Sequence-Controlled Delivery of Peptides from Hierarchically

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Sequence-Controlled Delivery of Peptides from Hierarchically Structured Nanomaterials (Carl) Wei He, Maria Parowatkin, Volker Mailaender, Marion FlechtnerMors, Ulrich Ziener, Katharina Landfester, and Daniel Crespy ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b13176 • Publication Date (Web): 04 Jan 2017 Downloaded from http://pubs.acs.org on January 5, 2017

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ACS Applied Materials & Interfaces

Sequence-Controlled Delivery of Peptides from Hierarchically Structured Nanomaterials Carl Wei He,1 Maria Parowatkin,1 Volker Mailänder,1 Marion Flechtner-Mors,3 Ulrich Ziener,2 Katharina Landfester,1 Daniel Crespy1,4*

1

Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany

2

University of Ulm, Institute of Organic Chemistry III, Albert-Einstein-Allee 11, D-89081,

Ulm, Germany 3

University of Ulm, Institute of Micro- and Nanomaterials, Albert-Einstein-Allee 47, D-

89081, Ulm, Germany Department of Materials Science and Engineering, School of Molecular Science and Engineering, Vidyasirimedhi Institute of Science and Technology (VISTEC), 21210 Rayong, Thailand Email: [email protected]

KEYWORDS Hierarchical nanostructures, polyelectrolyte complexation, nanoconfinement, miniemulsion, protein drug delivery, glycol chitosan, interfacial crosslinking

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Abstract Peptide drugs delivered orally need be protected from degradation for achieving their functions. To fulfill the complicated task of oral drug delivery, we present a hierarchically structured drug delivery system that can undertake structural changes so that multiple functions can be triggered by a sequence of stimuli. Such hierarchical system is achieved in a nanoparticle-in-nanofiber configuration, in which both the nanofibers and the nanoparticles are pH responsive and biocompatible. A model peptide is efficiently encapsulated under mild condition and the nanocarriers are further electrospun with a pH responsive mucoadhesive polymer. The nanoparticles are released from the nanofibers, and thereafter the peptides are released from the nanoparticles in a pH responsive manner. The nanoparticles are compatible with caco-2 cells and the endocytosis of the nanoparticles is described in detail.

Drugs delivered orally experience different environments in the body until they reach the targeted site. Especially, peptide drugs can be degraded before their functions are activated if they are not protected. The drug carriers should protect the peptide from degradation in the gastro-intestinal environment, release the peptide to a certain region of the intestine, transport the peptide through the epidermal cells, and release the peptide in the blood. To fulfill these tasks, such carriers should possess simultaneously four main properties. Firstly, the material embedding the drug, i.e. the drug carrier, should be pH-responsive so that it protects the drug at low pH value and release the drug at higher pH value near the regions of the duodenum or ileum´.1-2 Secondly, mucoadhesive properties for the drug carrier are suitable so that they can be preferentially adsorbed at the intestinal epidermal mucus layer. Thirdly, the drug needs to be selectively delivered at the targeted organ or in the blood.2-3 Finally, the endocytosis of the drug carrier is enhanced if the carriers are in the submicron range.4-5 Many different properties 2 ACS Paragon Plus Environment

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of an efficient drug carrier can therefore appear at first glance conflicting. However, such a drug carrier could be achieved by designing hierarchical structures with properties that are programed to be triggered in a sequential manner. Hierarchically structured drug delivery systems, including nano-in-nano,6-12 nano-inmicro,13-16 micro-in-micro,17 micro-in-macro,18-19 or nano-in-macro,20-22 systems are a current topic of research for maximizing the protection and efficient delivery of drugs in the human body.23-24 The multi-barrier nature of the hierarchical structures usually retards the release of the payload.7, 9, 12, 25-26 Meanwhile, different drugs can be loaded into each level of hierarchy to achieve sequential release.15, 21, 27-28 Furthermore, various stimuli responsive coatings have been utilized to regulate the release of peptide upon environmental triggers. For example, gold NPs grafted with protein drugs have been encapsulated into crosslinked gelatin capsules, which released the NPs upon ultrasound trigger.21 Porous silica nanoparticles were coated with an acid degradable coating, the degradation rate of which regulates the release of anticancer drugs.6 Similarly, porous silica nanoparticles loaded with an antidiabetic peptide drug have been coated with a pH responsive polymer containing enzyme inhibitor. The peptide drug and the enzyme inhibitor were released in a sequential manner upon an increase of environmental pH value.7 Multi-responsive systems were also fabricated by loading pH responsive NPs into gelatin capsules with a pH responsive coating. Encapsulated insulin could be released at pH 7.4 and be transported to the blood. An in vivo study on diabetic rats shows an increased insulin level and decreased glucose level after oral administration of the optimized formulation. 20 However, current drug nanocarriers lack the ability to retain peptide before they reach the targeted tissue. Therefore, they cannot take full advantage of a hierarchical structure. Besides, the overall encapsulation efficiency of peptides is limited because the peptides are either loaded after the formation of the particles7 or are partially not encapsulated during the 3 ACS Paragon Plus Environment


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formation of particles.20 These limitations can be overcome by the nanocarriers recently reported.29 The peptide can be encapsulated under mild conditions with high drug encapsulation efficiency, and the pH responsive nanocarriers sustain the release of peptide over five days. In this work, we present a hierarchical peptide delivery system for which two components in the hierarchy of structure are pH-responsive. This allows therefore for the release of peptide stimulated by a sequence of activation by changes of pH values encountered in the human body.

Results and Discussion Fabrication of a multi-stage pH-responsive system for peptide delivery The multi-stage pH-responsive peptide delivery system is composed of two levels of hierarchy (Scheme 1). The first level is represented by pH responsive nanoparticles. Crosslinked PEC nanoparticles (NPs) were prepared according to our previous report.29 The resulting nanocarriers had a z-average diameter of 229 ± 87 nm, which is in a reasonable range for the endocytosis of NPs by intestinal epidermal cells.30 The zeta potential of the nanocarriers was + 24.8 ± 3.8 mV. The positive surface charge enables colloidal stability in water and indicates that the surface is dominated by the presence of chitosan. Such property is suitable for oral drug delivery, because the chitosan moiety allows better mucous adhesiveness. The morphology of crosslinked PEC was further confirmed by SEM (Figure S1), showing that the crosslinked PEC particles are monolithic spherical particles. The majority of particles have a number-average size of 80 ± 34 nm as measured by SEM with very low amount of aggregates (~ 380 nm, 0.7% by number), which is verified by DLS measurements (Table 1).


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Scheme 1. Structural hierarchy of multi-stage pH-responsive peptide delivery system.

The second level of hierarchy is given by the pH responsive nanofibers that embed the nanoparticles. The electrospun EL55 nanofibers have an average diameter of 382 ± 65 nm, which is much larger than the size of the nanoparticles. The surface of the nanofibers is smooth and no nanoparticles can be detected at their surfaces (Figure 1a). Therefore, the nanoparticles are loaded inside the fiber and thus protected by the fiber´s matrix. The 5 ACS Paragon Plus Environment


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fluorescently labelled NPs were electrospun with EL55 and therefore can be visualized under CLSM (Figure 1b). The fluorescent NPs were separately distributed along the nanofiber without major aggregation, which is favorable for cellular uptake when the NPs are released in the vicinity of epidermal cells. The electrospinning procedure inherently provided simultaneously shearing force and fast drying of the polymer solution. Thus, the NPs were separated from each other during the elongation of the electrospinning jet and “frozen” when the fiber was dried.

Figure 1. Morphology of EL55 NFs containing crosslinked PEC NPs (a) SEM, (b) CLSM: The fluorescence signal (NPs) is overlapped with transmission signals (NFs).


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Release of NP from NF upon variation of pH value: The second level of hierarchy The next step was to verify that the nanoparticles could be released from the nanofibers at selected pH values. A droplet of buffer solution was casted on nanofibers. The dissolution of fibers and release of fluorescently labeled NPs was monitored in time by CLSM. The airliquid boundary of the droplet is shown in Figure 2. At low pH value, the nanofibers remained intact. Immersed in the buffer (pH value = 4.5) they showed no sign of swelling or dissolution and the NPs were constrained within the NFs throughout the experimental time (~ 30 min). At higher pH value, the fibers dissolve instantly upon contact with the buffer solution (pH value = 7.4). The droplet boundary proceeded toward dry NFs and led to the swelling of the NFs. The NPs were released without showing significant aggregation. Hence, the electrospun EL55 NFs serve as a smart matrix for the NPs. Indeed, they isolate the encapsulated NPs from external environment at lower pH value (pH value < 5.5) because EL55 is hydrophobic and insoluble. At higher pH value (pH > 5.5), EL55 NFs are dissolved and release the NPs.

Figure 2. Dissolution of NPs from NFs studied by CLSM. At pH 7.4, the fibers were dissolved within 5 min and all the NPs were released, and > 80% NP was released within 110 7 ACS Paragon Plus Environment


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s. At pH 6.0, the fibers dissolved gradually and the NPs were released slowly. Complete dissolution requires > 1 h. At pH 4.5, the fibers were not dissolved and no NP was released.

NFs containing fluorescently labeled NPs were immersed in various buffer solutions (pH = 4.5, 6.0, or 7.4) and the amount of released NPs was estimated by the fluorescence intensity of the released media (Figure 3). At pH 4.5, NFs were not wetted by the buffer and remained intact over the experimental time (~ 30 min). No fluorescent signal was detected in the media, which indicates that the NFs retained the NPs without leakage at lower pH value. At pH 6.0, the NFs dissolved slowly. 80% of NPs was released over 25 min. At pH 7.4, the NFs were easily wetted and completely dissolved in 5 min. 82% of NPs were released in 2 min and 98% in 12 min.

Figure 3. Release profile of fluorescently labeled NPs from NFs. 8 ACS Paragon Plus Environment

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It is known that the size of the drug nanocarriers strongly affects particles’ uptake in cells30 and hence the transportation efficiency of the payload. Therefore, it is favorable that the NPs released from NFs remain in a non-aggregated state. We studied the aggregation of NPs by DLS before electrospinning and after dissolution of the NFs (Table 1). The average size of the NPs released from EL55 NFs is 290 nm, which is higher than the size of the NP dispersion in water (229 nm) or in buffer solution (232 nm). The increase in polydispersity from 0.144 to 0.386 also suggests that the NPs partially aggregated. Despite a moderate aggregation of NPs after dissolution of the NFs, the size of the released NPs is still within the suitable range for endocytosis of chitosan NPs.

Table 1. Characteristics of the NPs before and after electrospinning. Entry

Diameter [nm]a

Polydispersity

ζ-potential [mV]

NPs in water (pH = 6.0)

229 ± 87

0.144

+24.8 ± 3.8

NPs in buffer (pH = 7.4)

232 ± 74

0.104

+2.5 ± 4.1

NPs in water containing EL55 (pH = 242 ± 119 6.0)

0.243

-37.2 ± 3.5

NPs released from NFs (pH = 7.4)

0.386

-38.0 ± 3.2

290 ± 180

a

Average hydrodynamic diameters as z-average diameter and measured by DLS. The calculated diameter was corrected based on the viscosity and refractive index of the Eudragit solution (Figure S2, S3)

To study the reason for the aggregation, a dispersion of nanoparticles was mixed with an EL55 aqueous solution and compared with NPs without EL55. An increase in both size and polydispersity accompanied with an inversion of the ζ-potential was observed. These trends were also observed with NPs released from NFs. Therefore, we believe that the negatively



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charged EL55 contributed to the aggregation by complexing with the positively charged NPs and partially bridging multiple NPs.

Release of peptide from the NP-in-NF system We investigated the release profiles of a fluorescently labeled model peptide rPYY-FITC from the NP-in-NF system in a medium simulating the gastrointestinal conditions at room temperature. In the first 2 h, the pH value was 2.0, mimicking the pH value in the stomach. Between 2 and 4 h after the beginning of the experiment, the pH was 6.0, which mimicked the condition in the lumen of the intestine. After 4 h, the pH value was adjusted to 7.4 for reproducing the environment on the surface of intestinal epidermal cells or the blood stream. Release profiles of rPYY-FITC from the NP-in-NF system were simultaneously recorded in the buffer solution with fixed pH values (4.5, 6.0, and 7.4) (Figure 4). In the first two hours (pH = 2), a small amount of peptide (3%) leaked from the NFs despite the fact that NPs were not released from the NFs. The release profile in the buffer at pH = 4.5 showed a similar slow leakage of peptide over 100 h. Such leakage indicated that the NF matrix is still permeable to an acidic environment although EL55 is practically insoluble at these conditions. At pH = 6.0, the NFs dissolved gradually with a fast release of 35% of the peptide within 2 h. Such fast release indicates that a fraction of the peptide was released to the media due to the dissolution of fibers, i.e. part of the peptide was released from the NPs to the NFs before the dissolution of the NFs. The majority of peptide was however retained in the NFs because of the hydrophobic nature of NFs under acidic conditions. By comparing Figure 3 to Figure 4, we can observe that the release rate of the NPs from NFs is faster than the release rate of rPYY-FITC from the NP-in-NF system at pH 6.0, which suggests that part of the peptide also leaked out of the NPs during the dissolution of the NFs. The two fractions of the released peptide made up in total 35% release of the initially encapsulated peptide. After 10 ACS Paragon Plus Environment

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the pH value was adjusted to 7.4, the release of peptide continued over 100 h, and an extra amount of peptide was released from dispersed NPs (10% after 24 h, 13% at 48 h, and 20% at 96 h). The continuous sustained release of peptide indicates that at least 20% PYY was still encapsulated in the NPs when they were released from the NFs. Such trend can also be found in the release profile of peptides in buffer solution at fixed pH values. Such property confers sustained release of peptide on the NP-in-NF system after dispersion of the NPs, thus taking full advantage of the hierarchically structured drug delivery system. Indeed, the peptide concentration is rapidly increased to the effective therapeutic concentration level and is maintained at the desired dosage for an extended period.

Figure 4. Release profile of the peptide from the NP-in-NF construct. Peptides were released from NP-in-NF systems at fixed pH (pH 4.5: red dots, pH 6.0: green triangles, pH 7.4: blue inverted triangles), or at varying pH (squares; 0 ~ 2 h, pH 2.0; 2 ~ 4 h, pH 6.0; 4 ~ 100 h, pH



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7.4). Data collected at pH 6.0, which correspond to the release of NPs from NFs are colored in magenta to guide the eyes.

The release profile of the NP-in-NF systems was influenced by the electrospinning conditions. One major factor was the contact time between the peptide loaded NPs and the EL55 solution before NF formation (Figure S4). The contact time between nanoparticles and EL 55 solution depends on the duration of the electrospinning process, which further scales with the amount of dispersion used for electrospinning. With the same crosslinking density, NFs prepared at a smaller scale (contact time = ~ 40 min) compared with the nanofibers with a larger scale (contact time = 4 h) displays suppressed leakage in acidic medium, and the NPs can sustain the release during the experimental time after being released from the NFs. We speculate that such difference is caused by the partial deprotonation of EL55 during electrospinning: EL55 in ethanol reduces the pH value upon mixing with the NP paste, which contains water, and the local acidic environment induces the leakage of payload during the electrospinning process. Therefore, a minimized contact time between NPs and EL55 solution achieved by spinning of the smaller batch is desired to ensure minimized leakage of peptide. Besides, the crosslinking density of the NPs also affects the retention ability of the NP-in-NF system. With the same spinning conditions, NFs loaded with NPs of 12% crosslinking degree shows a higher leakage of peptide, compared with those of 25% crosslinking. Therefore a certain level of crosslinking is necessary to regulate the swelling of the NPs.

Biocompatibility of nanoparticles with cells To determine the biocompatibility of the glycol chitosan nanoparticles (GCNPs), Caco-2 cells were incubated with a series of different ascending concentrations from 2.3 µg/mL to 600 µg/mL glycol chitosan nanoparticles. We chose Caco-2 as it is the industry and scientific standard in a transwell system for testing gut permeability of drugs. The magnitude of the chitosan effect was found to be not time dependent (Figure S5). The cytotoxicity assay 12 ACS Paragon Plus Environment

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(CellTiterGlo) showed that the GCNPs are not toxic even after 24 h incubation time. Even at increased concentration (up to 600 µg/mL), the Caco-2 cells stayed metabolically active (see Supporting Information for more details).

Cellular uptake and localization of nanoparticles analyzed by CLSM The intracellular uptake of the GCNP-RBITC was studied by confocal laser microscopy. The first step for better understanding of internalization was to compare the localization of free dye RBITC (free RBITC) and of GCNP-RBITC (GCNPs, in abbreviation). The Confocal microscopy images of the GCNPs shows some dots inside of cells additionally to filaments (Figure 5 A+B). In the sample with free RBITC only filaments were detected. This is an indication for localization of GCNPs in endosomes while the free RBITC is locating to other intracellular structures (Figure 5 A). It confirms the uptake and localization of GCNPs in the cell compartments that are relevant for transport of substances.

Figure 5. Intracellular uptake of GCNP-RBITC (GCNPs) (A) and free dye RBITC (B) to compare the localization. The Caco-2 cells were cultivated 3 days in µ-dishes and then incubated for 4 h with GCNPs or with free RBITC. The uptake was studied by CLSM as life 13 ACS Paragon Plus Environment


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cell imaging. Green: GCNPs, red: cell membrane stained with CellMask™ Orange, blue: cell nucleus stained with DRAQ5.

Obviously, free RBITC as well as RBITC released from the GC nanoparticles can locate to specific structures inside the cell. We see the fluorescence overlap in both cases: if we add non-encapsulated free RBITC and RBITC-GCNPs. The staining of mitochondria (Figure 6 A1+A2) showed that the fluorescence signal coming from RBITC (Figure 6 B1+B2) overlapped with the signal from mitochondria (Figure 6 C1+C2). This is an early event after starting the incubation of Caco-2 with nanoparticles. Already after 0.5 h of incubation with GCNPs, the spots coming from fluorescence signal inside of the cells are well visible (Figure 6 A1+B1+C1).


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Figure 6. Localization of GCNPs inside of Caco-2 cells after 0,5 h and 4 h incubation time. (A) Mitochondria. (B) GCNPs. (C) Merging of both signals from mitochondria and GCNPs. Green: GCNPs, red: mitochondria stained with MitoTracker® Green FM.



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We found evidence that the Caco-2 cells are able to take up the nanoparticles very fast. As Caco-2 cells represent a model for intestinal uptake of NPs, this experiment demonstrates that chitosan as NPs can be internalized in a very short time and therefore it appears to be an appropriate polymer for successful drug delivery.

As previously reported, the encapsulation of peptides is an important step to protect them against the acidic environment in the gastrointestinal tract. Furthermore, due to the adhesive properties of chitosan the peptides could be better absorbed to the intestinal mucus layer and thus taken up with higher efficiency.


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Figure 7. Cellular uptake of free rPYY-FITC (rPYY, in abbreviation) (A), mix of free rPYY and GCNPs (B) and GCrPYYNPs (C) after 2 h (1), 4 h (2) and 24 h (3) in the Caco-2 cells. The Caco-2 cells were seeded for 3 days in the µ-dishes, incubated with rPYY or NPs and imaged by CLSM. The concentrations were 600 µg/mL GC and 12 µg/mL rPYY. Red: rPYY labelled with FITC, Green: GCNPs labelled with RBITC, blue: cell nucleus stained with DRAQ5.



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The confocal microscopy images revealed that the Caco-2 cells were able to internalize the free rPYY-FITC (rPYY, in abbreviation) (Figure 7 A1+A2+A3), rPYY in mixture with GCNPs (Figure 7 B1+B2+B3) and rPYY loaded to the GCNPs as GCrPYYNPs (Figure 7 C1+C2+C3). Already after 2 h of incubation, the rPYY and NPs were found to be taken up (Figure 7 A1+B1+C1). In case of rPYY in mixture with GCNPs, only some dots close to the cell nucleus are visible (Figure 7 B1+B2+B3). An interesting aspect is that the sample with encapsulated rPYY in GCrPYYNPs shows higher number of spots coming from rPYY (Figure 7 C1+C2+C3). Therefore, a direct encapsulation of the PYY was needed for an effective delivery.

Quantitative determination of the uptake of rPYY The next step was the quantitative investigation of the peptide into the cells by flow cytometry. For this study, the rPYY-FITC (abbreviated rPYY) was used in three different forms: rPYY encapsulated in GCNPs (GCrPYYNP), rPYY mixed with GCNPs (rPYY in mix) and free rPYY. The fluorescent signal is increased four-fold after 24 h compared to 1 h incubation time. The peptide internalization is therefore a time dependent process. Furthermore, the flow cytometry analysis revealed that the encapsulated rPYY in GCrPYYNP could be taken up by the Caco-2 cells with higher efficiency (Figure 8) than free rPYY or as a mixture. The free rPYY shows the lowest uptake into the cells. The mucoadhesive properties of glycol chitosan seemed to play an important role 34 and could be a reason for the higher uptake signal of encapsulated rPYY compared to free rPYY. The chitosan enhanced the contact time between the cell surface and the peptide and therefore led to an increased uptake. These results correlate with the confocal microscopy analysis (Figure 7 C1+C2+C3). The encapsulation of rPYY in chitosan is an important method and provided not only


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protection against the acidic milieu in the intestinal tract but also is useful for enhanced contact and uptake into the cells.

Figure 8. Uptake of fluorescently labeled rPYY loaded to GCrPYYNPs, rPYY mixed with GCNPs and free rPYY-FITC. The used concentration of rPYY for all samples was 12 µg/mL. The detection of fluorescence intensity was measured by flow cytometry after 1 h, 2 h, 4 h and 24 h incubation time.

Quantitative determination of the uptake of GC nanoparticles The next experiment was to study the quantitative uptake of chitosan nanoparticles in the Caco-2 cells (Figure 9). After 1 h and 2 h, the same amount of GCrPYYNPs and GCNPs mixed with free rPYY could be taken up. Whereas after 4 h and 24 h a lower amount of GCrPYYNPs compared with GNPs mixed with free rPYY was internalized. In summary, the sample GCrPYYNP shows the best results for internalization and uptake.



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Figure 9. Uptake of fluorescently labeled glycol chitosan NPs loaded with rPYY (GCrPYYNPs), GCNPs mixed with free rPYY and GCNPs sole. The concentration of GCNPs for all samples was 600 µg/mL. The fluorescence intensity was measured by flow cytometry after 1 h, 2 h, 4 h and 24 h of incubation. The experiments demonstrated the positive encapsulation effect of the glycol chitosan on the uptake of PYY. The satiety hormone PYY loaded to the glycol chitosan NPs was well protected. The chitosan extended the contact time between the cell surface and the PYY and led to an improved uptake. Furthermore, the envelope enabled a release of peptide payload in a slow and sustained manner and thus supported a long-term effect of drug delivery.

Conclusions We successfully designed a hierarchically structured peptide delivery system, which can release multi-functional nanocarriers upon a first stimulus, while the peptide payload can be further released from the pH responsive nanocarriers in a sustained manner. Such step-wise encapsulation strategy has potential for complicated drug delivery tasks like oral delivery of peptide drugs. Further research such as in-vivo studies on the release profile and 20 ACS Paragon Plus Environment

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functionalization for multi-stimulus response of nanoparticle-in-nanofiber systems can be performed based on this system. Because the GCNPs are highly versatile, we believe gycol chitosan nanoparticles can be adjusted to a variety of hydrophilic drugs and is, thus, of general interest. Materials Rat PYY(1-36) (rPYY, > 95%, Bachem), glycol chitosan (GC, 77% by titration, Mw = 210 kDa measured by GPC, Santa Cruz Biotechnology), poly(γ-glutamic acid) sodium salt (PGA, > 70%, Mw = 100 kDa measured by GPC, Vedan), Eudragit® L100-55 (EL55, Evonik Industries), toluene diisocyanate (TDI, 95%, Sigma Aldrich), Rhodamine B isocyanate (RBITC, Sigma Aldrich), FITC antibody labelling kit (53027, Thermo Scientific), and Amicon centrifugal filters (UFC500396, Millipore) were used as received. Polyglycerol polyricinoleate (PGPR, 91%, E476, Danisco) was diluted in hexane and filtered (pore size: 0.45 µm, cellulose). PGPR was then concentrated by rotary evaporation and vacuum dried (40 mbar, 40 °C, overnight). Sterilized water (Aqua B. Braun) was used for all the experiments if not specifically mentioned. Buffer solution was prepared with monosodium phosphate, and acetic acid (Aldrich) at 0.1 M and was used for all the experiments if not specified. Cell Culture Caco-2 cells (ATCC® HTB-37™) were obtained from ATCC, UK and cultured in Eagle's Minimum Essential Medium (EMEM, Lonza, USA) supplemented with 10% fetal bovine serum (FBS, Invitrogen, USA), 1% penicillin/streptomycin (Life technologies, USA) and 1% L-glutamine (GlutaMAXTM, Life technologies, USA) in an incubator at 37 C°, 95% humidity and 5% CO2 (Labotec, Germany). Treatment with 0.25% trypsin-EDTA (Life technologies, USA) for 5 min was used to detach the cells for further assays. Preparation of crosslinked PEC nanocarriers



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PEC based nanocarriers (NPs) were prepared in miniemulsion according to the procedure described in the previous paper.29 Briefly, an aliquot of FITC labelled rPYY (rPYY-FITC) (ca. 50 µg) was thawed, dissolved in 0.6 mL of water, and mixed with a PGA stock solution (0.4 mL, 6 mg PGA/mL). After pre-emulsification (0.5 h, 1000 rpm magnetic stirring) in a PGPR solution (1 wt% in 7.5 mL cyclohexane), the coarse emulsion was ultrasonicated for 1 min with ice-water cooling bath (Branson sonifier W450, ½ inch probe). A GC stock solution (1 mL) was emulsified in the same way and was mixed with the PGA miniemulsion. An EL55 stock solution (3 mL, 2.5 wt%, pH = 6.0) was emulsified in the same way. The mixture was then ultrasonicated for 30 s. A predetermined amount of crosslinker (TDI) was diluted with toluene and injected in the emulsion. After 10 min of reaction at room temperature, the emulsion of EL55 (16 mL) was added and the mixture was ultrasonicated for 30 s. The final emulsion was diluted with 270 mL hexane and the oil phase was removed by centrifugation (2000 g, 15 °C, 5 min). The resulting crosslinked PEC was washed 3 times against ethyl acetate by centrifugation-redispersion. The resulting paste of NP was ready for the next step. The NP can be redispersed in an aqueous solution with slight sonication (5 min in ultrasound bath, r.t.). The final dispersion contained crosslinked PEC (ca. 1 mg/mL), and phosphate buffer (0.01 M, pH 6.0). The residual ethyl acetate was removed by vacuum evaporation (40 mbar, r.t. ca. 10 min). In the case of fluorescently labelled NPs (GCNP-RBITC), GC was fluorescently labelled with RBITC and the resulting GC-RBITC was used instead of GC. The details of the preparation of GC-RBITC are available in the Supporting Information. The encapsulation efficiency was 68% as previsouly reported.29 The final nanoparticles were dispersed in water by applying ultrasound in a sonication bath (1 mg NP/mL water, 5 min). The resulting water dispersion was analyzed by DLS (ALV CGS, data fitted with cumulant method) and zeta potential (Zetasizer, Malvern). The DLS 22 ACS Paragon Plus Environment

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results were corrected according to the viscosity and the refractive index of the Eudragit solution at different concentration (Figure S2, S3). For SEM sampling, the NP dispersion was casted onto a silicon wafer and left to dry. The wafer surface was rinsed with a water droplet 3 times to remove the soluble fraction. Diameters of the NPs were measured manually (n = 435) based on the SEM image. Electrospinning of pH responsive nanofibers EL55 nanofibers (NF) were produced by electrospinning. EL55 was dissolved in ethanol at 15 wt% and then degassed in a vial in a sonication bath for 5 min. A known amount of a paste consisting of nanoparticles dispersed in water (1 wt%) was mixed with the solution of EL55 by magnetic stirring or pipetting. The contact time between nanoparticles and EL 55 solution during electrospinning depended on the amount of the dispersion. In a larger- or smaller-batch preparation, where 10 mg or 1.7 mg nanoparticles were mixed with EL 55 solution at 1 wt%, the electrospinning takes 4 h and 40 min, respectively. The NP dispersion in EL55/ethanol was used immediately for electrospinning (IME Technologies, 16 kV voltage, 1 mL/h feed rate, 10 cm working distance, 65% humidity, 25 °C, 0.15 mL of injected solution per sample). After spinning, the nanofiber mat was peeled off and weighed. For long-term storage, the fiber mat was sealed in a glass vial and stored in a desiccator. Samples for SEM and LCSM were collected. The fiber diameter was measured based on SEM micrograph (n = 20). Monitoring the release of NP from pH-responsive NF NP dissolution from NF was monitored by confocal laser scanning microscopy (CLSM). NP containing NFs were collected onto a cover glass during electrospinning. A droplet of 0.1 M buffer solution (pH = 4.5 or pH = 7.4) was casted onto the NF. A CLSM micrograph was taken at the edge of the droplet to observe the dissolution process. For quantification of the release of NPs from NFs, the nonwovens containing NP-RBITC were weighed (∼ 20 mg) and immersed in 0.1 M buffer solutions (pH = 4.5 or pH = 6.0). At 23 ACS Paragon Plus Environment


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certain intervals, 200 µL of the sample was taken from the release medium and the fluorescence intensity of the dispersion was recorded. The relative release of particles (%) from the fibers was expressed as the ratio of the recorded intensity to the theoretical fluorescence intensity of the initial amount of GC-RBITC used for the synthesis of the NP. Multi-stage release of peptide from NP-in-NF system at different pH values rPYY-FITC was present in NPs and nanoparticle-in-nanofiber (NP-in-NF) systems, because it could be loaded during the synthesis of the particles. A fiber mat of known mass (∼ 20 mg) was weighed and immersed in the release medium (10 mL, pH = 4.5). At certain intervals of time, 200 µL of the sample was taken from the release medium and centrifuged (15 °C, 20 min, 40000 g). The relative release of peptide (%) from the NP-in-NF system was expressed as the ratio of the recorded intensity to the theoretical fluorescence intensity of the initial amount of rPYY-FITC used for the synthesis of the NPs. For the multi-stage release profile of peptide under a sequence of pH values, the fiber mat was first immersed in a 0.01 M HCl solution (pH = 2.0). After 2 h, the release medium was changed to phosphate buffer (pH = 6.0). After 4 h, the pH of the buffer was adjusted to 7.4 with a 1.0 M NaOH solution. NP uptake imaging by confocal laser scanning microscopy (CLSM) For CLSM studies, 1 × 104 Caco-2 cells per milliliter were seeded in 35 mm diameter µdishes (IBIDI, Germany) and cultured for 3 days in supplemented EMEM at 37 °C. Cell medium was then replaced by fresh EMEM containing 150 µg/mL GCNPs labelled with RBITC (GCNP-RBITC) or 600 µg/mL nanoparticles loaded with rPYY-FITC. The cells were incubated for 0.5, 2, 4 or 24 h and washed two times with Hank's Balanced Salt Solution (HBSS) with calcium, and magnesium, without phenol red (Life technologies, Germany) to remove the residual nanoparticles. Live cell images were carried out with a commercial setup (LSM SP5 STED Leica Laser Scanning Confocal Microscope, Leica, Germany), consisting of an inverse fluorescence microscope DMI 6000 CS equipped with a multi-laser combination, 24 ACS Paragon Plus Environment

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five detectors operating in the range of 400-800 nm. A HCX PL APO CS 63 x 1.4 oil objective was used for these studies and fluorochromes were excited and detected in a sequential mode under the following conditions: GCNP-RBITC were excited with a 561 nm laser and detected at 580-620 nm, rPYY-FITC was excited with a 488 nm laser and detected at 500-550 nm. The cell mitochondria were stained with MitoTracker® Green FM (Thermo Fisher Scientific; USA) excited with a 488 nm laser and detected at 500-540 nm. Flow cytometry The uptake studies were conducted using a flow cytometer CyFlow ML (Partec, Germany). Caco-2 cells were seeded in 12-well plates (Greiner Bio One, Germany) at a density of 1.0 × 105 cells per milliliter in supplemented EMEM per well and cultured for 3 days. Cell culture medium was then replaced by EMEM containing 600 µg/mL nanoparticles or nanoparticles loaded with rPYY-FITC at the used concentration of 12 µg/mL rPYY. To determine the uptake of GCNPs, 500 µL of GCNP-RBITCs diluted in supplemented EMEM were added to the cells and incubated for 1, 2, 4 h and 24 h. For preparation the cells were washed with supplemented EMEM and treated with 0.25% trypsin-EDTA for detaching them. To remove the free nanoparticle the cells were centrifuged for 3 min at 1000 g, resuspended in phosphate buffer solution (PBS) (Life technologies, Germany) and centrifuged one more time. The cell pellet was resuspended in PBS and cells detected using flow cytometry under the following conditions: GCNP-RBITC were excited with a 561 nm laser and detected by bandpass filter at 610 nm, rPYY-FITC was excited with a 488 nm laser and detected at 527 nm bandpass filter. The untreated cells were used as negative control and for determination of the cell population. The data analysis was performed by using FCS Express V4 software. Cell viability test The in vitro cytotoxicity assay was assessed by the CellTiter-Glo Luminescent Cell Viability Assay (Promega, USA), which determines the number of viable cells based on ATP 25 ACS Paragon Plus Environment


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quantitation as an indicator for metabolically active cells. CellTiter-Glo assay was performed according to the manufacturer’s protocol. Luminescence signal was detected using a Platereader Infinite M1000 (Tecan, Germany). Caco-2 cells were seeded in 96-well plates (Greiner Bio One, Germany) at a density of 1.0 × 104 cells resuspended in 200 µl supplemented EMEM per well and cultured for 48 h. To determine the cytotoxicity of glycol chitosan nanoparticles (GCNPs), 100 µl of NPs diluted in supplemented EMEM at certain concentration were added to the cells and incubated for 4 h and 24 h. For the cell viability determination 100 µl of CellTiter-Glo detergence were given into the wells. After 15 min cell lysis the luminescence signal was stable and could be measured. Cytotoxicity was expressed as the percentage of the readout compared to untreated control cells. Supporting Information available: Fluorescent labeling of the peptide rPYY and glycol chitosan, SEM micrograph of GC-PGA NPs, effects of contact time and dosage of crosslinker on the release profile, evolution of the viscosity and refractive index of Eudragit L, biocompatibility of nanoparticles with cells, cellular viability of Caco-2 cells. This material is available free of charge via the Internet at http://pubs.acs.org.


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Acknowledgements The authors are grateful for the support of the BMBF (project 01KX1237A).

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Sequence-Controlled Delivery of Peptides from Hierarchically

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