Lipid membrane-camouflaged biomimetic nanoparticle for MicroRNA based therapeutic delivery to intestinal epithelial cells

In addressing drug delivery systems targeted to the gut region, several things must be considered, as the delivery agent must withstand the large pH gradient in the gastrointestinal (GI) tract, especially the highly acidic environment of the stomach. The complexity and variability of the microbiome has a repertoire of factors capable of assimilating/neutralizing/degrading a wide range of therapeutic agents. Other significant hurdles before the nanocarrier can release its drug payload are the intestinal mucosal layer and its absorption capacity¹. Although encapsulation of nanocarriers must be performed to prevent their premature degradation and absorption in the gastrointestinal (GI) tract, tracing the delivery agent throughout the tract offers major advantages for precise site-specific targeting.

The morphology and size of nanoparticles also play an essential role in effective drug delivery. The nanoparticles having the morphology of the long nanorod type stay for a longer duration of time as compared to the spherical type of nanoparticles². The rod shape has a higher surface area for adsorbing a larger adsorbed payload as compared to spherical particles³. The major issue associated with the delivery of miRNA is its high affinity towards water, leading to its rapid diffusion. The specifically designed nanocarrier may improve the encapsulation efficiency of the miRNA⁴. The approach of the nanocarrier-based drug delivery may occur through receptor-mediated endocytosis, which could further reduce the drug dosage and the associated side effects manyfold⁴.

Targeting specific regions of the gut and achieving personalized precision therapy requires a combination of strategies. These include nanocarriers that respond to multiple physiological stimuli (such as pH and redox conditions), systems that adapt to the gut microbiome by responding to specific bacterial enzymes, and customizable delivery platforms tailored to an individual’s gastrointestinal profile⁵. For example, pH-responsive drug release may vary according to food intake, disease status, and, more importantly, the gut microbiome, which can influence the drug release profile, including the rate of release as well as the time of release⁶. On the other hand, non-invasive external stimuli such as temperature, magnetic field, light, ultrasound, can control the release of drugs more precisely and also release at the specific desired location with external control for effective localization of drugs, thereby reducing the off-target drug delivery. For example, core-shell zinc oxide–iron oxide nanoparticles (ZnO@Fe₃O₄) nanocomposites combine the photocatalytic and drug-loading capacity of ZnO with the magnetic responsiveness of Fe₃O₄. When exposed to an external magnetic field, these nanoparticles can accumulate at target sites (magnetic targeting) and trigger drug release via localized heating or mechanical disruption, enabling precise spatiotemporal control^7,8.

External stimuli can be more rationally regulated by the release of drugs at the targeted site as compared to the internal stimuli present in the GI tract. Therefore, magnetic nanoparticles could be effectively used as a delivery agent in the gut region because they can be traced to know when to apply the external stimulus to release the drug payload. Nowadays, various modern techniques are applied to monitor and detect magnetic nanoparticles, such as magnetic resonance imaging (MRI)⁹ and magnetic particle imaging (MPI)¹⁰. Recently, AC bioseptometry has been widely used to monitor magnetic nanoparticles specifically targeting the gut region^{11,12,13,14,15}.

There are many interrelated factors like size, shape, concentration¹⁶route of administration¹⁷, and type of synthesis method¹⁸ that are actually taken into consideration to avoid the side effects of ZnO nanoparticles for in vivo studies, as here biocompatibility is not a fixed attribute. However, it has been previously observed that certain modifications of ZnO nanoparticles, such as surface coating or incorporation of ZnO nanoparticles into chitosan-based nanofibrous mats, could improve their biocompatibility as well as antimicrobial properties¹⁹ Additionally, the pH of the GI region where ZnO nanoparticles are targeted can further affect biocompatibility, for example, in the acidic environment present in case of tumors, the rate of solubilization of ZnO nanoparticles is faster, resulting in rapid release of ions (Zn²⁺/Mn²⁺) that can also trigger oxidative stress and cytotoxicity. On the other hand, at neutral pH, the solubilization of ZnO nanoparticles is comparatively slow, which potentially reduces their cytotoxicity²⁰. We can interplay these factors to harness the therapeutic potential of ZnO nanoparticles.

ZnO nanoparticles size less than 100 nm are considered biocompatible, widely employed for in vivo studies, and are accepted in commercial cosmetic products²¹. The ZnO nanowires are also reported to be completely dissolved inside the body^22,23. In this context, ZnO-based magnetic nanoparticles could be considered as a potential candidate for drug delivery in the gut region.

In this context, ZnO-based magnetic nanoparticles, due to their pH-responsive dissolution and magnetic targeting capabilities, represent a promising platform for site-specific drug delivery in the GI tract, particularly in regions such as the colon where controlled release is essential. However, long term in vivo behavior would be essential for future prospects especially in terms of toxicity studies with the release of Mn²⁺ on the GI tract.

Furthermore, in case of GI targeted drug delivery, one of the barriers present in the GI tract is the mucous layer of the intestine, which does not allow the diffusion of drugs due to its viscoelastic microenvironment with adhesive nature. Hence, it is essential to modify the surface of nanoparticles for spatially localized delivery of drugs so that they can reach the targeted site. For example, Ensign et al. have shown that making the surface of nanoparticles more hydrophilic with a coating of PEG increases their probability of penetration through the mucus layer, making them more available for epithelial absorption with higher transcellular absorption²⁴. Similarly, Lai et al. have demonstrated that modified nanoparticles with mucus penetrating ability have a 10-fold increased probability of epithelial absorption compared to unmodified counterparts²⁵. These modifications are important in terms of the bioavailability of drugs applied via the oral route of drug administration for in vivo studies with increased solubility and penetration ability across the gut epithelial barrier²⁶.

Mucus-penetrating nanoparticles are postulated increase the cellular uptake at the intestinal lining for better absorption for more effective drug delivery²⁷. The application of AcMF increases the Brownian motion of nanoparticles by magneto-thermal effect, enhancing mucus-penetration and passing through the mucus barrier and shown to penetrate to the colon tumor site²⁸. In a widely used animal model of colitis (DSS-induced colitis), miR-200c was reported to be significantly enhanced in our previous report²⁹. After seven days of oral DSS (3%) administration, intestinal tissue miR-200c increased significantly (> 30 fold), while occludin mRNA levels decreased in tandem²⁹. Through recycling intestinal perfusion permeability assessment of isolated intestinal segments of live mice and intestinal tissue histology, the therapeutic administration (oral-gastric gavage) of high dose antisense antagomir-200c prevented the development of colitis and the increase in intestinal permeability to dextran 10kd that DSS caused in mice²⁹. According to these findings, intestinal permeability can be raised simply by increasing the expression of miR-200c-3p in mice intestinal epithelial cells.

The GI tract presents numerous absorption barriers that limit the effective delivery of drugs to target sites³⁰. The mucus layer presents a barrier to nanoparticle transport across intestinal tissue^31,32. To protect miRNAs from degradation in the harsh GI environment, encapsulation within nanocarriers is a promising approach^33,34. Various materials are used for encapsulation, the best methods are Liposomes. These lipid-based vesicles can encapsulate miRNAs and protect them from enzymatic degradation^35,36. Polymers like polyacrylates can be used to formulate nanoparticles that protect miRNAs and allow for controlled release³⁷. Milk-derived exosomes can deliver tumor-activated doxorubicin prodrugs and could be engineered for miRNA delivery to gut epithelium but it also targets other cell types e.g. hepatocytes³⁸. Previous examples include Laminarin, a polysaccharide, coating of nanogene complex can be used for oral delivery of miRNA-223 to target macrophage polarization in inflammatory bowel disease³⁹. Oral administration of miR-320-3p embedded in DiOleyl-Succinyl-Paromomycin (DOSP) can alter miRNA levels in the gut and brain⁴⁰. Oral administration of osa-miR168a, a plant miRNA, has shown to attenuate dextran sulfate sodium-induced colitis in mice⁴¹. There is no platform for delivering miRNA to gut epithelium or targeting epithelium specifically. Cell membrane-coated nanoparticles (CMNPs) are a promising strategy for targeted drug delivery and cell-specific targeting homotypic cells (cells of the same type), exploiting a phenomenon known as homotypic targeting⁴². Our approach to use a positively charged ZnO core to assemble miRNA payload as well as lipid from Caco-2 cells which is an intestinal epithelial line is to exploit ZnO nanoparticles surface area and affinity to negatively charged molecules, protecting and holding miRNA (agomir/antagomir) under liposome protection and biosimilar membrane based homotypic targeting to similar gut epithelial cells.

In the present study, magnetic ZnO nanoparticles doped with Manganese (Mn⁺⁺) ions nanosized in the range of 17 to 50 nm coated with miR-200c-3p agomir with Caco-2 cell membrane coating have been fabricated to apply the targeted miRNA delivery to the gut region.

We exploited known relationship between miR-200c agomir and occluding and used this to probe delivery efficacy of these bionic nanoparticles to the epithelial cells of the small intestine.

Synthesis of ZnO nanoparticles

Undoped ZnO nanoparticles and Mn-doped ZnO nanoparticles (ZnO: Mn) were synthesized by two different approaches viz. solid-state reaction method (SSR) (18) and precipitation method of the highly alkaline aqueous medium as previously described^43,44.

Briefly, in the case of the SSR method, the precursors zinc acetate dihydrate (Loba Chemie, India) and manganese acetate (Loba Chemie, India) were used to synthesize the nanoparticles of ZnO and ZnO: Mn. The dopant substitution fraction range was varied from x = 0.02 to 0.20. The aqueous blend of precursors was dried thoroughly in an oven; subsequently, the nanoparticles of ZnO and ZnO: Mn were synthesized in a thermal furnace at 500 °C in the presence of air⁴⁴.

The precipitation method involves synthesizing ZnO and ZnO: Mn nanoparticles at room temperature. In the aqueous solution of zinc acetate and manganese acetate, ammonia solution was added to create a highly aqueous environment of pH 10 to fabricate the nanoparticles through precipitation. Mn concentration in ZnO: Mn was varied from x = 0.02 to 0.1⁴⁴. MSA (Merck, India) was used as a capping agent to stabilize the active surface of the synthesized nanoparticles to prevent their agglomeration following the method as described previously^43,44.

Magnetic measurements were conducted using Vibrating-sample magnetometry (VSM) (Lakeshore 73504, USA) instrument. The measurements were conducted with the accuracy of 10⁻⁶ m emu/g, carried out at room temperature and in the magnetic field of up to 5000 Oe. The results were plotted after subtracting diamagnetic background of the sample holder. Magnetic measurements were performed for the nanoparticles of ZnO, ZnO: Mn (2%), ZnO: Mn (5%), ZnO: Mn (10%) and ZnO: Mn (15%) synthesized by solid state reaction method. ZnO, ZnO: Mn (2%), and ZnO: Mn (10%) were prepared by the precipitation method.

Cytotoxicity of zno: Mn nanoparticles

Human colon carcinoma Caco-2 cells were stored routinely in small aliquots in liquid nitrogen and the experimental cultures were prepared from the frozen stock cells and always kept in a sub-confluent state⁴⁵. The cells were cultured in DMEM with GlutaMax containing 1.5% glucose and supplemented with 10% FBS, 1% MEM non-essential amino acids and 10 mM HEPES buffer⁴⁶. The cells were cultured in a saturating humidified atmosphere of 5% CO₂ in air at 37 °C. The culture medium was changed every 3–4 days. The cultures were used for testing within 10 passages after the cells were received from ATCC. Cells were grown to 80% confluence and prepared for experimental procedures. They were washed with HBSS, harvested, and a single cell suspension was prepared. The cell suspension was added to 96-well plates and incubated for 24 h at 37 °C. Dosing solutions of 100-nm ZnO: Mn nanoparticles were prepared and added to the cells. The cells were exposed to ZnO: Mn nanoparticles at concentrations of 0.01, 0.1, 1.0, 10.0, and 20.0 µg mL–1. The cells were treated for 24 h at 37 °C in a saturated humidity atmosphere. The ZnO: Mn nanoparticles was tested in three independent culture experiments. The cytotoxicity of the ZnO-Mn nanoparticles was measured fluorometrically using the resazurin reduction assay⁴⁷. Using the Sigma Resazurin Assay kit, the cells were seeded in 96-well plates, treated with the vehicle control and the desired concentrations of the ZnO: Mn nanoparticles for 24 h at 37 °C, and then rinsed with sterile HBSS before being incubated with resazurin⁴⁸ for 30 min at 37 °C in a plate reader. Resorufin fluorescence was measured at 545 nm excitation and 590 nm emission to determine the viability as per kit Manuel.

AgomiR loading

miRNA was obtained from Qiagen (Qiagen, Germantown, MD). MiRNA (25 nM) was mixed with an equal volume of Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA) prepared in 25 µL Optimized Minimum Essential Medium (Opti-MEM), (Thermo Fisher Scientific, Waltham, MA) and incubated at room temperature for 10 min. Lipofectamine-miRNA complex solution 50 µL were incubated with Magnetic Nanoparticle Vesicles (MNV) (5 × 1012 particles/mL, 50 µL) for 30 min at room temperature (RT). The solution was diluted with 4 mL PBS and ultra-centrifuged at 100,000 g using a Type 60 Ti swing rotor for 70 min at 4 °C. The pellet was re-suspended in an appropriate volume of PBS for further studies.

Preparation and characterizations of Caco-2 cell membrane fragments and tCNVs/miR200c

Caco-2 cells (passage-18) were purchased from ATCC (Manassas, VA). Caco-2 cells were cultured in T650 large flasks to obtain enough cells. It took cells 14 days to reach a 70% confluency and were harvested by adding an EDTA-Trypsin solution (Gibco, USA), washed two times with PBS and collected by centrifugation at 5000 g for 10 min. 2 × 10⁶ macrophages per flask (4 flasks) were harvested.

Membranes of Caco-2 cells were isolated by repeated freeze thawing and sonication. Harvested cells were suspended in 10 times diluted PBS (0.1× PBS pH 7.4) with protease inhibitor cocktail (thermo USA). Samples were cyclically frozen in liquid nitrogen and then thawed at 37 °C 10 times. The sample was briefly sonicated to fragment the isolated membranes. Lipid fraction were isolated by centrifugation at 10,000 × g for 10 min followed by PBS wash. The fraction was resuspended in cold PBS by brief sonication and stored at 4 °C for maximally 1 day before further use. miR200c agomir coated or blank ZnO NPs were incubated with enterocyte membranes in 24-well plates. The coated particles were again centrifuged at 10,000 × g for 10 min and suspended in PBS till used.

AgomiR-200c delivery to the mice intestinal epithelial cells

All animal experiments were conducted following protocols approved by the Institutional Animal Care and Use Committee of The Pennsylvania State University College of Medicine (IACUC protocol no. PROTO201800216), Hershey, PA, USA. All procedures complied with the Animal Welfare Act and the recommendations of the Office of Laboratory Animal Welfare (OLAW). The full ARRIVE guideline checklist was submitted to the journal to demonstrate compliance with ARRIVE guidelines. Wild-type in-house bred male C57BL/6J mice (Original Stock No. 000664, Jackson Laboratory) were used for MIRACLE miRNAs Antagomir/Agomir as well as negative control (NC) Antagomir/Agomir were obtained from AcceGen Biotech (NJ, USA). All mice were housed in autoclaved polycarbonate cages (maximum 5 per cage) with autoclaved corncob bedding and nesting material for enrichment. Animals were maintained under specific pathogen-free (SPF) conditions in the Penn State College of Medicine animal facility with a 12-hour light/dark cycle, controlled temperature (22 ± 3 °C), and relative humidity (59 ± 5%). Mice had ad libitum access to autoclaved water and were fed a standard chow diet prior to weaning and an AIN-93 M purified diet post-weaning. Total RNA was isolated using the miRNeasy kit (Qiagen, Valencia, CA) and reverse transcription was performed using the QuantiTect reverse transcription kit (Qiagen, Valencia, CA) according to manufacturer’s protocol. The real-time PCRs were performed using PikoReal 96 Real-Time PCR system (Thermo Scientific, Waltham, MA). The Agomir-200c effect on intestinal permeability in an in vivo mouse model system was determined using a recycling intestinal perfusion method^29,49. Frozen mouse tissue sections were fixed and processed for Laser capture microdissection as previously described^29,49. On average, approximately 1000 cells were obtained per microdissection cap and total RNA was isolated^29,49.

Among all the transition metal ions, Mn shows more solubility in ZnO while preserving its nanocrystalline structure⁵⁰. Thus, Mn-doped ZnO nanoparticles were synthesized and studied for the possible targeted drug delivery to the gut region. The present study was conducted to reveal the preferred method to synthesize suitable magnetic ZnO nanoparticles for targeted drug delivery. We have selected ZnO nanoparticles due to its anticipated biocompatibility, antimicrobial activity, and pH responsive behavior with the ability of differential dissolution at different pH, these properties make the nanoparticles suitable for drug delivery into the GI tract^51,52. In addition, we have incorporated Mn nanoparticles, as their magnetic behavior is essential for controlled drug delivery by external stimuli, as well as, to make detection possible via imaging techniques such as magnetic resonance imaging (MRI) for future perspectives^53,54. Additionally, the incorporation of Mn may also provide the ability to reduce reactive oxygen species, which may increase the antioxidant activity of the nanoparticles to reduce the cytotoxicity associated with the use of metal oxide nanoparticles⁵⁵. Moreover, compared to commercially available nanoparticles such as gold and silver nanoparticles, which are of high cost with low biodegradability, these Mn doped ZnO nanoparticles can be more economical, have relatively high biodegradability and can be easily synthesized by the given methods⁵⁶.

Structural analysis using scanning electron microscopy (SEM analysis)

Scanning Electron microscopy (SEM) study indicated the nano spherical morphology of the ZnO nanoparticles synthesized by the SSR method (Fig. 1.a, b.) with an average size of 50 nm. Typical nanorods morphology could be observed for ZnO nanoparticles synthesized by alkaline aqueous solution method (Fig. 1.c, d) with the average nanocrystal size in the range of 17 to 40 nm. The synthesized ZnO nanoparticles exhibited the typical wurtzite hexagonal structure^43,44.

SEM image depicting spherical nanoparticle of ZnO (a) and ZnO: Mn (5%) (b) synthesized through solid state reaction method; while nanorod formation of ZnO nanoparticles could be visualized in both ZnO (c) and ZnO: Mn (2%) synthesized by alkaline aqueous method (d).

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The change in the crystal parameters indicated that dopant electronegativity and ionic radii influence the growth of nanoparticles, as previously described by Field^43,44. Theoretically, the rod shape of nanoparticles would have more available area for surface-adhesive interactions as compared to the spherical nanoparticles. Because of the high surface area, the nanorods of ZnO nanoparticles become more reactive; they perhaps can stay for longer periods of time and have high circulation time. Thus, they can be appropriately delivered to the deeper region of the tissue and can sustain for longer periods of time, enhancing their biological availability with relatively high clearance time⁵⁷. As reported elsewhere, due to the higher aspect ratio of ZnO nanorods, compared to their spherical morphology, they effectively align with the cell membrane resulting in higher endocytosis⁵⁸. As a result, we have preferred nanorod morphology of ZnO nanoparticles for penetration of the gut barrier including mucus layer and tight epithelial junctions especially to accomplish GI targeted drug delivery. For example, Sharma et al. have demonstrated that ZnO nanorods refereed higher drug absorption owing to their superior mucus penetrating ability due to their high transcellular uptake⁵⁹. Thus, it could be deduced that rod shape morphology could effectively deliver the drugs as compared to the spherical morphology of nanoparticles⁶⁰ by perhaps reducing the drug dosage and drug resistance.

Moreover, the nano size range of the synthesized nanoparticles (~ 50 nm) was found to be appropriate to deliver drugs in the gut region⁶¹. The magnetic properties of the as-synthesized nanoparticles of ZnO and ZnO: Mn were further studied to visualize the ferromagnetic behavior induced by the dopant.

Magnetic properties of the nanoparticles synthesized by solid state reaction method

In the case of the SSR method, the magnetic measurements of nanoparticles were conducted on the 2%, 5%, 10%, and 15% Mn-doped ZnO samples at room temperature.

In the present study, the result (Fig. 2a) characterized the diamagnetic behavior of ZnO nanoparticles as that of bulk ZnO. However, it has also been reported elsewhere that the change in electronic configuration also induces magnetism in ZnO nanoparticles without any doping⁶². However, here, the synthesized ZnO nanoparticles with an average size of 50 nm are relatively large and may not lie in the critical size range responsible for showing magnetic behavior; therefore, the undoped ZnO did not exhibit magnetic properties.

Magnetization curve of nanoparticles synthesized by solid state reaction (SSR) method (a to f), showing the ferromagnetic behavior of ZnO: Mn (5%) with typical hysteresis loop at room temperature (c) while diamagnetic behavior could be observed in ZnO nanoparticle both in capped (f) and bare ZnO (a); ferromagnetic behavior could not be observed in nanoparticles of ZnO: Mn (2%) (b), ZnO: Mn (10%) (d) and ZnO: Mn (15%) (e).

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Magnetic properties were also not observed at 2% doping of Mn ions (Fig. 2b), indicating that by using high thermal treatment of the synthesis, magnetism cannot be induced in ZnO nanocrystals with 2% Mn doping. However, at the higher concentration, as Mn ions begin to be incorporated into the ZnO nanocrystals, the ferromagnetic behavior can be clearly visible with a typical S-shaped hysteresis loop in the case of 5% Mn doping (Fig. 2c). This magnetic behavior was not found due to magnetic impurities as no signal of that was observed in the XRD study⁴⁴. The electronic structure of the ZnO semiconductor may be modified due to Mn doping and/or defects⁶³. The ferromagnetism in the 5% Mn-doped ZnO (Fig. 2c) is possibly due to the different valence states of the transition element of the Mn ions attributing to the double exchange^62,64,65. This provides compelling evidence, albeit indirect, that magnetization is not due to any precipitating secondary phase, as Mn-related secondary phases are antiferromagnetic.

Magnetization decreases rapidly with the further increase in Mn concentration (Fig. 2d, e), indicating that the magnetic moment of the transition metal ions is expected to show paramagnetic behavior at higher concentrations. The observed behavior arises due to spin alignment within a nanocrystal.

Although, it has been reported previously that the magnetic properties of the ZnO nanoparticles may not be only due to metal ions, but defects might also be responsible for imparting the magnetism⁶². Moreover, the type of capping agent was also found to be co-related with the type of defects and ferromagnetic properties at room temperature^66,67. The capping material, especially thiol containing capping agent, may lead to the transfer of the charge to the ZnO nanoparticles which might induce magnetic properties⁶⁸. However, in the present study, obtained magnetization curve was not found largely different for capped and uncapped ZnO nanoparticles (Fig. 2a and f). This may be due to the average size of nanoparticles (50 nm) which may not allow the change in electronic configuration without the presence of dopant.

Therefore, in our case, the undoped ZnO nanoparticles do not show any change in electronic structure even after capping with MSA; as a result, ferromagnetic behavior was absent. While magnetic measurements revealed that doped ZnO (5% Mn) showed typical ferromagnetic character, indicating that Mn doping induced the room temperature ferromagnetism to the ZnO nanoparticles.

Magnetic properties of the nanoparticles synthesized by alkaline aqueous solution method

In the case of the alkaline aqueous solution method, the magnetic measurements of nanoparticles were conducted on the 2% and 5% Mn-doped ZnO along with undoped ZnO samples at room temperature.

Similar to the SSR method, the undoped ZnO nanoparticles prepared by the alkaline aqueous solution method have shown diamagnetic behavior (Fig. 3a), while ferromagnetic behavior could be observed in 2% doping of Mn (Fig. 3b). Again, ferromagnetic behavior could not be observed at higher concentrations of Mn ions (Fig. 3c).

Magnetization curve of nanoparticles synthesized by alkaline aqueous method (a, b,c), showing the ferromagnetic behavior of ZnO: Mn (2%) with typical hysteresis loop at room temperature (b) while diamagnetic behavior could be seen in both bare ZnO (a) and ZnO: Mn(10%) (c).

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In the case of the SSR method, the ferromagnetic behavior was observed at 5% Mn doping in ZnO nanoparticles, while in the case of the alkaline aqueous solution method, the magnetic behavior was observed at 2% doping of Mn at room temperature. Different thermal annealing environments may influence the induced magnetic properties⁶³. Furthermore, inhomogeneity may be the result of the assembling of different Mn ions valences (Mn³⁺ and Mn⁴⁺) and differential magnetic interactions with nonmagnetic Zn²⁺ ions.⁶⁴ The nanorod morphology of the ZnO nanoparticles has a relatively more active surface area and, thus, is more receptive for the interphase reaction. As a result, in the alkaline aqueous solution method, the thermal desorption of Zn ⁺ ions and defects promote greater diffusion of ions and therefore the magnetic behavior could be seen at lower Mn concentrations. These parameters provide control for better diffusion of Mn ions hence resulting in magnetic properties⁶⁵. Interestingly, ferromagnetic behavior was not observed at extremely low or high Mn concentration in the ZnO nanoparticles.

Drug delivery agents could be effectively targeted to the desired region of the tissue if regulated by the external stimuli to release the drug’s payload⁶⁹. Here, we extrapolate that the ZnO magnetic nanoparticles could be traced using imaging techniques, and bio conjugated MSA-capped ZnO nanoparticles could be targeted to the site to release drug payload, reducing the side effects and drug dosage¹¹. Magnetic nanoparticles of iron oxide having super-paramagnetic behavior have been previously reported to be used for the diagnosis of metastasis and even used clinically for the surgical treatment of cancer-related to the esophagus and gastric area. However, in our case, the as-synthesized nanoparticles have diluted magnetic semiconductor behavior, so there would probably not be side effects as those of using stronger magnetic iron oxide nanoparticles^70,71,72.

Room temperature ferromagnetic Mn doped ZnO nanoparticle exhibit biocompatibility and non-cytotoxic behavior as reported elsewhere⁷³. Moreover, it has also been reported that Zn²⁺ ions and reactive oxygen species (ROS), if released in the application of targeted drug delivery, may have shown anticancer and antimicrobial activity due to electrostatic interactions occurring between the cell membrane and ZnO nanoparticles⁷⁴. The ZnO nanoparticles with intrinsic magnetic and photoluminescence properties with the capacity to integrate multiple moieties possibly enable the precise delivery of the drug and possibly could be used as therapeutics. Therefore, synthesized nanoparticles could be potentially used as nano-theragnostic, which denotes both “Therapeutics” and “Diagnostics” using nanoparticles^57,75.

The cytotoxicity of the nanoparticles could be further reduced by optimization of the nanoparticle dosage used in drug delivery vehicles. Their higher concentration could induce changes in the cell membrane properties as well as reduce the cellular adhesion capability. In addition, there may be unexpected interactions between biological molecules and nanoparticles that can be minimized by studying potential interactions in simulation analysis. Ferromagnetic Mn-doped ZnO nanoparticles exhibit biocompatibility⁷⁶ and, thus, could be applied as magnetically guided delivery vehicles for therapeutic applications. The prospect of magnetic nanoparticles is enormous; however, it depends on the systematic synergistic study of pharmacokinetics and dosage to formulate non-toxic nanocarriers together with computational analysis for the prediction of biological interactions with nanoparticles^76,77. We were not able to discern batch-to-batch variation in the nanoparticle synthesis. Another limitation of magnetism estimation is the possible diamagnetic background, which we were not able to subtract. There was no apparent toxicity of Mn-doped ZnO nanoparticles on Caco-2 cell lines in vitro. Future studies are required to assess toxicity in normal intestinal cells and other organ systems through in vivo and ex vivo studies.

Nanoparticle-AgomiR-200c-delivery causes an increase of miR-200 C expression and an increase in intestinal permeability

We next examined the use of these nanoparticles for delivery of Agomir 200c-3p in mouse intestinal permeability in vivo. We utilized these ZnO nanoparticles, to load the AgomiR-200c, and the size & shape of these nanoparticles were confirmed using Transmission electron microscopy (TEM. The miRNA-ZnO nanoparticle were coated with the intestinal epithelial cell membrane (Caco-2 cell membrane) fragments (Fig. 4). The nanoparticles were coated with Caco-2 cell membrane by sonication and breakage of Caco-2 cell membrane components and reconstitution of the cell membrane on the miRNA-ZnO nanoparticle. To validate the delivery of cell membrane-coated ZnO nanoparticles to the mouse intestinal mucosal surface, in these studies, we used high-resolution confocal microscopy to demonstrate the miRNA-ZnO nanoparticle delivery to the intestinal epithelial cells. The intestinal epithelial cell-coated, miRNA-loaded ZnO nanoparticles were localized in high concentration at the mouse intestinal cell surface compared to the ZnO nanoparticles without the bilipid cell membrane coating.

Schematic illustration of the construction of a Caco-2-cell membrane camouflaged nanoscale delivery system and its application in intestinal epithelial cells.

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Additionally, the effect of oral-gastric gavage of cell membrane-coated ZnO nanoparticles loaded with AgomiR-200c on mouse intestinal permeability was determined by recycling perfusion of an isolated segment (6 cm) of small intestine in-vivo, using fluorescein isothiocyanate–labeled dextran (10 kilodaltons) as a paracellular marker. Oral gavaging of AgomiR-200c coated nanoparticle caused a 28-29-fold increase in miR-200c expression in the intestinal enterocytes’ cells (Fig. 5a) and a corresponding decrease in intestinal enterocytes cells occludin mRNA (Fig. 5b). AgomiR-200c also caused an increase in small intestinal permeability to fluorescein isothiocyanate– dextran (Fig. 5c). Because intestinal tissue consists of a variety of cell types that could disproportionately affect tissue miR-200c or occludin mRNA level, laser capture microdissection was used to isolate a pure population of intestinal epithelial cells from the intestinal mucosal surface. 28 The enterocytes were captured by a 7.5-m diameter laser beam (pulsed at a duration of 0.5 milliseconds). 28. These in vivo studies indicated that the Oral gavage administration of AgomiR-200-coated nanoparticles was associated with an increase in enterocyte miR-200c miRNA activity and a decrease in occluding mRNA level.

The effect of Nanoparticle-agomiR-200c on mouse small intestinal permeability in-vivo. AgomiR-200c induced increase in microRNA-200c expression (A), induced decrease in occludin mRNA expression (B), and induced increase in mouse intestinal permeability(C). Data represent mean values from n = 3 animals per group. Unpaired two-tailed Student’s t-tests were used to compare treatment and control groups. A p-value < 0.05 was considered statistically significant. Due to the small sample sizes typical of pilot in vivo studies, degrees of freedom were limited and are available upon request.

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Nanoparticle-AgomiR-200c-delivery safety considerations

In our previous studies, we have administered a 200X concentration of antagomir, which we have now formulated. There was no toxicity, and we observed amelioration of colitis in a DSS injury mouse model²⁹. The carrier system used is not new, and numerous studies have employed the same⁷⁸. The limitation of this study is lack of complete safety profiling, metabolic fate and effect of this antagomir on other cell types. There is need of elaborate studies addressing the same.

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Authors and Affiliations

1. Division of Gastroenterology and Hepatology, Department of Medicine, The Penn State University College of Medicine, Hershey, PA, 17033, USA

   Manmeet Rawat, Yash Gupta & Thomas Y. Ma

2. Department of Biotechnology, Delhi Technological University, New Delhi, India

   Sharda Nara

3. Division of Gastroenterology and Hepatology, Department of Internal Medicine, University of New Mexico School of Medicine, Albuquerque, NM, 87131, USA

   Gulshan Parasher

4. Department of Medicine, Department of Molecular & Precision Medicine, The Penn State University College of Medicine, 500 University Drive, Hershey, PA, 17033, USA

   Manmeet Rawat

Contributions

Conceptualization, M.R., S.N.; data curation, M.R., S.N., Y.G.; formal analysis, M.R., S.N., Y.G. T.M., and G.P.; funding acquisition, M.R.; investigation, S.N., M.R.; methodology, M.R., S.N., Y.G.; supervision, M.R.; visualization, M.R., S.N.; writing-original draft, M.R., S.N., Y.G.; writing-review and editing, M.R., S.N., Y.G., T.M. and G.P.

Corresponding author

Correspondence to Manmeet Rawat.

Competing interests

The authors declare no competing interests.

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