Periodic Reporting for period 1 - SUPREME (Polymeric Cyclodextrin-Based Supramolecular Constructs Photoreleasing Nitric Oxide for Combating Antibiotic Resistance)
Reporting period: 2022-08-01 to 2024-11-30
Summary of the context and overall objectives of the project
Multidrug resistance (MDR) in bacteria is the prime example of bacterial adaptation and the pinnacle of evolution and remains the greatest challenge in public health care. The World Health Organization has named antibiotic MDR as one of the three most important public health threats of the 21st century and the EU has recognized and included in the Action Plan against the rising threats from antimicrobial resistance, “Infectious diseases represent a very significant burden for developing nations”. The slogan of one of the latest World Health Day was “Antimicrobial resistance: no action today no cure tomorrow.” This scenario, associated to the low turnover of new clinically approved antibiotic drugs, makes the development of novel therapeutic approaches to fight antibiotic resistance desperately needed and one of the most challenging goal in human health-care.
In this context, the General Objective of SUPREME has been to fight multidrug resistant bacteria creating innovation in the implementation of novel therapeutic approaches based on the controlled delivery “unconventional” antibacterial species. This challenge has been addressed by fabrication of unprecedented supramolecular platforms based on branched polymeric cyclodextrins (PCDs) integrating visible light-activatable nitric oxide (NO) photodonors (NOPDs) alone or in combination with antibiotics currently employed.
Towards this purpose, new devised PCDs have been used as suitable building blocks for the achievement of supramolecular nanogel and nanoparticle constructs integrating visible light activatable NOPDs. In addition, molecular hybrids integrating NOPD and conventional antibiotics are aimed to be fabricated. This global approach has aimed at circumventing the resistance machinery in MDR bacteria maximising the therapeutic efficacy and at limiting side effects typical for antibiotic drugs.
In this context, the General Objective of SUPREME has been to fight multidrug resistant bacteria creating innovation in the implementation of novel therapeutic approaches based on the controlled delivery “unconventional” antibacterial species. This challenge has been addressed by fabrication of unprecedented supramolecular platforms based on branched polymeric cyclodextrins (PCDs) integrating visible light-activatable nitric oxide (NO) photodonors (NOPDs) alone or in combination with antibiotics currently employed.
Towards this purpose, new devised PCDs have been used as suitable building blocks for the achievement of supramolecular nanogel and nanoparticle constructs integrating visible light activatable NOPDs. In addition, molecular hybrids integrating NOPD and conventional antibiotics are aimed to be fabricated. This global approach has aimed at circumventing the resistance machinery in MDR bacteria maximising the therapeutic efficacy and at limiting side effects typical for antibiotic drugs.
Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far
Supramolecular nanogels based on gamma-CDs have been prepared and used as nanocontainers to encapsulate NOPDs activatable with the highly biocompatible visible light. Both NOPDs used preserve in the nanogel their photochemical properties individually and also when co-entrapped, showing NO release regulated by blue and green light, associated to activatable green and persistent red fluorescence, respectively. The confinement in the nanogel leads to a significant increase of the NO photorelease efficiency of both compounds probably due to the active role of the CD scaffold as reactant in the radical-mediated NO photorelease mechanism. Antribacterial tests have been performed on Methicillin-resistant Staphylococcus aureus (MRSA) demonstrating that the supramolecular nanogel is very active in inhibiting the bacteria load. Fluorescence microscopy experiments provide an explanation about the interaction of the NOPDs with bacterial cells.
A hydrophobic nitric oxide (NO) photodonor integrating both nitroso- and nitro-functionalities within its chromophoric skeleton has been synthesized. Excitation of this compound with blue light triggers the release of two NO molecules from the nitroso and the nitro functionalities via a stepwise mechanism. Encapsulation of the NO photodonor within new generation of biocompatible neutral, cationic and anionic PCDs as suitable carriers leads to supramolecular nanoassemblies, which exhibit the same nature of the photochemical processes but enhanced NO photorelease performances by about one order of magnitude when compared with the free guest. Antibacterial tests carried out with Methicillin-resistant Staphylococcus aureus and Acinetobacter baumannii demonstrate an effective antibacterial activity exclusively under light activation and point out a differentiated role of the polymeric nanocarriers in determining the outcome of the antibacterial photodynamic action.
In the frame of new photopharmacological approach for multimodal antibacterial activity we designed a novel molecular hybrid consisting in a conventional antibiotic drug theporarily inactivated by the introduction of a nitroso functionality. In particular, we showed that nitrosation of the fluoroquinolone norfloxacin (NF), a broad-spectrum antibiotic, leads to the nitrosated bioconjugate NF-NO, which is inactive at the typical minimum inhibitory concentration of NF. Irradiation of NF-NO with visible blue light triggers the simultaneous release of NF and NO. The photouncaging process is accompanied by the revival of the typical fluorescence emission of NF, quenched in NF-NO, which acts as an optical reporter. This permits the real-time monitoring of the photouncaging process, even within bacteria cells where antibacterial activity is switched on exclusively upon light irradiation. The mechanism of photorelease seems to occur through a two-step hopping electron transfer mediated by the lowest triplet state of NF-NO and the phos-phate buffer ions or aminoacids such as tyrosine. Considering the well-known role of NO as an “unconventional” antibacterial, the NF-NO conjugate may represent a potential bimodal antibacterial weapon activatable on demand with high spatio-temporal control.
In an exhaustive review paper we have illustratd the most significant progress made over the last five years in achieving engineered materials including nanoparticles, gels and thin films, sharing the common feature to deliver NO under the exclusive control of the biocompatible Visible/Near Infrared light inputs. We have highlighted the logical design behind the fabrication of these systems, illustrating the potential therapeutic applications with particular emphasis on bacterial infections.
A hydrophobic nitric oxide (NO) photodonor integrating both nitroso- and nitro-functionalities within its chromophoric skeleton has been synthesized. Excitation of this compound with blue light triggers the release of two NO molecules from the nitroso and the nitro functionalities via a stepwise mechanism. Encapsulation of the NO photodonor within new generation of biocompatible neutral, cationic and anionic PCDs as suitable carriers leads to supramolecular nanoassemblies, which exhibit the same nature of the photochemical processes but enhanced NO photorelease performances by about one order of magnitude when compared with the free guest. Antibacterial tests carried out with Methicillin-resistant Staphylococcus aureus and Acinetobacter baumannii demonstrate an effective antibacterial activity exclusively under light activation and point out a differentiated role of the polymeric nanocarriers in determining the outcome of the antibacterial photodynamic action.
In the frame of new photopharmacological approach for multimodal antibacterial activity we designed a novel molecular hybrid consisting in a conventional antibiotic drug theporarily inactivated by the introduction of a nitroso functionality. In particular, we showed that nitrosation of the fluoroquinolone norfloxacin (NF), a broad-spectrum antibiotic, leads to the nitrosated bioconjugate NF-NO, which is inactive at the typical minimum inhibitory concentration of NF. Irradiation of NF-NO with visible blue light triggers the simultaneous release of NF and NO. The photouncaging process is accompanied by the revival of the typical fluorescence emission of NF, quenched in NF-NO, which acts as an optical reporter. This permits the real-time monitoring of the photouncaging process, even within bacteria cells where antibacterial activity is switched on exclusively upon light irradiation. The mechanism of photorelease seems to occur through a two-step hopping electron transfer mediated by the lowest triplet state of NF-NO and the phos-phate buffer ions or aminoacids such as tyrosine. Considering the well-known role of NO as an “unconventional” antibacterial, the NF-NO conjugate may represent a potential bimodal antibacterial weapon activatable on demand with high spatio-temporal control.
In an exhaustive review paper we have illustratd the most significant progress made over the last five years in achieving engineered materials including nanoparticles, gels and thin films, sharing the common feature to deliver NO under the exclusive control of the biocompatible Visible/Near Infrared light inputs. We have highlighted the logical design behind the fabrication of these systems, illustrating the potential therapeutic applications with particular emphasis on bacterial infections.
Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)
The results described in the previous section represent a significant step forward beyond the state of the art. In fact, it has been well demonstrated how the use of an unconventional antibiotic such as NO represent a valid alternative to conventional antibiotic. NO does not suffer MDR and does not present systemic effects common to antibiotics. The precise control of the appropriate concentration and site of action of NO by light stimuli is definitely a great advantage compared with the conventional treatment of bacterial infections. The use of highly biocompatible nanocarriers based on PCDs revealed a suitable strategy not only to solubilize highly hydrophobic NO photoprecursors but also to enhance their photochemical performances in terms of NO photorelease. This has been demonstrated to be attributable to the active role of the polymeric CDs scaffold in acting as rectant, which participate to the radical mediated mechanisms leading to the NO photorelease. In addition, we have proposed a completely new photopharmacological approach in which a conventional antiotic such as norfloxacin can act itself as antenna to trigger the NO release from its molecular skeleton, generating a new class og photoactivatable photo-drugs. The approach proposed, in principle, can be extended to other antibiotic drugs, which suffer the same limitation of norfloxacin, enhancing their antibacterial action and reducing all their main drawbacks. Finally, it should be enphasized that all supramolecular nanoconstruct proposed present higher biocompatibility, loading versatility and capacity superior to that of other amphiphilic host, with the additional advantage of potential higher skin penetration capability and bacteria membrane interactions.
Some of the systems developed are easy to be synthesized and offer great potential for a future scale up and commercialization after regulatory processes.
All the described works have been published in peer-reviewed journals and presented in the form of oral and invited communications as follows:
1. Martins, Tassia J.; Parisi, Cristina; Suzuki, Yota; Hashimoto, Takeshi; Nostro, Antonia; Ginestra, Giovanna; Hayashita, Takashi; Sortino, Salvatore. Supramolecular Assemblies of Fluorescent Nitric Oxide Photoreleasers with Ultrasmall Cyclodextrin Nanogels.
Molecules, v. 28(15), p. 2-11, 2023.
2. Martins, Tassia J.; Parisi, Cristina; Pinto, Juliana Guerra; Brambilla, Isabelle de Paula Ribeiro; Malanga, Milo; Ferreira-Strixino, Juliana; Sortino, Salvatore. Stepwise Nitric Oxide Release and Antibacterial Activity of a Nitric Oxide Photodonor Hosted within Cyclodextrin Branched Polymer Nanocarriers.
ACS Medicinal Chemistry Letters, v. 15, p. 857-863, 2024.
3. Martins, Tassia J.; Parisi, Cristina; Pinto, Juliana Guerra; Brambilla, Isabelle de Paula Ribeiro; Melilli, Barbara; Aleo, Danilo; Ferreira-Strixino, Juliana; Sortino, Salvatore. Simultaneous photoactivation of a fluoroquinolone antibiotic and nitric oxide with fluorescence reporting. Journal of Materials Chemistry B, v. 12, p. 7626-7634, 2024.
4. Parisi, Cristina; Laneri, Francesca; Martins, Tassia J.; Fraix, Aurore; Sortino, Salvatore. Nitric Oxide-Photodelivering Materials with Multiple Functionalities: From Rational Design to Therapeutic Applications.
ACS Applied Materials & Interfaces, v. 16 (44), p. 59697–59720, 2024.
1. Supramolecular assemblies of fluorescent NO photoreleasers with ultrasmall cyclodextrin nanogels with high photochemical performances: oral presentation at the 20th Congress of the European Society for Photobiology, held from August 27 – 30, 2023, Lyon, France.
2. Exploring novels antibacterial systems with a nitric oxide photodonor encapsulated in cyclodextrin polymers: oral presentation at Italian Photochemistry Meeting 2023, held from December 14 – 16, 2023, Sestri Levante, GE, Italy.
3. Polymeric Cyclodextrin-Based Supramolecular Constructs Photoreleasing Nitric Oxide for Combating Antibiotic Resistance: oral presentation at Giornata della Ricerca del Dipartimento di Scienze del Farmaco e della Salute, held on 22nd February,2024, in Catania.
4. Cyclodextrin polymer nanocarriers as hosts for nitric oxide photodonors: stepwise nitric oxide release and antibacterial activity: oral presentation at 21st International Cyclodextrin Symposium, held from June 10 – 14, 2024, on Dunkerque, France.
5. 18th International joint congress of Photobiology and Molecular and Experimental Pathology Society of Australasia, Pert, Australia, August 2024.
6. 20th Congress of the European Society for Photobiology, Lion, France, August 2023
Some of the systems developed are easy to be synthesized and offer great potential for a future scale up and commercialization after regulatory processes.
All the described works have been published in peer-reviewed journals and presented in the form of oral and invited communications as follows:
1. Martins, Tassia J.; Parisi, Cristina; Suzuki, Yota; Hashimoto, Takeshi; Nostro, Antonia; Ginestra, Giovanna; Hayashita, Takashi; Sortino, Salvatore. Supramolecular Assemblies of Fluorescent Nitric Oxide Photoreleasers with Ultrasmall Cyclodextrin Nanogels.
Molecules, v. 28(15), p. 2-11, 2023.
2. Martins, Tassia J.; Parisi, Cristina; Pinto, Juliana Guerra; Brambilla, Isabelle de Paula Ribeiro; Malanga, Milo; Ferreira-Strixino, Juliana; Sortino, Salvatore. Stepwise Nitric Oxide Release and Antibacterial Activity of a Nitric Oxide Photodonor Hosted within Cyclodextrin Branched Polymer Nanocarriers.
ACS Medicinal Chemistry Letters, v. 15, p. 857-863, 2024.
3. Martins, Tassia J.; Parisi, Cristina; Pinto, Juliana Guerra; Brambilla, Isabelle de Paula Ribeiro; Melilli, Barbara; Aleo, Danilo; Ferreira-Strixino, Juliana; Sortino, Salvatore. Simultaneous photoactivation of a fluoroquinolone antibiotic and nitric oxide with fluorescence reporting. Journal of Materials Chemistry B, v. 12, p. 7626-7634, 2024.
4. Parisi, Cristina; Laneri, Francesca; Martins, Tassia J.; Fraix, Aurore; Sortino, Salvatore. Nitric Oxide-Photodelivering Materials with Multiple Functionalities: From Rational Design to Therapeutic Applications.
ACS Applied Materials & Interfaces, v. 16 (44), p. 59697–59720, 2024.
1. Supramolecular assemblies of fluorescent NO photoreleasers with ultrasmall cyclodextrin nanogels with high photochemical performances: oral presentation at the 20th Congress of the European Society for Photobiology, held from August 27 – 30, 2023, Lyon, France.
2. Exploring novels antibacterial systems with a nitric oxide photodonor encapsulated in cyclodextrin polymers: oral presentation at Italian Photochemistry Meeting 2023, held from December 14 – 16, 2023, Sestri Levante, GE, Italy.
3. Polymeric Cyclodextrin-Based Supramolecular Constructs Photoreleasing Nitric Oxide for Combating Antibiotic Resistance: oral presentation at Giornata della Ricerca del Dipartimento di Scienze del Farmaco e della Salute, held on 22nd February,2024, in Catania.
4. Cyclodextrin polymer nanocarriers as hosts for nitric oxide photodonors: stepwise nitric oxide release and antibacterial activity: oral presentation at 21st International Cyclodextrin Symposium, held from June 10 – 14, 2024, on Dunkerque, France.
5. 18th International joint congress of Photobiology and Molecular and Experimental Pathology Society of Australasia, Pert, Australia, August 2024.
6. 20th Congress of the European Society for Photobiology, Lion, France, August 2023