Microfluidic Nanoassembly of Bioengineered Chitosan-Modified FcRn

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Biological and Medical Applications of Materials and Interfaces

Microfluidic Nanoassembly of Bioengineered Chitosan-Modified FcRn-Targeted Porous Silicon Nanoparticles @ Hypromellose Acetate Succinate for Oral Delivery of Anti-Diabetic Peptides João Martins, Dongfei Liu, Flavia Fontana, Mónica Ferreira, Alexandra Correia, Silvia Valentino, Marianna Kemell, Karina Moslova, Ermei M. Mäkilä, Jarno J. Salonen, Jouni Hirvonen, Bruno Sarmento, and Hélder A. Santos ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b20821 • Publication Date (Web): 07 Dec 2018 Downloaded from http://pubs.acs.org on December 8, 2018

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ACS Applied Materials & Interfaces

Microfluidic Nanoassembly of Bioengineered Chitosan-Modified FcRnTargeted Porous Silicon Nanoparticles @ Hypromellose Acetate Succinate for Oral Delivery of Anti-Diabetic Peptides João P. Martins a,*, Dongfei Liu a,b, Flavia Fontana a, Mónica P. A. Ferreira a, Alexandra Correia a, Silvia Valentino c, Marianna Kemell d, Karina Moslova d, Ermei Mäkilä a,e, Jarno Salonen e, Jouni Hirvonen a, Bruno Sarmento f,g,h, Hélder A. Santos a,b,* a Drug

Research Program, Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy,

University of Helsinki, Helsinki FI-00014, Finland b Helsinki

Institute of Life Science (HiLIFE), University of Helsinki, Helsinki FI-00014, Finland

c Department

of Drug Sciences, Università degli Studi di Pavia, Viale Taramello 12, 27100 Pavia, Itália

d Department

of Chemistry, University of Helsinki, Helsinki FI-00014, Finland

e Department

of Physics and Astronomy, University of Turku, Turku FI-20014, Finland

6 i3S

- Instituto de Investigação e Inovação em Saúde, University of Porto, 4200-135 Porto, Portugal

7 INEB

- Instituto de Engenharia Biomédica, University of Porto, 4200-135 Porto, Portugal

8 CESPU

- Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde, 4585-116

Gandra, Portugal

*Corresponding authors Prof. Hélder A Santos ([email protected]) João Pedro Martins ([email protected]) Keywords: microfluidics, nanoparticles, porous silicon, pH-responsive polymers, oral peptide delivery. Conflict of interest The authors declare no conflict of interest.

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Abstract Microfluidics technology is emerging as a promising strategy to improve the oral delivery of proteins and peptides. Herein, a multistage drug delivery system is proposed as a step forward in the development of non-invasive therapies. Undecylenic acid modified thermally hydrocarbonized porous silicon (UnPSi) nanoparticles (NPs) were functionalized with the Fc fragment of immunoglobulin G for targeting purposes. Glucagon like peptide-1 (GLP-1) was loaded into the NPs as a model anti-diabetic drug. Fc-UnPSi NPs were coated with mucoadhesive chitosan, and ultimately entrapped into a polymeric matrix with pHresponsive properties by microfluidic nanoprecipitation. The final formulation showed a controlled and narrow size distribution. The pH-responsive matrix remained intact in acidic conditions, dissolving only in intestinal pH, resulting in a sustained release of the payload. The NPs presented high cytocompatibility, and increased levels of interaction with intestinal cells when functionalized with the Fc fragment, which was supported by the validation of the Fc fragment integrity after conjugation to the NPs. Finally, the Fc-conjugated NPs showed augmented GLP-1 permeability in an intestinal in vitro model. These results highlight the potential of microfluidics as an advanced technique for the preparation of multistage platforms for oral administration. Moreover, this study provides new insights on the potential of the FcRn transcytotic capacity for the development of targeted therapies.


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1. Introduction Microfluidics is an innovative technique that is revolutionizing the pharmaceutical nanotechnology world.1 Such technique allows for the manipulation of nanoliter volumes inside of micrometer-sized fluidic channels,2 thereby allowing a controlled production of advanced drug delivery systems (DDS) with reduced batch-to-batch variations, as compared to conventional bulk methods.3 The current and emerging variety of microfluidic devices, materials, flow patterns and regimes is broadening the range of carriers that can be prepared with this technique, making it ideal for the development of DDS with exquisite precision and sensitivity.3 Particularly, microfluidics can be used to produce nanoparticles (NPs) by nanoprecipitation in glass capillary devices, in which two liquid phases are mixed, the first one containing the substance that forms the NPs in solution, and the second containing a non-solvent solution for the aforementioned substance.4 The exploratory research in microfluidics technologies has shed a light on their potential for developing robust multistage platforms also for oral drug administration. Oral administration unquestionably preferred due to the high patient compliance. However, despite the numerous ongoing investigations, the delivery of proteins and peptides by oral means, particularly of anti-diabetic drugs, still remains as one of the biggest challenges yet to overcome.5 Orally administered drugs face highly dynamic environmental changes throughout the gastrointestinal tract, including pH variations, enzymatic activities and, ultimately, the presence of mucus and the epithelial cell layers.5 Even though the use of NPs as drug carriers has been proven to efficiently overcome several of these barriers,6-7 the absorption of NPs at the intestinal microenvironment is still inefficient. The neonatal Fc receptor (FcRn) was firstly discovered in murine neonatal intestine, and identified as the main responsible facilitator of the diffuse transport of immunoglobulin G (IgG) throughout the colon.8 This receptor was later found to be expressed in the apical side of polarized epithelial barriers of adult humans in similar levels to the fetal expression.9 FcRn 3 ACS Paragon Plus Environment


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binds the Fc part of IgG according to a sharp pH-dependent mechanism, displaying nanomolar affinity at pH 6.5, and negligible binding at pH 7.4.10 Such characteristics prompted research on the therapeutic potential of FcRn targeting.11-14 Following the demonstrations that successful targeting can significantly improve the therapeutic outcome drug loaded nanosystems,12, 14-16 we hypothesize that Fc-functionalized NPs can cross the intestinal epithelium via FcRnmediated transcytosis, and therefore, increase the absorption of an anti-diabetic peptide in the intestinal microenvironment. In this study, we designed a multistage nanosystem, consisting of a core of porous silicon (herein abbreviated as UnPSi) NPs acting as drug nanocarrier, chemically conjugated with the Fc fragment of IgG for FcRn targeting, and then loaded with glucagon-like peptide-1 (GLP-1) as the model anti-diabetic drug (Scheme 1). UnPSi NPs were selected due to their remarkable versatility, featuring high loading capacity, high cytocompatibility, and tunable surface properties, among other advantages.17-18 Due to the poor solubility of Si at low pHvalues, these NPs can be loaded with different drugs in a wide variety of solutions suitable for dissolving the therapeutic compound.17,

19

The Fc-decorated UnPSi (Fc-UnPSi) NPs were

further coated with chitosan by physical adsorption to fine-tune the mucoadhesive properties of the nanocarrier,20 and ultimately entrapped into a pH-sensitive hypromellose acetate succinate (H grade fine powders; herein abbreviated as HF) matrix using glass capillary microfluidic nanoprecipitation (Scheme 1). The nanosystem was characterized for the physicochemical properties: particle size and size distribution, ζ–potential, and degree of surface modification upon functionalization with Fc. The integrity of the Fc fragment after conjugations to the surface to the NPs was studied. The dissolution of the pH-responsive matrix in different buffers, and the drug release profiles of GLP-1 from the NPs were evaluated. The cytocompatibility and cellular interactions were assessed with intestinal cells at different steps of the NP preparation. Finally, the FcRn expression was validated in a 2D in vitro intestinal model, and the intestinal permeability profiles of GLP-1 were evaluated. 4 ACS Paragon Plus Environment

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Scheme 1. Schematic representation of the preparation of the NPs developed in this study, as well as the microfluidics device setup used for nanoprecipitation (not to scale). The final nanocarrier system consisted of UnPSi NPs chemicaly conjugated with the Fc fragment of IgG, loaded with GLP-1 as the model anti-diabetic drug. The particles were coated with chitosan by physical adsorption for mucoadhesiveness, and entrapped into a pH-responsive HF matrix (CSFc-UnPSi@HF) by nanoprecipitation using glass cappilary microfluidics, aiming at FcRntargeted oral delivery of peptides.

2. Materials and methods 2.1. Materials The materials and reagents used are reported in detail in Supporting Information.

2.2. Fabrication of UnPSi NPs The preparation and characterization of UnPSi NPs have been widely reported.17, 21 The detailed protocol is described in Supporting Information.



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2.3. Fc fragment conjugation to UnPSi NPs The free amine groups of Alexa Fluor® 488-conjugated Fc fragment were chemically conjugated to the carboxylic groups on the surface of UnPSi NPs, according to previously optimized protocols.22-23 The detailed protocol is described in Supporting Information.

2.4. GLP-1 loading into Fc-UnPSi NPs GLP-1 was loaded by an immersion method into the Fc-UnPSi NPs.24 Firstly, 1 mg of NPs were added into 500 μL of water, tip-sonicated for 15 s at 30 % amplitude, and then dispersed into 2.25 mL of a 400 μg/L GLP-1 solution. After stirring the solution for 90 min at RT, the unloaded GLP-1 was removed by a 5 min centrifugation at 16100g (Micro Centrifuge, Model 5415D, Eppendorf®, USA), followed by one washing with Milli-Q water.

2.5. Chitosan coating of GLP-1-loaded Fc-UnPSi NPs In order to render the drug carrier mucoadhesive properties, the GLP-1-loaded FcUnPSi NPs were coated with chitosan.21, 25 Chitosan solution (CS) was prepared by dissolving 10 mg/mL chitosan in 1% (v/v) acetic acid, with pH adjustment to 5.5, and stirring overnight. Afterwards, the solution was centrifuged at 4020g (Hettich EBA 21, Tuttlingen, Germany), and kept at 4 °C for future use. For the NP coating, the GLP-1-loaded Fc-UnPSi NPs were dispersed into the CS (ratio 5:1 (w/w), and stirred for 6 h, allowing for polymer layer deposition onto the NP surface. The solution containing the CS-coated Fc-UnPSi (CS-Fc-UnPSi) NPs was then centrifuged at 27600g (Optima MAX, Beckmann Coulter, USA). A one-time wash with water was used to remove the excess CS.


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2.6. Glass capillary microfluidic assembly of pH-responsive NPs The GLP-1-loaded CS-Fc-UnPSi NPs were encapsulated into a HF matrix using the microfluidics glass capillary technique. HF is an enteric pH-sensitive coating polymer highly soluble at alkaline pH and that precipitates around pH ≤ 6.0. The setup of the glass capillary microfluidics platform was established based on previously reported protocols.4 Details of the microfluidic setup are described in the Supporting Information. In this study, the inner phase was injected through the inner capillary, and consisted of GLP-1-loaded CS-Fc-UnPSi NPs dispersed in 10 mg/mL HF solution in acetone. The outer solution phase was injected in between the inner and outer capillaries, and consisted of 1% PVA in Milli-Q water, pH 3.7. A 3D coaxial flow was achieved by simultaneously pumping the inner and outer fluids in the same direction,4 at flow rates controlled by two pumps (PHD 2000, Harvard Apparatus, USA). The inner solution was injected at 2 mL/h and the outer solution at 40 mL/h. The co-flow of and rapid mixing of both fluids, together with the acidic pH of the outer solution (pH 3.7), forces the precipitation of the pH-sensitive polymer, creating nanosized spherical polymer matrices. The samples were collected from the end of the outer borosilicate glass capillary.

2.7. Physicochemical characterization The physicochemical characterization of the developed NPs was performed as previously reported.23 The detailed protocols are described in the Supporting Information. Briefly, the hydrodynamic diameter (z-average), polydispersity index (PdI), and zeta (ζ)-potential of the NPs were assessed by dynamic and electrophoretic laser scattering (DLS and ELS). The chemical modifications on the surface of the NPs (Fc conjugation, CS coating and HF polymer matrix formation) were evaluated using attenuated total reflectance Fourier transformed infrared (ATR-FTIR) spectrometry. The amount of Fc chemically conjugated to the UnPSi NPs was determined by elemental analysis, based on the chemical structure of the fragment. The 7 ACS Paragon Plus Environment


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morphology of the different NPs was evaluated by transmission electron microscopy (TEM). The elemental compositions of CS-Fc-UnPSi@HF and bare HF NPs were measured by energy dispersive X-ray (EDX) spectrometry.

2.8. Integrity of the Fc fragment Due to the strong correlation between the Fc fragment structure and its biological activity, the capacity of the Fc fragment to bind to the receptor after being covalently conjugated to the UnPSi NPs was analyzed using the Human IgG ELISA kit (ab100547). This kit allows the detection of the Fc portion of the heavy chain of all subclasses of human IgG.26-28 For evaluation of the Fc fragment binding capacity after conjugation to the NPs, 100 µL samples of Fc-UnPSi NPs were added to the Human IgG-specific coated ELISA plate in several concentrations (25, 50, 100 and 250 µg/mL), and incubated for 2.5 h at RT. Non-conjugated particles were used as control. Afterwards, the NP solutions were discarded, and the wells were washed four times, according to the manufacturer instructions. PBS (100 µL) was added to each well, and the fluorescence of the NPs that remained in the plate via Fc binding was analyzed with Varioskan Lux Multimode Microplate Reader (ThermoFisher Scientific, USA). The intensity of the fluorescence of the Alexa Fluor® 488-conjugated Fc fragment in the surface of the NPs was measured at 488 nm. The same approach was used to evaluate whether the exposure of the NPs to acetone for encapsulation into the HF matrix could affect the integrity of the Fc fragment, and thus, its biological activity. Hence, Fc-UnPSi and CS-Fc-UnPSi NPs were exposed to acetone solution under the same conditions used for the microfluidic encapsulation. Afterwards, the acetone was washed away, the NPs were washed twice with water, added to the ELISA plate, and incubated for 2.5 h at RT. The fluorescence measurements were performed as described above.


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2.9. Dissolution behavior of the pH-responsive NPs To evaluate the dissolution behavior of the pH-responsive polymer in the final formulation, CS-Fc-UnPSi@HF NPs were dispersed in different buffer solutions (pH 1.2, 5.5 and 7.4), under magnetic stirring. Samples were collected at 2 min and 15 min after starting the incubation, and placed on copper-coated grids. After blotting away the excess amount of sample, the NPs left to dry over the grid at RT before observation under TEM microscopy.

2.10. Association efficiency (AE) and loading degree (LD) The amount of GLP-1 associated to the NPs was calculated by determining the AE (%) and LD (%) using equation (1) and equation (2). The amount of GLP-1 associated to the NPs was assessed by high performance liquid chromatography (HPLC), as previously described.22 The HPLC protocol used is described in detail in the Supporting Information.

𝐴𝐸 (%) =

𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝐺𝐿𝑃 ― 1 𝑎𝑑𝑑𝑒𝑑 (𝜇𝑔) ― 𝐹𝑟𝑒𝑒 𝑢𝑛𝑙𝑜𝑎𝑑𝑒𝑑 𝐺𝐿𝑃 ― 1 (𝜇𝑔) 𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝐺𝐿𝑃 ― 1 𝑎𝑑𝑑𝑒𝑑 (𝜇𝑔)

× 100

(1)

𝐿𝐷 (%) =

𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝐺𝐿𝑃 ― 1 𝑎𝑑𝑑𝑒𝑑 (𝜇𝑔) ― 𝐹𝑟𝑒𝑒 𝑢𝑛𝑙𝑜𝑎𝑑𝑒𝑑 𝐺𝐿𝑃 ― 1 (𝜇𝑔) 𝑇𝑜𝑡𝑎𝑙 𝑚𝑎𝑠𝑠 𝑜𝑓 𝑁𝑃𝑠 (𝜇𝑔) + 𝑇𝑜𝑡𝑎𝑙 𝑚𝑎𝑠𝑠 𝑜𝑓 𝑝𝑜𝑙𝑦𝑚𝑒𝑟 (𝜇𝑔)

× 100

(2)

2.11. GLP-1 in vitro release studies In order to mimic the passage of the NPs through the gastrointestinal tract, the GLP-1 release profile from CS-Fc-UnPSi@HF was performed in simulated gastric fluid (SGF) and in fasted state simulated intestinal fluid (FaSSIF). SGF solution consisted of 0.2% w/v sodium chloride, and 0.7% (v/v) hydrochloric acid, with pH adjusted to 1.2). FaSSIF solution consisted of 106 mM Sodium chloride, 28 mL monobasic sodium phosphate, 8.7 mM sodium hydroxide, 3 mM sodium taurocholate, and 0.75 mM lecithin. The pH of this solution was adjusted to 6.8. UV-Vis spectroscopy was performed in advance to validate the maintenance of the integrity of 9 ACS Paragon Plus Environment


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the drug throughout the experiment. For this purpose, a GLP-1 solution was initially prepared to a concentration of 30 µg/mL, and incubated with SGF and FaSSIF for 2 h and 6 h, respectively. The UV-Vis spectra of GLP-1 in SGF and FaSSIF was measured at time different points (0, 2 and 6 h) after incubation with the solutions using a UV-1600PC spectrophotometer (VWR, Radnor, PA, USA). The NPs were dispersed in SGF for 2 h. Afterwards, the NPs were removed from the SGF solution by centrifugation for 5 min at 16100g (Micro Centrifuge, Model 5415D, Eppendorf®, USA), and dispersed again in FaSSIF. The GLP-1 released from the NPs was monitored for the following 6 h. The experiment was conducted under stirring (300 rpm) at 37 °C. Samples of 100 μL were taken at several time points from 30 to 480 min, but replaced with the respective pre-warmed buffer solutions, in order to keep the volumes constant. After a 5 min centrifugation at 16100g (Micro Centrifuge, Model 5415D, Eppendorf®, USA), the supernatants were analyzed in HPLC.

2.12. Cell lines and culture conditions Human colon adenocarcinoma Caco-2 cells and Human goblet-like HT29-MTX were cultured according to previously optimized conditions.23 Further details can be found in Supporting Information.

2.13. Cell viability studies The compatibility of the developed NPs with Caco-2 and HT29-MTX cells was evaluated with CellTiter-Glo® Luminescent Cell Viability Assay, as previously reported.23 Specific details are described in Supporting Information.


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2.14. Cell–NP interaction studies The NPs interacting with the intestinal cells were studied by confocal fluorescence microscopy. Fc-UnPSi, CS-Fc-UnPSi, and CS-Fc-UnPSi@HF NPs were already fluorescently labeled, due to the use of Alexa Fluor® 488-conjugated Fc fragment as targeting ligand. The bare UnPSi NPs used as control were fluorescently labelled with Alexa Fluor® 488 using EDC/NHS chemistry, as described above. The ratio of Alexa Fluor® 488:Alexa Fluor® 488conjugated Fc fragment was optimized using a Varioskan Lux Multimode Microplate Reader (ThermoFisher Scientific, USA), in order to obtain the same fluorescence intensity for all the NPs.25 For 2D confocal imaging, Caco-2/HT29-MTX co-cultures (ratio of 90:10) were seeded at a concentration of 5  105 cells per well in Lab-Tek™ 8-chamber slides (Thermo Fisher Scientific, USA),29-30 and kept for 24 h at 37 °C and humidified atmosphere. Afterwards, the cells were washed twice with freshly prepared pre-warmed Hank’s balanced salt solution (HBSS)–2-(4-(2-hydroxyethyl)pipezarin-1-yl) ethanesulfonic acid (HEPES) (pH 7.4), and 250 µL of NP solution (250 µg/mL) were added to the each chamber and kept at 37 °C. After 3 h incubation, the NPs that were not interacting with the cells were gently removed by extensive washing with pre-warmed HBSS–HEPES (pH 6.5). The cell membranes were stained with CellMask® DeepRed by incubation for 4 min at 37 °C. The cells were washed again with prewarmed HBSS–HEPES (pH 6.5) and observed immediately with Leica SP5 II HCS-A confocal microscope (Leica Microsystems, Wetzlar, Germany). Images were analyzed using Fiji.31

2.15. FcRn expression in the Caco-2/HT29-MTX cell culture model The expression of FcRn in the 2D in vitro cell culture model was evaluated by immunocytochemistry. For this purpose, Caco-2/HT29-MTX co-culture monolayers were grown in 12-well Transwell™ filter membranes (3 µm pore-size; Corning Inc., USA) at a ratio of 90:10,29-30 respectively, and with a cell density of 68  104 cells per cm2. Cell culture medium was replaced every other day. At day 20 of co-culture, cell culture medium was replaced by 11 ACS Paragon Plus Environment


serum free medium, in order to avoid discrepancies caused by the occupation of the FcRn with constituents of the serum. After 21 days, Caco-2/HT29-MTX co-cultures were incubated with Alexa Fluor® 488-ChromPure Human IgG, Fc fragment (3.96 µg/mL) for 3 h at 37 °C and humidified 5% CO2 atmosphere. Caco-2/HT29-MTX co-cultures that were not exposed to the fluorescent Fc fragment were used as control. Then, cells were washed extensively, and fixed for 30 min at RT with 4% paraformaldehyde (PFA). After cutting, Transwell™ filter membranes were mounted in glass slides with Vectashield® antifade mounting medium with 4',6diamidino-2-phenylindole dihydrochloride (DAPI), which is used for counterstaining the nuclei, and finally observed at Leica SP5 II HCS-A confocal microscope (Leica Microsystems, Wetzlar, Germany).

2.16. GLP-1 permeation across intestinal cells The permeability studies were performed in Caco-2/HT29-MTX co-cultures, seeded in 12-well Transwell™ filter membranes (3 µm pore-size; Corning Inc., USA) at a ratio of 90:10,2930

respectively, and using a cell density of 6.8  104 cells per cm2. Cell culture medium was

replaced every other day. At the end of the culture period, the transepithelial electrical resistance (TEER) was measured using a Millicell® ERS-2 volt-ohm-meter with STX01 electrodes (Millipore, MA, USA), as previously described.32 The monolayers were rinsed once with HBSS–HEPES buffer solution in the apical (pH 6.8) and basolateral compartments (pH 7.4), and allowed to equilibrate for 15 min. The permeability of GLP-1-loaded CS-Fc-UnPSi@HF NPs was studied from the apical to the basolateral direction, with volumes of 0.5 mL and 1.5 mL HBSS–HEPES buffer solution in the respective compartments. FcRn non-targeted CSUnPSi@HF NPs and free GLP-1 were used as controls. The different NPs and the equivalent amount of free drug were added to the apical chamber, and the plates were maintained at 37 °C and shaken at 100 rpm. 200 μL samples were withdrawn from the basolateral chambers at different time points up to 180 min, and the same volume was replaced with pre-warmed 12 ACS Paragon Plus Environment

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HBSS–HEPES (pH 7.4) in order to maintain a constant volume. The amount of GLP-1 permeated was quantified using HPLC, as described above. At the end of the permeability studies, the monolayers were prepared for TEM analysis. Firstly, the monolayers were washed, in order to remove the excess non-interacting NPs, and fixed with glutaraldehyde for 15 min at RT. After washings, the membranes were dehydrated, followed by embedding in epoxy resin. Perpendicular ultrathin sections were cut and stained with uranyl acetate and lead citrate, and observed under TEM.

2.17. Statistical analysis All of the experiments were performed at least in triplicates, and the measured values are expressed as mean ± standard deviation. Results were evaluated by means of two-way analysis of variance (ANOVA) with Bonferroni post-test, and levels of significance set at probabilities of *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

3. Results and discussion 3.1. Fabrication and characterization of the NPs The UnPSi NPs used as core drug nanocarrier were prepared by electrochemical anodization of p+ type single-crystal Si wafers, using a top-down approach, followed by thermal hydrocarbonization, surface modification for –COOH termination by undecylenic acid treatment and milling, as described elsewhere.17 The physical properties of the UnPSi NPs were evaluated by N2 sorption at ― 196 ºC (Table S1). Alexa Fluor® 488-conjugated Fc fragment was covalently conjugated to the surface of UnPSi NPs by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide (EDC/NHS) chemistry. The inherent fluorescence of the Fc fragment can be used to obtain dynamic information on the localization and quantity of the NPs in confocal fluorescence microscopy studies. Elemental analysis was performed before and after chemical conjugation of Alexa Fluor® 488-conjugated Fc fragment onto the surface of 13 ACS Paragon Plus Environment


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UnPSi NPs (Table 1). The fact that the N element appeared only after functionalization with the Fc fragment, which is rich in primary amines, confirmed the conjugation of the ligand. Also, the increase observed in the amount of C element supported the successful functionalization of the NPs. Based on the chemical structure of the Fc region of IgG, there was ca. 0.17 ± 0.01 mg of Fc fragment per 1 mg of UnPSi NPs.

Table 1. Elemental analysis determination of the mass percentage of the N, C, and S elements in the UnPSi and Fc-UnPSi NPs. Results are expressed as mean ± s.d. (n = 3). Sample

N, %

C, %

S, %

UnPSi

Nu

15.65 ± 1.12

Su

Fc-UnPSi

2.63 ± 0.15

23.53 ± 2.10

Su

Nu = N amount below detection limit; Su = S amount below detection limit

The surface of Fc-UnPSi NPs was further coated with CS to render muchoadhesive properties to the drug carrier.20 Finally, to obtain a robust multistage platform for oral administration capable of facing the harsh gastrointestinal environment, and to improve the intestinal drug absorption, the CS-Fc-UnPSi NPs were encapsulated into a HF matrix by microfluidic nanoprecipitation. HF, a Food and Drug Administration approved enteric pHsensitive coating polymer, was used in this study for temporarily sealing the pores of the UnPSi NPs at acidic pH, before becoming deprotonated and dissolving at pH ≥ 6.0.33-34 The microfluidics platform consisted of two different glass capillaries aligned in a co-flow geometry.18 The inner borosilicate glass capillary was firstly tapered using a micropipette puller, then carefully sanded to an inner diameter of ca. 100 μm. This capillary was then inserted in the left end of an outer capillary (inner diameter of 1.1 mm), and coaxially aligned (Scheme 1). The CS-Fc-UnPSi NPs were dispersed into a HF solution in acetone and injected through the inner glass capillary, whereas a poly(vinyl alcohol) (PVA; 1%, w/v) solution was simultaneously injected from the outer glass capillary. Both inner and outer phases were flowed 14 ACS Paragon Plus Environment

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in the same direction, resulting in a 3D coaxial flow.4 The self-assembly of the HF polymeric matrices around the CS-Fc-UnPSi NPs started with the supersaturation, initiated by diffusion and mixing between the solvent (HF in acetone) and the anti-solvent streams (1% PVA, pH 3.7), resulting in the individualized entrapment of CS-Fc-UnPSi NPs into the HF matrices (CSFc-UnPSi@HF).35 DLS analysis, TEM images, EDX, and ATRFTIR analyses of the NPs at different steps of preparation are presented in Fig. 1. The core UnPSi NPs showed a size of ca. 194 ± 1 nm and a PdI of 0.1 (Fig. 1a), suggesting narrow size distribution of the NPs (Fig. S1). The negative surface ζ–potential of ― 33 ± 1 mV resulted from the negatively charged carboxylic groups present on the surface of the NPs (Fig. 1b). The particle size was consecutively increased upon functionalization with Fc, coating with CS and entrapment into the HF matrix, rendering the drug delivery system a final size of 603 ± 4 nm (Fig. 1a). The narrow size distribution of the overall system was maintained throughout the preparation steps (Fig. S1). A shift was observed on the surface ζ–potential of the NPs from highly negative to positive upon coating with CS (25 ± 0.5 mV), as expected.21 The presence of CS on the surface on the NPs is expected to reverse the repulsion faced by the negatively charged core UnPSi NPs by the also negatively charged mucin layer present on the intestinal cells, prolonging the retention time in the vicinity of the cells.36-37 The surface ζ–potential of the final formulation was virtually neutral ( ― 2 ± 0.02 mV), which was in accordance with the surface ζ–potential of bare HF capsules produced using the same microfluidics setup ( ― 1.00 ± 0.03 mV). TEM imaging of the NPs showed the expected irregular shape of the UnPSi NPs, and no significant morphological alterations were observed after functionalization with the Fc (Fig. 1c). By contrast, CS coating improved the smoothness of the nanosystem, by creating a thin layer of polymer adsorbed onto the surface of the UnPSi NPs (Fig. 1c). Ultimately, a HF matrix with spherical shape successfully entrapped the CS-Fc-UnPSi NPs by microfluidic nanoprecipitation (Fig. 1c), rendering pH-responsive capacity to the nanosystem. The TEM 15 ACS Paragon Plus Environment


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images supported the size measurements, the narrow size distribution, and the particle surface modifications observed by the DLS. EDX elemental composition analysis confirmed the presence of the CS-Fc-UnPSi NPs inside of the HF matrix, due to the appearance of the Si peak in the spectrum (Fig. 1d, black arrow), whereas no Si was detected in the spectrum of a bare HF capsule (Fig. 1d, blue arrow). The measurement points for TEM–EDX spectroscopy are shown in Fig. S2. The obtained NPs were characterized by ATR–FTIR spectroscopy to further evaluate the surface chemical modifications of the NPs at each step of preparation (Fig. 1e). The FTIR spectra of bare NPs showed clear absorption bands of UnPSi, with the carbonyl C=O stretching at 1714 cm-1, related to the carboxyl acid groups. This band was attenuated after covalent attachment of the free amine groups of the Fc fragment to the carboxylic groups of the UnPSi NPs, resulting in the formation of amides I (1641 cm-1) and II (1529 cm-1). The physical adsorption of CS showed only limited alterations in the surface of the NPs. Finally, the characteristic peaks of UnPSi and the resulting spectrum of Fc conjugation and CS coating could not be detected after the entrapment into the HF matrix. The spectrum of CS-FcUnPSi@HF NPs was very similar to the spectrum of HF polymer alone, which can be ascribed to the successful entrapment of the NPs into HF.


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Fig. 1. Characterization of the physicochemical properties of the developed NPs. (a) Size and PdI. (b) Surface charge. (c) TEM images of the NPs at different steps of preparation; scale bar represents 200 nm. (d) Elemental composition of CS-Fc-UnPSi@HF NPs and bare HF matrix by EDX analysis. (e) ATR–FTIR spectra of the NPs and HF polymer. Results are expressed as mean ± s.d. (n ≥ 3).

3.2. Fc fragment integrity and biological activity After having prepared and characterized the NP formulation developed in this study, the integrity of the Fc fragment and its biological activity when conjugated to the surface of the NPs were evaluated. It is known that conjugating biologics to nanoparticles increases their half17 ACS Paragon Plus Environment


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life.38-39 Also, there is a strong correlation between the integrity of a moiety and its biological activity. Therefore, the recognition and binding capacity of the Fc fragment on the surface of the NPs to its receptor was studied by ELISA (ab100547). Results showed a concentrationdependent binding effect of the Fc-conjugated NPs to the capture antibody on the bottom of the wells (Fig. 2a). The increasing amount of available NPs was reflected in an increased fluorescence, as a result of the binding of the NPs to the detection antibody. Furthermore, nonconjugated NPs revealed lack of binding capacity due to the absence of the Fc fragment on their surface, regardless of their concentration. Since the microfluidic nanoprecipitation technique for the entrapment of the core NPs into the pH-responsive polymer requires the use of acetone, and the use of organic solvents is widely known to interfere with the physicochemical properties of proteins,40 the capacity of the Fc fragment to be recognized by its receptor was studied after exposure of the Fc-UnPSi and CS-Fc-UnPSi NPs to acetone. The Alexa Fluor® 488-conjugated Fc fragment used in this study is hydrated in water and, thus the NPs exposed to water were used as control. Results showed that the binding efficacy of the Fc-conjugated NPs was not affected by the exposure to acetone when compared to those exposed in water (Fig. 2b). Here, the signal of the background and of UnPSi and UnPSi-CS NPs was subtracted to the measured values, in order to quantify exclusively the fluorescence of the ligand conjugated to the NPs. Additionally, it was also possible to observe that the CS increased the retention of the NPs in the bottom of the well, suggesting that part of the CS-Fc-UnPSi NPs were attached to the well and not bound to the capture antibody. Nonetheless, the use of NPs without CS supported their interaction exclusively via Fc binding, since non-conjugated NPs showed nearly no fluorescence. In summary, these results showed the biological activity of the Fc fragment after conjugation to the surface of the UnPSi NPs by a concentration-dependent binding effect, and revealed that the integrity of the molecule was not affected due to exposure of the conditions


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required by the microfluidic nanoprecipitation technique used to entrap the core NPs into the pH-responsive HF matrix.

Fig. 2. Integrity of the Fc fragment after conjugation to the NPs by ELISA. (a) Concentration-dependent binding effect of the Fc-conjugated NPs to the capture antibody on the bottom of the wells. (b) Maintenance of the Fc fragment integrity after exposure to water and to acetone during the preparation of the formulation. The level of significant differences was set at probabilities of ****p < 0.0001. Results are expressed as mean ± s.d. (n ≥ 3).

3.3. pH-dependent dissolution of the HF matrix To evaluate the behavior of the HF polymeric matrix dissolution, the final nanosystem consisting of CS-Fc-UnPSi@HF NPs was incubated with different buffers at pH 1.2, 5.5 and 7.4, mimicking the pH variations faced by the NPs after oral intake, and/or post-absorption by the intestinal cells.41 On one hand, results of TEM showed that the HF matrix remained intact throughout the time at the acidic pH of 1.2 (Fig. 3a, d), which is characteristic of the stomach. On the other hand, alterations on the nanosystem integrity started to be observed at mildly acidic conditions (pH 5.5) after 2 min (Fig. 3b), which progressed continuously to a higher degree of dissolution over 15 min (Fig. 3e). Such modifications suggested that the CS-Fc-UnPSi NPs will have no difficulty in being released from the polymeric matrix when exposed to the intestinal 19 ACS Paragon Plus Environment


cells (ca. pH 6.8) for further FcRn interaction. When moving up to pH 7.4, the HF matrix disappeared completely, exposing the CS-Fc-UnPSi NPs immediately after 2 min of incubation (Fig. 3c), and thus, were dissolved also after 15 min, as expected (Fig. 3f). The TEM images demonstrated the pH-responsive properties of the CS-Fc-UnPSi@HF NPs, which result from the dissolution of the polymeric matrix at neutral pH conditions. These images also showed that the NPs were able to maintain intact morphology at the acidic gastric conditions, and that the polymeric capsule started to dissolve progressively as the pH of the buffers were increased, exposing the CS-Fc-UnPSi NPs until they are completely released.

Fig. 3. Dissolution behavior of the pH-responsive HF matrix. TEM images of the HF degradation profile in CS-Fc-UnPSi@HF NPs after incubation in different buffer solutions (pH 1.2, 5.5 and 7.4), during 2 min (a-c) and 15 min (d-f). Scale bar represents 100 nm.

3.4. Drug loading and in vitro drug release studies After the physicochemical characterization of the developed NPs and the validation of the dissolution methodology of the pH-responsive polymeric matrix, the association efficiency (AE) and loading degree (LD) were assessed to determine the amount of GLP-1 associated to the NPs. Additionally, the pH-dependent in vitro drug release profiles were evaluated. GLP-1 was used as the model anti-diabetic drug,42 which was loaded onto the nanoparticle pores using 20 ACS Paragon Plus Environment

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an immersion method.24 GLP-1 is an incretin hormone, currently administrated by parenteral routes, and is in the pipeline for the treatment of type 2 diabetes mellitus (T2DM).43 GLP-1 is capable of promoting insulin secretion after meals, beyond stimulating the neogenesis and proliferation of pancreatic β cells, which can slow down the progression of T2DM.42 The loading of GLP-1 into the NPs was performed after the surface functionalization with the Fc fragment. Results for the CS-Fc-UnPSi@HF NPs showed an association efficiency of 30 ± 3% (w/w), and a loading degree of 0.55 ± 0.05% (w/w). The GLP-1 release profile from the pores of the NPs was also studied in SGF (pH 1.2) for the first 2 h, without pepsin, to avoid any discrepancies due to degradation of the drug in the presence of this endopeptidase. Afterwards, the NPs were incubated with FaSSIF (pH 6.8) for the following 6 h, to mimic their transit into the small intestine. To prevent that any putative degradation of the drug could interfere with the interpretation of the in vitro drug release studies, the maintenance of GLP-1 concentration in SGF and in FaSSIF throughout the incubation periods was studied using UV-Vis spectroscopy (Fig. S3). Results showed the same UV-Vis spectra for GLP-1 when measured immediately after preparation and after 2h incubation with SGF (Fig. S3). Also, the concentration of GLP1 in SGF at the beginning of the experiment was 28 ± 0.3 µg/mL, and after 2 h incubation 29 ± 0.9 µg/mL, suggesting that the drug concentration was not affected by the incubation conditions. The same effect was observed when analyzing the UV-Vis spectra of GLP-1 in FaSSIF both after preparation and after 6 h, showing concentrations of 29 ± 0.6 µg/mL and 30 ± 1 µg/mL, respectively. The maintenance of GLP-1 concentration over time suggested that the overall integrity of the drug was not compromised upon exposure to the buffers used for the drug release studies. Additionally, no distinct degradation peaks were observed in the HPLC spectra of GLP-1 in SGF and FaSSIF solutions, supporting the UV-Vis results. The GLP-1 release profiles from CS-Fc-UnPSi and CS-Fc-UnPSi@HF NPs were then evaluated (Fig. 4). The NPs that were not entrapped into the pH-responsive polymeric matrix 21 ACS Paragon Plus Environment


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started to release the drug immediately after incubation with SGF, which, in a living organism, could be translated to an unwanted exposure of the drug to the harsh stomach environment, compromising the structure and, subsequently, the therapeutic effect. By contrast, the CS-FcUnPSi NPs that were protected by the polymeric matrix did not release the drug at acidic pH, showing instead a burst release during the first 15 min after incubation with FaSSIF, which may be caused by diffusion of the peptide from the NP pores, followed by a sustained release profile up to ca. 60% for the next 6 h. When moving on to in vitro studies, this delayed drug release may contribute to the increased amount of active GLP-1 that will be released in the vicinity of the cells, and also the GLP-1 will be released from the NPs transcytosed via FcRn, increasing the overall amount of GLP-1 that could reach the basolateral side of intestinal mimicking cells. Furthermore, the encapsulation of the core NPs into HF did not affect the total amount of drug that could be released from the pores of the UnPSi NPs. The fact that not all GLP-1 was released from the NPs can be ascribed to the hydrophobic interactions between the drug and the surface of the pores of the UnPSi NPs, impairing the release of the peptide that was irreversibly adsorbed onto the pore surfaces, as previously reported.22 The in vitro drug release data suggested that the polymeric matrix formed by glass capillary microfluidic nanoprecipitation provided an easy way to tune the drug release profile by capping the CS-Fc-UnPSi NPs. GLP-1 integrity could be preserved by the intact form of the polymeric matrix at acidic conditions, with sustained release thereafter, due to the pHresponsiveness of the polymer. The relevant properties of UnPSi NPs, and the successful surface functionalization with the Fc fragment of IgG, turned this nanoparticle system into a multistage platform for FcRn-targeted therapy of orally administered GLP-1. Due to the versatility of the core UnPSi NPs, the applicability of this system could be easily translated to the improvement of therapeutic strategies based on the oral administration of other proteins or peptides, previously reported to be successfully loaded into the NPs.21


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Fig. 4. GLP-1 release profiles from CS-Fc-UnPSi and CS-Fc-UnPSi@HF NPs. The percentages of GLP-1 released from non-encapsulated and HF-encapsulated NPs were compared at time points from 0 to 480 min. The experiments were conducted at 37 °C and magnetic stirring (100 rpm), with the particles disperse in SGF solution (pH 1.2) for the first 2 h, and then transferred to FaSSIF (pH 6.8), and maintained for the following 6 h. Results are expressed as mean ± s.d. (n = 3).

3.5. Cytocompatibility studies Since the developed NPs are envisaged for increasing the intestinal absorption of orally administered drugs, their compatibility with the cells that constitute the intestinal epithelium was evaluated using a validated adenosine triphosphate (ATP)-based luminescence assay (Fig. 5).44 Human colon carcinoma Caco-2 cells were used to resemble the enterocytes, which represent ca. 90% of the total epithelial cells, whereas goblet-like HT29-MTX were used for being mucus producing cells, representing the remaining 10% of the intestinal epithelium.29-30 The cell lines were separately exposed to UnPSi, Fc-UnPSi, CS-Fc-UnPSi, and CS-Fc23 ACS Paragon Plus Environment


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UnPSi@HF NPs, for a period of 24 h, in concentrations of NPs ranging between 25 and 600 μg/mL. Different cell lines and concentrations were tested to demonstrate the potential of the NPs for further in vitro experiments, including concentration-dependent toxicity and highest safe concentrations to be used. The core UnPSi and Fc-UnPSi NPs induced toxicity in both cell lines for concentrations equal or greater than 100 µg/mL, nevertheless presenting cell viabilities close to 80%. When coated with CS, an enhancement in the cytocompatibility was observed, presenting a significant difference (**p < 0.01) only for the highest concentration of NPs in Caco-2, and for the two highest concentrations in HT29-MTX after 24 h. Again, the overall cell viability values were maintained close to 80%, demonstrating the cytocompatibility of the NPs with the intestinal cells. No significant cellular toxicity values were observed when exposing the Caco-2 cells to the final nanosystem. HT29-MTX seemed to respond slightly negatively to the highest concentration of the CS-Fc-UnPSi@HF NPs, still presenting cell viability of approximately 86%. Considering that the mucus producing cells constitute only 10% of the intestinal epithelium, a minimized cytotoxicity effect of the NPs is expected in future experiments. In summary, the toxicity induced in the intestinal cell lines by the UnPSi and Fc-UnPSi NPs seemed to be mostly induced by the nature of the particle surface. Due to their irregular shape, inherent hydrophobic nature, and subsequent hydrophobic interactions with the cell membranes,44 bare UnPSi NPs showed to have indeed the lowest compatibility with the intestinal cells. Additionally, the large UnPSi aggregates formed in aqueous medium may have also contributed to the reduced cell compatibility when comparing to the modified NPs. Nonetheless, the overall cell viability of the final NPs when in contact with Caco-2 and HT29MTX for 24 h supported the biocompatibility of the nanosystem developed in this study.


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Fig. 5. Cytocompatibility of the developed NPs. Cell viability values (%) of different intestinal cells when exposed to the different NP formulations (UnPSi, Fc-UnPSi, CS-Fc-UnPSi and CS-Fc-UnPSi@HF NPs) assessed by CellTiter-Glo® luminescence assay. The ATP content of Caco-2 and HT29-MTX was investigated after 24 h incubation with the NPs at different concentrations, and at the different steps of preparation. Cells incubated with HBSS–HEPES buffer; pH 7.4, as negative control. The incubations were conducted at 37 °C and 5% CO2. Two-way ANOVA followed by the Bonferroni post-test were used for the statistical analyses of the measured values. Each data set was compared to the negative control. The level of significant differences was set at probabilities of *p < 0.05, **p < 0.01 and ***p < 0.001. Results are expressed as mean ± s.d. (n ≥ 3).

3.5. FcRn-mediated cell–NP interactions As a preliminary study to evaluate the specificity of the FcRn-targeted NPs interactions with FcRn-expressing intestinal cells, the cell–NP interactions were studied by confocal fluorescence microscopy. FcRn has been reported to be expressed in the apical region of epithelial cells in both fetuses and adults in the small intestine and throughout the colon.9, 13 Therefore, a Caco-2/HT29-MTX co-culture (ratio 90:10, respectively)29-30 was used for 25 ACS Paragon Plus Environment


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evaluating the specific interactions of the developed NPs with the intestinal cells. After being in culture for 24 h, the co-cultures were exposed to UnPSi, Fc-UnPSi, CS-Fc-UnPSi and CSFc-UnPSi@HF NPs. The FcRn-targeted systems presented inherent fluorescence, due to the use of Alexa Fluor® 488-conjugated Fc fragment as the targeting ligand, which showed to be biologically active in the present formulation, as shown by the ELISA assay. The bare UnPSi NPs served as a control, and were fluorescently labelled by covalent conjugation with Alexa Fluor® 488 using the same EDC/NHS chemistry protocol. The ratio of dye versus dyeconjugated Fc fragment was optimized in advance, in order to obtain the same fluorescence intensity for all the NPs.25 The different NPs were dispersed in HBSS–HEPES buffer at intestinal pH and then incubated with the two cell lines. At this pH, the HF matrix in the final formulation was dissolved, exposing the core CS-Fc-UnPSi NPs. The cells were extensively washed after incubation in order to prevent signals from NPs that were not specifically interacting with the cells. Results of the cell–NP interaction studies are presented in Fig. 6, where the green fluorescence represents the Alexa Fluor® 488-conjugated NPs, whereas the red fluorescence represents the cell membranes stained with CellMask™ Deep Red. Images show that in the control group, in which the NPs were absent, only the cell membranes were observed. Also, minimal cell–NP interactions were observed when the cells were exposed to bare Alexa Fluor® 488-conjugated UnPSi NPs. By contrast, after incubation with FcRn-targeted UnPSi NPs, which are inherently fluorescent due to the use of Alexa Fluor® 488-conjugated Fc fragment, an increase in the interaction with the cells was observed, suggesting specific interactions of the Fc fragment on the surface of the NPs with the FcRn expressing intestinal cells, and supporting the biological activity of the molecule. After coating with CS, the CS-Fc-UnPSi NPs showed still more interactions with the cells than the UnPSi NPs, which can be potentiated by the strong adhesion properties of CS present on the surface of the NPs, thus increasing the electrostatic interactions between them.20, 36, 45 However, in this particular case, the NPs were 26 ACS Paragon Plus Environment

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more aggregated, which is in accordance with the slightly increased PdI observed in DLS at this step of the NP preparation. Such aggregation was overcome when incubating the cells with CS-Fc-UnPSi NPs after entrapment in the HF polymeric matrix, but the high levels of interactions between the cells and the NPs were maintained. Therefore, these results suggest that the interaction of FcRn-targeted NPs with the intestinal cells was substantially higher than that observed with non-targeted NPs, reinforcing the specific interactions of the NPs as a result of the surface functionalization with the Fc-fragment. This interaction is expected to be prolonged due to the presence of CS on the surface of the NPs.



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Fig. 6. FcRn-mediated cell–NPs interactions. Confocal fluorescence microscopy of Caco2/HT29-MTX co-cultures after incubation with UnPSi, Fc-UnPSi, CS-Fc-UnPSi and CS-FcUnPSi@HF NPs for 3 h at 37 °C. CellMask™ Deep Red (red) was used to stain the cell membranes, UnPSi NPs were labeled with Alexa Fluor® 488 (green), and Fc-UnPSi, CS-FcUnPSi, and CS-Fc-UnPSi@HF NPs where inherently labeled with Alexa Fluor® 488conjugated Fc fragment (green). 28 ACS Paragon Plus Environment

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3.7. Validation of FcRn expression Cell culture models provide an ideal system for studying drug absorption at early stages of drug development due to their high throughput feature and reproducibility.29 Caco-2/HT29MTX co-culture model currently benefits from the widespread popularity, being undoubtedly the most popular and rapid model for the study of the intestinal permeability of drug and/or drug carrier candidates.30 Caco-2 and HT29 cells, the most widely used cell lines for mimicking the intestinal microenvironment in vitro, were reported to individually express FcRn.46-48 Particularly, Caco-2 cells have been already used for transcytosis studies.12, 14, 49 However, it was also reported that the FcRn expression in Caco-2 cells varies according to the differentiation process.50 Moreover, no study has reported whether the HT29-MTX also express the receptor. HT29-MTX cells were treated with methotrexate to exhibit a fully differentiated goblet cell-like phenotype,51 being therefore the cell line of choice for representing the mucus producing cells in this study. Taking this into account, before evaluating the efficacy of the NPs in terms of intestinal absorption, the FcRn expression was evaluated in the in vitro Caco-2/HT29-MTX monolayers grown in Transwell™ filters for 21 days. For this purpose, Caco-2/HT29-MTX cells were cocultured in a ratio of 90:10, as previously reported.29-30 After 21 days, the epithelial cells are fully differentiated and polarized, and a mucus layer is formed, highly resembling the in vivo situation.52 To avoid any influence of the serum constituents in the FcRn transcytotic capacity, the co-cultures were maintained with serum-free medium overnight prior to the experiments. Afterwards, the 2D in vitro model was incubated with free Alexa Fluor®-labelled Fc fragment for 3 h at 37 °C in humidified atmosphere. Non-interacting Fc fragment was removed by extensively washing the co-cultures. Results of confocal fluorescence microscopy are presented in Fig. 7. Images show an intense and well-distributed yet heterogeneous FcRn immunofluorescence in the monolayer, whereas the co-cultures that were not incubated with 29 ACS Paragon Plus Environment


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the fluorescent Fc ligand showed no green fluorescence under the same conditions. The heterogeneous pattern of FcRn-expressing cells has been recently reported, in which single Caco-2 cells showed a similar surface FcRn expression pattern, presenting the receptor in ca. 60% of the cell population in culture.47 Hence, the 2D in vitro model used in this study was revealed to efficiently express FcRn, and can be used for evaluating the intestinal absorption of Fc engineered drugs and/or drug carriers.

Fig. 7. Expression of FcRn in the in vitro intestinal cell culture model. Confocal fluorescence microscopy of Caco-2/HT29-MTX cells co-cultured for 21 days in Transwell™ membranes after exposure to Alexa Fluor 488®-conjugated Fc fragment (3.96 µg/mL) for 3 h at 37 °C and humidified atmosphere. Fc fragment was used to stain FcRn (green), and DAPI was used to stain the nuclei (blue). Co-cultures non-treated with Fc fragment were used as control. Scale bar is 50 μm.


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3.8. GLP-1 permeability across the Caco-2/HT29-MTX monolayer To reach the blood circulation, and subsequently impart a therapeutic effect, GLP-1 must migrate from the apical to the basolateral side of the intestinal epithelium. Therefore, after the promising cell–NP interaction studies, and the validation of the FcRn expression by the 2D in vitro cell culture model used in this study, the permeability of GLP-1 across the intestinal cell monolayers was studied. Firstly, the functionality of the in vitro model was validated by checking the formation of tight junctions, which was studied by monitoring the TEER at the end of the culture period. The TEER is an indicator of the ion conductance across the monolayers, and it was measured as previously described.32 TEER values for the co-cultures were 224.90 ± 17.11 Ω cm-2, suggesting the formation of continuous tight junctions, and therefore, validating the functionality of the intestinal model, with a close resemblance to the in vivo scenario.32, 53 For the intestinal permeability studies, only non-FcRn targeted and FcRn-targeted NPs were used, since bare UnPSi NPs had already demonstrated minimal mucosal and cellular interactions.22, 54 Free GLP-1 in solution was used as a control. The different samples were incubated with the Caco-2/HT29-MTX monolayers for a period of 3 h, and the amount of drug that reached the basolateral side was quantified using HPLC. The pH values of the apical and basolateral sides were adjusted to 6.8 and 7.4, respectively. At pH 6.8 in the apical side, the pHresponsive polymeric capsule will be dissolved, exposing the FcRn-targeted and non-targeted NPs for the permeability studies. The GLP-1 cumulative permeability across the monolayers after incubation with the different samples is shown in Fig. 8. The results showed no significant variations in the permeability profiles of the free GLP-1 and the non-targeted NPs, with both increasing significantly (****p < 0.0001) during the first 15 min, and remaining stable throughout the rest of the experiment. In contrast, the curve of the FcRn-targeted NPs depicted an increase in the cumulative permeability of GLP-1 as compared to the free drug and CS-UnPSi@HF NPs. 31 ACS Paragon Plus Environment


Importantly, the permeation of the GLP-1 loaded into the FcRn-targeted NPs across the intestinal model was shown to be increasing over the incubation period, suggesting that the functionalization of the NPs with the Fc fragment plays a crucial role in the improvement of drug absorption at the intestinal microenvironment. Free GLP-1

Cumulative Permeability (%)

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CS-UnPSi@HF

CS-Fc-UnPSi@HF

20 15

** **

10 5 0

0

15

30

60

90

120

180

Time (min)

Fig. 8. In vitro GLP-1 permeability. Pemeability of free GLP-1, CS-UnPSi@HF, and CS-FcUnPSi@HF NPs loaded with GLP-1 across Caco-2/HT29-MTX monolayers. The experiments were conducted at 37 °C from the apical to the basolateral side of the Transwell™, pH 6.8 and 7.4, respectively, in HBSS–HEPES buffer. The level of significant differences was set at probabilities of ****p < 0.0001. Results are expressed as mean ± s.d. (n ≥ 3).

These results were corroborated with the TEM images of flat embedded ultrathin Caco2/HT29-MTX monolayers after the 3 h incubation with the free drug, CS-UnPSi@HF, and CSFc-UnPSi@HF NPs for the permeability studies (Fig. 9). The images did not show any alterations in the morphology of the cells when incubated with free drug (Fig. 9a), suggesting that the GLP-1 detected in the basolateral side crossed the monolayer by transport mechanisms.55 The CS coated, but not FcRn-targeted NPs, were found in the close vicinities of 32 ACS Paragon Plus Environment

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the cells, resulting from the mucoadhesive properties of CS physically adsorbed onto the surface of the NPs (Fig. 9b).21, 54 Still, on one hand, the CS coating was not enough to improve the amount of GLP-1 that was able to cross the intestinal cells as compared to the free drug. On the other hand, the CS-Fc-UnPSi@HF NPs were found to be in close contact with the brush border of the FcRn-expressing cells (Fig. 9c). Importantly, the NPs were found to be interacting with the cells and entrapped into vesicles (highlighted in the figure in red dash-lines), which is in accordance with the increased GLP-1 absorption detected in the basolateral compartment, thus supporting the successful development of this multistage platform for oral peptide delivery with increased drug absorption via FcRn transcytosis.



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Fig. 9. TEM images of the monolayers after the permeability studies. Representative TEM images of flat embedded ultrathin Caco-2/HT29-MTX monolayers grown for 21 days in Transwell™ filters, and incubated for 3 h with free GLP-1 (a), CS-UnPSi@HF (b), and CS-FcUnPSi@HF (c) NPs for in vitro permeation studies. Scale bar represents 1 μm.

4. Conclusion In this study, we reported the development of a multistage NP platform for oral peptide delivery using the glass capillary microfluidic nanoprecipitation technique. Such technique allowed the individual entrapment of CS-coated FcRn-targeted UnPSi NPs into a pHresponsive polymeric matrix in a highly reliable, efficient, and reproducible manner. The produced nanosystem showed monodisperse size distribution, optimal surface ζ–potential variations at different steps of preparation, and multistage pH-sensitive properties; precise ratiometric controlled drug loading, sustained and pH-responsive release kinetics of the payload. The pH-responsive capacity allowed for the protection of the drug at low pH values, and triggered a controlled drug delivery at intestinal pH. Binding assays validated the integrity of the Fc fragment and its biological activity after conjugation to the surface of the NPs, and after exposure to the conditions of the different steps of preparation of the formulation. Furthermore, this platform was screened with advanced in vitro cell culture models, in which the FcRn expression was validated. The NPs showed high cytocompatibility, and increased levels of interaction with the intestinal cells when functionalized with the Fc fragment. Enhanced GLP1 absorption across the intestinal monolayers as a result of the FcRn transcytotic capacity was demonstrated, representing a step forward in the study of the extremely underexplored FcRntargeted therapies for oral peptide delivery. Additionally, this platform showed potential to be used for the oral delivery of different drugs or drug combinations, and to overcome several limitations associated with the oral delivery of peptides.


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Supporting Information. Materials; Preparation of UnPSi NPs; analysis of UnPSi NPs with N2 sorption; DLS plots of size distribution of the prepared NPs; TEM image of CS-FcUnPSi@HF NPs for analysis of the elemental composition by EDX; UV-Vis spectra of GLP-1 solution in SGF and in FaSSIF.

Funding M. P. A. Ferreira received funding from Drug Research Programme of the University of Helsinki for a Ph.D. grant. B. Sarmento received funding from the project NORTE-01-0145FEDER-000012, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). FEDER - Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and the Portuguese funds through FCT - Fundação para a Ciência e a Tecnologia (POCI-01-0145-FEDER-007274). H.A. Santos received funding from the University of Helsinki Research Funds, the Sigrid Jusélius Foundation (decision no. 4704580), the HiLIFE Research Funds, and the European Research Council under the European Union’s Seventh Framework Programme (FP/2007-2013, grant no. 310892).

Acknowledgements The authors acknowledge Beatriz Martins for technical support on the ELISA studies. The authors acknowledge the following core facilities funded by Biocenter Finland: Electron Microscopy Unity of the University for providing the facilities for TEM imaging, and the Light Microscopy Unit of the Institute of Biotechnology for the confocal microscope.



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Graphical Abstract


Microfluidic Nanoassembly of Bioengineered Chitosan-Modified FcRn

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