Oral Administration and Selective Uptake of Polymeric Nanoparticles

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Oral Administration and Selective Uptake of Polymeric Nanoparticles in Drosophila Larvae as In Vivo Model Shan Jiang, Choon Peng Teng, Wee Choo Puah, Martin Wasser, Khin Yin Win, and Ming-Yong Han ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.5b00163 • Publication Date (Web): 14 Sep 2015 Downloaded from http://pubs.acs.org on September 21, 2015

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ACS Biomaterials Science & Engineering

Oral Administration and Selective Uptake of Polymeric Nanoparticles in Drosophila Larvae as In Vivo Model Shan Jiang,†,# Choon Peng Teng,†, §,# Wee Choo Puah,‡, # Martin Wasser,‡,* Khin Yin Win,†,* and Ming-Yong Han†, §,* †

Institute of Materials Research and Engineering, A*STAR, 3 Research Link, Singapore 117602

‡

Bioinformatics Institute, A*STAR, 30 Biopolis Street, Matrix, Singapore 138671

§

Department of Biomedical Engineering, National University of Singapore, 9 Engineering Drive

1, Singapore 117575 *Emails: [email protected], [email protected], [email protected] ABSTRACT: In this paper, Drosophila larvae are applied as an in vivo model to investigate the transport and uptake of polymeric nanoparticles in the larval digestive tract after oral administration. After feeding the larvae with food containing bare and chitosan-coated PLGA nanoparticles encapsulated with BODIPY, time-lapse imaging of live larvae is used to monitor the movement of fluorescent nanoparticles in the anterior, middle and posterior midgut of digestive tract. Also, the dissection of digestive tract enables analysis of cellular uptake in the midgut. Bare PLGA nanoparticles travel through the whole midgut smoothly while chitosancoated PLGA nanoparticles have a long retention in the posterior midgut. We identify this retention occurs in the posterior segment of the posterior midgut and it is termed as retention segment. During transport in the midgut, chitosan-coated nanoparticles pass through the nearneutral anterior midgut and become highly positively charged when entering into the highly acidic middle midgut. After traveling through the near-neutral anterior segment of posterior midgut, chitosan-coated nanoparticles have long retention of ~10 h in the retention segment,

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indicating the chitosan coating greatly enhances mucoadhesive ability and promotes cellular uptake in this part of midgut. The dynamic behavior of orally administered nanoparticles in Drosophila larvae is in agreement with studies in other animal models. Drosophila larva has the potential to evolve into a low-cost drug screening model through real time imaging, which will accelerate the development of improved nanoparticle formulations for oral drug delivery. Keywords: PLGA nanoparticles, chitosan, oral delivery, Drosophila larva, in vivo model

INTRODUCTION Administration of therapeutic drugs via the oral route, especially their transport and uptake along the gastrointestinal tract (also referred to as the digestive tract or the gut) of digestive system, represents an active challenge in modern pharmaceutical application.1 To achieve oral delivery at high efficacy, drugs must travel effectively through the digestive tract, adhere and permeate through mucus layer, transverse across intestinal epithelium, enter portal vein, and finally go into peripheral circulation.1 For example, peptides and proteins as drugs have poor oral bioavailability due to barriers such as harsh acidic environments in the stomach and enzymatic degradation in the digestive tract.2 Also, the absorption barriers such as mucosal layer and intestinal epithelium may prevent the drugs from entering systemic circulation. To achieve sufficient oral bioavailability of drugs, one of the most promising strategies is to develop oral delivery systems with the use of nanoparticle carriers that are made from biocompatible and biodegradable polymers.3 The major advantages of polymeric nanoparticle carriers are their capability to protect the encapsulated drugs and stability in the digestive tract, facilitating improved cellular uptake and controlled drug release at the targeted site.4


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Before clinical application, drug formulations of polymeric nanoparticles need to be screened through in vitro cell culture and in vivo animal studies. The majority of positive candidates that were identified in vitro were found to be ineffective when validated with in vivo animal models due to their complex and uncontrolled nature.5,6 There is thus a great demand to develop low-cost and easy-to-breed animal models for more effective screening of various formulated polymeric nanoparticles as drug carriers in a high throughput fashion. In this research, Drosophila melanogaster, henceforth referred to as Drosophila (i.e. fruit fly), is proposed as a short-lived model organism to achieve direct and non-invasive study of in vivo fate of formulated polymeric nanoparticles that are delivered through food. In Drosophila, food passes sequentially through foregut, anterior midgut, middle midgut and posterior midgut, and nutrient absorption takes place along the way. The food then reaches the hindgut and rectum, where water and electrolytes are exchanged, and finally, the anus for excretion.7 There is a large degree of similarity of the digestive tract of Drosophila to that of human.8,9 The foregut, midgut and hindgut-rectum-anus of Drosophila correspond analogously to the esophagus, small intestine and large intestine-rectum-anus of human, respectively.7 In addition, there is a layer of peritrophic matrix composed of chitin and glycoproteins that lines the insect gut lumen which acts as a physical barrier, similar to the mucus layer found in the intestinal tract of human.10,11 Moreover, there are different digestive enzymes in Drosophila’s gut to break down carbohydrates, proteins, and lipids for absorption. The digestion process is influenced by not only enzymatic activity but also pH, similar to human.12 Nearly 75% of human disease genes are estimated to have homologues in Drosophila, 13 and Drosophila can thus possibly be used to study various diseases such as cancer, Alzheimer’s disease and neurological disorders.


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As the larvae are transparent, it makes Drosophila an ideal system for real time monitoring of orally administered fluorescent polymeric nanoparticles of ~100 nm. This allows for ease of tracing in vivo with confocal microscopy to monitor their transport and distribution dynamics for understanding of the underlying processes at organism and cellular level. The aim of this study is to establish the effectiveness of Drosophila larvae as an animal model for potential screening of fluorescent polymeric nanoparticles as drug carriers under confocal laser scanning. This can help to achieve better understanding of nanoparticle transport under in vivo conditions and to improve nanoparticle design for efficient oral drug delivery. To study the fate of orally administered nanoparticles, fluorescent polymeric nanoparticles were synthesized with different surface coating materials, and mixed with Drosophila food to deliver to the gut. Time-lapse imaging of live larvae immobilized on a glass slide was conducted to analyze the dynamics of nanoparticles in the digestive tract. Furthermore, imaging of dissected larval midgut was performed using confocal microscope to monitor cellular absorption and subcellular localization of the orally delivered nanoparticles. The approach has the potential to evolve into a low-cost screening platform that will accelerate the development of nanoparticle formulations for oral drug delivery.

MATERIALS AND METHODS Materials. Poly(D,L-lactic-co-glycolic acid) (PLGA, L:G molar ratio 50:50, Mw 24,00038,000, Resomer® RG503H) was purchased from Boehringer Ingelheim. Polyvinyl alcohol (PVA, Mw 30,000-70,000), phosphate buffered saline (PBS, pH 7.4), chitosan (low molecular weight, 75-85% deacetylated), cornmeal, dextrose, low melting point agarose, glacial acetic acid, and sodium hydroxide were purchased from Sigma Aldrich. Yeast extract, BODIPY 630/650, 3-


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(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dichloromethane (DCM) were obtained from Becton Dickinson, Life Technologies, Molecular Probes and Merck, respectively. Anhydrous dimethyl sulfoxide (DMSO) was purchased from Tedia. All chemicals were used as received without further processing. Double-distilled water was used throughout experiments. Preparation of BODIPY-PLGA nanoparticles. BODIPY 630/650 was dissolved in anhydrous dimethyl sulfoxide (DMSO) to form a stock solution of 2.5 mg/mL. Then, 60 µL of BODIPY stock solution was added to 8 mL of DCM containing 100 mg of PLGA and then emulsified in 65 mL of aqueous PVA solution (2% w/v) to form an oil-in-water emulsion using a probe sonicator (VibracellTM Ultrasonic Processor) at maximum amplitude for 2 min. The emulsion was stirred at 400 rpm for 4 h to evaporate the organic solvent. The BODIPYencapsulated PLGA nanoparticles were recovered by centrifugation at 12,000 rpm for 30 min, followed by washing twice with ethanol/water (10/90, v/v %). The purified nanoparticles were dispersed in distilled water and then freeze dried at -50 °C for 24 h. The yield of PLGA nanoparticles was calculated by the weight of freeze-dried nanoparticles divided by the original amount of PLGA used. Preparation of chitosan-BODIPY-PLGA nanoparticles. An aqueous solution of chitosan (0.5 w/v%) was first prepared in 1% glacial acetic acid, adjusted to pH 5 with sodium hydroxide, and filtered. The chitosan-coated BODIPY-encapsulated PLGA (i.e. chitosan-BODIPY-PLGA) nanoparticles were synthesized using a similar protocol described above, except that 831 µL of the chitosan solution was added to the aqueous PVA solution (65 mL, 2% w/v) prior to emulsification with PLGA solution.


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Size, morphology and surface charge of PLGA nanoparticles. Scanning electron microscopy (SEM) images of PLGA nanoparticles were obtained on a scanning electron microscope (JEOL SEM 6360LA) after sputtering gold on samples using fine coater (JEOL JFC1200) for 20 s. The average size of PLGA nanoparticles was obtained by measuring the diameter of one hundred PLGA nanoparticles in high-magnification SEM images and calculating their average size ± SD using SmileView software. The surface charge of PLGA nanoparticles was determined by measuring zeta potential with ZetaPlus (Zeta Potential Analyzer, Brookhaven Instruments Corporation). Encapsulation efficiency of BODIPY in PLGA nanoparticles. Experimentally, 1 mg of freeze-dried PLGA nanoparticles were dispersed in 4 mL of DCM and stirred overnight. The PLGA nanoparticles were dissolved to extract the encapsulated BODIPY. The amount of the extracted BODIPY was determined by measuring the peak emission intensity at 650 nm with excitation wavelength of 630 nm using a spectrofluorophotometer (Shimadzu RF5301). The concentration was obtained according to the standard curve of BODIPY. The encapsulation efficiency was estimated by multiplying the amount of BODIPY in 1 mg of PLGA nanoparticles with the yield of PLGA nanoparticles and dividing it by the original amount of BODIPY added. Quantum yield measurement. The quantum yield of BODIPY in PLGA nanoparticles was determined relative to that of BODIPY in water. The quantum yield of BODIPY is nearly 100% in water, as given by the manufacturer (Life Technologies). The peak emission intensities of BODIPY-PLGA and chitosan-BODIPY-PLGA nanoparticles (200 µg/mL) were measured by spectrofluorophotometer and compared to the peak emission intensity of an equivalent amount of free BODIPY. The quantum yield of BODIPY in polymeric nanoparticles was calculated by


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dividing the peak fluorescence intensity of nanoparticles with that of free BODIPY and multiplying it with the quantum yield of BODIPY. MTT assay of PLGA nanoparticles. Human normal colon cells (CCD-112CoN cells were seeded at 2.0×104 cells/cm2 in the 96-well plates and cultured as a monolayer. Cells were incubated with the culture medium containing BODIPY-PLGA nanoparticles, or chitosanBODIPY-PLGA nanoparticles at different concentrations (62.5, 125, 250, 500 and 1000 µg/mL) in 5 replicates for 48 h at 37 °C in a humidified atmosphere containing 5% CO2. After 48 h incubation, the nanoparticles were removed and MTT reagent was added to further incubate for 4 h. The formazan precipitates were dissolved in DMSO to measure cell viability by measuring optical density at 570 nm. The optical density is directly proportional to the number of viable cells. The viability is calculated by the optical density of the nanoparticle-treated samples divided by that of control cells without nanoparticle treatment. Drosophila culture. UAS-GAL4 expression system was used to express green fluorescent protein (GFP) in the digestive tract of Drosophila. Female Drosophila carrying a UAS responder (UAS-GFP) are first mated to male Drosophila carrying a GLA4 driver, and the resulting progeny can express GFP.14 By meiotic recombination, a marker line was created to robustly expresses GFP in the foregut, midgut and hindgut of Drosophila larvae. Feeding larvae of Drosophila with PLGA nanoparticles. The Drosophila food was prepared with 1.75 g of cornmeal, 1.50 g of dextrose, 0.70 g of yeast extract, 0.30 g of low melting point agarose, and 30 mL of distilled water with stirring and heating at 90 °C for 20 min, followed by the addition of 900 µL of 10% nipagin to the cooled mixture. To analyze the oral delivery of nanoparticles in larvae, nanoparticles at a concentration of 500 µg/mL were uniformly mixed


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with the freshly prepared Drosophila food under warm condition (~ 40 °C) and then poured in a 35 mm petri dish to solidify the mixed food for feeding the 3rd-instar larvae of Drosophila at 25 °C. Live imaging and tracking of nanoparticles after larvae ingestion. Five-day old 3rd-instar larvae were fed with Drosophila food containing nanoparticles at 500 µg/mL for 15 min and viewed/monitored under a stereomicroscope (Olympus MVX10 macroview). After feeding for 15 min with food containing nanoparticles, Drosophila larvae were checked under stereomicroscope to ensure the fluorescent nanoparticles have been ingested into the gut. The larvae with nanoparticles in the gut were used for further observation under confocal microscope. The brightness of fluorescence is directly proportional to the amount of food ingested. The larvae were then washed three times in distilled water to remove traces of food on the skin and processed for imaging. Each larva was further immobilized on a glass slide with one cover slip on the top without affecting peristaltic movements. PBS solution was pre-added around the larva to allow for hydration during imaging. At 30 min, time-lapse data of nanoparticle movements in the live larvae were recorded using laser scanning confocal microscope (Carl Zeiss LSM 5 Live) with 10x dry objective lens. The multi-location time series macro was used to capture the entire digestive tract of a larva, with a minimum of three locations per larva. The fluorescence of GFP and nanoparticles were excited with laser at 488 nm and 635 nm respectively. Image stacks of two-color channels were collected at each of the different locations at 1 min intervals for 24 h. Cellular uptake of nanoparticles in larval gut. After feeding the 3rd-instar larvae with nanoparticles at 500 µg/mL for 1 day, the whole digestive tract was dissected out of


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anaesthetized larva and placed straight on a cover slip. As the digestive tract is a convoluted tube of ~16 mm that is 4 times longer than the larva, the tissue dissections helped in familiarizing the anatomy of the digestive system and identifying anatomical landmarks highlighted by the reporter protein, GFP. The subcellular localization of fluorescent nanoparticles in gut was investigated under confocal microscope with a 63× oil objective lens.

RESULTS AND DISCUSSIONS In the studies of Drosophila, the gut is divided into foregut, midgut and hindgut. The foregut is used to store food while the hindgut is the major site of water re-absorption by concentrating excrement prior to elimination. As one of the largest insect organs, the midgut is the main site of digestion and nutrient absorption, and this is the focus of the study in this research. The midgut is traditionally subdivided into three segments: the anterior, middle, and posterior midgut. When Drosophila ingests food, food gradually passes from the mouth to the anus by peristalsis through the hollow organs of the entire digestive tract. Experimentally, five-day old 3rd-instar larvae were fed with Drosophila food containing the nanoparticles at 500 µg/mL for 15 min at 25 °C and then viewed under a laser scanning confocal microscope to track the movement and absorption of nanoparticles in the digestive tract. Bare and chitosan-coated PLGA nanoparticles were selected to study the influence of difference in surface charge and mucoadhesive property of polymeric nanoparticles on their transport along digestive tract and cellular uptake in gut cells. Cells of the larval digestive tract were labelled using UAS-GAL4 GFP expression system which can assist to locate the fluorescent polymeric nanoparticles delivered orally with the food. The GFP expression in larvae allowed easy identification of landmarks in the digestive tract in


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the regions of middle midgut, posterior midgut and hindgut when observed under laser scanning confocal microscopy.14 The different sections of digestive tract including anterior, middle and posterior midgut, and hindgut were identified based on the different GFP labeling patterns. For example, the middle midgut contains a pool of highly differentiated copper cells, where the reporter GFP are strongly expressed.15 The midgut and hindgut are separated by a bright junction. For easy tracking of polymeric nanoparticles to study their fate in the gut, BODIPY 630/650 was chosen as model label and encapsulated in the polymeric nanoparticles. This is because BODIPY emits red fluorescence, which does not overlap with green fluorescence of GFP and the autofluorescence of the larval digestive tract. Preparation and characterization of polymeric nanoparticles. The single emulsion solvent evaporation (oil-in-water) method was used to encapsulate BODIPY in PLGA nanoparticles to form BODIPY-PLGA nanoparticles, which have spherical shape with narrow size distribution (Figure 1A). BODIPY-PLGA nanoparticles have an average size of 136.1±14.8 nm with negatively charged surface and zeta potential value of -23.2±1.29 mV. Meanwhile, the modified solvent evaporation method was successfully employed to synthesize chitosan-BODIPY-PLGA nanoparticles which have an increased mean diameter of 139.3±14.8 nm with positively charged surface and zeta potential value of +26.5±2.05 mV (Figure 1B). Chitosan is embedded inside and on the surface of PLGA nanoparticles due to the electrostatic interaction of cationic chitosan molecules with anionic PLGA polymer.16,17 These BODIPY-PLGA and chitosan-BODIPYPLGA nanoparticles exhibited bright fluorescence under confocal microscope with an excitation wavelength of 635 nm (inset of Figure 1A and B). The photoluminescence spectra of these BODIPY-encapsulated nanoparticles showed they emitted red fluorescence at 642 nm


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(Supplementary Figure S1). The production yields of the BODIPY-PLGA and chitosanBODIPY-PLGA nanoparticles are 53.6% and 47.6%, and their encapsulation efficiencies of BODIPY are 30.0% and 31.7%, respectively. The quantum yields of BODIPY in PLGA and chitosan-PLGA nanoparticles are 94.3% and 84.6%, respectively as compared to that of free BODIPY. Biocompatibility of the BODIPY-PLGA and chitosan-BODIPY-PLGA nanoparticles was assessed by tetrazolium-based colorimetric MTT assay after incubating nanoparticles with human normal colon cells (CCD-112CoN cells) for 48 hrs. No significant reduction in cell viability was observed at increased concentrations of bare and chitosan-coated PLGA nanoparticles from 62.5 to 1000 µg/mL, as seen in Supplementary Figure S2. Thus, both bare and chitosan-coated nanoparticles are used safely in the study of nanoparticles transport and cellular uptake. Feeding and imaging of polymeric nanoparticles. The 3rd-instar larvae were raised at 25 °C on the Drosophila food in the presence of bare and chitosan-coated PLGA nanoparticles. In order to study the movement of the polymeric nanoparticles through the digestive tract of larvae and identify the absorption segments of digestive tract, time-lapse images of live larvae fed with BODIPY-PLGA nanoparticles and chitosan-BODIPY-PLGA nanoparticles were captured. The digestive tract of larva was dissected and the cellular uptake of nanoparticles in the posterior midgut was analyzed. The overview of experiment and schematic midgut of Drosophila larva are shown in Supplementary Figure S3. The pH of the different segments is also indicated. GFP were strongly expressed in the middle midgut, which can be clearly differentiated from the posterior midgut. It is noted that Drosophila was used to investigate in vivo toxicity of various inorganic nanoparticles such as silver nanoparticles and functionalized carbon nanotubes,18-20


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and this is the first time Drosophila is used to investigate transport and uptake of polymeric nanoparticles. Live imaging of polymeric nanoparticles when passing through larval midgut. After feeding for 15 min with food containing the fluorescent nanoparticles of interest at a concentration of 500 µg/mL, the larva was immobilized and mounted on a cover slip (without further feeding). The time-lapse images of live larva were captured every 1 min under laser scanning confocal microscope. Figure 2 shows the schematic and confocal images taken at the time points of 0.5, 4, 8 and 14 h for one of the typical larvae fed with BODIPY-PLGA nanoparticles. As observed, the nanoparticles travelled down the digestive tract via peristaltic contractions. The nanoparticles passed quickly through anterior and middle midgut to reach posterior midgut in about 0.5 h (Figure 2B). The start and end of posterior midgut was indicated with S and E, respectively. The nanoparticles further took 13.5 h to exit posterior midgut before entering hindgut and excreted out from the larva (Figure 2C-E). The BODIPY-PLGA nanoparticles travelled smoothly in the midgut without obvious retention in any region. On the other hand, chitosan-BODIPY-PLGA nanoparticles took a longer period of time to pass through the midgut. Figure 3 shows the schematic and confocal images taken at the time points of 0.5, 6.5, 11.5 and 17.5 h for one of the larvae fed with chitosan-BODIPY-PLGA nanoparticles. Similar to the time required for BODIPY-PLGA nanoparticles to be transported through the anterior and middle midgut, the chitosan-BODIPY-PLGA nanoparticles also reached the posterior midgut in about 0.5 h (Figure 3B). They then took another 17 h to pass through the whole posterior midgut before entering the hindgut and excreted out of the larva (Figure 3C-E). During the whole travelling process, it was observed that the nanoparticles were retained in a


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specific region of the posterior midgut (i.e. retention segment) for ~10 h (Supplementary Figure S4). In comparison, chitosan-BODIPY-PLGA nanoparticles showed long retention in the retention segment observed by strong fluorescence while BODIPY-PLGA nanoparticles did not show any retention in the posterior midgut. The results demonstrated that chitosan coating enhanced the mucoadhesive ability of nanoparticles and thus chitosan-coated nanoparticles have longer retention than bare nanoparticles in the retention segment which may in turn promote them to be absorbed by gut cells. This is in agreement that chitosan containing delivery system prolongs the residence time in the small intestine in the animal due to its mucoadhesive property.21 Transport of polymeric nanoparticles in the posterior midgut. The larval midgut of Drosophila has at least three segments: an anterior near-neutral midgut, a short and narrow acidic middle midgut, and a long and wider posterior midgut that is near-neutral in the anterior segment of posterior midgut and alkaline in the posterior segment of posterior midgut.22 From the GFP labelling pattern, we have identified the retention segment to be the posterior segment of the posterior midgut. The pH of the different segments plays a role in the interaction of the nanoparticles and the digestive tract which in turn affects the nanoparticle transport after oral administration. Chitosan-BODIPY-PLGA nanoparticles are positively charged in near neutral pH so they can easily pass through the anterior near-neutral midgut to reach strongly acidic middle midgut. Chitosan becomes highly positively charged because it has a large number of –NH2 that is protonated to –NH3+ in the highly acidic region. In the highly acidic environment, the peritrophic matrix covering the surface of epithelium layer also becomes highly positively charged. The peritrophic matrix is composed mainly of proteins embedded in proteoglycan


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matrix which is a major component of the extracellular matrix.23,24 The large amount of proteins and collagen in the extracellular matrix present in the peritrophic matrix are changed from –NH2 to –NH3+ under acidic condition. Thus, the chitosan-coated nanoparticles and matrix proteins repel each other, causing the nanoparticles to pass quickly through middle midgut to reach posterior midgut. After passing the middle midgut, the highly positively charged nanoparticles stay in the anterior near-neutral segment of posterior midgut for a short period of time before travelling to the posterior alkaline segment of posterior midgut. Upon entering into the highly alkaline (pH>10) region,22 the highly positively charged nanoparticles remain in this region for a long period of time (10 h). This is because the peritrophic matrix of epithelium layer in this region also becomes highly negative charged and the chitosan-coated nanoparticles quickly bind on it by electrostatic attraction, resulting in the long retention as observed by their strong fluorescence in Supplementary Figure S4. Interestingly, the nanoparticles only stay stably in the first part of the posterior alkaline segment of posterior midgut. With time, these highly positively charged nanoparticles are gradually neutralized due to the deprotonation of –NH3+ to –NH2 again and then released slowly from the epithelium as observed by continuous movement to the hindgut in the next 7 h. This can be explained by the isoelectric point of chitosan (~pH 8.7 as seen in Supplementary Figure S5). Chitosan-coated nanoparticles become neutral at this pH, and above which they become negatively charged nanoparticles. In comparison, bare PLGA nanoparticles pass through the whole midgut and hindgut smoothly with no retention in the retention segment, resulting in weak fluorescence of nanoparticles.


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The cellular uptake of fluorescent chitosan-coated nanoparticles in the retention segment was observed to help understand the absorption of orally delivered nanoparticles in digestive tract. After feeding the 3rd-instar larvae with chitosan-coated nanoparticles at 500 µg/mL for 1 day, the larvae were dissected. Their digestive tracts were stretched straight and observed under the confocal microscope with a 63x objective lens for differentiation between luminal and cellular localization of fluorescent nanoparticles. The digestive tract in the live 3rd-instar larva can only be observed at cellular level under high magnification. Due to the limited effective working distance of high magnification objective lens, it cannot be used for tracking of nanoparticles in live larva. The subcellular localization of chitosan-BODIPY PLGA nanoparticles in gut was investigated under confocal microscope while the anatomical landmarks highlighted by GFP helped to facilitate identification of the location of nanoparticles. Confocal imaging of the dissected midgut at higher magnification showed clearly that the chitosan-coated nanoparticles were uptaken in retention segment of posterior midgut (Figure 4). The positively charged nanoparticles were effectively internalized into epithelial cells, indicated by clear intracellular bright fluorescent spots while the released BODIPY may be observed by diffused fluorescence. In contrast, no significant fluorescence was observed in the retention segment of the larvae fed with negatively charged BODIPY-PLGA nanoparticles (Figure 4). This further reinforced that the positive charge and mucoadhesive property of chitosan coating of nanoparticles enhance the cellular internalization of the nanoparticles in the posterior midgut. The fate of nanoparticles at different regions of the midgut is presented in the schematic and confocal image of the dissected midgut (Figure 5).


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Chitosan plays an important role for uptake of polymeric nanoparticles into midgut, similar to absorption of small molecule nutrients in the same region that has been studied extensively. It is in agreement that the posterior midgut is the main region of nutrient absorption.25 There are abundant amino groups (one in each repeating unit) on chitosan-coated PLGA nanoparticles, resulting in them become highly positively charged in highly acidic middle midgut. This helps to facilitate cellular uptake upon entering the retention segment of posterior midgut due to strong electrostatic attraction with the highly negatively charged peritrophic matrix of the epithelium. On the other hand, bare PLGA nanoparticles, which have few carboxylic groups on the surface, are negatively charged, thus resulting in little cellular uptake in middle midgut and posterior midgut. Our observations using the transparent Drosophila larvae as an in vivo model have enabled us to identify a retention segment in the posterior midgut as the main nutrient absorption of nanoparticles. In this region, the epithelium consists of nutrient-absorbing cells,25 which are responsible in absorbing digested food with smaller molecular weight. Similar findings of chitosan-coated PLGA nanoparticles were also observed in other well established animal models such as rat and rabbits.26,27 The administration of chitosan-coated PLGA nanoparticles to rats resulted in longer retention times and a higher affinity for cellular uptake than PLGA nanoparticles, which was attributed to interactions between the surface of positively charged nanoparticles and negatively charged cell membrane.26 Adhesion and uptake of chitosan-PLGA nanoparticles were also visible in the mucosal epithelial cells of rabbits when observed under fluorescence microscopy.27


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The digestive systems of Drosophila and human share many similarities though there are still some differences such as less complex microbiota composition and immune system. Drosophila assays are superior to mammalian-cell-based approach in screenings as they possess complex cellular composition which is a better replicate of the in vivo digestive system.9 Drosophila larvae can be used to screen large collections of potential candidates, reducing them to a smaller pool of higher quality leads before validating with established animal models to improve the rate of discovery.28 The assimilation of Drosophila into the drug formulation discovery process enables high throughput and cost-effective screening through real-time imaging of live transparent larvae.28

CONCLUSIONS Transport and uptake of nanoparticles were studied using the Drosophila larva model with bare and chitosan-coated BODIPY-PLGA nanoparticles. Transparent Drosophila larvae facilitate live imaging of the dynamics of fluorescent nanoparticle transit through the midgut. Dissection of the gut allows for the study the absorption of nanoparticles in larval digestive tract using confocal microscopy. From the time-lapse images of live larvae, we have identified the retention segment to be the posterior segment of posterior midgut where significantly longer retention of chitosancoated nanoparticles was observed as compared to bare nanoparticles. This was attributed to the electrostatic attraction between the positively charged chitosan-coated nanoparticles and the negatively charged peritrophic matrix in the retention segment. In addition, the chitosan coating also enhanced the mucoadhesive ability of nanoparticles and promoted interaction with the epithelium layer of the posterior midgut for cellular uptake. The Drosophila larvae model


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enables the study of nanoparticle transport, interaction with cells and cellular uptake along the digestive tract to be conducted in straightforward, time- and cost-effective ways, offering advantages in screening of nanoparticle formulations for oral administration. In conclusion, Drosophila larvae can potentially be developed as a screening platform for orally delivered nanoparticle drug formulations to narrow down a large pool of potential candidates to a smaller pool of leads before validating with established mammalian models.

AUTHOR INFORMATION Corresponding Authors *Emails: [email protected], [email protected], [email protected] Author Contributions #

S. J., C. P. T. and W. C. P. contributed equally to this work and should be considered as co-first authors.

S. J., W. C. P. and C. P. T. designed, planned and executed the study. M. W., K. Y. W. and M.Y. H. contributed to design and data analysis of the study. W. C. P. performed imaging studies. S. J. and C. P. T. performed nanoparticle studies and data analysis. All authors contributed toward preparation of the manuscript. Funding Sources


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The authors acknowledge the financial support from A*STAR Joint Council Office and technical support from the Institute of Materials Research and Engineering, and Bioinformatics Institute. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS The authors acknowledge the support from Institute of Materials Research and Engineering, and Bioinformatics Institute, Agency for Science, Technology and Research (A*STAR).

ASSOCIATED CONTENT Supporting Information Supplementary figures S1 to S5 can be found in the supporting information. This information is available free of charge via the Internet at http://pubs.acs.org/.

REFERENCES: 1. Chen, M. C.; Sonaje, K.; Chen, K. J.; Sung, H. W. A review of the prospects for polymeric nanoparticle platforms in oral insulin delivery. Biomaterials 2011,32, 9826-9838. 2. Hamman, J. H.; Enslin, G. M.; Kotze, A. F. Oral delivery of peptide drugs: barriers and developments. BioDrugs 2005, 19, 165-177. 3. Shi, J.; Votruba, A. R.; Farokhzad, O. C.; Langer, R. Nanotechnology in drug delivery and tissue engineering: From discovery to applications Nano. Lett. 2010, 10, 3223–3230.


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4. des Rieux, A.; Fievez, V.; Garinot, M.; Schneider, Y. J.; Preat, V. Nanoparticles as potential oral delivery systems of proteins and vaccines: a mechanistic approach. J. Control Release 2006, 116, 1-27. 5. Patel, V. F.; Liu, F.; Brown, M. B. Modeling the oral cavity: In vitro and in vivo evaluations of buccal drug delivery systems. J. Control Release 2012, 161, 746-756. 6. Aqil, F.; Munagala, R.; Jeyabalan, J.; Vadhanam, M. V. Bioavailability of phytochemicals and its enhancement by drug delivery systems. Cancer Lett. 2013, 334, 133–141. 7. Apidianakis, Y.; Rahme, L. G. Drosophila melanogaster as a model for human intestinal infection and pathology, Dis. Models. Mech. 2011, 4, 21-30. 8. Pitsouli, C.; Apidianakis, Y.; Perrimon, N.; Homeostasis in infected epithelia: stem cells take the lead. Cell Host Microbe 2009, 6, 301-307. 9. Rubin, D. C. Intestinal morphogenesis. Curr. Opin. Gastroenterol 2007, 23, 111-114. 10. Kuraishi, T.; Binggeli, O.; Opota, O.; Buchon, N.; Lemaitre, B. Genetic evidence for a protective role of the peritrophic matrix against intestinal bacterial infection in Drosophila melanogaster. Proc. Natl. Acad. Sci. U S A. 2011, 108, 15966-15971. 11. Lehane, M. J. Peritrophic matrix structure and function. Ann. Rev. Entomo, 1997, 42, 525520. 12. Lemaitre, B.; Miguel-Aliaga, I. The Digestive Tract of Drosophila melanogaster. Annu. Rev. Genet. 2013. 47, 377-404.


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13. Deepa, P. V.; Akshaya, A. S.; Solomon, F. D. Wonder animal model for genetic studies – Drosophila melanogaster- its life cycle and breeding methods – a review. S. Ramachandra J. Med. 2009, 2, 33-38. 14. Duffy, J. B. GAL4 system in Drosophila: a fly geneticist’s swiss army knife. Genesis 2002, 34, 1–15. 15. Dubreuil, R. R. Copper cells and stomach acid secretion in the Drosophila midgut. Int. J. Biochem. Cell Biol. 2004, 36, 745–752. 16. Sanna, V.; Roggio, A. M.; Siliani, S.; Piccinini, M.; Marceddu, S.; Mariani, A.; Sechi, M. Development of novel cationic chitosan-and anionic alginate–coated poly(d,l-lactide-coglycolide) nanoparticles for controlled release and light protection of resveratrol. Int. J. Nanomedicine 2012, 7, 5501–5516. 17. Liang, G. F.; Zhu, Y. L.; Sun, B.; Hu, F. H.; Tian, T.; Li, S. C.; Xiao, Z. D. PLGA-based gene delivering nanoparticle enhance suppression effect of miRNA in HepG2 cells. Nanoscale Res. Lett. 2011, 6, 447. 18. Gorth, D. J.; Rand, D. M.; Webster, T. J. Silver nanoparticle toxicity in Drosophila: Size does matter. Int. J. Nanomed. 2011, 6, 343–350. 19. Philbrook, N. A.; Walker, V. K.; Afrooz, A. R. M. N.; Saleh, N. B.; Winn, L. M. Investigating the effects of functionalized carbon nanotubes on reproduction and development in Drosophila melanogaster and CD-1 mice. Reprod. Toxicol. 2011, 32, 442–448.


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20. Liu, X.; Vinson, D.; Abt, D.; Hurt, R. H.; Rand, D. M. Differential toxicity of carbon nanomaterials in Drosophila: larval dietary uptake is benign, but adult exposure causes locomotor impairment and mortality. Environ. Sci. Technol. 2009, 43, 6357-6363. 21. Chaudhury A.; Das S. Recent advancement of chitosan-based nanoparticles for oral controlled delivery of insulin and other therapeutic agents. AAPS PharmSciTech. 2011, 12, 10-20. 22. Shanbhag, S.;Tripathi, S. Epithelial ultrastructure and cellular mechanisms of acid and base transport in the Drosophila midgut. J. Exp. Biol. 2009,212, 1731-1744. 23. Tellam, R. L.; Wijffels, G.; Willadsen, P. Peritrophic matrix proteins, Insect Biochem. Mol. Biol. 1999, 29, 87-101. 24. Hegedus, D.; Erlandson, M.; Gillott, C.; Toprak, U. New Insights into peritrophic matrix synthesis, architecture, and function. Annu. Rev. Entomol. 2009, 54, 285-302. 25. Park, J. H.; Kwon, J. Y. Heterogeneous expression of Drosophila gustatory receptors in enteroendocrine cells. PLoS ONE 2011, 6, e29022. doi: 10.1371/journal.pone.0029022. 26. Wang, M.; Zhang, Y.; Feng, J.; Gu, T.; Dong, Q.; Yang, X.; Sun, Y.; Wu, Y.; Chen, Y.; Kong, W. Preparation, characterization, and in vitro and in vivo investigation of chitosan-coated poly (d,l-lactide-co-glycolide) nanoparticles for intestinal delivery of exendin-4. Int. J. Nanomedicine 2013, 8, 1141-1154.


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27. Murugeshu, A.; Astete, C.; Leonardi, C.; Morgan, T.; Sabliov, C. M. Chitosan/PLGA particles for controlled release of α-tocopherol in the GI tract via oral administration. Nanomedicine (Lond) 2011, 6, 1513-1528. 28. Pandey, U. B.; Nichols, C. D. Human disease models in Drosophila melanogaster and the role of the fly in therapeutic drug discovery. Pharmaco. Rev. 2011, 63, 411-436.


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A

B

C

D

BODIPY-PLGA nanoparticles

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Chitosan-BODIPY-PLGA nanoparticles

Figure 1. SEM images of (A) BODIPY-PLGA nanoparticles and (B) chitosan-BODIPY-PLGA nanoparticles. Insets are confocal fluorescent microscopic images of the nanoparticles. Schematic of BODIPY-PLGA nanoparticles are shown in (C) and chitosan-BODIPY-PLGA nanoparticles in (D).


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A

Anterior/Middle midgut

S

Feeding

S

0.5 h

B

E

C

S

4h

Hindgut

Posterior midgut

0.5 h

0h BODIPYPLGA NPs

E

E

S

D

14 h

4h

8h

E

S

E

E

14 h

Anterior midgut Middle midgut

Posterior midgut

Figure 2. (A) Schematic of BODIPY-PLGA nanoparticles during transition though the posterior midgut. (B-E) Time-lapse images of live larva fed with BODIPY-PLGA nanoparticles and the posterior midgut was labelled with green GFP. The images taken at (B) 0.5 h, (C) 4h, (D) 8 h, and (E) 14 h post-feeding to monitor the transport of BODIPY-PLGA nanoparticles in posterior midgut. ‘S’ indicates the start of posterior midgut and ‘E’ indicates the end of posterior midgut. The nanoparticles (NPs) are in magenta color indicated with white arrows. Scale bar is 200 µm.


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A

Feeding

Anterior/Middle midgut

0h ChitosanBODIPYPLGA NPs

S

B

0.5 h

Posterior midgut

E 6.5 h

S

0.5 h

E

S

S

E

C

6.5 h

Hindgut

11.5 h

E

S

D

11.5 h

17.5h

S

E

E

17.5 h

E

E

E E

E S

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S

S

S

Retention segment of posterior midgut

Figure 3. (A) Schematic of chitosan-BODIPY-PLGA nanoparticles transport during transit though the posterior midgut. (B-E) Time-lapse images of live larva fed with chitosan-BODIPYPLGA nanoparticles and the posterior midgut was labelled with green GFP. The images were taken at (B) 0.5 h, (C) 6.5 h, (D) 11.5 h and (E) 17.5 h post-feeding to monitor the transport of chitosan-BODIPY-PLGA nanoparticles in posterior midgut. ‘S’ indicates the start of posterior midgut and ‘E’ indicates the end of posterior midgut. Yellow double arrowed line in (C) indicates the retention segment of posterior midgut. The nanoparticles (NPs) are in magenta color indicated with white arrows. Scale bar is 200 µm.


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Figure 4. Fluorescent microscopic images of posterior midgut cells of larvae fed with BODIPYPLGA nanoparticles and chitosan-BODIPY-PLGA nanoparticles for 1 day. PLGA nanoparticles were not seen in the GFP labelled posterior midgut while chitosan-coated PLGA nanoparticles (magenta) were successfully uptaken into the retention segment of posterior midgut cells. The scale bar is 20 µm.


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A Near neutral

Acidic

Near neutral

Alkaline

Anterior segment Anterior midgut

B

Middle midgut

Posterior segment

Posterior midgut

Hindgut

Retention segment

Figure 5. (A) Schematic and fluorescent microscopic image of dissected digestive tract. The schematic shows the transit of chitosan-BODIPY-PLGA nanoparticles and their interactions with the cells under different pH conditions along the midgut. (B) Confocal image shows the dissected digestive tract, the white box indicates the retention segment of the posterior midgut.


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For Table of Contents Use Only

Oral Administration and Selective Uptake of Polymeric Nanoparticles in Drosophila Larvae as In Vivo Model Shan Jiang, Choon Peng Teng, Wee Choo Puah, Martin Wasser, Khin Yin Win, and Ming-Yong Han


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Oral Administration and Selective Uptake of Polymeric Nanoparticles

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