Delivery of Monoclonal Antibodies to the Brain: The Impact of Nanocarrier Structure

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López-Estévez, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6558955/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Sep, 2025 Read the published version in Drug Delivery and Translational Research → Version 1 posted 5 You are reading this latest preprint version Abstract Monoclonal antibodies (mAbs) are promising therapeutic agents for neurological disorders due to their high specificity. However, their clinical application is significantly hindered by their poor transport across the blood-brain barrier (BBB) and their limited diffusion within the brain parenchyma. While significant efforts have been oriented to tackle the first barrier, the challenge of efficient brain diffusion remains largely underexplored. To address this, we have developed and evaluated two structurally distinct nanosystems for mAb delivery to the brain: PEGylated polyglutamic acid nanocapsules (PGA-PEG NCs) and PGAC14-based nanoassemblies (PGAC14 NAs). Both formulations encapsulated efficiently the model mAb bevacizumab (BVZ) while they exhibited different physicochemical properties. Namely, PGA-PEG NCs displayed a size of 80 nm and a neutral zeta potential, whereas PGAC14 NAs featured an ultra-small size of 40 nm and a negative surface charge. After assessing their diffusion capacity using immunofluorescence, we concluded that PGAC14 NAs exhibited the highest brain diffusion. This was likely driven by their small size and negative charge, along with a selective ability to interact with and deliver BVZ intracellularly to neuronal cells upon intraparenchymal administration. These findings provide key insights into optimizing nanocarrier design for improved mAb delivery to the brain. Monoclonal antibodies nanomedicine brain delivery diffusivity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. INTRODUCTION Monoclonal antibodies (mAbs) hold immense potential for treating neurological disorders, as evidenced by the recent FDA approval of three mAb-based therapies for Alzheimer disease and numerous ongoing clinical trials [1,2]. However, their effective delivery to the brain faces substantial challenges [3,4]. While much of the current research focuses on overcoming the blood-brain barrier (BBB), a critical obstacle for central nervous system (CNS) therapies, relies on the limited diffusivity of mAbs through the brain parenchyma. These barriers limit mAbs' ability to reach their target sites within the brain, thereby reducing therapeutic efficacy. To address these issues, nanotechnology has emerged as an enable technology for enhancing the delivery and distribution of therapeutic molecules within brain tissue [5–7]. While most studies have focused on the functionalization of nanocarriers for enhancing their transport across the BBB, [8–11], the number studies addressing the diffusion of mAbs-associated nanocarriers across the brain tissue remains limited. Pioneering work by Hanes, Suck and co-workers has shown that polymeric nanoparticles, particularly those incorporating polyethylene glycol (PEG), exhibit an efficient diffusion from the injection site into surrounding brain tissue [11–13]. This enhanced nanoparticle mobility within the brain parenchyma was attributed to their reduced opsonization and interactions with extracellular matrix components [14]. In this context, block copolymers composed of a biodegradable polypeptide blocks, such as poly(glutamic acid) (PGA) or poly(lysine) (PLL), combined with hydrophilic polymers, mostly PEG, have also emerged as promising polymeric therapeutics consisting of micellar formulations with various biomedical applications [15,16]. Polypeptides, including PGA, possess advantageous properties such as biocompatibility, biodegradability, non-immunogenicity, and low toxicity, making them attractive candidates for drug delivery systems [17,18]. Some examples of their potential are cisplatin-loaded PEG-PGA micelles, which have progressed to Phase III clinical trials for the treatment of advanced pancreatic cancer (NCT02043288) [19]. On the other hand, PGA-PEG nanocarriers have previously been explored for protein [20], and RNA delivery [21,22]. Despite this previous work, to the best of our knowledge, this is the first report on the development of PGA-PEG-based nanosystems specifically designed for mAb delivery, particularly for brain-targeted applications. In the design of our PGA-PEG carrier, our hypothesis was that particle size and surface charge play a crucial role in the diffusion of nanosystems through the brain parenchyma's extracellular matrix (ECM). This hypothesis relies on the reported information suggesting that to facilitate brain diffusion, the nanosystems should display a particle size below 100 nm, a dense PEG coating and a neutral or negative surface charge [23–25]. Moreover, although less studied, nanocarrier elasticity has also been suggested to influence diffusivity [8]. Based on this background information, one of the objectives of this work was to explore the utility of PGA-PEG nanocapsules (NCs) for the encapsulation and delivery of mAb to the brain. These NCs feature a hydrophilic PEGylated shell for prolonged stability and diffusivity, a vitamin E core to enhance deformability and brain parenchyma diffusion, and a positively charged phospholipid (DOTAP) to stabilize the structure and promote electrostatic interactions with the PGA shell. Using the model mAb bevacizumab (BVZ), we optimized the formulation to achieve high encapsulation efficiency, controlled release, and suitable physicochemical properties for CNS delivery. On the other hand, building on previous work from our lab [26,27], we explored a structurally distinct approach to mAb delivery. This approach was based on the assembling of PGA-C14 with mAbs and with the help of lecithin as a stabilizer. While both systems were engineered to encapsulate mAbs and facilitate brain delivery, their differences in structural composition offered a unique opportunity to investigate how nanosystem design influences brain diffusion and cellular uptake. Through this comparative study, we aimed to sed light into the impact of nanosystem architecture and physicochemical properties on mAb brain diffusion and intracellular delivery, as well as to provide key insights into the design principles that could optimize nanosystem-based mAb therapies for CNS disorders. 2. MATERIALS AND METHODS 2.1. Materials The humanized monoclonal antibody bevacizumab was kindly donated by mAbxience (Spain). Polyglutamic acid (PGA, nBuPGA(100)[Na]; molecular weight (Mp) determined by SEC-RI-MALS: 14.7 kDa; polydispersity index (Mw/Mn) by SEC-RI-MALS: 1.05) was provided by Curapath (Valencia, Spain). PGA-PEG (PEG (5 kDa)-b-PGA (10) [Na]) (polyethylene glycol (5 kDa)-block-poly(α-glumatic acid) (10u)sodium salt) was obtained from Polypeptide Therapeutic Solutions (Valencia, Spain). 1,2-dioleoyl-3-trimethylammonium-propane chloride (DOTAP) was obtained from Avanti Polar Lipids (Alabaster, AL, USA). Polysorbate 80 (Tween® 80), vitamin E (D, L-α-tocopherol), Accutase® solution, formaldehyde, penicillin/streptomycin solution, Amicon centrifuge filters (30–100 kDa), and glial fibrillary acidic protein (GFAP, 1:500, MAB360) were obtained from Merck (Darmstadt, Germany). Phosphatidylcholine from soybean (Lipoid S100) and N-(Carbonyl-methoxypolyethylene glycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG2K) were purchased from Lipoid GmbH (Germany). Dulbecco’s Modified Eagle Medium (DMEM) and Opti-MEM were obtained from Gibco (Gaithersburg, MD, USA). Fetal bovine serum (FBS) and 24-well plates were purchased from Corning (New York, NY, USA). AlamarBlue Cell Viability Reagent were obtained from Invitrogen (Massachusetts, MA, USA). NeuN antibody (clone A60, MAB360) was purchased from Sigma-Aldrich (San Luis, MO, USA). Ionized calcium-binding adaptor molecule 1 (Iba-1; ab178846) was obtained from Abcam (Cambridge, UK). Alexa Fluor 568-conjugated donkey anti-rabbit IgG or donkey anti-mouse IgG were purchased from Molecular Probes (Eugene, OR, USA). Oriole fluorescent gel stain was obtained from Bio-Rad (USA). GMP-grade zirconium-89 as [ 89 Zr]Zr-oxalate in 1 M oxalic acid was purchased from PerkinElmer (BV Cyclotron, VU Amsterdam, Netherlands). Isothiocyanate derivative of Deferoxamine was obtained from CheMatech (Dijon, France). Oxalic acid (98%) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). PD Imaging studies were conducted using a Bruker BioSpec 3T PET/MRI scanner (bore diameter: 17 cm) equipped with actively shielded gradients (450–900 mT/m). Centrifugal devices (300 kDa) were purchased from Pall Life Sciences (Port Washington, NY, USA). 2.2. Preparation of BVZ-loaded nanosystems 2.2.1. Preparation of PGA-PEG NCs NCs were formulated using a microfluidic approach (NanoAssemblr) in two steps. First, the organic phase, consisting of vitamin E (6.75 mg/mL), Tween® 80 (1 mg/mL), and DOTAP (0.25 mg/mL), all dissolved in ethanol, was mixed with an aqueous phase containing BVZ (0.5 mg/mL in water). Subsequently, PGA-PEG (4 mg/mL) was added to the preformed formulation to complete the NCs formation. Finally, BVZ-loaded NPs were concentrated up to a final BVZ concentration of 1 mg/mL using a nitrogen source. 2.2.2. Preparation of PGAC14 NAs Self-assemblies incorporating the PGAC14 amphiphilic polymer were prepared by adding 125 µL of an aqueous solution of BVZ (4 mg/mL) to 500 µL of an aqueous solution of PGAC14 (1 mg/mL) under magnetic stirring at 1100 rpm and RT. Subsequently, 50 µL of an ethanolic solution containing Lipoid S100 (20 mg/mL) and DSPE.PEG2K (2 mg/mL) in a ratio 1:1 v/v were added over the above mixture. Finally, BVZ-loaded NPs were concentrated up to a final BVZ concentration of 1 mg/mL using a nitrogen source. 2.3. Physicochemical characterization of nanosystems The physicochemical properties of all the prototypes were performed with a Malvern Zeta-Sizer (NanoZS, ZEN 3600, Malvern Instruments, Worcestershire, United Kingdom). The particle size and polydispersity index (PDI) were determined using Dynamic Light Scattering (DLS) after diluting accordingly the samples in H 2 O. The zeta potential was measured after dilution in PBS by Laser Doppler Anemometry (LDA), using the same device. For a further physicochemical characterization, PGA-PEG NCs were analyzed by Nanoparticle Tracking Analysis (NTA) after diluting the samples 10000 times in H 2 O (NanoSight NS3000, Amesbury, United Kingdom). 2.4. Quantification of the mab association efficiency (AE) and loading capacity (LC) by using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) The amount of associated mAb was determined by SDS-PAGE for separation under reducing conditions. PGA-PEG NCs were isolated by ultracentrifugation following the previously described conditions (i.e., 35000 rpm – 1.5 hour – 15 ºC). Then, the amount of non associated mAb present in the supernatant was quantified. Briefly, the supernatant was diluted accordingly to fit in the calibration curve. The mAb standard solutions of known concentrations (from 1 to 0.2 μg/lane) were prepared in PBS. Treated samples were diluted in a volume ratio 1:1 with 2X Laemmli buffer and vortexed for several seconds. Then, the samples were boiled at 99.5 ºC for 5 minutes. 20 μL of the denatured samples were resolved using stacking and resolving polyacrylamide gels of 4 and 12 %, respectively, in the presence of 0.1 % SDS in the running buffer. After 1 hour with variable milliamps and a voltage ranging from 100 to 120 V. Finally, samples were stained using Oriole fluorescent gel stain solution for 1 hour at RT in head-to-tail rotation. Prior to analysis, the gel was washed twice with ultrapure water. GelAnalyzer 19.1 software (www.gelanalyzer.com) by Istvan Lazar Jr., PhD and Istvan Lazar Sr., PhD, was used to process and analyze the samples. Total bands areas of the calibration curve were referred to the theoretical value and those ranging between 80 and 120% were considered. Samples’ bands intensities referred to the calibration curve. The AE and LC were determined as follows: AE (%) = [1- (mass of free mAb) / theoretical mass of mAb] x 100 LC (%) = [mass of associated mAb / total theoretical mass of the nanosystems] x 100 2.5. Stability in simulated biological media The colloidal stability of the PGA-PEG NCs and PGAC14 NAs was evaluated by diluting them 3-fold in PBS, followed by incubation at 37ºC under orbital shaking at 300 rpm. At specific time points (0, 2, 4, 8, and 24 hours), the particle size and PDI were measured using DLS. 2.6. AlexaFluor 488 - BVZ conjugation A total of 250 µL of BVZ (25 mg/mL) was mixed with AlexaFluor 488 (10 mg/mL) in a molar ratio of 8:1 (AlexaFluor 488 : BVZ), using 1 M NaHCO₃ buffer (pH 8.5) to achieve a final volume of 300 µL. The reaction was mixed at RT for 5 minutes under low magnetic stirring, followed by overnight incubation at 4°C. The conjugate was purified using Centripure MINI Desalt Z-50 columns (1000 g, 2 min) with 1x PBS, followed by concentration with 10 kDa MWCO centrifugal filters (14,000 g, 10 min, 15°C) to a final volume of 100 µL. Samples were stored at 4°C for short-term use or at -20°C for long-term storage. The protein concentration (M) was determined by measuring absorbance (A) at 280 nm and 495 nm using a Beckman Coulter® Du730 UV/VIS spectrophotometer, following the instructions from the supplier. 2.7. DFO-BVZ conjugation For radiolabeling the mAb, a first conjugation reaction was done between the mAb and the DFO [28]. Briefly, 1 mL of PBS containing 5 mg of mAb was mixed with 0.1 M Na 2 CO 3 for an acceptable pH range of 8.9 – 9.1 and homogenized by vortex. Over this solution, 20 µL of 5 mM DFO dissolved in DMSO were added in 5µL steps. After 30 minutes at 37ºC, the final product was purified by 30-100 kDa centrifugal filters and washed with ultrapure water. Finally, the purified DFO-mAb conjugate was made up to 1 mL with ultrapure water. 2.8. Radiolabeling with 89 Zr 2.8.1. Radiolabeling of PGA-PEG NPs A solution of 89 Zr in oxalic acid 1M was transferred into a metal-free tube, followed by the addition of Na 2 CO 3 2 M to increase the pH until 4-5. Then, an appropriate amount of HEPES 0.5 M (pH 7.4) buffer was added to neutralize the pH to 7 [29]. A solution of 150 – 200 µL of NCs was slightly added over the 89 Zr solutions and the reaction mixture was kept under orbital stirring at 550 rpm for 1 h at 25 °C. The final product was purified using Amicon® Ultra 0.5 mL Centrifugal Filters 100 K to remove free 89 Zr. 2.8.2. Radiolabeling of PGAC14 NAs The chelation of 89 Zr to the DFO-BVZ was performed as follows; 200 µL of 1M oxalic acid containing 89 Zr were neutralized with 90 µL of 2 M Na 2 CO 3 pH 7-8 and incubated for 3 minutes at RT. Then, 500 µL of 0.5 M HEPES buffer pH 7.2, 700 µL of DFO-BVZ, and 500 µL of 0.5 M HEPES buffer pH 7.2 were consecutively added over the previous solution and mixed at RT under horizontal agitation at 350 rpm. After 1 hour of incubation, the 89 Zr radiolabeled-BVZ was concentrated using 30-100 kDa centrifugal filters to its original volume. The 89 Zr radiolabeled-BVZ entrapment into the nanoassemblies was performed as described in section 2.2.2. Briefly, a non-radiolabeled BVZ was shifted by 89 Zr radiolabeled-BVZ. Afterwards, the NAs were concentrated to a final mAb concentration of 1 mg/ by using a centrifugal device of 300 kDa (12000 g, 1.5 min, RT). 2.9. Radiochemical characterization After radiolabeling with 89 Zr, the radiochemical yield (RCY%) was calculated as the percentage of radioactivity retained in the 89 Zr-labeled nanosystems after purification compared to initial radioactivity used in the reaction (Figure S1A). The percentage of radiochemical purity (RCP%) was assessed using instant thin-layer chromatography (iTLC) (Figure S1B). Briefly, 1.5 μl of 89 Zr-labeled nanosystems was added in a glass microfiber chromatography paper impregnated with silica gel (instant TLC-SG Chromatography paper, Agilent, Santa Clara, CA, USA) and the paper was eluted with a solution of citric acid 20 mM + 60 mM EDTA: Acetonitrile (9:1 v/v). The radioactivity was measured by a TLC radioactivity detector system (RITA, Elysia-Raytest, Angleur, Belgium). Gina Star Software (RITA, Elysia-Raytest, Angleur, Belgium) was used to determine the 89 Zr-labeled nanosystems which remained at the bottom of the paper and the free 89 Zr, which is chelated by the EDTA and eluted with the eluents. To evaluate radiochemical stability (RCS%), 89 Zr-labeled nanosystems were incubated in PBS supplemented with 10% of FBS at 37ºC for 4, 24 and 48 hours (Figure S1C). Following incubation, RCS% was determined as the percentage of radioactivity retained in the 89 Zr-labeled nanosystems compared to free radioactivity after purification. 2.10. Release profile of BVZ The amount of released 89 Zr-BVZ was quantified by gamma counter. The samples were radiolabeled with 89 Zr and diluted 3 times in PBS at pH 7.4 and incubated under orbital agitation at 37ºC. Each time point was prepared in an independent low binding Eppendorf and at different time points, 500μL of the samples were centrifuged (10000 g, 15 min, 20ºC). The free antibody in the permeate and the BVZ-entrapped into the nanosystems were recovered and quantified by Gamma counter. As control, free BVZ was treated in the same conditions and quantified. The release pattern was expressed as the % of cumulative BVZ released calculated as follows: Cumulative BVZ released (%) = free BVZ at t1 - free BVZ at t0 t0 indicates the condition before incubation, t1 corresponds to the condition at the time point under study. 2.11. Cell viability assays The toxicity profile of both prototypes was evaluated using the Resazurin assay (AlamarBlue Cell Viability Reagent). Primary monolayer cultures of astrocytes were kindly provided by Miguel López group [30]. Neurons (GT1-7 cells) and astrocytes were seeded in a 24-well plate at a density of 7 and 5 x 10 4 cells per well and allowed to adhere overnight for 24 hours at 37°C with 5% CO 2 . Dulbecco’s Modified Eagle Medium (DMEM) (high glucose) containing 10% fetal bovine serum, and 5% penicillin/streptomycin was used as the cell culture medium. When the cells reached 70-80% confluence, the culture medium was removed and replaced with 400 µL of BVZ loaded nanosystems at various concentrations in DMEM. Cells co-cultured with nanosystems were incubated for 4 hours. Finally, Resazurin (1.22 mg/mL) was diluted in the cell culture medium to a final concentration of 12.2 µg/mL and added to each well. After 45 minutes of incubation, the fluorescence was measured using a microplate reader (Promega, Madison, WI, USA). Untreated cells served as positive controls, while cells treated with 0.5% (v/v) Triton X-100 diluted in cell culture medium were used as negative controls. The percentage of cell viability was calculated by subtracting the values of negative control from those of the samples and dividing by the fluorescence of the positive control. 2.12. Cell uptake Neurons (GT1-7 cells) and primary cultures of astrocytes were seeded in a 24-well plate at densities of 7 and 5 x 10 4 cells and allowed to adhere overnight for 24 hours at 37°C with 5% CO 2 . DMEM (high glucose) containing 10% fetal bovine serum, and 5% penicillin/streptomycin was used as the cell culture medium. When the cells reached 70-80% confluence, the culture medium was removed and replaced with 400 µL of BVZ loaded nanosystems at various concentrations in DMEM. Cells co-cultured with nanosystems were incubated for 4 hours. After 4 h of incubation, the cell supernatant was discarded, and cells were washed with cold PBS three times. Then, cells were detached from the wells with Accutase for 5 min at 37ºC and collected in FACS tubes. Thereafter, cells suspensions were washed with 3mL of cold FACS washing buffer (PBS 2% FBS) and centrifugated at 1000 rpm for 5 min. Finally, cells were fixed with 100μL of paraformaldehyde (PFA 4%) for 30 min. Treated cells were analyzed by flow cytometry in a FACScalibur instrument (10000 events) and resulting data were analyzed by FlowJo software BDLifescience. 2.13. Animal studies The animal study protocol was carried out following the European Communities Council Directive 2010/63/EU, Directive 86/609/EEC, and Spanish RD 526/2014, and was approved by the corresponding committee at the University of Santiago de Compostela (protocol 14,715,012/2021/012; last version 16 April 2021). 2.14. Immunofluorescence study after intracranial administration BVZ, PGA-PEG NCs, and PGAC14 NAs were administered intrastriatally to male Sprague-Dawley rats (8–10 weeks old, n=6). Four animals were assigned to the PGA-PEG NCs/PGAC14 NAs group, receiving a PGA-PEG NCs injection in one striatum and a PGAC14 NAs injection in the contralateral striatum. Meanwhile, two animals were included in the control group, receiving BVZ in one striatum and PBS in the other. Before the surgical procedure, the animals were deeply anesthetized with ketamine (50 mg/kg) and medetomidine (0.4 mg/kg) and positioned in a stereotaxic frame (Kopf Instruments, CA, USA). A total volume of 2 μL of 1 mg/mL AlexaFluor 488 -BVZ, either encapsulated in PGA-PEG NCs, PGAC14 NAs, or in free form, was injected using a 10-μL Hamilton syringe attached to a motorized injector (Stoelting) at a rate of 0.5 μL/min. The stereotaxic coordinates used for the injections were A/P: 0.8 mm; M/L: 3.0 mm (right striatum) or -3.0 mm (left striatum) and D/V: 5.0 mm from dura. To minimize reflux, the needle was left in place for an additional 5 minutes before withdrawal. After 48 hours, the animals were euthanized with an anesthetic overdose, followed by transcardial perfusion with 4% paraformaldehyde. Their brains were rapidly removed, cryoprotected, and cut into 30-μm thick coronal tissue sections using a cryostat. Brain tissue sections were collected as five series of adjacent sections, which were processed for histological analysis. For diffusion quantification studies, one series of equally spaced (120 μm) sections covering the entire rostro caudal extent of the striatum were mounted on gelatin-coated slides and cover-slipped with Immu-Mount (Thermo-Shandon). Fluorescence images of each striatum were captured using a Nikon Optiphot-2 microscope with a 4x objective, a digital camera DXM1200 and ACT-1software. For double immunofluorescence labeling, free-floating tissue sections were pre-incubated in KPBS-1% BSA containing 5% normal donkey serum (Sigma-Aldrich, MO, USA) and 0.03% Triton X-100 for 60 minutes at room temperature. Tissue sections were then incubated overnight at 4 °C with the corresponding primary antibody: NeuN antibody (clone A60, 1:500, MAB360, Sigma-Aldrich, MO, USA) as a neuronal marker; glial fibrillary acidic protein (GFAP, 1:500, MAB360, Merk Millipore, MA, USA) as astrocytic marker; and Ionized calcium-binding adaptor molecule 1 (Iba-1; 1:500; ab178846, abcam) as microglial marker. The immunoreaction was visualized with the corresponding fluorescent secondary antibodies: Alexa Fluor 568-conjugated donkey anti-rabbit IgG (1:200; Molecular Probes) or donkey anti-mouse IgG (1:200; Molecular Probes). Finally, tissue sections were incubated for 30 min at RT with the DNA-binding dye Hoechst 33342 (1:2000, 62249, Sigma-Aldrich, MO, USA) mounted on gelatin-coated slides, coverslipped with Immu-Mount (Thermo-Shandon) and were visualized with a confocal laser-scanning microscope (AOBS-SP5X; Leica Microsystems Heidelberg GmbH, Mannheim, Germany). 2.14.1. Quantification of fluorescence images For diffusion quantification studies, a series of equally spaced (120 μm) sections covering the entire rostrocaudal extent of the striatum (30 μm thickness) were analyzed. The fluorescence area in each consecutive brain slice was precisely outlined and quantified using ImageJ. The total diffusion volume was then calculated using the following formula: 2.15. Statistical analysis All statistical analyses were conducted using GraphPad Prim version 9.3.0. Gaussian distribution was determined using a Shapiro-Wilk test. The differences were considered significant for * p< 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 with a confidence level of 0.05. The statistical analysis details are provided in the corresponding figure legends 3. RESULTS AND DISCUSSION To assess the impact of the composition and physicochemical properties of the selected nanocarriers, PEG-PGA NCs and PGAC14 NAs, on their diffusion capacity, we conducted a comparative analysis of their physicochemical properties, drug loading and cellular uptake in vitro , and in vivo behavior, including cellular interactions and brain diffusion. 3.1. Development and physicochemical characterization of PGA-PEG nanocapsules Building on our lab's expertise in NCs development [31–34], we designed NCs tailored for the encapsulation and delivery of mAbs to the CNS, with an emphasis on promoting widespread brain tissue distribution and effective interaction with brain cells. Previous studies have shown that PGA-based nanoconjugates bearing propargylamine moieties and bisdemethoxycurcumin not only were able to cross the BBB but also diffuse through the brain parenchyma and ultimately being internalized by brain cells [35,36]. These findings, along with the exceptional properties of polypeptides [17], motivated us to develop PGA-based nanocarriers for the efficient encapsulation of the mAb BVZ. In particular, we developed PGA-PEG NCs with a vitamin E core surrounded by a PGA-PEG shell (Figure 1). The hypothesis was that the PEGylated shell in combination with Tween ® 80 would enhance stability and favor the diffusion across the brain tissue [37]. Additionally, DOTAP was incorporated in the oily core to facilitate the electrostatic attachment of PGA-PEG to the NC structure [38], while vitamin E was expected to confer the NCs with an adequate deformability [8]. Hence, different combinations of PGA-PEG, DOTAP, vitamin E and Tween ® 80 were initially screened leading to the identification of a final prototype with a particle size of 98 nm, PDI<0.2 and a ζ-Potential of -6 mV (Table 1). Given that particle sizes below 100 nm have been described to ensure adequate distribution within the brain parenchyma [39,40], we explored microfluidic technology as a strategy to reduce the particle size of our final prototype. Table 1. Physicochemical properties, association efficiency (AE%) and loading capacity (LC %) of PGA-PEG NCs after at a final BVZ concentration of 1 mg/mL. Data are expressed as mean ± SD, n≥3. Statistical analysis was evaluated by using an unpaired t-test. Prototype Final composition Physicochemical properties AE (%) LC (%) PGA-PEG DOTAP Vitamin E Tween 80® Particle size (nm) PDI ζ potential (mV) PGA-PEG NC 4 0.25 6.75 1 93 ± 6 0.10 -1 ± 1 44 ± 12 2 PGA-PEG NC microfluidics 78 ± 3 0.06 -1 ± 1 99 ± 14** 4 As shown in Table 1, the use of microfluidics resulted in a reduction in particle size, obtaining NCs of 78 nm. Notably, the most significant optimization was observed in the AE%, which exhibited a marked increase, exceeding 90%. This improvement could be a result of the precise, continuous, and rapid mixing facilitated by microfluidics, promoting uniform formulation and improving nanoparticle self-assembly while minimizing compound loss [41,42]. In consequence, the LC% was doubled, highlighting the efficacy of microfluidics in optimizing nanoparticle performance. An additional characterization of the particle size of PGA-PEG NCs was conducted using NTA. The results in Figure 2A show their consistency with those obtained by DLS, confirming the presence of a monodisperse and stable (high dilution) NP population. In addition, PGA-PEG NCs were analyzed by scanning transmission electron microscopy (STEM), revealing a spherical morphology with a distinct dark core surrounded by a lighter shell, likely composed of PGA-PEG (Figure 2B). Particle size analysis showed a larger size, 117 ± 37 nm, compared to NTA/DLS measurements. This discrepancy may be attributed to sample preparation procedures, as drying and staining processes can influence nanoparticle characterization. 3.2. Development and physicochemical characterization of PGAC14 nanoassemblies To evaluate how the nanosystem’s structure affects diffusion through the brain parenchyma, as well as its interaction with brain cells and intracellular mAb delivery, we selected a second prototype, PGAC14 NAs, for comparison (Figure 3). These nanosystems have been previously developed in our group for the oral delivery of mAbs (A.M López-Estévez et al., In press). The primary reason for selecting this nanosystem was its similarity in terms of surface composition to PGA-PEG NCs, as both incorporate the PGA polymer as the key component driving mAb interaction and assembly. Additionally, PGAC14 NAs contained PEG, which, as previously mentioned, enhance nanoparticle diffusion through the brain parenchyma by reducing interactions with extracellular matrix components [14]. The main difference between these systems lies in their core composition and physicochemical properties. As shown in Figure 3, PGAC14 NAs possessed a hydrophilic core enclosed by a bilayer, with the mAb playing a crucial role in assembly and conformation, while PGA-PEG NCs featured a vitamin E core surrounded by a PGA-PEG polymer shell, as described in Section 3.1. Additionally, PGAC14 NAs exhibit ultra-small particle sizes, homogeneous populations, and a slightly negative surface charge (Table 2). Table 2. Physicochemical properties, association efficiency (AE%) and loading capacity (LC %) of PGA-PEG NCs AND PGAC14 NAs. Data are expressed as mean ± SD, n≥3. Statistical analysis was evaluated by using an unpaired t-test for comparison of particle size, PDI and ζ potential; and Mann-Whitney test for comparison of AE% and LC% Prototype Physicochemical properties AE (%) LC (%) Particle size (nm) PDI ζ potential (mV) PGA-PEG NCs 78 ± 3 0.06 -1 ± 1 99 ± 14 4 ± 1 PGAC14 NAs 44 **** ± 4 0.24 ** -10 ± 2 61 * ± 9 20 * ± 3 Regarding the AE%, PGAC14 NAs exhibited a lower encapsulation efficiency compared to PGA-PEG NCs. However, their LC reached 20% (w/w), an outstanding value compared to other protein-delivery nanocarriers, which typically do not exceed an LC of 10% [43–45]. Altogether, these features enabled a direct comparison of how structural and physicochemical differences influence brain diffusion and cellular uptake, further guiding the optimization of nanocarrier-based strategies for mAb delivery. 3.3. Colloidal stability in simulated biological medium and release profile in vitro To better understand the in vivo behavior of PGA-PEG NCs and PGAC14 NAs following intracranial administration, as well as the BVZ release profile, both nanosystems were incubated in PBS at 37°C to simulate physiological conditions, given that these nanocarriers will be directly administered into the striatum. First, their colloidal stability was evaluated. As shown in Figure 4A, both nanosystems remained stable for up to 24 hours. PGA-PEG NCs exhibited a slight initial increase in particle size before stabilizing, whereas PGAC14 NAs showed no significant alterations throughout the incubation period. Beyond stability, the ability to efficiently encapsulate BVZ and enable controlled release in biological fluids is a critical advantage of nanocarrier-based drug delivery [46,47]. As shown in Figure 4B, BVZ release from PGA-PEG NCs followed a controlled and gradual pattern, with an initial release of 9 ± 4%, progressively increasing to 51 ± 4% over 1 week. This sustained release profile minimizes burst release, potentially reducing the rapid clearance of free BVZ from the brain and enhancing intracellular delivery to target cells distant from the injection site. In contrast, PGAC14 NAs exhibited a rapid initial burst release of 37 ± 30%, which then plateaued with minimal additional release up to 1 week, ultimately reaching 49 ± 31%. Despite similar final release values, these results indicate that PGA-PEG NCs are able to better control the release of BVZ overtime. However, these findings should be interpreted with caution, as in vitro results do not always correlate with in vivo behavior. In vitro conditions provide an initial understanding of how effectively BVZ is entrapped within the nanosystem and its release over time, but they cannot be considered as definitive predictions for in vivo performance. 3.4. Impact of nanosystems on brain cell viability and mAb internalization Assessing the impact of nanosystems on brain cell viability and their ability to facilitate mAb internalization is important for determining their therapeutic potential. While efficient cellular uptake is essential for intracellular drug delivery, ensuring minimal cytotoxicity is equally important. In this study, both prototypes were prepared with AlexaFluor 488 -conjugated BVZ (Table S1) to enable this in vitro evaluation in neurons and astrocytes. To evaluate cytotoxicity, a resazurin reduction assay was performed to assess metabolic activity 4 hours post-treatment in neurons (Figure 5A) and astrocytes (Figure 5C). Cells were exposed to increasing BVZ concentrations (1-60 µg/mL), with a 30% reduction in cell viability considered toxic (ISO 10993-5 standards)[48]. As shown in Figure 5, both nanosystems exhibited distinct toxicity profiles across the tested cell types at equivalent BVZ concentrations. PGAC14 NAs demonstrated consistently low toxicity across all tested doses whereas PGA-PEG NCs exhibited a dose-dependent decrease in cell viability, remaining above 70% at BVZ concentrations ≤1 µg/mL in both cell types. This decrease is likely driven by the composition of PGA-PEG NCs, particularly the presence of cationic lipids, which has been associated with increased cytotoxicity at high concentrations [49]. However, cytotoxicity should be considered alongside cellular uptake, which was significantly higher in PGA-PEG NCs compared to PGAC14 NAs (Figures 5B and 5D). The high uptake of PGA-PEG NCs, even at low BVZ doses (0.5 µg/mL), may have contributed to increased cytotoxicity, potentially via elevated reactive oxygen species (ROS) production [50]. In contrast, PGAC14 NAs required a BVZ dose 120 times greater to achieve similar integrated mean fluorescence intensity (iMFI) levels as PGA-PEG NCs. The enhanced BVZ internalization observed for PGA-PEG NCs may be attributed to the composition of these NCs, which include compounds such as Tween® 80, known to facilitate cellular uptake [51,52]. Additionally, while particle size is often cited as a key factor in cellular uptake [53,54], our findings do not support a direct correlation, as PGAC14 NAs, despite their smaller size, exhibited lower internalization. Another important property that might contribute to their uptake is their elastic behavior. Notably, PGAC14 NAs maintained high cell viability even at elevated doses, suggesting that increasing their treatment concentration could enhance BVZ intracellular delivery without compromising cell viability in vitro . 3.5. In vivo assessment of mAb-loaded nanosystems diffusion in the brain by fluorescence The objective of this study was to compare the efficiency of two nanosystems with distinct nanostructures but sharing some similar components, in terms of their capacity to diffuse across the brain and deliver the model mAb BVZ inside the brain cells. To achieve this, the nanosystems, along with free BVZ as a control, were administered directly into the striatum of healthy rats. After 48 hours, immunolabeling of brain coronal sections was performed to assess their distribution and cellular internalization in the striatum (Figure 6). The images in Figure 6 showed distinct cellular interactions between the nanosystems and brain cells, with low correlation to in vitro results. PGA-PEG NCs exhibited significant uptake by microglia, suggesting strong recognition and potential degradation of PGA-PEG NCs. However, a low level of colocalization with neurons and no detectable uptake by astrocytes was observed, contrasting with the in vitro data (Figure 5). This discrepancy could be attributed to the rapid recognition and clearance of PGA-PEG NCs from brain tissue by microglia, thereby limiting their ability to interact with other brain cell types. Conversely, PGAC14 NAs displayed strong neuronal uptake along with moderate internalization by astrocytes and microglia (Figure 6A). This pattern indicates a preferential neuronal interaction while avoiding significant phagocytosis by microglia, in contrast to PGA-PEG NCs. This reduced microglial recognition highlights PGAC14 NAs as a promising candidate for neurodegenerative disorders, where neuronal targeting is a key objective [55,56]. The higher neuronal uptake of PGAC14 NAs compared to PGA-PEG NCs is likely attributed to their surface composition. Previous studies by Silvia Dante and colleagues have reported that negatively charged nanoparticles can preferentially interact with and internalize into neurons due to the electrical activity of neuronal cells, which may create a "charge-driven effect" attracting anionic nanosystems, whereas neutral or positively charged particles exhibit minimal interaction [57]. It should be noted that no colocalization was observed when free BVZ was evaluated (Figure S2), which demonstrates that the internalization of BVZ into brain cells is mediated by its encapsulation within the PGAC14 NAs. Beyond cellular interaction, a key focus of this study was the diffusion ability of both nanosystems within the brain parenchyma. To assess this, coronal slices were collected to evaluate the volume of distribution for each prototype. As shown in Figure 7, free BVZ remained largely confined to the injection site after 48 hours across all examined sections, whereas both BVZ-loaded nanosystems exhibited improved dispersion compared to free BVZ. However, a distinct difference in distribution profiles was observed between the two prototypes. As illustrated in Figure 7, PGA-PEG NCs remained distributed near the infusion site, suggesting their limited diffusion across the brain tissue. However, some fluorescence beyond the injection area was detectable, indicating a moderate distribution volume of approximately 4 mm³ (Figure 8). These results suggest that while some individual nanosystems may have diffused through the brain parenchyma, the overall distribution remained suboptimal. Given that similar oil-core nanosystems have previously facilitated the diffusion of mRNA-based nanoemulsions up to 3.4 mm from the injection site [8], these findings suggest that PGA-PEG NCs may be subject to rapid microglial recognition and phagocytosis, thereby limiting their diffusion. In contrast, PGAC14 NAs exhibited significantly enhanced diffusion from the injection site, as shown in Figure 7. Compared to free BVZ, PGAC14 NAs demonstrated a fourfold increase in distribution volume, reaching 10 mm 3 , confirming widespread dispersion across all examined brain sections (Figure 8). The superior distribution of PGAC14 NAs compared to PGA-PEG NCs can be attributed to their distinct physicochemical properties. Specifically, PGAC14 NAs possess an ultra-small size of 40 nm, compared to the 80 nm of PGA-PEG NCs. This difference is likely a key factor, as smaller nanoparticles have been reported to diffuse more efficiently through the brain’s interstitial space, which ranges from 38 to 64 nm in a normal brain [53,58]. Given that the extracellular matrix (ECM) can act as a physical barrier to larger particles, the reduced size of PGAC14 NAs may facilitate their movement through the brain tissue. However, the correlation between particle size and diffusion remains a subject of debate, as nanoparticles larger than 100 nm have also demonstrated robust diffusion following intracranial administration [59]. Therefore, in addition to size, the more negative surface charge of PGAC14 NAs (-10 mV) likely contributed to their enhanced mobility within the brain parenchyma. The brain ECM contains negatively charged components such as glycosaminoglycans and proteoglycans, which can strongly interact with positively charged nanoparticles, leading to their sequestration and limited diffusion [60]. In contrast, the negative zeta potential of PGAC14 NAs likely reduced these electrostatic interactions, allowing for greater freedom of movement through the brain tissue [26]. Furthermore, the lower sequestration of PGAC14 NAs by microglia, as evidenced by the lack of colocalization in Figure 6A, preventing excessive clearance and further promoting nanoparticle distribution. Notably, high concentrations of small nanoparticles have been shown to improve brain diffusion by saturating available binding sites within the extracellular matrix, allowing nanoparticles to migrate further from the infusion site [25,61]. Given the low toxicity observed in vitro and the high BVZ loading capacity of PGAC14 NAs, future studies could explore higher BVZ concentrations to further enhance brain parenchyma diffusion. Together, these properties highlight PGAC14 NAs as a promising platform for enhancing the diffusion of therapeutic macromolecules in the brain. 4. CONCLUSIONS In this study, we successfully developed and compared two different nanosystems, PGA-PEG NCs and PGAC14 NAs, to evaluate their ability to diffuse through the brain parenchyma and intracellular mAb delivery. Our findings showed that the physicochemical properties of these nanosystems play a crucial role in determining both their diffusion within the brain and their interaction with brain cells. PGA-PEG NCs exhibited limited neuronal and astrocytic uptake while showing a high degree of clearance by microglia, which we hypothesized to be a key factor in their restricted brain distribution. In contrast, PGAC14 NAs exhibited a high neuronal uptake and an enhanced brain diffusion. The diffusion was likely driven by their ultra-small size, which enabled them to remain in the extracellular space and penetrate deeper into brain tissue. These results emphasize the importance of nanosystem architecture in optimizing both brain diffusion and cellular targeting. Overall, PGAC14 NAs emerge as a promising platform for neuronal-targeted delivery and enhanced mAb distribution within the brain. Future studies should investigate how these structural differences impact therapeutic efficacy and explore further optimization of nanocarriers to enhance mAb-based treatments for neurological disorders. Declarations ACKNOWLEDGMENTS This work was supported by the government of Xunta de Galicia (Competitive Reference Groups, Consellería de Educación e Ordenación Universitaria, Xunta de Galicia, Ref: ED431C 2021/17); and by the FEDER/Spanish Ministry of Science, Innovation and Universities (AEI/Project PID2020-119368RB-I00); and (AEI/Project PID2023-150743OB-I00). Laura Piñeiro Alonso acknowledges a grant (ED481A 2021/098) from Xunta de Galicia “Axudas de apoio á etapa predoutoral 2021”. Inés Rubio-Prego acknowledges a grant (IFI23/00035) from Instituto de Salud Carlos III “Contratos i-PFIS: Doctorados ISS-empresa en Ciencias y Tecnologías de la Salud”. 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Supplementary Files Supplementarymaterial.docx GA.png GRAPHICAL ABSTRACT Cite Share Download PDF Status: Published Journal Publication published 02 Sep, 2025 Read the published version in Drug Delivery and Translational Research → Version 1 posted Editorial decision: Minor Revisions Needed 05 Jun, 2025 Reviewers agreed at journal 01 Jun, 2025 Reviewers invited by journal 02 May, 2025 Editor assigned by journal 30 Apr, 2025 First submitted to journal 29 Apr, 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-6558955","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":451055437,"identity":"8cab1444-881b-419c-b041-5df38e516b69","order_by":0,"name":"Laura Pineiro-Alonso","email":"","orcid":"","institution":"University of Santiago de Compostela: Universidade de Santiago de Compostela","correspondingAuthor":false,"prefix":"","firstName":"Laura","middleName":"","lastName":"Pineiro-Alonso","suffix":""},{"id":451055438,"identity":"cec13d9e-59ba-496a-b2a0-59d80b95bc7b","order_by":1,"name":"Inés Rubio-Prego","email":"","orcid":"","institution":"University of Santiago de Compostela: Universidade de Santiago de Compostela","correspondingAuthor":false,"prefix":"","firstName":"Inés","middleName":"","lastName":"Rubio-Prego","suffix":""},{"id":451055439,"identity":"966367f4-9bab-4197-a56b-841cec817bcc","order_by":2,"name":"Ana M. López-Estévez","email":"","orcid":"","institution":"University of Santiago de Compostela: Universidade de Santiago de Compostela","correspondingAuthor":false,"prefix":"","firstName":"Ana","middleName":"M.","lastName":"López-Estévez","suffix":""},{"id":451055440,"identity":"01da0571-3940-4d70-b307-13c4cbb10fd0","order_by":3,"name":"Pablo Garrido-Gil","email":"","orcid":"","institution":"University of Santiago de Compostela: Universidade de Santiago de Compostela","correspondingAuthor":false,"prefix":"","firstName":"Pablo","middleName":"","lastName":"Garrido-Gil","suffix":""},{"id":451055441,"identity":"55e11f99-06d0-43ba-b628-5b3de3f5333e","order_by":4,"name":"Rita Valenzuela","email":"","orcid":"","institution":"University of Santiago de Compostela: Universidade de Santiago de Compostela","correspondingAuthor":false,"prefix":"","firstName":"Rita","middleName":"","lastName":"Valenzuela","suffix":""},{"id":451055442,"identity":"b44d0a98-71e6-4908-80bd-78ef24b70099","order_by":5,"name":"José L. Labandeira-García","email":"","orcid":"","institution":"Universidade de Santiago de Compostela - Campus de Santiago: Universidade de Santiago de Compostela","correspondingAuthor":false,"prefix":"","firstName":"José","middleName":"L.","lastName":"Labandeira-García","suffix":""},{"id":451055443,"identity":"b13a581c-5468-4058-a687-76b16e02f6aa","order_by":6,"name":"Pablo Aguiar","email":"","orcid":"","institution":"University of Santiago de Compostela: Universidade de Santiago de Compostela","correspondingAuthor":false,"prefix":"","firstName":"Pablo","middleName":"","lastName":"Aguiar","suffix":""},{"id":451055444,"identity":"a523570a-c933-4ade-bf83-ca276cf8d648","order_by":7,"name":"Ana I. Rodríguez-Pérez","email":"","orcid":"","institution":"University of Santiago de Compostela: Universidade de Santiago de Compostela","correspondingAuthor":false,"prefix":"","firstName":"Ana","middleName":"I.","lastName":"Rodríguez-Pérez","suffix":""},{"id":451055445,"identity":"a411861b-7d9b-4303-97d6-2b5460dcbd50","order_by":8,"name":"Maria J Alonso","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIiWNgGAWjYJCCAww8DAx8YGYFKVrYwMwzpFgF1sLYRoRK3fazDw/8kGGQZ2PvPfzh47zD9vwNzIc/4NNidibd4GAPD4NhG8+5NMmZ2w4nzjjAliaBV8uBNIYDPDxAJ0nkmDHzbjucYMDAY4bXYWbnnzEc/MPDYA/UYvyZd85hewMG/s/4HXYjjeEw0JZEoBYDad6Gw4wbgOGH32E3njEcluGRSG7jOWMmOeNYeuKMw2xm+LWcT2P++LbHxrafvcf4w4caa3v+9ubHeB0GBow9yMYyE1QPAj+IUjUKRsEoGAUjFQAAf51EFYQS/xAAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-7187-9567","institution":"Universidade de Santiago de Compostela","correspondingAuthor":true,"prefix":"","firstName":"Maria","middleName":"J","lastName":"Alonso","suffix":""}],"badges":[],"createdAt":"2025-04-29 18:42:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6558955/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6558955/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s13346-025-01957-y","type":"published","date":"2025-09-02T15:57:30+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82158362,"identity":"96e1360b-e25c-4c01-9750-1c6a0a449404","added_by":"auto","created_at":"2025-05-07 08:09:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":915246,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic representation of the formulation method carried out for the development of PGA-PEG NCs.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6558955/v1/ed7fda68e00935cdce71c33f.png"},{"id":82158358,"identity":"b79fa0b8-d564-48e7-85d0-fc0515cecc7d","added_by":"auto","created_at":"2025-05-07 08:09:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":337764,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphs size distribution of BVZ-loaded PGA-PEG NCs by NTA (A) and STEM (B). Data is expressed as mean ± SD (mode), n ≥ 2\u003c/strong\u003e. \u003cstrong\u003eScale bar 200 nm.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6558955/v1/09e1eaf206a052cb840c82ae.png"},{"id":82158635,"identity":"44a88e2b-8080-44e0-a3bc-6fbbbb698956","added_by":"auto","created_at":"2025-05-07 08:17:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":737745,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic representation (A) and representative TEM images (B) of BVZ-loaded PGAC14 NAs. Scale bar: 50 nm\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6558955/v1/82800b3e1ae9602131bc7cc5.png"},{"id":82158637,"identity":"da5a2026-6ca2-46e7-8881-8440fea71f56","added_by":"auto","created_at":"2025-05-07 08:17:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":25867,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eColloidal stability of PGA-PEG NCs and PGA NA and BVZ release overtime upon incubation in PBS at 37ºC. A) Colloidal stability of both nanosystems was monitored overtime by DLS. B) The cumulative amount of released \u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e89\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eZr-labeled BVZ was quantified overtime by Gamma counter. As a control, free BVZ was treated in the same conditions. Data is represented as mean ± SEM, n≥2. Statistical analysis was done using a multiple unpaired t-test for comparison of PGA-PEG NCs vs PGAC14 NAs and multiple Mann-Whitney test for comparison of PGA-PEG NCs or PGAC14 NAs vs free BVZ.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6558955/v1/20bf78614df3670d151cd92a.png"},{"id":82158633,"identity":"fde4a78c-e63c-4527-af08-4e8a7e9f3040","added_by":"auto","created_at":"2025-05-07 08:17:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":71489,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMetabolic activity and cellular uptake of PGA-PEG NCs and PGAC14 NAs loaded with AlexaFluor\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e488\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e-labeled BVZ in neurons (A-B) and astrocytes (C-D) after 4 hours of exposure. Cells were exposed to increasing concentrations of blank and BVZ-loaded nanosystems. Cell viability values below 70% were considered toxic (dashed lines). Data are expressed as mean ± SD, n≥3. Statistical analysis for comparison between groups was done using a unpaired t-test (A and C). iMFI integrated mean fluorescence intensity.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6558955/v1/1b729725167c26b03312663e.png"},{"id":82158363,"identity":"59bfcad9-9ec8-487e-8f30-1157349de89a","added_by":"auto","created_at":"2025-05-07 08:09:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":994935,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInternalization of NPs in neuronal and glial cells in brain tissue following administration of PGA14 NAs\u0026nbsp; or PGA-PEG NCs. Triple immunofluorescent labeling for different cellular markers in red (NeuN as neuronal marker [A]; IBA-1 as microglial marker [E] and GFAP as astroglial marker [I]), the nuclear counterstaining Hoechst (in blue; [C, G, K]), and\u0026nbsp; AlexaFluor\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e488\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e-labeled PGA14 NAs\u0026nbsp; (top panel; [B, F, J]) or PGA-PEG NCs (bottom panel [B, F, J] (in green). Merged images (D, H, L) demonstrate co-localization of nanoparticles with various cell populations, indicating intracellular uptake. Scale bar: 20 µm.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-6558955/v1/b0e2875a2d03bd3d25f60229.png"},{"id":82158367,"identity":"58e6d389-1cdc-48ef-85c1-0db726eaad73","added_by":"auto","created_at":"2025-05-07 08:09:02","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":850332,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepresentative images from coronal striatal sections following administration of free AlexaFluor\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e488\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e-labeled\u0026nbsp; BVZ (A), and AlexaFluor\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e488\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e-labeled BVZ (green) encapsulated in PGAC14 NA (B) and PGA-PEG NCs (C) nanosystems. Free AlexaFluor\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e488\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e-labeled BVZ was used as a control. Coronal brain sections (30µm thick) were collected at 120µm intervals. Scale bar: 200 µm.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-6558955/v1/764fed759af2e3cfdfc1df7b.png"},{"id":82158377,"identity":"191e6db0-3dd3-4370-935a-0f122619634c","added_by":"auto","created_at":"2025-05-07 08:09:03","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":8916,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eQuantification of the volume of distribution for each nanosystem prototype and free BVZ, calculated based on the total diffusion area and the cumulative depth spanned by the brain sections. Coronal brain sections (30µm thick) were collected at 120µm intervals. Data are represented as mean ± SD; n≥2. Statistical analysis was performed using an unpaired t-test.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-6558955/v1/68596d83c7c014dfbf0f897f.png"},{"id":90828016,"identity":"2cbccae8-ba7c-4f23-9e7d-37dea93b356a","added_by":"auto","created_at":"2025-09-08 16:05:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5763895,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6558955/v1/9fee41ec-472d-461b-b539-3b9abd749705.pdf"},{"id":82159481,"identity":"5bcdfb58-f984-42e1-87d6-40640b6b9364","added_by":"auto","created_at":"2025-05-07 08:25:02","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4708256,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6558955/v1/a5adc9496b4d4b863b1bf86a.docx"},{"id":82158365,"identity":"a5e6383a-c5c6-48ff-be4c-9a22736434e2","added_by":"auto","created_at":"2025-05-07 08:09:02","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":188211,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGRAPHICAL ABSTRACT\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"GA.png","url":"https://assets-eu.researchsquare.com/files/rs-6558955/v1/1192036388638c8b7ec64fea.png"}],"financialInterests":"","formattedTitle":"Delivery of Monoclonal Antibodies to the Brain: The Impact of Nanocarrier Structure","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eMonoclonal antibodies (mAbs) hold immense potential for treating neurological disorders, as evidenced by the recent FDA approval of three mAb-based therapies for Alzheimer disease and numerous ongoing clinical trials [1,2]. However, their effective delivery to the brain faces substantial challenges [3,4]. While much of the current research focuses on overcoming the blood-brain barrier (BBB), a critical obstacle for central nervous system (CNS) therapies, relies on the limited diffusivity of mAbs through the brain parenchyma. These barriers limit mAbs\u0026apos; ability to reach their target sites within the brain, thereby reducing therapeutic efficacy.\u003c/p\u003e\n\u003cp\u003eTo address these issues, nanotechnology has emerged as an enable technology for enhancing the delivery and distribution of therapeutic molecules within brain tissue [5\u0026ndash;7]. While most studies have focused on the functionalization of nanocarriers for enhancing their transport across the BBB, [8\u0026ndash;11], the number studies addressing the diffusion of mAbs-associated nanocarriers across the brain tissue remains limited.\u003c/p\u003e\n\u003cp\u003ePioneering work by Hanes, Suck and co-workers has shown that polymeric nanoparticles, particularly those incorporating polyethylene glycol (PEG), exhibit an efficient diffusion from the injection site into surrounding brain tissue [11\u0026ndash;13]. This enhanced nanoparticle mobility within the brain parenchyma was attributed to their reduced opsonization and interactions with extracellular matrix components \u0026nbsp;[14]. In this context, block copolymers composed of a biodegradable polypeptide blocks, such as poly(glutamic acid) (PGA) or poly(lysine) (PLL), combined with hydrophilic polymers, mostly PEG, have also emerged as promising polymeric therapeutics consisting of micellar formulations with various biomedical applications [15,16]. Polypeptides, including PGA, possess advantageous properties such as biocompatibility, biodegradability, non-immunogenicity, and low toxicity, making them attractive candidates for drug delivery systems [17,18]. Some examples of their potential are cisplatin-loaded PEG-PGA micelles, which have progressed to Phase III clinical trials for the treatment of advanced pancreatic cancer (NCT02043288) [19]. On the other hand, PGA-PEG nanocarriers have previously been explored for protein [20], and RNA delivery [21,22].\u003c/p\u003e\n\u003cp\u003eDespite this previous work, to the best of our knowledge, this is the first report on the development of PGA-PEG-based nanosystems specifically designed for mAb delivery, particularly for brain-targeted applications. In the design of our PGA-PEG carrier, our hypothesis was that particle size and surface charge play a crucial role in the diffusion of nanosystems through the brain parenchyma\u0026apos;s extracellular matrix (ECM). This hypothesis relies on the reported information suggesting that to facilitate brain diffusion, the nanosystems should display a particle size below 100 nm, a dense PEG coating and a neutral or negative surface charge [23\u0026ndash;25]. Moreover, although less studied, nanocarrier elasticity has also been suggested to influence diffusivity [8].\u003c/p\u003e\n\u003cp\u003eBased on this background information, one of the objectives of this work was to explore the utility of PGA-PEG nanocapsules (NCs) for the encapsulation and delivery of mAb to the brain. These NCs feature a hydrophilic PEGylated shell for prolonged stability and diffusivity, a vitamin E core to enhance deformability and brain parenchyma diffusion, and a positively charged phospholipid (DOTAP) to stabilize the structure and promote electrostatic interactions with the PGA shell. Using the model mAb bevacizumab (BVZ), we optimized the formulation to achieve high encapsulation efficiency, controlled release, and suitable physicochemical properties for CNS delivery.\u003c/p\u003e\n\u003cp\u003eOn the other hand, building on previous work from our lab [26,27], we explored a structurally distinct approach to mAb delivery. This approach was based on the assembling of PGA-C14 with mAbs and with the help of lecithin as a stabilizer. While both systems were engineered to encapsulate mAbs and facilitate brain delivery, their differences in structural composition offered a unique opportunity to investigate how nanosystem design influences brain diffusion and cellular uptake.\u003c/p\u003e\n\u003cp\u003eThrough this comparative study, we aimed to sed light into the impact of nanosystem architecture and physicochemical properties on mAb brain diffusion and intracellular delivery, as well as to provide key insights into the design principles that could optimize nanosystem-based mAb therapies for CNS disorders.\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003e2.1.\u0026nbsp;Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe humanized monoclonal antibody bevacizumab was kindly donated by mAbxience (Spain). Polyglutamic acid (PGA, nBuPGA(100)[Na]; molecular weight (Mp) determined by SEC-RI-MALS: 14.7 kDa; polydispersity index (Mw/Mn) by SEC-RI-MALS: 1.05) was provided by Curapath (Valencia, Spain). PGA-PEG (PEG (5 kDa)-b-PGA (10) [Na]) (polyethylene glycol (5 kDa)-block-poly(\u0026alpha;-glumatic acid) (10u)sodium salt) was obtained from Polypeptide Therapeutic Solutions (Valencia, Spain). 1,2-dioleoyl-3-trimethylammonium-propane chloride (DOTAP) was obtained from Avanti Polar Lipids (Alabaster, AL, USA). Polysorbate 80 (Tween\u0026reg; 80), vitamin E (D, L-\u0026alpha;-tocopherol), Accutase\u0026reg;\u0026nbsp;solution, formaldehyde, penicillin/streptomycin solution, Amicon centrifuge filters (30\u0026ndash;100 kDa), and glial fibrillary acidic protein (GFAP, 1:500, MAB360) were obtained from Merck (Darmstadt, Germany). Phosphatidylcholine from soybean (Lipoid S100) and N-(Carbonyl-methoxypolyethylene glycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG2K) were purchased from Lipoid GmbH (Germany). Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM) and Opti-MEM were obtained from Gibco (Gaithersburg, MD, USA). Fetal bovine serum (FBS) and 24-well plates were purchased from Corning (New York, NY, USA). AlamarBlue Cell Viability Reagent were obtained from Invitrogen (Massachusetts, MA, USA). NeuN antibody (clone A60, MAB360) was purchased from Sigma-Aldrich (San Luis, MO, USA). Ionized calcium-binding adaptor molecule 1 (Iba-1; ab178846) was obtained from Abcam (Cambridge, UK). Alexa Fluor 568-conjugated donkey anti-rabbit IgG or donkey anti-mouse IgG were purchased from Molecular Probes (Eugene, OR, USA). Oriole fluorescent gel stain was obtained from Bio-Rad (USA).\u003c/p\u003e\n\u003cp\u003eGMP-grade zirconium-89 as [\u003csup\u003e89\u003c/sup\u003eZr]Zr-oxalate in 1 M oxalic acid was purchased from PerkinElmer (BV Cyclotron, VU Amsterdam, Netherlands). Isothiocyanate derivative of Deferoxamine was obtained from CheMatech (Dijon, France). Oxalic acid (98%) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). PD Imaging studies were conducted using a Bruker BioSpec 3T PET/MRI scanner (bore diameter: 17 cm) equipped with actively shielded gradients (450\u0026ndash;900 mT/m). Centrifugal devices (300 kDa) were purchased from Pall Life Sciences (Port Washington, NY, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.\u0026nbsp;Preparation of BVZ-loaded nanosystems\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.1.\u0026nbsp; \u0026nbsp;Preparation of PGA-PEG NCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNCs were formulated using a microfluidic approach (NanoAssemblr) in two steps. First, the organic phase, consisting of vitamin E (6.75 mg/mL), Tween\u0026reg;\u0026nbsp;80 (1 mg/mL), and DOTAP (0.25 mg/mL), all dissolved in ethanol, was mixed with an aqueous phase containing BVZ (0.5 mg/mL in water). Subsequently, PGA-PEG (4 mg/mL) was added to the preformed formulation to complete the NCs formation. Finally, BVZ-loaded NPs were concentrated up to a final BVZ concentration of 1 mg/mL using a nitrogen source.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.2.\u0026nbsp; \u0026nbsp;Preparation of PGAC14 NAs\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSelf-assemblies incorporating the PGAC14 amphiphilic polymer were prepared by adding 125 \u0026micro;L of an aqueous solution of BVZ (4 mg/mL) to 500 \u0026micro;L of an aqueous solution of PGAC14 (1 mg/mL) under magnetic stirring at 1100 rpm and RT. Subsequently, 50 \u0026micro;L of an ethanolic solution containing Lipoid S100 (20 mg/mL) and DSPE.PEG2K (2 mg/mL) in a ratio 1:1 v/v were added over the above mixture. Finally, BVZ-loaded NPs were concentrated up to a final BVZ concentration of 1 mg/mL using a nitrogen source.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.\u0026nbsp;Physicochemical characterization of nanosystems\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe physicochemical properties of all the prototypes were performed with a Malvern Zeta-Sizer (NanoZS, ZEN 3600, Malvern Instruments, Worcestershire, United Kingdom). The particle size and polydispersity index (PDI) were determined using Dynamic Light Scattering (DLS) after diluting accordingly the samples in H\u003csub\u003e2\u003c/sub\u003eO. The zeta potential was measured after dilution in PBS by Laser Doppler Anemometry (LDA), using the same device. For a further physicochemical characterization, PGA-PEG NCs were analyzed by Nanoparticle Tracking Analysis (NTA) after diluting the samples 10000 times in H\u003csub\u003e2\u003c/sub\u003eO (NanoSight NS3000, Amesbury, United Kingdom).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4.\u0026nbsp;Quantification of the mab association efficiency (AE) and loading capacity (LC) by using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe amount of associated mAb was determined by SDS-PAGE for separation under reducing conditions. PGA-PEG NCs were isolated by ultracentrifugation following the previously described conditions (i.e., 35000 rpm \u0026ndash; 1.5 hour \u0026ndash; 15 \u0026ordm;C). Then, the amount of non associated mAb present in the supernatant was quantified. Briefly, the supernatant was diluted accordingly to fit in the calibration curve. The mAb standard solutions of known concentrations (from 1 to 0.2 \u0026mu;g/lane) were prepared in PBS. Treated samples were diluted in a volume ratio 1:1 with 2X Laemmli buffer and vortexed for several seconds. Then, the samples were boiled at 99.5 \u0026ordm;C for 5 minutes. 20 \u0026mu;L of the denatured samples were resolved using stacking and resolving polyacrylamide gels of 4 and 12 %, respectively, in the presence of 0.1 % SDS in the running buffer. After 1 hour with variable milliamps and a voltage ranging from 100 to 120 V. Finally, samples were stained using Oriole fluorescent gel stain solution for 1 hour at RT in head-to-tail rotation. Prior to analysis, the gel was washed twice with ultrapure water.\u003c/p\u003e\n\u003cp\u003eGelAnalyzer 19.1 software (www.gelanalyzer.com) by Istvan Lazar Jr., PhD and Istvan Lazar Sr., PhD, was used to process and analyze the samples. Total bands areas of the calibration curve were referred to the theoretical value and those ranging between 80 and 120% were considered. Samples\u0026rsquo; bands intensities referred to the calibration curve. The AE and LC were determined as follows:\u003c/p\u003e\n\u003cp\u003eAE (%) = [1- (mass of free mAb) / theoretical mass of mAb] x 100\u003c/p\u003e\n\u003cp\u003eLC (%) = [mass of associated mAb / total theoretical mass of the nanosystems] x 100\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5.\u0026nbsp;Stability in simulated biological media\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe colloidal stability of the PGA-PEG NCs and PGAC14 NAs was evaluated by diluting them 3-fold in PBS, followed by incubation at 37\u0026ordm;C under orbital shaking at 300 rpm. At specific time points (0, 2, 4, 8, and 24 hours), the particle size and PDI were measured using DLS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6.\u0026nbsp;AlexaFluor\u003csup\u003e488\u003c/sup\u003e - BVZ conjugation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 250 \u0026micro;L of BVZ (25 mg/mL) was mixed with AlexaFluor\u003csup\u003e488\u003c/sup\u003e (10 mg/mL) in a molar ratio of 8:1 (AlexaFluor\u003csup\u003e488\u003c/sup\u003e: BVZ), using 1 M NaHCO₃ buffer (pH 8.5) to achieve a final volume of 300 \u0026micro;L. The reaction was mixed at RT for 5 minutes under low magnetic stirring, followed by overnight incubation at 4\u0026deg;C. The conjugate was purified using Centripure MINI Desalt Z-50 columns (1000 g, 2 min) with 1x PBS, followed by concentration with 10 kDa MWCO centrifugal filters (14,000 g, 10 min, 15\u0026deg;C) to a final volume of 100 \u0026micro;L. Samples were stored at 4\u0026deg;C for short-term use or at -20\u0026deg;C for long-term storage. The protein concentration (M) was determined by measuring absorbance (A) at 280 nm and 495 nm using a Beckman Coulter\u0026reg; Du730 UV/VIS spectrophotometer, following the instructions from the supplier.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7.\u0026nbsp;DFO-BVZ conjugation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor radiolabeling the mAb, a first conjugation reaction was done between the mAb and the DFO [28]. Briefly, 1 mL of PBS containing 5 mg of mAb was mixed with 0.1 M Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e for an acceptable pH range of 8.9 \u0026ndash; 9.1 and homogenized by vortex. Over this solution, 20 \u0026micro;L of 5 mM DFO dissolved in DMSO were added in 5\u0026micro;L steps. After 30 minutes at 37\u0026ordm;C, the final product was purified by 30-100 kDa centrifugal filters and washed with ultrapure water. Finally, the purified DFO-mAb conjugate was made up to 1 mL with ultrapure water.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.8.\u0026nbsp;Radiolabeling with \u003csup\u003e89\u003c/sup\u003eZr\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.8.1.\u0026nbsp; \u0026nbsp;Radiolabeling of PGA-PEG NPs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA solution of \u003csup\u003e89\u003c/sup\u003eZr in oxalic acid 1M was transferred into a metal-free tube, followed by the addition of Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e 2 M to increase the pH until 4-5. Then, an appropriate amount of HEPES 0.5 M (pH 7.4) buffer was added to neutralize the pH to 7 [29]. A solution of 150 \u0026ndash; 200 \u0026micro;L of NCs was slightly added over the \u003csup\u003e89\u003c/sup\u003eZr solutions and the reaction mixture was kept under orbital stirring at 550 rpm for 1 h at 25 \u0026deg;C. The final product was purified using Amicon\u0026reg; Ultra 0.5 mL Centrifugal Filters 100 K to remove free \u003csup\u003e89\u003c/sup\u003eZr.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.8.2.\u0026nbsp; \u0026nbsp;Radiolabeling of PGAC14 NAs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe chelation of \u003csup\u003e89\u003c/sup\u003eZr to the DFO-BVZ was performed as follows; 200 \u0026micro;L of 1M oxalic acid containing \u003csup\u003e89\u003c/sup\u003eZr were neutralized with 90 \u0026micro;L of 2 M Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u0026nbsp;\u003c/sub\u003epH 7-8 and incubated for 3 minutes at RT. Then, 500 \u0026micro;L of 0.5 M HEPES buffer pH 7.2, 700 \u0026micro;L of DFO-BVZ, and 500 \u0026micro;L of 0.5 M HEPES buffer pH 7.2 were consecutively added over the previous solution and mixed at RT under horizontal agitation at 350 rpm. After 1 hour of incubation, the \u003csup\u003e89\u003c/sup\u003eZr radiolabeled-BVZ was concentrated using 30-100 kDa centrifugal filters to its original volume.\u003c/p\u003e\n\u003cp\u003eThe \u003csup\u003e89\u003c/sup\u003eZr radiolabeled-BVZ entrapment into the nanoassemblies was performed as described in section 2.2.2. Briefly, a non-radiolabeled BVZ was shifted by \u003csup\u003e89\u003c/sup\u003eZr radiolabeled-BVZ. Afterwards, the NAs were concentrated to a final mAb concentration of 1 mg/ by using a centrifugal device of 300 kDa (12000 g, 1.5 min, RT).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.9.\u0026nbsp;Radiochemical characterization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter radiolabeling with \u003csup\u003e89\u003c/sup\u003eZr, the radiochemical yield (RCY%) was calculated as the percentage of radioactivity retained in the \u003csup\u003e89\u003c/sup\u003eZr-labeled nanosystems after purification compared to initial radioactivity used in the reaction (Figure S1A).\u003c/p\u003e\n\u003cp\u003eThe percentage of radiochemical purity (RCP%) was assessed using instant thin-layer chromatography (iTLC) (Figure S1B). Briefly, 1.5 \u0026mu;l of \u003csup\u003e89\u003c/sup\u003eZr-labeled nanosystems was added in a glass microfiber chromatography paper impregnated with silica gel (instant TLC-SG Chromatography paper, Agilent, Santa Clara, CA, USA) and the paper was eluted with a solution of citric acid 20 mM + 60 mM EDTA: Acetonitrile (9:1 v/v). The radioactivity was measured by a TLC radioactivity detector system (RITA, Elysia-Raytest, Angleur, Belgium). Gina Star Software (RITA, Elysia-Raytest, Angleur, Belgium) was used to determine the \u003csup\u003e89\u003c/sup\u003eZr-labeled nanosystems which remained at the bottom of the paper and the free \u003csup\u003e89\u003c/sup\u003eZr, which is chelated by the EDTA and eluted with the eluents.\u003c/p\u003e\n\u003cp\u003eTo evaluate radiochemical stability (RCS%), \u003csup\u003e89\u003c/sup\u003eZr-labeled nanosystems were incubated in PBS supplemented with 10% of FBS at 37\u0026ordm;C for 4, 24 and 48 hours (Figure S1C). Following incubation, RCS% was determined as the percentage of radioactivity retained in the \u003csup\u003e89\u003c/sup\u003eZr-labeled nanosystems compared to free radioactivity after purification.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.10.\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eRelease profile of BVZ\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe amount of released \u003csup\u003e89\u003c/sup\u003eZr-BVZ was quantified by gamma counter. The samples were radiolabeled with \u003csup\u003e89\u003c/sup\u003eZr and diluted 3 times in PBS at pH 7.4 and incubated under orbital agitation at 37\u0026ordm;C. Each time point was prepared in an independent low binding Eppendorf and at different time points, 500\u0026mu;L of the samples were centrifuged (10000 g, 15 min, 20\u0026ordm;C). The free antibody in the permeate and the BVZ-entrapped into the nanosystems were recovered and quantified by Gamma counter.\u003c/p\u003e\n\u003cp\u003eAs control, free BVZ was treated in the same conditions and quantified. The release pattern was expressed as the % of cumulative BVZ released calculated as follows:\u003c/p\u003e\n\u003cp\u003eCumulative BVZ released (%) = free BVZ at t1 - free BVZ at t0\u003c/p\u003e\n\u003cp\u003et0 indicates the condition before incubation, t1 corresponds to the condition at the time point under study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.11. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Cell viability assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe toxicity profile of both prototypes was evaluated using the Resazurin assay (AlamarBlue Cell Viability Reagent). Primary monolayer cultures of astrocytes were kindly provided by Miguel L\u0026oacute;pez group [30]. Neurons (GT1-7 cells) and astrocytes were seeded in a 24-well plate at a density of 7 and 5 x 10\u003csup\u003e4\u003c/sup\u003e cells per well and allowed to adhere overnight for 24 hours at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM) (high glucose) containing 10% fetal bovine serum, and 5% penicillin/streptomycin was used as the cell culture medium. When the cells reached 70-80% confluence, the culture medium was removed and replaced with 400 \u0026micro;L of BVZ loaded nanosystems at various concentrations in DMEM. Cells co-cultured with nanosystems were incubated for 4 hours. Finally, Resazurin (1.22 mg/mL) was diluted in the cell culture medium to a final concentration of 12.2 \u0026micro;g/mL and added to each well. After 45 minutes of incubation, the fluorescence was measured using a microplate reader (Promega, Madison, WI, USA). Untreated cells served as positive controls, while cells treated with 0.5% (v/v) Triton X-100 diluted in cell culture medium were used as negative controls. The percentage of cell viability was calculated by subtracting the values of negative control from those of the samples and dividing by the fluorescence of the positive control.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cimg width=\"347\" height=\"36\" src=\"data:image/png;base64,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\" alt=\"image\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.12.\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Cell uptake\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNeurons (GT1-7 cells) and primary cultures of astrocytes\u0026nbsp;were seeded in a 24-well plate at densities\u0026nbsp;of 7 and 5 x 10\u003csup\u003e4\u003c/sup\u003e cells\u0026nbsp;and allowed to adhere overnight for 24 hours at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. DMEM (high glucose) containing 10% fetal bovine serum, and 5% penicillin/streptomycin was used as the cell culture medium. When the cells reached 70-80% confluence, the culture medium was removed and replaced with 400 \u0026micro;L of BVZ loaded nanosystems at various concentrations in DMEM. Cells co-cultured with nanosystems were incubated for 4 hours.\u0026nbsp;After 4 h of incubation, the cell supernatant was discarded, and cells were washed with cold PBS three times. Then, cells were detached from the wells with Accutase for 5 min at 37\u0026ordm;C and collected in FACS tubes. Thereafter, cells suspensions were washed with 3mL of cold FACS washing buffer (PBS 2% FBS) and centrifugated at 1000 rpm for 5 min. Finally, cells were fixed with 100\u0026mu;L of paraformaldehyde (PFA 4%) for 30 min. Treated cells were analyzed by flow cytometry in a FACScalibur instrument (10000 events) and resulting data were analyzed by FlowJo software BDLifescience.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.13.\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Animal studies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal study protocol was carried out following the European Communities Council Directive 2010/63/EU, Directive 86/609/EEC, and Spanish RD 526/2014, and was approved by the corresponding committee at the University of Santiago de Compostela (protocol 14,715,012/2021/012; last version 16 April 2021).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.14.\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Immunofluorescence study after intracranial administration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBVZ, PGA-PEG NCs, and PGAC14 NAs were administered intrastriatally to male Sprague-Dawley rats (8\u0026ndash;10 weeks old, n=6). Four animals were assigned to the PGA-PEG NCs/PGAC14 NAs group, receiving a PGA-PEG NCs injection in one striatum and a PGAC14 NAs injection in the contralateral striatum. Meanwhile, two animals were included in the control group, receiving BVZ in one striatum and PBS in the other. Before the surgical procedure, the animals were deeply anesthetized with ketamine (50 mg/kg) and medetomidine (0.4 mg/kg) and positioned in a stereotaxic frame (Kopf Instruments, CA, USA). A total volume of 2 \u0026mu;L of 1 mg/mL AlexaFluor\u003csup\u003e488\u003c/sup\u003e-BVZ, either encapsulated in PGA-PEG NCs, PGAC14 NAs, or in free form, was injected using a 10-\u0026mu;L Hamilton syringe attached to a motorized injector (Stoelting) at a rate of 0.5 \u0026mu;L/min. The stereotaxic coordinates used for the injections were A/P: 0.8 mm; M/L: 3.0 mm (right striatum) or -3.0 mm (left striatum) and D/V: 5.0 mm from dura. To minimize reflux, the needle was left in place for an additional 5 minutes before withdrawal. After 48 hours, the animals were euthanized with an anesthetic overdose, followed by transcardial perfusion with 4% paraformaldehyde. Their brains were rapidly removed, cryoprotected, and cut into 30-\u0026mu;m thick coronal tissue sections using a cryostat. Brain tissue sections were collected as five series of adjacent sections, which were processed for histological analysis.\u003c/p\u003e\n\u003cp\u003eFor diffusion quantification studies, one series of equally spaced (120 \u0026mu;m) sections covering the entire rostro caudal extent of the striatum were mounted on gelatin-coated slides and cover-slipped with Immu-Mount (Thermo-Shandon). Fluorescence images of each striatum were captured using a Nikon Optiphot-2 microscope with a 4x objective, a digital camera DXM1200 and ACT-1software.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor double immunofluorescence labeling, free-floating tissue sections were pre-incubated in KPBS-1% BSA containing 5% normal donkey serum (Sigma-Aldrich, MO, USA) and 0.03% Triton X-100 for 60 minutes at room temperature. Tissue sections were then incubated overnight at 4\u0026thinsp;\u0026deg;C with the corresponding primary antibody: NeuN antibody (clone A60, 1:500, MAB360, Sigma-Aldrich, MO, USA) as a neuronal marker; glial fibrillary acidic protein (GFAP, 1:500, MAB360, Merk Millipore, MA, USA) as astrocytic marker; and Ionized calcium-binding adaptor molecule 1 (Iba-1; 1:500; ab178846, abcam) as microglial marker. The immunoreaction was visualized with the corresponding fluorescent secondary antibodies: Alexa Fluor 568-conjugated donkey anti-rabbit IgG (1:200; Molecular Probes) or donkey anti-mouse IgG (1:200; Molecular Probes). Finally, tissue sections were incubated for 30 min at RT with the DNA-binding dye Hoechst 33342 (1:2000, 62249, Sigma-Aldrich, MO, USA) mounted on gelatin-coated slides, coverslipped with Immu-Mount (Thermo-Shandon) and were visualized with a confocal laser-scanning microscope (AOBS-SP5X; Leica Microsystems Heidelberg GmbH, Mannheim, Germany).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.14.1.\u0026nbsp;Quantification of fluorescence images\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor diffusion quantification studies, a series of equally spaced (120 \u0026mu;m) sections covering the entire rostrocaudal extent of the striatum (30 \u0026mu;m thickness) were analyzed. The fluorescence area in each consecutive brain slice was precisely outlined and quantified using ImageJ. The total diffusion volume was then calculated using the following formula:\u003c/p\u003e\n\u003cp\u003e\u003cimg width=\"571\" height=\"52\" 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\" alt=\"image\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.15.\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll statistical analyses were conducted using GraphPad Prim version 9.3.0. Gaussian distribution was determined using a Shapiro-Wilk test. The differences were considered significant for * p\u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, and **** p \u0026lt; 0.0001 with a confidence level of 0.05. The statistical analysis details are provided in the corresponding figure legends\u003c/p\u003e"},{"header":"3. RESULTS AND DISCUSSION","content":"\u003cp\u003eTo assess the impact of the composition and physicochemical properties of the selected nanocarriers, PEG-PGA NCs and PGAC14 NAs, on their diffusion capacity, we conducted a comparative analysis of their physicochemical properties, drug loading and cellular uptake \u003cem\u003ein vitro\u003c/em\u003e, and\u003cem\u003e\u0026nbsp;in vivo\u003c/em\u003e behavior, including cellular interactions and brain diffusion.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1.\u0026nbsp;Development and physicochemical characterization of PGA-PEG nanocapsules\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBuilding on our lab\u0026apos;s expertise in NCs development [31\u0026ndash;34], we designed NCs tailored for the encapsulation and delivery of mAbs to the CNS, with an emphasis on promoting widespread brain tissue distribution and effective interaction with brain cells.\u003c/p\u003e\n\u003cp\u003ePrevious studies have shown that PGA-based nanoconjugates bearing propargylamine moieties and bisdemethoxycurcumin not only were able to cross the BBB but also diffuse through the brain parenchyma and ultimately being internalized by brain cells [35,36]. These findings, along with the exceptional properties of polypeptides [17], motivated us to develop PGA-based nanocarriers for the efficient encapsulation of the mAb BVZ. In particular, we developed PGA-PEG NCs with a vitamin E core surrounded by a PGA-PEG shell (Figure 1). The hypothesis was that the PEGylated shell in combination with Tween\u003csup\u003e\u0026reg;\u003c/sup\u003e 80\u003csup\u003e\u0026nbsp;\u003c/sup\u003ewould enhance stability and favor the diffusion across the brain tissue [37]. Additionally, DOTAP was incorporated in the oily core to facilitate the electrostatic attachment of PGA-PEG to the NC structure [38], while vitamin E was expected to confer the NCs with an adequate deformability [8].\u003c/p\u003e\n\u003cp\u003eHence, different combinations of PGA-PEG, DOTAP, vitamin E and Tween\u003csup\u003e\u0026reg;\u003c/sup\u003e 80 were initially screened leading to the identification of a final prototype with a particle size of 98 nm, PDI\u0026lt;0.2 and a\u0026nbsp;\u0026zeta;-Potential of -6 mV (Table 1). Given that particle sizes below 100 nm have been described to ensure adequate distribution within the brain parenchyma [39,40], we explored microfluidic technology as a strategy to reduce the particle size of our final prototype.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e \u003cstrong\u003ePhysicochemical properties, association efficiency (AE%) and loading capacity (LC %) of PGA-PEG NCs after at a final BVZ concentration of 1 mg/mL.\u003c/strong\u003e \u003cstrong\u003eData are expressed as mean \u0026plusmn; SD, n\u0026ge;3. Statistical analysis was evaluated by using an unpaired t-test.\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"614\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 98px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePrototype\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" valign=\"top\" style=\"width: 224px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFinal composition\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 187px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePhysicochemical properties\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 67px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAE (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 37px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLC (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003ePGA-PEG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003eDOTAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003eVitamin E\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003eTween 80\u0026reg;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003eParticle size (nm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 46px;\"\u003e\n \u003cp\u003ePDI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e\u0026zeta; potential (mV)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePGA-PEG NC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 62px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 57px;\"\u003e\n \u003cp\u003e6.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 49px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e93 \u0026plusmn; 6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 46px;\"\u003e\n \u003cp\u003e0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e-1 \u0026plusmn; 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003e44 \u0026plusmn; 12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePGA-PEG NC\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003emicrofluidics\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e78 \u0026plusmn; 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 46px;\"\u003e\n \u003cp\u003e0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e-1 \u0026plusmn; 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003e99 \u0026plusmn; 14**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eAs shown in Table 1, the use of microfluidics resulted in a reduction in particle size, obtaining NCs of 78 nm. Notably, the most significant optimization was observed in the AE%, which exhibited a marked increase, exceeding 90%. This improvement could be a result of the precise, continuous, and rapid mixing facilitated by microfluidics, promoting uniform formulation and improving nanoparticle self-assembly while minimizing compound loss [41,42]. In consequence, the LC% was doubled, highlighting the efficacy of microfluidics in optimizing nanoparticle performance.\u003c/p\u003e\n\u003cp\u003eAn additional characterization of the particle size of PGA-PEG NCs was conducted using NTA. The results in Figure 2A show their consistency with those obtained by DLS, confirming the presence of a monodisperse and stable (high dilution) NP population.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn addition, PGA-PEG NCs were analyzed by scanning transmission electron microscopy (STEM), revealing a spherical morphology with a distinct dark core surrounded by a lighter shell, likely composed of PGA-PEG (Figure 2B). Particle size analysis showed a larger size, 117 \u0026plusmn; 37 nm, compared to NTA/DLS measurements. This discrepancy may be attributed to sample preparation procedures, as drying and staining processes can influence nanoparticle characterization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2.\u0026nbsp;Development and physicochemical characterization of PGAC14 nanoassemblies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate how the nanosystem\u0026rsquo;s structure affects diffusion through the brain parenchyma, as well as its interaction with brain cells and intracellular mAb delivery, we selected a second prototype, PGAC14 NAs, for comparison (Figure 3). These nanosystems have been previously developed in our group for the oral delivery of mAbs (A.M L\u0026oacute;pez-Est\u0026eacute;vez et al., In press).\u003c/p\u003e\n\u003cp\u003eThe primary reason for selecting this nanosystem was its similarity in terms of surface composition to PGA-PEG NCs, as both incorporate the PGA polymer as the key component driving mAb interaction and assembly. Additionally, PGAC14 NAs contained PEG, which, as previously mentioned, enhance nanoparticle diffusion through the brain parenchyma by reducing interactions with extracellular matrix components [14].\u003c/p\u003e\n\u003cp\u003eThe main difference between these systems lies in their core composition and physicochemical properties. As shown in Figure 3, PGAC14 NAs possessed a hydrophilic core enclosed by a bilayer, with the mAb playing a crucial role in assembly and conformation, while PGA-PEG NCs featured a vitamin E core surrounded by a PGA-PEG polymer shell, as described in Section 3.1. Additionally, PGAC14 NAs exhibit ultra-small particle sizes, homogeneous populations, and a slightly negative surface charge (Table 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u003c/strong\u003e \u003cstrong\u003ePhysicochemical properties, association efficiency (AE%) and loading capacity (LC %) of PGA-PEG NCs AND PGAC14 NAs.\u003c/strong\u003e \u003cstrong\u003eData are expressed as mean \u0026plusmn; SD, n\u0026ge;3. Statistical analysis was evaluated by using an unpaired t-test for comparison of particle size, PDI and \u0026zeta; potential; and Mann-Whitney test for comparison of AE% and LC%\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"554\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 140px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePrototype\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 267px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePhysicochemical properties\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 96px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAE (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 52px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLC (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eParticle size (nm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003ePDI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003e\u0026zeta; potential (mV)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 140px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePGA-PEG NCs\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e78 \u0026plusmn; 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003e-1 \u0026plusmn; 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e99 \u0026plusmn; 14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e4 \u0026plusmn; 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 140px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePGAC14 NAs\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e44\u003csup\u003e****\u003c/sup\u003e \u0026plusmn; 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e0.24\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003e-10 \u0026plusmn; 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e61\u003csup\u003e*\u003c/sup\u003e \u0026plusmn; 9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e20\u003csup\u003e*\u003c/sup\u003e \u0026plusmn; 3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eRegarding the AE%, PGAC14 NAs exhibited a lower encapsulation efficiency compared to PGA-PEG NCs. However, their LC reached 20% (w/w), an outstanding value compared to other protein-delivery nanocarriers, which typically do not exceed an LC of 10% [43\u0026ndash;45].\u003c/p\u003e\n\u003cp\u003eAltogether, these features enabled a direct comparison of how structural and physicochemical differences influence brain diffusion and cellular uptake, further guiding the optimization of nanocarrier-based strategies for mAb delivery.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3.\u0026nbsp;Colloidal stability in simulated biological medium and release profile \u003cem\u003ein vitro\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo better understand the \u003cem\u003ein vivo\u003c/em\u003e behavior of PGA-PEG NCs and PGAC14 NAs following intracranial administration, as well as the BVZ release profile, both nanosystems were incubated in PBS at 37\u0026deg;C to simulate physiological conditions, given that these nanocarriers will be directly administered into the striatum.\u003c/p\u003e\n\u003cp\u003eFirst, their colloidal stability was evaluated. As shown in Figure 4A, both nanosystems remained stable for up to 24 hours. PGA-PEG NCs exhibited a slight initial increase in particle size before stabilizing, whereas PGAC14 NAs showed no significant alterations throughout the incubation period.\u003c/p\u003e\n\u003cp\u003eBeyond stability, the ability to efficiently encapsulate BVZ and enable controlled release in biological fluids is a critical advantage of nanocarrier-based drug delivery [46,47]. As shown in Figure 4B, BVZ release from PGA-PEG NCs followed a controlled and gradual pattern, with an initial release of 9 \u0026plusmn; 4%, progressively increasing to 51 \u0026plusmn; 4% over 1 week. This sustained release profile minimizes burst release, potentially reducing the rapid clearance of free BVZ from the brain and enhancing intracellular delivery to target cells distant from the injection site.\u003c/p\u003e\n\u003cp\u003eIn contrast, PGAC14 NAs exhibited a rapid initial burst release of 37 \u0026plusmn; 30%, which then plateaued with minimal additional release up to 1 week, ultimately reaching 49 \u0026plusmn; 31%. Despite similar final release values, these results indicate that PGA-PEG NCs are able to better control the release of BVZ overtime. However, these findings should be interpreted with caution, as \u003cem\u003ein vitro\u003c/em\u003e results do not always correlate with \u003cem\u003ein vivo\u003c/em\u003e behavior. \u003cem\u003eIn vitro\u003c/em\u003e conditions provide an initial understanding of how effectively BVZ is entrapped within the nanosystem and its release over time, but they cannot be considered as definitive predictions for \u003cem\u003ein vivo\u003c/em\u003e performance. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4.\u0026nbsp;Impact of nanosystems on brain cell viability and mAb internalization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAssessing the impact of nanosystems on brain cell viability and their ability to facilitate mAb internalization is important for determining their therapeutic potential. While efficient cellular uptake is essential for intracellular drug delivery, ensuring minimal cytotoxicity is equally important. In this study, both prototypes were prepared with AlexaFluor\u003csup\u003e488\u003c/sup\u003e-conjugated BVZ (Table S1) to enable this \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003eevaluation in neurons and astrocytes.\u003c/p\u003e\n\u003cp\u003eTo evaluate cytotoxicity, a resazurin reduction assay was performed to assess metabolic activity 4 hours post-treatment in neurons (Figure 5A) and astrocytes (Figure 5C). Cells were exposed to increasing BVZ concentrations (1-60 \u0026micro;g/mL), with a 30% reduction in cell viability considered toxic (ISO 10993-5 standards)[48].\u003c/p\u003e\n\u003cp\u003eAs shown in Figure 5, both nanosystems exhibited distinct toxicity profiles across the tested cell types at equivalent BVZ concentrations. PGAC14 NAs demonstrated consistently low toxicity across all tested doses whereas PGA-PEG NCs exhibited a dose-dependent decrease in cell viability, remaining above 70% at BVZ concentrations \u0026le;1 \u0026micro;g/mL in both cell types. This decrease is likely driven by the composition of PGA-PEG NCs, particularly the presence of cationic lipids, which has been associated with increased cytotoxicity at high concentrations [49].\u003c/p\u003e\n\u003cp\u003eHowever, cytotoxicity should be considered alongside cellular uptake, which was significantly higher in PGA-PEG NCs compared to PGAC14 NAs (Figures 5B and 5D). The high uptake of PGA-PEG NCs, even at low BVZ doses (0.5 \u0026micro;g/mL), may have contributed to increased cytotoxicity, potentially via elevated reactive oxygen species (ROS) production [50]. In contrast, PGAC14 NAs required a BVZ dose 120 times greater to achieve similar integrated mean fluorescence intensity (iMFI) levels as PGA-PEG NCs.\u003c/p\u003e\n\u003cp\u003eThe enhanced BVZ internalization observed for PGA-PEG NCs may be attributed to the composition of these NCs, which include compounds such as Tween\u0026reg; 80, known to facilitate cellular uptake [51,52]. Additionally, while particle size is often cited as a key factor in cellular uptake [53,54], our findings do not support a direct correlation, as PGAC14 NAs, despite their smaller size, exhibited lower internalization. Another important property that might contribute to their uptake is their elastic behavior. Notably, PGAC14 NAs maintained high cell viability even at elevated doses, suggesting that increasing their treatment concentration could enhance BVZ intracellular delivery without compromising cell viability \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5. \u003cem\u003eIn vivo\u0026nbsp;\u003c/em\u003eassessment of mAb-loaded nanosystems diffusion in the brain by fluorescence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe objective of this study was to compare the efficiency of two nanosystems with distinct nanostructures but sharing some similar components, in terms of their capacity to diffuse across the brain and deliver the model mAb BVZ inside the brain cells. To achieve this, the nanosystems, along with free BVZ as a control, were administered directly into the striatum of healthy rats. \u0026nbsp;After 48 hours, immunolabeling of brain coronal sections was performed to assess their distribution and cellular internalization in the striatum (Figure 6).\u003c/p\u003e\n\u003cp\u003eThe images in Figure 6 showed distinct cellular interactions between the nanosystems and brain cells, with low correlation to \u003cem\u003ein vitro\u003c/em\u003e results. PGA-PEG NCs exhibited significant uptake by microglia, suggesting strong recognition and potential degradation of PGA-PEG NCs. However, a low level of colocalization with neurons and no detectable uptake by astrocytes was observed, contrasting with the \u003cem\u003ein vitro\u003c/em\u003e data (Figure 5). This discrepancy could be attributed to the rapid recognition and clearance of PGA-PEG NCs from brain tissue by microglia, thereby limiting their ability to interact with other brain cell types.\u003c/p\u003e\n\u003cp\u003eConversely, PGAC14 NAs displayed strong neuronal uptake along with moderate internalization by astrocytes and microglia (Figure 6A). This pattern indicates a preferential neuronal interaction while avoiding significant phagocytosis by microglia, in contrast to PGA-PEG NCs. This reduced microglial recognition highlights PGAC14 NAs as a promising candidate for neurodegenerative disorders, where neuronal targeting is a key objective [55,56]. The higher neuronal uptake of PGAC14 NAs compared to PGA-PEG NCs is likely attributed to their surface composition. Previous studies by Silvia Dante and colleagues have reported that negatively charged nanoparticles can preferentially interact with and internalize into neurons due to the electrical activity of neuronal cells, which may create a \u0026quot;charge-driven effect\u0026quot; attracting anionic nanosystems, whereas neutral or positively charged particles exhibit minimal interaction [57].\u003c/p\u003e\n\u003cp\u003eIt should be noted that no colocalization was observed when free BVZ was evaluated (Figure S2), which demonstrates that the internalization of BVZ into brain cells is mediated by its encapsulation within the PGAC14 NAs.\u003c/p\u003e\n\u003cp\u003eBeyond cellular interaction, a key focus of this study was the diffusion ability of both nanosystems within the brain parenchyma. To assess this, coronal slices were collected to evaluate the volume of distribution for each prototype. As shown in Figure 7, free BVZ remained largely confined to the injection site after 48 hours across all examined sections, whereas both BVZ-loaded nanosystems exhibited improved dispersion compared to free BVZ. However, a distinct difference in distribution profiles was observed between the two prototypes.\u003c/p\u003e\n\u003cp\u003eAs illustrated in Figure 7, PGA-PEG NCs remained distributed near the infusion site, suggesting their limited diffusion across the brain tissue. However, some fluorescence beyond the injection area was detectable, indicating a moderate distribution volume of approximately 4 mm\u0026sup3; (Figure 8). These results suggest that while some individual nanosystems may have diffused through the brain parenchyma, the overall distribution remained suboptimal. Given that similar oil-core nanosystems have previously facilitated the diffusion of mRNA-based nanoemulsions up to 3.4 mm from the injection site [8], these findings suggest that PGA-PEG NCs may be subject to rapid microglial recognition and phagocytosis, thereby limiting their diffusion.\u003c/p\u003e\n\u003cp\u003eIn contrast, PGAC14 NAs exhibited significantly enhanced diffusion from the injection site, as shown in Figure 7. Compared to free BVZ, PGAC14 NAs demonstrated a fourfold increase in distribution volume, reaching 10 mm\u003csup\u003e3\u003c/sup\u003e, confirming widespread dispersion across all examined brain sections (Figure 8). The superior distribution of PGAC14 NAs compared to PGA-PEG NCs can be attributed to their distinct physicochemical properties. Specifically, PGAC14 NAs possess an ultra-small size of 40 nm, compared to the 80 nm of PGA-PEG NCs. This difference is likely a key factor, as smaller nanoparticles have been reported to diffuse more efficiently through the brain\u0026rsquo;s interstitial space, which ranges from 38 to 64 nm in a normal brain [53,58]. Given that the extracellular matrix (ECM) can act as a physical barrier to larger particles, the reduced size of PGAC14 NAs may facilitate their movement through the brain tissue. However, the correlation between particle size and diffusion remains a subject of debate, as nanoparticles larger than 100 nm have also demonstrated robust diffusion following intracranial administration [59].\u003c/p\u003e\n\u003cp\u003eTherefore, in addition to size, the more negative surface charge of PGAC14 NAs (-10 mV) likely contributed to their enhanced mobility within the brain parenchyma. The brain ECM contains negatively charged components such as glycosaminoglycans and proteoglycans, which can strongly interact with positively charged nanoparticles, leading to their sequestration and limited diffusion [60]. In contrast, the negative zeta potential of PGAC14 NAs likely reduced these electrostatic interactions, allowing for greater freedom of movement through the brain tissue [26]. Furthermore, the lower sequestration of PGAC14 NAs by microglia, as evidenced by the lack of colocalization in Figure 6A, preventing excessive clearance and further promoting nanoparticle distribution.\u003c/p\u003e\n\u003cp\u003eNotably, high concentrations of small nanoparticles have been shown to improve brain diffusion by saturating available binding sites within the extracellular matrix, allowing nanoparticles to migrate further from the infusion site [25,61]. Given the low toxicity observed \u003cem\u003ein vitro\u003c/em\u003e and the high BVZ loading capacity of PGAC14 NAs, future studies could explore higher BVZ concentrations to further enhance brain parenchyma diffusion.\u003c/p\u003e\n\u003cp\u003eTogether, these properties highlight PGAC14 NAs as a promising platform for enhancing the diffusion of therapeutic macromolecules in the brain.\u003c/p\u003e"},{"header":"4. CONCLUSIONS","content":"\u003cp\u003eIn this study, we successfully developed and compared two different nanosystems, PGA-PEG NCs and PGAC14 NAs, to evaluate their ability to diffuse through the brain parenchyma and intracellular mAb delivery. Our findings showed that the physicochemical properties of these nanosystems play a crucial role in determining both their diffusion within the brain and their interaction with brain cells. PGA-PEG NCs exhibited limited neuronal and astrocytic uptake while showing a high degree of clearance by microglia, which we hypothesized to be a key factor in their restricted brain distribution.\u003c/p\u003e\n\u003cp\u003eIn contrast, PGAC14 NAs exhibited a high neuronal uptake and an enhanced brain diffusion. The diffusion was likely driven by their ultra-small size, which enabled them to remain in the extracellular space and penetrate deeper into brain tissue. These results emphasize the importance of nanosystem architecture in optimizing both brain diffusion and cellular targeting.\u003c/p\u003e\n\u003cp\u003eOverall, PGAC14 NAs emerge as a promising platform for neuronal-targeted delivery and enhanced mAb distribution within the brain. Future studies should investigate how these structural differences impact therapeutic efficacy and explore further optimization of nanocarriers to enhance mAb-based treatments for neurological disorders.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the government of Xunta de Galicia (Competitive Reference Groups, Conseller\u0026iacute;a de Educaci\u0026oacute;n e Ordenaci\u0026oacute;n Universitaria, Xunta de Galicia, Ref: ED431C 2021/17); and by the FEDER/Spanish Ministry of Science, Innovation and Universities (AEI/Project PID2020-119368RB-I00); and (AEI/Project PID2023-150743OB-I00). Laura Pi\u0026ntilde;eiro Alonso acknowledges a grant (ED481A 2021/098) from Xunta de Galicia \u0026ldquo;Axudas de apoio \u0026aacute; etapa predoutoral 2021\u0026rdquo;. In\u0026eacute;s Rubio-Prego acknowledges a grant (IFI23/00035) from Instituto de Salud Carlos III \u0026ldquo;Contratos i-PFIS: Doctorados ISS-empresa en Ciencias y Tecnolog\u0026iacute;as de la Salud\u0026rdquo;. Figures have been created with BioRender.com.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICT OF INTEREST\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM. J. Alonso is founder and shareholder of LiberaBio. The rest of the authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJ. Pellico, P.J. Gawne, R. T. M. De Rosales, Radiolabelling of nanomaterials for medical imaging and therapy, Chem Soc Rev 50 (2021) 3355\u0026ndash;3423. https://doi.org/10.1039/D0CS00384K.\u003c/li\u003e\n\u003cli\u003eJ. Cummings, Y. Zhou, G. Lee, K. Zhong, J. Fonseca, F. 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Suk, Strategies to enhance the distribution of nanotherapeutics in the brain, Journal of Controlled Release 267 (2017) 232\u0026ndash;239. https://doi.org/10.1016/j.jconrel.2017.07.028.\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":"drug-delivery-and-translational-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ddtr","sideBox":"Learn more about [Drug Delivery and Translational Research](https://www.springer.com/journal/13346)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ddtr/default.aspx","title":"Drug Delivery and Translational Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Monoclonal antibodies, nanomedicine, brain delivery, diffusivity","lastPublishedDoi":"10.21203/rs.3.rs-6558955/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6558955/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Monoclonal antibodies (mAbs) are promising therapeutic agents for neurological disorders due to their high specificity. However, their clinical application is significantly hindered by their poor transport across the blood-brain barrier (BBB) and their limited diffusion within the brain parenchyma. While significant efforts have been oriented to tackle the first barrier, the challenge of efficient brain diffusion remains largely underexplored. To address this, we have developed and evaluated two structurally distinct nanosystems for mAb delivery to the brain: PEGylated polyglutamic acid nanocapsules (PGA-PEG NCs) and PGAC14-based nanoassemblies (PGAC14 NAs). Both formulations encapsulated efficiently the model mAb bevacizumab (BVZ) while they exhibited different physicochemical properties. Namely, PGA-PEG NCs displayed a size of 80 nm and a neutral zeta potential, whereas PGAC14 NAs featured an ultra-small size of 40 nm and a negative surface charge. After assessing their diffusion capacity using immunofluorescence, we concluded that PGAC14 NAs exhibited the highest brain diffusion. This was likely driven by their small size and negative charge, along with a selective ability to interact with and deliver BVZ intracellularly to neuronal cells upon intraparenchymal administration. These findings provide key insights into optimizing nanocarrier design for improved mAb delivery to the brain.","manuscriptTitle":"Delivery of Monoclonal Antibodies to the Brain: The Impact of Nanocarrier Structure","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-07 08:08:53","doi":"10.21203/rs.3.rs-6558955/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor Revisions Needed","date":"2025-06-05T11:36:38+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-06-01T04:08:10+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-02T11:14:17+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-30T15:45:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"Drug Delivery and Translational Research","date":"2025-04-29T14:40:26+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"drug-delivery-and-translational-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ddtr","sideBox":"Learn more about [Drug Delivery and Translational Research](https://www.springer.com/journal/13346)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ddtr/default.aspx","title":"Drug Delivery and Translational Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"57a1957b-9deb-44cf-8686-1ddeff949616","owner":[],"postedDate":"May 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-09-08T16:02:17+00:00","versionOfRecord":{"articleIdentity":"rs-6558955","link":"https://doi.org/10.1007/s13346-025-01957-y","journal":{"identity":"drug-delivery-and-translational-research","isVorOnly":false,"title":"Drug Delivery and Translational Research"},"publishedOn":"2025-09-02 15:57:30","publishedOnDateReadable":"September 2nd, 2025"},"versionCreatedAt":"2025-05-07 08:08:53","video":"","vorDoi":"10.1007/s13346-025-01957-y","vorDoiUrl":"https://doi.org/10.1007/s13346-025-01957-y","workflowStages":[]},"version":"v1","identity":"rs-6558955","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6558955","identity":"rs-6558955","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}