Lyophilization and characterization of Poly (lactic acid) (PLA) nanoparticles containing anti-leishmania drug candidates
INTRODUCTION Leishmaniasis is a parasitic disease which, worldwide, has more cases in Brazil than in other countries. It may be associated with economic and social issues, and is presented in two forms: mucocutaneous leishmaniasis and visceral leishmaniasis, the latter having a higher lethality rate (Barbosa et al., 2021). However, the drugs used to treat leishmaniasis are ineffective, have worrying adverse effects and contribute to parasite resistance. Current treatment includes pentavalent antimonial drugs: meglumine antimoniate and sodium stibogluconate, and second-line drugs with greater toxicity are also used, such as amphotericin B, pentamidine, paromomycin and miltefosine (Nafari et al., 2020). Thiophene derivatives are a group of aromatic heterocyclic compounds that have attracted the attention of the scientific community for their diverse pharmacological activities, including antifungal, antiproliferative, antibacterial and antileishmanial activities (Duvauchelle et al., 2022). Among 10 groups of 2-amino-thiophene derivatives, 2-[(5-bromo-1H-indol-3-ylmethylene)-amino]-4,5,6,7-tetrahydro-4Hcyclohexa[b]thiophene-3-carbonitrile (SB-83) showed promising activity on the amastigote and promastigote forms of the Leishmania amazonenses parasite, in addition to having greater efficiency and low toxicity (Rodrigues et al., 2018). Previous studies have shown that SB-83 has low water solubility; however, various techniques can be used to overcome this limitation, including polymeric nanoparticles, which have the properties of controlling the release of the encapsulated active ingredient, protecting the molecule and targeted delivery, as well as increasing the apparent solubility of the drug; in addition, the use of biodegradable polymers is advantageous as they are eliminated from the body through natural metabolic pathways (Silva et al., 2016; Tyler et al., 2016). All in all, polymeric nanoparticles are produced in aqueous suspension, which poses a major challenge in terms of their stability. When subjected to storage, they can suffer aggregation, particle fusion, chemical instability and drug leakage, which is a major limitation for their application in the pharmaceutical industry (Maraldi et al., 2020). Lyophilization is a drying method commonly used to overcome the chemical, physical and biological instability of pharmaceutical products such as polymeric nanoparticles. This technique includes three stages: freezing, removal of water by sublimation (primary drying) and removal of thawed water by desorption in a vacuum (secondary drying) (Umerska et al., 2018). Although this technique is promising for increasing the shelf life of nanoparticles during the freezing and water removal processes, it can cause stress on the nanoparticles and lead to instability. In order to overcome this problem, it is necessary to add cryoprotectants that form a glassy matrix around the nanoparticles and from this immobilization reduce impacts on the physical and chemical characteristics and reduce aggregation between the particles, avoiding tension (Fonte et al., 2016).
AIMS The aim of this work was to improve the chemical and physical stability of polymeric nanoparticles made of poly(lactic acid) containing SB-83, a leishmanicidal drug candidate, using the lyophilization method.
METHODOLOGY PLA nanoparticles were obtained using the nanoprecipitation method. The nanoparticles were lyophilized in a freeze-dryer using trehalose as a cryoprotectant at a concentration of 5%. The PLA nanoparticles with SB-83 were reconstituted with distilled water and then the particle size, polydispersity index and zeta potential were determined using the dynamic light scattering method. In addition, the in vitro release kinetics of the three groups were analyzed: PLA nanoparticles containing SB-83 (NP SB-83) in suspension, and lyophilized with and without trehalose.
RESULTS AND DISCUSSION During the lyophilization process, the samples did not collapse (NP-SB83 without cryoprotectant and NP-SB83 with cryoprotectant) and presented as a yellow powder. The freeze-dried product was resuspended in distilled water with manual stirring for 30 seconds. This result is in line with the literature, and it has already been reported that trehalose is an excellent cryoprotectant for nanoparticles, as well as being easy to resuspend nanoparticles in aqueous media (Arya et al., 2022). The results for zeta potential, particle size and polydispersity index show that the lyophilization process did not significantly alter the zeta potential values, which fluctuated between 18,1 and 20,1. The freeze-dried sample with the addition of 5% trehalose showed little variation in terms of average particle size and polydispersity index. However, the freeze-dried sample without the addition of the cryoprotectant fluctuated in size from 196,6 nm to 273,2 nm and its polydispersity index value from 0,335 to 0,425. These results therefore demonstrate the importance of adding excipients such as cryoprotectants to maintain the size and polydispersity properties of NP-SB83 (Prata, 2011). With regard to the study of release kinetics, we observed that during the first hours of the study there was a slower release of the nanoparticles with SB-83 in suspension, and only after 6 hours did a more significant release begin, with around 72% of the drug being released after 96 hours of analysis. When we compare the study of the release kinetics of PLA nanoparticles containing SB-83 in suspension with the release kinetics after lyophilization without the use of trehalose, we see that during the first 4 hours there was practically no release of the drug, after this period and over the predetermined time a slow release was maintained, reaching 68% of SB-83 released after 96 hours. Although the lyophilization process is used to overcome problems related to stability, it can cause stress to the nanoparticles, which makes the addition of cryoprotectants crucial, given that cryoprotectants such as trehalose improve the stability of nanoparticles through mechanisms already described in the literature, which are vitrification and particle isolation (Trenkeschuh et al., 2021). With the addition of 5% trehalose in the lyophilization process of nanoparticles containing SB-83, the release kinetics study showed a sustained release over 96 hours with around 90% of the active ingredient released at the end of the in vitro release kinetics study.
CONCLUSION The lyophilization technique proved to be effective in overcoming the limitations associated with suspending the nanoparticles in an aqueous medium, ensuring improved stability. Lyophilization allowed nanoparticles containing the SB-83 derivative with antileishmanial activity to be successfully obtained with and without the addition of the cryoprotectant. In addition, the results presented revealed that the lyophilization process without the use of cryoprotectant influenced the average diameter and polydispersity index of the nanoparticles containing SB-83, while the addition of trehalose preserved the average diameter and polydispersity index of the nanoparticle after the drying process. The study of the in vitro release kinetics of nanoparticles containing SB-83 revealed that the use of trehalose accelerated the process of releasing SB-83 from the nanoparticles, increasing its release from 72% to 90% after 96 hours. Therefore, further studies are needed to better characterize the nanoparticles obtained after lyophilization.
ACKNOWLEDGEMENTS CAPES- Coordination for the Improvement of Higher Education Personnel for granting the scholarship, process no. 88887.873293/2023-00
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