Marion Donnier-Marechal

Nicolas Galanos

Teddy Grandjean

Yoann Pascal

Ding-Kun Ji

Lei Dong

Emilie Gillon

Xiao-Peng He

Anne Imberty

Eric Kipnis

Marion Donnier-Maréchal

Rodrigue Dessein

Sébastien Vidal

Perylenediimide-based glycoclusters as high affinity ligands of bacterial lectins: Synthesis, binding studies and anti-adhesive properties

come

Introduction

Perylenediimide-based organic dyes are now widely studied 1 due to their π-π stacking aggregation properties [2][3][4] that led to application in field effect transistors [5][6][7][8][9] or other biologically relevant systems. [10][11][12][13][14] Nevertheless, applications are sometimes troublesome due to their specific prone to poor solubility in water and aggregation. The introduction of carbohydrate moiety onto the PDI scaffold was reported [15][16][17][18][19][20][21][22][23] as a method to provide watersoluble PDI-based glycomaterials for further applications in biology. Several other approaches for the conjugation of carbohydrates to aromatic cores 24 or fluorescent dyes 18, 19, 25-32 have also been reported in this context. We and other described that the multivalent glycoclusters with high affinity for bacterial lectins could represent a new way to treat bacterial infection especially infection due to the bacteria Pseudomonas aeruginosa. 33, 34 P. aeruginosa is a pathogenic bacterium involved in acute and chronic lung infection especially in immune-compromised patients or patients suffering from cystic fibrosis. Two lectins have been identified as implicated in the adhesion of the bacteria to the host cells, namely the galactosespecific LecA and the fucose-specific LecB. 35, 36 We have also previously designed a large series of glycoclusters for applications in anti-adhesive strategies against P. aeruginosa infection. [37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52][53] We have especially demonstrated the excellent anti-adhesive properties of calixarene-based glycoclusters in animal models of lung infection leading to protection of mice against P. aeruginosa infection.

45

In the present study, we designed a series of PDI-based glycoclusters with different and complementary properties towards the (1) multivalent binding to bacterial lectins and also (2) their anti-adhesive properties against bacterial infection. The goal was to take advantage of the intrinsic fluorescence properties of the PDI core for diagnostic tools or a better understanding at the biomolecular level of the anti-adhesive mechanism.

Results and discussion

Synthesis of the PDI-based glycoclusters

The regioselective 1,6,7,12-tetrachlorination of 3,4,9,10-perylene tetracarboxylic dianhydride 1 to the corresponding 1,6,7,12tetrachloroperylene-3,4,9,10-tetracarboxylic dianhydride 2 were mostly adapted from chlorination of perylenediimide scaffolds using chlorosulfonic acid and iodine under heating and during 20 h. 54 The reaction conditions required refluxing in chlorosulfonic acid as a solvent for 20 h. [54][55][56][57][58] Nevertheless, reducing the heating to 15 h was not detrimental. 59 A methodology study indicated that the reaction performed during 5 h afforded the desired tetrachlorinated compound while longer reaction times of 10 h and 20 h provided the penta-and hexa-chlorinated derivatives as by-products. 60 We therefore decided to use these conditions (5 h reflux) for the preparation of compound 2 which provided the best reproducible results in our hands (Scheme 1). The isolated yield of 85% required a long soxhlet extraction (11 days) due to the poor solubility of compound 2.

Scheme 1. Synthesis of tetra-and hexa-propargylated PDI-based cores Formation of the perylenediimide (PDI) moiety was then performed using either propargylamine or t-butyl glycinate [61][62][63][64][65] to afford the desired diimides 3 and 4 (Scheme 1). Condensation 16 with p-propargyloxyphenol 66 provided the hexa-propargylated core 5 17, 18 and tetra-propargylated core 6. The introduction of two glycine residues was used to provide improved water solubility to the glycocluster through the carboxylic acid moieties.

67

Six azido-functionalized carbohydrates 7a-f were then used for the conjugation to the alkynylated PDI-based cores. The azidotriethyleneglycol-based linkers have been largely reported as good spacer arms for the design of high affinity lectin ligands.

34, 37-39, 43, 45 Glycosides 7a-d were synthesized according to revisited and robust large scale (> 5 g) procedures.

42, 68-70 A more hydrophobic linker was also investigated to take advantage of a hydrophobic group in the periphery of the anomeric position which improved the binding properties to LecA 42, 43, 46, 71-73 and LecB. 74 The synthesis was performed both in the galactose and fucose series for this study. The glucoside 7a will be used to prepare glucosylated glycoclusters as negative control in ITC experiments. The mannose derivative 7c was used to provide a further glycocluster with potential applications with other lectins. The mono-cycloadducts 7e-f were obtained from the corresponding propargyl glycosides using a large excess of 1,5-diazido-3oxapentane (25 equivalents) and the glycoside was added slowly with a syringe pump (~ 16 h) in a heated (90°C) and diluted solution of reagents. Selective mono-functionalization was readily achieved in high isolated yields (> 90%).

R 2 R 2 O O O O O O O O N N N N N N N N N N N N N N N N N N R R R R R R N3 R N N N R N N N R N N N R N N N R R = O OR 1 R 1 O R 1 O OR 1 O O O O OR 1 R 1 O R 1 O OR 1 O O O O R 1 O R 1 O R 1 O OR 1 O O O O Me R 1 O OR 1 OR 1 O O O O OR 1 R 1 O R 1 O OR 1 O O Me R 1 O OR 1 OR 1 O N N N O N N N O 8a-f R 1 = Ac 9a-f R 1 = H a (GlcEG 3 ) b (GalEG 3 ) c (ManEG 3 ) d (FucEG 3 ) e (GalTzEG 2 ) f (FucTzEG 2 ) NaOMe MeOH r.t. / 16 h 10a-f R 1 = Ac, R 2 = tBu 11a-f R 1 = R 2 =

ITC binding studies

The glycoclusters were then evaluated as multivalent ligands of LecA and LecB by isothermal titration microcalorimetry (ITC), a bioanalytical technique providing the thermodynamics parameters for the interactions such as the stoichiometry (N) of the complex, the enthalpy (∆H) and also the association constant (K a = 1/K d ) in a single measurement.

77, 78

The binding properties toward LecA were evaluated for the galactosylated glycoclusters 9b, 9e, 11b and 11e (Table 1). The The increase in galactose valency resulted in higher enthalpy of binding, but the entropy cost is also stronger, resulting in equivalent affinity. These galactosylated glycoclusters are good candidates for further applications in anti-adhesive strategies against Pseudomonas aeruginosa. 34 Titration curves obtained with LecB are more complex (Figure 1) and, except for glycocluster 9d, they could not be fitted with a single site model (Table 2). For tetravalent compounds 11d and 11f, a two-binding site model was used, resulting in determination of low affinity (K d ~ 500 nM) and high affinity (K d ~ 100 nM) binding sites. Hexavalent glycoclusters 9d and 9f also displayed affinity in the 300-500 nM range. The low affinity binding site displays affinity in the same range as methyl α-L-fucopyranoside (α-FucOMe) used as the monovalent ligand reference. The second binding site may result from change in lectin conformation (allosteric effect) or contact with aglycon of already bound ligand. This second binding site has higher affinity, which may be promising and should be further investigated. The glucosylated glycocluster 11a did not display any binding toward LecB as expected.

Kd1 (nM) β1 [b] N2 [a] -∆H2 (kJ/mol) -T∆S2 (kJ/mol) Kd2 (nM) β2 [b]

PDI-(EG3Glc)4 (11a)

No binding observed (see Supporting Information Figure S10) [a] Stoichiometry of binding defined as the number of glycoclusters per monomer of LecB. [b] Calculated using methyl α-L-fucopyranoside (α-FucOMe) as a monovalent reference with Kd = 0.43 µM. [START_REF] Sabin | [END_REF] Please do not adjust margins Please do not adjust margins

PDI

Fluorescence assays

The UV-vis absorption and fluorescence emission properties of representative PDI-based glycoclusters were tested in different solvents. Typical absorbance band of PDI monomer with multiple absorbance peaks was observed for all glyco-PDIs, 80 suggesting the successful introduction of the carbohydrate moieties to the perylene diimide scaffold (Figure 2a). A red shift in absorbance was observed for the glyco-PDIs in EtOH and water with respect to that in other solvents. This might be a result of aggregation (forming micelle-like architectures due to their amphiphilic property) of the compounds in these solvents to increase the intermolecular interactions of the hydrophobic perylene molecules. Typical PDI fluorescence emission was observed for the glyco-PDIs in DMF, DMSO and MeOH upon excitation at 410 nm (Figure 2b). However, the fluorescence emission was largely quenched in EtOH and H 2 O (the emission maxima also slightly red-shifted in EtOH), probably because of an aggregation-induced-quenching in these two solvents. This solvent effect is similar to that observed in the UV-vis spectra of the glyco-PDIs. The UV-vis absorption and fluorescence emission for all tested glyco-PDIs appeared to be similar irrespective of the valency and carbohydrate units clicked to PDI.

Cell adhesion assays

We have evaluated the ability of PDI-based glycoclusters to counteract P. aeruginosa infection in an in vitro model using the human pulmonary cell lines A549 as previously described. 45 A significant decrease of the cytotoxicity mediated by P. aeruginosa was observed with PDI-(EG 3 Gal) 4 (Figure 3A), PDI-(TzEGTzFuc) 4 , PDI-(TzEGTzFuc) 6 and PDI-(EG 3 Fuc) 4 (Figure 3C). This decrease of the cytotoxicity was not dependent on the linker arm or carbohydrate moieties, but rather on the valency of the glycoclusters. Moreover, an important decrease of bacterial adhesion in a range between 50-60% was observed with PDI-

Conclusions

The synthesis of PDI-based glycoclusters was readily performed from hexa-and tetra-propargylated cores through azide-alkyne "click" conjugation. These multivalent macromolecules were then evaluated as lectin ligands against LecA and LecB as the two proteins involved in the adhesion of Pseudomonas aeruginosa to host cells. A high affinity was measured by microcalorimetry (ITC) in both cases with K d values in the nanomolar range for both lectins. Although two valencies (4 or 6) and several linker arms were combined, the 8 glycoclusters performed almost equally as lectin ligands. These encouraging nanomolar affinities prompted for the evaluation of their anti-adhesive properties in a cell-based assay for adhesion to human epithelial cells. The PDI-based glycoclusters were demonstrated as potent anti-adhesive agents against Pseudomonas aeruginosa with only 30-40% residual adhesion observed with such glycoclusters. The fluorescence properties of the PDI core were then investigated by confocal microscopy on cellbacteria cultures. However, the red fluorescence signal of the tetravalent fucosylated PDI-based glycocluster selected for the confocal study was too weak to provide significant data. Altogether, the present study provides interesting data for another type of antiadhesive glycocluster against bacterial infection with a large aromatic PDI core.

Scheme 3 .

 3 Scheme 3. Synthesis of the PDI-based glycoclusters

  glucosylated PDI-based glycocluster 11a was first evaluated as a negative control. No binding could be detected with this ligand indicating that no nonspecific binding occurred with the lectin due to the PDI-based core. The tetravalent compounds 11b and 11e displayed the same binding toward LecA with K d values of nearly 200 nM. The stoichiometry for compounds 11b and 11e indicated the binding of three lectin monomers to each cluster. The four galactosides are therefore not all involved in lectin binding suggesting that steric hindrance would occur around the PDIbased core for a maximal binding of lectins. This steric hindrance was further observed with PDI-based glycoclusters 9b and 9e for which four contacts were evidenced (N = 0.25) while six galactose were available. The binding properties (K d values in the 150 nM range) were only barely improved in comparison to the tetravalent glycoclusters 11b and 11e (K d values in the 200 nM range).

Figure 1 .

 1 Figure 1. ITC binding studies. Left: Raw ITC data (top) for LecA (0.1 mM) obtained by injections of glycocluster 9b: PDI-(EG3-Gal)6 (0.24 mM) and the corresponding integrated titration curve (bottom) obtained with a one-site model. Right: Raw ITC data (top) for LecB (0.1 mM) obtained by

Figure 2 .

 2 Figure 2. (a) UV-vis absorption spectra of glyco-PDIs (50 μM) in different solvents. (b) Fluorescence emission spectra of glyco-PDIs (12.5 μM) in different solvents with excitation of 410 nm. All experiments have been repeated at least three times, and representative data are shown.

TzEGTzGal) 4 , 4 Figure 3 .

 443 Figure 3. Bacterial adhesion to A549 cells in the presence or absence of different PDI-based glycoclusters. All results are compared to the first column without adhesion inhibitors (PAO1, positive control): (A,B) galactosylated glycoclusters and (C,D) fucosylated glycoclusters. (A,C) LDH release following cell death, (B,D) inhibition of adhesion of wild-type PAO1. All experiments were performed in triplicate. Results are mean ± SEM (*p < 0.05). NS: not significant. * P < 0.05; ** P < 0.01; *** P < 0.001 compared to control. The tetra-fucosylated PDI-based glycocluster PDI-(EG 3 Fuc) 4 appeared as the most potent anti-adhesive candidate against P. aeruginosa. Its fluorescence properties have been used in a confocal microscopy experiment to investigate if the glycocluster would colocalize at the cell-bacteria interface or around swimming bacteria. Nevertheless, the fluorescence emission of GFP overlapped the fluorescence emission of PDI-(EG 3 Fuc) 4 . This characteristic of the compound does not allow to establish definitely if the PDI-(EG 3 Fuc) 4 colocalized with P. aeruginosa (Figure4). While bacteria and A549 cells can be readily identified through their green and blue fluorescence respectively, the red fluorescence of the PDI-(EG 3 Fuc) 4 was rather weak but could still be colocalized with bacteria when noticeable (top right hand corner), thus demonstrating the actual interaction of the glycoclusters with the bacteria in the cell culture medium.

Figure 4 .

 4 Figure 4. Confocal microscopy of A549 alveolar epithelial cells. Nuclei stained with 4′,6′-diamidino-2-phenylindole (DAPI) infected by PAO1 strain with a GFP plasmid at MOI 10 in the presence of PDI-(EG3Fuc)4 at 20 mM concentrations.

Table 1 .

 1 ITC measurements for binding of PDI-based glycoclusters 9b, 9e, 11a, 11b and 11e to LecA.

		Ligand		N [a]	-∆H	-T∆S	Kd	β [b]
					(kJ/mol)	(kJ/mol)	(nM)
		PDI-(EG3Glc)4 (11a)	No binding observed (see Supporting Information Figure S9)
		PDI-(EG3Gal)4 (11b)	0.34±0.03	71.2±1.6	31.1	202±68	346
		PDI-(TzEGTzGal)4 (11e)	0.41±0.02	79.2±0.6	41.0	208±19	336
		PDI-(EG3Gal)6 (9b)	0.25±0.01	113.9±0.7	75.3	172±5	406
		PDI-(TzEGTzGal)6 (9e)	0.26±0.01	104.7±2.7	65.6	143±3	489
		[a] Stoichiometry of binding defined as the number of glycoclusters per monomer of LecA. [b] Calculated
		using methyl β-D-galactopyranoside (β-GalOMe) as a monovalent reference with Kd = 70 µM.
	Ligand	N1 [a]	-∆H1	-T∆S1
			(kJ/mol)	(kJ/mol)

37

Table

2

. ITC measurements for binding of PDI-based glycoclusters 9d, 9f, 11a, 11d and 11f to LecB.

  Kd values. Fitting with 1-site model was used.

	-(EG3Fuc)4 (11d)	0.18±0.01	29.2±1.5	-12.4	527±122	0.8	0.14±0.01	103.2±2.4	59	137±47	3.1
	PDI-(TzEGTzFuc)4 (11f)	0.23±0.03	45.6±2.2	4	534±9	0.8	0.12±0.01	98.4±7.6	52.2	88±26	4.9
	PDI-(EG3Fuc)6 (9d)	0.19±0.01	79±3	43	473±2	0.9		Single site binding observed on titration curve
	PDI-(TzEGTzFuc)6 (9f)	0.25±0.01	44.5±2.8	5.2	325±141	1.3	Two sites binding observed on titration curve. Fitting with 2-site model
							could not provide 2

Organic & Biomolecular Chemistry

Published on 15 November 2017. Downloaded by CEA VALRHO on 16/11/2017 16:23:27.View Article Online

Published on 15 November 2017. Downloaded by CEA VALRHO on 16/11/2017 16:23:27. View Article Online DOI: 10.1039/C7OB02749D

Acknowledgements

The authors thank the Université Claude Bernard Lyon 1, Université Grenoble Alpes and the CNRS for financial support. Vaincre la Mucoviscidose is gratefully acknowledged for financial support under grant number RF20160501652. A.I. acknowledges support from the ANR projects Glyco@Alps (ANR-15-IDEX-02) and Labex ARCANE (ANR-11-LABX-003). N.G. and Y.P. are grateful to the Région Rhône-Alpes (ARC 1 Santé) and L.D. to China Scholarship Council for their PhD stipend. Dr A. Berlioz-Barbier and C. Duchamp are gratefully acknowledged for mass spectrometry analyses. D.-K.J. and X.-P.H. are grateful to the National Natural Science Foundation of China (Grant No. 21722801).

Conflicts of interest

There are no conflicts to declare. Please do not adjust margins Please do not adjust margins

ARTICLE


Cyril Routier

Lorenzo Vallan

Yohann Daguerre

Marta Juvany

Emin Istif

Daniele Mantione

Cyril Brochon

Georges Hadziioannou

Torgny Åsa Strand

Torgny Nasholm

Eric Cloutet

Eleni Pavlopoulou

Eleni Stavrinidou

Chitosan-modified polyethyleneimine nanoparticles for enhancing the carboxylation reaction and plants' CO2 uptake

Keywords: nanoparticles, CO2 capture, polyethyleneimine, chitosan, photosynthesis

During photosynthesis, plants capture carbon dioxide (CO2) and, by utilizing light energy, convert it to carbohydrates, which is the energy source of most heterotrophic organisms but also a source of fibers and biofuels. With the growing population, saturation of resources and the climate crisis, there is a great need to improve plants photosynthetic efficiency and increase crop yield. One of the major limiting steps of photosynthesis occurs during the initial carboxylation reaction for carbon fixation in the chloroplasts. During this step of the Calvin cycle, CO2 is converted to the organic acid 3-PGA (3-phosphoglycerate) catalyzed by the RuBisCO (Ribulose 1,5-bisphosphate carboxylase/oxygenase) enzyme. [START_REF] Martin | The Calvin Cycle and Its Regulation[END_REF][START_REF] Raines | The Calvin Cycle Revisited[END_REF] While RuBisCO has naturally poor affinity for CO2, its dual nature as oxygenase also leads to a competing reaction with O2, creating the wasteful pathway of photorespiration that reduces the Calvin cycle's efficiency by decreasing the photosynthetic carbon fixation of more than 25%. [START_REF] Sharkey | Estimating the Rate of Photorespiration in Leaves[END_REF][START_REF] Shi | Photorespiration: The Futile Cycle?[END_REF] Furthermore, to reach the RuBisCO enzyme, atmospheric CO2 has to follow a resistive pathway and diffuse through the leaf stomata, the intercellular space, the cell membrane, cytoplasm, and finally the chloroplast envelopes. [START_REF] Borovsky | Purification and Properties of Potato 1,4-Alpha-d-Glucan: 1,4-Alpha-d-Glucan 6-Alpha-(1,4-Alpha-Glucano)-Transferase. Evidence against a Dual Catalytic Function in Amylose-Branching Enzyme[END_REF][START_REF] Kaldenhoff | Mechanisms Underlying CO2 Diffusion in Leaves[END_REF] Thus, in order to sustain their growth, plants synthesize a large number of RuBisCO enzymes which requires high nitrogen use, a process that can be problematic for the plant development when nitrogen availability is limited.

Despite great progress in understanding RuBisCO biosynthesis and catalytic action, but also advances on plastid transformation [START_REF] Martin-Avila | Modifying Plant Photosynthesis and Growth via Simultaneous Chloroplast Transformation of Rubisco Large and Small Subunits[END_REF] and RuBisCO recombinant expression, [START_REF] Aigner | Plant RuBisCo Assembly in E. Coli with Five Chloroplast Chaperones Including BSD2[END_REF][START_REF] Lin | Small Subunits Can Determine Enzyme Kinetics of Tobacco Rubisco Expressed in Escherichia Coli[END_REF] improving RuBisCO's catalytic activity and specificity to CO2 in crop plants via genetic engineering remains a complex and challenging endeavor. [START_REF] Whitney | Advancing Our Understanding and Capacity to Engineer Nature's CO2-Sequestering Enzyme, Rubisco[END_REF][START_REF] Sharwood | Engineering Chloroplasts to Improve Rubisco Catalysis: Prospects for Translating Improvements into Food and Fiber Crops[END_REF] Apart from direct enzyme engineering, another promising route is to increase the amount of CO2 in the vicinity of RuBisCO via a CO2concentrating mechanism, [START_REF] Hanson | Towards Engineering Carboxysomes into C3 Plants[END_REF] either biochemically, such as C4 photosynthesis [START_REF] Von Caemmerer | The Development of C 4 Rice: Current Progress and Future Challenges[END_REF][START_REF] Bar-Even | Design and Analysis of Synthetic Carbon Fixation Pathways[END_REF] or through the expression of inorganic carbon transporters such as those for bicarbonate. [START_REF] Furbank | Improving Photosynthesis and Yield Potential in Cereal Crops by Targeted Genetic Manipulation: Prospects, Progress and Challenges[END_REF] Beyond genetic engineering, other approaches to enhance or augment plant functions based on materials science and nanotechnology have emerged. [START_REF] Buriak | Nano and Plants[END_REF][START_REF] Dufil | Plant Bioelectronics and Biohybrids: The Growing Contribution of Organic Electronic and Carbon-Based Materials[END_REF][START_REF] Lew | The Emergence of Plant Nanobionics and Living Plants as Technology[END_REF] Synthetic materials can be rationally designed with desired properties that are optimized independently of the biological environment.

However, it is challenging to introduce the synthetic materials into the biological organism in a seamless manner and incorporate the synthetic functionality into the biological machinery. In the literature, several examples at the proof-of-concept level exist. Functionalized nanotubes have been introduced to plants for in vivo detection of endogenous analytes such as H2O2 [START_REF] Lew | Real-Time Detection of Wound-Induced H2O2 Signalling Waves in Plants with Optical Nanosensors[END_REF] or to convert plants to environmental sensors for detecting exogenous analytes such as arsenic. [START_REF] Lew | Plant Nanobionic Sensors for Arsenic Detection[END_REF] Quantum dots have been used for in vivo targeted delivery of biochemicals, [START_REF] Santana | Targeted Delivery of Nanomaterials with Chemical Cargoes in Plants Enabled by a Biorecognition Motif[END_REF] while conjugated polymer nanoparticles were used for controlling plant functions via light stimulation. [START_REF] Tullii | A Prototypical Conjugated Polymer Regulating Signaling in Plants[END_REF] Our group, on the other hand, focused on the development of plant biohybrids using the biochemistry of plants to polymerize in vivo conducting polymers that can be used for energy storage. [START_REF] Stavrinidou | In Vivo Polymerization and Manufacturing of Wires and Supercapacitors in Plants[END_REF][START_REF] Dufil | Enzyme-Assisted in Vivo Polymerisation of Conjugated Oligomer Based Conductors[END_REF][START_REF] Parker | Biohybrid Plants with Electronic Roots via in Vivo Polymerization of Conjugated Oligomers[END_REF] Enhancing photosynthesis with synthetic materials has been attempted by mostly focusing on improving the light-dependent reactions. Semiconducting nanoparticles that localized in the proximity of the thylakoids have shown to improve the electron transport rates [START_REF] Kwak | Chloroplast-Selective Gene Delivery and Expression in Planta Using Chitosan-Complexed Single-Walled Carbon Nanotube Carriers[END_REF][START_REF] Chandra | High Throughput Electron Transfer from Carbon Dots to Chloroplast: A Rationale of Enhanced Photosynthesis[END_REF] and/or act as antennas, extending the light absorption spectrum of the plant. [START_REF] Xu | PVA-Coated Fluorescent Carbon Dot Nanocapsules as an Optical Amplifier for Enhanced Photosynthesis of Lettuce[END_REF][START_REF] Sai | Nontoxic Fluorescent Carbon Nanodot Serving as a Light Conversion Material in Plant for UV Light Utilization[END_REF] To date, leveraging nanomaterials for enhancing the CO2 uptake of plants has not been demonstrated.

Polyethyleneimine-based materials are promising candidates as they have an excellent CO2 capture ability as it was demonstrated by several studies focusing on the design of new CO2 absorbents for post-combustion processes. [START_REF] Shen | Polyethylenimine Applications in Carbon Dioxide Capture and Separation: From Theoretical Study to Experimental Work[END_REF][START_REF] Min | Oxidation-Stable Amine-Containing Adsorbents for Carbon Dioxide Capture[END_REF][START_REF] Xu | Novel Polyethylenimine-Modified Mesoporous Molecular Sieve of MCM-41 Type as High-Capacity Adsorbent for CO2 Capture[END_REF] The highly dense amines of polyethyleneimine (PEI) react with CO2, forming zwitterionic species such as carbamic acids and carbamates or, in the presence of water, the corresponding ammonium hydrogen carbonate salts. Furthermore, PEI or PEI-coated nanomaterials can cross plant cellular barriers and localize in the cytosol or organelles, as it was demonstrated both in vitro [START_REF] Zolghadrnasab | Ultrasound-Mediated Gene Delivery into Suspended Plant Cells Using Polyethyleneimine-Coated Mesoporous Silica Nanoparticles[END_REF][START_REF] Li | Transient Expression of Exogenous Gene into Plant Cell Mediated by PEI Nanovector[END_REF] and in vivo studies. Particularly, PEI has been used as carrier to deliver plasmids to plants for transient gene expression, [START_REF] Zhang | Expression of PEI-Coated Gold Nanoparticles Carrying Exogenous Gene in Periwinkle Mesophyll Cells and Its Practice in Huanglongbing Research[END_REF] as well as to deliver of small interfering RNA, siRNA, for gene silencing. [START_REF] Zhang | Gold-Nanocluster-Mediated Delivery of SiRNA to Intact Plant Cells for Efficient Gene Knockdown[END_REF] PEI's high positive charge condensates negatively charged polynucleotides and, at the same time, protects them from degradation, thus improving transfection efficiency. Yasumoto et al. demonstrated that small biogenic polyamines, such as piperazine and putrescine, can capture atmospheric CO2 which can then be used as a substrate for the carboxylation reaction. [START_REF] Yasumoto | Atmospheric CO2 Captured by Biogenic Polyamines Is Transferred as a Possible Substrate to Rubisco for the Carboxylation Reaction[END_REF] However, they have not studied their introduction to plants or their phytotoxicity. Other polyamines, such as PEI, can be highly toxic to plants depending on their molecular structure and concentration used. [START_REF] Rychter | Ecotoxicological Impact of Selected Polyethylenimines toward Their Potential Application as Nitrogen Fertilizers with Prolonged Activity[END_REF] In this work, we designed phytocompatible green-fluorescent PEI-based nanoparticles that can capture and transfer CO2 to RuBisCO for the carboxylation reaction. The phytotoxicity of the nanoparticles is inhibited by the presence of protective chitosan oligomers while the fluorophore functionalization enables CO2 loading-dependent fluorescence that does not overlap with the leaf autofluorescence. The nanoparticles were introduced to the plant via infiltration, and they spontaneously localized within the chloroplasts where photosynthesis takes place.

Results and Discussion

Branched PEI of 2 kDa molecular weight was chosen as an initial potential candidate for enhancing plants' CO2 uptake and carbon fixation. However, infiltration of branched PEI in Nicotiana Tabacum (tobacco) leaves revealed its toxicity for the plant as it will be discussed in detail in the next section. The synthesis of polymers containing both ethylenamide and glycoside units recently proved to be a promising strategy for the preparation of non-toxic gene vectors. [START_REF] Liu | New Poly(D-Glucaramidoamine)s Induce DNA Nanoparticle Formation and Efficient Gene Delivery into Mammalian Cells[END_REF][START_REF] Smith | Diblock Glycopolymers Promote Colloidal Stability of Polyplexes and Effective PDNA and SiRNA Delivery under Physiological Salt and Serum Conditions[END_REF][START_REF] Van Bruggen | Nonviral Gene Delivery with Cationic Glycopolymers[END_REF] Inspired by this approach, we modified 2 kDa PEI with chitosan oligomers.

Chitosan is obtained from the partial deacetylation of chitin, the constituent of several crustaceans and insects exoskeleton, and therefore it is the second most abundant polymer in nature after cellulose. As an emerging biocompatible, biodegradable and cheap material, chitosan and chitosan nanoparticles have found use in several fields, such as packaging, [START_REF] Oladzadabbasabadi | Recent Advances in Extraction, Modification, and Application of Chitosan in Packaging Industry[END_REF] gene delivery, [START_REF] Cao | Recent Advances in Chitosan-Based Carriers for Gene Delivery[END_REF] enzyme immobilization, [START_REF] Acet | O-Carboxymethyl Chitosan Schiff Base Complexes as Affinity Ligands for Immobilized Metal-Ion Affinity Chromatography of Lysozyme[END_REF] water treatment [START_REF] Olivera | Potential Applications of Cellulose and Chitosan Nanoparticles/Composites in Wastewater Treatment: A Review[END_REF] and agriculture. [START_REF] Malerba | Chitosan Effects on Plant Systems[END_REF] Chitosan based materials have been used in plant applications for enhancing seedling growth, 47 controlled release of nutrients [START_REF] Joshi | Controlled Release Action of Chitosan Nanoparticles to Improve Nutrient Use Efficiency[END_REF] and for antifungal and antibacterial purposes. [START_REF] Khayrova | Evaluation of Antibacterial and Antifungal Properties of Low Molecular Weight Chitosan Extracted from Hermetia Illucens Relative to Crab Chitosan[END_REF] First, chitosan oligomers (oligochitosan), of average 2-3 sugar units (Figure S1), were prepared by nitrous acid-promoted depolymerization of chitosan (MW 15 kDa), exploiting a previously reported procedure that allows to roughly control the size of the oligomers through the stoichiometry of the reaction. [START_REF] Moussa | Reducing-End "Clickable" Functionalizations of Chitosan Oligomers for the Synthesis of Chitosan-Based Diblock Copolymers[END_REF] In contrast with the chitosan polymer, these oligosaccharides are highly soluble in water even at neutral pH. Moreover, the cleavage of the glycosidic linkage during the depolymerization reaction forms a 2,5-anhydro-D-mannofuranose reducing-end, whose aldehyde group does not participate in intramolecular hemiacetals and can be exploited for further reactions. In the second step, we performed a reductive amination reaction between the amines of PEI and the aldehydes of oligochitosans. By forming a C-N covalent bond between PEI and oligochitosan, this synthetic route converts the primary amines of PEI to secondary amines and the secondary amines to tertiary amines. In this way, PEI-Chi was obtained, consisting of a PEI branched molecule covered by numerous oligochitosan functionalities (Figure 1A). It should be noted that this functionalization strategy does not consume PEI amine groups, thus not reducing the polymer's ability to capture CO2. PEI-Chi structure was investigated by nuclear magnetic resonance (NMR) spectroscopy. In the proton ( 1 H) and carbon ( 13 C) APT (Attached Proton Test) NMR spectra of PEI-Chi, the signals of the oligochitosan and PEI parts are present, whereas the aldehyde signal, located at 5.01 ppm in the 1 H spectrum of oligochitosan, has disappeared in the spectrum of PEI-Chi, demonstrating that the functionalization has taken place (Figure S2 to S6). Finally, we labeled PEI-Chi with a small amount of fluorescein isothiocyanate (FITC), whose fluorescence can be exploited for in vivo localization studies. By washing with ethanol and acetone, the free dye was completely removed, and we isolated the green-fluorescent PEI-Chi (gPEI-Chi). The NMR spectra of gPEI-Chi showed no significant differences from those of PEI-Chi , except for weak proton signals between 7.7 and 6.5 ppm, which suggest the presence of bound FITC (Figure S7 to S9). Infrared spectroscopy (IR) analysis of oligochitosan, PEI-Chi and gPEI-Chi showed the typical vibrational features of polysaccharides (Figure S10). In detail, the spectrum of gPEI-Chi shows wide and multiple absorption bands between 3600-3300 cm -1 , which correspond to O-H and N-H stretching, free or involved in hydrogen bonds. Between 2928 and 2868 cm -1 it is observed the C-H symmetric and asymmetric stretching of the glycans and PEI skeleton, while the bands at 1649 and 1560 cm -1 correspond to amide I and amide II vibrational modes. Even N-H bending probably contributes to the intensity of those bands, as features in the same area are present in the spectrum of bare PEI. The signals between 1414 and 1317 cm -1 were attributed to O-H bending, while C-OH and C-O-C stretching mainly contributes to the peaks located at 1149, 1069 and 1024 cm -1 . An additional contribution to the absorption in this range is given by the C-N stretching, which also appears in the spectrum of bare PEI at 1117 and 1042 cm -1 . [START_REF] Fernandes Queiroz | Does the Use of Chitosan Contribute to Oxalate Kidney Stone Formation?[END_REF] Atomic force microscopy (AFM) characterization of gPEI-Chi (Figure S11) showed that the sample is composed of individual nanoparticles with 2.0-2.5 nm size and of larger particles, which could consist of aggregates of about 5 nm. For comparison, the individual nanoparticles of bare PEI show an average size of 1.0-1.5 nm, therefore the larger size of gPEI-Chi is due to the oligochitosan functionalization. Size exclusion chromatography (SEC) also confirmed the increase of size, estimating a molecular weight of gPEI-Chi more than doubled with respect to bare PEI (Figure S12 and Table S13). In addition, the dispersity of gPEI-Chi is higher than that of bare PEI, as a result of the functionalization.

Next, we proceeded to characterize the phytotoxicity of gPEI-Chi and compared it with bare branched PEI. Both polymers were infiltrated in the apoplast of 6 weeks old Nicotiana Tabacum (tobacco) leaves (Figure 1B). To maintain cell homeostasis, the materials were dissolved in a typical aqueous buffer used for infiltration, consisting of 2-(Nmorpholino)ethanesulfonic acid (MES) (pH 5.6) and MgCl2, both in the concentration of 10 mM. [START_REF] Norkunas | Improving Agroinfiltration-Based Transient Gene Expression in Nicotiana Benthamiana[END_REF][START_REF] Goodin | PGD Vectors: Versatile Tools for the Expression of Green and Red Fluorescent Protein Fusions in Agroinfiltrated Plant Leaves[END_REF][START_REF] Liu | The Promoter of a Rice Glycine-Rich Protein Gene, Osgrp-2, Confers Vascular-Specific Expression in Transgenic Plants[END_REF] The plants were visually inspected for a minimum of seven days after infiltration.

As shown in Figure 1C, the leaves infiltrated with bare PEI stopped growing with clear toxicity effects. The infiltrated area dried out and became brittle, while the adjacent tissue exhibited chlorosis. Indeed, it has been found that PEI in PEI-functionalized nanotubes induce transcriptional reprogramming in plants, leading to stress, immune responses, and senescence that may lead to cell death and tissue damage depending on the concentration of PEI and structure. [START_REF] González-Grandío | Carbon Nanotube Biocompatibility in Plants Is Determined by Their Surface Chemistry[END_REF] In contrast, the plants infiltrated with gPEI-Chi did not show any visible sign of toxicity for more than a week and the infiltrated leaves continued to grow normally. To verify the tissue integrity of the infiltrated leaves, we performed histochemical staining with Propidium Iodide (PI). PI is a fluorescent dye that binds to plant cell wall and cannot cross the intact cell membrane (insets in Figure 1C). The area of leaves infiltrated with PEI could not be stained with PI as it dried out with no intact cells for efficient staining. The adjacent discolored tissue was stained with the dye entering the cells, indicating that the membranes were damaged and the cells dying. Instead, in the case of gPEI-Chi infiltrated leaves, the PI did not cross the cell membranes, not even very close to the infiltration point, indicating that the cells are intact and that the oligochitosan functionalization effectively improved the phytocompatibility of PEI.

com). (C) Comparison

of the effects of the infiltration of PEI (above) and PEI-Chi (below) on the tobacco leaf. PEI infiltration leads to a quick damage in the infiltrated area and around it while the infiltration of PEI-Chi does not show any visible sign of toxicity even one month after infiltration (Figure S14). The insets on the right show fluorescent imaging pictures obtained after staining of the samples with propidium iodide, a cationic dye that does not cross intact membranes but binds to cell walls, forming an outline of living cells. Scale bar: 50 μm.

The ability of PEI-Chi to bind carbon dioxide and store it in the form of hydrogen carbonate ions was studied by NMR spectroscopy (Figure 2A and2B). CO2 was bubbled in a deuterium oxide solution of PEI-Chi (30 mg/ml) and the carbon APT spectrum was recorded. A strong peak appears at 160 ppm after CO2 bubbling, corresponding to the HCO3 -anion. A smaller peak at 125 ppm corresponds instead to carbon dioxide, which is in equilibrium with the hydrogen carbonate. The pH of the solution shifted from 8-9 to 5-6 after CO2 bubbling, reflecting the conversion of amines into ammonium hydrogen carbonate groups. The APT experiment was repeated in absence of PEI-Chi under identical experimental conditions, finding that in pure D2O no signal of bicarbonate appeared after CO2 bubbling (Figure S15), thus demonstrating the essential role of PEI-Chi in storing CO2 as bicarbonate.

Then, we investigated the optical properties of gPEI-Chi with UV-Vis absorption and fluorescence emission spectroscopy (Figure 2C and2D). Fluorescence emission with a maximum at 518 nm was observed for λexc=490 nm, corresponding to the FITC fluorescence. This emission wavelength is particularly convenient for in vivo detection and localization studies as it does not interfere with the plant tissue autofluorescence.

Next, we studied the change in fluorescence due to the CO2 loading in solution. Interestingly, both absorption and fluorescence decreased significantly after CO2 loading. We hypothesize that prior to CO2 bubbling, fluorescein is present as a dianion where both the carboxylic acid and the phenol group are deprotonated due to the strongly basic environment generated by the abundant surrounding amines of the gPEI-Chi structure. When gPEI-Chi reacts with carbon dioxide, the pH of the solution decreases and the phenol is restored, causing a loss of emission intensity since the monoanion form has a lower quantum yield compared to the dianion form. [START_REF] Sjöback | Absorption and Fluorescence Properties of Fluorescein[END_REF] The decrease in fluorescence intensity due to CO2 loading was observable even when we used the infiltration buffer as the solvent, indicating that even local pH changes induce a change in the nanoparticles' fluorescence (Figure S16). The CO2-dependent fluorescence of gPEI-Chi is very promising for in vivo studies as it may enable monitoring CO2 concentration dynamics, providing insight on the CO2 uptake and transport within the leaf. After validating the ability of gPEI-Chi to uptake CO2, we proceeded to investigate whether the loaded CO2 can participate in biochemical reactions and specifically the carboxylation reaction. During this first phase of the Calvin cycle, CO2 reacts with the RuBisCO which converts ribulose-1,5-bisphosphate (RuBP) into 3-phosphoglycerate (3-PGA) (Figure 3A). [START_REF] Tommasi | The Mechanism of Rubisco Catalyzed Carboxylation Reaction: Chemical Aspects Involving Acid-Base Chemistry and Functioning of the Molecular Machine[END_REF] CO2 is electrostatically bound to PEI-Chi in the form of hydrogen carbonate which cannot react directly with RuBisCO. However, CO2 and hydrogen carbonate exist in thermodynamic equilibrium, and they dynamically interchange through reaction with water. We hypothesize that PEI-Chi will increase the hydrogen bicarbonate concentration and consequently the amount of CO2 available for RuBisCO. The ability of RuBisCO to use the CO2 delivered by PEI-Chi as substrate for the synthesis of 3-PGA in vitro was evaluated with an enzymatic assay adapting the method previously described by Yasumoto et al [START_REF] Yasumoto | Atmospheric CO2 Captured by Biogenic Polyamines Is Transferred as a Possible Substrate to Rubisco for the Carboxylation Reaction[END_REF] . We evaluated the activity of RuBisCO in five different aqueous solutions: gPEI-Chi in water (1 mg/ml) bubbled with N2 (gPEI-Chi-N2) or CO2 (gPEI-Chi-CO2), deionized water (DIw) bubbled with N2 (DIw-N2) or CO2 (DIw-CO2) as negative controls, and 2 mM NaHCO3 as positive control.

These solutions were added in a buffered solution with RuBisCO and were let to incubate for 10 minutes at room temperature in order to activate the enzyme. Next, RuBP was introduced, triggering the RuBisCO/CO2 catalyzed synthesis of 3-PGA. After 6 minutes, the reaction was stopped by addition of formic acid. Finally, the 3-PGA concentration in each solution was quantified with liquid chromatography-mass spectrometry (LC-MS) and normalized so that the amount of 3-PGA produced by the positive control NaHCO3 correspond to 100%. As shown in Figure 3B, the concentration of 3-PGA was 45% higher in the gPEI-Chi-CO2 solution compared to the gPEI-Chi-N2. Furthermore, the 3-PGA concentration in the gPEI-Chi-CO2 solution was 20% higher than in DIw-CO2, verifying that gPEI-Chi-CO2 can effectively increase the CO2 amount that reacts with RuBisCO via the hydrogen carbonate -CO2 conversion in water. When 2 mM NaHCO3 was used, the concentration of 3-PGA produced by RuBisCO was higher than that produced in the presence of gPEI-Chi, probably due to different concentration of captured CO2. However, in contrast to gPEI-Chi, NaHCO3 cannot be infiltrated in plants as it is highly toxic. Thus, these results suggest that gPEI-Chi could potentially improve the CO2 conversion of the plant by increasing CO2 concentration in the RuBisCO environment. After demonstrating the ability of gPEI-Chi to efficiently increase the CO2 that reacts with RuBisCO in vitro, we investigated the localization of the nanoparticles in the leaf tissue to gain insight on their potential for reaching the chloroplasts and increasing the CO2 amount at the photosynthetic sites in vivo. According to the lipid exchange envelope penetration (LEEP) model, infiltrated nanoparticles can spontaneously localize in the chloroplast depending on their size and surface charge. Nanoparticles must be within a specific range of size and zeta potential, to penetrate the negatively charged membrane of the chloroplasts and localize within their stroma. [START_REF] Wong | Lipid Exchange Envelope Penetration (LEEP) of Nanoparticles for Plant Engineering: A Universal Localization Mechanism[END_REF] We quantified the ζ-potential of the gPEI-Chi nanoparticles in the aqueous infiltration buffer (pH 5.6) and found a small shift from 35.8 mV before CO2 bubbling to 37.4 mV after. The size and ζ-potential distributions, and possibility of aggregation of the nanoparticles, at the pH used for infiltration, indicate that some of the nanoparticles may passively enter the chloroplasts according to the LEEP model (Figure S17). We infiltrated CO2 loaded gPEI-Chi (gPEI-Chi-CO2) in tobacco leaves and, using confocal microscopy, we observed that the nanoparticles mainly localized in the leaf apoplast but could also penetrate the mesophyll cells in the infiltrated area and reach the chloroplasts (Figure 4). The colocalization of the gPEI-Chi-CO2 nanoparticles and chloroplasts was confirmed by recording the emission spectra of the selected regions (Figure 4A-iv and 4B-iv) and by calculating the Pearson and Mander's colocalization coefficients (Figure S18). Furthermore, we could observe that the fluorescence of gPEI-Chi-CO2 followed the patterns of the discs stacks (grana) formed by the thylakoids inside the chloroplasts, which are the sites of photosynthetic reactions containing the chlorophyll which appear as the fluorescent red dots in our microscopy images (Figure 4C-iv). The confocal imaging confirms that the CO2-loaded nanoparticles can reach the photosynthetic sites and thus have the potential to increase the local CO2 concentration at the RuBisCO site and enhance the carboxylation reaction. In addition, we performed transmission electron microscopy (TEM) imaging of chloroplasts from plants infiltrated with gPEI-Chi-CO2 and found indications of the internalisation of the nanoparticles in the chloroplasts, especially in the starch grains and around the stromal thylakoids (Figure S19). We then assessed whether the unloaded nanoparticles can uptake CO2 after infiltration to the plant as this will enable the reloading of the nanoparticles with atmospheric CO2 and the formation of a sustainable CO2-concentrating mechanism in planta. We first infiltrated both N2-bubbled and CO2-loaded gPEI-Chi in the apoplast of tobacco leaves (Figure S20) and confirmed that gPEI-Chi fluorescence maintained its CO2 dependence in the in vivo environment i.e., gPEI-Chi fluorescence decreases when it is CO2-loaded. Next, to study the capacity of the nanoparticles to capture atmospheric CO2 while in the leaves, we infiltrated gPEI-Chi previously bubbled with N2 and then monitored the leaves fluorescence over time.

The plants were imaged one day after infiltration to let any excessive infiltrated solvent to evaporate. The infiltrated leaves, still attached to the plant, were placed in a CO2-rich environment under a fluorescent microscope (Figure 5A) and the change in fluorescence intensity was evaluated after 2 hours. As a control, we imaged infiltrated leaves that remained in atmospheric conditions. Figure 5B and 5C show that the fluorescence intensity decreased 54% for the leaves exposed to high CO2 concentration in comparison to decrease of 22% for the leaves let in atmospheric conditions, indicating that the nanoparticles in the apoplast of the plant are able to capture CO2. The small decrease in intensity in the control sample could also indicate that the nanoparticles can capture some CO2 when placed in atmospheric conditions, however, there is also the possibility of photobleaching of the dye after repetitive measurements at the same location. Furthermore, we measured the assimilation rate of infiltrated leaves at saturating light (A: the rate of CO2 uptake per unit time per unit leaf area (μmol of CO2 m -2 s - 1 )) and at different CO2 levels using the LI-6800 portable photosynthesis system and found that the infiltrated areas show higher CO2 uptake than controls 2 days after the infiltration (Figure S21). While these results are promising, more studies beyond the scope of this work are required to prove that the nanoparticles enhance plants photosynthetic activity in vivo.

Conclusions

In conclusion, we designed chitosan-modified polyethyleneimine nanoparticles, gPEI-Chi, which can capture atmospheric CO2 in the form of bicarbonate and increase the CO2 that reacts with RuBisCO enzyme enhancing the carboxylation reaction by 20% in in vitro assay. The gPEI-Chi nanoparticles were infiltrated in tobacco leaves and, even after an extended period of time, they did not cause any adverse effect to the plant as PEI's toxicity was obviated by the conjugated oligochitosan moieties. The CO2-loaded nanoparticles localized within the extracellular space, but most importantly, they could also pass cellular barriers and localize within the chloroplast organelles which are the sites of photosynthetic activity. Using the gPEI-Chi CO2-dependent fluorescence, we also demonstrated the nanoparticles' ability to uptake CO2 in vivo when the leaf is placed in a CO2-rich environment while, in future studies, the fluorescence modulation can be extended to monitor internal CO2 concentrations. Overall, our results show that gPEI-Chi is a promising nanobiotechnological approach for integrating a CO2-concentrating mechanism in plants. CO2-loaded nanoparticles could potentially increase the CO2 concentration in the vicinity of the RuBisCO and then be reloaded by atmospheric CO2 when in planta. Furthermore, plants possess the carbonic anhydrase enzyme that converts bicarbonate to CO2, which can potentially increase the contribution of the gPEI-Chi-loaded nanoparticles since the conversion of bicarbonate to CO2 will be accelerated. [START_REF] Floryszak-Wieczorek | The Multifunctional Face of Plant Carbonic Anhydrase[END_REF][START_REF] Dimario | Plant Carbonic Anhydrases: Structures, Locations, Evolution, and Physiological Roles[END_REF] However, to demonstrate the nanoparticles potential in planta, it will require their extended distribution throughout the plant's photosynthetic sites. Although leaf infiltration is commonly used for introducing materials to plant leaves, the materials are usually distributed close to the infiltration site. Using vacuum infiltration in a detached leaf, we observed an extended distribution of the nanoparticles in the whole leaf area (Figure S22). Still, this method cannot be applied to intact plants apart from small seedlings that can fit into a vacuum chamber. [START_REF] Chincinska | Leaf Infiltration in Plant Science: Old Method, New Possibilities[END_REF][START_REF] Koman | Emerging Investigator Series: Linking Nanoparticle Infiltration and Stomatal Dynamics for Plant Nanobionics[END_REF] Therefore, to evaluate the effect of the gPEI-Chi nanoparticles on RuBisCO activity and photosynthetic efficiency in vivo, another strategy for extended distribution of the nanoparticles to the plant photosynthetic sites has to be developed, such as uptake via root or foliage.

Experimental Section

Synthesis of oligochitosan: 3.0 g of chitosan (MW=15 kDa, deacetylation degree=85%, Polyscience) were added to 150 ml of ultrapure water. Then, 1.8 ml of HCl 37% were added and the mixture was stirred for 1 h until complete dissolution of the material. Afterwards, 800 mg of NaNO2 were added and immediate N2 gas evolution is observed. After 16 h, the pH was neutralized with AmberLite™ IRN78 OH. After filtration, the addition of acetone (300 ml) and centrifugation (8000 rpm, 5 min) allowed the recovery of the chitosan oligomers (oligochitosan). The material was redispersed in a minimum amount of water, precipitated again by acetone addition, and collected by centrifugation. Finally, it was washed with methanol and with acetone. After vacuum-drying, 1.8 g of oligochitosan in the form of a white powder was obtained.

Synthesis of PEI-Chi:

In a round-bottom flask, 200 mg of branched polyethyleneimine (MW=2 kDa, Polyscience) were dissolved in 30 ml of 1.5% acetic acid solution in water (v/v), then, 600 mg of oligochitosan were added and the stirring solution was heated up to 40°C. Three separate additions of sodium cyanoborohydride of 150 mg each were performed after 1 h, 6 h and 24 h. After three days, the solution was poured in a larger vessel, 1 ml of triethylamine was added first and then, after 10 minutes stirring, 60 ml of acetone, achieving the precipitation of PEI-Chi. The solid was recuperated by centrifugation (8000 rpm, 5 min), thus, it was redispersed in a minimum amount of water, precipitated again by acetone addition, and collected by centrifugation. Finally, it was washed with methanol and with acetone. After vacuum-drying, 540 mg of PEI-Chi in the form of a beige powder were obtained.

Functionalization of PEI-Chi with fluorescein 5-isothiocyanate: In a round-bottom flask, 130 mg of PEI-Chi were dispersed in 50 ml of water, followed by the addition of 0.3 ml of triethylamine. A second solution was prepared by dissolving 5 mg of fluorescein 5isothiocyanate in 10 ml of dimethyl sulfoxide. Then, the fluorescein solution was added slowly to the PEI-Chi solution while stirring. After one day, gPEI-Chi was precipitated by the addition of acetone. The product was collected by centrifugation and washed with a mixture of acetone/ethanol 2:1, where free fluorescein is highly soluble. When no more fluorescein was detected in the supernatant, the precipitate was washed with acetone only and, after vacuumdrying, 96 mg of gPEI-Chi in the form of an orange powder were obtained.

NMR spectra were recorded at 298 K with a 400.33 MHz Bruker Avance Spectrometer. Deuterated solvents and NMR tubes were purchased from Eurisotop. Branched polyethyleneimine (MW 2 kDa) and Chitosan (MW 15 kDa, deacetylation degree 85%) were purchased from Polyscience. Sodium cyanoborohydride (95%), sodium nitrite (97%), triethylamine (99.5%), acetic acid (glacial, 99%) were purchased from Sigma Aldrich.

Fluorescein 5-Isothiocyanate (isomer I, 97%) was purchased from TCI.

Size exclusion chromatography: Acetic acid aqueous solution was used as the mobile phase at a flow rate of 1.00 ml/min and glycerol as a flow rate marker. All polymers were injected (100 μl of solution) at a concentration of 5 mg/ml after filtration through a 0.45 μm pore-size membrane. The separation was carried out on two Agilent columns [2 × PLgel 5 μm Mixed C (300 × 7.5 mm)] and a guard column (PL gel 5 μm). Columns and detectors were maintained at 40°C. Relative Mw was determined thanks to a conventional calibration obtained with polyethylene glycol narrow standards.

UV-vis-NIR absorption spectra were obtained using an Agilent Cary 5000 instrument. Spectra were recorded between 200 nm and 800 nm, using a 10 mm side quartz cuvette. The absorption spectra were measured both before and after CO2 loading of the nanoparticles. The loading was carried out by gently bubbling carbon dioxide through a needle plunged in the polymer water solutions for five minutes.

Steady-state fluorescence measurements were carried out with a Horiba FluoroMax 4 spectrofluorimeter using a 10 mm side quartz cuvette. The excitation and emission spectra were measured both before and after CO2 loading of the nanoparticles The loading was carried out by gently bubbling carbon dioxide through a needle plunged in the polymer water solutions for five minutes.

Fourier Transform Infrared Spectroscopy (FTIR) measurements were performed on a Nicolet iS 5N FTIR spectrometer. Spectra were collected from 4000 to 400 cm -1 with the following settings: 16 scans per sample, and spectral resolution: 4 cm -1 .

Atomic force microscopy (AFM) (Dimension Fast Scan, Bruker) was used in a tapping mode using a silicon cantilever (Fastscan-A) with a nominal tip radius of 5 nm. The samples were prepared by drop-casting solution of polymer (0.1 mg/ml, 3000 pm, 120 s) on a mica substrate, which was subsequently dried at room temperature for 1 day and on a hot plate at 60°C for 5 min.

ζ-potential measurements were determined using a Zetasizer (NanoZS90, Malvern, United Kingdom). First, nanoparticles were suspended in morpholinoethanesulfonic acid (MES, Sigma Aldrich) (pH 5.6 adjusted with KOH) and the nanoparticle surface charge was determined using zeta potential measurement (averaged over 10 measurements each one having 20 runs).

Plant material and growth condition: Nicotiana Tabacum SR1 (tobacco) seeds were sown in the soil and grown in a growth chamber (Percival, CLF PlantClimatics GmBH, Wertingen, Germany) at 120 μmol.m -2 .s -1 light intensity, 60% relative humidity, and a 12/12 h day/night regime at 24°C during the day and 18°C during night-time. All experiments and measurements were performed using 6 weeks old plants.

Infiltration of Nicatiana Tabacum (tobacco):

Plants were illuminated with light of high intensity of ~500 μmol.m -2 .s -1 for 1 hour to make sure the stomata are open. Then, a small hole was made on the abaxial side of the tobacco leaf with the tip of a sterile 0.8x50 mm HENKE-JECT syringe needle (1 hole per leaf infiltrated). It is important to note that the hole does not completely puncture through the leaf but only passes the epidermal layer in order to facilitate the infiltration of the solution in the plant tissue. Once the hole was made, 200-300 µl of a 0.1 mg/mL gPEI-Chi solution in 10 mM MES, 10 mM MgCl2, pH 5.6 (or just 10mM MES, 10 mM MgCl2, pH 5.6 as control) was infiltrated from the hole with a 1 ml needleless syringe.

In order to control the applied pressure and not to destroy the plant tissue the researcher put their finger on the adaxial side of the leaf to maintain in place the syringe on the abaxial side of the leaf and then by gently pushing the plunger of the syringe to infiltrate the solution in the leaf. Any solution administered on the surface of the leaf was rinsed with water and dried gently with a piece of clean tissue. For microscopy experiments, the infiltrated plants were let to stand 24 to 48 h post-infiltration in normal growth conditions to allow remaining solvent to evaporate, while for toxicity experiments and staining they were let to stand in normal growth conditions up to 1 to 2 weeks.

Propidium Iodide staining was performed by cutting, with a clean razor blade, the leaf of interest and dipping in for 20 min in a 20 µmol aqueous solution of propidium iodide (Sigma Aldrich, St. Louis, MO, USA) in a 50 m conical tube (Sarstedt, Nümbrecht, Germany). The leaf was then removed from the solution and excess staining was removed by rinsing in water. The stained leaves were then directly used for microscopy.

Fluorescence microscopy: Widefield fluorescence imaging was performed with a Ni-E Upright Motorized Microscope (Nikon) equipped with a Zyla sCMOS camera (Andor Technology). The source of illumination was a Lambda DG-4 ultra-high speed wavelength switching illumination system (Sutter Instrument). The Propidium Iodide emission was detected between 580 and 650 nm using a TRITC filter, a 50x/0.60 TU PLAN ELWD objective and an exposure time of 1s. The gPEI-Chi emission was detected between 510 and 560 nm using a FITC filter, a 50x/0.60 TU PLAN ELWD objective and an exposure time of 200 ms. The images were treated using the software NIS-Elements (Nikon) and ImageJ. The samples used for microscopy were prepared by cutting a small leaf section of the infiltrated or stained leaf tissue and inserting it between a glass slide (VWR international GmbH, Darmstadt, Germany) and Menzel-Gläzer coverslip (VWR international GmbH, Darmstadt, Germany) using a 30% glycerol aqueous solution as mounting medium to keep the sample moisturized during imaging.

RuBisCO assays: the enzymatic assays for determining the activity of RuBisCO were realized following the method described by Yasumoto et al.. [START_REF] Yasumoto | Atmospheric CO2 Captured by Biogenic Polyamines Is Transferred as a Possible Substrate to Rubisco for the Carboxylation Reaction[END_REF] The chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA).

3-Phosphoglyceric acid determination by LC-MS was performed at the Swedish Metabolomics

Center in Umeå, Sweden. Before the analysis, 200 µl of freeze-dried sample was re-suspended in 50 + 50 µl methanol and water. The chromatographic separation was performed on an Agilent 1290 Infinity UHPLC-system (Agilent Technologies, Waldbronn, Germany). 1 μl of each sample were injected onto an Atlantis Premier BEH-HILIC, 1.7 µm, 2.1 x 50 mm, held at 40°C. The gradient elution buffers were composed of (A) 10mmol/L ammonium acetate in water of pH 9 and (B) 10 mmol/l ammonium acetate in 10/90 water/acetonitrile (v/v) of pH 9 (pH of aqueous buffer before mixing with methanol). Ammonium hydroxide was used to adjust pH of the mobile phase. A final 5 μM concentration of methylenediphosphonic acid was spiked into the solvents for the analysis. The flow rate was 0.35 ml/minute, and the compounds were eluted with a linear gradient consisting of 95-30% B over 7 minutes, B was held at 30% for 2 minutes; B was increased to 95% for 0.5 minutes and held for 1.5 minutes, then the flow-rate was increased to 0.6 ml.min -1 for 1.5 minutes; these conditions were held for 0.2 minutes, after which the flow-rate was reduced to 0.35 ml.min -1 for 0.1 minutes before the next injection.

The compounds were detected with an Agilent 6546 Q-TOF mass spectrometer equipped with a jet stream electrospray ion source operating in negative ion mode. A reference interface was connected for accurate mass measurements; the reference ions purine (4 μM) and HP-0921 (Hexakis(1H, 1H, 3H-tetrafluoropropoxy)phosphazine) (1 μM) were infused directly into the MS at a flow rate of 0.05 ml.min -1 for internal calibration, and the monitored ions were purine m/z 119.03632; HP-0921 m/z 966.000725. The gas temperature was set to 150°C, the drying gas flow to 8 l.min -1 and the nebulizer pressure 35 psig. The sheath gas temp was set to 350°C and the sheath gas flow 11 l.min -1 . The capillary voltage was set to 4000 V. The nozzle voltage was 300 V. The fragmentor voltage was 120 V, the skimmer 65 V and the OCT 1 RF Vpp 750 V. The collision energy was set to 0 V. The m/z range was 70-1700, and data was collected in centroid mode with an acquisition rate of 4 scans.s -1 (1977 transients/spectrum). Data were processed and analysed using MassHunter Qualitative Analysis, Quantitative Analysis (QqQ; Agilent) and Excel (Microsoft) software.

Methanol, HPLC-grade was obtained from Fischer Scientific (Waltham, MA, USA) Acetonitrile, HPLC-grade was obtained from Fischer Scientific (Waltham, MA, USA) 2-

Figure 1 :

 1 Figure 1: Phytocompatible infiltration of PEI-Chi in Nicotiana Tabacum (tobacco) plants. (A) Functionalization of PEI with oligochitosan by reductive amination. (B) Scheme of the infiltration method: a solution contained in a needleless syringe is infiltrated in the abaxial part of a leaf by gently applying pressure. The solution is pushed in the intercellular space existing

Figure 2 .

 2 Figure 2. Functionalization of PEI-Chi for fluorescent detection in vivo and CO2 uptake emission dependence. (A) Scheme of the ammonium bicarbonate formation from the reaction between amine and CO2 in aqueous environment (PEI contains primary, secondary, and tertiary amine and all three kinds can react with CO2. We here used primary amines only as a showcase). (B) APT NMR spectra of PEI-Chi in D2O before and after bubbling carbon dioxide. After bubbling, strong peaks corresponding to hydrogen carbonate and CO2 appear. (C) Absorption and (D) excitation and emission spectra of gPEI-Chi (0.05 mg/ml) in water before and after saturating the solution with carbon dioxide.

Figure 3 .

 3 Figure 3. Evaluation of 3-phosphoglycerate production using gPEI-Chi as a CO2-giver to activate RuBisCO. (A) Simplified Calvin cycle showing the importance of RuBisCO as catalyst for the conversion of RuBP to 3-PGA using CO2 (created with BioRender.com). (B) Either CO2 gas or N2 gas, to remove CO2, was bubbled for 30 minutes in 10 ml of either 1 mg/ml of gPEI-Chi or distilled water. The solutions loaded with CO2 were then used as substrates for the carboxylation reaction between RuBP and CO2 catalyzed by RuBisCO and compared to their equivalent without CO2 treatment and to NaHCO3 (2 mM) as a carbon source for positive control. The amount of 3-PGA produced by the reaction was then analyzed by LC-MS. Bars indicate the standard errors (n = 3). Treatments not labeled by the same letter are significantly different. Tukey-Kramer HSD, P-value < 0,001. JMP Pro software was used to run the analysis.

Figure 4 .

 4 Figure 4. Confocal microscopy imaging to evaluate the chloroplast accessibility to gPEI-Chi-CO2. Imaging of column (A) a control tobacco leaf, column (B) and (C) a leaf infiltrated with gPEI-Chi-CO2. The top red panel (i) shows the autofluorescence of the chlorophyll, the middle green panel (ii) shows the gPEI-Chi-CO2 fluorescence, and the panel (iii) is a merging of the two previous. The graphs (A-iv) and (B-iv) are emission spectra of the circles in the merged images, obtained with a monochromator, and serve to evaluate the spatial distribution of the gPEI-Chi-CO2 nanoparticles in respect to the chlorophyll. The schematic (C-iv) represents the structure of a chloroplasts containing thylakoids forming stacks of disks (grana)

Figure 5 .

 5 Figure 5. In vivo CO2 uptake of gPEI-Chi. (A) Scheme of the experimental setup where a leaf, still attached to its plant, is isolated in a CO2 incubator and imaged under fluorescence microscopy (created with BioRender.com). (B) Fluorescence microscopy of a Nicotiana Tabacum (tobacco) leaf infiltrated with gPEI-Chi, with or without incubation in a CO2-rich environment for 2 hours. Scale bar: 50 μm. (C) Fluorescence intensity evolution under atmospheric CO2 conditions or in a CO2-rich environment. Bars indicate the standard errors (n = 4 plants)

Acknowledgments

This work was supported by the European Union's Horizon 2020 research and innovation program under Grant Agreement No. 800926 (FET-OPEN-HyPhOE) and by the Swedish Research Council (VR-2017-04910). Additional funding was provided by the Swedish Government Strategic Research Area in Materials Science on Advanced Functional Materials at Linköping University (Faculty Grant SFO-Mat-LiU No. 2009-00971). Swedish Metabolomics Centre, Umeå, Sweden (www.swedishmetabolomicscentre.se) is acknowledged for the LC-MS determination of 3-phosphoglyceric acid. The authors acknowledge the facilities and technical assistance of the Umeå Core Facility Electron Microscopy (UCEM) at the Chemical Biological Centre (KBC), Umeå University, a part of the National Microscopy Infrastructure NMI (VR-RFI 2019-00217). The authors thank Dr. C. Hermida-Carrera from the Department of Plant Physiology, Umeå Plant Science Centre, Sweden, for her assistance in establishing the assimilation-CO2 response curves (A-Ci).

Propanol, HPLC-grade was obtained from VWR (Radnor, PA, USA) H2O, Milli-Q, Ammonia solution, 35% HPLC-grade was obtained from Fischer Scientific (Waltham, MA, USA).

Reference and tuning standards: Purine, 4 μM, Agilent Technologies (Santa Clara, CA, USA) HP-0921 (Hexakis(1H, 1H, 3H-tetrafluoropropoxy)phosphazine), 1 μM, Agilent Technologies We evaluated the contribution of the polymer at each wavelength using the LSM lambda scan spectrophotometer, and compared images of the green and red channels by using both the confocal and the Airyscan2 super-resolution modes. We captured images in 16 bits with a bidirectional acquisition, an averaging of 4 images, an image size of 1863 x 1863 pixels (pixel size = 0.043 µm) and a zoom of 1.3. Both the fluoresceine isocyanate (FITC) and chlorophyll were excited using a 488 nm laser at an intensity of 0.4% with a pinhole of 5 AU, a detector gain at 700 V and a pixel time of 1.13 µs. The Alexa Fluorophore 520 (emission wavelength 520 nm) emission filter was used for the detection of gPEI-Chi, and the Alexa Fluorophore 700 (emission wavelength 719 nm) emission filter was used for the detection of the chlorophyll.

The samples used were prepared by cutting a small leaf section of tobacco tissue infiltrated with gPEI-Chi preliminary loaded with CO2 (gPEI-Chi-CO2) and inserting it between a glass slide (VWR international GmbH, Darmstadt, Germany) and Menzel-Gläzer coverslip (VWR international GmbH, Darmstadt, Germany) using a 30% glycerol aqueous solution as mounting medium to keep the sample moisturized during imaging. We gently removed via capillarity the extra mounting medium between the glass slide and the coverslip in order to create a suction effect that allows to turn the samples upside down for imaging with the inverted microscope.

CO2 incubation: 24 to 48 h post-infiltration with gPEI-Chi, the plants were fixed upside down on the fluorescent microscope platform and the infiltrated leaf was isolated in a closed petri dish having an entry tube for CO2 and a small hole allowing air circulation. The fluorescence of gPEI-Chi was first imaged in atmospheric conditions, then, we filled the petri dish with CO2 (coming from a CO2 tank) for 2 hours and captured the fluorescence again.

Conflict of Interest

The authors declare no conflict of interest.

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