Bioinspired Orthogonal-shaped protein–biometal Nanocrystals Enable Oral Protein Absorption

Bioinspired orthogonal-shaped protein–biometal nanocrystals enable oral protein absorption Matilde Dur´ an-Lobato a,b,c,d , Sulay Tovar a,b,e , Juan Cu˜ narro a,b , Rocío Ramos-Membrive f,g , Iv´ an Pe˜ nuelas f,g , Ilaria Marigo h,i , Federico Benetti j , Miguel Chenlo k , Clara V. ´ Alvarez k , Vashegyi Ildik´ o l , Rudolf Urbanics l,m , J´ anos Szebeni n,o,p , María Jos´ e Alonso a,b,c,* a Center for Research in Molecular Medicine & Chronic Diseases (CIMUS), University of Santiago de Compostela, Campus Vida, 15782 Santiago de Compostela, Spain b Health Research Institute of Santiago de Compostela (IDIS), University of Santiago de Compostela, Campus Vida, 15782 Santiago de Compostela, Spain c Department of Pharmacology, Pharmaceutics and Pharmaceutical Technology, School of Pharmacy, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain d Departamento de Farmacia y Tecnología Farmac´ eutica, Facultad de Farmacia, Universidad de Sevilla, Prof. García Gonz´ alez, 2, 41012 Seville, Spain e CIBER Fisiopatología de la Obesidad y Nutrici´ on (CIBERobn), Madrid, Spain f Radiopharmacy Unit, Department of Nuclear Medicine, Clínica Universidad de Navarra, Instituto de Investigaci´ on Sanitaria de Navarra (IdiSNA), Av. Pío XII 36, 31008 Pamplona, Spain g Translational Molecular Imaging Unit, Department of Nuclear Medicine, Clínica Universidad de Navarra, Instituto de Investigaci´ on Sanitaria de Navarra (IdiSNA), Av. Pío XII 36, 31008 Pamplona, Spain h Department of Surgery, Oncology and Gastroenterology, University of Padova, Padova, Italy i Veneto Institute of Oncology IOV-IRCCS, Padova, Italy j ECSIN-European Center for the Sustainable Impact of Nanotechnology, ECAMRICERT SRL, Padova, Italy k Neoplasia & Endocrine Differentiation P0L5, Centre for research in Molecular Medicine and Chronic Disease (CIMUS), Av Barcelona s/n, 15782 Santiago de Compostela, Spain l SeroScience Ltd, Budapest, Hungary m Nanomedicine Research and Education Center, Department of Pathophysiology, Semmelweis University, Budapest, Hungary n Nanomedicine Research and Education Center, Department of Translational Medicine, Semmelweis University, Budapest 1089, Hungary o Department of Nanobiotechnology and Regenerative Medicine, Faculty of Health Sciences, Miskolc University, Miskolc 2880, Hungary p School of Chemical Engineering and Translational Nanobioscience Research Center, Sungkyunkwan University, Suwon 16419, Republic of Korea ARTICLE INFO Keywords: Peptide delivery Protein delivery Oral delivery Transmucosal Biologicals Nanocomplexes ABSTRACT With the growing number of marketed biological drugs, the development of technological strategies for their oral systemic absorption, becomes increasingly important. The harsh gastrointestinal environment and low permeability of the intestinal epithelium, represent a huge challenge for their systemic delivery. Herein, bioinspired in the physiological insulin-Zn interaction, the design of orthogonal-shaped protein-biometal hybrid nanocrystals, further enveloped by a bilayer of functional biomaterials, is reported. The nanocrystals exhibited a size of 80 nm, a neutral surface charge and a high insulin loading. In vitro studies showed the capacity of the nanocomplexes to control the release of the associated insulin, while preserving its stability. In vivo evaluation showed sustained blood glucose reductions in both healthy and diabetic rats (up to 40 % and 80 %, respectively), while chronic immunotoxicity studies in mice indicated no toxicity effect. Preliminary efficacy studies in healthy awake pigs following oral capsule administration showed over 20 % absolute bioavailability. 1. Introduction Protein/peptide therapeutics have a prominent role in current and future healthcare market scenarios, mainly because their macromolecular structures make them more specific and potent than small molecules [1]. However, their structural complexity makes them vulnerable in physiological environments and inefficient at overcoming biological barriers to systemic delivery [1,2]. As a result, peptide * Corresponding author at: Center for Research in Molecular Medicine & Chronic Diseases (CIMUS), University of Santiago de Compostela, Campus Vida, 15782 Santiago de Compostela, Spain. E-mail address: [email protected] (M.J. Alonso). Contents lists available at ScienceDirect Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel https://doi.org/10.1016/j.jconrel.2024.11.016 Received 30 March 2024; Received in revised form 13 October 2024; Accepted 7 November 2024 Journal of Controlled Release 377 (2025) 17–36 Available online 17 November 2024 0168-3659/© 2024 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/bync-nd/4.0/ ). therapeutics are currently administered mainly via injection [3]. which limits their use in chronic treatments. The oral route is by far the most useful and acceptable modality of drug administration [4]. Consequently, significant efforts have been devoted to overcoming each of the major barriers identified for oral protein delivery, namely, the harsh gastrointestinal environment, the mucus layer and the underlying epithelial barrier [5]. The strategies developed thus far include the use of protease inhibitors and/or permeation enhancers, the chemical modification of peptides for tuning their physicochemical profiles [6], and a wide range of polymeric-, lipidand complex-based nanocarriers [3,7]. Furthermore, technologies previously applied in other fields have been explored for oral-route applications, such as ionic liquids [8] and iontophoresis [9], while complex microneedle capsule designs have emerged [10–14]. Nevertheless, success has been elusive thus far; there has been extensive preclinical research but currently very few technologies in early clinical evaluation [5,7], and only two formulations, based on permeation enhancers, have been recently approved for commercialization [15,16]. The ultimate remaining obstacle is low oral bioavailability along with high intraand intersubject response variability [7]. On the other hand, translational aspects, such as biomaterial quality and safety, robust design performance, conversion into a final solid dosage form, production technology scalability and cost/benefit balance frequently remain underrated, hampering clinical translation [7,17]. In order to overcome both biological and technological barriers, this work focused on designing a new generation of oral protein nanomedicines inspired by physiological phenomena. Specifically, our inspiration derived from the observation of natural insulin-Zn complexes in the pancreatic storage deposits [18]. This extensively studied protein–biometal interaction [19,20] has been widely exploited for prolonging insulin release following parenteral administration, leading to the co-addition of Zn salts to encapsulate insulin in microand nanocarriers [21–24], where an excess of Zn decreased the solubility of insulin [24]. However, the potential of such an interaction has never been exploited as a nanocarrier in itself, and more specifically for the formation of ultrasmall (below 100 nm), orthogonally shaped insulin-Zn nanocrystals, and their use for oral insulin delivery. Hence, herein, we report a potential nanomedicine candidate consisting of tunable insulinZn orthogonal nanocomplexes endowed with the capacity to overcome the biological barriers associated with the oral route. To enhance their stability and promote interaction with enterocytes, the nanocrystals were coated with a bilayer of amphiphilic materials, specifically lauroyl arginate ester (LAE) and polyethylene glycol stearate (PEGst). This bilayer was designed to mimic the phospholipid architecture of a cell membrane while exposing a neutral hydrophilic surface. The nanocarriers were evaluated in terms of their key physicochemical properties and colloidal stability, controlled release and protection against enzymatic degradation in biologically relevant media. Additionally, the freeze-dried form of the nanocarriers was evaluated to assess the preservation of nanocarrier properties and the bioactivity of the processed protein. Furthermore, in vivo efficacy studies were conducted in both healthy and diabetic rat models and a 28-day chronic immunotoxicity study in mice was performed. In a final step, an enteric capsule dosage form containing the freeze-dried powder of the formulation was administered to healthy awake pigs and the insulin bioavailability was determined. 2. Results and discussion 2.1. Rational design and production of enveloped insulin-Zn nanocomplexes The design was based on the hypothesis that the new nanoformulation would protect insulin from intestinal enzymes, navigate through the mucus layer and facilitate the interaction with the underlying epithelium. The key quality attributes of a targeted insulin nanocarrier include a reproduceable non-spherical nanoscale size, high protein association and loading, sustained release, colloidal stability in intestinal fluids, mucodiffusive character and the ability to promote the interaction with the epithelium. Additionally, the technological requisites include biomaterial safety, production scalability and reproducibility, and the generation of a final solid dosage form suitable for oral administration. Given the required insulin dose and the dilution in the orally administered dried form, significant attention was focused on achieving a high insulin loading (quantity of insulin/quantity of nanocarrier). To this aim, our bioinspiration relied on the insulin-Zn complex naturally occurring in the pancreas and involved in physiological insulin metabolism [25]. This interaction has served as the basis for some sustainedrelease formulations already on the market [21,23,26], and Zn salts and insulin mixtures have been widely employed as encapsulated materials in microand nanocarriers [21–23] for protein aggregation, thus favoring insulin encapsulation and delaying its release. However, the technological approach adopted here is radically different. Indeed, the aim was to design and develop hybrid orthogonal-shaped insulin-Zn nanocrystals stabilized by a bilayer of functional biomaterials with penetration enhancing properties. Such configuration was achieved in a first stage by adopting a bottom-up methodology, that allowed to produce structured insulin-Zn nanocomplexes with tunable physicochemical properties controlled by selected process conditions. We identified the following parameters as critical for achieving optimal nanosystem properties: i) the optimal insulin:Zn molar ratio was 1:6, ensuring the complete hexameric complexation of the protein and maximized association (Fig. 1A); ii) the optimal incubation media pH was 5.1, resulting in a low particle size (<100 nm) and polydispersion index (PDI) of <0.3 (Fig. 1B, C, Table S1), which was attributed to optimized protein ionization and subsequent interaction with Zn cations; and iii) stabilization of the complexation step at 4 ◦C, leading to a reduced particle size and more homogenous system crystallization (Table S2). The nanocomplexes presented an ~80 nm size, a 0.3 PDI and a negative zeta potential (ZP) (Fig. 1E). Their small size was expected to favor intestinal cell interactions and mucodiffusion [27], and the negatively charged surface was attributed to negatively charged insulin residues at the final pH of 6.8 of the nanocomplex suspension media [28]. This is consistent with the hexameric structure of the insulin-Zn complex, where Zn ions are found in an inner axial position coordinated with histidine side chains [29]. A remarkable association efficiency of close to ~100 % with a final loading of over 90 % was obtained (Fig. 1E). Such exceptional drug loading is crucial for achieving the required oral dose after final packaging into oral solid dosage forms. Transmission electron microscopy (TEM) imaging (Fig. 1F) interestingly revealed a population of non-spherical polygonal nanostructures with a significantly smaller size of approximately 50 nm and lower polydispersion compared to dynamic light scattering (DLS) analysis. The orthogonal shape of the structures was attributed to the crystal nature of the pure insulin-Zn complex [30]. This shape was considered to be the cause of the overestimation of the particle size and polydispersity values obtained by DLS analysis, as this technique is adapted to spherical particles [31]. Nanoparticle geometry has been recently identified as a parameter with substantial impact on oral uptake and transport [32], influencing the retention time in the gastrointestinal tract [33], mucus permeation, cellular uptake, intracellular processing and transmembrane transport [33–35]. Overall, the improved performance of non-spherical particles has been attributed to their larger contact surface area [32], and modified Brownian movement, rotation around the mucus networks and shear flow [34,36]. 2.2. Surface envelopment of the nanocomplexes with a cosurfactant bilayer Next, the nanocomplexes were enveloped by a hydrophilic neutral surfactant bilayer to provide colloidal stability, improved mucodiffusion M. Dur´ an-Lobato et al. Journal of Controlled Release 377 (2025) 17–36 18 Fig. 1. Bottom-up production of enveloped nanocomplexes. A, pH values adjusted during the production process and resulting physicochemical parameters of the formulations as a function of the employed insulin:Zn ratio. Several ratios [37] were assayed to ensure maximized protein hexameric complexation, and a ratio of 1:6 was finally selected as optimal (over 90 % association). B, pH values in the production process, particle size and PDI measured by DLS of screened formulations as a function of the employed acid-base volume ratios (NaOH 0.1 ɴ: HCl 0.01 ɴ). C, Graphic representation of the particle size and PDI of the formulations as a function of pH at the incubation step. A pH value of 5.1 (NaOH:HCl volume ratio 0.08) was selected for further development, since it allowed us to maintain a small particle size (<100 nm) along with an acceptable PDI value (0.3). D, ZP and PDI values of the nanocomplexes upon addition of increasing concentrations of LAE-PEGst coating, leading to neutral ZP values and increased PDI. E, Physicochemical parameters of formulations before and after coating and upon increasing batch size (10and 100fold) after adapting the production process, validating the scaling-up trial. F, TEM images of plain nanocomplexes. G, TEM images of enveloped nanocomplexes. All measurements and error bars are displayed as the mean values ±SDs (n =3). M. Dur´ an-Lobato et al. Journal of Controlled Release 377 (2025) 17–36 19 [38], protection against enzymatic degradation [39] and enhanced epithelial penetration [40]. For this purpose, a combination of neutral and positively charged amphiphilic molecules was selected, specifically PEGst, a widely employed FDA-approved excipient, and LAE, an arginine-based biologically derived generally-regarded-as-safe (GRAS) excipient [41]. Both molecules were expected to act synergistically based on their reported capacity to interact with cell membranes and alter their permeability [42]. Briefly, PEGst and LAE were sequentially added to a nanocomplex suspension at an 8:1 PEGst:LAE mass ratio, since a higher relative amount of LAE compared to PEGst (lower PEGst: LAE mass ratio) led to nanocomplex aggregation. By increasing the amount of both PEGst and LAE added to the formulation, while maintaining the optimized PEGst:LAE mass ratio, the ZP of the formulation increased to neutral values (Fig. 1D) while the mean particle size was maintained, thus indicating the surfactant interaction with the nanosystem surface. Interestingly, the PDI values increased, particularly when the ZP values attained neutrality (Fig. 1D), due to an excess of surfactant molecules forming micelles. TEM imaging confirmed that the nanocomplex size and morphology remained unaltered after the enveloping step (Fig. 1G). A concentration of 0.8 mg/mL LAE–6.4 mg/mL PEGst in the final nanocomplex suspension was selected on account of the resulting neutral ZP, indicating the complete coverage of the nanocomplex surface. The resulting formulation was successfully freezedried (Fig. S1 and S2). 2.3. Architectural organization of the orthogonal crystalline nanocomplexes enveloped by a surfactant bilayer Freeze-dried samples of the enveloped nanocomplexes were analyzed by X-ray diffraction (XRD), where the widening of the highest intensity peaks in a diffractogram is related to the size of the crystalline domains. Overall, wide peaks or bands attributable to nanocrystalline domains were identified for both trehalose and PVP-mannitol formulation powders (Fig. 2B, C), while control samples presented either no peaks or narrow peaks indicative of nonnanocrystalline domains, altogether suggesting the presence of nanocrystallites in the formulation. The cosurfactant envelope architecture, initially understood as depicted in Fig. 3A based on experimental results, was further investigated. For instance, when only LAE was added to the nanocrystal suspension, the nanoparticles immediately aggregated due to the interaction of positively charged LAE molecules with the negatively charged nanocomplexes. On the other hand, when only PEGst was added to the nanocomplexes, no change in the physicochemical properties of the nanocomplexes was observed. However, when PEGst and LAE were Fig. 2. Characterization of the inner crystalline nanocomplex structure. X-ray analysis was performed to assess whether the nanocomplexes presented the known crystalline structure [30] of the insulin-Zn hexameric complex. A, TEM imaging of nanocomplexes showing inner structural patterns. B, X-ray diffractogram of nanocomplexes freeze-dried with PVP-mannitol (purple line) and control sample of the freeze-dried solution of PVP-mannitol and zinc (green line) at the same concentrations as in the formulations. The diffractogram of the nanocomplexes (purple line) presented several narrow peaks coincident with those of the zinc control sample (green line), which indicated the nonnanocrystalline domains attributed to mannitol. However, the formulation powder also presented wider peaks attributable to nanocrystalline domains, which were not present in the zinc control sample diffractogram, where higher narrower peaks appeared instead (14 to 18◦2Th and 23 to 24◦2Th). C, X-ray diffractogram of nanocomplexes freeze-dried with trehalose (purple) and control samples of freeze-dried trehalose and zinc (green line). The absence of crystalline phases for the control trehalose‑zinc sample (green line) allowed more accurate identification of crystalline domains for the freeze-dried formulation (purple line). The diffractogram of nanocomplexes freeze-dried with trehalose (purple line) presented several wide peaks or bands indicative of nanocrystalline domains, attributed to the nanocomplex core structure. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) M. Dur´ an-Lobato et al. Journal of Controlled Release 377 (2025) 17–36 20 adequately combined (8:1 mass ratio) and added, LAE molecules supposedly interacted with the nanocomplexes, while their aggregation was prevented by the subsequent attachment of PEGst molecules. In fact, the overall neutral ZP of the resultant enveloped nanocomplexes suggested that the hydrophilic PEG portions of PEGst were oriented toward the outer media. The interaction of both surfactants would hypothetically occur through their hydrocarbon chains, constituting a surfactant bilayer (Fig. 3A). To confirm this hypothesis, 1 H NMR analysis (Fig. 3B, S4 and S5) and water-ligand observed via gradient spectroscopy (waterLOGSY) experiments (Fig. 3B and S6) were carried out on the enveloped nanocomplexes, where bulk solvent (H 2 O) protons were selectively saturated, and this magnetization was transferred via cross-relaxation to free molecules in contact with water media. As a result, the resonances of molecular regions interacting with water appeared with opposite signs to those interacting within a structure, such as a nanoparticle [43]. The resulting spectrum (Fig. 3B) clearly showed a 3.71 ppm inverted peak corresponding to the PEG regions of PEGst, confirming their external location on the nanocomplex surface in contact with the aqueous medium and with higher mobility compared to the stearate region. On the Fig. 3. Characterization of the surfactant bilayer envelope. A, Schematic picture showing the hypothesized conformation of the bilayer of amphiphilic biomaterials based on experimental results. B, 1 H NMR and waterLOGSY (water-ligand observed via gradient spectroscopy) analysis of the enveloped nanocomplexes. Detailed 1 H NMR signal assignment and diffusion-ordered spectroscopy (DOSY) experiments disregarding the influence of surfactant molecules not attached to the nanocomplex surface are available in the SI section. The waterLOGSY spectrum clearly showed a 3.71 ppm inverted peak corresponding to PEG regions, while the signal corresponding to bulk aliphatic chains from both surfactants (1.27 ppm) remained noninverted. The observed changes in the signals were proportional to the length of the applied pulse (from 3 to 1 ms, SI section), thus confirming that they were due to the waterLOGSY effect. This observation indicated the external location of the PEG regions and internal location of the aliphatic chains, supporting the hypothesized envelope bilayer architecture. M. Dur´ an-Lobato et al. Journal of Controlled Release 377 (2025) 17–36 21 other hand, the signal corresponding to bulk aliphatic chains from both PEGst and LAE (1.27 ppm) remained noninverted, indicating their internal location within the structure. Altogether, these results supported the hypothesized envelope bilayer architecture, where PEG regions remain oriented toward the external aqueous media, while aliphatic chains from both surfactants are located inside the nanostructure. 2.4. In vitro performance of enveloped nanocomplexes and their potential for scalability and translation The high ionic force and complex composition of intestinal fluids represent one of the barriers for oral delivery, where adequately tuning the nanoparticle surface may drastically determine the capacity of the carriers to maintain their physicochemical properties under physiological conditions [44,45]. The evaluation of our nanocomplexes in biorelevant fluids revealed that their enveloping shell provided adequate colloidal stability [44] (Fig. 4A), while protection against enzymatic degradation (Fig. 4B) and sustained release (Fig. 5C) were attributed to Zn because of its enzymatic inhibitory [46] and insulin complexation [24,47] properties, respectively. Additionally, the batch size was increased by 100-fold (Fig. 1E), indicating the successful identification and control of key process control parameters. Notably, scaling-up potential constitutes a highly important added value that current nanotechnology-based drug delivery approaches rarely have [48,49]. In addition, given the particularities of the insulin-Zn interaction, the possibility of translating this technology to other biomacromolecules was uncertain. Thus, we explored the nanocomplexation of an RNA model molecule with biometals by adapting the production procedure and the biomolecule–biometal mass ratios. Nanocomplexes with tunable surface charges coated with several polymers were obtained (Table S5, S6 and S7), offering possibilities for the further modulation of nanocarrier properties. While the preliminary character of these studies is noted, the results suggest that producing coated nanocomplexes of biometals and biomacromolecules is feasible, which opens up new avenues for formulation development based on this strategy. Particularly, exploring combinations of biomacromolecules and biometals between which specific physiological interactions are known to take place could be an approach of interest. Fig. 4. In vitro physicochemical characterization of the coated nanocomplexes. A, Colloidal stability of enveloped nanocomplexes as a function of average particle size and count rate measured by DLS (mean value ±S.D. (n =3)). The formulation proved to be stable for up to 4 h, as opposed to the noncoated nanocomplexes, which immediately aggregated (data not shown). B, Protection of insulin against pancreatic degradation as a function of remaining nondegraded insulin. Both coated and noncoated formulations displayed similar profiles, increasing insulin t 1/2 by 4-fold with respect to that of free insulin (mean values ±SDs (n =3)). C, Insulin release from coated nanocomplexes. A sustained release over the incubation time (up to 4 h) was displayed (mean values ±SDs (n =3)). D, Insulin bioactivity evaluation in pSynSRE-T-luc-transfected cells as a function of luciferase activity over a transfection control promoter (beta-galactosidase). Fresh solutions of free insulin of increased concentrations as a positive control induced dose–response luciferase activity in pSynSRE-T-luc-transfected cells, while no statistically significant effect was observed in pSynSRE-Mut-T-luc-transfected cells, bearing four-point mutations in the insulin-responsive element, therefore validating the assay. Reconstituted freeze-dried coated nanocomplexes at insulin concentrations equivalent to those of the positive control resulted in the specific activation of the insulin receptor, shown as delta luciferase, compared to the control. The luciferase activity values obtained were in the same range as those obtained with fresh insulin solutions, confirming the preserved bioactivity of insulin after formulation processing and freeze-drying. Cell study results are presented as the means ±SDs, n =5 independent experiments with 6–8 replicates per condition in each (one-way ANOVA followed by Tukey’s multiple comparison test; significance levels compared to the control *p ≤0.05; ***p ≤0.001, ****p ≤0.001). M. Dur´ an-Lobato et al. Journal of Controlled Release 377 (2025) 17–36 22 An ultimate constraint specific to oral administration arises from the necessity to generate a solid dosage form as the end product. This usually requires the freeze-drying of formulations produced in aqueousbased media, which may compromise the preservation of the initial physicochemical properties of nanoparticles as well as the bioactivity of the loaded drug [17,50]. To assess whether the lyophilized nanocomplexes maintained their key properties, samples from freeze-dried, small and large batches were evaluated in vitro, showing preserved insulin bioactivity on human INSR in human HepG2 hepatocytes [51,52] (Fig. 4D) and physicochemical properties (Fig. S2) after 2 months of storage. Next, the freeze-dried nanocomplexes were loaded into gelatin capsules along with a composition intended to generate carbon dioxide (CO 2 ) gas bubbles that would promote a fast resuspension of the nanocomplexes and their projection toward the intestinal wall, comprising tartaric acid (TA), sodium bicarbonate (SBC) and Poloxamer 199 (P188). This acid-base reaction has been employed in a similar approach using citric acid and SBC to promote tablet disintegration [53,54]. It was also employed to promote the in situ assembling and/or stabilization of nanostructures [55–57], where intestinal permeation was ultimately dependent upon the action of permeation enhancers such as trimethyl chitosan (TMC) [54] or sodium dodecyl sulphate (SDS) [55–57] or the performance of nanocarriers [53] in this composition, TA and SBC were selected to generate the CO 2 bubbles, P188 was employed as surfactant to decrease surface tension and therefore increase the intensity of the reaction effect [58], and intestinal permeation was expected to be attained on account of the rationally designed nanocomplexes. The capsules were further coated with an enteric polymer to prevent gastric degradation, and the integrity of the coating and the triggering of the gas producing reaction after coating dissolution were assessed in simulated gastric and intestinal fluids respectively. In the latter, the acid-base reaction was observed to take place within 2 min upon coating dissolution, with an intense gas production (Fig. S7). The capsules were stored at RT protected from humidity up to 1.5 months before administration to pigs, when their content homogeneity and drug bioactivity was previously evaluated by s.c. administration to healthy rats. 2.5. In vivo interaction of enveloped nanocomplexes with the intestinal wall The nanocarriers were radiolabeled with technetium-99 m ( 99m Tc) via linkage to the PEG chains on the nanocarrier envelope [59], followed by oral administration to rats and subsequent single photon emission computed tomography-computed tomography (SPECT-CT) evaluation [60]. Prior to the study, the stability and lack of release of the radioisotope in simulated gastric fluid (SGF) were assayed (Fig. S8 and S9). The results (Fig. 5) showed that, while the free technetium control (sodium pertechnetate solution) remained localized in the stomach, the Fig. 5. Biodistribution of technetium-99 m radiolabeled nanocarriers in rats. A, % Radioactivity detected per organ after oral administration of the radiolabeled formulation. B, % Radioactivity detected per organ after oral administration of [ 99m Tc]‑sodium pertechnetate (control). C, SPECT-CT images of the rats after oral administration of the radiolabeled formulation and free 99m Tc control; s =stomach; I =intestine; c =cecum; cl =colon; r =rectum. The radiolabeled carriers remained mostly in the small bowel (up to 2 h) and in the cecum (4 h, 8 h and 11 h) for the duration of the study, whereas the free technetium control remained in the stomach. The results are presented as the mean values of n =4. M. Dur´ an-Lobato et al. Journal of Controlled Release 377 (2025) 17–36 23 radiolabeled carriers remained mostly in the small bowel (up to 2 h) and cecum (4 h, 8 h and 11 h) for the duration of the study. This prolonged intestinal transit profile is in agreement with similar experiments carried out with nanoparticulate formulations [59,61] and is supported by the lower transport velocity in distal segments of the gastrointestinal (GI) tract [62] and the fact that isoflurane anesthesia is known to decrease GI motility and prolong transit times due to muscle relaxation [63]. Overall, the 99m Tc-labeled formulation significantly interacted with the intestine, having been retained for up to 26 h, in contrast to the technetium solution control, which remained mostly in the stomach. This enhanced intestinal interaction of the formulation could be attributed to the nanocarrier features, namely, i) their small size for favoring cellular interactions and mucodiffusion [32]; ii) a PEGylated surface for facilitating mucodiffusion [38]; and iii) the presence of LAE, a surfactant with a shown capacity to disrupt cellular membranes [42], hypothetically potentiated by PEGst. For instance, PEG-fatty acid esters have shown enhanced skin drug penetration [64], while other nonionic polyoxyethylated surfactants have been widely studied as topical ocular penetration enhancers [65]. Interestingly, no systemic absorption of 99m Tc was observed. This lack of absorption of the nanosystem was expected based on previous literature pertaining to nanoparticle transport across the intestinal wall. The key issue was whether the enhanced interaction of the nanosystem with the intestinal wall may be translated into greater insulin absorption. The absence of the systemic absorption of the nanosystem itself may be considered a positive feature in terms of pharmaceutical purposes, since it would seem to imply reduced toxicological concerns [61]. 2.6. Pharmacological performance of the enveloped nanocomplexes The enveloped nanocomplex efficacy was first evaluated following subcutaneous (s.c.) administration to healthy rats [44] (Fig. 6A). Fig. 6. In vivo efficacy evaluation in small and large animal models. Efficacy results are displayed as % blood glucose values with regard to the baseline values at time 0 h. Plasma insulin results are displayed as plasma insulin concentrations (ppb (ng/L)). A, Blood glucose after s.c. administration of freshly prepared and freezedried reconstituted formulation, vs. insulin control to healthy rats. B, Blood glucose after s.c. administration of insulin extracted from enteric capsule powder after 2 months of storage vs. insulin control to healthy rats. C, Blood glucose profile after IJ administration of the formulation (n =5) vs. an insulin solution (negative control) (n =6) to healthy rats. D, Blood glucose profile after IJ administration of the formulation (n =9) vs. an insulin solution (negative control) (n =8) to diabetic rats. E, Blood glucose profile after oral administration of enteric capsule formulation vs. oral administration of PBS (negative control) and SC administration of an insulin solution (positive control) (n =3) to awake domestic pigs. Oscillations in levels from 6 h onward were due to i.v. administrations of glucose in response to hypoglycemic shock signs. F, Plasma insulin analysis by LC–MS resulting from the administration of the enteric capsule vs. negative and positive control to awake domestic pigs. No plasma samples were withdrawn after 6 h due to concern about the healthy state of the animals. All results are displayed as the mean values ± SEMs. A two-way ANOVA followed by a Holm–Sidak multiple comparison test was applied; significance levels compared to the control *p ≤0.05; **p ≤0.01; ***p ≤ 0.001, ****p ≤0.001). M. Dur´ an-Lobato et al. Journal of Controlled Release 377 (2025) 17–36 24 Subsequently, the formulation was assayed for its efficacy in vivo by intestinal direct injection [44,66–69], which avoids the variability associated with gastric transit times, both in healthy and diabetic rats [69]. The healthy rat model maintains the physiological insulin autoregulation mechanisms [70] and avoids the high variability associated with STZ diabatization [68,71]. As a counterpart, only modest responses are to be expected [44,68], and fasting periods need to be decreased [69]. Hence, the intrajejunal (IJ) administration of the coated nanocomplexes to healthy rats (Fig. 6C) yielded an expected modest, but significant, 42 % decrease in the initial blood glucose levels. Importantly, the effect was maintained for up to 6 h. Similar responses were reported for other delivery carriers [68,72,73]. This finding confirmed that the insulin associated with the nanocarrier was absorbed in a sufficient amount to exert a prolonged blood glucose reduction response [68,70,74]. Hypothetically, this could be due to the enhanced nanostructure interaction with and penetration into the intestinal epithelium, as noted for the radiolabeled formulation. On the other hand, β-cell deficiency in the diabetic model allows low amounts of absorbed insulin to elicit a markedly decreased glucose response [68], and hence, potential effects of an oral formulation would have a higher chance of being identified. Following direct IJ injection of the formulations, a remarkable blood glucose decrease of up to 80 % was obtained (Fig. 6D), with clear differences regarding the negative control even at the end of the measurement period (8 h). This high and prolonged response was comparable to or higher than those reported in references in the field [75–77]. As expected, the results showed a clearer effect in comparison with those of the healthy rat model, further confirming insulin intestinal absorption. Along with biodistribution studies, the results indicated that the nanocomplexes enabled insulin absorption without nanocarrier translocation. 2.7. Preliminary assessment of the lack of chronic immunotoxicity in mice Chronic exposure to insulin treatment requires the consideration of Fig. 7. Preliminary chronic immunotoxicity evaluation of the formulation in mice. The results from FACS analysis of the immune inflammatory status of lymphoid organs after 28 days of treatment. A, Percentage of myeloid cells in spleens following chronic administration of formulation vs. insulin for up to 28 days. Different myeloid subpopulations (inflammatory monocytic MDSCs (MO-MDSCs), granulocytic-MDSCs (PMN-MDSCs), macrophages and dendritic cells (DCs)) were evaluated for each treatment group. B, Evaluation of DC maturation after formulation vs. insulin administration in terms of percentage. C, Evaluation of DC maturation in terms of fluorescence intensity for each treatment group. D, Percentage of lymphocytes after chronic administration of formulation vs. insulin; percentage of T lymphocytes CD3 total, CD4 or CD8. The T-cell percentages from each treatment group were similar. E, Immunohistochemistry of intestinal regions after formulation administration with CD3 and CD68. In the Fig., colocalization with DAPI is reported (magnification 20×with dry objective). No increases in lymphocyte (CD3) or macrophage (CD68) signals were observed at the level of the small intestine after chronic administration of insulin (data not shown) or the nanocarriers. Bar graphs represent the means ±SDs; n =5 mice per group. *p ≤0.05; **p ≤0.01; ***p ≤0.001, by one-way ANOVA. M. Dur´ an-Lobato et al. Journal of Controlled Release 377 (2025) 17–36 25 s.c. administration to rats along with free insulin for comparison [44]. To this aim, male Sprague Dawley rats (250 g) were fasted for 4 h before the study with free access to water. A solution of free insulin, freshly prepared formulation and previously freeze-dried, reconstituted formulation were administered at a dose of 1 IU/kg. Before administration (0 h), blood samples were withdrawn from the tail vein, and initial blood glucose levels were measured with a hand-held glucometer (Glucocard™ G +meter, Arkray Factory, Japan) and monitored every hour thereafter. 4.2.15. In vivo efficacy studies in pigs In vivo efficacy studies in pigs were conducted on 12to 14-week-old neutered male domestic pigs (Sus scrofa domesticus) (15–20 kg) from the Research Institute of Animal Breeding and Nutrition, Herceghalom, Hungary. They were housed in individual caging (1 m ×2.5 m), fed a standard powder diet for domestic pigs twice a day with access to water ad libitum, and allowed to acclimatize for 1 week prior to the study. The studies were designed in accordance with accepted pharmacological principles to meet the requirements of the principles of Hungarian Act 1998: XXVIII regulating animal protection (latest modified by Act 2011 CLVIII) and Government Decree 40/2013 on animal experiments; EEC Directive 2004/27/EC of the European Parliament and of the Council of March 31, 2004 amending Directive 2001/83/EC on the Community code relating to medical products for human use (Official Journal L-136, 30/04/2004, pp. 34-57). Animal handling and care were conducted according to the Guide for the Care and Use of Laboratory Animals, NRC, 2011 and Directive 2010/63/EU (European Parliament and Council, took full effect on 1 January 2013). Special permission for animal studies under number PEI/001/3948-6/2014 was issued by the Pest County Government Office of Food Safety and Animal Health Directorate. All procedures carried out on animals were approved by the local ethical committee of Semmelweis University. 4.2.15.1. Surgical procedure. On the day of instrumentation, animals were preanesthetized intramuscularly with Calypsol/Dexdomitor (2–3/ 0.2-0.3 mL, based on body weight) injections in the stalls to avoid stress and were transported to the operating room sedated. The anesthesia was maintained using isoflurane inhalation narcosis (2–3 %) with oxygen through a muzzle mask. Animals were allowed to breathe spontaneously. Their respiration was monitored using a pulse-oximeter (fixed on the tail), measuring the blood oxygen saturation (SpO2), and their temperature was measured rectally (both parameters were monitored by InnoCare-T Anesthesia Monitor - Innomed Medical Co. Hungary). A capnograph was connected to the muzzle mask to monitor etCO2 (% of end-tidal CO2) and the respiratory rate (CAP10 Medlab, Medlab Medizinische Diagnosegerate GmbH, Karlsruhe, Germany) during the operation. The pigs were instrumented with a central venous single-lumen catheter set (BBraun Certofix Mono V330, 5 Fr, 30 cm, 16G, Cat. 4,160,290 N) introduced into the vena cava cranialis through the vena maxillaris externa ➔ vena jugularis externa. The catheters’ tails with sampling ports were led out subcutaneously through the back, followed by fixation at the exit sites with sutures. All incision areas were shaved and disinfected by liberal application of povidone‑iodine 10 % prior to the operation and closed after the fixation of cannulas. The cannulas were washed daily with heparin-saline during the recuperation period. Following the operation, the pigs were transported back to the stalls and observed until spontaneous awakening. Preventive antibiotic treatment (Syvaquinol at a dose of 2.5 mg/kg body weight) and analgesia (rheumocam at a dose of 0.4 mg/kg body weight) were applied. 4.2.15.2. Formulation administration and sampling. Prior to formulation administration, the pigs were fasted overnight prior to per os administration of the enteric capsules with a plunger applicator. Before and following administration, pre- (0’, control) and posttreatment whole blood samples were collected at predetermined intervals for blood cell and blood glucose analysis, and K3EDTA plasma samples were collected for insulin analysis. Convulsions and hypoglycemic shock signs 6 h after administration in response to the unexpectedly intense effect of the formulations were treated by i.v. injections of 10 % w/v glucose. The animals were further monitored until their glycemic profiles stabilized. K3EDTA samples were centrifuged immediately after collection (1500g, 10 min at 4 ◦C), aliquoted, frozen at −20 ◦C and subsequently transferred to −80 ◦C for storage until analysis. Whole blood samples were analyzed immediately by a Diatron Vet hematology analyzer (Diatron MI PLC, Budapest, Hungary) for the following parameters: white blood cells (WBC), red blood cells (RBC), hemoglobin (Hgb), platelets (PLT), lymphocytes (Ly) and granulocytes (Gr). After completion of the study, the pigs were euthanized by i.v. administration of Euthasol (1 mL/10 kg body weight) followed by i.v. injection of cc. KCl solution (15 mL/10 kg body weight). Euthanasia was confirmed before disposing the carcass by observing that there was no respiratory movement for at least 3 min and that the heartbeat had ceased. 4.2.15.3. Pharmacokinetic evaluation by LC–MS. The insulin content in the plasma samples obtained from the pig studies was analyzed by liquid chromatography/tandem mass spectrometry (LC/MS) following a sample extraction and analysis methodology described elsewhere [100] with some modifications. Stock solutions of bovine insulin (internal standard, IS) and human insulin for the construction of calibration curves were initially prepared in HCl 0.01 N at 5 mg/mL and subsequently diluted in a freshly prepared premixed solvent of ultrapure water/methanol (80/20) with 0.1 % v/v acetic acid. Plasma samples of 250 μ L were added to 25 μ L of 500 ppb IS stock solution; 0–25 μ L human insulin stock solutions (25 ppb and 100 ppb) were used when applicable for the construction of calibration curves; and up to 100 μ L of the said solvent was used for a constant final volume of 350 μ L in all samples. Next, 750 μ L of an acetonitrile–methanol mixture (1:1 v/v) was added to each sample as a precipitant. The samples were vortexed and maintained under stirring in an incubator (Heidolph Instruments GmbH & Co. KG, Schwabach, Germany) for 10 min (37 ◦C, 600 rpm) and then centrifuged (21,000 rcf, 15 ◦C, 10 min, Eppendorf Centrifuge 5430 R, Eppendorf Ib´ erica S.L.U., Madrid, Spain). The obtained supernatant was filtered through 0.22 μ m PVDF hydrophilic filters (4 mm, SLGVR04NL, Merck Millipore, Germany) and vacuumed at 37 ◦C until completely evaporated with the aid of a SpeedVac. Finally, the dry samples were reconstituted with 90 μ L of a freshly prepared premixed solvent of ultrapure water/methanol (80/20) with 0.1 % v/v acetic acid and subsequently analyzed. All solvents employed were of LC/MS grade with the exception of acetic acid, which was USP, pH. Eur. reagent grade, and lowbinding materials and recipients were used at all times. The UPLC system consisted of an Acquity UPLC® H-class system (Waters Corp, Milford, USA) and a column compartment (Acquity UPLC® CSH™ C18 (2.1 ×50 mm, 1.7 μ m; Part number 186005296, Serial no. 01463731715175, Cienytech, Santiago de Compostela, Spain). The experimental analytical conditions were as follows: the mobile phase consisted of 0.1 % formic acid aqueous solution (A) and 0.1 % formic acid acetonitrile solution (B). A gradient program was used as follows: 80 % to 63 % A from 0 to 3.8 min; 63 % to 2 % A from 3.8 to 3.9 min; 2 % A maintained from 3.9 to 4.9 min; 2 % to 80 % A from 5.9 to 5 min, and kept constant up to 7 min to allow the system to equilibrate. The total run time was 7 min. The column temperature was maintained at 40 ◦C, and the autosampler was thermostatized at 10 ◦C. The injected volume was 50 μ L. Under these conditions, human insulin was eluted at 3.34 ±0.02 min, and bovine insulin was employed as an internal standard at 3.19 ±0.02 min. The UPLC system was coupled to a Xevo® Triple Quadrupole Detector (TQD) (Waters Corp, Milford, USA) with an electrospray ionization (ESI) interface. A temperature of 525 ◦C was selected as the source temperature, and 60 ◦C was selected as the desolvation temperature. The capillary voltage was 2.0 kV, and the cone M. Dur´ an-Lobato et al. Journal of Controlled Release 377 (2025) 17–36 32 voltage and collision energy were set at 50 V and 3 V, respectively. Nitrogen was used for desolvation and as the cone gas at flow rates of 1000 L/h and 80 L/h, respectively. Argon was used as the collision gas. Mass spectrometric detection was operated in positive mode and set up for multiple reaction monitoring (MRM) to monitor the human insulin transitions of m/z 969.0 >> 1133.0 (cone voltage 50 V, collision energy 25 V); 1162.6 >> 143.2 (cone voltage 60 V, collision energy 40 V); 1162.6 >> 219.0 (cone voltage 60 V, collision energy 50 V); 1162.6 >> 226.0 (cone voltage 60 V, collision energy 40 V), and bovine insulin transitions of m/z 956.5 >> 1114.8 (cone voltage 50 V, collision energy 18 V); 956.6 >> 315.2 (cone voltage 50 V, collision energy 50 V); 956.6 >> 1121.2 (cone voltage 50 V, collision energy 18 V); and 1147.5 >> 315.5 (cone voltage 70 V, collision energy 52 V). Calibration curves were constructed over a range of 0.25–5 ppb, with an LOQ of 0.5 ppb. Data acquisition and analysis were performed using TargetLynx v4.1 software (Waters Corp., Milford, USA). 4.2.16. Production and physicochemical characterization of biometal–tRNA nanocomplexes Nanocomplexes of tRNA-Zn were prepared by adding a 20 mg/mL zinc acetate dihydrate aqueous solution over 500 μ L of a 0.1 mg/mL tRNA solution in HCl 0.01 ɴ to attain a charge ratio (Zn mol–nucleotide) of either 5:1 or 10:1. The mixture was maintained under magnetic stirring at 300 rpm while 0.1 ɴ NaOH was added to adjust the pH to 7.4. Next, the mixture was incubated at 4 ◦C for 3 h. Subsequently, the mixture was allowed to temper at RT for 4 min, vortexed for 10 s and then characterized in terms of particle size and ZP. Regarding the tRNA–Fe nanocomplex production, two types of nanocomplexes were produced, namely, Type I nanocomplexes, exhibiting a negative surface charge and ability to be coated with a positively charged material, and Type II nanocomplexes, exhibiting a positive surface charge and ability to be coated with a negatively charged material. To produce Type I nanocomplexes, a 0.1 mg/mL tRNA solution in RNAse-free water and a 0.246 mg/mL ferric chloride hexahydrate solution in acetate buffer (100 mᴍ) (pH 6) were prepared. Next, the ferric chloride hexahydrate solution was added dropwise over the tRNA solution under stirring (500 rpm) at a 1:1 v/v ratio, and the mixture was stirred for 10 min. To coat the resulting nanocomplexes, the obtained suspension was immediately added dropwise over a 0.1 mg/mL solution of the selected positively charged polymer (chitosan or polyarginine (PARG)) under stirring (500 rpm) at a 1:1 v/v ratio, and the mixture was stirred for 10 min. To produce Type II nanocomplexes, a 0.1 mg/mL tRNA solution in RNAse-free water and a 2.216 mg/mL ferric chloride hexahydrate solution in acetate buffer (100 mM) (pH 6) were prepared. Next, the tRNA solution was added dropwise over the ferric chloride hexahydrate solution under stirring (500 rpm) at a 1:1 v/v ratio, and the mixture was stirred for 10 min. To coat the resulting nanocomplexes, the obtained suspension was immediately added dropwise over a 2 mg/mL solution of the selected negatively charged polymer ((HA), CS, PEG(5 k)- PGA(10) or PSA) under stirring (500 rpm) at a 1:1 v/v ratio, and the mixture was stirred for 10 min. The particle size distribution and PDI were determined by DLS, and the ZP was determined from the electrophoretic mobility values obtained by LDA using Malvern Zetasizer equipment (NanoZS ZEN 3600, Malvern Instruments, Worcestershire, UK) equipped with a red laser light beam (λ =632.8 nm). The formulations were directly measured without dilution at 25 ◦C with at least three different batches and triplicate analysis of each batch. 4.2.17. Statistics All experiments were performed at least in triplicate, and data are presented as the means ±standard deviations (SDs) in physicochemical studies and the standard errors of the mean (SEMs) for in vivo studies. Statistical analysis was carried out using one-way analysis of variance (ANOVA) with a multiple comparisons test (GraphPad Prism, GraphPad software Inc., CA, USA). All other analyses were performed using oneway analysis of variance (ANOVA). The level of significance was set at probabilities of *p <0.05, **p <0.01, ***p <0.001 and **** p < 0.0001. Funding statement This work was supported by the European TRANS-INT Consortium, which received funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement NO. 281035. M. Dur´ an-Lobato acknowledges a postdoctoral fellowship (Contrato de Acceso al Sistema Espa˜ nol de Ciencia, Tecnología e Innovaci´ on (grant number USE-19533-Y)) granted by “VI Plan Propio” from the University of Seville. CRediT authorship contribution statement Matilde Dur´ an-Lobato: Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Sulay Tovar: Writing – review & editing, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization. Juan Cu˜ narro: Validation, Investigation, Formal analysis, Data curation. Rocío Ramos-Membrive: Validation, Investigation, Formal analysis, Data curation. Iv´ an Pe˜ nuelas: Writing – review & editing, Supervision, Resources, Methodology, Investigation, Funding acquisition, Conceptualization. Ilaria Marigo: Writing – review & editing, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Federico Benetti: Writing – review & editing, Validation, Resources, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Miguel Chenlo: Validation, Supervision, Resources, Methodology, Formal analysis, Data curation. Clara V. ´ Alvarez: Writing – review & editing, Supervision, Resources, Methodology, Funding acquisition, Conceptualization. Vashegyi Ildik´ o: Validation, Investigation, Formal analysis, Data curation, Conceptualization. Rudolf Urbanics: Validation, Supervision, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. J´ anos Szebeni: Writing – review & editing, Supervision, Resources, Funding acquisition, Conceptualization. María Jos´ e Alonso: Writing – review & editing, Supervision, Resources, Funding acquisition, Conceptualization. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data availability Data will be made available on request. Acknowledgments The authors are especially grateful for Dr. ´ Alvaro Antelo for his assistance with LC–MS method development and analysis, Dr. Manuel Martín-Pastor (Nuclear Magnetic Resonance Unit, RIADT, Universidade de Santiago de Compostela) for his assistance with NMR experimental design and interpretation, Dr. Bruno Dacu˜ na Mari˜ no for his help with Rx experimental design and interpretation (X-ray Unit, Crystalline Powder Section, RIAIDT, Universidade de Santiago de Compostela, Maria Suarez-Fari˜ na for the technical help with cell-based bioactivity assays, and Sanofi for providing the human insulin peptide as well as assistance with peptide handling and analysis. M. Dur´ an-Lobato et al. Journal of Controlled Release 377 (2025) 17–36 33 Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.jconrel.2024.11.016. References [1] A.N. Zelikin, C. Ehrhardt, A.M. 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