Yeast Microcapsule-Mediated Targeted Delivery of Diverse

Jan 11, 2017 - *E-mail: [email protected]., *E-mail: [email protected]., *E-mail: [email protected]. [email protected]. ... tissues, and finall...
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Yeast Microcapsule-Mediated Targeted Delivery of Diverse Nanoparticles for Imaging and Therapy via the Oral Route Xing Zhou,†,‡ Xiangjun Zhang,†,§ Songling Han,† Yin Dou,†,‡ Mengyu Liu,†,‡ Lin Zhang,∥ Jiawei Guo,† Qing Shi,† Genghao Gong,† Ruibing Wang,§ Jiang Hu,*,⊥ Xiaohui Li,*,‡ and Jianxiang Zhang*,†,‡ †

Department of Pharmaceutics, College of Pharmacy, Third Military Medical University, Chongqing 400038, China Institute of Materia Medica, College of Pharmacy, Third Military Medical University, Chongqing 400038, China § State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Taipa, Macau, China ∥ Department of Radiation, Southwest Hospital, Third Military Medical University, Chongqing 400038, China ⊥ Department of Biologic and Materials Sciences, University of Michigan, Ann Arbor, Michigan 48109, United States ‡

S Supporting Information *

ABSTRACT: Targeting of nanoparticles to distant diseased sites after oral delivery remains highly challenging due to the existence of many biological barriers in the gastrointestinal tract. Here we report targeted oral delivery of diverse nanoparticles in multiple disease models, via a “Trojan horse” strategy based on a bioinspired yeast capsule (YC). Diverse charged nanoprobes including quantum dots (QDs), iron oxide nanoparticles (IONPs), and assembled organic fluorescent nanoparticles can be effectively loaded into YC through electrostatic force-driven spontaneous deposition, resulting in different diagnostic YC assemblies. Also, different positive nanotherapies containing an anti-inflammatory drug indomethacin (IND) or an antitumor drug paclitaxel (PTX) are efficiently packaged into YC. YCs containing either nanoprobes or nanotherapies may be rapidly endocytosed by macrophages and maintained in cells for a relatively long period of time. Post oral administration, nanoparticles packaged in YC are first transcytosed by M cells and sequentially endocytosed by macrophages, then transported to neighboring lymphoid tissues, and finally delivered to remote diseased sites of inflammation or tumor in mice or rats, all through the natural route of macrophage activation, recruitment, and deployment. For the examined acute inflammation model, the targeting efficiency of YC-delivered QDs or IONPs is even higher than that of control nanoprobes administered at the same dose via intravenous injection. Assembled IND or PTX nanotherapies orally delivered via YCs exhibit remarkably potentiated efficacies as compared to nanotherapies alone in animal models of inflammation and tumor, which is consistent with the targeting effect and enhanced accumulation of drug molecules at diseased sites. Consequently, through the intricate transportation route, nanoprobes or nanotherapies enveloped in YC can be preferentially delivered to desired targets, affording remarkably improved efficacies for the treatment of multiple diseases associated with inflammation. KEYWORDS: Yeast, microcapsule, nanoparticle, oral targeting, drug delivery, imaging

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internalization and translocation into the gut-associated lymphoid tissues.11,12 Thereafter, they are transported to the circulation and diseased sites via the lymphatic system in a variety of animal models.12,13 Utilizing this specific invasion route, bacteria have been exploited as therapeutics or delivery vehicles for targeted cancer therapy and anti-inflammation treatment of sepsis.13−17 Furthermore, nucleic acids and proteins were orally delivered by bacteria or yeast shells for immunization or treatment of infectious diseases, tumors, and

anoparticles have been widely employed as probes or drug delivery vehicles for diagnosis and therapy of many severe diseases,1−4 and their targeted delivery via patientfriendly and cost-effective oral administration is the holey grail of nanomedicine. While current strategies based on delicate control over the biophysicochemical properties of nanoparticles themselves can improve oral bioavailability of some therapeutics to a certain degree,5−10 oral delivery of a sufficient amount of nanoparticles and their payloads to remote diseased sites remains highly challenging. By contrast, microorganisms such as bacteria and fungi can be recognized by the apical membrane receptors expressed on M cells overlying the lymphoid follicles of intestinal Peyer’s patches, and subsequently undergo rapid © 2017 American Chemical Society

Received: October 29, 2016 Revised: January 4, 2017 Published: January 11, 2017 1056

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Figure 1. Schematic diagram of a yeast capsule (YC)-mediated oral targeting of nanoparticles to diseased sites of inflammation-associated diseases distant from the gastrointestinal tract. (a) Translocation of nanoparticle-loaded YCs to diseased sites. (b) Preparation of nanoparticle-loaded YCs by an electrostatic force-mediated spontaneous self-deposition approach.

gene deficiencies.18−21 Notably, oral delivery of small interfering RNA via a yeast capsule (YC) effectively suppressed systemic inflammation by attenuating pro-inflammatory activities of macrophages.20 We therefore hypothesize that the YC, which is mainly composed of β-glucan that can be recognized by Dectin-1 on M cells,22 can “mail” diverse nanoparticles precisely and safely to remote inflammatory sites via intricate macrophage-mediated transportation, thereby negotiating many biological barriers currently preventing the effective oral delivery of most nanomedicines invented so far (Figure 1a). However, to achieve this goal two crucial issues need to be addressed. First, although nucleic acids can be efficiently entrapped into YC by an in situ layer-by-layer approach,20,23 this method cannot be easily converted to efficiently deliver payloads in distinct forms,24,25 limiting its versatility and potential in clinical translation. Second, although YC delivery has been demonstrated effective in systemically suppressing inflammation, the need remains to clarify whether or not the orally administered laden YC can be properly delivered to the remote diseased sites and exert local detective and/or therapeutic functions. To address these challenges, we developed a facile while highly robust approach, based on electrostatic force-driven selfdeposition, to package nanoparticulate cargoes with different physiochemical properties into empty YCs (Figure 1b). YC was prepared according to the previously reported method with minor modification,23 giving rise to microcapsules with a mean size of 4.7 μm in diameter and zeta-potential of −6.5 mV (Figure S1a,b). Transmission electron microscopy (TEM) observation indicated a dramatical loss of cytoplasmic

components in obtained YC products as compared to intact yeasts (Figure 2a). Fluorescence imaging of YCs stained with calcofluor-white (a dye selectively binding to the yeast wall) revealed a typical capsular structure, which was cross-examined and confirmed by FITC-labeling and imaging. These results also demonstrated the integrity structure of YCs, in line with observation by scanning electron microscopy (SEM, Figure S1c). A positively charged quantum dot with maximal emission at ∼620 nm (QD620, with a hydrodynamic size of 20 nm and zeta-potential > 50 mV) was efficiently packaged into YC over 1 h of incubation at pH 9.2 (Figure S1d). The total loading content increased with enhanced feeding or prolonged incubation time (Figure S1e,f). TEM visualization further confirmed the existence of QD620 in final YC assemblies (Figure 2b). Real-time monitoring on zeta-potential changes during the loading process showed an abrupt increase upon addition of QD620 into the YC suspension (Figure 2c), suggesting the instant absorption of positive QD620 onto YC surface. After ∼700 s, zeta-potential gradually decreased to the basal level, reflecting the diffusion of QD620 inside YC from periphery to interior. After complete loading, fluorescence imaging showed bright red fluorescence representative of QD620 inside YC (Figure 2d). While positive QD620 was efficiently packaged into YC, negatively charged or PEGylated QDs failed using the same protocol (Figure S1g,h). Thus, a modification was reinforced, wherein YC was preincubated with a cationic polymer of polyethylenimine (PEI) before the loading of negative QD620, leading to its effective packaging (Figure S1i). To expand the spectrum and utility, a variety of positive QDs with different emission wavelengths (such as 1057

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Figure 2. Preparation and characterization of nanoparticle-loaded yeast capsules (YCs). (a) TEM images of intact yeast (top left) and YCs (top right) prepared by serial alkaline and solvent extractions; fluorescence staining of YCs with calcofluor white (bottom left, blue) or FITC (bottom right, green) showing the typical capsular structure. (b) TEM images of QD620-loaded YC (QD620/YC). (c) Zeta-potential changes over the time of QD620 loading. (d,e) Fluorescence images of YCs loaded with QD620 (d) and other quantum dots (e) including QD540, QD525, or QD450. (f) TEM images of Cy7.5-labeled nanoparticles (Cy7.5 NP) and Cy7.5 NP-loaded YC (Cy7.5 NP/YC). (g) TEM images of indomethacin (IND)containing nanoparticles (IND NP) and IND NP-loaded YC (IND NP/YC). (h) Fluorescence images of IND NP/YC illuminated with IND autofluorescence (green).

QD540, QD525, and QD450) were separately or simultaneously incorporated into YC, producing multicolored fluorescent YC assemblies when combined (Figure 2e and Figure S1j). Following a similar procedure, a positive iron oxide nanoparticle (IONP) (diameter, 15 nm; zeta-potential, 53 mV) was successfully loaded, yielding a magnetic YC assembly (Figure S2a−c). Magnetic resonance imaging (MRI) showed that T2 relaxivity was notably improved when IONP was packaged inside YC (Figure S2d,e), likely due to the formation

of IONP clusters in the confined YC space, which can induce a magnetic relaxation switching effect.26 Besides inorganic nanoprobes, an organic near-infrared (NIR) fluorescent nanoparticle Cy7.5 NP (diameter, 260 nm; zeta-potential, 25.7 mV) assembled from Cy7.5-conjugated PEI, was efficiently packaged into YC as well (Figure 2f and Figure S3a). To package therapeutic nanoparticles into YC, positive nanomedicines with ultrahigh loading contents were first fabricated. According to our previous studies, PEI may interact 1058

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Figure 3. Gastrointestinal transportation and translocation of YC after oral administration in mice. (a,b) Fluorescent stereomicroscopy images illustrating transportation of QD620 in the ileum (a) and localization of QD620 in MLN (b). The black arrows in the image a indicate lymphatic vessels. A typical Peyer’s patch is indicated by the white arrow, while the white circles show diffuse lymphatic tissues. The white asterisks in the image b show lymphatic sinuses. (c) Co-localization of QD620 (red) and macrophage marker CD68 (green) in MLN cryosections. QD620/YC was locally injected in the intestinal lumen at a dose of 3 pmol QD620 in each BALB/c mouse. At 1 h after injection, different tissues were collected for examination. (d,e) Representative in vivo images (d) and quantitative analysis (e) showing the roles of Dectin-1 in transportation and translocation of QD620/YC. The white arrow heads indicate inflamed paws. Acute paw inflammation in mice was induced by i.d. injection of carrageen. After 12 h, QD620/YC was orally administered at 3.0 nmol/kg of QD620 with or without pretreatment with laminarin at 150 mg/kg 1 h before QD620/YC administration. The same dose of QD620/YC was administered in normal mice. At 24 h post treatment, real-time fluorescence imaging was conducted and quantitatively analyzed. Scale bars, 1 cm for the images a,b, and 10 μm for the image c. Error bars, mean ± SD (n = 4) of independent experiments. *P < 0.05, **P < 0.01.

clinically used for cancer chemotherapy) were formulated (Figure S3c−e). In this case, IND/PEI nanoassemblies functioned as nanovehicles for PTX, leading to a PTX nanotherapy of IND-PTX NP. After incubation of IND NP with YC suspension, TEM observation revealed densely loaded YC assemblies with mean size of 4.0 μm (Figure 2g and Figure S3f), which was further validated by fluorescence microscopy of autofluorescence from IND molecules (Figure 2h). Quantitative analysis revealed an IND loading content as high as of 20.6 wt %. Likewise, assembled IND-PTX NP was efficiently packaged into YC (Figure S4a,b) with a drug loading content

with carboxyl-containing guest molecules to afford nanoparticles by a one-pot self-assembly process mediated via multiple noncovalent forces. These nanoparticles may serve as efficient vehicles for loading and delivery of other carboxyldeficient hydrophobic drugs.27,28 Nanoparticles containing indomethacin (IND, which has a carboxyl group), a first-line anti-inflammatory drug, were first created by coassembly with PEI, resulting in spherical IND-loaded nanoparticles (IND NP) with an average diameter of 98 nm and zeta-potential of 60.7 mV (Figure 2g and Figure S3b). Through similar procedures, positive nanoparticles containing paclitaxel (PTX, a drug 1059

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Observation by microscopy suggested that lymphatic sinuses of MLNs exhibited strong fluorescence intensities with a disseminated distribution pattern reflecting the primary lymphoid follicle structures (Figure 3b). Immunofluorescence imaging validated the existence of QD620 in isolated CD68+ macrophages (Figure 3c). Flow cytometric analysis measured that 28.4 ± 8.1% isolated cell population was QD620+ in which 95.7 ± 4.0% cells were F4/80+ macrophages (Figure S10e). We also observed the in situ distribution of QD620 in glycoprotein 2 (GP-2)-positive M cells in the villi and subepithelial domes of Payer’s patches, and in CD68+ macrophages in the lamina propria (Figure S11). When pretreated with laminarin, QD620/YC was largely retained in the lumen with much less accumulation at Peyer’s patches (Figure S12). These observations disclosed that QD620/YC was mainly absorbed at Payer’s patches, ferried by M cells, subsequently endocytosed by macrophages, and finally carried to MLNs and other lymphatic tissues. The transportation of YC was then examined in animals under an acute inflammatory condition, induced by intradermal (i.d.) injection of carrageen in the mouse unilateral hind paws. At 8 h after oral gavage (i.g.) of QD620/YC, the inflamed paws displayed significantly stronger fluorescence signals as compared to the noninflamed contralateral paws (Figure 3d) with comparable fluorescence strength observed in collateral paws of noninflamed control mice. Oral dosing of laminarin prior to i.g. QD620/YC dramatically reduced fluorescence intensities in both inflamed and noninflamed paws (Figure 3e). Notably, QD620/YC-treated inflamed paws exhibited remarkably higher fluorescence signals as compared to those pretreated with laminarin. In line with this observation, we detected reduced absorption of YC at Peyer’s patches and MLNs, as well as decreased distribution in the spleen (Figure S13a−d) but not in the liver and kidneys (Figure S13c,e,f). Moreover, laminarin treatment significantly lowered the QD620 internalization into monocyte/macrophage populations in circulation or residing in the spleen, liver, and inflamed paws (Figure S14) but had no detectable impact on blood erythrocytes and lymphocytes. Splenic monocytes/macrophages have been reported to be exclusively involved in the regulation of inflammation,32 and we observed the inhibitory effect of laminarin treatment on splenic tropism, thus we further interrogated the role of spleen in transportation of absorbed YC. Splenectomized mice exhibited very low fluorescence signals in both inflamed and noninflamed paws with significantly weaker fluorescence signals detected in the inflamed paws as compared to those of sham-operated animals (Figure S15). Collectively, these observations implied that QD620/YC was first transported to the lymphoid tissues after being transcytosed by M cells and endocytosed by resident and spleen-derived monocytes/macrophages then ferried to inflammatory sites through the deployment of macrophages. However, in healthy mice recruitment of macrophages to specific tissue destinations was considerably suppressed due to homeostasis,33,34 resulting in a low translocation rate of i.g. QD620/YC and improved safety. On the basis of the above findings, we then explored the inflamed site-targeting capability and applications of diverse nanoprobe/YC assemblies. After induction of acute inflammation in the right-hind paw of mice by i.d. injection of carrageen, either unpackaged QD620 or QD620/YC was orally administered and PEGylated QD620 (QD620-PEG) was intravenously (iv) injected as a conventional administration route control. In vivo imaging showed gradually enhanced

of 7.5%. In vitro release tests demonstrated that both IND and PTX could be released from loaded YCs in simulated gastric and intestinal fluids (Figure S5). In both cases, the drug release rate in the simulated intestinal fluid was considerably higher than that in the simulated gastric fluid. This may be because IND NP and IND-PTX NP were more stable in acidic solutions as compared to buffers with high pH values, resulting from notably increased solubility of IND at pH higher than its pKa of ∼4.5.10 Nevertheless, it should be noted that drug molecules should be more slowly released in the gastrointestinal tract, due to the absence of large volume of water under in vivo conditions. Collectively, these data showed that charged nanoparticles including nanoprobes and nanotherapies can be directly and efficiently packaged into YC by electrostatic force-driven self-deposition. Using size standards of FITClabeled and charged polystyrene latex beads, we demonstrated that particles with a diameter up to ∼750 nm could be successfully loaded into YC (Figure S4c). To examine the cellular uptake of nanoparticle-loaded YCs, both macrophage cell line and primary culture were tested, which can recognize β-1,3-glucan via the membrane phagocytic pattern-recognition receptor Dectin-1.29,30 Incubation of QD620/YC with murine macrophage cell line RAW264.7 resulted in a time-dependent internalization of QD620/YC (Figure S6a,b), through an endolysosomal endocytosis and trafficking pathway (Figure S6c). The presence of laminarin (a soluble type of β-1,3-glucan that can also be recognized by Dectin-1) significantly inhibited QD620/YC uptake by either RAW264.7 cells or primary peritoneal macrophages collected from BALB/c mice (Figure S7), suggesting that YC internalization by macrophages was mainly attributed to Dectin-1 recognition. The internalized QD620/YC was found to retain inside the cells over 3 days of observation (Figure S6d), corroborating a previous finding that thus engulfed β-glucan particles can remain intact in macrophages for 3−5 days.31 Likewise, YC loaded with nanotherapy IND-PTX NP could be effectively endocytosed by macrophages and present for a prolonged time period (Figure S6e). In addition, YC internalization itself had no detectable influence on sequential endocytosis of other particles like FITC-labeled polystyrene beads (500 nm in diameter) by RAW264.7 cells (Figure S8a,b) and it did not affect the migration rate of cells either (Figure S8c−e). These observations are critical for the following in vivo studies, as the cargoes are intended to be uptaken and delivered by macrophages without any damage or loss to their major physiological functions. To establish the oral administration route, we first validated the stability of assemblies using inorganic nanoprobe-loaded YCs subject to a strong acidic environment. QD620- and IONP-loaded YCs were stable in a simulated gastric fluid at pH 1.2, largely due to the protection by their own polymer coating as well as the enveloping YC walls (Figure S9). Then the transportation and translocation of YC in intestinal tissues were studied. QD620/YC was locally injected into the mouse intestinal lumen. Ex vivo imaging revealed an initial fluorescence signal distribution in the intestinal epithelium and mesenteric lymph nodes (MLNs) at 1 h after injection (Figure S10a), followed by a diffusion phase with gradually distributed fluorescence into the mesenteric lymph duct and Peyer’s patches (Movie S1). QD620/YC was finally found to be mainly present in Peyer’s patches and diffuse lymphoid tissues of the ileum (Figures 3a and S10b−d) with lymphatic vessels close to blood vessels displaying the brightest fluorescence. 1060

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Figure 4. Inflammation and tumor targeting by orally delivered nanoprobes packaged in YCs. (a,b) Representative in vivo images at 24 h (a) and quantitative analysis (b) illustrating the accumulation of QD620 in inflamed paws of mice after oral administration of QD620/YC at 1.5, 3.0, or 6.0 nmol/kg of QD620. Control QD620 or QD620-PEG was administered by i.g. or iv injection at 3.0 nmol/kg of QD620, respectively. (c) Fluorescence images of tissue sections indicating the distribution of QD620 (red) in inflamed paws. (d) Immunofluorescence images showing internalization of QD620 (red) in macrophages (green) isolated from inflamed paws. For images c,d, paws were from mice treated with QD620/YC at 3.0 nmol/kg of QD620. (e) Flow cytometric quantification of QD620+ cells isolated from inflamed paws and collateral controls after oral administration of QD620/YC at 1.5 nmol/kg of QD620 daily for 3 days. (f,g) Representative real-time fluorescence images (f) and quantitative analysis (g) indicating the biodistribution of Cy7.5 NP in nude mice bearing MCF-7 xenografts after oral administration of Cy7.5 NP or Cy7.5 NP/ YC at 5.0 mg/kg of Cy7.5. (h) Representative images (top) and quantification (bottom) of Cy7.5 accumulated in tumors excised from mice 48 h after one i.g. treatment with saline, Cy7.5 NP, or Cy7.5 NP/YC. (i) Immunofluorescence images of tumor sections showing the colocalization of QD620 (red) with macrophage marker CD68 (green). QD620/YC was orally administered daily to MCF-7 xenografts-bearing mice at 1.5 nmol/kg of QD620 for three sequential days, and then tumors were excised for further analysis. Scale bars, 10 μm for the image c, 5 μm for the images d and i. Error bars, mean ± SD (n = 4) of independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

fluorescent signals in the inflamed right-hind paws of all the mice treated with QD620 in different formulations (Figures S16 and 4a). QD620-PEG exhibited a maximal accumulation at 8 h post iv injection, while fluorescence signals continued to increase for all orally delivered groups (Figure 4b). Of note, QD620/YC treatment displayed significantly higher intensity than that of unpackaged QD620 treatment. Surprisingly, i.g. QD620/YC at 3.0 nmol/kg showed stronger fluorescence signals than iv QD620-PEG at the same dosage after 24 h. Ex vivo imaging revealed tropism of QD620 in the Peyer’s patch, inguinal lymph node (ILN), MLN, liver, and spleen of mice treated with QD620/YC at 3 and 6 nmol/kg (Figure S17a,b). Notably, unpackaged QD620 afforded the strongest fluorescence in the kidneys (Figure S17c,d), implying active renal metabolism and/or excretion of smaller nanoparticles.35 The rapid renal filtration and urinary excretion may also explain the observed low signals of QD620-PEG in both the liver and spleen.36 Fluorescence imaging of tissue cryosections by microscopy affirmed the existence of QD620 in the inflamed paws of mice treated with QD620/YC (Figure 4c) and the presence of QD620 in CD68+ macrophages in particular (Figure 4d). Moreover, the inflamed paws had much more QD620+ cell counts than the normal paws (8.7 ± 3.9% versus 1.7 ± 0.3%) of which 86.5 ± 8.7% were F4/80+ macrophages (Figures S17e and 4e). Likewise, the majority of QD620+ cells

were contributed from F4/80+ monocyte/macrophage populations in whole blood, liver, and spleen of mice treated with QD620/YC (Figure S18). These results altogether demonstrated that QD620/YC assemblies can effectively target peripheral inflamed sites following oral administration, carried by macrophages destined to the diseased inflammatory tissues. To expand the spectrum of applications, we further explored the potential of IONP/YC assemblies in MRI observation of inflamed tissues. After oral administration of IONP/YC in rats with carrageen-induced acute hind paw inflammation, T2weighted images displayed a dose-dependent T2 hypointensity in the inflamed paws (Figure S19). It is worth noting that i.g. IONP/YC at 0.5 mg/kg of Fe showed significantly higher relaxivity values as compared to iv IONP-PEG at the same dose, while ig unloaded IONP showed no difference from saline control. Both the cluster formation of IONP packed in YC cavity and the enhanced targeting mediated by macrophage deployment may explain the observed enhanced effectiveness of i.g. IONP/YC. Taken together, YC can be harnessed to effectively package different nanoprobes for diverse applications, which were ultimately delivered and targeted to remote inflammatory sites through an oral and macrophage-mediated route. As well documented, safety is one of the major concerns limiting clinical translation of contrast agents based on nanomaterials that are generally administered by iv injec1061

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Figure 5. Targeted therapy of acute and chronic inflammatory diseases as well as tumor by macrophage-mediated oral delivery of nanotherapies. (a) IND levels in inflamed paws and collateral controls of rats induced by i.d. injection of carrageen and followed by treatment with oral gavage of free IND, IND NP, or IND NP/YC at 10 mg/kg of IND over 24 h. Drug concentrations of free IND and IND NP groups were determined at 24 h. (b,c) Therapeutic efficacy of orally administered IND NP/YC in acute (b) and chronic (c) inflammation models in rats. (d,e) Therapeutic effects of a PTX nanotherapy UDCA-PTX NP delivered by YC in mice bearing B16F10 melanoma tumors. (d) Changes in the relative tumor volume during 15 days of treatment. (e) Relative tumor weights at day 15. Tumor weight at the end point was normalized to that of the control group. (f) PTX concentrations in excised tumor tissues. (g−i) Efficacy of antitumor PTX nanotherapies delivered by YC in mice bearing MCF-7 xenografts. (g) Changes in the relative tumor volume. (h) Gross appearance of excised tumors. (i) Relative tumor weight normalized to the control group. (j) PTX concentrations in excised tumor tissues. Error bars, mean ± SD (a,b,g, n = 5; c−f, n = 6; i,j, n = 4 for the PTX group, and n = 5 for other groups). *P < 0.05, **P < 0.01, ***P < 0.001.

afforded significantly higher fluorescence intensity than that of control unpackaged Cy7.5 NP (Figure 4g). Ex vivo imaging of excised tumor tissues at 48 h exhibited fluorescence intensity in the Cy7.5 NP/YC group twice as that in the Cy7.5 NP control group (Figure 4h). Closer examination on cellular distribution at the tumor sites using QD620/YC as the indicator verified the presence of QD620 in CD68+ macrophages (Figure 4i). Flow cytometric analysis revealed that 11.6 ± 4.7% cells isolated from tumors were QD620+, in which 81.1 ± 27.9% cells were F4/80+ macrophages (Figure S20). Similarly, we found an enhanced accumulation of Cy7.5 NP in B16F10 melanoma when the nanoprobe was packaged in YC (Figure S21). Moreover, tumor targeting by i.g. nanoprobe/YC was substantiated by MRI observation in Sprague−Dawley rats bearing Walker 256 carcinoma. T2-weighted images showed remarkably increased T2 hypointensity at 2 h after oral administration of IONP/YC at 0.5 mg/kg of Fe (Figure S22a). Of note, at the same dosage,

tion.37,38 These nanomedicines must be subjected to strict regulations for clinical trials, because they may be directly exposed to a large number of cellular and molecular components of blood and therefore affect different organs and tissues after iv administration. The relative poor patient compliance is another shortcoming for iv injected contrast agents, particularly in specific patient populations. By contrast, orally delivered nanoprobes via YCs are largely transported to the diseased sites by monocytes/macrophages-mediated translocation, which is a more safe and patient-friendly strategy. Macrophages have been reported to be exclusively involved in tumor progression.37,39−41 We thus interrogated tumor targeting capability of nanoprobe/YC assemblies. After 2 h of oral gavage of Cy7.5 NP/YC to mice bearing MCF-7 human breast cancer xenografts, real-time imaging showed strong fluorescence signals in tumor tissues over the period of observation (Figure 4f). At the same dosage, Cy7.5 NP/YC 1062

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T2-star relaxation enhancement at 4 h through YC delivery was comparable to that of iv IONP-PEG (Figure S22b). Collectively, nanoprobes packaged in and orally delivered by YC demonstrated their potential in targeting tumoral sites for noninvasive diagnosis using either fluorescence imaging or MRI. To exploit its therapeutic potential, nanotherapy-loaded YC was first examined in a rat acute paw inflammation model. After 2 h of oral administration, the inflamed paws of rats treated with IND NP/YC had significantly higher levels of IND as compared to those of noninflamed paws (Figure 5a). The IND NP/YC group also had a significantly higher IND content in inflamed paws compared with those of IND NP or free IND treated groups. Of note, no differences were observed between inflamed paws and noninflamed paws for animals from both IND NP and free IND treated groups. After 24 h, higher drug levels were observed in macrophages isolated from MLN, liver, spleen, peripheral blood, and inflamed paws from the IND NP/ YC group, compared with that from the IND NP or free IND group (Figure S23a). Particularly, in the IND NP/YC-treated group the observed difference of IND levels between inflamed and noninflamed paws was paralleled with the increased IND content measured in splenic macrophages (Figure S23b). These data demonstrated that nanotherapies were preferentially delivered to inflamed sites when they were packaged in YC. Because of this superior capability of site-specific drug distribution, IND NP/YC treatment inhibited paw edema to a much greater extent than that of unpackaged IND NP (Figure 5b). Moreover, IND NP/YC showed much better therapeutic performance in a chronic inflammation model in rats induced by complete Freund’s adjuvant (Figure 5c). Nevertheless, it should be noted that no significant differences between IND NP and IND NP/YC were oberved in the chronic inflammation model, which might be largely attributed to premature release of IND before the delivery system reached the inflamed site in this case. Finally, we examined the therapeutic potential of nanotherapy/YC assemblies in cancer therapy. To this end, PTX nanotherapies including IND-PTX NP and UDCA-PTX NP were first fabricated by self-assembly as aforementioned. As for UDCA-PTX NP, it was assembled by PTX/PEI with ursodeoxycholic acid (UDCA), a natural compound of bile acids.27 Whereas both iv administered PTX and i.g. delivered UDCA-PTX NP inhibited tumor growth to a certain degree in mice bearing B16F10 melanoma tumors, orally administered UDCA-PTX NP/YC afforded more significant efficacy (Figure 5d), resulting in notably reduced tumor mass at day 15 (Figure 5e). A significantly higher level of PTX was detected in tumors collected from mice treated with UDCA-PTX NP/YC, as compared to those from free PTX or UDCA-PTX NP treated groups (Figure 5f). Similar therapeutic efficacy was observed in mice bearing MCF-7 breast cancer xenografts (Figure 5g−j), when IND-PTX NP or UDCA-PTX NP was packaged into YCs. In summary, we have demonstrated the yeast-derived bioinspired microcapsule can act as an effective carrier for oral delivery of various nanoprobes and nanotherapies targeting remote diseased sites. The capability to precisely and conveniently deliver nanoparticles through oral administration opens a new avenue for diagnosis and treatment of many diseases including arthritis, diabetes, cancer, and cardiovascular disease, wherein macrophages are intimately involved and play an important role in disease progression.

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.6b04523. The detailed experimental section and supplementary results including Figure S1 to Figure S23 (PDF) Gradually distributed fluorescence into the mesenteric lymph duct and Peyer’s patches (AVI)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. [email protected]. ORCID

Ruibing Wang: 0000-0001-9489-4241 Jianxiang Zhang: 0000-0002-0984-2947 Author Contributions

X.Z., X.J.Z., and S.L.H. contributed equally to this work. Author Contributions

J.X.Z. conceived the project, and J.X.Z., X.Z., X.J.Z., S.L.H., Y.D., L.Z., and J.H. designed the experiments. X.Z., X.J.Z., S.L.H., J.X.Z., Y.D., M.Y.L., Q.S., J.W.G., L.Z., and G.H.G. performed all the experiments. J.X.Z., X.Z., X.J.Z., J.H., X.H.L., and R.B.W. analyzed the data and composed the manuscript. All authors discussed the results and reviewed the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the National Natural Science Foundation of China (No. 81471774), the Research Foundation of Third Military Medical University (No. 2014XJY04), and the Program for New Century Excellent Talents in University (No. NCET-13-0703).

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REFERENCES

(1) Veiseh, O.; Tang, B. C.; Whitehead, K. A.; Anderson, D. G.; Langer, R. Nat. Rev. Drug Discovery 2014, 14, 45. (2) Blanco, E.; Shen, H.; Ferrari, M. Nat. Biotechnol. 2015, 33, 941. (3) Cheng, Z. L.; Al Zaki, A.; Hui, J. Z.; Muzykantov, V. R.; Tsourkas, A. Science 2012, 338, 903. (4) Yang, X.; Yang, M. X.; Pang, B.; Vara, M.; Xia, Y. N. Chem. Rev. 2015, 115, 10410. (5) Pridgen, E. M.; Alexis, F.; Kuo, T. T.; Levy-Nissenbaum, E.; Karnik, R.; Blumberg, R. S.; Langer, R.; Farokhzad, O. C. Sci. Transl. Med. 2013, 5, 213ra167. (6) Bakhru, S. H.; Furtado, S.; Morello, A. P.; Mathiowitz, E. Adv. Drug Delivery Rev. 2013, 65, 811. (7) Roger, E.; Lagarce, F.; Garcion, E.; Benoit, J. P. Nanomedicine 2010, 5, 287. (8) Jia, Y.; Tang, Y.; He, H. M.; Li, S. H.; Che, L.; Zhou, X.; Dou, Y.; Zhang, J. X.; Li, X. H. Nanomedicine 2013, 9, 408. (9) Zhu, Y. X.; Che, L.; He, H. M.; Jia, Y.; Zhang, J. X.; Li, X. H. J. Controlled Release 2011, 152, 317. (10) Zhou, X.; Zhao, Y.; Chen, S. Y.; Han, S. L.; Xu, X. Q.; Guo, J. W.; Liu, M. Y.; Che, L.; Li, X. H.; Zhang, J. X. Biomacromolecules 2016, 17, 2540. (11) Prescott, R. J.; Harris, M.; Banerjee, S. S. J. Clin. Pathol. 1992, 45, 806. (12) Berg, R. D.; Garlington, A. W. Infect. Immun. 1979, 23, 403. (13) Loessner, H.; Weiss, S. Expert Opin. Biol. Ther. 2004, 4, 157. 1063

DOI: 10.1021/acs.nanolett.6b04523 Nano Lett. 2017, 17, 1056−1064

Letter

Nano Letters (14) Forbes, N. S. Nat. Rev. Cancer 2010, 10, 785. (15) Arribas, B.; Rodriguez-Cabezas, M. E.; Camuesco, D.; Comalada, M.; Bailon, E.; Utrilla, P.; Nieto, A.; Concha, A.; Zarzuelo, A.; Galvez, J. Br. J. Pharmacol. 2009, 157, 1024. (16) Yoo, J. W.; Irvine, D. J.; Discher, D. E.; Mitragotri, S. Nat. Rev. Drug Discovery 2011, 10, 521. (17) Luo, C. H.; Huang, C. T.; Su, C. H.; Yeh, C. S. Nano Lett. 2016, 16, 3493. (18) De Smet, R.; Demoor, T.; Verschuere, S.; Dullaers, M.; Ostroff, G. R.; Leclercq, G.; Allais, L.; Pilette, C.; Dierendonck, M.; De Geest, B. G.; Cuvelier, C. A. J. Controlled Release 2013, 172, 671. (19) Tesz, G. J.; Aouadi, M.; Prot, M.; Nicoloro, S. M.; Boutet, E.; Amano, S. U.; Goller, A.; Wang, M.; Guo, C. A.; Salomon, W. E.; Virbasius, J. V.; Baum, R. A.; O’Connor, M. J.; Soto, E.; Ostroff, G. R.; Czech, M. P. Biochem. J. 2011, 436, 351. (20) Aouadi, M.; Tesz, G. J.; Nicoloro, S. M.; Wang, M. X.; Chouinard, M.; Soto, E.; Ostroff, G. R.; Czech, M. P. Nature 2009, 458, 1180. (21) Hu, Q. L.; Wu, M.; Fang, C.; Cheng, C. Y.; Zhao, M. M.; Fang, W. H.; Chu, P. K.; Ping, Y.; Tang, G. P. Nano Lett. 2015, 15, 2732. (22) Taylor, P. R.; Tsoni, S. V.; Willment, J. A.; Dennehy, K. M.; Rosas, M.; Findon, H.; Haynes, K.; Steele, C.; Botto, M.; Gordon, S.; Brown, G. D. Nat. Immunol. 2007, 8, 31. (23) Soto, E. R.; Ostroff, G. R. Bioconjugate Chem. 2008, 19, 840. (24) Soto, E.; Kim, Y. S.; Lee, J. H.; Kornfeld, H.; Ostroff, G. R. Polymers 2010, 2, 681. (25) Soto, E. R.; Caras, A. C.; Kut, L. C.; Castle, M. K.; Ostroff, G. R. J. Drug Delivery 2012, 2012, 143524. (26) Perez, J. M.; Josephson, L.; O’Loughlin, T.; Hogemann, D.; Weissleder, R. Nat. Biotechnol. 2002, 20, 816. (27) Zhou, X.; Che, L.; Wei, Y.; Dou, Y.; Chen, S.; He, H.; Gong, H.; Li, X.; Zhang, J. Acta Biomater. 2014, 10, 2630. (28) Cheng, J.; Feng, S. B.; Han, S. L.; Zhang, X. J.; Chen, Y. D.; Zhou, X.; Wang, R. B.; Li, X. H.; Hu, H. Y.; Zhang, J. X. ACS Nano 2016, 10, 9957. (29) Brown, G. D.; Gordon, S. Nature 2001, 413, 36. (30) Brown, G. D.; Taylor, P. R.; Reid, D. M.; Willment, J. A.; Williams, D. L.; Martinez-Pomares, L.; Wong, S. Y.; Gordon, S. J. Exp. Med. 2002, 196, 407. (31) Hong, F.; Yan, J.; Baran, J. T.; Allendorf, D. J.; Hansen, R. D.; Ostroff, G. R.; Xing, P. X.; Cheung, N. K.; Ross, G. D. J. Immunol. 2004, 173, 797. (32) Swirski, F. K.; Nahrendorf, M.; Etzrodt, M.; Wildgruber, M.; Cortez-Retamozo, V.; Panizzi, P.; Figueiredo, J. L.; Kohler, R. H.; Chudnovskiy, A.; Waterman, P.; Aikawa, E.; Mempel, T. R.; Libby, P.; Weissleder, R.; Pittet, M. J. Science 2009, 325, 612. (33) Geissmann, F.; Manz, M. G.; Jung, S.; Sieweke, M. H.; Merad, M.; Ley, K. Science 2010, 327, 656. (34) Hashimoto, D.; Chow, A.; Noizat, C.; Teo, P.; Beasley, M. B.; Leboeuf, M.; Becker, C. D.; See, P.; Price, J.; Lucas, D.; Greter, M.; Mortha, A.; Boyer, S. W.; Forsberg, E. C.; Tanaka, M.; van Rooijen, N.; Garcia-Sastre, A.; Stanley, E. R.; Ginhoux, F.; Frenette, P. S.; Merad, M. Immunity 2013, 38, 792. (35) Shuhendler, A. J.; Pu, K. Y.; Cui, L.; Uetrecht, J. P.; Rao, J. H. Nat. Biotechnol. 2014, 32, 373. (36) Choi, H. S.; Liu, W.; Misra, P.; Tanaka, E.; Zimmer, J. P.; Itty Ipe, B.; Bawendi, M. G.; Frangioni, J. V. Nat. Biotechnol. 2007, 25, 1165. (37) Weissleder, R.; Nahrendorf, M.; Pittet, M. J. Nat. Mater. 2014, 13, 125. (38) Choi, H. S.; Frangioni, J. V. Mol. Imaging 2010, 9, 291. (39) Murdoch, C.; Muthana, M.; Coffelt, S. B.; Lewis, C. E. Nat. Rev. Cancer 2008, 8, 618. (40) Germano, G.; Frapolli, R.; Belgiovine, C.; Anselmo, A.; Pesce, S.; Liguori, M.; Erba, E.; Uboldi, S.; Zucchetti, M.; Pasqualini, F.; Nebuloni, M.; van Rooijen, N.; Mortarini, R.; Beltrame, L.; Marchini, S.; Fuso Nerini, I.; Sanfilippo, R.; Casali, P. G.; Pilotti, S.; Galmarini, C. M.; Anichini, A.; Mantovani, A.; D’Incalci, M.; Allavena, P. Cancer Cell 2013, 23, 249.

(41) Miller, M. A.; Zheng, Y. R.; Gadde, S. Nat. Commun. 2015, 6, 8692.

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DOI: 10.1021/acs.nanolett.6b04523 Nano Lett. 2017, 17, 1056−1064