Noncompetitive Active Transport Exploiting ... - ACS Publications

Feb 3, 2017 - Raghu Ganugula, Meenakshi Arora, Melissa Guada, Prabhjot Saini, and Majeti N. V. Ravi Kumar*. Department of Pharmaceutical Sciences, ...
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Noncompetitive Active Transport Exploiting Intestinal Transferrin Receptors for Oral Delivery of Proteins by Tunable Nanoplatform Raghu Ganugula, Meenakshi Arora, Melissa Guada, Prabhjot Saini, and Majeti N. V. Ravi Kumar* Department of Pharmaceutical Sciences, College of Pharmacy, Texas A&M University, Reynolds Medical Building, TAMU Mailstop 1114, College Station, Texas 77843, United States S Supporting Information *

ABSTRACT: Here we present a “thinking-outside-the-box”, tunable nanoplatform for oral delivery of proteins using insulin as a model protein. These nanosystems offer noncompetitive active transport exploiting transferrin receptors present in the intestine and permit tailored release in vivo. Such delivery approaches have the potential to individualize insulin therapy to a regimen that is compatible with the patient’s glucose profile.

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Here, we present a tunable nanoplatform offering tailored insulin release in vivo, suitable for personalized oral dosage forms. The GA-functionalized PLGA (PLGA-GA) was synthesized using ethylenediamine (EDA) linker by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. Similarly, we have prepared fluorescein-coupled PLGA for trafficking studies (details in SI, Figure 1a,b). For trafficking studies, fluorescein-coupled PLGA (4% w/w) is mixed with PLGA-GA for making fluorescent nanosystems (F-NS). The hydrophilic end would maneuver the GA to the surface during the emulsification process in an aqueous environment with surfactant, resulting in nanosystems of ∼140 nm in size (Figure 1c,d; details in SI).11 To demonstrate the noncompetitive receptor binding of the nanosystems, we have used rat small intestinal tissue section to better mimic in vivo scenario (details in SI). Our results demonstrate the noncompetitive behavior of PLGA-GA nanosystems evident by colocalization of particles with TfR despite the TfR being blocked by transferrin (Tf; Figure 2). Intracellular trafficking studies hold the key in understanding the fate of the nanosystems after internalization. The trafficking studies were conducted in caco-2 cells preincubated with F-NS for 1 h, followed by incubation with lysotracker for 30 min to label lysosomes (Figure 3; details in SI). Our results indicate both PLGA/PLGA-GA nanosystems on 90 min incubation time (60 min particle + 30 min lysotracker) show colocalization within lysosomes, while a majority are in the cytosol. The

uccessful oral delivery of peptides/proteins is a therapeutic “Holy Grail” due to ease of administration, offers high patient compliance,1,2 and importantly, proves very effective for health care programs in low-resource settings. However, high enzymatic and nuclease activity, in addition to the physical barrier, presents stiff challenges, particularly for labile peptides and proteins, resulting in extremely low bioequivalence in comparison to invasive routes.1,2 Reformulating drugs to address stability, pharmacokinetics (PK)/pharmacodynamics (PD) issues is a common strategy, and biodegradable nanosystems are hailed as a breakthrough technology for this purpose.3 These nanosystems, because of their size, are route independent, generally improving bioavailability, therapeutic efficacy, and safety of the carried drug and can deliver drugs of distinct physicochemical attributes.1−3 However, studies from our own group as well as from others suggest that the passive nanosystems are incompletely absorbed in the intestine.4−6 While approaches using permeation enhancers are coming under increasing scrutiny over safety concerns.7 Recently, ligand-functional polymer nanosystems have shown promising potential as carriers for transepithelial delivery of therapeutic peptides and proteins, exploiting intestinal receptors.8,9 However, the current approaches using endogenous mimic ligands for targeting could be outcompeted by physiological ligands.2,10 In an attempt to address the limitations of competitive active drug targeting, we have developed a noncompetitive strategy utilizing gambogic acid (GA), a xanthanoid. The GA prefunctionalized poly(lactide-coglycolide) (PLGA), upon emulsification, forms well-defined nanosystems with surface expressing GA for transferrin receptor (TfR) mediated transport in vitro and in vivo.11 © XXXX American Chemical Society

Received: January 18, 2017 Accepted: February 1, 2017

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DOI: 10.1021/acsmacrolett.7b00035 ACS Macro Lett. 2017, 6, 161−164

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Figure 1. (a) Schematic of PLGA-GA and PLGA-fluorescein synthesis; (b) Cartoon depicting surface expression of GA. (c) DLS particle size; and (d) Scanning electron micrograph of PLGA-GA NS.

Figure 3. Live cell imaging of nanosystems intracellular trafficking. Representative confocal images of caco-2 cells with and without preincubation of F-NS and images acquired at 40 × 2.5× magnification. Green represents the fluorescence of F-NS, red represents lysotracker, and blue represents nuclei stained with Hoechst. The insets show a magnified view of the marked areas, demonstrating colocalization in PLGA/PLGA-GA F-NS. The colocalization analysis was performed with default M1 and M2 coefficients in ImageJ software. All data are presented as mean + SEM: (a) Lysosomal percent colocalization; (b) Percent fluorescein intensity. Statistical comparisons were made with t test using Mann−Whitney U-test, medians significant difference of two-tailed p value (p < 0.05).

Figure 2. Representative confocal images of small intestine sections subjected to appropriate protocols. Protocol 1: without blocking the transferrin receptor (TfR), (a) control, (b) PLGA F-NS, and (c) PLGA-GA F-NS (tissue section stained for actin (red); nucleus (blue) with F-NS seen in green and images acquired at 20× magnification). Protocol 2: after blocking TfR with purified rat transferrin (Tf), (d) control, (e) PLGA F-NS, and (f) PLGA-GA F-NS and sections stained for TfR (red), F-NS seen in green, showing highly visible colocalization in case of PLGA-GA F-NS (images acquired at 40 × 1.8× magnification). The colocalization analysis was performed with default M1 and M2 coefficients in ImageJ software. The upper panels with arrows in (e) and (f) indicate colocalization puncta through Zstacking (x−z). (g) All data are presented as mean ± SEM of three independent experiments. Statistical comparisons were made with t test using Mann−Whitney U-test, medians significant difference of two-tailed p value (p < 0.05).

in varying percentages: PLGA (100%); PLGA (75%):PLGAGA (25%); PLGA (50%):PLGA-GA (50%); PLGA (25%):PLGA-GA (75%); and PLGA-GA (100%). The nanosystems will henceforth be termed as PLGA100, PLGA-GA25, PLGA-GA50, PLGA-GA75, and PLGA-GA100 (details in SI, Figure S2a). A single dose pharmacokinetic study was conducted in male Sprague−Dawley rats (n = 4) in order to understand the ability of GA to facilitate transport of the nanosystems and the associated insulin across the intestinal barriers (details in SI). Our results indicate that a tailored insulin release in vivo is possible by adjusting the surface GA density (Figure 4).

fluorescent and colocalization intensities represent cytosol and lysosomal concentrations of the F-NS. To evaluate the effect of surface GA density on the transport of insulin carrying PLGA-GA nanosystems across the intestinal barriers, five versions of nanosystems were prepared with increasing amount of surface GA associated with the nanosystems. This was achieved by blending PLGA-GA with PLGA 162

DOI: 10.1021/acsmacrolett.7b00035 ACS Macro Lett. 2017, 6, 161−164

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ACS Macro Letters

However, to be therapeutically viable, these systems need to escape from the secondary endosomes. The data supports endocytosis pathway of particle transport as both PLGA/ PLGA-GA F-NS shown colocalization in the lysosomes at 90 min incubation. However, lot more green fluorescence can be seen in the cytoplasm in the case of PLGA-GA F-NS compared to PLGA F-NS, indicating better receptor-mediated transportability of the former. A longer incubation of the particles with cells might see all the particles escaping lysosomes, while particles are known to getting in as quick as 10 min incubation, and this can vary between cell types.16−18 An increasing trend in the Cmax of the insulin was observed with increase in GA surface density. Another notable feature is the time it takes to peak the insulin concentration, which is also an important aspect when we target postprandial glucose reduction. It is also interesting to note a distinct double-peak with PLGA-GA100, with the first one appearing at 30 min and second at 6 h, while in PLGA-GA25−75 groups it was not that prominent. Such double peaks in plasma concentration after oral administration are not unusual or exceptional and are believed due to the two different sites of absorption or the enterohepatic circulation.19 While other possibilities such as the irregular patterns of gastric emptying are also expected to influence the absorption.20 Based on the t1/2 data presented in the table (Figure 4b), we could rule out the possible enterohepatic circulation, as the PLGA-GA nanosystems did not show longer half-life in plasma concentration−time profile, characteristics of enterohepatic circulation. All the animals were treated in fed-state, and the differences in the pharmacokinetics does not appear to be the effect of gastric emptying. Therefore, in the present case, we believe the double peaks could be due to different sites of absorption depicting differences in the TfR presence in duodenum and jejunum. In the current study, the PLGA systems appear to have peaked at 12 h (35.5 μIU/mL), but the difference is very marginal at 24 h (33.8 μIU/mL), which is in agreement with our prior findings, a Cmax at 24 h.21 A delivery system with such tailored pharmacokinetics can be very beneficial in addressing variable glucose levels in different patients.22 In conclusion, the data represents a unique noncompetitive delivery platform that can facilitate oral peptide/protein delivery, and in doing so, there is tremendous opportunity to head forward for personalized dose regimen.

Figure 4. Blood persistence properties of insulin as a function of surface density of GA on the nanosystems. AUC was calculated by linear trapezoidal rule. Frel, was calculated by the ratio of AUC for each orally administered nanosystems over AUC for subcutaneously administered insulin (sc). Cmax, maximum plasma concentration; Tmax, time to reach maximum plasma concentration; AUC, area under the plasma concentration−time curve; Frel, relative oral bioavailability; sc, subcutaneous; iv, intravenous. Data expressed as mean ± sd (n = 4). *p < 0.05 compared to PLGA (Mann−Whitney U test). Cmax and Tmax were directly obtained from the plotted data and Kel= In(Cpeak) − In(Ctrough)/tinterval and t1/2 = 0.693/kel. PIL = plasma insulin level.

The GA coupling to PLGA using EDC chemistry is robust and scalable. The polymers were thoroughly characterized confirming GA association with PLGA (details in SI, Figure S1). The insulin-loaded nanosystems were prepared by a double emulsion-diffusion-evaporation method (details in SI). The UV-vis spectroscopy of lyophilized insulin-loaded nanosystems revealed peaks at 290 and 360 nm that are characteristic wavelength for GA, suggesting GA on the surface (SI, Figure S2b). The surface GA increased proportionally to the PLGA-GA ratio in the blend due to higher proportions of GA in the blend. The preparations led to ∼56 μg insulin/mg polymer. This loading, in general, is much higher than that achieved in competing solutions.12,13 The particle characteristics of all the blends had similar particle sizes ranging from 233.45 ± 3.93−295.13 ± 13.40 nm and entrapment efficiencies 33.52 ± 5.67−36.90 ± 2.71% (SI, Figure S2c). Such uniformity in the insulin content becomes essential for understanding the influence of surface density on the bioavailability. The use of 4 μm transverse section of rat jejunum (SI, Figure S3) allowed us to better understand the noncompetitive interaction of GA-coupled nanosystems with TfR in the presence of purified rat Tf. In the first part of the study (Figure 2a−c) with actin and nucleus staining, significant green fluorescence from PLGA-GA F-NS can be seen throughout, with excessive accumulation at the submucosal area.14,15 In a second study where TfR was labeled, a very high colocalization was observed, suggesting particles interacting with the receptor, and this happens in the presence of Tf. Such noncompetitive binding will help overcome the problems of the carrier being outcompeted by physiological ligand, a problem associated with all targeted systems being explored in literature.2 The protocol developed in this study can be adapted to test various delivery strategies exploiting other receptors and tissues. In general, the particles once transported into the cell are trafficked to enter either recycling endosomes to undergo exocytosis or late endosomes to undergo degradation.



ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.7b00035.



Experimental and characterization data (PDF).

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: (979) 436-0721. ORCID

Majeti N. V. Ravi Kumar: 0000-0001-5606-401X Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. 163

DOI: 10.1021/acsmacrolett.7b00035 ACS Macro Lett. 2017, 6, 161−164

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ACS Macro Letters Funding

(20) Amekyeh, H.; Billa, N.; Yuen, K. H.; Lim, S. C. AAPS PharmSciTech 2016, 17 (5), 1060−1066. (21) Sharma, G.; van der Walle, C. F.; Kumar, M. N. V. R. Int. J. Pharm. 2013, 440 (1), 99−110. (22) Raz, I.; Riddle, M. C.; Rosenstock, J.; Buse, J. B.; Inzucchi, S. E.; Home, P. D.; Del Prato, S.; Ferrannini, E.; Chan, J. C.; Leiter, L. A.; Leroith, D.; Defronzo, R.; Cefalu, W. T. Diabetes Care 2013, 36 (6), 1779−1788.

Authors would like to acknowledge the College of Pharmacy, TAMU, for a seed grant. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS We would like to acknowledge Dr. Batteas, Department of Chemistry, for assistance with ATR-FTIR, Materials Characterization Facility (MCF) at TAMU for access to SEM; Dr. Ficht, HSC, for providing access to particle size analyser; Dr. Mouneimne, Image Analysis Laboratory, for the help with the analysis of confocal images, Dr. Capareda, Biological and Agricultural Engineering-TAMU, for providing access to KF Titrino for moisture content analysis, and Dr. Wylie, TAMUNMR facility, for assistance with NMR.



ABBREVIATIONS Cmax concentration maximum; DLS dynamic light scattering; EDA ethylenediamine; EDC 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide; F-NS fluorescent nanosystems; PLGAGA GA-functionalized PLGA; GA gambogic acid; NMR nuclear magnetic resonance; PK pharmacokinetics; NS nanosystems; PLGA poly(lactide-co-glycolide); SEM scanning electron microscope; Tmax time at which Cmax is observed; Tf transferrin; TfR transferrin receptor



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DOI: 10.1021/acsmacrolett.7b00035 ACS Macro Lett. 2017, 6, 161−164