer (JASCO V-560) with 5 nm resolution and scanning speed of 400 nm/min. The nanocomplexes Alg and Alg-AmB were dispersed in distilled water (1 mg of sample/mL). The material was diluted again to 10 \u0026micro;M in distilled water in order to avoid saturation of the test. AmB control was performed using 1082 \u0026micro;M diluted in borate buffer (0.1 M, pH 11). The solution was also diluted to 10 \u0026micro;M with distilled water. To perform the measurement, 200 \u0026micro;L of the solutions were analyzed in a quartz cuvette. The absorbance ratio of peak I (315-350) to peak IV (408-410) was used to monitor the aggregation status of AmB and the results were evaluated for the quantification of AmB in the sample.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vitro\u003c/em\u003e toxicity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the \u003cem\u003ein vitro\u003c/em\u003e toxicity tests, a commercial product (AmBisome\u0026reg;) was taken as a control. This drug is for injectable use and it has a liposomal formulation containing AmB. AmBisome\u0026reg; prescription is mentioned in several articles due to its low toxicity \u003csup\u003e6,7,34\u003c/sup\u003e. Hemolysis and cytotoxicity were the testes chosen to perform the safety of the new nanocomplexes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHemolysis test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHemolysis test assesses the percentage of erythrocyte rupture. Both the protocol and the animal's blood were derived from Silva-Carvalho et al. (2020) \u003csup\u003e5\u003c/sup\u003e. We used ordinary blood samples (dog) from a blood bank. The whole blood was centrifuged for 10 min (4 \u0026deg;C, 1200 g) and the supernatant was discarded. Red blood cells were resuspended in phosphate buffer solution (pH 7.4).\u003c/p\u003e\n\u003cp\u003eResuspended red blood cells were counted in a Neubauer chamber. In a 48-well plate, 450 \u0026micro;L of red blood cells at a concentration of 1 x 10\u003csup\u003e8\u003c/sup\u003e cells / mL were placed in contact with 50 \u0026micro;L of the samples, at concentrations of 1, 2, 4, 8, 16 and 32 \u0026micro;M AmB, Alg, Alg -AmB, Alg-AmB + NCC and AmBisome\u0026reg;. The plate was incubated with shaking for 30 minutes (37 \u0026deg;C, 120 rpm).\u003c/p\u003e\n\u003cp\u003eAfter the period, the solutions were collected and centrifuged for 10 min (4 \u0026deg; C, 1200 G). The supernatants were collected and analyzed by UV-Vis spectrophotometry, with absorbance at 540 nm (referring to hemoglobin). Complete lysis (100%) was admitted by hemoglobin released with 1% triton X-100 (positive control).\u003c/p\u003e\n\u003cp\u003eCytotoxicity\u003c/p\u003e\n\u003cp\u003eHuman Embryonic Kidney (HEK) cell line was selected to perfume citotoxity due to the toxicity of AmB in renal cells. The HEK 293T monolayer (1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cell/well) was incubated for 24 h (37 \u0026deg;C in a 5% CO\u003csub\u003e2 \u003c/sub\u003eatmosphere) with AmB, Alg, Alg-AmB, Alg-AmB + NCC and AmBisome\u0026reg; at concentrations of 0.78, 1, 17, 1.76, 2.63, 3.95, 5.93, 8.89, 13.33, 20, 30, 45 and 67.5 \u0026mu;M. After the incubation period, 10% (v / v) of a 2.5 mM resazurin solution was added to each well and the plates were incubated again under the same conditions as above for 4 hours.\u003c/p\u003e\n\u003cp\u003eFluorescence was measured (\u0026lambda; 560 / \u0026lambda; 590) in a SpectraMAX GeminiXS microplate reader (Molecular Devices LLC, California, USA). The results were expressed as mean percentage \u0026plusmn; SD of viable cells in relation to the positive control (condition considered to be 100% viable cells).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData from toxicity tests were expressed as mean \u0026plusmn; standard deviation. Analysis of Variance (ANOVA) followed by the Duncan test were used to verify differences among treatment protocols, and p values \u0026lt;0.05 were considered significant. Results were analyzed using Statistica\u0026reg; v. 8.0 (Dell, Round Rock, TX, USA) and GraphPad Prism\u0026reg; v. 6.0 (San Diego, CA, USA).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge financial support from Coordination for Higher Level Graduate Improvements (CAPES/Brazil, finance code 001), National Council for Scientific and Technological Development (CNPq/Brazil Process #428751/2016-4), and the State of S\u0026atilde;o Paulo Research Foundation (FAPESP/Brazil, processes #2019/22626-5 and #2018/10508-5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVSS: Investigation, Methodology, Formal analysis, Writing - original draft; RSC: Investigation, Methodology, Formal analysis, Writing - review \u0026amp; editing; DM: Methodology develop; PP: Methodology develop; DG: Validation, Writing - review \u0026amp; editing; MVC: Writing - review \u0026amp; editing; FMG: Conceptualization, Writing - review \u0026amp; editing, Supervision; AFJ: Conceptualization, Writing - review \u0026amp; editing, Supervision.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests Statement:\u003c/strong\u003e the authors declare that they have no competing interests\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGray, K. 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Pharm.\u003c/em\u003e \u003cstrong\u003e447\u003c/strong\u003e, 38\u0026ndash;46 (2013).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Bacterial cellulose, Nanocrystals, Amphotericin B. Alginate, Nanocomplexes","lastPublishedDoi":"10.21203/rs.3.rs-105741/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-105741/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAmphotericin B (AmB) is a drug with anti-leishmanial and anti-fungal ability, but it has low water solubility and permeability, limiting its therapeutic use. Therefore, the incorporation of AmB into nanostructured systems could be profit. Nanostructured systems without surfactants have pharmacotechnical advantages such as amplify the water solubility and decrease the toxicity. For this reason, the present study aimed to produce a nanocomplex of alginate (Alg) with AmB covered by NCC in order to decrease the toxicity of AmB. This straightforward process allowed to obtain water soluble particles Furthermore, the ionic adsorption of the NCC into the Alg-AmB nanocomplex surface was confirmed by an increase in the particle size and a small surface charge decrease. The amorphous inclusion of AmB complex into the polysaccharide chain network in both formulations. AmB in the nanocomplexes was in supper-aggregated form and showed good biocompatibility, being significantly less cytotoxic \u003cem\u003ein vitro\u003c/em\u003e against kidney cells and significantly less hemolytic compared to the free-drug. The results indicated the Alg-AmB nanocomplex can be considered a non-toxic alternative to improve the AmB therapeutic effect. Furthermore, NCC coating of the nanocomplexes brought additional protection to the system without compromised the advantages attributed to the developed formulation.\u003c/p\u003e","manuscriptTitle":"Alginate-Amphothericin B Nanocomplexes Covered By Nanocrystals From Bacterial Cellulose: Physico-Chemical Characterization And In Vitro Toxicity","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2021-02-15 12:54:55","doi":"10.21203/rs.3.rs-105741/v2","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2021-09-29T10:25:04+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-09-09T01:48:50+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-07-03T10:55:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"538d7731-2777-4643-8523-1cbfc3ae4b62","date":"2021-06-22T11:27:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"f6b3d5b6-4f74-48ed-ade4-3bd08486b740","date":"2021-05-12T02:47:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"d976c4f7-1e79-40ec-8fb6-29f66b1ccd05","date":"2021-04-30T16:48:47+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-02-19T19:21:26+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2021-02-18T16:58:05+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2021-02-08T10:00:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2021-02-08T06:37:43+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2021-01-27T19:50:34+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"098ed48e-9583-45c4-83b2-979edf903c41","owner":[],"postedDate":"February 15th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":2417844,"name":"Biological sciences/Biotechnology/Biomaterials"},{"id":2417845,"name":"Nanoscience"}],"tags":[],"updatedAt":"2021-12-01T10:14:15+00:00","versionOfRecord":[],"versionCreatedAt":"2021-02-15 12:54:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-105741","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-105741","identity":"rs-105741","version":["v2"]},"buildId":"rHA-KDH7Qsr4HCuvH75dn","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}