| Due to the excellent safety and convenience of oral administration, oral delivery is the most common route for drug administration. In recent years, with the rapid development of nanotechnology, materials science, and pharmacy, the research of novel oral drug delivery systems has made extensive progress, with respect to oral controlled release, oral colon targeted delivery, intestinal adhesion release and transmucosal transport. On the other hand, the development of novel drug targeted delivery systems in the past decades has demonstrated that targeted drug delivery can highly improve the efficacy of drug and significantly reduce off-target effects, adverse reactions, and side effects. However, the gastrointestinal(GI) tract is characterized with complex p H environment and the presence of food residues, digestive enzymes, and continuous secretion of mucus, which makes it is difficult to design orally targeted drug delivery systems for diseased sits remote from the GI tract.To achieve orally targeted delivery of imaging reagents and therapeutics, we hypothesize that novel macrophage targeted oral drug delivery systems can be constructed by simulating the way utilized by microbes upon infection in the GI tract. To this end, yeastderived yeast shell(YS), which is mainly composed of β-1,3-D-gulcan, was investigated. The YS-based systems can be realized targeting to inflammation and tumor sites by the recruitment of monocytes/macrophages in these diseased locations.Methods1. Preparation of yeast shellBaker’s yeast was alkaline-and-acid-extracted, followed by alcohol and acetone extraction, and then dried at 20-25°C.2. Preparation and characterization of positively charged nanoparticles(PNP) loaded YSAbout 1 mg of dry YS was firstly incubated in carbonate buffer(CBS, p H 9.2) at 37 °C for 30 min, then 10 μL quantum dots with polydiallydimethylammounium chloride(PDDA-QD) or iron oxide nanoparticles with polydiallydimethylammounium(PDDA-IONP) suspension was added. After 4 h of incubation at 37 °C, precipitate of PDDA-QD/YS(QD YS) or PDDA-IONP/YS(IONP YS)was collected without further treatment. Morphology of YS and PNP-loaded YS was characterized by scanning electron microscopy(SEM), confocal laser scanning microscopy(CLSM), and transmission electron microscopy(TEM). Particle size and zeta potential were measured by a Nano Zetasizer(Nano ZS). The QD loading content was determined by fluorescence spectroscopy, while the IONP loading was quantified by thermogravimetric analysis(TGA).YS was suspended in CBS(p H 9.2) solution, and PDDA-QD or PDDA-IONP suspension was quickly added at a certain amount with shaking, then the process of PNP loading into YS was monitored by the CLSM or magnetic resonance imaging(MRI), Nano ZS, and differential scanning calorimetry(DSC) to study the underlying mechanism.Factors including incubation time, temperature, solvent, and PNP feeding were examined for PDDA-QD and PDDA-IONP, respectively.3. Effects of YS uptake on the biological activity of macrophageAfter Raw264.7 macrophages were co-incubated with YS for 2 h at the cell/YS ratio of 1:50, the phagocytosis and migration capability of Raw264.7 cells were evaluated.4. Acute toxicity evaluationMale Kunming mice were employed to evaluate possible acute toxicity of YS. After oral administration of YS in mice at 5 g/Kg, their body weight and general behaviors were observed for any signs of illness. After 14 d, animals were sacrificed. Blood samples were collected for the quantification of hematological parameters and biochemical markers relevant to liver/kidney functions. Organs including heart, liver, kidney, lung, and spleen were harvested and weighed to calculate the organ index. They were also fixed to prepare histopathological sections for hematoxylin and eosin(HE) staining. GI tissues were collected for histopathological examination based on HE stained sections.5. Intracellular uptake study of QD YSRaw264.7 macrophages and QD YS were co-incubated at the cell/QD YS ratio of 1:50. After cells were incubated for the indicated time periods at 37 °C /with 5 % CO2 and washed with PBS, cells were removed to observe and determine the uptake of QD YS in macrophages by CLSM and flow cytometry(FCM), respectively. Then,the mechanism study of macrophage recognizing YS is studied.Raw264.7 cells were firstly co-incubated with laminarin at various concentrations for 0.5 h, and then QD YS was added to coincubate with Raw264.7 at the cell/QD YS ratio of 50:1. After 1 h, cells were washed with PBS and removed to observe and determine the uptake of QD YS in macrophages by CLSM and FCM, respectively. Similar procedures were employed in the case of peritoneal macrophages collected from mice.6. Study on QD YS biodistribution in mice with acute inflammationQD YS was employed to follow in vivo distribution of YS after oral administration in inflammatory mice with dynamic fluorescence imaging using an in vivo imaging system(IVIS) and data were acquired serially for up to 24 h. The major organs and swelling paws of mice were collected after 24 h for ex vivo imaging and tissues including liver, spleen, paw, and mesenteric LN(MLN) were fixed and digested for immunofluorescence(IF) staining and FCM, respectively.7. Study on QD YS biodistribution in mice bearing tumorCyanine7.5(Cy7.5)-conjugated polyethyleneimine was dialyzed to prepare Cy7.5 nanoassemblies(Cy7.5-PEI). Then Cy7.5-PEI was encapsulated in YS to form Cy7.5 loaded YS(Cy7.5 YS), which was employed to follow in vivo distribution of YS after oral administration in mice bearing human breast cancer MCF-7 xenograft tumor, by dynamic fluorescence imaging. Data were acquired continuously for up to 48 h. The major organs and swelling paws were collected after 24 h for ex vivo imaging and tumors were fixed and digested for IF staining and FCM, respectively.8. IONP YS in the rat with acute inflammation for MRIAfter SD rats with inflammatory paws were given various iron oxide nanoparticle formulations, T2 star intensities at swelling paws were observed at predetermined time points by MRI.9. IONP YS in the rat bearing xenograft tumor for MRISD rats were inoculated with Walker 256 breast cancer cells to establish tumor-bearing rats. When the tumor size was about 1 cm3, various IONP formulations were administered to study the application of IONP YS in tumor MRI by monitoring the T2 star at tumor sites.10. Preparation of positively charged drug nanoassemblies(PNA).A dialysis approach was adopted to prepare PNA based on branched polyethyleneimine(b PEI) and various hydrophobic carboxyl-bearing dugs(CBD). Briefly, both CBD and b PEI was co-dissolved in a common solvent at predetermined weight ratios, and the obtained solution was dialyzed against deionized water for 24 h at room temperature. Unless stated otherwise, b PEI concentration in the organic solvent was 10 mg/m L. The outer aqueous solution was exchanged every 2 h. The dialysate was directly used for characterization without further treatment. Similar procedures were employed to produce PNA containing no carboxyl-bearing hydrophobic dugs(NCBD), in which b PEI with 25 k Da molecular weight(b PEI25), CBD, and NCBD were co-dissolved in DMSO. Morphology of nanoassemblies was characterized by CLSM and TEM. Particle size and zeta potential was measured by Nano ZS. The drug loading and entrapment efficiency of CBD-loaded PNA and NCBDloaded PNA was determined by ultraviolet spectroscopy(UV) and high performance liquid chromatography(HPLC), respectively. The stability of nanoassemblies was evaluated in PBS(p H 7.4) as well as in PBS(p H 7.4) containing trypsinase or chymotrypsin incubated at 37oC. Digital photos and TEM images were taken for various samples at selected time points, and the size of nanoassemblies was also determined. Interactions between b PEI and CBC were theoretically assessed by molecular modeling containing molecular docking through Autodock 4.2 and Molecular Operating Environment(MOE) software and characterized by Fourier transform infrared spectra(FT-IR), 1 h and 1 h-1 h Roesy NMR, DSC measurements.11. Evaluation of in vitro antitumorMurine melanoma B16F10, human hepatocellular carcinoma Hep G2, human cervical cancer Hela, MCF-7, and human breast cancer MDA-MB-231 cells were treated with the medium containing indomethacin(IND)-loaded PNA(IND/b PEI25), paclitaxel(PTX)-loaded PNA(PTX/IND/b PEI25), or pristine PTX(solubilized in the aqueous solution containing Cremophor EL® and ethanol) at different doses for 24 and 48 h. The cell viability was quantified by MTT method, and IC50 values were calculated using Originpro 7.0.12. Preparation and characterization of PNA-loaded YSAbout 10 mg of dry YS was mixed with 100 μL PNA suspension, incubated at 37 °C for 12 h, and then the suspension was centrifuged at 4000 rpm for 10 min. PNA-loaded YS was lyophilized after washing twice with 1 m L water. Morphology of PNA-loaded YS was characterized by CLSM and TEM. The drug loading and entrapment efficiency of CBD and NCBD was determined by UV and HPLC, respectively.13. In vitro release studyLyophilized samples were dispersed into PBS(0.01 M, p H 7.4) and solutions simulating p H conditions in the GI tract for in vitro release study. Drug concentration in the release buffer was quantified by UV and HPLC for CBD and NCBD, respectively.14. In vivo pharmacokinetic studyAfter male Sprague-Dawley(SD) rats(200-250 g) were orally administered with various IND formulations, blood samples were collected at predetermined time points to determine IND concentrations in plasma. Pharmacokinetic(PK) parameters were calculated based on a non-compartmental model using the software of DAS 3.0.15. Tissue distribution and in vivo cell distributionTo evaluate IND distribution profiles, after SD rats with swelling paws were orally given IND formulations, various tissues, including heart, liver, spleen, lung, kidney, stomach, ileum, Peyer’s Patch, and lymph node(LNs) were dissected. Monocytes/ macrophages in blood and tissues including liver, spleen, paw and MLN were isolated by density gradient centrifugation and disrupted by repeated freeze-thawing. Then IND concentrations in tissues and cells were determined by HPLC.16. In vivo efficacy of IND formulations in inflammatory disease models.Therapeutic effects of IND were evaluated in both acute and chronic models established by intradermal injection of 100 μL of carrageenan solution(0.1 wt. % in saline) and complete Freund’s adjuvant in the right paw of rats, respectively. The anti-inflammatory efficacy was evaluated by the swelling degree defined as the difference between the paw volume before and after inflammation. After treatment, all mice were sacrificed, and GI tissues and main organs were collected for further analysis.17. Assessment of antitumor activity of PTX formulationsTumor xenografts were generated by inoculating B16F10 cells into the right limb armpits of athymic nude mice. When the average volume of the xenograft tumors reached 10 mm3, the mice were given various PTX formulations. The tumor volume was determined to evaluate the anti-tumor activity. After 15 days, all mice were sacrificed, and the collected tumors were weighed. Blood samples, GI tissues, and main organs were also collected for further analysis.18. Study of oral absorption and transportation of QD YS at ileumQD YS was directly injected into the specified ileum lumen, ileum lumen and MLNs were taken out for observation by stereo fluorescence microscopy(SFM), CLSM, IF staining, FCM, and IVIS.Results1. YS were successfully prepared by alkaline-and-acid-extraction, with size of 4.5 μm and zeta-potential of-6.5 m V, which were composed primarily of β-1,3-D-gulcan. PNPloaded YS such as QD YS and IONP YS were successfully fabricated by electrostatic interaction between positively charged NPs and YS. The process of PNP-loading into YS was achieved with two steps. QD was firstly enriched near the YS surface, and then it was transferred into the interior of YS by potential shift.2. YS at the high dose 5g/Kg showed no obvious side effects and pathological changes in Kunming mice after oral administration. In vitro evaluation showed that migration and phagocytosis of macrophages were not influenced by YS.3. YS could be rapidly recognized and phagocytosed by macrophage based on Dectin-1 receptor. YS had a high stability in macrophages, and the intact yeast microstructure was observed 7 days after phagocytosis.4. QD YS showed targeted distribution profiles in swelling paws and tumor sites after oral administration, and exhibited high distribution in the liver and spleen. In liver, spleen, swelling paw, and tumor, QD YS was showed to be co-located with monocytes/macrophages, as determined by IF staining and FCM.5. IONP YS was orally targeted to swelling paws and tumor sites after oral administration. The loading of YS could significantly improve the T2 relaxation enhancement of IONP. At the tumor sites and in swelling paws, IONP YS showed no significant difference at T2 relaxation enhancement in comparison to PEG-IONP, a passively targeted nanoparticle.6. Taking advantage of multiple interactions between carboxyl-containing drugs and PEI, PNA with size from tens to hundreds nanometers were facilely prepared by dialysis. The existence of hydrogen bonding, hydrophobic interaction, electrostatic interaction between drugs and b PEI25 was confirmed by molecular simulation, FT-IR and 1 h- NMR measurements. Based on the formation of hydrophobic region in nanoassemblies of CBD/ b PEI25, NCBD was successfully encapsulated to offer positively charged nanoassemblies such as PTX/IND/b PEI25.7. PNA exhibited good biological effects in vitro that the IC50 values of PTX nanoformulations were significantly smaller than those of the PTX solution formulation against both sensitive and resistant cancer cells after 24 h of incubation, especially for multi-drug resistant cells.8. PNA-loaded YS such as IND YS and PTX YS were facile prepared by encapsulation of PNA(IND/b PEI25 or PTX/IND/b PEI25). PTX YS was rapidly recognized and phagocytosed by macrophages, and a high concentration of PTX was determined 1 h after co-incubation of PTX YS with Raw264.7.9. In vitro release and in vivo pharmacokinetic tests suggested that IND YS can significantly improve the release rate of insoluble drugs and highly improve the bioavailability of IND, affording remarkably enhanced values of area under the plasma drug concentration-time curve(AUC), which was even superior over IND/b PEI25.10. After oral administration of IND YS, IND YS can significantly increase IND concentration in monocytes/ macrophages, and showed targeted drug distribution in swelling paws based on the recruitment of macrophages during inflammation.11. To illuminate the therapeutic significance of IND YS, we implemented in vivo pharmacodynamic(PD) studies in both acute and chronic inflammatory models. Whereas oral administration of various IND formulations was able to mitigate the swelling degree of acute paw edema induced by carrageen and chronic arthritis model induced by the complete Freund’s adjuvant in SD rats, the YS delivery system showed more superior activity even than IND nanoassemblies, which showed a stronger therapeutic than raw IND.12. Studies on in vivo antitumor activities of various PTX formulations were conducted in MCF-7 xenograft-bearing nude mice. After oral administration, we found that PTX YS had obvious tumor targeting effect. After continuous oral administration at the same dose, the concentration of PTX of PTX-loaded YS(PTX YS) was 7.8 folds higher than that of PTX solution, leading to more excellent tumor inhibition.13. QD YS was mainly absorbed at Peyer’s Patch and firstly taken up by microfold cells(M cells), and then phagocytosed by macrophage that could subsequently deliver YS to MLNs and lymph system.14. Eight hours after QD YS oral administration, non-specific QD distribution was found in splenectomized mice, while QD YS in normal mice showed significant targeting effect in swelling paws and monocytes/macrophages. Surprisingly, we found that QD YS did not present specific macrophage distribution in healthy mice.15. Laminarin given by oral administration and intravenous injection could significantly inhibit QD YS distribution in swelling paws and monocytes/macrophages. Further, we found that oral administration of laminarin can inhibit the intestinal absorption of QD YS at Peyer’s Patches.Conclusions1. YS composed of β-1,3-D-gulcan was successfully obtained. PNP and PNA can be successfully loaded in YS to form PNP-loaded YS and PNA-loaded YS by electrostatic attraction. YS can be specifically recognized and phagocytosed by macrophages based on Dectin-1 receptor.2. YS can be orally targeted to monocytes/macrophages in vivo, showing enhanced imaging in inflammation and tumor sites based on the recruitment of macrophages to the diseased locations. This indicated that YS-based systems are promising for cell-mediated targeted imaging.3. We discovered a facile, convenient, cost-effective, and easily scalable one-pot assembly strategy to formulate various lipophilic therapeutics into PNA based on multiple interactions between CBDs and PEI. PNA can be self-deposited in YS to form PNA-loaded YS with high drug loading capability.4. PNA-loaded YS can deliver drugs to inflammation and tumor locations by the recruitment of macrophages to the diseased sites, which can highly improve in vivo antiinflammatory efficacy and anti-tumor activity of loaded drugs.5. After oral administration, QD YS was mainly adhered and absorbed at Peyer’s Patches by Dectin-1 receptor on M cells. Then QD YS was transported into macrophages at the intrinsic layer of the intestinal wall and MLNs by Dectin-1 receptor on macrophages. In MLNs, QD YS was delivered to the spleen through the lymphatic system and then delivered to peripheral blood as well as inflammation and tumor sites by the migration of spleenderived monocytes and macrophages.In summary, we discovered a facile, convenient, safe and efficient macrophage targeted oral drug delivery system by stimulating the way employed by microbes upon infection in the GI tract. By the recruitment of monocytes/macrophages in inflammation and tumor, fluorescent agents, MRI contrast agents, and various therapeutics can be successfully targeted to diseased sites. The preparation of this oral targeted drug delivery system is simple, efficient, easily scalable, and widely suitable for various drugs, which is promising for future applications. In addition, the present study provides new insights into the design of novel oral delivery systems for targeted imaging and therapy of other macrophage-related diseases. |
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