POLYMERIC NANOPARTICLES CONTAINING TITHONIA DIVERSIFOLIA (HEMSL) A. GRAY FLOWERS ALCOHOLIC EXTRACT AND COATED BY HYALURONIC ACID: DEVELOPMENT, SYNTHESIS, AND CHARACTERIZATION

1Graduation Student Pharmacy Course CCBS Mackenzie Presbyterian University. São Paulo, SP, Brazil. 2Pharmacist Semi-industrial Laboratory Pharmacy Course CCBS Mackenzie Presbyterian University. São Paulo, SP, Brazil. 3Teacher – Laboratory of Teaching and Research of the Pharmacy Course, University Health Center, ABC (CUSABC), Santo André, SP, Brazil. 4Teacher Semi-industrial Laboratory Pharmacy Course CCBS Mackenzie Presbyterian University. São Paulo, Brazil. e-mail: marcelo.guimaraes@mackenzie.br. Authors: Fernanda Arriel Pedrozo Rezende1; Anna Luiza de Aveiro Ruocco1; Bruno Batista da Silva2; Robson Miranda da Gama3; José Armando-Jr3, Marcelo Guimarães4,A. POLYMERIC NANOPARTICLES CONTAINING TITHONIA DIVERSIFOLIA (HEMSL) A. GRAY FLOWERS ALCOHOLIC EXTRACT AND COATED BY HYALURONIC ACID: DEVELOPMENT, SYNTHESIS, AND CHARACTERIZATION Original Article


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Introduction Over the last decade, there has been an intensification in researches involving vectors that can control the alcoholic plant extract and neutralization reaction with NaOH. Stability parameters, such as hydrodynamic diameter, polydispersity index, zeta potential, spectroscopy (IV), Scanning Electron Microscopy (SEM), thermogravimetry (TG) and Differential Exploration Calorimetry (DSC) were analysed. The nanoparticle system was evaluated taking into consideration particle stability prior and posterior to the addition of the extract, as well as coated and uncoated particles. The results demonstrated the good reactivity of the monomers of cyanoacrylates, as well as effectiveness of hyaluronic acid in relation to the proposed objective, evidenced in the results obtained by Infrared Spectroscopy, by SEM, DSC and, also, TG. The study demonstrated that there are possible future applications for this method.
drug release at specific sites of action, improving both the rate of distribuition and the dosage regime. The main systems studied for these purposes have been microparticles and coloidal systems (liposomes and nanoparticles). Nanoparticles are systems formed by biodegradable, synthetic or natural polymers, that have gained significant importance in different industry segments, as well as being the highlight of essential researches. These tiny particles are applicable in the health segment due to the easiness of controlling chemical structures, surface functionalities and particle diameter (AKAGI et al., 2007).
Nanostructures present various promising characteristics, such as: site-specific and gradual drug release, as well as improvement of active principle solubility and stability, enhancement of the desired action of a formulation, and allowing the combination of active substances of varying hydrophilic/lipofilic degrees (SINTOV & SHAPIRO, 2004;CUNHA et al., 2003).
Several nanotechnological stratagies, such as polymeric nanoparticles, allow substances with different properties to be applied in the same formulation, making it possible to alter the properties and behaviour of a given substance in a biological environment (BON-IFÁCIO et al., 2014;GUTERRES, et al., 2012).
Polymeric nanoparticles present diameters ranging from 10 to 1000 nanometers, and can be synthesized through various methods. The most common methods used for the production of polymeric nanoparticles are: in situ polymerization (with dispersed monomers) and precipitation method of preformed polymers. Regardless of the chosen method, the products are obtained as colloidal dispersions or aqueous microemulsions, generating thermodynamically stable systems (BEHAN, et al., 2001;BERTHO-LON, et al., 2006).
Compared with conventional formulations, polymeric nanoparticles are capable of increasing the solubility of constituents, reducing the therapeutic dose and improving absorption of active componentes. Furthermore, when circulating in the bloodstream, nanoparticles have the advantages of being stable, non-toxic, non-thrombogenic, non-immunogenic and non-inflammatory (BONIFÁCIO et al., 2014, SIMEONOVA, 2009.
With the intention of enhancing the stability of the nanoparticles, the present study uses the alternative of hyaluronic acid coating, in an attempt of increasing the effectiveness of the nanosystem as a whole.
In parallel, the use of plants for medicinal purposes has aroused a growing interest in several sci-entific communities, as it allows the development of innovative products with lower potential side effects (COSTA et al., 2010). Plant extracts provide several beneficial properties with different applications, however they are not physical-chemically stable, which may lead to the loss of their antioxidant properties (DAUDT et al., 2013). Thus, the combination of nanotechnology with medicinal plants might be able to enhance the action of plant extracts, such so that many innovative drug carriers have emerged, including polymeric nanoparticles.

Objective
The objective of this study was to develop, synthesize and characterize polymeric nanoparticles coated with hyaluronic acid, containing Tithonia diversifolia (Hemsl) A. Gray flowers alcoholic extract, aiming to improve the nanoparticle's stability, for possible future applications.

Alcoholic extract preparation
The alcoholic extract is obtained using the flowers of the Tithonia diversifolia (Hemsl) A. Gray plant. The flowers undergo a week long drying process using a hot air chamber at approximately 50ºC. These flowers are then reduced by a mill, remaining in a 100% ethanol solution, under stirring and at room temperature for 24 hours. This mixture is then subjected to a filtration process and through the use of a rotary evaporator conected to a vacum pump, the extract is condensed to a concentration of 0.1g/mL (GAMA, R.M. et al., 2014).

HCL and NaOH solutions
Hydrocloric acid solution (HCl) and sodium hydroxide solution (NaOH) were previously prepared. The ideal HCl solution should have a hydrogen potential (pH) close to 2.5, as the polymerization reaction is pH dependent, occurring best in an acidic medium. The NaOH solution is basic, being able to neutralize the acidic pH of the reaction medium and thus interrupt the polymerization reaction (SCHAFFAZIK et al., 2003;VAUTHIER et al., 2003).

Polymerization
The nanoparticles were synthesized through the process of emulsion polymerization, at 800 rpm, where the monomer n-butyl-cyanoacrylate together with 100 mg of Dextran® were incorporated into 10 mL of the aqueous HCl solution (0,01M, pH 2,5), with posterior addition of the alcoholic plant extract and final reaction neutralization with NaOH (pH 7,0 ± 0,3). The resulting polymer poly (n-butyl-cyanoacrylate) (PBCA) was then filtered and stored in an Eppendorf, being labeled as ´Plain´PBCA.
For the incorporation of the extract, the same process was repeated, but after one hour of the continuous stirring, 1mL of PBCA was removed and 1 mL of the alcoholic plant extract was added to the mixture present in the conical flask. The stirring carried on for three hours and then process was then terminated with the addition of the NaOH solution and posterior filtration with a glass funnel and filter paper. The resulting mixture was stored and identified as PB-CA-Extract (polymer containing the alcoholic plant extract) (GUIMARÃES, M., 2015).

Hyaluronic acid coating
The process of coating was based on the work of HE and collaborators (2009), where a 1% (w/v) hyaluronic acid dispersion is prepared using purified water, under stirring. The obtained dispersion was posteriorly added, drop by drop, under stirring (800 rpm) to 5 mL of the PBCA-Extract (GUIMARÃES, 2015). After one hour, a sample was collected and labeled as Coated PBCA containing Extract. The same method was applied to 5 mL of the ´Plain´PBCA and the resulting mixture obtained was labeled as Coated PBCA. The final stored samples were posteriorly lyophilized in order to carry out the absorption spectroscopy in the infrared region and the scanning electron microscopy.

Nanoparticle characterization pH and macroscopic characterization
pH readings were carried out in triplicates for each reading and the mean value for each measurement was calculated. Macroscopic characterization was based on the visual color of the obtained samples, as well as possible presence of precipitates or phase separation posterior to the end of the polymerization reaction.

Medium hydrodynamic diameter, nanoparticle size distribution and zeta potential
The polydispersivity index, hydrodynamic diameter and the particle size distribution were performed with the aid of a Zetasizer equipment (3000 HS (Malvern Instruments), at 25 °C and 90°, through the process of light scattering. Zeta potential readings were based on the electrophoretic mobility, utilizing a force field of 20 V/cm.
The analyses were performed in triplicate, aiming to reduce the possible errors associated with the technique.

Infrared absorption spectroscopy (FTIR)
Spectroscopy provides information on the concentration of specific compounds and their chemical and morphological structure. Vibrational spectroscopies, more specifically infrared spectroscopy, allow the identification of functional groups corresponding to the absorption bands generated by the vibration of the molecule/atom. The infrared band most commonly used is the medium infrared (400-4000 cm -1 ), due to the easiness of associating a band with a specific functional group (CUFFINI, 2009).
For the proposed study, pellets were obtained through sample mixture with potassium bromide (KBr). Spectrums were performed utilizing a Fourier Transform Infrared spectrometer (FT-IR).

Characterization of nanoparticles by scanning electron microscopy (SEM)
SEM has been widely used to evaluate the structures of nanoparticles, including their shape and size, and depending on their formulation, average particle size variation, as well as general eff ects of the drug in the reactional medium (SCHAFFAZICK et al., 2003). Samples were analyzed with a JEOL microscope, model JSM, 6510LV series and a magnifi cation of 30x -5000x.

Diff erential scanning calorimetry (DSC)
Samples of ´Plain´PBCA, PBCA-extract, Coated PBCA coated and Coated PBCA containing Extract were analyzed. Th e analyses were performed in a DSC 7020 scanning calorimeter (Exstar, SII Nano Technology Inc., Japan), 25 -400 °C, utilizing approximately 2 mg of each sample, heating ratio of 10°C/min and dynamic atmosphere of nitrogen with fl ow rate of 50 ml/min.

Th ermogravimetry (TG)
Th ermogravimetry is contemplated within the thermal analysis methods and aims to verify the weight variation of a certain substance(s), in relation to the temperature and/or time variation, under controlled conditions of temperature and atmosphere (VO-GEL, 2017).
Th e resulting graphs can be used to obtain information regarding the thermal stability of the substance, its composition and intermediate products/ residues formed (SILVA et al., 2007). TG analysis was carried out in a Netzsch cell (STA 449F3a) with nitrogen atmosphere. Samples of ´Plain´PBCA, PBCA--extract, Coated PBCA and Coated PBCA containing extract were analyzed

Results and discussion
Regarding macroscopic aspects, the nanoparticle emulsion presented itself as homogeneous, with a whitish color and milky aspect, without the presence of precipitate or phase separation (Figure 1). Th e emulsion remained with its whitish tint even aft er the addition of the yellow alcoholic plant extract. Th e aspect of the formulation remained constant throughout the experiment. Th e non-evidence of phase separation suggests that the combination of extract and polymer obtained a good stability. In terms of the pH variation (Figure 2), it is possible to see that both ´Plain´PBCA and PBCA-Extract formulations started with a pH 7.0 ± 0.3, since this is the pH required for the completion of the polymerization reaction. pH value remained relatively stable throughout the weeks, within the variation of ± 0.3.

Figure 2.
Nanoparticle after extract addition (PBCA-Extract) Nanoparticle before extract addition (´Plain´PBCA) Th e fact that the pH remained close to neutrality is a good indicator of formulation stability, as an increase in acidity could indicate possible polymer degradation, due to the hydrolysis of the polymeric chain, generating carboxyl groups (SANTOS, 2010).
The nanoparticles presented a mean diameter (151.6 nm for the ´Plain´PBCA and 180.0 nm for the PBCA-Extract), consistent with the range described in literature of 100 -300 nm ( Table 1). The values obtained for PBCA without the extract are similar to those described by Bertholon et al. (2006) also using the emulsion polymerization method with Dextran. An increase in the mean diameter, as well as in the polydispersivity index was observed, which could be explained by the addition of the extract before the end of the polymerization reaction, since it may lead to the formation of larger particles (SCHAFFAZICK, 2003). This average hydrodynamic increase can be indicative of the adsorption of the extract to the Polymer, according to Table 1. PBCA-Extract 180,0 ± 2,2 0,10 ± 0,33 -0,70 ± 1,27 The zeta potential is associated with the surface potential of particles. Results obtained showed slightly negative values, which may be associated with the fact that the reaction occurs in an acidic medium (lower pH), contributing to a reduced dissociation of the free acrylic acid groups (REDDY & MURTHY, 2004).
The polydispersivity index is associated with the distribution of the particle weight, being contemplated on a scale from 0 to 1. Both results obtained for the samples (0.06 for the ´Plain´PBCA and 0.10 for the PBCA-Extract) demonstrated a good distribution, given that values below 0.2 present a narrow distribution of size, that is, a system closer to being monodispersed (DAS et al., 2012). The standard deviation was low, suggesting that the method showed good reproducibility.
According to Figure 3, a relatively similar behavior can be perceived between the three samples, with only a small variation in transmittance. The sample presented by a red line on the graph referes to the nanoparticle containing the plant extract but without the hyaluronic acid coating. One can observe that the sample presented significant peaks, demonstrating a greater instability when compared to the other curves. This result suggests that the hyaluronic acid coating of the nanoparticle containing the extract is essential in the process of stabilizing them.       The images provided by SEM demonstrated that the nanoparticles were not completely spherical, but rather varied in format (Figures 4, 5, and 6). Although no particle size alteration was observed posterior to the addition of the plant extract, a change in the particle agglomeration state could be noticed. Particles were perceived as being more dispersed, possibly due to the presence of the plant extract inside the nanoparticle ( Figure 5).
Coated nanoparticles presented different superficial characteristics (Figure 6) when compared to the other two samples. Particles appeared to have a spongy characteristic to them due to clearly visible superficial pores.
The thermal stability of nanoparticles was evaluated through differential scanning calorimetry (DSC) and thermogravimetry (TG) (Figure 7).
The curve of the DSC analysis (Figure 7) obtained for the nanoparticle containing the plant extract and the coating presents an endothermic peak in the range of 300-450° C, which is characteristic of simultaneous and/or subsequent events. One can infer that this event occurred due to loss of moisture during the analysis. The beginning of the process of degradation of the polymeric structure referring to the sample with extract and with coating is approximately at 200 ° C.
TG analysis of the main formulation developed in this study (nanoparticles containing plant extract and with coating) demonstrated the absence of an exo-thermic peak (Figure 8). This result may be indicative of possible adsorption of the hyaluronic coating to the surface of the nanoparticles, protecting the latter against degradation.  Furthermore, the endothermic events of the nanoparticles containing plant extract and coating appear wider and/or displaced, indicating possible interactions between particle, extract and the hyaluronic acid coating. This observation also reinforces evidence of nanoparticle coating.

Conclusion
Considering the objectives of this work, one can conclude that the development of the nanoparticles of poly (n-butyl-cyanoacrylate) allowed the addition of the alcoholic extract of the plant Tithonia diversifolia (HEMSL) A. Gray, as well as the use of hyaluronic acid as a coating medium. This process generated a nanostructure, which remained stable in relation to its physical, and morphological characteristics. Results evidenced by spectroscopy, SEM, DSC and also by TG, demonstrate the effectiveness of hyaluronic acid as a coating medium, supporting the qualitative aspect of the reasearch to improve the physicochemical stability of the nanosystem. Future studies taking into consideration these analyses may add to the set of nanotechnological subsidies for the pharmaceutical área.