Arajo 2015

Page 1 of 40 ACS Nano
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7 Microfluidic Assembly of a Multifunctional Tailorable Composite
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10 System Designed for Site Specific Combined Oral Delivery of
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13 Peptide-Drugs
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Francisca Araújo1,2,3,4, Neha Shrestha4, Mohammad-Ali Shahbazi4, Dongfei Liu4, Bárbara
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20 Herranz-Blanco4, Ermei M. Mäkilä4,5, Jarno J. Salonen5, Jouni T. Hirvonen4, Pedro L. Granja1,2,3,
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22 Bruno Sarmento1,2,6,*, Hélder A. Santos4,*
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26 Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal
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29 INEB – Instituto de Engenharia Biomédica, 4150-180 University of Porto, Portugal
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ICBAS – Instituto Ciências Biomédicas Abel Salazar, 4150-180 University of Porto, Portugal
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36 Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, FI-00014
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38 University of Helsinki, Finland
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42 Laboratory of Industrial Physics, FI-20014 University of Turku, Finland
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45 CESPU, Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde,
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47 4585-116 Gandra, Portugal
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51 *Corresponding authors: Division of Pharmaceutical Chemistry and Technology, Faculty of
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53 Pharmacy, P.O. Box 56 (Viikinkaari 5E), FI-00014 University of Helsinki, Finland. E-mails:
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bruno.sarmento@ineb.up.pt (B. Sarmento), helder.santos@helsinki.fi (H.A. Santos)
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ACS Paragon Plus Environment
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ACS Nano Page 2 of 40
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ABSTRACT
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Multifunctional tailorable composite systems, specifically designed for orally dual-deliver of a
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8 peptide (glucagon-like peptide-1) and an enzymatic inhibitor (dipeptidyl peptidase 4 (DPP4)),
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10 were assembled through the microfluidics technique. Both drugs were co-loaded into these
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13 systems for a synergistic therapeutic effect. The systems were composed of chitosan and cell-
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15 penetrating peptide modified poly(lactide-co-glycolide) and porous silicon nanoparticles as
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17 nanomatrices, further encapsulated in an enteric hydroxypropylmethylcellulose acetylsuccinate
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20 polymer. The developed multifunctional systems were pH-sensitive, inherited by the enteric
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22 polymer, enabling the release of the nanoparticles only in the simulated intestinal conditions.
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24 Moreover, the encapsulation into this polymer prevented the degradation of the nanoparticles’
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27 modifications. These nanoparticles showed strong and higher interactions with the intestinal cells
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29 in comparison with the non-modified ones. The presence of DPP4 inhibitor enhanced the peptide
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permeability across intestinal cell monolayers. Overall, this is a promising platform for
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34 simultaneously deliver two drugs from a single formulation. Through this approach peptides are
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36 expected to increase their bioavailability and efficiency in vivo both by their specific release at
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39 the intestinal level and also by the reduced enzyme activity. The use of this platform, specifically
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41 in combination of the two antidiabetic drugs, has clinical potential for the therapy of type 2
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43 diabetes mellitus.
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50 Keywords: chitosan; dual-delivery; microfluidics; PLGA; porous silicon
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Nanoparticles composed of biocompatible and biodegradable materials have been claimed as
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6 promising candidates towards the oral administration of peptides.1, 2 These nanoparticles enhance
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8 the oral bioavailability of peptides and control their release, as well as provide a preserved
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environment for the encapsulated drugs.3-5
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14 Among the mostly studied materials, poly(lactic-co-glycolic acid) (PLGA) and mesoporous
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16 silicon (PSi) have attracted a lot of attention becoming the most desirable materials in the drug
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delivery field for the administration of macromolecules.6, 7 In one hand, PLGA features can be
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21 modulated by the ratio between the monomers which constitute the polymer, exhibiting a wide
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23 range of erosion times, favourable degradation characteristics and tuneable mechanical
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26 properties. It can be used to encapsulate numerous drugs with different physicochemical
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28 properties and also to sustain their release.8, 9 Moreover, it has extensive clinical applications,
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30 being already approved by the Food and Drug Administration (FDA) and the European Medicine
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33 Agency (EMA) for parenteral administration.9 On the other hand PSi nanoparticles have large
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35 surface area, pore volumes with adjustable diameters (2-50 nm), as well as higher drug loading
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37 capacity compared to the majority of the other materials.10-12 Furthermore, the drug loading is
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40 usually a simple process where the drug is retained inside the mesopores by physical adsorption
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42 or electrostatic interactions.10 Nevertheless, the oral delivery of PLGA and PSi nanoparticles is
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not in such advanced state. The negative surface charge of the nanoparticles tends to limit their
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47 interaction with the negatively charged cell’s surface, which is further intensified by the rapid
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49 turnover of the mucus and the intestinal cells.4
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To tackle the limitations of these oral drug nanocarriers, the nanoparticles can be tailored with
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55 other materials to yield a variety of physical properties to overcome the main intestinal barriers,
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57 such as the mucus layer, epithelium and enzymatic degradation.13, 14
Chitosan (CS) and cell
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penetrating peptides (CPPs) are polycationic molecules extensively used in the drug delivery
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6 field. CS is a biopolymer that has been mainly used to modify nanoparticles due to its
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8 mucoadhesive characteristics and its properties as an intestinal permeability enhancer by
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transiently open the tight junctions existing between the epithelial cells.15-17 CPPs were originally
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13 considered as a “Trojan horse” because of their ability of entering cells without causing damage
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15 or eliciting a cellular response,18, 19
increasing the transcellular transport.20 Yet, a major
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18 challenge still remains for the oral delivery of peptides in order to overcome the harsh conditions
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20 of the stomach. In this regard, pH-sensitive polymers have been frequently employed in the drug
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22 formulation and/or coating of nano- and microparticles to protect them from the very acidic
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25 gastric pH.11, 21
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28 In this study, we developed novel multifunctional systems that can simultaneously load peptides
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30 and enzymatic inhibitors in a single carrier with the aim to resist the conditions of the stomach,
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33 to enhance the nanoparticle’s interactions with the intestinal mucus and epithelium, and to
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35 protect the peptides from enzymatic degradation after the release. An antidiabetic peptide,
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37 glucagon-like peptide-1 (GLP-1), is in the pipeline for the type 2 diabetes mellitus (T2DM)
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40 therapy, and was used here as a model peptide. Due to its poor intestinal permeability, GLP-1
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42 needs to be administrated by parenteral route, resulting in poor patient compliance.22, 23
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Moreover, the success of the GLP-1 therapy is hindered by its rapid degradation (< 2 min) by the
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47 dipeptidyl peptidase 4 (DPP4) enzyme produced in the intestine.24 Therefore, in order to achieve
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49 an efficient release and permeability across the intestinal epithelium of active GLP-1, the
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52 antidiabetic peptide was loaded into different nanoparticles composed of PLGA and PSi
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54 biomaterials. The PLGA and PSi nanoparticles were further modified with the mucoadhesive
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56 polymer CS and with an oligoarginine CPP to increase the permeability of nanoparticles across
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the intestinal cells. Afterwards, the nanoparticulate systems were encapsulated within an enteric
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6 polymer hydroxypropylmethylcellulose acetylsuccinate (HPMC-AS) loaded with the DPP4
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8 inhibitor, using the microfluidic technique (Scheme 1). To the best of our knowledge, this is the
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first time that the combination of these two drugs was formulated in a single delivery system.
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13 This innovative approach produced monodisperse and uniform particulate structures with the
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15 desired composition.11, 21
In comparison to the conventional preparation methods with
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18 microfluidics, by tuning the flow rates of the immiscible fluids, emulsions are formed with an
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20 exquisite degree of control and rather high encapsulation efficiency.25, 26
The resultant
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22 multifunctional systems were then characterized for size, morphology, pH-responsiveness, drug
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25 release, and synergistic antidiabetic peptide GLP-1 permeability across a triple intestinal cell co-
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27 culture model in the presence of the enzymatic DPP4 inhibitor. The inhibition capacity of the
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DPP4 enzyme activity was also evaluated.
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Scheme 1. Schematic representation of the microfluidics approach used to produce the pH-
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6 responsive systems, co-loaded with GLP-1 and DPP4 inhibitor. The inner fluid consisted of
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8 modified PLGA and PSi nanoparticles and the enteric polymer dissolved in ethyl acetate. The
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antidiabetic peptide GLP-1 was loaded into the modified nanoparticles, whereas the enzymatic
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13 DPP4 inhibitor was dissolved directly in the inner fluid. The outer continuous fluid was an
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15 aqueous solution of F127 (2 % w/v) which could efficiently stabilize the oil/water (O/W)
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18 interface.
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RESULTS AND DISCUSSION
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28 Characterization of the Multifunctional systems. After the production of the nanoparticles and
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30 the CS−CPP surface functionalization, the nanoparticles were characterized in respect to mean
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33 size, polydispersity index (PdI), surface charge, association efficiency (AE) and loading degree
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35 (LD), as shown in Table 1. Both the unmodified PLGA and PSi nanoparticles presented a Z-
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average size of ca. 200 nm with a PdI below 0.1, and negative surface charges. In order to
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40 increase the interaction of the nanoparticles with the negatively charged intestinal mucus layer
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42 and to improve the permeability of the antidiabetic peptide GLP-1 across the intestinal cells, the
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nanoparticles were coated firstly with CS to form PLGA+CS and PSi+CS nanoparticles.7, 8, 28
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47 The CS-modified nanoparticles showed an increase in their size and conversion of the zeta-
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49 potentials to positive values, demonstrating the successful CS coating of the nanoparticles.7, 8, 13,
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52 In order to improve the transcellular transport, the CS-coated nanoparticles were further
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54 modified by covalent attachment of polyarginine R9 CPP to the amine groups of the CS. CPPs,
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56 such as R9, are small peptides of few acidic amino acid residues with a high positive charge.20
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Such CPPs are known to have a positive role in the translocation of some drugs across the
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6 intestinal epithelium, increasing their oral bioavailability.29-33 Overall, when modified with CS
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8 and CPP (to form PLGA+CS-CPP and PSi+CS-CPP particles), the nanoparticles presented
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higher particle sizes than the unmodified ones, without significant change in the zeta-potential
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13 values (Table 1). Together with the size and charge, the similarity in the AE and LD values,
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15 indicated the high homogeneity of the nanoparticles from batch-to-batch.7
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The CS-CPP modified nanoparticles were further encapsulated within an enteric polymer,
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21 HPMC-AS, through the microfluidics technique, for their protection, as well as for the protection
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23 of the CS and CPP, at the low pH conditions of the gastric environment, originating the H-
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26 PLGA+CS-CPP and H-PSi+CS-CPP particles. The microfluidics technique has brought
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28 revolutionary impact in the pharmaceutical technology field by manipulating nanoliter volumes,
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30 in microscale fluidic channels, with high precision and reduced consumption of reagents.34, 35 In
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33 contrast with the conventional production methods, which yield a polydisperse population with
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35 drug loading levels less than ideal, with the microfluidic technique the efficiency of
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37 encapsulation is near 100%, the particles formed are stable, homogeneous and can be size-
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40 controlled.11, 36
Moreover, it can be applicable for diverse constituent fluids, with various
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42 chemical compositions and to different cargos, making it a promising technique for the
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production of several drug delivery systems.37 The flow-focusing geometry used in this work is a
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47 droplet-based method widely used to produce different types of fluid entities on a continuous
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49 basis by applying an extensional co-flow.37 In this method, fluids are forced through a narrow
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52 orifice where high shear and capillary instability break bubbles off the tip, forming monodisperse
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54 emulsion droplets.36
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As shown by the scanning electron microscopy (SEM) (Figure 1), the produced particles
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6 (control group, pH 4.0) had a regular and spherical shape with smooth surface and size around
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8 59.44 ± 8.01 µm. The SEM images also showed a homogeneous size distribution, suggesting the
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11 production of monodisperse particles by the microfluidic technique.11 The DPP4 inhibitor
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13 encapsulation efficiency was 20 ± 5 %. Considering the amount (mass) of DPP4 inhibitor in the
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final formulation and that the IC50 of the DPP4 inhibitor is of 14 nM, the amount of inhibitor
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18 present in the current formulation should be more than enough to be active and to inhibit the
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20 enzyme activity efficiently. However, higher values can certainly be obtain in the final
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formulation, for example, by increasing the viscosity of the system (e.g., the viscosity of the
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25 HPMC-AS polymer) in order to retain strongly the DPP4 inhibitor in the polymer matrix; by
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27 increasing the osmolality of the collection solution or the solution viscosity; or by encapsulating
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30 the DPP4 in the nanoparticles either together with the GLP-1 or alone.
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33 Table 1. Characterization of the nanoparticles in respect to their size, PdI, zeta-potential (ζ
ζ-
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35 potential), AE and LD of GLP-1 of different nanoparticles. Results are presented as mean ±
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38 standard deviation, n ≥ 3.
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41 Size ζ-potential AE LD
42 Sample PdI
43 (nm) (mv) (%) (%)
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46 PLGA 199.0 ± 4.1 0.08 ± 0.02 −23.6 ± 0.2 58.9 ± 6.9 0.14 ± 0.03
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48 243.0 ± 2.2 0.26 ± 0.01 24.8 ± 2.7 59.7 ± 0.7 0.07 ± 0.01
PLGA+CS
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51 PLGA+CS-
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277.2 ± 3.8 0.32 ± 0.02 21.6 ± 3.8 59.7 ± 0.7 0.07 ± 0.01
CPP
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55 PSi 193.7 ± 3.0 0.09 ± 0.03 −16.3 ± 0.3 75.0 ± 0.6 15.00 ± 0.05
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4 PSi+CS 282.9 ± 8.0 0.34 ± 0.02 19.2 ± 0.4 75.0 ± 0.5 7.50 ± 0.03
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7 PSi+CS-CPP 320 ± 9.8 0.33 ± 0.02 19.1 ± 1.0 75.0 ± 0.5 7.50 ± 0.03
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Figure 1. Dissolution behaviour of the CS-CPP modified nanoparticles encapsulated in the
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43 HPMC-AS polymer at different pH conditions of 1.2 and 5.5 for 2 h, and at pH 6.0 and 6.8 for
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45 10 min and 2 h. The SEM images show the morphology of the prepared particles. The control
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48 group was composed by particles at pH 4.0. At pH values below 6.0 the structural integrity of the
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50 particles was maintained. At pH 6.0 and 6.8 the structure of the polymeric matrix was destroyed
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52 by the dissolution of the polymer.
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pH-Sensitive Response of the Multifunctional Systems. HPMC-AS is a polymer insoluble in
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6 acidic conditions and highly soluble at neutral or alkaline pH (pH > 6.0).11 To evaluate whether
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8 the prepared nanoparticles were successfully encapsulated in the polymer and whether the
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multifunctional system could stand the harsh conditions of the stomach, the particles were
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13 observed under SEM at different pH conditions. As shown in Figure 1, at pH 1.2 and 5.5, the
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15 shape and surface morphology of particles were regular and smooth, similarly to the observed
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18 control particles, showing that the HPMC-AS encapsulation using the microfluidics technique
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20 was effective and that the nanoparticles were protected at the acidic conditions. When the
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22 multifunctional particulate system was in contact with buffer at pH 6.0, the polymer started to
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25 dissolve and the particles structure started to be compromised already after the first 10 min. After
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27 2 h of incubation, the surface of the particles appeared rough with some visible holes as an
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indication for the dissolution of the polymer. At pH 6.8, this behaviour was even more
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32 pronounced with greater changes in the particles’ structure during the first 10 min. After 2 h the
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34 polymer was completely dissolved. These results are in accordance with the pH responsive
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37 characteristics of the polymer, as described elsewhere.11, 21
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40 Cell Viability Studies. The viability studies were performed in three different cell lines at two
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42 different time points and using different concentrations of the particles. AGS is a gastric
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45 epithelial cell line originated from human gastric adenocarcinoma,38 and the cell viability was
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47 assessed after 3 h of incubation with the particulate systems. Caco-2 and HT29-MTX are
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intestinal cell lines originated from colon adenocarcinomas, representing the enterocytes and
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52 mucus producing goblet cells,27, 39 respectively, and their viability was measured after 12 h of
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54 incubation with the particles. Testing these three cell lines in the two different time points
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covered the maximum transit time that the particles would be in the stomach and intestine when
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orally administered, making this a suitable assay to understand whether there is any
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6 concentration or component dependent toxicity.7, 40
Non-modified nanoparticles, CS-CPP-
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AS particles were tested.
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14 With regards to the PLGA systems, it was observed in the three cell lines studied, that none of
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16 the non-modified nanoparticles, CS-CPP modified nanoparticles and CS-CPP modified
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nanoparticles encapsulated with HPMC-AS presented toxicity at the tested concentrations, as
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21 depicted in Figure 2. PLGA is commonly accepted for its low cytotoxic and good
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23 biocompatibility and biodegradability properties, and has already been approved by the FDA for
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26 clinical use.18, 29
On the other hand, positively charged particles are usually described as
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28 interacting more with the surface of the cell membranes than the negatively charged particles,
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30 which may lead to higher cellular cytotoxicity.41, 42 However, in this study, despite the positive
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33 surface charge of the CS-CPP modified nanoparticles, no decrease in cell viability was observed
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35 in all the cell lines tested. This indicates that the amounts of CS and CPP used did not trigger
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37 cytotoxicity reaction, and thus, could be considered as safe for oral drug administration purposes.
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40 Previously, some studies have also demonstrated that certain CPPs, including oligoarginine, did
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42 not cause any harm to the intestinal epithelial cells.33, 43
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45 HPMC-AS has been used as a pharmaceutical excipient and has a median lethal dose (LD50)
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48 higher than 2.5 g kg-1, which is much higher than the amounts used in our particle formulation.44
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50 Moreover, the organic solvent used to dissolve the polymer, ethyl acetate (EA), is considered a
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rather non-toxic organic solvent,45 and it is believed that no residues of the solvent would be
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55 present in the final formulation. In fact, due to its high solubility in water, EA presented a fast
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diffusion rate to this solvent which allowed the fast solidification of the particles in the collecting
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6 solution, after their formation in the microfluidics device.21, 45
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9 For the PSi particles, it was observed that the non-modified nanoparticles, CS-CPP modified
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11 nanoparticles, and the CS-CPP modified nanoparticles encapsulated with HPMC-AS presented
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14 concentration dependent cytotoxicity values. The CS-CPP modified nanoparticles presented
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16 higher viability values than the non-modified ones with the exception of the HT29-MTX cells.
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This behaviour may be related to the presence of CS on the surface of the nanoparticles, which
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21 would increase the interaction of the nanoparticles with the mucus secreting cells due to the
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23 mucoadhesive properties,40 as described elsewhere.7 When the pH-sensitive polymer was used to
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26 encapsulate the CS-CPP modified nanoparticles, there were no statistically significant changes in
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28 the viability of the three cell lines. Although there was a tendency for improved cell viability
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30 values by the encapsulated nanoparticles compared to the non-encapsulated ones.
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Figure 2. Cell viability levels of the gastric and intestinal cells after exposure to the particles
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6 assessed by the CellTiter-Glo® luminescence assay. Viability of AGS after 3 h of incubation with
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8 different particles concentrations at 37 °C (a and b). Viability of Caco-2 (c and d), and HT29-
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MTX (e and f) cells after 12 h incubation with different particle concentrations at 37 °C. All the
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13 data sets were compared to the negative control (Hank’s Balanced Salt Solution, HBSS) using a
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15 Student’s t-test with an unpaired post-test. Error bars represent mean ± standard deviation (n ≥
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18 3), and the difference probabilities were set at *p < 0.05, **p < 0.01, and ***p < 0.001.
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21 In vitro Release Studies. To confirm the successful protection of the CS-CPP modified
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23 nanoparticles and the loaded drugs from the acidic gastric conditions, as well as to predict the
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26 release profiles of the antidiabetic peptide GLP-1 and the enzymatic DPP4 inhibitor, in vitro
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28 release studies were performed. In order to mimic the environment of the stomach, the study was
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30 conducted at pH 1.2 for 2 h. Similarly, to mimic the intestinal environment, the study was
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33 performed using simulate fasted state intestinal fluid (FaSSIF) at pH 6.8 for 6 h. The time points
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35 chosen are similar to the estimated time for the gastric and intestinal transit, respectively.7
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38 The release profiles of GLP-1 from the H-PLGA+CS-CPP and H-PSi+CS-CPP particles at pH
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41 1.2 and 6.8 are shown in the Figures 3a and 3b, respectively. At pH 1.2, during the 2 h, the
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43 release of GLP-1 was less than 2 % and 5 % from the PLGA and PSi systems, respectively.
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45 However, at pH 6.8 the amount of GLP-1 released from particles was higher, reaching 40 %
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48 release after 6 h for both the systems. For the H-PLGA+CS-CPP particles, the release was
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50 sustained along with the time, unlike in the case of H-PSi+CS-CPP release profile, in which
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there was a GLP-1 burst release, with 40 % of the peptide released in the first 30 min and a very
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55 slow release thereafter. These biphasic release patterns dependent on the pH-value confirmed the
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57 controlled properties of encapsulation with the enteric polymer, and thus confirmed the fact that
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the CS-CPP modified nanoparticles and GLP-1 were indeed protected from the acidic conditions.
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6 These results are also in agreement with previous studies using the enteric polymer for oral drug
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8 delivery.11 The differences observed in the amounts that were released from H-PLGA+CS-CPP
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and H-PSi+CS-CPP over time were related with the characteristics of each nanoparticle and the
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13 methods used to load the GLP-1 in both of them. In PLGA nanoparticles, the GLP-1 was
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15 encapsulated inside of a polymer matrix, while in the PSi nanoparticles the GLP-1 was loaded
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18 mainly through physical adsorption in the pores of the nanoparticles which allowed a faster
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20 release.7
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23 With regards to the enzymatic DPP4 inhibitor the release was not pH dependent, unlike to what
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26 was observed for GLP-1. As it can be observed in Figures 3c and 3d, 80 % of the DPP4
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28 inhibitor was released at pH 1.2 during the first 30 min for both the formulations, reaching 100
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30 % release in less than 1 h. This can be explained by the very high solubility of the inhibitor in
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33 both aqueous solutions and in the organic solvents.45 The double emulsion technique could retain
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35 the aqueous solution in the organic solvent, and thus, retain the CS-CPP modified nanoparticles
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37 dispersed in water, until the complete polymer dissolution. However, this was not efficient in
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40 retaining the enzymatic DPP4 inhibitor and it started to diffuse into the organic solvent. When
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42 the particles were collected, the organic solvent started to diffuse to the water due to its high
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solubility. With the fast solidification process of the microdroplets, the DPP4 inhibitor was
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47 trapped in the outer layer of HPMC-AS matrix leading to the release of the enzymatic DPP4
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49 inhibitor.45 Nevertheless, the enzymatic DPP4 inhibitor has been reported to be successfully
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52 administrated orally by adding it into the food and water, without compromising its activity,
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54 meaning that the enzymatic DPP4 inhibitor can resist the harsh conditions of the stomach.46, 47
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35 Figure 3. Release profiles of the GLP-1 and the enzymatic DPP4 inhibitor from H-PLGA+CS-
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CPP and H-PSi+CS-CPP particles at pH 1.2 (a and c) for 2 h and at pH 6.8 (b and d) for 6 h. All
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40 the experiments were performed at 37 °C and 100 rpm. The statistical analysis was done using a
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42 one-way analysis of variance (ANOVA) with a Bonferroni post-test. Error bars represent the
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45 mean ± standard deviation (n = 3).
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51 Cell–Particle Interaction Studies. Once in the small intestine, the CS-CPP modified
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53 nanoparticles were completely unconfined due to the dissolution of the pH-sensitive polymer.
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56 The interactions of the CS-CPP modified nanoparticles were analysed qualitatively using
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confocal fluorescence microscopy and quantitatively by flow cytometry experiments. For the
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6 confocal microscopy and flow cytometry experiments, the nanoparticles were labelled with
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8 Alexa Fluor® 488 hydrazine (green fluorescence emission), as detailed in Supporting
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Information (SI), and the cell membranes were labelled with CellMask™ Red (red fluorescence
12
13 emission). In line with the viability experiments, the Caco-2 and HT29-MTX cells, which are
14
15 representative of the majority of the intestinal cells, were used to perform the cell–particle
16
17
18 interaction studies. A ratio of 90:10 for Caco-2 and HT29-MTX cells, respectively, was used in
19
20 order to mimic the physiological conditions of the intestine.27 The confocal fluorescence
21
22 microscopy results are shown in Figure 4a.
23
24
25
26 It was observed that for the unmodified nanoparticles there were no significant interactions with
27
28 the cells for either the PLGA or the PSi nanoparticles. When the nanoparticles were modified
29
30 with CS, the cell–nanoparticle interactions were greatly increased and many of the nanoparticles
31
32
33 were observed in the close vicinity of the cell membranes. This increase in interaction could be
34
35 due to the cationic nature and the mucoadhesive properties of the CS, causing strong electrostatic
36
37 interactions with the mucus producing cells of the co-culture,8, 48
as described elsewhere.7, 40
38
39
40 After the modifications with CS and CPP, the cell–nanoparticle interactions were even higher as
41
42 compared to the non-modified nanoparticles, as more nanoparticles were associated to the cells’
43
44
45
surface. In this case, the nanoparticles were interacting with the cell membranes and were also
46
47 taken up by the cells. Even after the encapsulation with the enteric polymer, and once the
48
49 polymer was dissolved at a higher pH, the nanoparticles presented a high capacity to interact and
50
51
52 to be internalized by the cells. The control groups were just cells in co-culture without any
53
54 particles.
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3
Quantitative studies of the cell-particles interaction with flow cytometry demonstrated that the
4
5
6 CS-CPP modified PLGA and CS-CPP modified PSi nanoparticles presented a 5.6-fold and 1.3-
7
8 fold increase in the interaction with the intestinal cells in the co-culture (control group),
9
10
11
compared to the unmodified nanoparticles, respectively (Figure 4b). The cationic nature of the
12
13 CS and the CPP were important features that led to high cell–nanoparticle interactions.18
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Figure 4. Interactions between the different nanoparticles and the Caco-2:HT29-MTX (90:10)
4
5
6 co-culture after incubation for 3 h at 37 °C. (a) Confocal microscopy images of the cell
7
8 membranes stained in red by CellMask® Red, and the nanoparticles in green conjugated with
9
10
11
Alexa Fluor® 488. (b) Flow cytometry quantification of the interactions by PLGA and PSi
12
13 nanosystems. The results were related to the fold increase of the percentage of cells interacting
14
15 with the nanoparticles. Data sets were compared to the control (*p < 0.05, **p < 0.01, and ***p
16
17
18 < 0.001) and between them (#p < 0.05, ##p < 0.01, and ###p < 0.001) using a Student’s t-test with
19
20 an unpaired post-test. Error bars represent mean ± standard deviation (n ≥ 3).
21
22
23
24
25
26
GLP-1 Permeability Across the Intestinal Cell Monolayers in the Presence of DPP4
27
28
29 Inhibitor. The permeability of the antidiabetic peptide GLP-1 was studied for H-PLGA+CS-
30
31 CPP and H-PSi+CS-CPP particles in the presence and absence of the DPP4 inhibitor. As
32
33
34
observed in Figures 5a and S1, the GLP-1 permeation across the monolayers was higher for the
35
36 H-PSi+CS-CPP particles than for the H-PLGA+CS-CPP particles. The difference between the
37
38 amounts of GLP-1 permeated across the cell monolayers by the two systems may be due to the
39
40
41 differences observed in the release profiles of GLP-1 loaded in both the particles. For the PSi
42
43 system, it was observed a burst release of GLP-1 during the first 30 min, while for the PLGA
44
45 system the release was more sustained over time. Thus, the amount of GLP-1 available to
46
47
48 permeate across the cell monolayers in the presence of the PSi system was 2-fold compared to
49
50 the amount of the PLGA system, which justifies the observed permeability results after 3 h.7 In
51
52
the presence of the enzymatic DPP4 inhibitor, the permeability of GLP-1 across the cell
53
54
55 monolayers was even higher, with ~1.5-fold increase for the PSi systems and ~5-fold increase
56
57 for the PLGA systems when compared to the permeability values without the enzymatic DPP4
58
59
60
18
1
2
3
inhibitor (Figure 5b). The synergistic effect of GLP-1 permeation in the presence the enzymatic
4
5
6 DPP4 inhibitor is probably due to the inhibition of the DPP4 enzyme, which helped to protect the
7
8 GLP-1 from degradation when permeating across the cell monolayer. Studies conducted in
9
10
11
animal models have shown that when the DPP4 inhibitor was orally administrated, the plasma
12
13 levels of intact and active GLP-1 were increased, which indicated that it was not rapidly
14
15 degraded by the DPP4 enzyme.47, 49
16
17
18 When analysing the enzymatic activity of DPP4 (Figure 5c) after the 4 h incubation with the
19
20 particles, it was observed that the enzymatic activity of DPP4 in the cells exposed to the inhibitor
21
22 was significantly lower for both the PLGA (>20 µU min-1) and PSi systems (40 µU min-1) than
23
24
25 in the cells with no exposure to the inhibitor (~1062 µU min-1) (control group). When compared
26
27 to the free form of the inhibitor, the activity of the inhibitor loaded in the particles was not
28
29
affected, and despite of no statistically significant difference, these values were lower than in the
30
31
32 case of the free form of DPP4 inhibitor. The similar permeability profiles of the enzymatic DPP4
33
34 inhibitor (Figure 5d) across the cell monolayers corroborate these results. It has been shown in
35
36
37 in vivo studies that in the presence of DPP4 inhibitor the enzymatic activity was changed from
38
39 3.26 ± 0.19 mU mL-1 to 0.01 ± 0.03 mU mL-1, and that the inhibition of the enzyme was dose
40
41 dependent.47, 49
42
43
44 Because DPP4 inhibitor has also been demonstrated has having a positive effect in decreasing
45
46 the blood glucose levels, it will have an additional benefit of our system for future in vivo
47
48 applications. 47, 49-51
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31 Figure 5. (a) GLP-1 permeability of H-PLGA+CS-CPP and H-PSi+CS-CPP across the Caco-
32
33 2/HT29-MTX cell monolayers in the presence of DPP4 inhibitor. (b) Differences between GLP-
34
35
36 1 permeability across the Caco-2/HT29-MTX cell monolayers in the presence and absence of the
37
38 DPP4 inhibitor. (c) DPP4 enzymatic activity after cellular incubation with particles loaded with
39
40 the DPP4 inhibitor. (d) Permeability profiles of DPP4 across the Caco-2/HT29-MTX cell
41
42
43 monolayers in the free form and when loaded in both the multifunctional particulate systems.
44
45 The statistical analysis was done using a Student’s t-test and one-way analysis of variance
46
47
(ANOVA) with unpaired and Bonferroni post-test, respectively. Error bars represent mean ±
48
49
50 standard deviation (n ≥ 3), set at probabilities of *p < 0.05, **p < 0.01, and ***p < 0.001.
51
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3
CONCLUSIONS
4
5
6
7 In this study, two novel multifunctional composite systems were prepared in a highly
8
9 reproducible, efficient and reliable manner using the innovative microfluidics technique. The
10
11 systems consisted of CS-CPP modified PLGA and PSi nanoparticles used as nanomatrices
12
13
14 encapsulated into an enteric polymer, which were co-loaded with an antidiabetic peptide GLP-1
15
16 and an enzymatic DPP4 inhibitor. The dual-delivery of these two drugs by the same formulation
17
18
19
was for the first time described. Due to the characteristics of the enteric polymer, the
20
21 multifunctional composite systems were able to protect the CS-CPP modified nanoparticles and
22
23 consequently prevent the premature release of the peptide and its degradation in the adverse
24
25
26 conditions of the gastrointestinal tract. By precisely releasing the CS-CPP modified nanoparticles
27
28 and subsequently the peptide in the upper intestine, the bioavailability of peptide and its
29
30 permeation would be greatly improved. Moreover, due to the modifications of the nanoparticles,
31
32
33 the interactions and the retention time of the nanoparticles with the intestinal cells were also
34
35 greatly improved. It was also shown that the release of both the drugs had a synergistic effect
36
37 since the presence of the enzymatic DPP4 inhibitor decreased drastically the activity of the
38
39
40 enzyme, improving further the amount of active peptide permeated across the intestinal cells.
41
42 Thus, taking particularly into account the GLP-1 peptide and the enzymatic DPP4 inhibitor,
43
44
45
these multifunctional particulate systems might be very promising for clinical applications in the
46
47 therapy of T2DM, due to the potential to increase in GLP-1 half-life in an in vivo situation.
48
49 Overall, due to the flexibility of the techniques employed here, and the multifunctional character
50
51
52 of the particulate systems, they have potential to orally deliver sensitive biomolecules in
53
54 combination with other drugs with different physicochemical properties.
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1
2
3
MATERIALS AND METHODS
4
5
6 Materials and Cell culture. GLP-1 was purchased from United Peptides (USA). DPP4 inhibitor
7
8 (NVP DPP 728 dihydrochloride) was purchased from Tocris Bioscience (UK) and CPP R9 was
9
10
11
purchased from GenicBio (China). PLGA 50:50 was obtained from Purac Biomaterials,
12
13 Purasorb® PDLG 5004A, The Netherlands. Polyvinyl alcohol (PVA), medium molecular weight
14
15 CS, 2-(N-morpholino)-ethanesulfonic acid (MES), 1-ethyl-3-(3-dimethylaminopropyl)-
16
17
18 carbodiimide (EDC), N-hydroxysuccinimide (NHS), 4-(2-hydroxyethyl)-1-
19
20 piperazineethanesulfonic acid (HEPES) and glutaraldehyde were purchased from Sigma-Aldrich
21
22 (USA). HPMC-AS was obtained by Shin-Etsu (Japan). Pluronic® F127 was purchased from
23
24
25 BASF (Germany). Hank’s balanced salt solution (HBBS), phosphate buffered saline (PBS),
26
27 Alexa Fluor® 488 hydrazine, Versene and heat inactivated foetal bovine serum (FBS) were
28
29
purchased from Life Technologies (USA). Dulbecco’s Modified Eagle medium (DMEM),
30
31
32 Roswell Park Memorial Institute medium (RPMI 1640), L-glutamine, non-essential amino acids,
33
34 penicillin (100 IU mL-1) and streptomycin (100 mg mL-1), ethylenediamine tetraacetic acid
35
36
37 (EDTA) and trypsin–EDTA were purchased from HyClone (USA). Human colon carcinoma
38
39 (Caco-2), lymphocytic Raji B cells and human gastric adenocarcinoma (AGS) cell lines were
40
41 obtained from American Type Culture Collection (ATCC, USA), and the human colorectal
42
43
44 adenocarcinoma modified with methotrexate (HT29-MTX) cells was kindly provided by Dr. T.
45
46 Lesuffleur (INSERM U178, Villejuif, France).
47
48
49
50
51
52
53
54
55
56
57
58
59
60
22
1
2
3
Preparation of PLGA and PSi Nanoparticles. The nanoparticles composed of PLGA and PSi
4
5
6 were prepared as described elsewhere.7, 52, 53
The procedure can be found in detail in the SI.
7
8 Briefly, PLGA nanoparticles were prepared through modified solvent
9
10
11 emulsification−evaporation method, based on the water-in-oil-in-water (w/o/w) double emulsion
12
13 technique using an aqueous solution of PVA 2% as a surfactant. For the PSi nanoparticles, an
14
15
electrochemical anodization method was used followed by surface modification using
16
17
18 undecylenic acid to produce undecylenic modified thermally hydrocarbonized PSi nanoparticles.
19
20 Both the nanoparticle types, PLGA and PSi, were coated with CS in a ratio of 2:1 (w/w)
21
22
23
(CS:nanoparticles) through physical adsorption overnight at room temperature, to form
24
25 PLGA+CS and PSi+CS nanoparticles, respectively.7
26
27
28 CPP Conjugation to the CS-coated Nanoparticles. The covalent conjugation between the free
29
30
amine groups in the CS structure with the carboxylic group of CPP was made through the
31
32
33 EDC/NHS coupling chemistry. For chemical conjugation, 1 mg of PLGA+CS nanoparticles and
34
35 300 µg of PSi+CS were separately dispersed in 1 mL of 10 mM MES containing EDC (final
36
37
38 concentration 10 mM) and NHS (final concentration 17 mM) at pH 5.5. CPP was added to this
39
40 dispersion in a ratio of 1:10 (CPP:nanoparticles) and the nanoparticles were allowed to conjugate
41
42 overnight in the dark, stirring at 300 rpm originating PLGA+CS-CPP and PSi+CS-CPP,
43
44
45 respectively. Afterwards, the nanoparticles were collected by centrifugation at 34300 × g for 20
46
47 min for PLGA+CS-CPP nanoparticles and at 27600 × g for 5 min for PSi+CS-CPP, and then
48
49 washed three times with MilliQ-water.
50
51
52
53
54
55
56
57
58
59
60
23
1
2
3
Enteric Coating of Nanoparticles Using Microfluidics. The GLP-1 loaded nanoparticles were
4
5
6 encapsulated into a pH-sensitive polymer (HPMC-AS) using a double emulsion technique,
7
8 through a microfluidic flow-focusing glass device (Scheme 1),11 described in detail in the SI. The
9
10
11
device consisted of two types of glass capillaries with different diameters, in which the outer
12
13 diameter of the inner cylindrical tapered capillary fitted the inner dimensions of the outer
14
15 capillary, facilitating the alignment of their axes.
16
17
18
19
To prepare the co-drug loaded multifunctional systems, 0.5 mg of DPP4 inhibitor was added into
20
21 1 mL of 4% of HPMC-AS dissolved in ethyl acetate (oil phase). To this solution, 100 µL of
22
23 MilliQ-water containing 20 mg of PLGA+CS-CPP or 200 µg of PSi+CS-CPP (water phase)
24
25
26 were added dropwise and homogenised for 30 s using a Vibra-Cell™ ultrasonic processor
27
28 (Sonics®, Sonics and Matrials, Inc., USA), originating to the first emulsion (w/o). This solution
29
30 was then poured into a syringe to be injected in the microfluidic device as the inner fluid (oil
31
32
33 phase). The outer fluid used was an aqueous solution of 2% Pluronic® F127 (water phase). The
34
35 inner and outer fluids were both pumped into the microfluidic device in opposite directions at 10
36
37 mL h-1 and 230 mL h-1, respectively. This flow-focusing geometry forces the inner fluid to
38
39
40 breakdown, forming the second monodisperse emulsion (w/o/w) droplets at the entrance orifice
41
42 of the tapered cylindrical glass capillary. The droplets were collected in a cylindrical beaker
43
44
45
containing 80 mL of an aqueous sucrose solution (100 mOsm L-1) in order to facilitate the
46
47 particles deposition. These particles were solidified through the diffusion of ethyl acetate to the
48
49 external aqueous phase. As a result, multifunctional systems containing both GLP-1 loaded CS-
50
51
52 CPP modified PLGA and PSi nanoparticles and DPP4 inhibitor into a pH-sensitive polymer were
53
54 obtained, to form the H-PLGA+CS-CPP and H-PSi+CS-CPP respectively.
55
56
57
58
59
60
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2
3
Particle Characterization. The nanoparticles average size (Z-average), PdI and surface charge
4
5
6 were analysed by dynamic light scattering using a Malvern Zetasizer Nano ZS instrument
7
8 (Malvern Instruments Ltd, UK).
9
10
11 The AE and LD of GLP-1 in the developed nanoparticles were calculated by the difference
12
13
14 between the total amount of GLP-1 used to prepare the loaded nanoparticles and the amount of
15
16 GLP-1 that remained in the aqueous phase, after the nanoparticles isolation by centrifugation, as
17
18
19
described elsewhere.7 The amount of GLP-1 was determined by high performance liquid
20
21 chromatography (HPLC) (Agilent 1260, Agilent Technologies, USA), using a C18 column (4.6 ×
22
23 150 mm, 5 µm, Supelco Discovery® C18, USA). The mobile phase consisted of 0.1%
24
25
26 trifluoroacetic acid (pH 2.0) and acetonitrile initially set at the ratio of 70:30 (v/v), which linearly
27
28 changed to 60:40 (v/v) as a gradient over 5 min. Afterwards, the ratio was kept constant for 5
29
30
more minutes. The flow rate was 1.0 mL min-1 and the injected volume of the sample was 75 µL.
31
32
33 The column temperature was set at room temperature and the detection wavelength at 240 nm.
34
35 The total area under the curve was used to quantify the GLP-1.
36
37
38
The morphology and surface topography, shape and size of the enteric encapsulated particles
39
40
41 were assessed by SEM (SEM, Zeiss DSM 962, Germany).
42
43
44 pH-Sensitive Response of the Multifunctional particulate systems. After collection and
45
46 washing of the particles, they were placed on the SEM supports with double sided carbon
47
48
49 adhesive tape. The excess of water was removed with filter paper and different buffer solutions
50
51 at pH values of 1.2, 4, 5.5, 6.0 and 6.8, were added on top of the particles for 2 h. For pH of 6.0
52
53
54
and 6.8, the 10 min time point was also analysed. After these time points, the excess of buffer
55
56 solutions were removed with filter paper and the particles were allowed to dry at room
57
58
59
60
25
1
2
3
temperature overnight. Afterwards, the particles were sputter coated with platinum before
4
5
6 visualizing under the SEM.
7
8
9 In Vitro Release Studies. The multifunctional system particles, corresponding to 50 µg of GLP-
10
11 1, were added separately to 14 mL of pH 1.2 buffer (50 mM KCl) to simulate the gastric fluid
12
13
14 and to FaSSIF pH 6.8 (50 mM KH2PO4, 15 mM NaOH, 1.0% (w/v) pancreatin) to simulate the
15
16 fasted state fluid of the intestinal milieu. At specific time points, aliquots of 750 µL were
17
18
19
collected and the withdrawn volume was replaced with fresh medium, keeping the volume
20
21 constant. All the collected aliquots were centrifuged at 27600 × g and the supernatant was used
22
23 for HPLC analysis in order to quantify the GLP-1 and DPP4 inhibitor released from the systems
24
25
26 over time. All the tests were performed at 37 ºC and at 100 rpm under sink conditions.
27
28
29 GLP-1 was quantified by HPLC as described above. DPP4 inhibitor was quantified by the total
30
31 area under the curve through HPLC (Agilent 1260, Agilent Technologies, USA), using a Kinetex
32
33
34 2.6u XB-C18 100A column (4.6 × 75 mm, Phenomenex®, USA). The mobile phase consisted of
35
36 0.025% of ammonium hydroxide (pH 9.5 adjusted with 50% of phosphoric acid) and acetonitrile
37
38
39 set at the ratio of 82:18 (v/v) for 5.5 min. The flow rate was 1.0 mL min-1 and the injected
40
41 volume of the sample was 5 µL. The column temperature was set at room temperature and the
42
43 detection wavelength at 275 nm.
44
45
46
47 Cell Culturing. AGS (passage numbers of 10−15) grew in a complete medium consisting of
48
49 RPMI 1640 supplemented with 10% (v/v) FBS, 1% (v/v) L-glutamine, 1% (v/v) NEAA, and 1%
50
51
(v/v) antibiotic–antimitotic mixture (final concentration of 100 U mL-1 Penicillin and 100 U mL-1
52
53
54 Streptomycin). Caco-2 (passage numbers of 31−40) and HT29-MTX (passage numbers of 20–
55
56 50) cells grew separately in culture flasks in a complete medium consisting of DMEM
57
58
59
60
26
1
2
3
supplemented in the same way as described before for RPMI. The cells were sub-cultured once a
4
5
6 week using 0.25% trypsin–EDTA to detach the cells from the flasks and seeded at a density of
7
8 0.5 × 106 cells per 75 cm2 flask. The culture medium was replaced every other day. Cells were
9
10
11 maintained in an incubator (BB 16 gas incubator, Heraeus Instruments GmbH, Germany) at 37
12
13 ºC and 5- % CO2 and 95 % relative humidity. Raji B cells (passage numbers of 26–35) were
14
15
cultured in flasks with supplemented DMEM and with the same conditions as described above.
16
17
18
19 In Vitro Cell Viability Studies. The cell viability studies were performed on gastric AGS cells,
20
21 intestinal enterocytes like Caco-2 cells, and mucus-producing goblet HT29-MTX cells. About
22
23 50,000 cells were seeded individually in each well of the 96-well plates (Corning Inc., USA) and
24
25
26 were left to attach overnight. The cells were washed twice with pre-warmed and fresh
27
28 HBSS−HEPES buffer (pH 7.4), and then 100 µL of different particles were added to cells at
29
30
31 different concentrations (25−400 µg/mL). For the AGS cells, the NPs were incubated with the
32
33 cells for 3 h at 37 °C, and for the intestinal cells the incubation time was 12 h at 37 °C. After the
34
35
36 incubation period, the cells were washed twice with fresh HBSS−HEPES buffer. Then, 100 µL
37
38 of CellTiter-Glo® assay reagent (Promega Corporation, USA), previously diluted with
39
40
41
HBSS−HEPES buffer in a ratio of 1:1, was added to the wells. 1% of Triton X-100 solution and
42
43 HBSS−HEPES buffer solution were used as the positive and negative controls, respectively, and
44
45
both controls were treated in similar manner as the samples. The resulting luminescence from the
46
47
48 treated cells was recorded using Varioskan Flash Multimode Reader (Thermo Fisher Scientific,
49
50 USA).
51
52
53
54 Cell Viability Studies. The viability tests were conducted using the AGS, Caco-2 and HT29-
55
56 MTX cell lines. Typically, 100 µL of 0.5 × 106 cells mL-1 were seeded separately in 96-well
57
58
59
60
27
1
2
3
plates and were allowed to attach overnight. Afterwards, the medium was aspirated and the wells
4
5
6 were washed twice with 100 µL of pre-warmed HBSS−HEPES buffer (pH 7.4). After washing,
7
8 100 µL of the nanoparticle solutions at concentrations of 0.1, 0.25, 0.5 and 1 mg mL-1 were
9
10
11 added to each well and the plates were incubated at 37 ºC for a period of 3 h for AGS, and for 12
12
13 h for Caco-2 and HT29-MTX cells. Afterwards, the plates were equilibrated at room temperature
14
15
16
for about 30 min and then washed twice with 100 µL of fresh HBSS−HEPES buffer (pH 7.4) at
17
18 room temperature. 50 µL of the reagent assay CellTiter-Glo® (Promega Corporation, USA) were
19
20 added to 50 µL of HBSS−HEPES (pH 7.4) in each well. Negative (HBSS−HEPES buffer, pH
21
22
23 7.4) and positive (1% Triton X-100) control wells were also used and treated similarly as
24
25 described above. The solutions were mixed and protected from the light for approximately 10
26
27
28 min on an orbital shaker at room temperature. Finally, the luminescence was measured using a
29
30 Varioskan Flash plate reader (Thermo Fisher Scientific Inc., USA). All data sets were compared
31
32 with a negative control, considered as 100% viability.
33
34
35
36
Cell–Nanoparticle Interactions.
37
38
39 Confocal Microscopy Studies. The intestinal cell−nanoparticle interactions were evaluated
40
41
through confocal microscopy using a Leica SP5 confocal microscope (Leica Microsystems,
42
43
44 Germany). Caco-2 and HT29-MTX were co-cultured in a 90:10 ratio (Caco-2:HT29-MTX), in
45
46 Lab-Tek 8-chamber slides (Thermo Fisher Scientiﬁc Inc., USA) and allowed to attach
47
48
49 overnight.7 Then, the cells were washed twice with pre-warmed fresh HBSS−HEPES buffer (pH
50
51 7.4). 50 µL of Alexa Fluor 488™ labeled nanoparticles (see SI) at a concentration of 200 µg mL-1
52
53
54
were added to the cells and incubated at 37 °C for 3 h. After incubation, the cells were washed
55
56 twice with pre-warmed fresh HBSS−HEPES buffer (pH 7.4). Afterwards, the plasma membrane
57
58
59
60
28
1
2
3
was stained by adding 200 µL of CellMask™ Orange (Invitrogen, USA) and incubated for 3 min
4
5
6 at 37 °C. The excess of staining solution was washed twice with pre-warmed fresh
7
8 HBSS−HEPES buffer (pH 7.4) and the cells were fixed using 2.5% glutaraldehyde for 20 min.
9
10
11
12 Flow Cytometry Studies. The quantification of the nanoparticles that are associated to the
13
14 cells was evaluated using flow cytometry. 1 mL of nanoparticles at a concentration of 300 µg
15
16
mL-1 were added to 0.7 × 106 Caco-2:HT29-MTX co-culture cells, in a ratio 90:10, in pre-
17
18
19 warmed HBSS−HEPES buffer (pH 7.4) and incubated at 37 °C for 3 h. Afterwards, cells were
20
21 washed three times with HBSS−HEPES buffer (pH 7.4) and detached with Versene for the
22
23
24 measurements. Then, the cells were re-suspended in basic sorting buffer that consisted of 1× PBS
25
26 (Ca/Mg2+ free) with 5 mM of EDTA, 25 mM of HEPES and 2% of FBS, to avoid cells
27
28
29 aggregation, and analysed right after using a Beckman Coulter Galios™ Flow Cytometer with a
30
31 laser excitation wavelength of 488 nm. The results were analysed using the software Kaluza
32
33 Analysis Version 1.3.
34
35
36
37
Permeability Experiments. For the permeability experiments a triple co-culture cell model was
38
39 used. For that, 7 × 104 cells cm-3 of Caco-2 and HT29-MTX cells in a ratio of 90:10 were seeded
40
41
42
in 12-Transwell cell culture inserts and were allowed to grow and differentiate for 14 days with
43
44 medium replacement every other day. Then, 1.0 × 105 Raji B cell were added to the basolateral
45
46
chamber for 7 days more in order to induce the phenotype change of Caco-2 cells into M cells
47
48
49 and to obtain a triple co-culture model.27. The permeability experiments across the cell
50
51 monolayers were performed in the apical-to-basolateral direction in HBSS−HEPES (pH 7.4) at
52
53
54 37 ºC using an orbital shaker (100 rpm). After removing the cell culture medium, the
55
56 Transwells were washed twice with pre-warmed fresh HBSS−HEPES (pH 7.4) buffer and
57
58
59
60
29
1
2
3
equilibrated for 30 min. Then, 0.5 mL of nanoparticles corresponding to an amount of 6 µg mL-1
4
5
6 of GLP-1 and 40 µg mL-1 of DPP4 inhibitor were pipetted into the apical side of the inserts. At
7
8 different time points (15, 30, 60, 120 and 180 min), 100 µL of samples were taken from the
9
10
11 basolateral side of the inserts and the same volume of pre-warmed fresh HBSS−HEPES (pH 7.4)
12
13 buffer was added to replace the withdrawn volume. Sample concentrations were quantified for
14
15
GLP-1 by EIA GLP-1 Kit (Sigma-Aldrich®, USA) according to manufacturer’s instructions and
16
17
18 for DPP4 inhibitor by HPLC, as previously described. The integrity of the cell monolayers was
19
20 checked before and after the permeability experiments by measuring the transepithelial electric
21
22
23
resistance (TEER) using Millicell®-Electrical Resistance System (Millipore, USA).
24
25
26 DPP4 Enzymatic Activity. The enzymatic activity of the DPP4 was measure according to the
27
28 manufacturer’s instructions using a DPP4 Activity Assay Kit (Sigma-Aldrich®, USA). After the
29
30
31
permeability experiments, the cells were washed once with pre-warmed HBSS−HEPES buffer
32
33 (pH 7.4). Then, the cells were lysed with 100 µL of ice-cold Assay Buffer. After lysis, 10 µL of
34
35 each sample was diluted with Assay Buffer to have a final volume of 50 µL. To each test
36
37
38 samples 10 µL of the Assay Buffer was added. To the blank samples, 10 µL aliquots of DPP4
39
40 inhibitor were added, mixed well and incubated for 10 min at 37 °C. Afterwards, 40 µL of the
41
42
Master Reaction Mix was added to each test sample and properly mixed and protected from the
43
44
45 light. The samples were then incubated for 5 min at 37 °C before starting the fluorescent
46
47 measurements (λex = 360 and λem = 460). The plate was kept at 37°C and protected from the
48
49
50
light, and the measurements were taken every 5 min for 1 h using a Varioskan Flash plate reader
51
52 (Thermo Fisher Scientific Inc., USA).
53
54
55 Statistical Analysis. All the experiments were performed in triplicate and represented as mean ±
56
57
58
standard deviation (SD). A Student t-test and one-way analysis of variance (ANOVA) with
59
60
30
1
2
3
Unpaired and Bonferroni post-test (GraphPadPrism, GraphPad software Inc., CA, USA) were
4
5
6 used to analyse the data, respectively. The level of significance was set at probabilities of *p <
7
8 0.05, **p < 0.01, and ***p < 0.001.
9
10
11 Acknowledgement. This work was financed by the European Regional Development Fund
12
13
14 (ERDF) through the Programa Operacional Factores de Competitividade – COMPETE, by
15
16 Portuguese funds through Fundação para a Ciência e a Tecnologia (FCT) in the framework of the
17
18
19
project PEst-C/SAU/LA0002/2013, and co-financed by the North Portugal Regional Operational
20
21 Programme (ON.2 – O Novo Norte) in the framework of project SAESCTN-PIIC&DT/2011,
22
23 under the National Strategic Reference Framework (NSRF). Francisca Araújo would like to
24
25
26 thank to FCT for financial support (SFRH/BD/87016/2012). Dr. Hélder A. Santos acknowledges
27
28 financial support from the Academy of Finland (decision no. 252215 and 2813001), the
29
30 University of Helsinki Research Funds, the Biocetrum Helsinki, the Finnish Center for
31
32
33 International Mobility (grant no TM-13-9048), and the European Research Council under the
34
35 European Union's Seventh Framework Programme (FP/2007–2013, grant No. 310892).
36
37
38 Supporting Information Available: Different preparation techniques of the PLGA and PSi
39
40
41 nanoparticles, fabrication of a glass-capillary microfluidic flow-focusing device and Alexa
42
43 Fluor® 488 conjugation to nanoparticles are given detail in the supporting information. The
44
45 permeability profiles of GLP-1 from H-PLGA+CS-CPP and H-PSi+CS-CPP particles in the
46
47
48 presence and absence of DPP4 inhibitor in a cellular triple co-culture model comprised of Caco-
49
50 2, HT29-MTX and Raji B cells are also presented in this section. This material is available free
51
52
of charge via the Internet at http://pubs.acs.org.
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