Drug-free Carboxymethyl cellulose Hydrogels with extract from Morinda citrifolia for enhancing antimicrobial activity

ABSTRACT

Currently, it is crucial to develop innovative materials for treating various diseases. Thus, this research produces innovative hydrogels incorporating a natural extract from Morinda citrifolia leaves from the semiarid region of Brazil, demonstrating antibacterial and antifungal activity. These hydrogels were characterized by spectroscopic analysis (Raman, UV–VIS), Zeta potential, and Dynamic Light Scattering (DLS). Additionally, the kinetic release of the extract was performed. Moreover, antibacterial and antifungal activities were performed against Escherichia coli and Candida albicans, respectively. The results showed that nanoencapsulation of the colloidal extract modified the physicochemical properties of the hydrogels. In addition, the nano-encapsulation was homogeneously dispersed within the hydrogel. Moreover, the kinetic release of the natural extract demonstrated effective release kinetics due to the different molecules released over time. Furthermore, the antibacterial and antifungal activity showed great results after 24 hours. These results confirm the potential of using these hydrogels as therapeutic biomaterials for “Antibiotic-free” and “Antifungal-free’ health problems.

Keywords:
Colloids; Noni; Nanomedicine; Carbohydrate; 3D matrix

1. INTRODUCTION

The number of populations worldwide affected by diverse types of infections has been growing and is emerging as a significant global threat [¹]. Nevertheless, with the introduction of antimicrobial agents after the 1950s decreased for a while over the years, an incredible number of new microorganisms appearing present a great threat in recent years, even more so with the indiscriminate abuse of drugs like antibiotic and antifungal for humanity in various part of Planet provoking an evolution of microorganism because of cross-resistant/multi-­resistant strains [²,³,⁴]. Additionally, this scenario has caused many deaths in the health system worldwide (about 85 %) [⁵].

In this context, it is essential to develop innovative tools for antimicrobial activity, such as new materials for ‘antibiotic-free’ or ‘antifungal-free’ applications [⁶]. These materials can be produced by various sources and in diverse ways, utilizing synthetic, natural, recycled, mineral, and other materials. One of the drug’s basic strategies of production is based on natural substances, with subsequent improvement of the hit molecules [⁷]. Many of these natural compounds are derived from plants with high potential for preventing, treating, and curing various health problems. Due to their easy acceptability, availability, and low costs, they are largely produced by extracting their natural compounds using various solvents, such as alcohol (ethanol or methanol) or water [⁸].

Moreover, the World Health Organization (WHO) reports that about 80% of the world’s population uses plant extracts for medicinal purposes, mainly in developing countries [⁸]. These plant extracts were used in popular culture resources for various complementary health practices. For a long time, the use of medicinal plants to cure various diseases and inflammatory processes in living beings has been reported in all societies. This popular knowledge provided positive results, but also negative ones, with side effects resulting from the lack of scientific knowledge about the byproducts present in these plants [⁸].

In this scenario, some exotic plants, such as Morinda citrifolia (M. citrifolia), have been studied for phyto-therapeutic benefits. Popularly known by the name noni, M. citrifolia is a small tree belonging to the Rubiácea family, originating in southwest Asia and being spread by Indian peoples to the islands of French Polynesia. The applications of noni by the Polynesians are attributed to the plant’s phytotherapeutic effects, which include antibacterial, antiviral, antifungal, antitumor, anthelmintic, analgesic, anti-inflammatory, hypotensive, and immune-stimulating properties. These properties have been utilized by them for over two thousand years [⁹,¹⁰,¹¹]. However, the effective use of natural extracts directly in human tissue is uncertain, as the adequate concentration, dilution in fluid, and other parameters can affect the extract properties.

One alternative is incorporating these natural compounds in small concentrations, nano-encapsulated in a three-dimensional matrix, such as hydrogels [¹²]. These hydrogels are three-dimensional materials with incredible fluid/liquid adsorption capacity and remain chemically stable [¹³]. Additionally, the soft matrix enables them to mimic various human tissues, a particularly interesting characteristic for biomaterial applications. Moreover, literature proposes its applications in various areas due to its biodegradability, mechanical properties, chemical stability, and polymer-polymer interactions [¹⁴]. Furthermore, these three-dimensional materials can be enhanced by incorporating diverse compounds and materials into their matrix, such as drugs, nano- and microparticles, and natural extracts from plants [¹⁵].

Thus, the present research performed an innovative hydrogel based on carboxymethyl cellulose with a colloidal extract from Morinda citrifolia (M. citrifolia) nano-encapsulated as an alternative for antibiotic and antifungal drugs. Thus, to the best of the authors’ knowledge, this is the first time the extract from M. citrifolia is nano-encapsulated in a hydrogel matrix of carbohydrates. Additionally, these hydrogels were fully characterized using various techniques. The kinetic release of the colloidal extract was performed, and its antimicrobial activity was analyzed, yielding promising results for biological and biomedical applications.

2. MATERIALS AND METHODS

2.1. Materials

Sodium salt of carboxymethyl cellulose (≥ 99.5 %, Synth, Brazil, Mw = 250,000 g.mol^–1, DS = 0.7) hydrochloric acid (Neon, Brazil, 36.5–38.0 %, MM = 36.46 g.mol^–1, HCl), Sodium hydroxide (≥ 99 %, NaOH), Methanol (99.8 %, CH₃CH₂OH), supplied by Synth (Brazil), citric acid (C₆H₈O₇, ≥ 99.5 %, Synth, Brazil), ethanol (Synth, Brazil, C₂H₆O, ≥ 99.9 %, MM = 46 g.mol^–1). Deionized water (DI-water) (Millipore Simplicity, Merck, USA) with a resistivity of 18 MΩ·cm was used. Moreover, the leaves were collected in the northern region of Minas Gerais, Brazil, in the municipality of Janaúba, at 15° 48′ 10″ S, 43° 18′ 32″ W, during the winter and fall seasons (the optimal time for leaf collection in this area).

2.2. Extraction of colloidal extract from Morinda citrifolia

The leaves of Morinda citrofolia tree were collected in the north region of Minas Gerais, Brazil, in the municipality of Janaúba at 15° 48′ 10″ S 43° 18′ 32″ O during the winter and fall and similar to literature [¹⁶, ¹⁷], it was put in adequate recipient and transferred to laboratory under controlled conditions for extraction of colloidal extract. The colloidal extract was obtained via an alcoholic route with methanol. First, leaves were washed several times using deionized water, followed by sodium hypochlorite and alcohol (90 %) to remove any residues that could contaminate the extract. Then, the leaves were dried in an oven at 70 ºC for 24 hours. After, 45 g of dried leaves were macerated with 405 mL of methanol and left in a water bath at 70 ºC. Subsequently, the obtained colloidal solution was filtered. Finally, the materials were placed in a rotary evaporator at 45 ± 2 ˚C to remove all remaining alcohol. The final colloidal suspensions were poured into plastic tubes (1.5 mL in Eppendorf tubes) and stored at 4 ± 2 ˚C until further use.

2.3. Incorporation of extract in carboxymethyl cellulose hydrogel

The incorporation of colloidal extract into hydrogels was performed using sodium salt, carboxymethyl cellulose (CMC), and citric acid, using two colloidal extract concentrations (500 µL and 1000 µL). Then, 2 g of CMC was dissolved in deionized water for 24 hours under moderate stirring. After, the colloidal extract in proportions of 500 µL or 1000 µL was added to the CMC solution and left under moderate stirring for 24 hours to homogenize the system. Thus, 10 % (m/m of polymer) of citric acid was added to the solution and left under moderate stirring for 20 minutes. Finally, the solution was placed in a polystyrene petri dish and incubated in an oven at 40 ºC for 24 hours to remove the water. Then, at 80 ºC, crosslink the citric acid to the CMC chain, producing the hydrogel.

2.4. Characterization of extract and hydrogels

The colloidal extract and hydrogels were characterized by ultraviolet-visible spectroscopy with a BEL UV Visible spectrometer (BEL UV-MX, Italy) from 190 to 800 nm. Additionally, Raman spectroscopy analysis was performed on the sample using a CORA 5001 Direct 532/785 Raman spectroscopy system (Anton Paar, Austria) with an excitation laser at 532 nm and an output power of 50 mW for 1500 ms. Moreover, Zeta Potential and Dynamic Light Scattering were performed using a NanoBrook 90Plus PALS of Brookhaven (Mississippi, USA) using a water solution.

Moreover, physiologic adsorption in tissue and kinetic dissolution of the hydrogel, swelling (SD), and gel fraction (GF) procedures were conducted using similar methods as those described in the literature [¹⁸]. Then, all hydrogels were placed in a PBS solution (pH 7.0). To reach equilibrium for both procedures, the kinetic intervals were 1, 2, 3, 4, 6, 8, 24, and 50 h. Altogether, 21 samples were used for each system (n = 21, 7 samples of 3 different syntheses). The results were statistically equivalent (ANOVA, one-way included Tukey’s test, p < 0.05, software Origin v.8.1, OriginLab Corporation, USA).

2.5. In vitro Accumulative kinetics extracted delivery in carboxymethyl cellulose hydrogels with colloidal extract incorporated

Kinetic experiments were conducted to determine the time required for colloidal extract delivery to reach equilibrium in tissue. The cumulative kinetics of extract delivery were determined in triplicate at 37.0 ± 0.1 °C in deionized water, consistent with the literature [¹⁸]. Hydrogels with an area of 1 cm² were placed inside a plastic basket immersed in 15 mL of deionized water under magnetic stirring, and the release of extracts was monitored for 96 h (n = 3). At each time interval, 1 mL of solution was collected and analyzed by UV–VIS to determine the extract concentration based on the Beer-Lambert correlation curve (λ = 264 nm) (Fig. S1). Moreover, the volume of solution removed at each time was replaced to guarantee the same volume in all procedures. Additionally, the pH and temperature were maintained at all times, as these parameters can significantly affect the results.

2.6. Antimicrobial activity of hydrogels with incorporation of colloidal extract

The hydrogels were sterilized in an autoclave at 120 °C under 1 kg.cm^–2 for 15 min, using a procedure similar to that described in the literature [¹⁹]. As for the negative control samples (CN), a hydrogel without extract (CN1) was used. Thus, the antimicrobial activity of the hydrogel with 500 µL and the hydrogel with 1000 µL was evaluated. The antibiotics and antifungals used were gentamicin (CP, 100 µg) (brand name, SENSIBIODISC-CECON) or Ketoconazole (CP, 100 µg) on a disk, used as a positive control (CP). After 24 hours in the bacteriological oven at 37 °C, the halos (both vertical and horizontal) were removed and measured. Then, the Gram-negative human bacterial pathogens Escherichia Coli (E. coli, ATCC 15597) and Candida albicans human fungus (Candida albicans, C. albicans, ATCC 90029) obtained from the American Type Culture Collection (ATCC) were tested on hydrogels. The inoculums of the test organism were incubated at 37 °C in Muller–Hinton medium until reaching the logarithmic phase. The optical density of bacterial suspensions was measured at λ = 620 nm using a microplate reader (Spectra II Microplate Reader, Tecan) in absorbance mode.

3. RESULTS AND DISCUSSION

3.1. Extract and hydrogels characterization results

Morinda citrifolia contains a variety of organic and inorganic molecules, including proteins, vitamins, polysaccharides, and other long-chain compounds, in each part of the tree, including the leaves [²⁰]. These factors contribute to an increase in the hydrodynamic diameter. Considering that Zeta Potential (ZP) and Dynamic Light Scattering (DLS) were performed on colloidal extract and hydrogels to determine the surface charge and hydrodynamic size, respectively. The analysis carried out by zeta potential demonstrated that the colloidal extract of Morinda citrifolia contained in the CMC hydrogels presented a negative surface charge (–11.18 ± 4.11) mV, indicating a stable surface charge based on ASTM D6703. A negative surface charge in the hydrogels is attributed to specific chemical groups in the Morinda citrifolia extract, including carboxylates, sulfates, and phosphates. These groups tend to ionize when exposed to the aqueous solution (lose positive ions, such as H⁺), resulting in a negative charge on the surface of the hydrogel. In the case of DLS, the diameter value was (26886 ± 1) nm, a high value but expected, as DLS measures the hydrodynamic value. In the case of a colloidal extract, molecules with long, conjugated chains, such as carbohydrates and proteins, increase the hydrodynamic size. Therefore, the polydispersity was greatly valued (0.178), indicating a highly monodisperse solution.

Raman spectroscopy showed the main bands in the colloidal extract and hydrogels (Figure 1). Bands at 2202 cm^–1 and 1161 cm^–1 are perceived in the CMC related to hydroxyl groups in its chain [²⁰,²¹,²²]. At 1614 cm^–1 and 1495 cm^–1, bands linked to COO^– symmetric and asymmetric vibrations are visualized relative to the carboxylic group, respectively [²⁰,²¹,²²]. In 1161 cm^–1, a band related to the alcoholic group (C-O-C) in polysaccharides is perceived [²⁰,²¹,²²]. Additionally, CH₂ stretching is observed at 1231 cm^–1 and 1349 cm^–1 [²⁰,²¹,²²]. In the case of colloidal extract, a band at 1164 cm^–1 linked to the carotenoid is visualized [²³]. At 1961 cm^–1, vibrations of hydroxyl groups (OH) are perceived. At 1335 cm^–1, bands related to u(C–O) + δ(C–O–H) linked to carbohydrates are visualized [²⁴]. In 824 cm^–1, the band in the pyranose region is perceived to be associated with sulphonate carbohydrates or proteins in the extract [²⁴]. In addition, bands at 645 cm^–1, 540 cm^–1, 143 cm^–1 and 201 cm^–1 is visualized, related to C-C-O, gC-C-C + gC-C-O, respectively [²⁴]. Moreover, at 2210 cm^–1, a band related to CN stretching is observed, which is attributed to proteins and vitamins in the extract [²⁴]. In both hydrogels (hydrogels with 500 µL and 1000 µL of colloidal extract), bands associated with carboxymethyl cellulose and extract were visualized with slight differences in intensity and with red-shift of bands when the extract decreased, related to uC-O-C, C-O-S, CH₂ + u(C–O) + δ(C–O–H), and gC-C-C + gC-C-O groups, and with blue-shift in the case of groups CN and uCOO^–.

Figure 1
Raman spectra of CMC (a), Colloidal extract (b), Hydrogel with 1000 µL of extract nano-encapsulated (c), and Hydrogel with 500 µL of extract nano-encapsulated (d).

Hydrogels are a versatile material for various biological and biomedical applications. In these areas, physiologic fluid adsorption and degradation/dissolution are important parameters to evaluate. Thus, swelling behavior (SB) and gel fraction (GF) (dissolution into medium) were performed in the hydrogels with and without colloidal extract. Figure 2 shows the results. The SB (Figure 2A) exhibited distinct behavior for the two hydrogels with colloidal extract. In the case of the hydrogel with a higher nano-encapsulated extract, a similar behavior was observed, with the swelling increasing until 25 hours, then decreasing after that, and stabilizing at 50 hours. This behavior may be associated with the presence of more carbohydrate polymers in the hydrogel matrix, which is linked to the amount of nano-encapsulated extract containing carbohydrates in its colloids, as described in the Raman analysis. However, in the SB of hydrogel with 500 µL, the behavior is quite diverse; after 25 hours, the swelling increased until 50 hours, when it stabilized. In the GF analysis (Figure 2B), a similar diverse behavior is perceived between the two hydrogels with colloidal extract. However, in this case, the hydrogel with 500 µL showed similar behavior to the hydrogel without extract, decreasing slightly after 25 hours and then stabilizing at 50 hours. Nevertheless, the GF of the hydrogel with 1000 µL maintained its stabilization at all times analyzed, only increasing in the first minutes. Therefore, all the SB and GF analyzed presented good behavior and chemical stability, comparable to the results in the literature [²⁵,²⁶,²⁷,²⁸,²⁹], indicating their suitability for use in biological and biomedical applications.

Figure 2
Swelling behavior of hydrogels with and without colloidal extract (A) Gel-fraction behavior of hydrogels with and without colloidal extract (B).

3.2. In vitro Colloidal extract kinetic release

Morinda citronella is recognized for its therapeutic effects on diabetes [³⁰], its antioxidant activity [³¹], potential anticancer properties [³²], analgesic and anti-inflammatory effects [³³, ³⁴], and other pharmaceutical benefits. Therefore, to the authors’ knowledge, it is the first time that its extract has been nano-encapsulated in a hydrogel matrix; thus, it becomes important to evaluate its kinetic liberation in a medium suitable for biological and biomedical applications. Therefore, in vitro extract delivery kinetics were performed using two hydrogels with colloidal extract nanoencapsulated (Figure 3). The release of extract from the hydrogel matrix showed three diverse behaviors over time. In the first hours (25 hours), minor molecules, such as vitamins and carotenoids, were released until they reached the equilibrium and remained until 50 hours. Then, the hydrogels began to release molecules higher than vitamins and carotenoids, such as proteins, until they reached equilibrium at 100 hours. This second behavior is more pronounced in the hydrogel with a higher colloidal extract nano-encapsulation. In the third release, after 150 hours, the hydrogels reached equilibrium at 210 hours, releasing high-molecular-weight compounds, such as carbohydrates. GONG et al. [³⁵] demonstrated that this behavior is expected in materials with different types of molecules. Larger molecules tend to be retained longer in the hydrogel, resulting in a more prolonged release. On the other hand, in the initial stages, the release kinetics are less pronounced due to the presence of smaller molecules, such as flavonoids and vitamins, which are degraded into smaller fractions and released more quickly [³⁶].

Figure 3
Kinetics of hydrogels with colloidal extract nano-encapsulated release in vitro over time (inset: examples of molecules released at the time highlighted).

3.3. Antimicrobial activity of the hydrogel of the colloidal extract incorporated

The growth of pathogens that affect human health poses a significant global threat. Although this situation is important to highlight, the most varied pathogens account for about 85% of mortality worldwide [³⁷]. Moreover, there has been an overwhelming increase in the resistance of widespread pathogens to commonly used drugs [³⁸]. Most of this resistance is caused by the excessive and inappropriate use of antibiotics, antiseptics, and disinfectants to combat infectious agents, which promotes the evolution/selection of microorganisms and the consequent development of cross-resistant and multi-resistant strains [³⁹].

Although a wide range of antimicrobial agents and antibiotics is commercially available, effectively combating bacterial infections is particularly challenging due to increased microbial resistance caused by the misuse of such drugs [¹⁹, ³⁹]. In this context, studies on antimicrobial agents, such as hydrogel polymers, have been utilized in various biological and biomedical applications [⁴⁰].

In this context, the morphological and physicochemical characteristics of hydrogels, including surface charge and chemical groups, are important properties that can provide longer half-lives and specific biodistribution profiles compared to the free drug, thereby improving antimicrobial capacity [⁴¹,⁴²,⁴³]. Considering that this study evaluated the antimicrobial activity of hydrogels using the agar-well diffusion method. The results are shown in Figure 4 after 24 hours of contact, when the antimicrobial activity stabilized.

Figure 4
Antifungal activity of hydrogels against Candida albicans (A); Antibacterial activity of hydrogels against Escherichia coli. (CP is the antibiotic or antifungal, and CN is a negative control; in this case, a filter with deionized water).

All hydrogels with colloidal extract effectively attacked both antimicrobial strains (E. coli and C. albicans). It was observed that as the colloidal extract concentration increased in the hydrogel, the antimicrobial activity improved, and the average diameter of the halo increased. Therefore, the antibacterial activity was more intense than the antifungal activity for both hydrogels with colloidal extract. However, even in concentrations smaller than the antibiotic and antifungal drug used as the Positive control (CP), the hydrogels showed good results for use as a potential antibiotic-free and antifungal-free biomaterial for biological and biomedical applications.

The literature [⁴³, ⁴⁴] proposes that various factors may promote biofilm formation in Candida albicans and E. coli, which in turn diminishes the activity of these microorganisms. One proposal is the formation of oxidative compounds, which can be produced by molecules with high hydroxyl groups in their chain [⁴³, ⁴⁴]. Raman spectroscopy revealed the presence of hydroxyl groups, which form diverse molecules in the colloidal extract and are also present in carboxymethyl cellulose within the hydrogel matrix. This chemical group may promote a decrease in biofilm activity, thereby enhancing antimicrobial activity. Additionally, since the antimicrobial activity was assessed at 24 hours, the main compounds that may contribute to antimicrobial activity are associated with vitamins and carotenoids, as demonstrated by the kinetics of the colloidal extract analyzed.

4. CONCLUSION

The present research involved the production of an innovative hydrogel incorporating a colloidal extract from Morinda citrifolia. Additionally, it evaluated the release kinetics of its extract and antimicrobial activity for various biological and biomedical applications. As the potential characteristics of Morinda citrifolia extract are well-documented in the literature, its incorporation into a carbohydrate hydrogel matrix using carboxymethyl cellulose and citric acid can enhance its properties, enabling its application as an exceptional biomaterial for various biomedical applications. Thus, these hydrogels were fully characterized based on their morphological, chemical, and surface charge properties. Additionally, the kinetics of colloidal extract release and antimicrobial activity were evaluated using two microorganism models (E. coli and C. albicans). The results showed that the chemical groups on hydrogels and colloidal extracts, as determined by Raman, are mainly composed of carotenoids, vitamins, and carbohydrate-related chemical groups, such as carbonyl, carboxylic, and hydroxyl groups. Furthermore, the size of the molecules in the colloidal extract was very large (26886 ± 1) nm, with a negative surface charge (–11.18 ± 4.11) mV, but the suspension was very stable (PDI 0.178). Additionally, the swelling and gel-fraction behavior were quite diverse, depending on the extract concentration in the hydrogel matrix, indicating the chemical stability of the hydrogel over time. Moreover, the kinetics of colloidal extract release from the hydrogel over time exhibited different behavior associated with varying molecular sizes in the extract. Furthermore, an environment with high oxygen groups in hydrogels has been shown to decrease biofilm activity in microorganisms due to oxidative stress, as described in the literature [⁴⁴,⁴⁵,⁴⁶], which in turn promotes the antimicrobial activity of hydrogels with the colloidal extract. Additionally, to the authors’ knowledge, this is the first time a colloidal extract has been nanoencapsulated in a carboxymethyl cellulose hydrogel for potential biological and biomedical applications.

SUPPLEMENTARY MATERIAL

The following online material is available for this article.

Figure S1 –Calibration curve of colloidal extract.

5. ACKNOWLEDGMENTS

The authors acknowledge and express their gratitude for the financial support and technical analyses to the BIOSEM-LESMA from Universidade Federal dos Vales do Jequitinhonha e Mucuri (UFVJM), The authors acknowledge the FAPEMIG (APQ-02565-21), FINEP/MCTI (0 1 22 0528 00), CAPES, and CNPq for financial support.

6. BIBLIOGRAPHY

• ^[1] KIRTANE, A.R., VERMA, M., KARANDIKAR, P., et al., “Nanotechnology approaches for global infectious diseases”, Nature Nanotechnology, v. 16, n. 4, pp. 369–384, 2021. doi: https://doi.org/10.1038/s41565-021-00866-8. PubMed PMID: 33753915.
  » https://doi.org/10.1038/s41565-021-00866-8
• ^[2] BOUTAYEB, A., “The burden of communicable and non-communicable diseases in developing countries”, In: Preedy, V.R., Watson, R.R., (eds), Handbook of disease burdens and quality of life measures, New York, Springer, pp. 531–546, 2010. doi: https://doi.org/10.1007/978-0-387-78665-0_32.
  » https://doi.org/10.1007/978-0-387-78665-0_32
• ^[3] JAMPILEK, J., KRALOVA, K., “Advances in nanostructures for antimicrobial therapy”, Materials, v. 15, n. 7, pp. 2388, 2022. doi: https://doi.org/10.3390/ma15072388. PubMed PMID: 35407720.
  » https://doi.org/10.3390/ma15072388
• ^[4] SEGURA-EGEA, J.J., GOULD, K., ŞEN, B.H., et al., “Antibiotics in endodontics: a review”, International Endodontic Journal, v. 50, n. 12, pp. 1169–1184, 2017. doi: https://doi.org/10.1111/iej.12741. PubMed PMID: 28005295.
  » https://doi.org/10.1111/iej.12741
• ^[5] LAXMINARAYAN, R., VAN BOECKEL, T., FROST, I., et al., “The Lancet Infectious Diseases Commission on antimicrobial resistance: 6 years later”, The Lancet. Infectious Diseases, v. 20, n. 4, pp. e51–e60, 2020. doi: https://doi.org/10.1016/S1473-3099(20)30003-7. PubMed PMID: 32059790.
  » https://doi.org/10.1016/S1473-3099(20)30003-7
• ^[6] JAN, H., GHAYAS, S., HIGAZY, D., et al., “Antibacterial and anti-biofilm activities of antibiotic-free phosphatidylglycerol/docosahexaenoic acid lamellar and non-lamellar liquid crystalline nanoparticles”, Journal of Colloid and Interface Science, v. 669, pp. 537–551, 2024. doi: https://doi.org/10.1016/j.jcis.2024.04.186. PubMed PMID: 38729002.
  » https://doi.org/10.1016/j.jcis.2024.04.186
• ^[7] MENDELSON, M., SHARLAND, M., MPUNDU, M., “Antibiotic resistance: calling time on the ‘silent pandemic’”, JAC-Antimicrobial Resistance, v. 4, n. 2, pp. dlac016, 2022. doi: https://doi.org/10.1093/jacamr/dlac016. PubMed PMID: 35310572.
  » https://doi.org/10.1093/jacamr/dlac016
• ^[8] SOUZA, A.J.M., LIMA, C.T., GONÇALVES, M.P., et al., “First steps on comprehensive understanding of biosafety and toxicity of natural extract from Calotropis procera seeds, new insights towards sustainability”, Journal of Hazardous Materials Advances, v. 5, pp. 100042, 2022. doi: https://doi.org/10.1016/j.hazadv.2021.100042.
  » https://doi.org/10.1016/j.hazadv.2021.100042
• ^[9] CLAFSHENKEL, W.P., KING, T.L., KOTLARCZYK, M.P., et al., “Morinda citrifolia (noni) juice augments mammary gland differentiation and reduces mammary tumor growth in mice expressing the unactivated c-erb B2 transgene”, Evidence-Based Complementary and Alternative Medicine, v. 2012, pp. 1–15, 2012. doi: https://doi.org/10.1155/2012/487423.
  » https://doi.org/10.1155/2012/487423
• ^[10] ILOKI, A.S.B., LEWIS, L.L.M., RIVERA-CASTAN˜EDA, E.G., et al., “Effect of maturity and harvest season on antioxidant activity, phenolic compounds and ascorbic acid of Morinda citrifolia L. (noni) grown in Mexico (with track change)”, African Journal of Biotechnology, v. 12, pp. 4630–4639, 2013.
• ^[11] PALU, A.K., WEST, B.J., JENSEN, J., “Noni-based nutritional supplementation and exercise interventions influence body composition”, North American Journal of Medical Sciences, v. 3, n. 12, pp. 552–556, 2011. doi: https://doi.org/10.4297/najms.2011.3552. PubMed PMID: 22363077.
  » https://doi.org/10.4297/najms.2011.3552
• ^[12] SOUZA, A.J.M., RODRIGUES, J.S., BERNARDES, M.S., et al., “Sustainable starch-calotropis procera extract bio-packing for protection against fungus attack and fruit fast maturation”, Fine Chemical Engineering, v. 4, pp. 242–256, 2023. doi: https://doi.org/10.37256/fce.4220232930.
  » https://doi.org/10.37256/fce.4220232930
• ^[13] NOGUEIRA, N., CONDE, O., MIÑONES, M., et al., “Characterization of poly(2-hydroxyethyl methacrylate) (PHEMA) contact lens using the Langmuir monolayer technique”, Journal of Colloid and Interface Science, v. 385, n. 1, pp. 202–210, 2012. doi: https://doi.org/10.1016/j.jcis.2012.06.015. PubMed PMID: 22841707.
  » https://doi.org/10.1016/j.jcis.2012.06.015
• ^[14] THANKAM, F.G., MUTHU, J., SANKAR, V., et al., “Growth and survival of cells in biosynthetic poly vinyl alcohol-alginate IPN hydrogels for cardiac applications”, Colloids and Surfaces. B, Biointerfaces, v. 107, pp. 137–145, 2013. doi: https://doi.org/10.1016/j.colsurfb.2013.01.069. PubMed PMID: 23475061.
  » https://doi.org/10.1016/j.colsurfb.2013.01.069
• ^[15] BORSAGLI, F.G.L.M., PAIVA, A.E., “Eco-friendly luminescent ZnO nanoconjugates with thiol group for potential environmental photocatalytic activity”, Journal of Environmental Chemical Engineering, v. 9, n. 4, pp. 105491, 2021. doi: https://doi.org/10.1016/j.jece.2021.105491.
  » https://doi.org/10.1016/j.jece.2021.105491
• ^[16] KEERTHIRATHNA, L., SIGERA, S., RATHNAYAKE, M., et al., “Chnoospora minima polysaccharide-mediated green synthesis of silver nanoparticles: potent anticancer and antimicrobial activities”, Biology, v. 14, n. 7, pp. 904, 2025. doi: https://doi.org/10.3390/biology14070904. PubMed PMID: 40723461.
  » https://doi.org/10.3390/biology14070904
• ^[17] RAGAVI, R., SIGERA, P.S., NIRMANI, A.A.E.B., et al., “Green synthesis of silver nanoparticles using Padina commersonii: Characterization, hypoglycemic effects, and antimicrobial potential against human pathogenic bacteria”, Journal of Genetic Engineering and Biotechnology, v. 23, n. 2, pp. 100493, 2025. doi: https://doi.org/10.1016/j.jgeb.2025.100493. PubMed PMID: 40390489.
  » https://doi.org/10.1016/j.jgeb.2025.100493
• ^[18] SILVA, N.C., SILVA, C.J.T., GONÇALVES, M.P., et al., “Carboxymethyl-cellulose-based hydrogels incorporated with cellulose nanocrystals loaded with vitamin D for controlled drug delivery”, Processes, v. 12, n. 7, pp. 1437, 2024. doi: https://doi.org/10.3390/pr12071437.
  » https://doi.org/10.3390/pr12071437
• ^[19] MADUREIRA, J.A., MARINHO, B.M., CARVALHO, S.M., et al., “Sustainable calcium oxide nanoparticles based on agroindustry waste for potential endodontics applications”, Materials Chemistry and Physics, v. 320, pp. 129426, 2024. doi: https://doi.org/10.1016/j.matchemphys.2024.129426.
  » https://doi.org/10.1016/j.matchemphys.2024.129426
• ^[20] WANG, M.-Y., WEST, B.J., JENSEN, C.J., et al., “Morinda citrofolia (Noni): a literature review and recent advances in Noni research”, Acta Pharmacologica Sinica, v. 23, n. 12, pp. 1127–1141, 2002. PubMed PMID: 12466051.
• ^[21] SCARDACI, V., COMPAGNINI, G., “Raman spectroscopy investigation of graphene oxide reduction by laser scribing”, J Carbon Res, v. 7, n. 2, pp. 48, 2021. doi: https://doi.org/10.3390/c7020048.
  » https://doi.org/10.3390/c7020048
• ^[22] BORSAGLI, F.G.L.M., PAIVA, A.E., “Eco-friendly luminescent ZnO nanoconjugates with thiol group for potential environmental photocatalytic activity”, Journal of Environmental Chemical Engineering, v. 9, n. 4, pp. 105491, 2021. doi: https://doi.org/10.1016/j.jece.2021.105491.
  » https://doi.org/10.1016/j.jece.2021.105491
• ^[23] BERNARDES, M.S., ALLIPRANDINI FILHO, P., RAMOS, W.T.S., et al., “Carboxymethyl cellulose/graphene oxide nanocomposite semiconductor for potential energy applications”, International Journal of Biological Macromolecules, v. 284, n. Pt 2, pp. 138090, 2025. doi: https://doi.org/10.1016/j.ijbiomac.2024.138090. PubMed PMID: 39615717.
  » https://doi.org/10.1016/j.ijbiomac.2024.138090
• ^[24] AGARWAL, U.P., “Analysis of cellulose and lignocellulose materials by Raman Spectroscopy: a review of the current status”, Molecules, v. 24, n. 9, pp. 1659, 2019. doi: https://doi.org/10.3390/molecules24091659. PubMed PMID: 31035593.
  » https://doi.org/10.3390/molecules24091659
• ^[25] LEGNER, R., VOIGT, M., SERVATIUS, C., et al., “A four-level maturity index for hot peppers (Capsicum annum) using non-invasive automated mobile raman spectroscopy for on-site testing”, Applied Sciences, v. 11, n. 4, pp. 1614, 2021. doi: https://doi.org/10.3390/app11041614.
  » https://doi.org/10.3390/app11041614
• ^[26] PTAK, S.H., SANCHEZ, L., FRETTÉ, X., et al., “Complementarity of Raman and Infrared spectroscopy for rapid characterization of fucoidan extracts”, Plant Methods, v. 17, n. 1, pp. 130, 2021. doi: https://doi.org/10.1186/s13007-021-00830-6. PubMed PMID: 34930361.
  » https://doi.org/10.1186/s13007-021-00830-6
• ^[27] RODRIGUES, J.S., BRANDÃO, P., DUARTE, S.O.D., et al., “Sustainable carbon dots loaded into carboxymethylcellulose based hydrogels for uterine cancer bioimaging”, Pharmaceutics, v. 16, n. 12, pp. 1500, 2024. doi: https://doi.org/10.3390/pharmaceutics16121500. PubMed PMID: 39771480.
  » https://doi.org/10.3390/pharmaceutics16121500
• ^[28] SCHMITZ, T., GRABOVAC, V., PALMBERGER, T.F., et al., “Synthesis and characterization of a chitosan-N-acetyl cysteine conjugate”, International Journal of Pharmaceutics, v. 347, n. 1–2, pp. 79–85, 2008. doi: https://doi.org/10.1016/j.ijpharm.2007.06.040. PubMed PMID: 17681439.
  » https://doi.org/10.1016/j.ijpharm.2007.06.040
• ^[29] CHEN, Y., ZHANG, Y., MENSAHA, A., et al., “A plant-inspired long-lasting adhesive bilayer nanocomposite hydrogel based on redox-active Ag/Tannic acid-Cellulose nanofibers”, Carbohydrate Polymers, v. 255, pp. 117508, 2021. doi: https://doi.org/10.1016/j.carbpol.2020.117508. PubMed PMID: 33436257.
  » https://doi.org/10.1016/j.carbpol.2020.117508
• ^[30] ALIZADEH, R., ZARRINTAJ, P., KAMRAVA, S.K., et al., “Conductive hydrogels based on agarose/alginate/chitosan for neural disorder therapy”, Carbohydrate Polymers, v. 224, pp. 115161, 2019. doi: https://doi.org/10.1016/j.carbpol.2019.115161. PubMed PMID: 31472854.
  » https://doi.org/10.1016/j.carbpol.2019.115161
• ^[31] INADA, A.C., SILVA, G.T., SILVA, L.P.R., et al., “Therapeutic effects of Morinda citrifolia Linn. (Noni) aqueous fruit extract on the glucose and lipid metabolism in high-fat/high-fructose-fed Swiss mice”, Nutrients, v. 12, n. 11, pp. 3439, 2020. doi: https://doi.org/10.3390/nu12113439. PubMed PMID: 33182564.
  » https://doi.org/10.3390/nu12113439
• ^[32] FONTES, R.F., ANDRADE, J.K.S., RAJAN, M., et al., “Chemical characterization of different parts of noni (Morinda citrifolia) fruit and its freeze-dried pulp powder with emphasis on its bioactive compounds and antioxidant activities”, Food Science and Technology, v. 43, pp. e103722, 2023. doi: https://doi.org/10.1590/fst.103722.
  » https://doi.org/10.1590/fst.103722
• ^[33] KUMAR, H.C., LIM, X.Y., MOHKIAR, F.H., et al., “Efficacy and safety of Morinda citrifolia L. (Noni) as a potential anticancer agent”, Integrative Cancer Therapies, v. 21, pp. 15347354221132848, 2022. PubMed PMID: 36448674.
• ^[34] ASSI, R.A., DARWIS, Y., ABDULBAQI, I.M., et al., “Morinda citrifolia (Noni): A comprehensive review on its industrial uses, pharmacological activities, and clinical trials”, Arabian Journal of Chemistry, v. 10, n. 5, pp. 691–707, 2017. doi: https://doi.org/10.1016/j.arabjc.2015.06.018.
  » https://doi.org/10.1016/j.arabjc.2015.06.018
• ^[35] GONG, T., ZHANG, H., LI, X., et al., “Swelling behavior and biodegradability of hydrogels for biomedical applications”, Journal of Biomedical Materials Research. Part A, v. 106, pp. 122–129, 2018.
• ^[36] KIRTANE, A.R., VERMA, M., KARANDIKAR, P., et al., “Nanotechnology approaches for global infectious diseases”, Nature Nanotechnology, v. 16, n. 4, pp. 369–384, 2021. doi: https://doi.org/10.1038/s41565-021-00866-8. PubMed PMID: 33753915.
  » https://doi.org/10.1038/s41565-021-00866-8
• ^[37] JAMPILEK, J., KRALOVA, K., “Advances in nanostructures for antimicrobial therapy”, Materials, v. 15, n. 7, pp. 2388, 2022. doi: https://doi.org/10.3390/ma15072388. PubMed PMID: 35407720.
  » https://doi.org/10.3390/ma15072388
• ^[38] LAXMINARAYAN, R., VAN BOECKEL, T., FROST, I., et al., “The Lancet Infectious Diseases Commission on antimicrobial resistance: 6 years later”, The Lancet. Infectious Diseases, v. 20, n. 4, pp. e51–e60, 2020. doi: https://doi.org/10.1016/S1473-3099(20)30003-7. PubMed PMID: 32059790.
  » https://doi.org/10.1016/S1473-3099(20)30003-7
• ^[39] SACCO, P., TRAVAN, A., BORGOGNA, M., et al., “Silver-containing antimicrobial membrane based on chitosan-TPP hydrogel for the treatment of wounds”, Journal of Materials Science. Materials in Medicine, v. 26, n. 3, pp. 128, 2015. doi: https://doi.org/10.1007/s10856-015-5474-7. PubMed PMID: 25693676.
  » https://doi.org/10.1007/s10856-015-5474-7
• ^[40] RODRIGUES, J.S., SILVA, C.J.T., SILVA, N.C., et al., “Carbohydrate polymer hydrogels: an environmental and ecofriendly choice for biomedical applications?”, Journal of Polymer Research, v. 30, n. 10, pp. 382, 2023. doi: https://doi.org/10.1007/s10965-023-03764-w.
  » https://doi.org/10.1007/s10965-023-03764-w
• ^[41] MOHAMMADI, Z., JAFARZADEH, H., SHALAVI, S., et al., “Recent advances in root canal disinfection: a review”, Iranian Endodontic Journal, v. 12, n. 4, pp. 402–406, 2017. PubMed PMID: 29225632.
• ^[42] PANT, A., MACKRAJ, I., GOVENDER, T., “Advances in sepsis diagnosis and management: a paradigm shift towards nanotechnology”, Journal of Biomedical Science, v. 28, n. 1, pp. 6, 2021. doi: https://doi.org/10.1186/s12929-020-00702-6. PubMed PMID: 33413364.
  » https://doi.org/10.1186/s12929-020-00702-6
• ^[43] FARROKHI, Y., AL-SHIBLI, B., AL-HAMEEDAWI, D.F.-J., et al., “Escherichia coli enhances the virulence factors of Candida albicans, the cause of vulvovaginal candidiasis, in a dual bacterial/fungal biofilm”, Research in Microbiology, v. 172, n. 4–5, pp. 103849, 2021. doi: https://doi.org/10.1016/j.resmic.2021.103849. PubMed PMID: 34089837.
  » https://doi.org/10.1016/j.resmic.2021.103849
• ^[44] BERNARDES, M.S., SALES, J.D.L., BORSAGLI, F.G.M., “Antioxidant and antimicrobial activity of innovative carboxymethyl cellulose/graphene oxide nanocomposite for biological proposals”, Journal of Molecular Liquids, v. 422, pp. 126938, 2025. doi: https://doi.org/10.1016/j.molliq.2025.126938.
  » https://doi.org/10.1016/j.molliq.2025.126938
• ^[45] FARROKHI, Y., AL-SHIBLI, B., AL-HAMEEDAWI, D.-F.-J., et al., “Escherichia coli enhances the virulence factors of Candida albicans, the cause of vulvovaginal candidiasis, in a dual bacterial/fungal biofilm”, Research in Microbiology, v. 172, n. 4–5, pp. 103849, 2021. doi: https://doi.org/10.1016/j.resmic.2021.103849. PubMed PMID: 34089837.
  » https://doi.org/10.1016/j.resmic.2021.103849
• ^[46] JAMPILEK, J., KRALOVA, K., “Advances in nanostructures for antimicrobial therapy”, Materials, v. 15, n. 7, pp. 2388, 2022. doi: https://doi.org/10.3390/ma15072388. PubMed PMID: 35407720.
  » https://doi.org/10.3390/ma15072388

Publication Dates

• Publication in this collection
  02 Feb 2026
• Date of issue
  2026

History

• Received
  08 Aug 2025
• Accepted
  09 Dec 2025