Bioinspired Yeast Microcapsules Loaded With Self-Assembled Nanotherapies For Targeted Treatment of Cardiovascular Disease

Materials Today Volume 00, Number 00 July 2017 RESEARCH
Bioinspired yeast microcapsules loaded
RESEARCH: Original Research

with self-assembled nanotherapies for
targeted treatment of cardiovascular
disease
Xiangjun Zhang1,2,4, Xiaoqiu Xu1,3,4, Yidan Chen1, Yin Dou1,2, Xing Zhou1,2,
Lanlan Li1, Chenwen Li1, Huijie An1, Hui Tao1, Houyuan Hu3,*,
Xiaohui Li2 and Jianxiang Zhang1,2,*
1
Department of Pharmaceutics, College of Pharmacy, Third Military Medical University, Chongqing 400038, China
2
Institute of Materia Medica, College of Pharmacy, Third Military Medical University, Chongqing 400038, China
3
Department of Cardiology, Southwest Hospital, Third Military Medical University, Chongqing 400038, China
We developed a biomimetic approach for targeted treatment of cardiovascular disease, which is

mediated by a yeast-derived microcapsule (YC). Different positively charged nanoprobes and
nanotherapies can be efficiently packaged into YC via electrostatic force-driven spontaneous deposition.
These nanoparticle-loaded YCs may be rapidly endocytosed by macrophages and maintained in cells for
up to one week. After oral delivery of YCs containing fluorescent nanoprobes, their accumulation in aortic
plaques and macrophages was observed in apolipoprotein E-deficient (ApoE / ) mice with established
atherosclerotic lesions. This YC-mediated atherosclerotic targeting was realized by transcytotic absorption
in the gastrointestinal tract via M cells in Peyer’s patches, followed by subsequent endocytosis in resident
monocytes/macrophages, and final translocation through recruitment to diseased sites via the lymphatic
system. Correspondingly, oral delivery of assembled anti-atherosclerotic nanotherapies packaged in YCs
afforded notably potentiated efficacies in ApoE / mice. These findings demonstrated that this Trojan
horse-like YC mediated nanomedicinal approach may be employed to orally deliver a diverse array of
therapies for the management of atherosclerosis and other vascular disorders.
Introduction well as microRNA and siRNA therapeutics [10,11], have been

Cardiovascular disease remains the leading cause of morbidity and clinically used or pre-clinically investigated for the management
mortality worldwide [1]. Atherosclerosis, a chronic inflammatory of atherosclerosis. Unfortunately, current pharmaceutical treat-
condition characterized by the formation of intimal atheromatous ments are insufficient to adequately reduce the risk of atheroscle-
plaques, is the most manifest contributor to myocardial infarction, rosis. For the majority of currently used or studied therapeutics,
ischemic stroke, and peripheral vascular disease [2]. Therapeutic their systemic administration generally leads to indiscriminate
approaches are generally employed to reduce the risk of athero- drug distribution throughout the body. This, on the one hand,
sclerosis-related diseases. In this context, a diverse array of thera- causes relatively low drug accumulation in atherosclerotic pla-
peutics including lipid-lowing drugs [3], compounds facilitating ques, while on the other hand, may raise concerns of undesired
cholesterol efflux [4], inhibitors of cholesterol biosynthesis or dose-dependent side effects in normal tissues or organs, particu-
absorption [5], antioxidants [6], phospholipase A2 inhibitors [7], larly after long-term treatment.
anti-inflammatory biologics [8], CD47-blocking antibodies [9], as Recently, the nanoparticle-based targeting strategy has been
considered to be a new approach for molecular imaging and
*Corresponding authors:. Hu, H. (houyuanhu@hotmail.com), Zhang, J. therapeutic intervention of atherosclerosis [12–16]. It has been
(jxzhang1980@gmail.com), (jxzhang@tmmu.edu.cn) demonstrated that nanoparticles can target atherosclerotic
4
These authors contributed equally to this work.
1369-7021/ß 2017 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mattod.2017.05.006

1
Please cite this article in press as: X. Zhang, et al., Mater. Today (2017), http://dx.doi.org/10.1016/j.mattod.2017.05.006
MATTOD-943; No of Pages 13
RESEARCH Materials Today Volume 00, Number 00 July 2017
plaques through different pathways. In most cases, nanoparticles a proof of concept, in vivo targeting of atheromatous lesions was
are able to directly enter plaques via the leaky endothelium near first demonstrated using orally administered YCs loaded with
atherosclerotic vessels [17,18]. Alternatively, nanoparticles may different fluorescent nanoprobes. Subsequently, we evaluated in
reach lesioned sites via the neovessels originating from vasa vivo efficacies of various nanotherapies after being packaged in YCs
vasorum in the adventitia and extending into the base of plaques in an experimental model of atherosclerosis established in apoli-
[15,19]. Besides this nonspecific strategy, targeting of atheroscle- poprotein E-deficient (ApoE / ) mice, using rapamycin (RAP) as a
rosis can be realized by specific targeting of the vasculature and/or candidate drug, which has been demonstrated effective for anti-
components directly related to the development of vascular in- atherosclerotic therapy previously [41,42].
flammation and atheromatous plaques [20–22]. Additionally, in-
travenously administered nanoparticles may be endocytosed by Results and discussion
circulating or splenic monocytes and/or macrophages that subse- Preparation of nanoprobes- or nanotherapies-containing YCs
quently migrate to atherosclerotic lesions [23,24]. For targeted YCs were prepared by sequential treatment with an aqueous
therapy of atherosclerosis, different nanotherapies have been solution of sodium hydroxide and an acid solution as well as
developed and investigated, including liposomes [19,25], lipid- washing with isopropyl alcohol and acetone (Fig. 1), according
based nanoparticles [11,20], polymer nanoparticles [22,26], lipid- to the previously established method with minor revisions [43].
polymer hybrid nanoparticles [18], high-density lipoprotein nano- Whereas plump and solid microparticles could be clearly observed
particles [27,28], and multifunctional nanotheranostics [29,30], in by scanning electron microscopy (SEM) and transmission electron
which therapeutics varying from small molecular drugs, peptides, microscopy (TEM) of the untreated yeast (Fig. 2a), most contents
proteins, and nucleic acids are loaded. Moreover, nanomaterials were removed for the obtained YC samples, resulting in signifi-
themselves have been studied as anti-atherosclerotic nanothera- cantly wizened structure in the SEM image and notably decreased
pies [31]. contrast in the TEM micrograph (Fig. 2b). Initially, the obtained
Although these studies have unambiguously demonstrated the YCs were stained by Calcofluor white, a fluorescent probe gener-
effectiveness of nanomedicines for targeted therapy of atheroscle- ally used for staining of yeasts, fungi, and other organisms by
rosis, the development of effective and safe anti-atherosclerotic binding to their cell walls. Observation by confocal laser scanning
nanotherapies with clinical significance remains highly challeng- microscopy (CLSM) revealed a typical capsule structure for the
ing [13,32–35]. First, intravenous (i.v.) injection is required for the core-removed YCs (Fig. S1a). In addition, pores with mean diame-
majority of currently developed anti-atherosclerotic nanomedi- ter of 755 126 nm could be clearly observed on the surface of
cines. However, their targeting efficiency to atherosclerotic lesions Calcofluor white-stained YCs (Fig. 2c, left), which was further
is low, largely due to the rapid clearance of i.v. administered affirmed by top-down observation via CLSM (Fig. S1b). CLSM
nanoparticles by the mononuclear phagocyte system [18,19]. imaging of fluorescein isothiocyanate (FITC)-labeled YC also indi-
Second, the retention time of nanotherapies accumulated in pla- cated the capsular structure as well as the existence of pores
ques is too short for the treatment of this chronic disease of (Fig. 2c, right). The similar pore structure could be found in
atherosclerosis [35]. To maintain the minimum effective concen- SEM and TEM images shown in Fig. 2b. The presence of these
tration, repeated i.v. dosing is essential, which may lead to poor relatively large pores may be resulted from bud scars, which were
patient compliance over a long time, and raise the risk of potential previously observed in Saccharomyces cerevisiae cells [44]. Yeast cells
physiological impacts after repeated or continuous injections. may bud during the intervals of S/G2/M phases [45]. Under opti-
Additionally, the repeated dosing regimen may bring considerable mal conditions, about 20% cells are unbudded [46]. According to
economic and mental burden on patients’ daily lives. Collectively, observation by SEM and TEM, we found about 70–80% of total YCs
these issues might cause negative consequences that outweigh have large pores due to bud scars. The mean diameter of YCs was
therapeutic benefit [36]. Accordingly, the undesirable risk-benefit 4.6 0.6 mm, while the zeta-potential value of YC was
ratio has greatly limited the bench-to-bedside translation of cur- 13.7 0.4 mV.
rently developed anti-atherosclerotic nanotherapies. According to previous studies, water-soluble nucleic acids and
On the other hand, oral delivery has been considered to be the proteins can be loaded into YCs through electrostatic interactions
most desirable administration route for management of many [43,47,48]. Nevertheless, it remains difficult to efficiently encage
chronic disorders, in view of its advantages such as convenience, different small molecular drugs and nanoparticles into YCs
relatively good safety profile, high patient acceptance and adher- [49,50]. Most recently, we found positively charged nanoparticles
ence, as well as easy modulation on dosing [37–39]. Nevertheless, could be effectively loaded into YCs by electrostatic forces-medi-
currently there have been tremendous challenges in developing ated spontaneous deposition [40]. Herein real-time observation by
effective and orally accessible nanomedicines for targeted therapy CLSM showed a dynamic process of the diffusion of a quantum dot
of atherosclerosis. Most recently, we found that microcapsules (QD525, with fluorescence emission at 525 nm, hydrodynamic
derived from Saccharomyces cerevisiae yeast can efficiently load diameter of 15 nm, and zeta-potential >+50 mV) from the periph-
different charged nanoparticles [40]. After oral administration, eral into the interior of YC (Fig. 2d, left). Clearly, fluorescent
nanoparticles packaged in the yeast capsule (YC) can be preferen- signals of QD525 in YC were gradually enhanced and saturated
tially delivered to diseased sites of arthritis and tumor. Herein we within 3 h. This was further supported by semi-quantitative
hypothesize that self-assembled nanotherapies may be trans- analysis of CLSM images (Fig. 2d, right). The loading efficiency
ported to atherosclerotic plaques after they are loaded in YC of QD525 was 82.1% according to quantification by fluorescence
and administered via the oral route, and therefore this approach spectroscopy. After loading, QD525-loaded YC (QD525/YC) dis-
can be employed for targeted therapy of atherosclerosis (Fig. 1). As played a densely charged core as illustrated by typical TEM
2
FIGURE 1
Schematic illustration of atherosclerosis targeting by nanotherapies orally delivered via a yeast capsule (YC). (a) Self-assembly of positive nanotherapies. (b)
Packaging of positive nanotherapies into YC. (c) Oral absorption, translocation, and targeting of atherosclerotic plaques by YC-delivered nanotherapies.
images (Fig. 2e). Following similar procedures, QDs with differ- examine whether other positive nanoparticles can be successful-
ent emission wavelengths could be simultaneously entrapped in ly loaded into YC, we firstly fabricated different positive fluo-
YC, leading to multicolor YC (Fig. 2f). For different QDs, their rescent nanoprobes based on organic materials by self-assembly.
loading efficiency was above 80%. These results demonstrated To this end, Cyanine 5 (Cy5) was conjugated onto a cationic
that positive QDs can be effectively entrapped into YC. To polymer polyethyleneimine (PEI) with Mw of 25 kDa, resulting
3

FIGURE 2
Fabrication of diverse fluorescent yeast capsules (YCs). (a and b) SEM (left) and TEM (right) images showing intact yeast cells (a) and YCs (b). (c) CLSM
images of either Calcofluor white-stained (left) or FITC-labeled (right) YCs indicating the presence of large pores due to bud scars on the surface of yeast
cells. (d) Time-lapse CLSM images showing spontaneous deposition of green quantum dots (QD525) into YC. The right panel illustrates quantitative data
based on fluorescence images as well as loading efficiency calculated by fluorometric quantification. (e) A TEM image of QD525-loaded YC. (f ) CLSM images
of YC simultaneously loaded with different QDs including QD450, QD525, QD540, and QD620. (g–h) TEM and CLSM images of assembled organic fluorescent
NPs and YCs containing Cy5 NP (g) or Cy5-Cy7.5 NP (h).
in an amphiphilic copolymer Cy5-PEI (Fig. S2a). Cy5-PEI could According to our previous studies, carboxyl-containing hydro-
assemble into positively charged nanoparticles (Cy5 NP) with phobic compounds can serve as guest molecules to mediate self-
mean diameter of 270 nm and zeta-potential of +33.6 mV (Fig. assembly of PEI, giving rise to positively charged spherical nano-
S3a and the left panel of Fig. 2g). Incubation of Cy5 NP with YC particles with extremely high payload contents [51,52]. These
afforded fluorescent Cy5 NP/YC assemblies (the middle and right assembled nanoparticles may also function as nanocarriers for
panels of Fig. 2g). Similarly, Cy7.5-conjugated PEI (Cy7.5-PEI) or loading and delivery of other hydrophobic drugs. Herein, this
Cy5/Cy7.5 simultaneously conjugated PEI (Cy5-Cy7.5-PEI) were self-assembly strategy was employed to fabricate RAP nanothera-
synthesized (Fig. S2b and c), which can assemble into fluorescent pies. Firstly, a RAP nanotherapy was fabricated by co-assembly of
nanoparticles Cy7.5 NP (279 nm; +34.4 mV) or Cy5-Cy7.5 NP RAP with PEI and indomethacin (IND), a carboxyl-containing
(220 nm; +41.0 mV), respectively (Fig. S3a, Fig. S4a, and the left compound, which is also an anti-inflammatory drug. A typical
panel of Fig. 2h). Both Cy7.5 NP and Cy5-Cy7.5 NP could be nanotherapy IND-RAP NP thus obtained exhibited a well-defined
effectively loaded into YCs (Fig. S4a–c, the middle and right panels spherical shape (Fig. 3a and Fig. S3b), with mean size of 65.1 nm
of Fig. 2h). Additionally, Cy5-Cy7.5 NP/YC showed significant and zeta-potential of +55.9 mV. Incubation of IND-RAP NP with
fluorescent signals at excitation/emission wavelengths of both YC offered RAP nanotherapy-containing YC, and the RAP content
Cy5 and Cy7.5 (Fig. S4d). Consequently, in combination with in YC increased in a time-dependent pattern (Fig. S5a). After
sophisticated techniques for engineering of functional nanomater- loading of IND-RAP NP, TEM observation showed densely filled
ials, nanoprobes covering a wide spectrum of materials varying from core in YC, which is in line with the fluorescence image (Fig. 3b).
visible to near-infrared (NIR) fluorescence can be encased into YCs. Also, we found the loading efficiency was related to temperature
4

FIGURE 3
Loading of different RAP nanotherapies into YCs. (a) TEM images (left) and size distribution profiles (right) of a RAP nanotherapy IND-RAP NP assembled in
the presence of PEI and IND. The weight ratio of PEI/IND/RAP was 1:2:2, and the mean diameter was 65.1 nm. (b) Representative TEM (left) and CLSM (right)
images of YCs loaded with IND-RAP NP (IND-RAP NP/YC). The RAP content in IND-RAP NP/YC was 3.8%. (c) TEM images (left) and size distribution profiles
(right) of a RAP nanotherapy SUL-RAP NP assembled in the existence of PEI and SUL. The weight ratio of PEI/SUL/RAP was 1:3:2, while the mean diameter
was 304 nm. (d) Typical TEM (left) and CLSM (right) images of YCs loaded with SUL-RAP NP (SUL-RAP NP/YC). The RAP content in SUL-RAP NP/YC was 4.5%.
(e) TEM images (left) and size distribution profiles (right) of a RAP nanotherapy UDCA-RAP NP assembled in the presence of PEI and UDCA. The weight ratio
of PEI/UDCA/RAP was 1:4:2, and the mean diameter was 210 nm. (f ) Representative TEM (left) and CLSM (right) images of YCs loaded with UDCA-RAP NP
(UDCA-RAP NP/YC). The RAP content in UDCA-RAP NP/YC was 3.1%.
(Fig. S5b), and this may be related to swelling of the yeast wall in loading content was 4.5% and 3.1% for SUL-RAP NP/YC and
aqueous solution [53]. At 308C, the highest loading efficiency was UDCA-RAP NP/YC, respectively.
achieved for IND-RAP NP, and therefore this optimal temperature Together, independent of their components, positive nanopar-
was adopted in following experiments. Likewise, positive RAP ticles can be successfully packaged into YC, affording fluorescent
nanotherapies could be assembled in the presence of PEI and or therapeutic YCs. By contrast, negative nanoparticles cannot be
carboxyl-containing sulindac (SUL, an anti-inflammatory com- directly loaded into YC, as indicated by negative QD620 and
pound) or ursodeoxycholic acid (UDCA, a natural compound from polystyrene nanoparticles of different sizes (Fig. S8). However,
bile acids), offering different sphere-shaped SUL-RAP NPs or observation by TEM, SEM, and CLSM revealed that negative nano-
UDCA-RAP NPs at various formulations (Fig. S6a–c, Fig. S7a–c, particles with diameter up to 700 nm may be efficiently packaged
Fig. 3c and 3e, Fig. S3b). Notably, both SUL-RAP NP and UDCA- into YCs pre-incubated with PEI (Fig. S9a and b). Further quantifi-
RAP NP could be packaged into YCs (Fig. S6d, Fig. S7d, Fig. 3d and cation by fluorescence spectroscopy revealed decreased loading
3f), as revealed by both TEM and fluorescence images. The RAP efficiency when the particle size increased (Fig. S9c). This further
5
substantiated that loading of different nanoparticles into YCs is macrophages (Fig. S13). These results demonstrated that YCs con-
mainly mediated by the electrostatic force. It should be noted that, taining either nanoprobes or nanotherapies may be effectively
in addition to large pores resulting from bud scars, nanoparticles endocytosed by macrophages, and the engulfed nanoparticle-load-
may be loaded into YCs through the porous wall primarily com- ed YC can be maintained in cells for a relatively long period of time.
posed of b-1,3-D-glucan, as implied by previous studies on entrap- It has been well documented that recruitment and accumula-
ment of siRNA or DNA [43,48]. This was also demonstrated by TEM tion of monocytes/macrophages from the circulation and spleen
and CLSM observation of YCs loaded with polystyrene nanoparti- are critically related to the pathogenesis in different stages of
cles, in which multiple nanoparticles were successfully entrapped in atherosclerosis [58]. Therefore, a preliminary Transwell cell migra-
capsules without bud scar-derived pores (Fig. S9a and b). The exact tion assay was carried out to examine anti-migration capability of
mechanisms, however, remain to be elucidated in future studies. RAP nanotherapy-loaded YC. Whereas induction with monocyte
Importantly, RAP molecules loaded into YC could be released in chemoattractant protein-1 (MCP-1) caused considerable migra-
simulated intestinal fluid, while drug release was largely inhibited tion of macrophages, treatment with free RAP remarkably reduced
in simulated gastric fluid (Fig. S5c). This pH-responsive release the number of migrated cells (Fig. 4f). More significant effect was
profile is closely related to the characteristics of IND. At acidic pH achieved by IND-RAP NP/YC. In this case, we even did not observe
1.2, carboxyl of IND is protonated since its pKa is 4.5, leading to crystal violet-stained cells in the lower chamber, indicating that
extremely low solubility. Accordingly, IND-RAP NP is stable and macrophage migration was completely inhibited. Accordingly,
drug release is mainly attenuated in the stomach-relevant acidic YC-mediated intracellular delivery of RAP nanotherapy can also
solution. By contrast, IND dissociation at pH 7.4 can notably enhance its activity.
enhance its dissolution due to significantly increased solubility at
pH higher than its pKa. Furthermore, the dissolution of IND may Targeting atherosclerotic plaques in mice by nanoparticles orally
lead to erosion of assembled IND-RAP NP, accelerating drug release. delivered via YC
According to our recent finding that orally delivered YC can
Cellular uptake and in vitro activity of YCs containing different effectively ferry loaded nanoparticles to the diseased sites of acute
nanoparticles inflammation by macrophage-mediated translocation, we hy-
Since YC-mediated therapeutic delivery was supposed to be real- pothesize that YC may also deliver nanoparticle payloads to
ized by macrophage-mediated translocation, we examined cellular chronic inflammatory sites via the oral route. Herein we first
uptake profiles of YC by macrophages. Initially, 5-(4,6-dichloro- examined targeting capability of nanoparticles orally delivered
triazinyl)aminofluorescein (DTAF)-labeled YC (DTAF-YC) was ex- via YC in atherosclerosis, a chronic inflammatory disease that is
plored in RAW264.7 macrophage cell line (Fig. S10). After also closely related macrophages [2,58].
incubation of DTAF-YC with macrophages for different periods After continuous daily oral administration of Cy7.5 NP/YC at
of time, quantification by flow cytometry showed gradually in- 2.4 mg/kg of Cy7.5 for four times, ex vivo imaging showed signifi-
creased fluorescent signals (Fig. 4a). Also, observation by CLSM cant NIR fluorescent signals in atherosclerotic plaques of the aortic
revealed endocytosis of YC by macrophages in a time-dependent root and arch in apolipoprotein E-deficient (ApoE / ) mice sub-
manner (Fig. 4b). Real-time optical microscopy imaging demon- jected to Western diet for 2 months (Fig. 5a, the left panel). Of
strated very rapid internalization of YC into cells (Fig. S11). This note, fluorescent intensity of the Cy7.5 NP/YC group was dramat-
rapid and effective uptake is in line with the fact that the b-glucan ically higher than that of the Cy7.5 NP group (Fig. 5a, the right
receptor, Dectin-1, is predominantly expressed on macrophages panel). In addition, immunofluorescence analysis revealed the
[54], while b-glucan is the main component of yeast cell walls [55]. distribution of DTAF-YC in macrophages isolated from the aorta
In addition, macrophages can phagocytose yeast via cell surface of atherosclerotic plaque-bearing ApoE / mice, which were orally
pattern recognition receptors, such as scavenger receptors class A- administered with DTAF-YC, since Cy7.5 fluorescence cannot be
I/II [56]. Further, fluorescent staining of late endosomes and observed by CLSM (Fig. 5b). In line with this, higher accumulation of
lysosomes indicated intracellular trafficking of YC through the Cy7.5 fluorescent signals could be detected in the intestine, liver,
endolysosomal pathway. Similarly, time-dependent endocytosis and spleen of Cy7.5 NP/YC-treated mice when compared with those
and endolysosomal transportation were observed for QD525/YC administered with Cy7.5 NP (Fig. S14a–d). In the case of different
(Fig. 4c). More importantly, we found that endocytosed QD525/ lymphatic tissues, we found significantly higher Cy7.5 fluorescent
YC was maintained in RAW264.7 cells even after 7 days of incu- signals in mesenteric lymph node (MLN) and thymus of the Cy7.5
bation (Fig. 4d). This agrees with a previous study that typically NP/YC group, while no significant differences were detected in
ingested yeast particles remained intact for 3–5 days in a murine armpit lymph node (ALN) and subcutaneous lymph node (SLN),
macrophage line J774, and complete degradation of b-1,3-glucan as compared to the free Cy7.5 NP group (Fig. S14e–i). Additionally,
particles required >13 days [57]. These findings implied that the almost no absorption occurred in the stomach, as evidenced by
recognition capability of b-1,3-glucan on the YC wall was well rapidly decreased Cy7.5 fluorescent signals after 2 h of oral admin-
retained, although the yeast sample was treated at a high temper- istration of Cy7.5 NP/YC (Fig. S15). These results demonstrated that
ature of 808C to prepare YC. Thermogravimetric analysis suggested oral delivery of nanoparticles via YC can increase their intestinal
that both yeast and YC samples were stable below 2458C (Fig. S12). absorption and facilitate subsequent transportation via the lymphat-
Consistently, we found sustained RAP levels for 12 h in RAW264.7 ic system, thereby enhancing targeting to atherosclerotic plaques.
cells after incubation with IND-RAP NP/YC (Fig. 4e). Furthermore, Subsequently, we performed preliminary studies to explore
in vitro cell culture experiments revealed that blank YC and YCs mechanisms underlying YC-mediated absorption and transloca-
loaded with QDs or nanotherapies showed low cytotoxicity in tion in the gastrointestinal (GI) tract. After continued daily oral
6
FIGURE 4
Cellular uptake and in vitro activity of nanoparticle-loaded YCs in RAW264.7 cells. (a) Typical flow cytometric curves (upper panel) and quantitative analysis
(lower panel) of intracellular internalization of DTAF-labeled YC in a time dependent pattern. (b) CLSM images showing time-dependent endocytosis of
DTAF-YC. (c,d) Representative fluorescence microscopy images illustrating time-dependent endocytosis of QD525/YC (c) and intracellular retention of QD525/
YC (d). (e) Intracellular RAP concentrations after incubation with IND-RAP NP/YC for different periods of time. (f ) Typical optical microscopy images (left) and
quantitative data (right) indicating the migrated macrophages stained by crystal violet. For all CLSM images, scale bars represent 10 mm. All data are
mean SD (n = 3); *P < 0.05, ***P < 0.001.
administration of blank YC at 5 mg (about 1 109) in each mouse, Peyer’s patches (Fig. 5c). This increase is beneficial for enhanced
Peyer’s patches in the GI tract and MLNs were collected. As transepithelial transport, since M cells are generally present in the
compared to the control mice treated with saline, mice adminis- gut-associated lymphoid tissue of Peyer’s patches [59]. In addition,
tered with YC showed the significantly increased number of flow cytometric analysis revealed notably decreased populations of
7

FIGURE 5
In vivo targeting of atherosclerotic plaques by nanoparticles orally delivered by YCs. (a) Representative ex vivo images (left) and quantitative results (right)
illustrating distribution of Cy7.5 NP/YC in aortas with plaques established in ApoE / mice. (b) Immunofluorescence images showing co-localization of DTAF-
YC in macrophages of aortic tissues isolated from ApoE / mice after two times of oral gavage. Cy3-labeled antibody was used to stain macrophages, while
nuclei were stained with DAPI. (c) The number of Peyer’s patches after oral administration of YC. (d, e) Populations of Ly-6Clow monocytes and macrophages
after oral delivery of YC. For CLSM images, scale bars represent 5 mm. All data are mean SD (n = 4, a; n = 6, c-e); *P < 0.05, ***P < 0.001.
macrophages and Ly-6Clow monocytes (which also called ‘‘patrol- approach. Oral gavage of IND-RAP NP/YC was performed every 3
ling’’ monocytes) in Peyer’s patches (Fig. 5d). Likewise, both days in ApoE / mice after one month of Western diet. Post two
macrophage and Ly-6Clow monocyte populations were dramati- months of therapy, the entire aortas were isolated and atheroscle-
cally decreased in MLNs (Fig. 5e). These results implied that rotic plaques were stained by Oil Red O (ORO). As compared to the
resident macrophages and Ly-6Clow monocytes in both Peyer’s model control, we found considerably lower ORO-stained area in
patches and MLNs may have participated in the translocation of aortas collected from IND-RAP NP/YC-treated mice (Fig. 6a), while
orally administered YC, while loss of these cell populations have IND-RAP NP itself only slightly reduced the lesion area. Quantita-
not been replenished by the circulating or splenic cells at the tive analysis revealed that IND-RAP NP/YC more significantly
examined time points. decreased the plaque area when compared with IND-RAP NP
Taken together, these preliminary findings revealed that in- (Fig. 6b). Similar results could be observed in ORO-stained cryo-
creased targeting of atherosclerotic plaques by nanoparticles orally sections of aortic roots collected from various groups (Fig. 6c).
delivered via YC may be due to the enhanced transepithelial Examination on hematoxylin and eosin (H&E)-stained sections of
transport and absorption via Peyer’s patches, which is followed aortic roots indicated that necrotic cores were considerably re-
by cellular translocation via monocytes/macrophages in Peyer’s duced in mice treated with IND-RAP NP/YC (Fig. S16). Further
patches and lymphatic tissues. As well known, the lymphatic analysis by immunohistochemistry suggested that macrophages
system drains extracellular fluid from the peripheral tissues, such in plaques were notably lowered after therapy with IND-RAP NP/
as the gut-associated lymphoid tissue and Peyer’s patches, through YC (Fig. 6d). Consistent with the reduced macrophage population,
the lymph nodes, and into the thoracic duct, which empties into we found the level of matrix metalloproteinase-9 (MMP-9) was
the left subclavian vein. On the other hand, macrophages can be remarkably decreased after IND-RAP NP/YC treatment (Fig. 6e),
recruited from the lymphoid tissues or blood to atherosclerotic while the collagen content around plaques was higher in the IND-
plaques [60]. Consequently, macrophage-mediated transportation RAP NP/YC group as compared to that in other groups. Of note,
through the lymphatic system mainly contributes to targeting of both macrophages and MMP-9 may promote the development of
atherosclerotic plaques by YC after oral delivery. vulnerable lesions, while fibrous caps, predominantly composed
of collagen, may contribute to plaque stability [61]. Additionally,
Treatment of atherosclerosis by an orally delivered nanotherapy quantification by flow cytometry indicated that therapy by IND-
Based on the above promising results, in vivo studies were per- RAP NP/YC significantly decreased the population of monocytes/
formed to evaluate the therapeutic effect of nanotherapies deliv- macrophages in the circulation, in particular the pro-inflammatory
ered via YC. First, an assembled nanotherapy IND-RAP NP was Ly-6Chigh monocytes (Fig. 7a and b). Moreover, the levels of typical
employed, which was loaded into YC through the aforementioned pro-inflammatory cytokines including tumor necrosis factor-a
8
FIGURE 6
Targeted therapy of atherosclerosis by YC-mediated oral delivery of a nanotherapy IND-RAP NP. (a) Typical photographs of en face ORO-stained entire aortas
from ApoE / mice post various treatments. (b) Quantification of the plaque area. (c) ORO-stained cryosections of aortic roots from various groups. (d,e)
Immunohistochemistry analyses on sections of aortic roots by separately staining with anti-CD68 antibody (d) as well as anti-MMP-9 and Masson’s trichrome
(e). **P < 0.01, ***P < 0.001.
9
be achieved by i.v. delivered nanoparticles [12,13], YC-directed

oral delivery offered additional advantages such as good safety
profile and patient compliance, since long-term treatment is gen-
erally required for the management of this chronic inflammatory
setting.
Safety profile of orally administered IND-RAP NP/YC

Further, we examined potential side effects after the long-term

treatment with IND-RAP NP/YC. Analysis on the organ index of
typical major organs including heart, liver, spleen, lung, and
kidney showed insignificant differences between different groups
(Fig. S17a). Measurement of representative hematological param-
eters showed they were in normal ranges for mice treated with
different formulations (Fig. S18). As compared to the control
group, the IND-RAP NP/YC group did not display statistically
significant changes in the levels of lipids including total choles-
terol (TCH), triglyceride (TG), high-density lipoprotein (HDL), and
low-density lipoprotein (LDL) (Fig. S17b). Quantification of bio-
markers relevant to hepatic and kidney functions revealed no
abnormal variations in different treatment groups (Fig. S17c
and d). Consistent with these results, further examination on
H&E stained histological sections of organs including liver, spleen,
and kidney, which are closely related to absorption and excretion
of orally administered IND-RAP NP/YC, showed no discernable
injuries in these organs (Fig. S17e). Particularly, long-term oral
administration of IND-RAP NP/YC did not cause irritation or
induce ulceration in GI tissues such as stomach and different
intestinal segments (Fig. S17f). In all groups, normal mucosae
and microvilli could be clearly observed in epithelial tissues of
the stomach and intestines. All these results suggested that IND-
RAP NP/YC displayed good safety profile for oral administration.
Therapeutic potentials of different RAP nanotherapies orally

FIGURE 7 delivered via YC
Changes of pro-inflammatory factors after therapy with IND-RAP NP orally We also investigated in vivo efficacies of RAP nanotherapies assem-
delivered by YC. (a,b) Representative flow cytometry profiles (a) and
bled in the presence of different carboxyl-containing compounds.
quantitative histograms (b) indicating populations of pro-inflammatory
monocytes/macrophages in blood samples collected from ApoE / mice In this context, nanotherapies UDCA-RAP NP and SUL-RAP NP
treated with saline or IND-RAP NP/YC. (c) Levels of pro-inflammatory were examined. After 9 weeks of oral administration of YCs con-
cytokines after treatment with IND-RAP NAP/YC. Data are mean SD taining UDCA-RAP NP or SUL-RAP NP at 3.0 mg/kg of RAP (one
(n = 6); **P < 0.01, ***P < 0.001. time every three days), aortas were isolated and stained using ORO.
Direct observation revealed considerably reduced ORO-stained
(TNF-a) and interferon-g (INF-g) were strikingly lowered post area in both UDCA-RAP NP/YC and SUL-RAP NP/YC treated
treatment by IND-RAP NP/YC (Fig. 7c). groups, when compared with that in the control group
These results implicated that oral treatment with IND-RAP NP/ (Fig. 8a). Further quantitative analysis demonstrated the plaque
YC can notably delay the progression of atherosclerosis and effec- area was strikingly reduced by treatment with either UDCA-RAP
tively stabilize atherosclerotic plaques. Accordingly, by efficiently NP/YC or SUL-RAP NP/YC (Fig. 8b). Also, the levels of pro-inflam-
transporting orally administered YC to atherosclerotic plaques, YC matory cytokines such as TNF-a and INF-g were remarkably de-
can considerably enhance the anti-atherosclerotic activity of load- creased after treatment with different nanotherapies orally
ed therapeutic nanoparticles. By contrast, either free RAP or IND- delivery via YC (Fig. 8c). These results demonstrated that the
RAP NP alone showed limited beneficial outcomes. As well docu- anti-atherosclerotic activity could be achieved for different RAP
mented, macrophages play a pivotal role in the initiation and nanotherapies after oral delivery via YC, and their in vivo efficacies
development of atherosclerosis [58], and their accumulation in were largely independent of the chemical structure of carboxyl-
plaques has been observed at different stages. Also, i.v. adminis- containing compounds used for assembly.
tered nanoparticles may reach atherosclerotic plaques by mono- Likewise, possible toxicity was examined during and after treat-
cyte/macrophage-mediated transportation [23]. Accordingly, the ment. During the whole period of treatment, comparable body
ferrying effect of macrophages should have contributed to plaque weight values were found for mice treated with saline (the control
targeting and therapeutic effects of nanoparticle-loaded YC. group) or YC delivered nanotherapies (Fig. S19a). No significant
Whereas effective targeting and therapy of atherosclerosis can changes in the organ index of typical major organs were observed
10

FIGURE 8
Therapy of atherosclerosis by nanotherapies UDCA-RAP NP and SUL-RAP NP orally delivered via YC in ApoE / mice. (a) Typical digital photos of ORO-
stained aortas from ApoE / mice post various treatments for 2 months. (b) Quantification of the plaque area. (c) Concentrations of TNF-a and INF-g in
serum samples collected from ApoE / mice. Data are mean SD (n = 6); *P < 0.05, **P < 0.01, ***P < 0.001.
for both UDCA-RAP NP/YC and SUL-RAP NP/YC groups (Fig. contributed by the increased transepithelial absorption via M cells
S19b). Also, nanotherapy-treated groups exhibited hematological in Peyer’s patches and subsequently facilitated translocation by
parameters comparable to those of the control group (Fig. S19c). monocytes/macrophages through the lymphatic system. After
Histological analysis of H&E-stained sections of the GI tissues two months of treatment in ApoE / mice with established athero-
showed no detectable injuries in two nanotherapy-treated groups sclerosis, different nanotherapies orally delivered via YC significant-
(Fig. S19d). Besides, we did not find abnormal variations in the ly reduced plaque area and notably enhanced the stability of
microstructure from H&E sections of liver, spleen, and kidney (Fig. atherosclerotic lesions, thereby considerably delaying the progres-
S19e). All these results implied that RAP nanotherapy-containing sion of atherosclerosis to more complicated plaques. Of note, YC-
YCs may be developed as effective and safe therapeutics for the delivered nanotherapies were much more effective than unpack-
treatment of atherosclerosis via oral delivery. It is worth noting aged nanomedicines and pristine RAP. Importantly, preliminary
that i.v. injection of different nanotherapies at 3.0 mg/kg of RAP examination on possible toxicity suggested nanotherapy-packaged
every three days may lead to severe side effects, such as tail necrosis YCs displayed good safety profile after long-term oral administra-
and animal death within 10 days (Fig. S20). Accordingly, i.v. tion, which is extremely important for the management of chronic
administration of nanotherapies cannot be used as controls to diseases. As a conceptual proof study, demonstration of atheroscle-
compare their long-term in vivo efficacy with YC-delivered rosis targeting through the oral route paves a way for effective and
nanotherapies. safe delivery of a large number of different therapeutics including
small molecular drugs, peptides, proteins, and nucleic acids.
Summary
We demonstrated different fluorescent nanoprobes and RAP Materials and methods
nanotherapies can be effectively packaged into YCs via electrostatic Preparation of yeast capsules
force-mediated spontaneous deposition. YCs containing different Yeast capsules (YCs) were prepared according to the previously
nanoparticles may be rapidly endocytosed in macrophages and reported method with minor modification [48]. In brief, 20 g yeast
intracellularly transported via the endolysosomal pathway. The was suspended in 200 mL of 1 M NaOH, and the obtained suspen-
engulfed nanoparticle-loaded YCs can be retained for up to 7 days sion was heated at 808C for 1 h. After the sample was collected by
in macrophages. Treatment with YC loaded with an assembled centrifugation at 2200 g for 15 min, and rinsed twice with deio-
nanotherapy IND-RAP NP almost completely suppressed macro- nized water, it was dispersed in aqueous solution with pH 4.5 and
phage migration, in line with the sustained level of RAP after incubated at 558C for 1 h. The yeast sample was collected after
cellular uptake of IND-RAP NP/YC. Oral delivery of Cy7.5 NP via centrifugation and thorough washing with deionized water. Sub-
YC showed significantly higher distribution of nanoparticles in sequently, the obtained sample was rinsed with 40 mL of isopropyl
atherosclerotic plaques in aortas of ApoE / mice, when compared alcohol four times. After additional washing with 40 mL of ace-
with free Cy7.5 NP. This enhanced targeting of atherosclerosis was tone twice, the yeast sample was collected and dried under vacuum
11
at 20–258C, which contains about 2 1011 particles per gram IND-RAP NP/YC every 3 days for additional 2 months. For all RAP
sample. formulations, the dose of RAP was 3 mg/kg. At the end of treat-
ment, mice were euthanized. The blood, whole aorta, aortic sinus,
Preparation of YCs loaded with different fluorescent and main organs were harvested to assess the degree of atheroscle-
nanoparticles rosis and possible adverse effects resulted from various treatments.
To prepare YC loaded with QD525, a certain amount of dry YC was In another cohort of studies, efficacies of UDCA-RAP NP/YC and
first incubated in 100 mL of carbonate buffer (pH 9.2) at 378C for SUL-RAP NP/YC were examined through the similar procedures
30 min, and then 10 mL of aqueous solution containing QD525 (n = 6 in this case).

was added. After 4 h of incubation at 378C, QD525-loaded YC
(QD525/YC) was collected by centrifugation at 2057 g for 10 min, Quantification of atherosclerotic plaques
and then thoroughly washed with deionized water to remove un- After ApoE / mice were euthanized, the extent of pathological
loaded QD525. Finally, QD525/YC was harvested after lyophiliza- changes was quantified by measuring lesion area of aortas from the
tion. QD525 entrapped in YC was determined by quantifying un- heart to the iliac bifurcation. Briefly, the aorta was fixed by perfu-
loaded QD525 in aqueous solution by fluorescence spectrometry. sion with formalin (10% in PBS) for 50 min. After the peri-adven-
In a separate experiment, the loading process of QD525 into YC titial tissue was cleaned, the aorta was opened longitudinally and
was directly observed by confocal laser scanning microscopy. stained with ORO to evaluate plaque area. To assess the degree of
Other QDs were packaged into YCs through the similar proce- atherosclerosis at the aortic origin, tissues embedded in Tissue-Tek
dures. O.C.T. Compound were cross-sectioned serially at 8-mm intervals
To fabricate Cy7.5 NP-loaded YC (Cy7.5 NP/YC), Cy7.5 NP and stained by ORO. Quantitative analysis was carried out with
containing 600 mg Cy7.5 was added into 1 mL deionized water Nis-Elements BR 3.2 software.
containing 10 mg YC, and stirred at 378C for 12 h under the dark.
Cy7.5 NP/YC was obtained after centrifugation and rinsing with Acknowledgments
deionized water three times. This study was supported by the National Natural Science
Foundation of China (No. 81471774), the Program for New
Preparation of YCs packaged with RAP nanotherapies Century Excellent Talents in University (No. NCET-13-0703), the
First, 100 mg YC was suspended in 10 mL deionized water, and Research Foundation of Third Military Medical University (No.
stirred at 408C for 30 min. Then, IND-RAP NP containing 15 mg 2014XJY04), the Science and Technology Innovation Program in
RAP was added and stirred at 308C for 12 h. IND-RAP NP-loaded YC Military Medicine of Southwest Hospital (No. SWH2016LHYS-05),
(IND-RAP NP/YC) was obtained after centrifugation at 2000 g for and the Innovation Program for Key Technologies of Southwest
10 min, washing with deionized water, and freeze-drying. SUL- Hospital (No. SWH2016ZDCX1016).
RAP NP or UDCA-RAP loaded YCs were fabricated through similar
procedures. In addition, time and temperature dependent loading Appendix A. Supplementary data
was examined in the case of IND-RAP NP to optimize loading Supplementary data associated with this article can be found, in
parameters. The RAP content loaded in YCs was quantified by high the online version, at doi:10.1016/j.mattod.2017.05.006.
performance liquid chromatography, after RAP was extracted in
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Bioinspired Yeast Microcapsules Loaded With Self-Assembled Nanotherapies For Targeted Treatment of Cardiovascular Disease

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Materials Today  Volume 00, Number 00  July 2017 RESEARCH