Impaginato 133 Adv. Hort. Sci., 2023 37(1): 133­137 DOI: 10.36253/ahsc­13870 Nanosponges and CPPU: a scoping review and a pre­test to assess the potentiality for shelf­life prolongation of cut carnations L. Battisti 1 (*), F. Caldera 2, G. Hoti 2, F. Trotta 2, M. Devecchi 3 1 Department of Cultures, Politics and Society, University of Turin, Lungo Dora Siena, 100/A, 10153 Torino, Italy. 2 Department of Chemistry, University of Turin, Via P. Giuria, 9, 10125 Torino, Italy. 3 Department of Agricultural, Forest and Food Sciences, University of Turin, Largo Paolo Braccini, 2, 10095 Grugliasco (TO), Italy. Key words: Dianthus caryphyllus L., forchlorfenuron, growth regulators. Abstract: Nanosponges can favour the gradual release of molecules over a pro­ longed time, increasing the bioavailability and action of preservatives and phy­ toregulators, reducing the concentrations usually adopted. In floriculture, they have previously been proposed for the delivery of anti­ethylene compounds to improve the shelf­life of cut flowers. However, the potential of nanosponges is not only limited to these compounds. The present scoping review evaluated the effects of β­cyclodextrin­based nanosponges and growth regulators on the post­harvest longevity of cut flowers of ornamental species. One novelty was the use of Forchlorfenuron (CPPU), a growth regulator belonging to the group of cytokinins predominantly used in fruit cultivation, to evaluate its potential to increase the shelf­life of cut carnations (Dianthus caryophyllus L). In particular, an in­depth analysis of a pre­test involving the use of nanosponges and CPPU is proposed. Specifically, as far as post­harvest longevity is concerned, the treat­ ments involved the use of: deionised water; nanosponges and deionised water; nanosponges loaded with CPPU; nanosponges loaded with a classic solution for cut flowers, composed of sucrose, aluminium sulphate and 8­hydroxyquinoline sulphate. Preliminary results show that the nanosponge and deionised water complex and the nanosponge and classical solution complex prolonged the longevity of the cut flower by up to 20 days, compared to the control (17 days). In contrast, the CPPU­nanosponge complex showed similar results to the con­ trol. Replication of the research is necessary to validate the results. 1. Introduction Cyclodextrin Nanosponges (CDNSs) are cage­like network polymers prepared by connecting cyclodextrin molecules with various kinds of bi­ or polyfunctional linking agents. The term “nanosponge” was first intro­ (*) Corresponding author: luca.battisti@unito.it Citation: BATTISTI L., CALDERA F., HOTI G., TROTTA F., DEVECCHI M., 2023 ­ Nanosponges and CPPU: a scoping review and a pre‐test to assess the poten‐ tiality for shelf‐life prolongation of cut carnati. ­ Adv. Hort. Sci., 37(1): 133­137. Copyright: © 2023 Battisti L., Caldera F., Hoti G., Trotta F., Devecchi M. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 26 October 2022 Accepted for publication 10 January 2023 AHS Advances in Horticultural Science Short note https://doi.org/10.36253/ahsc-13870 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2023 37(1): 133­137 134 duced by Ma and Li, in a paper focusing on the sequestering capacity of polyurethane crosslinked CD polymers, to highlight the nanometric porosity of this class of materials (Ma and Li, 1999). In addition to the central lipophilic cavity of CD molecules, CDNSs exhibit a second type of pores, which are the empty spaces among CD molecules, whose polarity and size can be modulated by varying the chemical structure of the linking agent and its concentration. Such fea­ tures make CDNSs highly versatile materials with potential applications as nanostructured containers and delivery systems for a broad set of active princi­ ples. Furthermore, CDNSs are low­cost, easy to pre­ pare, safe and sustainable materials, as they are mainly composed of starch derivatives and, in some cases, even the linker comes from renewable sources (e.g., citric acid) (Caldera et al., 2017). The application of CDNSs in the pharmacological field has been widely explored. Several classes of drugs have been successfully encapsulated, stabilized and released with controlled kinetics, including anti­ cancer, anti­inflammatory, antiviral, antibacterial, antifungal drugs and many others. Recently, the func­ tionalization of CDNSs with specific moieties has led to the development of a new generation of stimuli­ responsive CDNSs, capable of releasing drugs on­ demand and highly selective CDNSs for targeted drug delivery. In the environmental field, CDNSs have been used as absorbing agents for the removal of organic pollu­ tants as well as heavy metal cations for water decon­ tamination (Krabicova et al., 2020). Although CDNSs have been extensively studied for biomedical and environmental applications, their full potential in the horticultural field remains largely unknown. To date, only a few studies have explored the use of CDNSs for the encapsulation and release of the herbicide ailanthone and some anti­ethylene molecules (Demasi et al., 2021). Forchlorfenuron [N­(2­chloro­4­pyridyl)­Nʹ­pheny­ lurea], whose acronym is CPPU, is a plant growth reg­ ulator (synthetic cytokinin) that promotes the pericli­ nal cell division and is often used in agriculture to increase the berry size, the quality and the yield of grapes and kiwifruit (Peppi and Fidelibus, 2008; Cruz­ Castillo et al., 2014). Although several studies have evaluated the effi­ cacy of CPPU in postharvest in fruits (Zhang et al., 2017; Chang, 2021), few studies have focused on cut foliage and flowers, in contrast to a similar molecule, Thidiazuron, which has been used for years (Ferrante et al., 2002; Chamani et al., 2006). The aim of this scoping review is to highlight the application potential of nanosponges in the posthar­ vest context. In addition, an in­depth study is pro­ posed on the production and testing of nanosponges and CPPU in order to assess their effectiveness in prolonging the shelf‐life of cut carnations. More specifically, we intend to describe the pre­test phase of the two products listed above, which is also useful from an economic point of view in order to produce a quantity of nanosponges in the laboratory suitable for performing an experimental test with replica­ tions, as well as to proceed with the purchase of CPPU in larger quantities. 2. Materials and Methods In the coming sections, the following will be reported: a scoping review on the use of nanosponges in floriculture; a first trial of the use of CPPU and nanosponges on cut flower carnation. Synthesis of a carbonate cyclodextrin nanosponge A carbonate cyclodextrin nanosponge was synthe­ sized by heating β­cyclodextrin (β­CD, 6.500 g, 5.73 mmol) and 1,1’­carbonyldiimidazole (CDI, 3.715 g, 22.92 mmol) in 39 mL of N,N­dimethylformamide at 90°C for 4 h. In the end, the solid gel formed during the crosslinking reaction was crushed and washed with approximately 2 L of deionized water via Buchner filtration. The nanosponge was further puri­ fied in a Soxhlet extractor with ethanol for 20 h and, once dry, finely ground in a planetary ball mill. CPPU loading in the nanosponge The loading of CPPU into the nanosponges was performed subsequent to the synthesis and purifica­ tion of the nanosponge. Briefly, 450 mg of nanosponge was added to a solution of 50 mg of CPPU dissolved in 25 mL ethanol. Then, the disper­ sion was stirred at room temperature for 24 h. Finally, the CPPU­loaded nanosponge was collected by removing the entire volume of ethanol in a rotary evaporator at 50°C under vacuum. The complex was stored in a desiccator at room temperature until use. Pre‐test setting The pre­test was conducted in March and April 2022, in the laboratories of the Department of Chemistry and Department of Agricultural, Forest and Food Sciences, University of Turin. The carnations were collected, packaged and pur­ https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/cytokinin https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/kiwifruit Battisti et al. ‐ Nanosponges and CPPU in postharvest 135 chased at the flower market in Sanremo (Italy) on 27 March at 6 p.m. and were transported to Turin and kept in the transported condition until 3 p.m. on 28 March, when the pre­test began. The four packages, purchased from ‘Cooperativa Tre Ponti’ (intermedi­ ary), contained 20 flowers each. The first phase consisted of eliminating flowers that visually showed diseases, lesions and flowers at a different stage of flowering from the others. A total of 70 flowers were selected and the stems were cut into the water leaving a length of 30 cm. The pre­test comprised seven different treat­ ments (solutions with deionized water), each applied to 10 flowers. Each flower was placed in a glass tube containing 50 ml of solution. On day 8 and 16, 10 ml of the specific solution was added to all tubes. Measurements were conducted daily, and data were acquired for the following parameters: cut carnation weight (g) and flower diameter (cm). The pre­test lasted 20 days, with the following environmental conditions: 12 hours of light and 12 hours of dark­ ness; temperature 19±2°C. The treatments are shown in Table 1. 3. Results and Discussions Pre‐test results Preliminary results (Fig. 1) show that the treat­ ment A and the treatment C prolonged the longevity of the cut flower by up to 20 days, compared to the Control (17 days). In contrast, the treatment E com­ plex showed similar results to the control. There was no inhomogeneity in the diameters of the flowers within the various treatments; however, the largest diameters (9.5 cm) were more frequently reached by the treatment F. Lateral shoot development was a common side effect of CPPU and commercial solution treatments. The pre­test yielded interesting results that allow planning the production of nanosponges and the pur­ chase of CPPU in order to set up a scientific trial. Of particular interest could be the application in solu­ tion and spray of nanosponges and CPPU on cut foliage. Final discussions and next steps The application of CDNSs in floriculture, and more specifically post­harvest preservation, is an emerging line of research still widely unexplored. To date, only few scientific papers focusing on the encapsulation of anti­ethylene compounds in CDNSs have appeared in the literature. Of these ethylene inhibitors, 1­methyl­ cyclopropene (1­MCP) is one of the most studied, especially in combination with CDs and CD­deriva­ tives. In 1994, Serek et al. described for the first time the ability of 1­MCP to prolong the post­harvest shelf­life of plant material (Serek et al., 1994). Since then, 1­MCP has been extensively studied as a non­ toxic anti­ethylene agent to inhibit the senescence Code Treatments A Nanosponges (5 g/l) B Sucrose 30 g/l + aluminium sulphate (200 mg/Kg) + 8­hydroxyquinoline sulphate (200 mg/l) C Sucrose 30 g/l + aluminium sulphate (200 mg/Kg) + Nanosponges (5g/l) D CPPU (200 mg/l) E CPPU in Nanosponges (200 mg/l) F Commercial solution ­ Chrisal prof. 2 (10 ml/l) Control Deionised water Table 1 ­ Pre­test treatments Fig. 1 ­ Average values of weights (g) and standard error bars of cut carnations during the pre­test. Adv. Hort. Sci., 2023 37(1): 133­137 136 process of cut flowers, vegetables and fruits at the receptor level (Blankenship et al., 2003; Nasiri et al., 2020). In 2000, 1­MCP appeared on the market in two different formulations commercialized by Floralife, Inc. (Walterboro, SC) and AgroFresh, Inc. (Spring House, PA). The Floralife product is named EthylBloc® and it is meant to be applied on ornamen­ tals only. While, Agrofresh provides a formulation, named SmartFresh®, that can be used with edible crops. However, both products are based on the inclusion complex of 1­MCP gas in α­CD (Gehla et al., 2003). The efficacy of these formulations is time­limited, as most of 1­MCP is released within 20­30 minutes from application, under normal temperature and pressure conditions (Blankenship et al., 2003). To overcome such limitations, new materials such as CDNSs have been tested for the encapsulation and prolonged release of 1­MCP. In a first attempt, 1­MCP, 2,5­norbornadiene and silver nitrate were encapsulated in a carbonate CDNS and added to two flower species, specifically Dianthus caryophyllus ‘ Idra di Muraglia’ and Ranunculus asiaticus ‘Elegance’. While the formula­ tion containing 1­MCP allowed to extend the vase life of Dianthus caryophyllus up to 23 days, the other for­ mulations did not show significant effects and none of the formulations was able to improve the longevi­ ty of ranunculus (Devecchi et al., 2009). With the aim of understanding the role played by the central cavity of CD molecules in the encapsula­ tion and controlled release of 1­MCP, carbonate NSs based on α­CD (6 glucose units, diameter of the cavi­ ty: 4.7­5.3 Å) were used in comparison with the anal­ ogous carbonate NSs prepared with β­CD (7 glucose units, diameter of the cavity 6.0­6.5 Å). The tests were performed on Dianthus caryophyllus ‘Idra di Muraglia’. Results demonstrated that β­CD is remark­ ably more effective than α­CD in extending the post­ harvest life of the Dianthus flowers, as no evidence of deterioration was observed up to nearly 11 days. Whilst, the 1­MCP­loaded NSs based on α­CD did not show any significant anti­ethylene activity (Sceglie et al., 2011 a). The next step was to determine the influence of the NS’s degree of crosslinking on the encapsulation and slow release of 1­MCP. A series of samples of β­ CDNS were synthesized with different carbonate to CD molar ratio (i.e., 2, 4 and 8), resulting in different degree of crosslinking. After loading with 1­MCP, the NSs were used to extend the vase life of carnation cut flowers. All the NS formulations exhibited anti­ ethylene activity. However, the NS with the highest degree of crosslinking (i.e., linker/β­CD molar ratio of 8), and therefore the most densely crosslinked poly­ mer structure, showed the highest efficacy for pro­ longed time, even at low concentration (Sceglie et al., 2011 b). The protective effect of the 1­MCP­loaded β­CDNS with monomer ratio 1:8 against infection by Botrytis cinerea on Dianthus caryophyllus L. ‘Idra di Muraglia’ was studied as well. After eleven days of treatment, the NS at low dosage reduced the spreading of the grey mould by approximately 60 %. At higher dosage, the NS formulation outperformed the commercial 1­ MCP product, used as a reference, reaching a value above 90%, while the commercial formulation stopped at 76% (Sceglie et al., 2012). As a final step, the effectiveness of the 1­MCP/ β­ CDNS(1:8) complex in delaying the senescence process of cut flowers was evaluated in different flo­ ral species. Specifically, Anemone coronaria L. multi‐ color , Paeonia lactiflora L. ‘Sarah Bernhardt’, Helianthus annuus L. ‘Sunrich Orange’, Ranunculus asiaticus L. ‘Minou Abrown’, Papaver nudicaule L. multicolor and Rosa hybrida L. ‘Jupiter’ were treated with the NS formulation in the presence of exoge­ nous ethylene. Although the mechanism of action was different, depending on the flower species, the CDNS increased the anti­ethylene activity of 1­MCP in all the tested samples (Sceglie et al., 2013). 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