Evaluation of cationic peptide-based nanogels as delivery systems for negatively charged molecules: a formulative study

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Hamley, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7158671/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Oct, 2025 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract Peptide-based nanogels (NGs) represent a cutting-edge class of nanoscale drug delivery systems. Due to their structural properties, NGs platforms can encapsulate and protect therapeutic agents ( e.g. peptides, proteins, and nucleic acids), while allowing for controlled and stimuli-responsive release. These pharmacokinetic/pharmacodynamic features can be specifically modified including peptide functional elements as NGs component. This study explores the formulation, decoration strategies, and structural properties of NGs derived from mixed hydrogel matrices of Fmoc-diphenylalanine (Fmoc-FF) with cationic amphiphilic peptides (CAPs). CAPs, composed by cationic hexapeptide (GK)3 sequence decorated at its N -terminus with alkyl chain, were found able to confer a net positive charge to Fmoc-FF NGs. Fmoc-FF/C16-(GK)3 and Fmoc-FF/C18-(GK)3 NGs were obtained using polysorbate 80 (TWEEN ® 80) and sorbitan monostearate 80 (SPAN ® 80) colloidal stabilizing surfactants and characterized in terms of size, secondary structure, superficial charge and shelf stability by Dynamic Light Scattering (DLS), Circular Dichroism (CD), Fourier Transform Infrared (FTIR) and SAXS technique. Different formulative routes were applied and mutually compared to encapsulate/adsorb Alexa Fluor TM 430 (succinimidyl ester), used as model of an anionic, pharmaceutical agent. In vitro experiments demonstrate a good cytocompatibility of these systems and the release of Alexa Fluor TM 430 was also evaluated. Biological sciences/Biotechnology Physical sciences/Chemistry Biological sciences/Drug discovery Physical sciences/Nanoscience and technology peptide nanogels peptide-based nanoplatforms drug delivery anionic active pharmaceutical ingredients formulation study Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Supramolecular nanostructures like micelles [ 1 , 2 ] liposomes [ 3 , 4 ], and polymeric nanoparticles [ 5 , 6 ] have been extensively investigated as innovative platforms for the delivery of a wide range of active pharmaceutical ingredients (APIs). The success of these nanosystems is related to their capability to improve APIs bioavalability and the biodisponibility, by changing its pharmacokinetic and pharmacodynamic profiles, while also offering physical protection against in vivo degradation[ 7 , 8 ] However, the inherent anionic nature of liposomes and polymeric micelles generally hinders the encapsulation of negatively charged molecules, such as nucleic acid-based biopharmaceuticals (e.g., siRNA, miRNA, and gene-silencing agents) [ 9 ]. As a result, these APIs are often adsorbed onto the outer surface of cationic liposomes, forming lipoplexes [ 10 ], which do not shield them from enzymatic degradation in vivo. In this context, the development of alternative nanostructures capable of efficiently deliver biotechnological active principles is of particular interest.[ 11 , 12 ] Hydrogel (HG) nanoparticles, also named nanogels (NGs), have been recently proposed as potential nanosized injectable delivery systems for negative molecules.[ 13 ] Structurally, NGs are formed of an interior hydrogel-like network ( core ) stabilized by an external surfactant coating ( shell ). This supramolecular structuration allows preserving the inner interconnected hydrogelated network they derive from. [ 14 , 15 ] NGs can be prepared for mechanical submicronization and stabilization of the macroscopic HG by top-down approach. [ 14 , 16 ] Recently a small library of multicomponent peptide-based HGs, prepared by mixing in a 1/1 mol/mol ratio Fmoc-diphenylalanine (Fmoc-FF, Fmoc = fluorenylmethoxycarbonyl) gelator with cationic amphiphilic peptides (CAPs), was described. [ 17 ] Primary peptide sequences were designed as functional charge modifiers of anionic Fmoc-FF matrices, introducing positive charges for matrix/API electrostatic interaction. A common cationic hexapeptide (GK)3 sequence, alternating glycine (G) and lysine (K) residues, was decorated at its N-terminus with an alkyl chain, differing for their length from C8 to C18. The multiscale structural and morphological characterization of these matrices highlighted the guiding gelation rule of Fmoc-FF and a partial immobilization of CAPs in gel matrix. [ 17 ] Applying a top-down strategy, Fmoc-FF/C16-(GK)3 and Fmoc-FF/C18-(GK)3 NGs externally stabilized by polysorbate 80 (TWEEN®80, Polyethylene glycol sorbitan monooleate) and sorbitan monostearate 80 (SPAN®80, Sorbitan monooleate) have been here formulated (see Fig. 1 ) and fully characterized. In particular, the NGs size, superficial charge and shelf stability were assessed by Dynamic Light Scattering (DLS). Circular Dichroism (CD), and Fourier Transform Infrared (FTIR) spectroscopies were carried out to visualize homologies or differences in secondary structure of NGs and the corresponding HGs. [ 17 ] Moreover, the NGs organization has been further investigated by Small-angle X-ray scattering (SAXS) technique. Different formative routes were applied to decorate Fmoc-FF/C16-(GK)3 and Fmoc-FF/C18-(GK)3 formulations with the negatively charged AlexaFluor TM 430 (succinimidyl ester), used as model molecule. AlexaFluor TM 430 was alternatively encapsulated into the NG core or adsorbed into surfactant shell. The comparison of the encapsulation efficiency percentage (EE%) and the encapsulation ratio percentage (ER%), obtained with the different procedures and different amount of Alexa Fluor TM 430, allows identifying a general preparation procedure, reinforcing the proposing rule of NGs as potential platforms for negatively charged APIs delivery. Results and Discussion Formulation of empty NGs Fmoc-FF have gained attention for its ability to form well-ordered nanostructures (like micelles and HGs) through non-covalent interactions, driven by hydrogen bonding and electrostatic forces. Fmoc-FF was also explored in combination with different molecular elements ( eg. other peptide sequences, proteins or polymers) for developing multicomponent systems. [ 18 – 21 ] It was observed that the inclusion of both hydrophilic and hydrophobic components into Fmoc-FF HG is perfectly compatible with the common gelation procedure of the “ solvent-switch ”. This gelation route involves dissolving the dipeptide monomers at a high concentration (100 mg/mL) in an organic solvent (DMSO, ethanol, or 1,1,1,3,3,3- hexafluoro-isopropanol), followed by stock dilution with water. Dilution triggers gel formation, creating a three-component system (peptide/solvent/water).[ 22 , 23 ] Using this approach, Fmoc-FF/C n -(GK)3 1/1 mol/mol gel matrices were formulated and analyzed ( n from 8 to 18 carbon atoms). The corresponding NGs were formulated according to the top-down procedure, previously optimized for preparation of pure Fmoc-FF NG.[ 14 ] This protocol is based on the submicronization of the corresponding macroscopic HG by homogenization and tip-sonication. The resulting nanoparticles are composed by an inner core, externally stabilized by a TWEEN® 80 and SPAN® 80 surfactant coating, added at 53/47 w/w (total number of mol = 3.0 ∙10 − 5 mol) in order to have a hydrophilic-lipophilic balance (HLB) of 10.[ 14 ] NGs preparation were initially studied by Dynamic Light Scattering (DLS) analysis at θ = 173°. Appling the top-down protocol, capric (C10), lauric (C12) and myristic (C14)-(GK)3 containing NGs resulted as unstable and polydisperse preparations (polydispersity index PDI > 0.350), with main diameter ( d ) dimensions incompatible with potential injectability (see Figure S1 as exemplificative example). This evidence suggests that C10-, C12- and C14 derivatives alter the total HLB, modify the TWEEN® /SPAN® surfactant and stabilize performance. On the contrary, Fmoc-FF/C16-(GK)3 and Fmoc-FF/C18-(GK)3 shows a monomodal DLS profile (Fig. 2 a), indicating the presence of homogeneous aggregate population (PDI = 0.202 and 0.243 for C16 and C18-(GK)3 containing systems, respectively). These results suggest that the length of the CAPs alkyl chain can affect the formation of the NG. According to this suggestion, we previously observed that macroscopical matrices of C10-, C12-, and C14- peptides led to the formation of more rigid HGs, meanwhile alkyl chain elongation causes a notable reduction in the storage modulus (G′ = 794 Pa for C16 and 73 Pa for C18). This difference may have a rule in micronization step, favoring NGs formation for palmitic (C16) and stearic (C18) mixed matrices. Apparent translational diffusion coefficients are reported in Table 1 . As well-known at infinite dilution, the mean diameter ( D ), may be evaluated by using the Stokes–Einstein–Sutherland (SES) [ 24 ]. The mean diameter of Fmoc-FF/C16-(GK)3 and Fmoc-FF/C18-(GK)3 is 139 and 102 nm, respectively. These values are smaller than the analogue one of pure Fmoc-FF (mean diameter ~ 187 nm), advising that the intercalation of the amphiphilic peptides into the Fmoc-FF causes a contraction of the nanoparticles. No significant variation in formulation size can be detected up to one month (Fig. 2 b and 2 c), thus indicating good stability features. This evidence can be correlated to the positive values found for the zeta potential ( ζ ) of both the NGs systems (+ 38 and + 51 mV for C16- and C18-derivatives, respectively), preventing flocculation/sedimentation for the lyophilic colloids NG In vitro cytotoxicity assay In order to assess the potential capability of these NGs to be used in vivo as delivery systems, their cytotoxicity profiles were evaluated in vitro on HEK-293 cells. Cells were incubated for 72 h at 37°C with different concentrations of NGs (NG formulation was diluted in the range 1:160 ÷ 1:320000 corresponding to a 23.3 µmol/L ÷ 11.68 nmol/L total peptide concentration range). The cell viability was evaluated by MTT assay, which typically evaluates metabolically active cells via MTT reduction to formazan by mitochondrial dehydrogenases. As clearly shown in Figure S2, no significant toxicity was observed for NGs at all the tested concentrations. [ 17 ] Structural characterization of NGs: CD, FT-IR and SAXS To deeply inspect the formulation features, the secondary structure of NGs was evaluated by CD spectroscopy. Being optically active, peptides and peptide aggregates exhibit disparity in circular polarized light absorption. Consequently, CD spectra of peptides and proteins are predominantly related to amide group excitation transitions, as function of secondary structure. From the inspection of Fig. 3 A and 3 B, it can be observed that both the NGs spectra are characterized by two leading signals. The first positive band, located in the 220–230 nm region (218 and 222 nm for C16-(GK)3 and 220 nm for C18-(GK)3 HG l and NG, respectively), is generally related to peptides β-sheet suprastructuration. [ 25 , 26 ] The dichroic inversion of this signal, coupled with its increased intensity in NGs formulation with respect to HGs ones, can be probably attributed to a global different chiral environment in the NG as compared with the HG. However, it is worth noting that this tri-dimensional surrounding does not alter the fundamental-sheet organization of the NG. Instead, the second broad peak, corresponding to the distinctive signature of the Fmoc moiety, appears slightly red-shifted in the NG formulations (269 and 268 nm for C16-(GK)3 and C18-(GK)3, respectively) respect to the corresponding HG ones (265 and 264 nm).[ 17 ] The bathochromic effect can be a consequence of a different dielectric constant in the NG core covered by surfactants with respect to the macroscopic HG. It is worthwhile noting that the two samples are also different in their physical state, and thus the scattering phenomena could also contribute to the shifted behavior [ 27 ]. The Fourier-transform infrared spectroscopy (FT-IR) analysis carried out on samples supported the CD secondary structure assignment. The FT-IR spectra of NGs were deconvoluted in absorbance in Amide I region (1700 − 1600 cm − 1 , Fig. 3 c), that, controlled by C-O and C-N stretching vibrations, is the most sensitive spectral region for secondary structure of peptides and proteins. [ 28 ] The presence of a prevalent C = O stretching band at ∼1640 cm − 1 reinforces the β-sheet organization, as the weighted percentages of each secondary structure functions ( Table S1 ). As expected, no significative differences can be detected between C16- and C18-(GK)3 containing formulations. Peptide-based NGs were further characterized by SAXS measurements. These provide information on the form factor, i.e. on the shape and dimensions of the fibrils that underpin the NG structures. The SAXS data for the Fmoc-FF NG, or the mixed NGs containing alternatively C16-(GK)3 or C18-(GK)3 show similar behavior, with an intensity scaling at low wavenumber q , I ~ q − 2 , which is characteristic of layered structures [ 29 ]. The data can be well fitted using a model for nanotape structures based on bilayers (solid lines shown in Fig. 4 , fit parameters listed in SI Table S2 ). The bilayer electron density profile is represented by three Gaussian functions, two representing the ‘headgroups’ and the central one representing the inner hydrophobic layer [ 30 ]. Here, the inner part of the bilayer will comprise the Fmoc units as well as potentially the phenylalanine residues, while the ‘headgroups’ are the charged C-termini and the lysine residues in the case of the NGs containing the cationic lipopeptides. The effective half thickness of the bilayer is reduced from 77.7 Å for the Fmoc-FF NG to 42.0 Å or 31.3 Å for the mixed NGs with C16-(GK)3 or C18-(GK)3 respectively. The data suggest that the addition of lipopeptides leads to better defined and thinner bilayers, whereas Fmoc-FF nanotapes seem to comprise multilayers since the thickness from the SAXS data fitting is much larger than two molecular lengths. The data for Fmoc-FF show a less well- defined high q form factor maximum which was modelled by incorporating a contribution to the fitted SAXS data of a monomer form factor represented as a generalized Gaussian coil with radius of gyration 10 Å, this was not required to fit the data for the two NGs containing lipopeptides. The SAXS data thus indicate that all Fmoc-FF NGs contain nanotape fibrils, and that these are better defined in the NGs containing the lipopeptides, i.e. the molecules are fully aggregated, and the layer thickness corresponds to interdigitated bilayers, considering the molecular length of the lipopeptides. Formulation and characterization of Alexa Fluor TM 430 filled NGs The NGs formulation were evaluated about their potential use as drug delivery platforms. Alexa Fluor TM 430, was selected as model dye for NGs analysis, due to its water-soluble anionic nature and its spectroscopic features (λ abs = 430 nm). The versatility formulative procedure, as NGs structure, allows a different dye localization (Fig. 5 ). The dye was alternatively included (encapsulation route) in mixed peptide gel matrix or added as functional surface dye via electrostatic interactions (adsorption strategy). Increasing amounts of Alexa Fluor TM 430 (0.05 ÷ 3.0 mmol/L concentration range) were alternatively encapsulated into the core (formulations indicated as NG A ÷ NG E ) or adsorbed onto the NGs shell (formulations indicated as NG F ÷ NG K ). The procedure of encapsulation was easily achieved by preparing the corresponding Alexa Fluor TM 430 filled HG (0.05 ÷ 3.0 mmol/L concentration range), which successively underwent top-down protocol. No syneresis effects were evidenced for both C16 and C18 containing dye matrices, thus suggesting an efficient localization into the fibrillary gel network for all the tested concentrations. Self-supporting properties for all the samples were verified via inverted test tube (data not shown). As well known, the inclusion of additional chemical entities in HGs matrices can deeply alter the gel mechanical response as consequence of interactions, solubility, molecular weight in relationship with supramolecular gel mesh size.[ 31 ] Previous rheological studies on empty 0.5 wt % HGs of Fmoc-FF/C n -(GK)3 allowed to estimate an elastic modulus (G’) of 794 and 73 Pa for Fmoc-FF/C16-(GK)3 and Fmoc-FF/C18-(GK)3 HGsmatrices, respectively. The inclusion of the Alexa Fluor TM 430 in the HGs causes a significative decrease G’ value < 10 Pa for all the tested concentrations without a specific trend, as deducible from dynamic oscillation strain testes ( Figure S3 , Table 1 ). These evicedences indicate an impact on the rheological features of the matrices, conduting to a viscous-like behaviour. More specifically, it seems that the inclusion of Alexa Fluor TM 430 induces an increase of the mash size via repulsion interaction between supramolecular fibers, with a consequent decrease of G’ according to the theory of rubber elasticity [ 32 ]. All these matrices were efficiently transformed in the correspetive NGs preparations. Table 1 Rheological parameters of HGs encapsulating different dye concentrations. G’ and G’’ are extrapolated at γ = 1.26% in the linear viscoelastic region (LVR). Tanδ = G’’/G’ [AlexaFluor TM 430] (mmol/L) G’ (Pa) G’’ (Pa) tanδ 0.05 4.79 3.01 0.63 0.25 3.22 2.91 0.90 0.50 2.16 1.30 0.60 0.75 1.56 1.65 1.06 1.0 3.71 2.07 0.56 3.0 1.35 1.50 1.11 An AlexaFluor TM 430 absorption route was also performed, supposing a favourable dye-shell electrostatic interaction pathway. To achieve absorption (0.05 ÷ 3.00 mmol/L concentration range), preformed empty NG formulations were incubated with lyophilized dye powders overnight under stirring at room temperature. The purification from free dye was performed via SEC (size-exclusion chromatography), with consequent analytical quantification of unretained AlexaFluor TM 430 via UV-vis spectroscopy. Spectroscopic features of adsorbed and loaded dye NGs were studied in comparison with free dye and nude formulation (Fig. 6 , C16-containing system). As expected, Alexa Fluor TM 430 shows a max λ abs at 430 nm. Otherwise, a blue shift phenomenon is detected for both NGs preparations (λ abs = 423 nm and λ abs = 413 nm for adsorbed and encapsulated Alexa preparation, respectively). The hypsochromic shift can be a consequence of the hydrogen bonding and of the solvent exposure [ 33 ]. On the other hand, the blue shift in the NGs preparation can be attributed to a more hydrophilic environment for encapsulated dye in the NG core respect to shell surfactant compartment. The same effect is verified in fluorescence spectral emission. The blue shift of the emission peak can be attributed to solvent polarity and relaxations decreasing for chromophores excited states in polar solvents. These phenomena stabilize the ground state, increasing the transition energy gap causing the emission peak to shift to shorter wavelengths [ 34 ]. The structural parameters of all the encapsulated/absorbed formulations, including size and the ζ potential, were assessed by DLS analyses (Table 2 ). Otherwise, intensity profiles and correlation functions of C16- and C18- containing formulations are grouped in Figure S4 - S7. All the formulations, excluding C16-NG K and C18-NG K (higher amount of adsorbed AlexaFluor TM 430) were found stable and further characterized. The intensity profiles of NGs indicate the prevalence of a monodisperse population for all the experimental conditions, with a mean diameter ranged between 123 and 163 nm. A lower polydispersity indexes (P.D.I.) is associated with samples prepared with the adsorption method with respect to encapsulation route ones. As verified for nude formulations, no substantial size variations were detected up to one month, thus corroborating the good stability for NG systems. This evidence can be again correlated to the positive values found for the zeta potential (ζ), for both encapsulated and adsorbed systems, thus preventing flocculation/sedimentation for the AlexaFluor TM 430 containing NGs. Table 2 Structural parameters, mean diameters, polydispersity indexes (P.D.I.), diffusion coefficients ( D ) and zeta potentials (ζ) of Alexa Fluor TM 430 loaded NGs measured by DLS technique. Sample Preparation method [Alexa Fluor TM 430] (mmol/L) Mean diameter (nm) P.D.I. D (m 2 s − 1 ) ∙10 − 24 ζ (mV) C16 empty top-down --- 139 ± 63.4 0.202 3.54 ± 1.61 + 38 C16 C16-NG A encapsulation 0.05 150 ± 116 0.438 3.28 ± 2054 + 24.9 C16-NG B encapsulation 0.25 123 ± 65.4 0.315 4.00 ± 2.13 + 12.8 /+32.7 C16-NG C encapsulation 0.50 131 ± 91.4 0.269 3.75 ± 2.61 + 35.7 C16-NG D encapsulation 0.75 134 ± 74.1 0.288 3.67 ± 2.03 + 7.4 /+30.5 C16-NG E encapsulation 1.00 147 ± 84.6 0.543 3.35 ± 1.93 + 6.0/ +33 C16-NG F adsorption 0.05 144 ± 78.0 0.297 3.42 ± 1.85 + 31.1 C16-NG G adsorption 0.25 137 ± 81.3 0.288 3.59 ± 2.13 + 38.6 C16-NG H adsorption 0.50 163 ± 81.8 0.285 3.02 ± 1.52 + 22.6 C16-NG I adsorption 0.75 150 ± 75.5 0.284 3.28 ± 1.65 + 27.5 C16-NG K adsorption 1.00 - - - - C18 empty top-down --- 102 ± 76.3 0.243 4.82 ± 3.60 + 51.0 C18 C18-NG A encapsulation 0.05 140 ± 99.4 0.413 3.51 ± 2.49 + 54.9 C18-NG B encapsulation 0.25 115 ± 79.9 0.284 4.28 ± 2.97 + 58.6 C18-NG C encapsulation 0.50 155 ± 95,7 0.319 3.17 ± 1.96 + 64.3 C18-NG D encapsulation 0.75 127 ± 87,2 0.275 3.87 ± 2.66 + 36.7 C18-NG E encapsulation 1.00 126 ± 74,0 0.258 3.90 ± 2.29 + 19.8/ +46.4 C18-NG F adsorption 0.05 145 ± 90,2 0.300 3.39 ± 2.11 + 51.5 C18-NG G adsorption 0.25 138 ± 81,9 0.260 3.56 ± 2.11 + 54.7 C18-NG H adsorption 0.50 159 ± 102 0.274 3.09 ± 1.98 + 46.2 C18-NG I adsorption 0.75 160 ± 109 0.259 3.07 ± 2.09 + 37.6 C18-NG K adsorption 1.00 - - - - The amount of the loaded (encapsulated/adsorbed) dye for each formulation was again analytically extimated by UV-vis spectroscopy, measuring the absorbance of free Alexa Fluor TM 430, after its separation from NG by SEC. The loaded dye amount for each formulation is reported as encapsulation efficiency percentage ( EE %) and of the encapsulation ratio percentage ( ER %) and collected in Table 3 . From the comparison of the ER% and EE% values of both C16 and C18 NG formulations, it can be observed a dependence with the loading methodology (encapsulation versus adsorbtion). Specifically, in the set of encaptulating formulations (NG A -NG E ), a linear trend of the ER% and EE% as a function of the initial Alexa Fluor TM 430 amount was found (see Fig. 7 A). On the contrary, no significant variation can be detected for NG F -NG K samples. Table 3 Amount of loaded AlexaFluor TM 430 for each NG formulation, expressed as encapsulation efficiency percentage ( EE %) and encapsulation ratio percentage ( ER %). Sample Preparation method AlexaFluor TM 430 (µg) AlexaFluor TM 430 loaded (µg) ER (%) EE (%) C16 C16-NG A encapsulation 67 12.1 18.0 0.0931 C16-NG B encapsulation 133 67.8 50.8 0.522 C16-NG C encapsulation 200 86.3 43.2 0.664 C16-NG D encapsulation 267 146 54.7 1.12 C16-NG E encapsulation 800 736 91.8 5.66 C16-NG F adsorption 67 50.5 71.9 0.388 C16-NG G adsorption 133 125 89.0 0.962 C16-NG H adsorption 200 176 83.5 1.35 C16-NG I adsorption 267 232 82.9 1.78 C16-NG K adsorption 800 --- -- --- C18 C18-NG A encapsulation 67 11.3 16.9 0.0870 C18-NG B encapsulation 133 17.4 13.1 0.134 C18-NG C encapsulation 200 104 51.9 0.800 C18-NG D encapsulation 267 218 81.9 1.68 C18-NG E encapsulation 800 709 87.4 5.44 C18-NG F adsorption 67 59.7 85.1 0.452 C18-NG G adsorption 133 108 76.9 0.831 C18-NG H adsorption 200 158 74.9 1.22 C18-NG I adsorption 267 219 78.1 1.68 C18-NG K adsorption 800 --- --- --- Dye release from NGs formulations The in vitro Alexa Fluor TM 430 release was evaluated using a dynamic dialysis approach in water up to 120 h. Two different NGs systems (formulations C and G, Table 2 ) for palmitic and stearic containing components were selected, to evidence the impact of both C n -peptide component and dye decoration strategy. Reported as cumulative percentage, release curve are collected in Fig. 7 B. The dye released was evaluated via fluorescence spectroscopy, using a calibration curve in the same experimental conditions. As presumable, the dye release kinetics is priority affected by Alexa Fluor TM 430 decoration approach, with final percentages of ~ 97 and ~ 26% released after 120 h for adsorbed and encapsulated formulations, respectively. An Alexa Fluor TM 430 sustained and constant release profile can be visioned for adsorbed formulation, with a total release at 120 h. The extremely slow-release profile for encapsulating NGs can be attributed to a slow water equilibrium between internal NG core and bulk solution, thus limiting the dye diffusion. No substantial differences are detectable for C16- and C18- containing samples, reinforcing the structural homology between NGs. Materials and Methods Lyophilized Fmoc-FF powder was purchased from Bachem (Bubendorf, Switzerland). TWEEN®80 (polysorbate 80), SPAN®80 (sorbitan monostearate 80), n-hexane and oil mineral were purchased from Sigma-Aldrich, Fluka (Bucks, Switzerland) or LabScan (Stillorgan, Dublin, Ireland) and were used as received unless otherwise stated. AlexaFluorTM430 ([9-[6-(2,5-dioxopyrrolidin-1-yl)oxy-6-oxohexyl]-8,8-dimethyl-2-oxo-4-(trifluoromethyl)pyrano[3,2-]quinolin-6-yl]methanesulfonate) as purchased from Molecular Probes (Eugene, Oregon, USA). Absorbance measurements on the Alexa FluorTM430 solutions were carried out on a nanodrop 2000c spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, DE, USA) equipped with a 1.0 cm quartz cuvette (Hellma) using as molar absorptivity (ε) the value of 17740 mol − 1 ∙L∙cm − 1 at 430 nm. A MICCRA D-9 homogenizer (MICCRA GmbH, Germany) and a tip sonicator Branson SFX150, (Branson, Germany) were used for homogenization and tip sonication formulative steps. Peptide synthesis and nanogels (NGs) formulation procedures Peptide synthesis, purification and their chemical characterization was already reported. [ 17 ] C16-(GK)3/Fmoc-FF and C18-(GK)3/Fmoc-FF (1/1 mol/mol ) based NGs were formulated according to the top-down procedure, using their respective HG (400 µL, 0.5 wt%) prepared via solvent switch method (DMSO/H 2 O) as previously described.[ 18 ] 1.6 mL of an aqueous, filtered solution of TWEEN®80/SPAN®80 with a Hydrophilic-Lipophilic Balance (HLB) of 10 (53/47, w/w ratio, total content of 3.0 ∙10 − 5 mol) were added to the preformed HGs. The resulting suspensions were homogenized at 25,000 min − 1 for 5 min and then tip sonicated for 5 min at 9 W. Formulation of AlexaFluor™ 430 containing NGs Increasing concentrations of the Alexa Fluor TM 430 dye (0.05, 0.25, 0.50, 0.75, 1.0 and 3.0 mmol/L) were alternatively encapsulated (see procedure A ) or absorbed on NGs (see procedure B ), prepared according to the top-down methodology above described. The amount of the encapsulated/adsorbed dye was quantitatively determined as difference by measuring the absorbance at λ = 430 nm via UV-vis spectroscopy (ε 430 = 17740 mol − 1 ∙L∙cm − 1 ). Quantification was performed after exclusion chromatography purification of formulations on a gel column (Sephadex G50 column). Procedure A: Encapsulation 360 µL of the dye water solution, at different concentrations, was added to 40 µL of peptides stock solution (100 mg/mL) in DMSO. The suspension was vortexed for a few seconds and left at room temperature until the complete gel formation. Then the HG underwent to the top-down methodology above described to obtain NGs. Procedure B: Adsorption Lyophilized powder of the dye was dehydrated with peptide NGs at room temperature for 24 h under a gentle stirring for 24 h. Dynamic Light Scattering (DLS) measurements The DLS measurements, in triplicate, were carried out on all the aqueous samples. Hydrodynamic radii ( r H ), diffusion coefficients ( D ) and zeta potential ( ζ ) of all the peptide NG formulations were measured by Dynamic Light Scattering (DLS) using a Zetasizer Advance- Malver Light Scattering Technology (Malvern Panlytical, Westborough, MA, USA). Instrumental settings for the measurements were a backscatter detector at 173° in automatic modality, room temperature, and disposable sizing cuvette as cell. Cell cultures and in vitro NGs toxicity assay HEK-293 (human embryonic kidney) cells were cultured in were cultured in Dulbecco’s modified Eagle Medium (DMEM) (Gibco, Thermo Fisher Scientific Waltham, Massachusetts, US), supplemented with 10% Fetal Bovine Serum (FBS) (Gibco), 2 mM Glutamine, 100 U mL⁻¹ Penicillin, and 100 µg mL⁻¹ Streptomycin (Gibco) at 37°C in a 5% CO 2 . Cell confluence was maintained at 60–80%, and subculturing was performed at a 1:3 ratio twice a week using 25 cm² flasks (Nunc, Thermo Fisher Scientific, US). HEK-293 (human embryonic kidney) cells were seeded in 96-well plates at a density of 5.0 × 10 5 cells per well. [ 35 ] Formulations of C16 and C18 NGs were dissolved in cultured medium. Cells were incubated in the presence of different dilutions of NGs, ranging from 1:160 ÷ 1:320000, for 72h. Cell viability was measured by the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay according to the manufacturer’s instructions. Briefly, 20 mL of 5 mg/mL MTT solution in PBS was added to each well and then incubated for 4 h at 37°C, 5% CO2. Cell viability dependent absorbance of the dark blue formazan crystals was measured in a spectrophotometer (λ max = 570nm). All the analyses were performed in triplicate. Cell survival was expressed as percentage of viable cells in the presence of hydrogels or nanogels, compared to control cells grown in their absence. MTT assay was repeated twice with similar results. Circular Dichroism (CD) For CD spectroscopy, NGs were placed in 0.1 mm quartz cells and Far-UV CD spectra were collected through a Chirascan spectropolarimeter equipped with a thermal controller. Spectra were recorded from 290 to 190 nm. 0.5 nm step, 1.0 nm bandwidth, 1 s collection time per step were used as experimental settings for the measures. Each spectrum was obtained by averaging three scans and corrected for the blank. Fourier Infrared (FT-IR) Spectroscopy A JascoFT/IR 4100 spectrometer was used for acquiring, in an attenuated total reflection (ATR) mode, spectra of all the peptide-based xerogels, using a Ge single-crystal at a resolution of 4 cm − 1 . A total of 120 scans of each sample were recorded with a rate of 2 mm∙s − 1 against a KBr background. After collection in transmission mode, amide I deconvolutions (in the 1600–1700 cm − 1 region) were automatically returned as emission by Jasco SSE instrument integrated software using a method of principal component regression (PCR). Small-Angle X-ray Scattering (SAXS) SAXS experiments were performed on beamline B21 [ 36 ] at Diamond (Didcot, UK). The sample solutions were loaded into the 96-well plate of an EMBL BioSAXS robot and then injected via an automated sample exchanger into a quartz capillary (1.8 mm internal diameter) in the X-ray beam. The quartz capillary was enclosed in a vacuum chamber, to avoid parasitic scattering. After the sample was injected into the capillary and reached the X-ray beam, the flow was stopped during the SAXS data acquisition. Beamline B21 operates with a fixed camera length (3.9 m) and fixed energy (12.4 keV). The images were captured using a PILATUS 2M detector. Data processing was performed using dedicated beamline software ScÅtter. Rheological characterization Rheological measurements were performed at 25°C by using a rotationally controlled stress rheometer Modular Advanced Rheometer System (Haake Mars) from ThermoScientific equipped with a 15.0 mm flat-plate geometry (P35-Ti). A freshly prepared HG sample (1.0 mL) with different concentrations of dye were used for measurements using a gap of 1.00 mm. Strain (0.1–100%) sweeps were conducted to identify the regime of linear viscoelasticity. Final analyses are reported as G′ (storage elastic modulus) and G″ (shear loss or viscous modulus) in Pascal [Pa]. AlexaFluor TM 430 release Release of AlexaFluor™ 430 from four different NG formulations was studied using 1.0 mL of each purified formulation using dialysis bags (Float-A-Lyzer® 3.5-5 kDa cut-off, Spectra/Por, USA) in 10 mL of pure water (under stirring at 37° C). At each selected time-point (30 min, 1, 2, 4, 8 24, 48, 72, 96 and 120 h), 2.0 mL of solution were collected and replaced with an equal amount of water. GloMax Multimode Microplate Reader (Promega Corporation, Milan, Italy) was used to quantify the released dye, using fluorescence filters (λ ex = 428 nm; λ em = 500–550 nm) and an Alexa Fluor TM 430 calibration curve. The amount of the released dye was reported as released percentage with respect to the total quantity encapsulated or adsorbed. Conclusions Peptide-based materials have been evaluated as alternative delivery platforms of chemically different APIs, including small molecules and diagnostic agents. De-novo designed peptide elements can be efficiently included in formulation, modulating the cargo features and affecting its chemical responsiveness, topological properties, morphology, or electrostatic nature. Considering this prospective, multicomponent Fmoc-FF NGs were developed and studied. Fmoc-FF, able to include amphiphilic peptide functional charge modifiers of hexapeptide (GK)3 sequence, underwent a top-down strategy, producing C16- and C18-(GK)3 containing NGs. These systems were efficiently stabilized by TWEEN and SPAN surfactant shell, with no colloidal instability up to one month and an internal supramolecular β-sheet arrangement. Alexa Fluor TM 430 molecule, selected as model anionic fluorescent dye, was alternatively encapsulated in (core) or adsorbed on (shell) NGs compartments. The decoration methodology was found able to affect the release behaviour, with different release regimes. This formulative study supports the evidence of NG peptide systems as tunable, modulable and promising platform for negatively charged APIs, including biotechnology drugs (siRNA, miRNA, DNA). Their physicochemical properties (including stability and reproducibility) enable improved APIs stability and bioavailability, often limited by enzymatic degradation and poor cellular uptake. Furthermore, the chemical accessibility to NG functionalization underscores their future potentialities in treating a wide range of diseases, from cancer to chronic inflammatory conditions. Declarations Acknowledgements and Funding Declaration C.D. M.R. and E.R. acknowledge the grant CN00000041 “National Center for Gene Therapy and Drugs based on RNA Technology” (concession number 1035 of 17 June 2022-PNRR MUR - M4C2 - Investment 1.4 Call “National Centers”,financed by EU- NextGenerationEU), project codeMUR:CN00000041−CUP UNINA: E63C22000940007. C.D.as the principal investigator acknowledges the project PRIN_2022TSLMHR titled “Biomaterials from peptide self-assembling generated by mimicking protein amyloid-like structures” - Ministero dell’Università e della Ricerca -NextGenerationEU (European Union). Author contributions Conceptualization, C.D., E.R., A.A. and G.M.; methodology, all authors; sample preparation and characterization M.R, E.R., A.A and C.D.; software, M.R., E.R., C.D. and V.C.; validation, M.R., A.A., I.W., V.C. and C.D.; investigation, formal analysis and figure preparation all authors; resources, A.A., I.W. and C.D. ; release curve M.R and C.D; data curation, writing-original draft preparation, review and editing all authors;, visualization, C.D., A.A. and I.W.; supervision A.A, I.W, G.M and C.D.; funding acquisition, A.A., G.M. I.W. and C.D. All authors have read and agreed to the published version of the manuscript. Competing interests The author(s) declare no competing interests. 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Supplementary Files SupplementaryMaterialsSciRepRosa.docx Cite Share Download PDF Status: Published Journal Publication published 22 Oct, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 08 Sep, 2025 Reviews received at journal 15 Aug, 2025 Reviews received at journal 14 Aug, 2025 Reviewers agreed at journal 13 Aug, 2025 Reviewers agreed at journal 08 Aug, 2025 Reviewers agreed at journal 05 Aug, 2025 Reviewers invited by journal 05 Aug, 2025 Editor assigned by journal 05 Aug, 2025 Editor invited by journal 05 Aug, 2025 Submission checks completed at journal 01 Aug, 2025 First submitted to journal 01 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7158671","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":496329364,"identity":"40589bef-a2d2-4f77-b0ff-ee398d4dd0f8","order_by":0,"name":"Mariangela Rosa","email":"","orcid":"","institution":"University of Naples “Federico II”","correspondingAuthor":false,"prefix":"","firstName":"Mariangela","middleName":"","lastName":"Rosa","suffix":""},{"id":496329365,"identity":"562dcf2c-d641-4c44-9832-1cefc35d8136","order_by":1,"name":"Elisabetta Rosa","email":"","orcid":"","institution":"University of Naples “Federico II”","correspondingAuthor":false,"prefix":"","firstName":"Elisabetta","middleName":"","lastName":"Rosa","suffix":""},{"id":496329367,"identity":"6b85cbad-ee71-40b7-bfd8-e240d33437a8","order_by":2,"name":"Valeria Castelletto","email":"","orcid":"","institution":"University of Reading","correspondingAuthor":false,"prefix":"","firstName":"Valeria","middleName":"","lastName":"Castelletto","suffix":""},{"id":496329369,"identity":"b1a8c549-3a3c-4868-945d-a641dddea811","order_by":3,"name":"Ian W. Hamley","email":"","orcid":"","institution":"University of Reading","correspondingAuthor":false,"prefix":"","firstName":"Ian","middleName":"W.","lastName":"Hamley","suffix":""},{"id":496329370,"identity":"561812bb-87f0-48a7-8cbc-91326fcfb950","order_by":4,"name":"Giancarlo Morelli","email":"","orcid":"","institution":"University of Naples “Federico II”","correspondingAuthor":false,"prefix":"","firstName":"Giancarlo","middleName":"","lastName":"Morelli","suffix":""},{"id":496329371,"identity":"67810026-f0e4-46d7-b90e-fe8eeba9063c","order_by":5,"name":"Antonella Accardo","email":"","orcid":"","institution":"University of Naples “Federico II”","correspondingAuthor":false,"prefix":"","firstName":"Antonella","middleName":"","lastName":"Accardo","suffix":""},{"id":496329372,"identity":"9d75212e-2bb5-4054-a45b-ef56d46f4014","order_by":6,"name":"Carlo Diaferia","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9klEQVRIiWNgGAWjYHACxgMMDMw8DMxAKoHBBsgGCRrg1wPRwpYA0pIG04JfzwGwMpAWBobDMEHcWvjbzz448IHBWoa/jfnYhwc15xO3s/Me/MBQ8AenFokz6QYHZzCk80gcY0uekXDsduLOZr5kCXwOMwC6/jAPCN3vMWZIYLuduOEwjwF+LfzPIFrkj/F/Zkj4dw6kxfgHXi0SUFsMjvEwMyS2HQBpMcNri8SNZwwHZxik8xgeYzNmSOxLNgZpsUgwMMaphb8/jfHBhwpre7ljzI8Zf3yzk91w/ozxjQ9/5HBqgQUCGkggoGEUjIJRMApGAX4AAMixTfpJ32szAAAAAElFTkSuQmCC","orcid":"","institution":"University of Naples “Federico II”","correspondingAuthor":true,"prefix":"","firstName":"Carlo","middleName":"","lastName":"Diaferia","suffix":""}],"badges":[],"createdAt":"2025-07-18 14:38:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7158671/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7158671/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-20945-3","type":"published","date":"2025-10-22T16:17:20+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":88499764,"identity":"8966043d-28c7-4d66-97f7-b5f48afb5466","added_by":"auto","created_at":"2025-08-07 06:45:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":197657,"visible":true,"origin":"","legend":"\u003cp\u003eChemical formulas, schematic representation of Fmoc-FF and C\u003cem\u003en\u003c/em\u003e-(GK)3 peptides and multicomponent hydrogels and nanogels. The chemical formula of TWEEN\u003csup\u003e®\u003c/sup\u003e80 and SPAN\u003csup\u003e®\u003c/sup\u003e80 surfactants are also reported.\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7158671/v1/f3f296c1da29a434c6ddeea7.png"},{"id":88499759,"identity":"599a4170-a492-4e32-bb15-dc2d2cc93809","added_by":"auto","created_at":"2025-08-07 06:45:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":785203,"visible":true,"origin":"","legend":"\u003cp\u003eDLS characterization. a) Intensity profiles of C16-(GK)3 (blue line) and C18-(GK)3 NGs (green line). Correlation coefficient over time for b) C16-(GK)3 and c) C18-(GK)3 containing NGs.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7158671/v1/13217a11afb432f32e68fde8.png"},{"id":88505352,"identity":"9614a8f1-ae75-4c42-99cd-8e9f8b0590bb","added_by":"auto","created_at":"2025-08-07 07:25:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":139477,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSecondary structure characterization.\u003c/strong\u003e CD spectra of hydrogels and nanogels of a) C16-(GK)3 and b) C18-(GK)3. c) FT-IR deconvolution for C16-(GK)3 (blue line) and C18-(GK)3 (green line) nanogels containing formulations.\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7158671/v1/6520d8e380d85f6cb76e9657.png"},{"id":88500844,"identity":"4542f8c9-8d59-4302-a767-4a030ea7f37b","added_by":"auto","created_at":"2025-08-07 06:53:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":173072,"visible":true,"origin":"","legend":"\u003cp\u003eSAXS data for nanogels as indicated. Open symbols: measured data, solid lines: form factor fits described in the text. For ease of visualization, the data for C16-(GK)3 containing NG are shifted by division by a factor of 5 and that for C18-(GK)3 containing NG by division by a factor of 25, and for all data sets only every 5\u003csup\u003eth\u003c/sup\u003e data point is plotted.\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7158671/v1/ae0dc0013c666fc2ed58c3b4.png"},{"id":88499767,"identity":"246f0ffc-dffe-48e8-9937-8f6998175019","added_by":"auto","created_at":"2025-08-07 06:45:25","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":381128,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation for encapsulation or adsorption of Alexa FluorTM430 in NGs formulations.\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7158671/v1/3bed8aff232f997dc6153cd3.png"},{"id":88500857,"identity":"5d83274b-8cfc-4275-a473-158c03a4d5b6","added_by":"auto","created_at":"2025-08-07 06:53:26","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":301118,"visible":true,"origin":"","legend":"\u003cp\u003eUV-vis (a) and fluorescence (b) spectra of AlexaFluor\u003csup\u003eTM\u003c/sup\u003e430 and NGs formulations.\u003c/p\u003e","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7158671/v1/b6c82623502fe7621e3ebf76.png"},{"id":88499777,"identity":"26bb8027-6875-44e5-9f4e-4ab228286db8","added_by":"auto","created_at":"2025-08-07 06:45:25","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":10589258,"visible":true,"origin":"","legend":"\u003cp\u003eUV-vis (a) and fluorescence (b) spectra of AlexaFluor\u003csup\u003eTM\u003c/sup\u003e430 and NGs formulations.\u003c/p\u003e","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7158671/v1/10b2cfdfa43e987bdfd24a93.png"},{"id":88499789,"identity":"63354a70-29fa-45a5-9e79-3c69419628dc","added_by":"auto","created_at":"2025-08-07 06:45:26","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":168836,"visible":true,"origin":"","legend":"\u003cp\u003ea) Encapsulation efficiency of NGs as a function of the amount of Alexa FluorTM430 expressed in μg. b) Cumulative percentage dye release for C16-(GK)3 (blue) and C18-(GK)3 containing NGs. Encapsulating and adsorbing formulations are reported as filled and empty symbols, respectively.\u003c/p\u003e","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7158671/v1/6461b71860f63d0809334a4d.png"},{"id":94490231,"identity":"89f266ba-6f38-47eb-a7c4-76f4a393df03","added_by":"auto","created_at":"2025-10-27 17:08:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13999836,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7158671/v1/80f81121-c822-480e-9ece-448ca2ffcc8b.pdf"},{"id":88499762,"identity":"87672982-109b-47c9-a063-ac8675369d83","added_by":"auto","created_at":"2025-08-07 06:45:25","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":387971,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterialsSciRepRosa.docx","url":"https://assets-eu.researchsquare.com/files/rs-7158671/v1/911a4e5e57efebd6c467eb20.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evaluation of cationic peptide-based nanogels as delivery systems for negatively charged molecules: a formulative study","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSupramolecular nanostructures like micelles [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] liposomes [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], and polymeric nanoparticles [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] have been extensively investigated as innovative platforms for the delivery of a wide range of active pharmaceutical ingredients (APIs). The success of these nanosystems is related to their capability to improve APIs bioavalability and the biodisponibility, by changing its pharmacokinetic and pharmacodynamic profiles, while also offering physical protection against \u003cem\u003ein vivo\u003c/em\u003e degradation[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] However, the inherent anionic nature of liposomes and polymeric micelles generally hinders the encapsulation of negatively charged molecules, such as nucleic acid-based biopharmaceuticals (e.g., siRNA, miRNA, and gene-silencing agents) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. As a result, these APIs are often adsorbed onto the outer surface of cationic liposomes, forming lipoplexes [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], which do not shield them from enzymatic degradation in vivo. In this context, the development of alternative nanostructures capable of efficiently deliver biotechnological active principles is of particular interest.[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] Hydrogel (HG) nanoparticles, also named nanogels (NGs), have been recently proposed as potential nanosized injectable delivery systems for negative molecules.[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] Structurally, NGs are formed of an interior hydrogel-like network (\u003cem\u003ecore\u003c/em\u003e) stabilized by an external surfactant coating (\u003cem\u003eshell\u003c/em\u003e). This supramolecular structuration allows preserving the inner interconnected hydrogelated network they derive from. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] NGs can be prepared for mechanical submicronization and stabilization of the macroscopic HG by top-down approach. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] Recently a small library of multicomponent peptide-based HGs, prepared by mixing in a \u003cem\u003e1/1 mol/mol\u003c/em\u003e ratio Fmoc-diphenylalanine (Fmoc-FF, Fmoc\u0026thinsp;=\u0026thinsp;fluorenylmethoxycarbonyl) gelator with cationic amphiphilic peptides (CAPs), was described. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] Primary peptide sequences were designed as functional charge modifiers of anionic Fmoc-FF matrices, introducing positive charges for matrix/API electrostatic interaction. A common cationic hexapeptide (GK)3 sequence, alternating glycine (G) and lysine (K) residues, was decorated at its N-terminus with an alkyl chain, differing for their length from C8 to C18. The multiscale structural and morphological characterization of these matrices highlighted the guiding gelation rule of Fmoc-FF and a partial immobilization of CAPs in gel matrix. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eApplying a top-down strategy, Fmoc-FF/C16-(GK)3 and Fmoc-FF/C18-(GK)3 NGs externally stabilized by polysorbate 80 (TWEEN\u0026reg;80, Polyethylene glycol sorbitan monooleate) and sorbitan monostearate 80 (SPAN\u0026reg;80, Sorbitan monooleate) have been here formulated (see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and fully characterized. In particular, the NGs size, superficial charge and shelf stability were assessed by Dynamic Light Scattering (DLS). Circular Dichroism (CD), and Fourier Transform Infrared (FTIR) spectroscopies were carried out to visualize homologies or differences in secondary structure of NGs and the corresponding HGs. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] Moreover, the NGs organization has been further investigated by Small-angle X-ray scattering (SAXS) technique. Different formative routes were applied to decorate Fmoc-FF/C16-(GK)3 and Fmoc-FF/C18-(GK)3 formulations with the negatively charged AlexaFluor\u003csup\u003eTM\u003c/sup\u003e430 (succinimidyl ester), used as model molecule. AlexaFluor\u003csup\u003eTM\u003c/sup\u003e430 was alternatively encapsulated into the NG core or adsorbed into surfactant shell. The comparison of the encapsulation efficiency percentage (EE%) and the encapsulation ratio percentage (ER%), obtained with the different procedures and different amount of Alexa Fluor\u003csup\u003eTM\u003c/sup\u003e430, allows identifying a general preparation procedure, reinforcing the proposing rule of NGs as potential platforms for negatively charged APIs delivery.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003e\u003cb\u003eFormulation of empty NGs\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFmoc-FF have gained attention for its ability to form well-ordered nanostructures (like micelles and HGs) through non-covalent interactions, driven by hydrogen bonding and electrostatic forces. Fmoc-FF was also explored in combination with different molecular elements (\u003cem\u003eeg.\u003c/em\u003e other peptide sequences, proteins or polymers) for developing multicomponent systems. [\u003cspan additionalcitationids=\"CR19 CR20\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] It was observed that the inclusion of both hydrophilic and hydrophobic components into Fmoc-FF HG is perfectly compatible with the common gelation procedure of the \u0026ldquo;\u003cem\u003esolvent-switch\u003c/em\u003e\u0026rdquo;. This gelation route involves dissolving the dipeptide monomers at a high concentration (100 mg/mL) in an organic solvent (DMSO, ethanol, or 1,1,1,3,3,3- hexafluoro-isopropanol), followed by stock dilution with water. Dilution triggers gel formation, creating a three-component system (peptide/solvent/water).[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] Using this approach, Fmoc-FF/C\u003csub\u003e\u003cem\u003en\u003c/em\u003e\u003c/sub\u003e-(GK)3 1/1 \u003cem\u003emol/mol\u003c/em\u003e gel matrices were formulated and analyzed (\u003cem\u003en\u003c/em\u003e from 8 to 18 carbon atoms). The corresponding NGs were formulated according to the top-down procedure, previously optimized for preparation of pure Fmoc-FF NG.[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] This protocol is based on the submicronization of the corresponding macroscopic HG by homogenization and tip-sonication. The resulting nanoparticles are composed by an inner core, externally stabilized by a TWEEN\u0026reg; 80 and SPAN\u0026reg; 80 surfactant coating, added at 53/47 \u003cem\u003ew/w\u003c/em\u003e (total number of mol\u0026thinsp;=\u0026thinsp;3.0 ∙10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e mol) in order to have a hydrophilic-lipophilic balance (HLB) of 10.[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eNGs preparation were initially studied by Dynamic Light Scattering (DLS) analysis at θ\u0026thinsp;=\u0026thinsp;173\u0026deg;. Appling the top-down protocol, capric (C10), lauric (C12) and myristic (C14)-(GK)3 containing NGs resulted as unstable and polydisperse preparations (polydispersity index PDI\u0026thinsp;\u0026gt;\u0026thinsp;0.350), with main diameter (\u003cem\u003ed\u003c/em\u003e) dimensions incompatible with potential injectability (see \u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e as exemplificative example). This evidence suggests that C10-, C12- and C14 derivatives alter the total HLB, modify the TWEEN\u0026reg; /SPAN\u0026reg; surfactant and stabilize performance. On the contrary, Fmoc-FF/C16-(GK)3 and Fmoc-FF/C18-(GK)3 shows a monomodal DLS profile (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), indicating the presence of homogeneous aggregate population (PDI\u0026thinsp;=\u0026thinsp;0.202 and 0.243 for C16 and C18-(GK)3 containing systems, respectively). These results suggest that the length of the CAPs alkyl chain can affect the formation of the NG. According to this suggestion, we previously observed that macroscopical matrices of C10-, C12-, and C14- peptides led to the formation of more rigid HGs, meanwhile alkyl chain elongation causes a notable reduction in the storage modulus (G\u0026prime; = 794 Pa for C16 and 73 Pa for C18). This difference may have a rule in micronization step, favoring NGs formation for palmitic (C16) and stearic (C18) mixed matrices.\u003c/p\u003e\u003cp\u003eApparent translational diffusion coefficients are reported in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. As well-known at infinite dilution, the mean diameter (\u003cem\u003eD\u003c/em\u003e), may be evaluated by using the Stokes\u0026ndash;Einstein\u0026ndash;Sutherland (SES) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The mean diameter of Fmoc-FF/C16-(GK)3 and Fmoc-FF/C18-(GK)3 is 139 and 102 nm, respectively. These values are smaller than the analogue one of pure Fmoc-FF (mean diameter\u0026thinsp;~\u0026thinsp;187 nm), advising that the intercalation of the amphiphilic peptides into the Fmoc-FF causes a contraction of the nanoparticles. No significant variation in formulation size can be detected up to one month (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eb and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ec), thus indicating good stability features. This evidence can be correlated to the positive values found for the zeta potential (\u003cem\u003eζ\u003c/em\u003e) of both the NGs systems (+\u0026thinsp;38 and +\u0026thinsp;51 mV for C16- and C18-derivatives, respectively), preventing flocculation/sedimentation for the lyophilic colloids NG\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vitro cytotoxicity assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn order to assess the potential capability of these NGs to be used in vivo as delivery systems, their cytotoxicity profiles were evaluated in vitro on HEK-293 cells. Cells were incubated for 72 h at 37\u0026deg;C with different concentrations of NGs (NG formulation was diluted in the range 1:160\u0026thinsp;\u0026divide;\u0026thinsp;1:320000 corresponding to a 23.3 \u0026micro;mol/L\u0026thinsp;\u0026divide;\u0026thinsp;11.68 nmol/L total peptide concentration range). The cell viability was evaluated by MTT assay, which typically evaluates metabolically active cells via MTT reduction to formazan by mitochondrial dehydrogenases. As clearly shown in Figure S2, no significant toxicity was observed for NGs at all the tested concentrations. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/p\u003e\u003cp\u003e\u003cb\u003eStructural characterization of NGs: CD, FT-IR and SAXS\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo deeply inspect the formulation features, the secondary structure of NGs was evaluated by CD spectroscopy. Being optically active, peptides and peptide aggregates exhibit disparity in circular polarized light absorption. Consequently, CD spectra of peptides and proteins are predominantly related to amide group excitation transitions, as function of secondary structure. From the inspection of Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, it can be observed that both the NGs spectra are characterized by two leading signals. The first positive band, located in the 220\u0026ndash;230 nm region (218 and 222 nm for C16-(GK)3 and 220 nm for C18-(GK)3 HG l and NG, respectively), is generally related to peptides β-sheet suprastructuration. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] The dichroic inversion of this signal, coupled with its increased intensity in NGs formulation with respect to HGs ones, can be probably attributed to a global different chiral environment in the NG as compared with the HG. However, it is worth noting that this tri-dimensional surrounding does not alter the fundamental-sheet organization of the NG. Instead, the second broad peak, corresponding to the distinctive signature of the Fmoc moiety, appears slightly red-shifted in the NG formulations (269 and 268 nm for C16-(GK)3 and C18-(GK)3, respectively) respect to the corresponding HG ones (265 and 264 nm).[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] The bathochromic effect can be a consequence of a different dielectric constant in the NG core covered by surfactants with respect to the macroscopic HG. It is worthwhile noting that the two samples are also different in their physical state, and thus the scattering phenomena could also contribute to the shifted behavior [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe Fourier-transform infrared spectroscopy (FT-IR) analysis carried out on samples supported the CD secondary structure assignment. The FT-IR spectra of NGs were deconvoluted in absorbance in Amide I region (1700\u0026thinsp;\u0026minus;\u0026thinsp;1600 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003ec), that, controlled by C-O and C-N stretching vibrations, is the most sensitive spectral region for secondary structure of peptides and proteins. [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] The presence of a prevalent C\u0026thinsp;=\u0026thinsp;O stretching band at \u0026sim;1640 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e reinforces the β-sheet organization, as the weighted percentages of each secondary structure functions (\u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). As expected, no significative differences can be detected between C16- and C18-(GK)3 containing formulations.\u003c/p\u003e\u003cp\u003ePeptide-based NGs were further characterized by SAXS measurements. These provide information on the form factor, \u003cem\u003ei.e.\u003c/em\u003e on the shape and dimensions of the fibrils that underpin the NG structures. The SAXS data for the Fmoc-FF NG, or the mixed NGs containing alternatively C16-(GK)3 or C18-(GK)3 show similar behavior, with an intensity scaling at low wavenumber \u003cem\u003eq\u003c/em\u003e, \u003cem\u003eI\u003c/em\u003e\u0026thinsp;~\u0026thinsp;\u003cem\u003eq\u003c/em\u003e\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, which is characteristic of layered structures [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The data can be well fitted using a model for nanotape structures based on bilayers (solid lines shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003e, fit parameters listed in SI \u003cb\u003eTable S2\u003c/b\u003e). The bilayer electron density profile is represented by three Gaussian functions, two representing the \u0026lsquo;headgroups\u0026rsquo; and the central one representing the inner hydrophobic layer [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Here, the inner part of the bilayer will comprise the Fmoc units as well as potentially the phenylalanine residues, while the \u0026lsquo;headgroups\u0026rsquo; are the charged C-termini and the lysine residues in the case of the NGs containing the cationic lipopeptides. The effective half thickness of the bilayer is reduced from 77.7 \u0026Aring; for the Fmoc-FF NG to 42.0 \u0026Aring; or 31.3 \u0026Aring; for the mixed NGs with C16-(GK)3 or C18-(GK)3 respectively. The data suggest that the addition of lipopeptides leads to better defined and thinner bilayers, whereas Fmoc-FF nanotapes seem to comprise multilayers since the thickness from the SAXS data fitting is much larger than two molecular lengths. The data for Fmoc-FF show a less well- defined high \u003cem\u003eq\u003c/em\u003e form factor maximum which was modelled by incorporating a contribution to the fitted SAXS data of a monomer form factor represented as a generalized Gaussian coil with radius of gyration 10 \u0026Aring;, this was not required to fit the data for the two NGs containing lipopeptides. The SAXS data thus indicate that all Fmoc-FF NGs contain nanotape fibrils, and that these are better defined in the NGs containing the lipopeptides, \u003cem\u003ei.e.\u003c/em\u003e the molecules are fully aggregated, and the layer thickness corresponds to interdigitated bilayers, considering the molecular length of the lipopeptides.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eFormulation and characterization of Alexa Fluor\u003c/b\u003e\u003csup\u003e\u003cb\u003eTM\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e430 filled NGs\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe NGs formulation were evaluated about their potential use as drug delivery platforms. Alexa Fluor\u003csup\u003eTM\u003c/sup\u003e430, was selected as model dye for NGs analysis, due to its water-soluble anionic nature and its spectroscopic features (λ\u003csub\u003e\u003cem\u003eabs\u003c/em\u003e\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;430 nm). The versatility formulative procedure, as NGs structure, allows a different dye localization (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The dye was alternatively included (encapsulation route) in mixed peptide gel matrix or added as functional surface dye \u003cem\u003evia\u003c/em\u003e electrostatic interactions (adsorption strategy).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIncreasing amounts of Alexa Fluor\u003csup\u003eTM\u003c/sup\u003e430 (0.05\u0026thinsp;\u0026divide;\u0026thinsp;3.0 mmol/L concentration range) were alternatively encapsulated into the core (formulations indicated as NG\u003csub\u003eA\u003c/sub\u003e \u0026divide; NG\u003csub\u003eE\u003c/sub\u003e) or adsorbed onto the NGs shell (formulations indicated as NG\u003csub\u003eF\u003c/sub\u003e \u0026divide; NG\u003csub\u003eK\u003c/sub\u003e). The procedure of encapsulation was easily achieved by preparing the corresponding Alexa Fluor\u003csup\u003eTM\u003c/sup\u003e430 filled HG (0.05\u0026thinsp;\u0026divide;\u0026thinsp;3.0 mmol/L concentration range), which successively underwent top-down protocol. No syneresis effects were evidenced for both C16 and C18 containing dye matrices, thus suggesting an efficient localization into the fibrillary gel network for all the tested concentrations. Self-supporting properties for all the samples were verified \u003cem\u003evia\u003c/em\u003e inverted test tube (data not shown). As well known, the inclusion of additional chemical entities in HGs matrices can deeply alter the gel mechanical response as consequence of interactions, solubility, molecular weight in relationship with supramolecular gel mesh size.[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] Previous rheological studies on empty 0.5 wt % HGs of Fmoc-FF/C\u003csub\u003e\u003cem\u003en\u003c/em\u003e\u003c/sub\u003e-(GK)3 allowed to estimate an elastic modulus (G\u0026rsquo;) of 794 and 73 Pa for Fmoc-FF/C16-(GK)3 and Fmoc-FF/C18-(GK)3 HGsmatrices, respectively. The inclusion of the Alexa Fluor\u003csup\u003eTM\u003c/sup\u003e430 in the HGs causes a significative decrease G\u0026rsquo; value\u0026thinsp;\u0026lt;\u0026thinsp;10 Pa for all the tested concentrations without a specific trend, as deducible from dynamic oscillation strain testes (\u003cb\u003eFigure S3\u003c/b\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These evicedences indicate an impact on the rheological features of the matrices, conduting to a viscous-like behaviour. More specifically, it seems that the inclusion of Alexa Fluor\u003csup\u003eTM\u003c/sup\u003e430 induces an increase of the mash size \u003cem\u003evia\u003c/em\u003e repulsion interaction between supramolecular fibers, with a consequent decrease of G\u0026rsquo; according to the theory of rubber elasticity [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. All these matrices were efficiently transformed in the correspetive NGs preparations.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eRheological parameters of HGs encapsulating different dye concentrations. G\u0026rsquo; and G\u0026rsquo;\u0026rsquo; are extrapolated at γ\u0026thinsp;=\u0026thinsp;1.26% in the linear viscoelastic region (LVR). \u003cem\u003eTanδ\u0026thinsp;=\u0026thinsp;G\u0026rsquo;\u0026rsquo;/G\u0026rsquo;\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003e[AlexaFluor\u003csup\u003eTM\u003c/sup\u003e430] (mmol/L)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eG\u0026rsquo; (Pa)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eG\u0026rsquo;\u0026rsquo; (Pa)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003etanδ\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e4.79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.63\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.91\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.90\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.60\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.06\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3.71\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.56\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.11\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eAn AlexaFluor\u003csup\u003eTM\u003c/sup\u003e430 absorption route was also performed, supposing a favourable dye-shell electrostatic interaction pathway. To achieve absorption (0.05\u0026thinsp;\u0026divide;\u0026thinsp;3.00 mmol/L concentration range), preformed empty NG formulations were incubated with lyophilized dye powders overnight under stirring at room temperature. The purification from free dye was performed \u003cem\u003evia\u003c/em\u003e SEC (size-exclusion chromatography), with consequent analytical quantification of unretained AlexaFluor\u003csup\u003eTM\u003c/sup\u003e430 \u003cem\u003evia\u003c/em\u003e UV-vis spectroscopy.\u003c/p\u003e\u003cp\u003eSpectroscopic features of adsorbed and loaded dye NGs were studied in comparison with free dye and nude formulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003e, C16-containing system). As expected, Alexa Fluor\u003csup\u003eTM\u003c/sup\u003e430 shows a max λ\u003csub\u003e\u003cem\u003eabs\u003c/em\u003e\u003c/sub\u003e at 430 nm. Otherwise, a blue shift phenomenon is detected for both NGs preparations (λ\u003csub\u003e\u003cem\u003eabs\u003c/em\u003e\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;423 nm and λ\u003csub\u003e\u003cem\u003eabs\u003c/em\u003e\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;413 nm for adsorbed and encapsulated Alexa preparation, respectively). The hypsochromic shift can be a consequence of the hydrogen bonding and of the solvent exposure [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eOn the other hand, the blue shift in the NGs preparation can be attributed to a more hydrophilic environment for encapsulated dye in the NG core respect to shell surfactant compartment. The same effect is verified in fluorescence spectral emission. The blue shift of the emission peak can be attributed to solvent polarity and relaxations decreasing for chromophores excited states in polar solvents. These phenomena stabilize the ground state, increasing the transition energy gap causing the emission peak to shift to shorter wavelengths [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe structural parameters of all the encapsulated/absorbed formulations, including size and the \u003cem\u003eζ\u003c/em\u003e potential, were assessed by DLS analyses (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Otherwise, intensity profiles and correlation functions of C16- and C18- containing formulations are grouped in \u003cb\u003eFigure S4\u003c/b\u003e-\u003cb\u003eS7.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAll the formulations, excluding C16-NG\u003csub\u003eK\u003c/sub\u003e and C18-NG\u003csub\u003eK\u003c/sub\u003e (higher amount of adsorbed AlexaFluor\u003csup\u003eTM\u003c/sup\u003e430) were found stable and further characterized. The intensity profiles of NGs indicate the prevalence of a monodisperse population for all the experimental conditions, with a mean diameter ranged between 123 and 163 nm. A lower polydispersity indexes (P.D.I.) is associated with samples prepared with the adsorption method with respect to encapsulation route ones. As verified for nude formulations, no substantial size variations were detected up to one month, thus corroborating the good stability for NG systems. This evidence can be again correlated to the positive values found for the zeta potential (ζ), for both encapsulated and adsorbed systems, thus preventing flocculation/sedimentation for the AlexaFluor\u003csup\u003eTM\u003c/sup\u003e430 containing NGs.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eStructural parameters, mean diameters, polydispersity indexes (P.D.I.), diffusion coefficients (\u003cem\u003eD\u003c/em\u003e) and zeta potentials (ζ) of Alexa Fluor\u003csup\u003eTM\u003c/sup\u003e430 loaded NGs measured by DLS technique.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSample\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePreparation method\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e[Alexa Fluor\u003csup\u003eTM\u003c/sup\u003e430] (mmol/L)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMean diameter (nm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eP.D.I.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cem\u003eD\u003c/em\u003e (m\u003csup\u003e2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003cem\u003e∙10\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u0026thinsp;24\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eζ (mV)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eC16 empty\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003etop-down\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e---\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e139\u0026thinsp;\u0026plusmn;\u0026thinsp;63.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.202\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e3.54\u0026thinsp;\u0026plusmn;\u0026thinsp;1.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e+\u0026thinsp;38\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"9\" rowspan=\"10\"\u003e\u003cp\u003e\u003cb\u003eC16\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC16-NG\u003csub\u003eA\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eencapsulation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e150\u0026thinsp;\u0026plusmn;\u0026thinsp;116\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.438\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e3.28\u0026thinsp;\u0026plusmn;\u0026thinsp;2054\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e+\u0026thinsp;24.9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC16-NG\u003csub\u003eB\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eencapsulation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e123\u0026thinsp;\u0026plusmn;\u0026thinsp;65.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.315\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e4.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e+\u0026thinsp;12.8 /+32.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC16-NG\u003csub\u003eC\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eencapsulation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e131\u0026thinsp;\u0026plusmn;\u0026thinsp;91.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.269\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e3.75\u0026thinsp;\u0026plusmn;\u0026thinsp;2.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e+\u0026thinsp;35.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC16-NG\u003csub\u003eD\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eencapsulation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e134\u0026thinsp;\u0026plusmn;\u0026thinsp;74.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.288\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e3.67\u0026thinsp;\u0026plusmn;\u0026thinsp;2.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e+\u0026thinsp;7.4 /+30.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC16-NG\u003csub\u003eE\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eencapsulation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e147\u0026thinsp;\u0026plusmn;\u0026thinsp;84.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.543\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e3.35\u0026thinsp;\u0026plusmn;\u0026thinsp;1.93\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e+\u0026thinsp;6.0/ 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align=\"left\" colname=\"c3\"\u003e\u003cp\u003eadsorption\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e137\u0026thinsp;\u0026plusmn;\u0026thinsp;81.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.288\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e3.59\u0026thinsp;\u0026plusmn;\u0026thinsp;2.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e+\u0026thinsp;38.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC16-NG\u003csub\u003eH\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eadsorption\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" 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colname=\"c8\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eC18 empty\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003etop-down\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e---\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e102\u0026thinsp;\u0026plusmn;\u0026thinsp;76.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.243\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e4.82\u0026thinsp;\u0026plusmn;\u0026thinsp;3.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e+\u0026thinsp;51.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"9\" rowspan=\"10\"\u003e\u003cp\u003e\u003cb\u003eC18\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC18-NG\u003csub\u003eA\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eencapsulation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e140\u0026thinsp;\u0026plusmn;\u0026thinsp;99.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.413\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e3.51\u0026thinsp;\u0026plusmn;\u0026thinsp;2.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e+\u0026thinsp;54.9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC18-NG\u003csub\u003eB\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eencapsulation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e115\u0026thinsp;\u0026plusmn;\u0026thinsp;79.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.284\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e4.28\u0026thinsp;\u0026plusmn;\u0026thinsp;2.97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e+\u0026thinsp;58.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC18-NG\u003csub\u003eC\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eencapsulation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e155\u0026thinsp;\u0026plusmn;\u0026thinsp;95,7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.319\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e3.17\u0026thinsp;\u0026plusmn;\u0026thinsp;1.96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e+\u0026thinsp;64.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC18-NG\u003csub\u003eD\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eencapsulation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e127\u0026thinsp;\u0026plusmn;\u0026thinsp;87,2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.275\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e3.87\u0026thinsp;\u0026plusmn;\u0026thinsp;2.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e+\u0026thinsp;36.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC18-NG\u003csub\u003eE\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eencapsulation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e126\u0026thinsp;\u0026plusmn;\u0026thinsp;74,0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.258\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e3.90\u0026thinsp;\u0026plusmn;\u0026thinsp;2.29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e+\u0026thinsp;19.8/ +46.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC18-NG\u003csub\u003eF\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eadsorption\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e145\u0026thinsp;\u0026plusmn;\u0026thinsp;90,2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.300\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e3.39\u0026thinsp;\u0026plusmn;\u0026thinsp;2.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e+\u0026thinsp;51.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC18-NG\u003csub\u003eG\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eadsorption\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e138\u0026thinsp;\u0026plusmn;\u0026thinsp;81,9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.260\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e3.56\u0026thinsp;\u0026plusmn;\u0026thinsp;2.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e+\u0026thinsp;54.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC18-NG\u003csub\u003eH\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eadsorption\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e159\u0026thinsp;\u0026plusmn;\u0026thinsp;102\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.274\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e3.09\u0026thinsp;\u0026plusmn;\u0026thinsp;1.98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e+\u0026thinsp;46.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC18-NG\u003csub\u003eI\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eadsorption\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e160\u0026thinsp;\u0026plusmn;\u0026thinsp;109\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.259\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e3.07\u0026thinsp;\u0026plusmn;\u0026thinsp;2.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e+\u0026thinsp;37.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC18-NG\u003csub\u003eK\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eadsorption\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe amount of the loaded (encapsulated/adsorbed) dye for each formulation was again analytically extimated by UV-vis spectroscopy, measuring the absorbance of free Alexa Fluor\u003csup\u003eTM\u003c/sup\u003e430, after its separation from NG by SEC. The loaded dye amount for each formulation is reported as encapsulation efficiency percentage (\u003cem\u003eEE\u003c/em\u003e%) and of the encapsulation ratio percentage (\u003cem\u003eER\u003c/em\u003e%) and collected in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. From the comparison of the ER% and EE% values of both C16 and C18 NG formulations, it can be observed a dependence with the loading methodology (encapsulation \u003cem\u003eversus\u003c/em\u003e adsorbtion). Specifically, in the set of encaptulating formulations (NG\u003csub\u003eA\u003c/sub\u003e-NG\u003csub\u003eE\u003c/sub\u003e), a linear trend of the ER% and EE% as a function of the initial Alexa Fluor\u003csup\u003eTM\u003c/sup\u003e430 amount was found (see Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). On the contrary, no significant variation can be detected for NG\u003csub\u003eF\u003c/sub\u003e-NG\u003csub\u003eK\u003c/sub\u003e samples.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAmount of loaded AlexaFluor\u003csup\u003eTM\u003c/sup\u003e430 for each NG formulation, expressed as encapsulation efficiency percentage (\u003cem\u003eEE\u003c/em\u003e%) and encapsulation ratio percentage (\u003cem\u003eER\u003c/em\u003e%).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSample\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePreparation method\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAlexaFluor\u003csup\u003eTM\u003c/sup\u003e430 (\u0026micro;g)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAlexaFluor\u003csup\u003eTM\u003c/sup\u003e430\u003c/p\u003e\u003cp\u003eloaded (\u0026micro;g)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cem\u003eER\u003c/em\u003e(%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cem\u003eEE\u003c/em\u003e (%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"9\" rowspan=\"10\"\u003e\u003cp\u003e\u003cb\u003eC16\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC16-NG\u003csub\u003eA\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eencapsulation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e12.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e18.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.0931\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC16-NG\u003csub\u003eB\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eencapsulation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e133\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e67.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e50.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.522\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC16-NG\u003csub\u003eC\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eencapsulation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e86.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e43.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.664\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC16-NG\u003csub\u003eD\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eencapsulation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e267\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e146\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e54.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.12\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC16-NG\u003csub\u003eE\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eencapsulation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e800\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e736\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e91.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e5.66\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC16-NG\u003csub\u003eF\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eadsorption\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e50.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e71.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.388\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC16-NG\u003csub\u003eG\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eadsorption\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e133\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e125\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e89.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.962\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC16-NG\u003csub\u003eH\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eadsorption\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e176\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e83.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.35\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC16-NG\u003csub\u003eI\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eadsorption\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e267\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e232\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e82.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.78\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC16-NG\u003csub\u003eK\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eadsorption\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e800\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e---\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e--\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e---\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"9\" rowspan=\"10\"\u003e\u003cp\u003e\u003cb\u003eC18\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC18-NG\u003csub\u003eA\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eencapsulation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e11.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e16.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.0870\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC18-NG\u003csub\u003eB\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eencapsulation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e133\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e17.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e13.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.134\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC18-NG\u003csub\u003eC\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eencapsulation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e104\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e51.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.800\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC18-NG\u003csub\u003eD\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eencapsulation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e267\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e218\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e81.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.68\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC18-NG\u003csub\u003eE\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eencapsulation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e800\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e709\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e87.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e5.44\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC18-NG\u003csub\u003eF\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eadsorption\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e59.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e85.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.452\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC18-NG\u003csub\u003eG\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eadsorption\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e133\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e108\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e76.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.831\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC18-NG\u003csub\u003eH\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eadsorption\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e158\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e74.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.22\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC18-NG\u003csub\u003eI\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eadsorption\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e267\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e219\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e78.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.68\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC18-NG\u003csub\u003eK\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eadsorption\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e800\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e---\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e---\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e---\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eDye release from NGs formulations\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e Alexa Fluor\u003csup\u003eTM\u003c/sup\u003e430 release was evaluated using a dynamic dialysis approach in water up to 120 h. Two different NGs systems (formulations C and G, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) for palmitic and stearic containing components were selected, to evidence the impact of both C\u003cem\u003en\u003c/em\u003e-peptide component and dye decoration strategy. Reported as cumulative percentage, release curve are collected in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e7\u003c/span\u003eB. The dye released was evaluated \u003cem\u003evia\u003c/em\u003e fluorescence spectroscopy, using a calibration curve in the same experimental conditions. As presumable, the dye release kinetics is priority affected by Alexa Fluor\u003csup\u003eTM\u003c/sup\u003e430 decoration approach, with final percentages of ~\u0026thinsp;97 and ~\u0026thinsp;26% released after 120 h for adsorbed and encapsulated formulations, respectively. An Alexa Fluor\u003csup\u003eTM\u003c/sup\u003e430 sustained and constant release profile can be visioned for adsorbed formulation, with a total release at 120 h. The extremely slow-release profile for encapsulating NGs can be attributed to a slow water equilibrium between internal NG core and bulk solution, thus limiting the dye diffusion. No substantial differences are detectable for C16- and C18- containing samples, reinforcing the structural homology between NGs.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eLyophilized Fmoc-FF powder was purchased from Bachem (Bubendorf, Switzerland). TWEEN\u0026reg;80 (polysorbate 80), SPAN\u0026reg;80 (sorbitan monostearate 80), n-hexane and oil mineral were purchased from Sigma-Aldrich, Fluka (Bucks, Switzerland) or LabScan (Stillorgan, Dublin, Ireland) and were used as received unless otherwise stated. AlexaFluorTM430 ([9-[6-(2,5-dioxopyrrolidin-1-yl)oxy-6-oxohexyl]-8,8-dimethyl-2-oxo-4-(trifluoromethyl)pyrano[3,2-]quinolin-6-yl]methanesulfonate) as purchased from Molecular Probes (Eugene, Oregon, USA). Absorbance measurements on the Alexa FluorTM430 solutions were carried out on a nanodrop 2000c spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, DE, USA) equipped with a 1.0 cm quartz cuvette (Hellma) using as molar absorptivity (ε) the value of 17740 mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e∙L∙cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at 430 nm. A MICCRA D-9 homogenizer (MICCRA GmbH, Germany) and a tip sonicator Branson SFX150, (Branson, Germany) were used for homogenization and tip sonication formulative steps.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePeptide synthesis and nanogels (NGs) formulation procedures\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePeptide synthesis, purification and their chemical characterization was already reported. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] C16-(GK)3/Fmoc-FF and C18-(GK)3/Fmoc-FF (1/1 \u003cem\u003emol/mol\u003c/em\u003e) based NGs were formulated according to the top-down procedure, using their respective HG (400 \u0026micro;L, 0.5 wt%) prepared \u003cem\u003evia\u003c/em\u003e solvent switch method (DMSO/H\u003csub\u003e2\u003c/sub\u003eO) as previously described.[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] 1.6 mL of an aqueous, filtered solution of TWEEN\u0026reg;80/SPAN\u0026reg;80 with a Hydrophilic-Lipophilic Balance (HLB) of 10 (53/47, \u003cem\u003ew/w\u003c/em\u003e ratio, total content of 3.0 ∙10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e mol) were added to the preformed HGs. The resulting suspensions were homogenized at 25,000 min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for 5 min and then tip sonicated for 5 min at 9 W.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFormulation of AlexaFluor\u0026trade; 430 containing NGs\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIncreasing concentrations of the Alexa Fluor\u003csup\u003eTM\u003c/sup\u003e430 dye (0.05, 0.25, 0.50, 0.75, 1.0 and 3.0 mmol/L) were alternatively encapsulated (see \u003cem\u003eprocedure A\u003c/em\u003e) or absorbed on NGs (see \u003cem\u003eprocedure B\u003c/em\u003e), prepared according to the top-down methodology above described. The amount of the encapsulated/adsorbed dye was quantitatively determined as difference by measuring the absorbance at λ\u0026thinsp;=\u0026thinsp;430 nm \u003cem\u003evia\u003c/em\u003e UV-vis spectroscopy (ε\u003csub\u003e430\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;17740 mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e∙L∙cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Quantification was performed after exclusion chromatography purification of formulations on a gel column (Sephadex G50 column).\u003c/p\u003e\u003cp\u003e\u003cem\u003eProcedure A: Encapsulation\u003c/em\u003e\u003c/p\u003e\u003cp\u003e360 \u0026micro;L of the dye water solution, at different concentrations, was added to 40 \u0026micro;L of peptides stock solution (100 mg/mL) in DMSO. The suspension was vortexed for a few seconds and left at room temperature until the complete gel formation. Then the HG underwent to the top-down methodology above described to obtain NGs.\u003c/p\u003e\u003cp\u003e\u003cem\u003eProcedure B: Adsorption\u003c/em\u003e\u003c/p\u003e\u003cp\u003eLyophilized powder of the dye was dehydrated with peptide NGs at room temperature for 24 h under a gentle stirring for 24 h.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDynamic Light Scattering (DLS) measurements\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe DLS measurements, in triplicate, were carried out on all the aqueous samples. Hydrodynamic radii (\u003cem\u003er\u003c/em\u003e\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e), diffusion coefficients (\u003cem\u003eD\u003c/em\u003e) and zeta potential (\u003cem\u003eζ\u003c/em\u003e) of all the peptide NG formulations were measured by Dynamic Light Scattering (DLS) using a Zetasizer Advance- Malver Light Scattering Technology (Malvern Panlytical, Westborough, MA, USA). Instrumental settings for the measurements were a backscatter detector at 173\u0026deg; in automatic modality, room temperature, and disposable sizing cuvette as cell.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell cultures and in vitro NGs toxicity assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eHEK-293 (human embryonic kidney) cells were cultured in were cultured in Dulbecco\u0026rsquo;s modified Eagle Medium (DMEM) (Gibco, Thermo Fisher Scientific Waltham, Massachusetts, US), supplemented with 10% Fetal Bovine Serum (FBS) (Gibco), 2 mM Glutamine, 100 U mL⁻\u0026sup1; Penicillin, and 100 \u0026micro;g mL⁻\u0026sup1; Streptomycin (Gibco) at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e. Cell confluence was maintained at 60\u0026ndash;80%, and subculturing was performed at a 1:3 ratio twice a week using 25 cm\u0026sup2; flasks (Nunc, Thermo Fisher Scientific, US). HEK-293 (human embryonic kidney) cells were seeded in 96-well plates at a density of 5.0 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well. [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] Formulations of C16 and C18 NGs were dissolved in cultured medium. Cells were incubated in the presence of different dilutions of NGs, ranging from 1:160\u0026thinsp;\u0026divide;\u0026thinsp;1:320000, for 72h.\u003c/p\u003e\u003cp\u003eCell viability was measured by the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay according to the manufacturer\u0026rsquo;s instructions. Briefly, 20 mL of 5 mg/mL MTT solution in PBS was added to each well and then incubated for 4 h at 37\u0026deg;C, 5% CO2. Cell viability dependent absorbance of the dark blue formazan crystals was measured in a spectrophotometer (λ\u003csub\u003emax\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;570nm). All the analyses were performed in triplicate. Cell survival was expressed as percentage of viable cells in the presence of hydrogels or nanogels, compared to control cells grown in their absence. MTT assay was repeated twice with similar results.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCircular Dichroism (CD)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFor CD spectroscopy, NGs were placed in 0.1 mm quartz cells and Far-UV CD spectra were collected through a Chirascan spectropolarimeter equipped with a thermal controller. Spectra were recorded from 290 to 190 nm. 0.5 nm step, 1.0 nm bandwidth, 1 s collection time per step were used as experimental settings for the measures. Each spectrum was obtained by averaging three scans and corrected for the blank.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFourier Infrared (FT-IR) Spectroscopy\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA JascoFT/IR 4100 spectrometer was used for acquiring, in an attenuated total reflection (ATR) mode, spectra of all the peptide-based xerogels, using a Ge single-crystal at a resolution of 4 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. A total of 120 scans of each sample were recorded with a rate of 2 mm∙s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e against a KBr background. After collection in transmission mode, amide I deconvolutions (in the 1600\u0026ndash;1700 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e region) were automatically returned as emission by Jasco SSE instrument integrated software using a method of principal component regression (PCR).\u003c/p\u003e\u003cp\u003e\u003cb\u003eSmall-Angle X-ray Scattering (SAXS)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSAXS experiments were performed on beamline B21 [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] at Diamond (Didcot, UK). The sample solutions were loaded into the 96-well plate of an EMBL BioSAXS robot and then injected via an automated sample exchanger into a quartz capillary (1.8 mm internal diameter) in the X-ray beam. The quartz capillary was enclosed in a vacuum chamber, to avoid parasitic scattering. After the sample was injected into the capillary and reached the X-ray beam, the flow was stopped during the SAXS data acquisition. Beamline B21 operates with a fixed camera length (3.9 m) and fixed energy (12.4 keV). The images were captured using a PILATUS 2M detector. Data processing was performed using dedicated beamline software Sc\u0026Aring;tter.\u003c/p\u003e\u003cp\u003e\u003cb\u003eRheological characterization\u003c/b\u003e\u003c/p\u003e\u003cp\u003eRheological measurements were performed at 25\u0026deg;C by using a rotationally controlled stress rheometer Modular Advanced Rheometer System (Haake Mars) from ThermoScientific equipped with a 15.0 mm flat-plate geometry (P35-Ti). A freshly prepared HG sample (1.0 mL) with different concentrations of dye were used for measurements using a gap of 1.00 mm. Strain (0.1\u0026ndash;100%) sweeps were conducted to identify the regime of linear viscoelasticity. Final analyses are reported as G\u0026prime; (storage elastic modulus) and G\u0026Prime; (shear loss or viscous modulus) in Pascal [Pa].\u003c/p\u003e\u003cp\u003e\u003cb\u003eAlexaFluor\u003c/b\u003e\u003csup\u003e\u003cb\u003eTM\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e430 release\u003c/b\u003e\u003c/p\u003e\u003cp\u003eRelease of AlexaFluor\u0026trade; 430 from four different NG formulations was studied using 1.0 mL of each purified formulation using dialysis bags (Float-A-Lyzer\u0026reg; 3.5-5 kDa cut-off, Spectra/Por, USA) in 10 mL of pure water (under stirring at 37\u0026deg; C). At each selected time-point (30 min, 1, 2, 4, 8 24, 48, 72, 96 and 120 h), 2.0 mL of solution were collected and replaced with an equal amount of water. GloMax Multimode Microplate Reader (Promega Corporation, Milan, Italy) was used to quantify the released dye, using fluorescence filters (λ\u003csub\u003eex\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;428 nm; λ\u003csub\u003eem\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;500\u0026ndash;550 nm) and an Alexa Fluor\u003csup\u003eTM\u003c/sup\u003e430 calibration curve. The amount of the released dye was reported as released percentage with respect to the total quantity encapsulated or adsorbed.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003ePeptide-based materials have been evaluated as alternative delivery platforms of chemically different APIs, including small molecules and diagnostic agents. \u003cem\u003eDe-novo\u003c/em\u003e designed peptide elements can be efficiently included in formulation, modulating the cargo features and affecting its chemical responsiveness, topological properties, morphology, or electrostatic nature. Considering this prospective, multicomponent Fmoc-FF NGs were developed and studied. Fmoc-FF, able to include amphiphilic peptide functional charge modifiers of hexapeptide (GK)3 sequence, underwent a top-down strategy, producing C16- and C18-(GK)3 containing NGs. These systems were efficiently stabilized by TWEEN and SPAN surfactant shell, with no colloidal instability up to one month and an internal supramolecular β-sheet arrangement. Alexa Fluor\u003csup\u003eTM\u003c/sup\u003e430 molecule, selected as model anionic fluorescent dye, was alternatively encapsulated in (core) or adsorbed on (shell) NGs compartments. The decoration methodology was found able to affect the release behaviour, with different release regimes. This formulative study supports the evidence of NG peptide systems as tunable, modulable and promising platform for negatively charged APIs, including biotechnology drugs (siRNA, miRNA, DNA). Their physicochemical properties (including stability and reproducibility) enable improved APIs stability and bioavailability, often limited by enzymatic degradation and poor cellular uptake. Furthermore, the chemical accessibility to NG functionalization underscores their future potentialities in treating a wide range of diseases, from cancer to chronic inflammatory conditions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements and Funding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC.D. M.R. and E.R. acknowledge the grant CN00000041 \u0026ldquo;National Center for Gene Therapy and Drugs based on RNA Technology\u0026rdquo; (concession number 1035 of 17 June 2022-PNRR MUR - M4C2 - Investment 1.4 Call \u0026ldquo;National Centers\u0026rdquo;,financed by EU- NextGenerationEU), project codeMUR:CN00000041\u0026minus;CUP UNINA: E63C22000940007. C.D.as the principal investigator acknowledges the project PRIN_2022TSLMHR titled \u0026ldquo;Biomaterials from peptide self-assembling generated by mimicking protein amyloid-like structures\u0026rdquo; - Ministero dell\u0026rsquo;Università e della Ricerca -NextGenerationEU (European Union).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, C.D., E.R., A.A. and G.M.; methodology, all authors; sample preparation and characterization M.R, E.R., A.A and C.D.; software, M.R., E.R., C.D. and V.C.; validation, M.R., A.A., I.W., V.C. and C.D.; investigation, formal analysis and figure preparation all authors; resources, A.A., I.W. and C.D. ; release curve M.R and C.D; data curation, writing-original draft preparation, review and editing all authors;, visualization, C.D., A.A. and I.W.; supervision \u0026nbsp;A.A, I.W, G.M and C.D.; funding acquisition, A.A., G.M. I.W. and C.D. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author(s) declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the Corresponding Author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTanbour, R., Martins, A.M., Pitt, W.G., Husseini, G.A. Drug delivery systems based on polymeric micelles and ultrasound: A review. \u003cem\u003eCurr. Pharm. Des.\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 2796-2807; 10.2174/1381612822666160217125215 (2016).\u003c/li\u003e\n\u003cli\u003eMadegard, L. et al. Bioorthogonal drug release from nanometric micelles in living cells. \u003cem\u003eChem. 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Synchrotron Radiat. \u003c/em\u003e\u003cstrong\u003e27\u003c/strong\u003e, 1438-1446; 10.1107/S1600577520009960 (2020). \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"peptide nanogels, peptide-based nanoplatforms, drug delivery, anionic active pharmaceutical ingredients, formulation study","lastPublishedDoi":"10.21203/rs.3.rs-7158671/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7158671/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePeptide-based nanogels (NGs) represent a cutting-edge class of nanoscale drug delivery systems. Due to their structural properties, NGs platforms can encapsulate and protect therapeutic agents (\u003cem\u003ee.g.\u003c/em\u003e peptides, proteins, and nucleic acids), while allowing for controlled and stimuli-responsive release. These pharmacokinetic/pharmacodynamic features can be specifically modified including peptide functional elements as NGs component. This study explores the formulation, decoration strategies, and structural properties of NGs derived from mixed hydrogel matrices of Fmoc-diphenylalanine (Fmoc-FF) with cationic amphiphilic peptides (CAPs). CAPs, composed by cationic hexapeptide (GK)3 sequence decorated at its \u003cem\u003eN\u003c/em\u003e-terminus with alkyl chain, were found able to confer a net positive charge to Fmoc-FF NGs. Fmoc-FF/C16-(GK)3 and Fmoc-FF/C18-(GK)3 NGs were obtained using polysorbate 80 (TWEEN\u003csup\u003e®\u003c/sup\u003e80) and sorbitan monostearate 80 (SPAN\u003csup\u003e®\u003c/sup\u003e80) colloidal stabilizing surfactants and characterized in terms of size, secondary structure, superficial charge and shelf stability by Dynamic Light Scattering (DLS), Circular Dichroism (CD), Fourier Transform Infrared (FTIR) and SAXS technique. Different formulative routes were applied and mutually compared to encapsulate/adsorb Alexa Fluor\u003csup\u003eTM\u003c/sup\u003e430 (succinimidyl ester), used as model of an anionic, pharmaceutical agent. \u003cem\u003eIn vitro\u003c/em\u003e experiments demonstrate a good cytocompatibility of these systems and the release of Alexa Fluor\u003csup\u003eTM\u003c/sup\u003e430 was also evaluated.\u003c/p\u003e","manuscriptTitle":"Evaluation of cationic peptide-based nanogels as delivery systems for negatively charged molecules: a formulative study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-07 06:45:20","doi":"10.21203/rs.3.rs-7158671/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-08T10:36:44+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-15T10:21:59+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-14T06:28:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"105220890013539058042199006837541389213","date":"2025-08-14T02:22:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"219442063230108557492990153221482647508","date":"2025-08-08T08:26:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"282579644371115271421267452319017136582","date":"2025-08-05T07:58:27+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-05T07:44:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-05T07:29:12+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-08-05T06:31:25+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-01T10:36:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-08-01T10:32:43+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a6cd32b1-dfe4-492d-b6f9-2de11bd58127","owner":[],"postedDate":"August 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":52714990,"name":"Biological sciences/Biotechnology"},{"id":52714991,"name":"Physical sciences/Chemistry"},{"id":52714992,"name":"Biological sciences/Drug discovery"},{"id":52714993,"name":"Physical sciences/Nanoscience and technology"}],"tags":[],"updatedAt":"2025-10-27T16:28:10+00:00","versionOfRecord":{"articleIdentity":"rs-7158671","link":"https://doi.org/10.1038/s41598-025-20945-3","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-10-22 16:17:20","publishedOnDateReadable":"October 22nd, 2025"},"versionCreatedAt":"2025-08-07 06:45:20","video":"","vorDoi":"10.1038/s41598-025-20945-3","vorDoiUrl":"https://doi.org/10.1038/s41598-025-20945-3","workflowStages":[]},"version":"v1","identity":"rs-7158671","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7158671","identity":"rs-7158671","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}