Physicochemical studies of chemically modified chitosans through Solid-State Nuclear Magnetic Resonance: from properties to applications

Author(s)

William Marcondes Facchinatto

• Document type: Doctoral Thesis
• Press: São Carlos.
• Institution: Universidade de São Paulo (USP). Instituto de Química de São Carlos (IQSC/BT)
• Defense date: 2021-04-30

Examining board members

Luiz Alberto Colnago; Sergio Paulo Campana Filho; Rosane Aguiar da Silva San Gil; Sonia Maria Cabral de Menezes; Tiago Bueno de Moraes

• Advisor: Luiz Alberto Colnago

Abstract

The main objective of this Thesis is to control the preparative condition for investigating the properties of chemically modified chitosans and the potential application of these materials in different fields of knowledge. For such a goal, different high- and low-NMR techniques were applied in order to elucidate intrinsic characteristics of chitins, chitosans and derivatives sometimes already verified through other methods. To fulfill the requirement of each specific objective, it were performed a series of characterizations, including non-spectroscopic and biological assays. In this sense, different studies were developed and sequentially presented in a clear and concise manner as possible, being divided into five distinguished Chapters, each one straightly related to some innovative proposal for a given research field. Although related to each other, specific state-of-the-art and applicabilities were explored. It was proposed in CHAPTER I a novel approach relied on high-resolution solid-state 13C NMR spectroscopy to quantify the crystallinity index of chitosans (Ch) prepared with variable average degrees of acetylation (DA) and average weight molecular weight (Mw). The results show that the short-range ordering can be assigned to C4/C6 signals on 13C CPMAS and, for our case, the deconvolution procedure between disordered and ordered phases revealed increasing crystallinity with DA, as confirmed by SVD multivariate analysis. In CHAPTER II, the same set of chitosans were analyzed by time-domain relaxometry (TD-NMR) using the RK-ROSE pulse sequence to acquire 1H NMR signal of solid-state materials. RK-ROSE data were modeled by using PLS multivariate regression, which showed a high correlation between the signal decay profile and average degrees of acetylation and crystallinity index. In CHAPTER III, it was systematically investigated the effects of particle size/ granulometry on the acidolysis of β-chitin (BCH) aiming the preparation of β-chitin nanowhiskers (CWH). The results shown that several features of nanowhiskers can be accessed by choosing properly the average powder size of parent chitin. In CHAPTER IV, two samples of N-(2-hydroxy)-propyl-3-trimethylammonium, O-palmitoyl chitosan (DPCat) with different average degrees of quaternization named as DPCat35 (DQ = 35 %) and DPCat80 (DQ = 80 %), were successfully synthesized by reacting GTMAC with O-palmitoyl chitosan (DPCh) derivative (DS = 12 %). Such amphiphilic derivatives of chitosan were fully water-soluble and showed significant electrostatic stability enhancement of a self-assembly micellar nanostructure due to its positively-charged out-layer. In vitro mucoadhesive and cytotoxicity essays toward mouse healthy fibroblast cells (Balb/C 3T3), and human prostate cancer (DU145) and liver cancer (HepG2/C3A) cell lines revealed that the biological properties of DPCat derivatives were strongly dependent on DQ. Additionally, DPCat35 had better interactions with the biological tissue and with mucin glycoproteins at pH 7.4. These DPCat derivatives were further used in CHAPTER V as nanocarriers of clotrimazole (CLT-NPs) for the topical treatment of vulvovaginal candidiasis (VVC). The nanosystem featured pH-independent sustained drug release up to 24 h, which affected both in vitro anti-Candida activity and cytotoxicity. The CLT-loaded nanostructured platform yielded favorable selectivity index values for a panel of standard strains and clinical isolates of Candida spp. and female genital tract cell lines (HEC-1-A, Ca Ski and HeLa), as compared to the free drug. CLT-NPs also improved in vitro drug permeability across HEC-1-A and Ca Ski cell monolayers, thus suggesting that the nanocarrier may provide higher mucosal tissue levels of the active compound. Overall, data support that CLT-NPs may be a valuable asset for the topical treatment of VVC. The authentic format of this document makes it especially useful as a starting point for the development of different research segments. In this way, alternative studies may broaden de focus and applicability of the materials and characterizations used here, extending the knowledge to distinct innovations. (AU)

• FAPESP's process: 16/20970-2 - Study of chitosan and hydrophilic derivatives by time domain NMR
• Grantee: William Marcondes Facchinatto
• Support Opportunities: Scholarships in Brazil - Doctorate