Patent Application
20220033767
Red blood cells (RBCs; also called erythrocytes), are the most abundant cellular constituent of blood and a natural drug delivery system in vertebrates (Yoo et al., 2011). Erythrocytes are special cells in various aspects: they are biconcave shaped, isolated cells without organelles, which exclusively serve as biological carriers. They only exist to distribute and transport various compounds contained within their volume and extended membrane surface. RBCs are responsible for oxygen delivery (O2) throughout the body. They show prolonged circulation through the vascular system for up to 3 months within mammals and have access to not only components within the blood (Yoo et al., 2011), but also the endothelium and reticuloendothelial system (RES) under physiological conditions. RBCs play a pivotal medical role in various fields including transfusion medicine, or the regulation of the adaptive immune system (e.g. by carrying anti-inflammatory agents or inhibitors of phagocytes). Due to their physiological impact and properties, including biocompatibility, abundance and longevity in circulation, RBCs have been explored over the past decades as carriers of various compounds and nanoparticles and served as an inspirational source of novel functional assemblies and advanced architectures for biomedical applications (Yan et al., 2017; Villa et al., 2016; Villa et al., 2015; Magnani et al., 2014; Wang et al., 2014). Examples range from coupling drugs onto the surface of erythrocytes to improve their delivery and therapeutic effects (Mukthavaram et al., 2014; Godfrin et al., 2012; Pierige et al., 2008), to the attachment of nanoparticles (NPs) onto RBC membrane to alter the circulation behavior of NPs (Brenner et al., 2018; Anselmo et al., 2013; Pan et al., 2018; Anselmo et al., 2015), the encapsulation of RBCs with nanometric films to modulate immune response (Mansouri et al., 2011; Wang et al., 2014; Park et al., 2017; Kim et al., 2016; Park et al., 2016), and the embedding of magnetic NPs in the interior of RBCs to enable magnetic alignment and guidance (Wu et al., Wang & Pumera, 2015).
To date, different strategies have been developed for RBC engineering: 1) Genetic engineering, which causes RBCs to express therapeutic proteins for the treatment of different diseases (Chao & Liu, 2018); 2) Surface grafting, which modifies the RBC membrane by coupling of drugs/targeting agents (Shi et al., 2014); 3) Hypotonic loading, which employs formation of transient pores in plasma membranes in hypotonic solutions to allow the subsequent loading of drugs or NPs in the RBC inner volume (Yan et al., 2017; Villa et al., 2016); 4) Surface hitchhiking, where functional NPs are noncovalently attached to the membrane (Brenner et al., 2018; Anselmo et al., 2013; Pan et al., 2018; Anselmo et al., 2015); 5) Cell-in-shell, which employs encapsulation of RBCs by a nanometric artificial shell, e.g. made of polyelectrolytes, polydopamine, or iron-phenolic networks (Mansouri et al., 2011; Wang et al., 2015; Park et al., 2017; Kim et al., 2016; Park et al., 2016; Park et al., 2014).
Nevertheless, RBCs remain highly sensitive and instable, biological structures that strongly depend on the environment such as the tonicity of chemicals and reaction conditions as well as handling, which strongly hampers current RBC engineering approaches. In order to truly turn RBCs into multifunctional supernanocarrier with externally tunable functions and properties, several limitations needs to be overcome: Current protocols are limited by (i) multiple processing steps and/or long preparation time, (ii) they strongly require the optimization of most material synthesis conditions (such as pH, temperature, precursor concentration) to avoid RBC lysis, and (iii) the lack of versatile capability to endow RBCs with multiple functionalities for biomedical applications.
To overcome these limitations, a general strategy was designed to generate stabilized, multifunctional cell-based supercarriers, such as RBC-based supercarriers, that are externally tunable based on a superassembly approach where nanometric metal-organic frameworks (MOFs) act as functional building blocks.
In one embodiment, a modified vertebrate cell, e.g., a viable vertebrate cell having an intact cellular membrane and one or more functions of an unmodified (native) corresponding vertebrate cell, is provided that comprises a coat of reversibly interlinked metal-organic framework (MOF) nanoparticles. In one embodiment, the modified cell comprises a mammalian cell. In one embodiment, the modified cell comprises a human cell. In one embodiment, the modified cell comprises a red blood cell. Red blood cells have a long life in the body, e.g., from about 100-120 days, and can traverse small capillaries, are about 40-50% of the blood volume, and can be easily purified, and so are desirable substrates for preparing armored cells. In one embodiment, the modified cell is a non-adherent cell. In one embodiment, the modified cell comprises a hematopoietic cell. In one embodiment, the modified cell comprises a B cell, T cell or macrophage cell. In one embodiment, the MOF nanoparticles comprise ZIF-8, MIL-100(Fe), UiO-66, UiO-66-NH2, magnetic iron oxide (Fe3O4) NPs@ZIF-8, or mesoporous silica NP@ZIF-8, or a combination thereof. In one embodiment, the nanoparticles have a diameter of about 100 nm to about 500 nm. In one embodiment, the nanoparticles have a diameter of about 100 nm to about 200 nm. In one embodiment, the nanoparticles have a diameter of about 200 nm to about 300 nm. In one embodiment, the nanoparticles have a diameter of about 400 nm to about 500 nm. In one embodiment, the nanoparticles have a diameter of about 450 nm to about 600 nm. In one embodiment, the nanoparticles further comprise covalently attached moieties. In one embodiment, a dye including a fluorescent or luminescent molecule may be attached to the nanoparticles to allow for detection (e.g., imaging or tracking in vivo), e.g., FITC, RITC, Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 647, or DyLight 800. In one embodiment, the mesopores in the nanoparticles further comprise a moiety, e.g., a diagnostic, e.g., DAR-1, or a therapeutic moiety, e.g., an anti-cancer agent such as doxorubicin. In one embodiment, the mesopores are loaded before the nanoparticles are linked. In one embodiment, the moiety comprises an optically detectable molecule, a diagnostic molecule or a therapeutic molecule. In one embodiment, the diagnostic molecule comprises a contrast agent, e.g., an agent comprising iodine, barium, gadolinium, manganese, or microbubbles, or a magnetic molecule.
In one embodiment, a method of making modified viable vertebrate cells comprising a coat of reversibly interlinked metal-organic framework (MOF) nanoparticles is provided that includes combining a population of vertebrate cells, e.g., intact, functional red blood cells, and a population of MOF nanoparticles under conditions that allow for attachment of the MOF nanoparticles to the surface of the cells, thereby providing a mixture; and adding an amount of a ligand to the mixture under conditions that allow for interconnecting the MOF nanoparticles. In one embodiment, the MOF nanoparticles have a negative charge ranging from −3.0 to −30 mV in 0.2× PBS. In one embodiment, the ligand comprises tannic acid, epigallocatechin gallate, epicatechin gallate myricetin, quercetin, quercetagetin, eupafolin, luteolin, scutellarein, dicaffeoylquinic, theaflavin, heaflavin-3′-gallate (TF1). theaflavin-3, 3′-digallate (TF2) or a combination thereof In one embodiment, the MOF nanoparticles are in an acidic isotonic buffer that alters the zeta potential of the MOF nanoparticles. In one embodiment, the MOF nanoparticles are in a buffer that ranges from pH 5 to pH 7. In one embodiment, the MOF nanoparticles are in a buffer that has a pH less than 7.4.
In one embodiment, the armored cells may be employed to deliver a drug, e.g., doxorubicin, olaparib, altretamine, capecitabine, cyclophosphamide, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, pemetrexed, topotecan, paclitaxel, or docetaxel, a sensor or any biomolecule cargo (e.g., a protein such as an enzyme, or nucleic acid including but not limited to DNA or RNA) which may be loaded in the pores of MOFs, such as those formed of Fe3O4 or Au, or which include MSNs. The MOFs may be further modified to provide for additional functionalities, e.g., so that the armored cells can be detected when they are in a body, can be used as a diagnostic or as a therapeutic delivery vehicle. For instance, if an antibody is attached to the MOF nanoparticles, the armored cell binds to the target of the antibody, or if a prodrug is attached to the MOF nanoparticles, the corresponding drug can be delivered, or a combination thereof which can lead to targeted drug delivery.
In one embodiment, a method of making vertebrate cells comprising a coat of reversibly interlinked metal-organic framework nanoparticles is provided that includes providing a population of viable vertebrate cells, a population of MOF nanoparticles and a ligand; and adding the MOF nanoparticles and ligand, e.g., sequentially, to the cells under conditions that allow for interconnecting the MOF nanoparticles. In one embodiment, the MOF nanoparticles have a negative charge ranging from −3.0 to 30 mV in 0.2× PBS. In one embodiment, the ligand comprises tannic acid, epigallocatechin gallate, epicatechin gallate myricetin, quercetin, quercetagetin, eupafolin, luteolin, scutellarein, dicaffeoylquinic, theaflavin, heaflavin-3′-gallate (TF1), theaflavin-3, 3′-digallate (TF2) or a combination thereof In one embodiment, the MOF nanoparticles are in an acidic isotonic buffer that alters the zeta potential of the MOF nanoparticles. In one embodiment, the buffer ranges from pH 5 to pH 7.
Also provided is a method to disrupt linkages in vertebrate cells comprising a coat of reversibly interlinked metal-organic framework (MOF) nanoparticles, comprising contacting a population of vertebrate cells comprising a coat of reversibly interlinked metal-organic framework nanoparticles with an agent that disrupts the linkages. In one embodiment, the agent comprises a metal chelator. In one embodiment, when armored RBCs squeeze through tiny capillaries in the vasculature (e.g., lung vasculature), the cardiac blood output causes shear force and directs RBC to endothelium contact, thus facilitating transfer of MOF nanoparticles from RBC surface to the pulmonary capillary endothelial cells.
Bio/artificial hybrid nanosystems based on biological matter and synthetic nanoparticles holds great promises to revolutionize the field of nanomedicine. Herein, armored cells such as ‘Armored Red Blood Cells’ (Armored RBCs), which are native cells super-assembled and protected by a functional exoskeleton of interlinked metal-organic framework nanoparticles (MOF NPs), are described herein. MOFs are periodic and atomically well-defined porous crystalline materials that are typically self-assembled by metal nodes and organic ligands, offering structural diversity, high surface area, tunable porosity, and due to their hybrid nature, the ability to independently functionalize the external and internal surfaces (Furukawa et al., 2013; Denny et al., 2016; Horcajada et al., 2012). This enables the design of MOFs for a spectrum of applications including gas storage and separation, water harvesting, sensing, energy, drug delivery, and acting as nanobuilding blocks for the construction of complex hierarchical nanoarchitectures.
For example, Armored RBCs described herein preserve the original properties of RBCs, and inherit those of MOFs NP, e.g., show enhanced resistance against external stressors. By modifying the physicochemical properties of MOF NPs, Armored RBCs provide diagnostic properties like blood nitric oxide sensing or contrast for multimodal imaging. The synthesis of Armored RBCs is reliable and reversible allowing for stepwise disassembly into distinct building blocks. Its general applicability allows its application to not only any kind of MOF nanoparticles but also to any cell type. The armored cells described herein enlarge the tool box of hybrid nanomedicines to unlock their potential for different fields ranging from biomedical imaging detection and therapy to targeted 3D micropattering in cells and even personalized medicine.
In one embodiment, RBCs are encapsulated, e.g., the surface engineered with functional, modular, MOF nanobuilding block-based exoskeletons (
The invention will be further described by the following non-limiting example.
Reagents. All chemicals and reagents were used as received. Zinc nitrate hexahydrate, 2-methylimidazole, zirconium(IV) chloride, 2-aminoterephthalic acid, dimethylformamide (DMF), iron(III) chloride hexahydrate, trimesic acid, tetraethyl orthosilicate (TEOS), (3-aminopropyl)triethoxysilane (APTES), ammonium hydroxide, ammonium nitrate, hexadecyltrimethylammonium bromide (CTAB), cyclohexane, tannic acid, ethylenediaminetetraacetic acid, fluorescein isothiocyanate (FITC), iron(III) acetylacetonate (Fe(acac)3), benzyl alcohol, methanol, Ham's F-12K (Kaighn's) medium, Iscove's modified Dulbecco's media (IMDM), formaldehyde solution (36.5-38% in H2O), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich. Alexa Fluor™ 647 NHS ester (succinimidyl ester) and DyLight 800 NHS ester were purchased from Thermo Fisher Scientific. Heat-inactivated fetal bovine serum (FBS), 10× phosphate-buffered saline (PBS), 1× trypsin-EDTA solution, and penicillin-streptomycin (PS) were purchased from Gibco (Logan, Utah). Dulbecco's modification of Eagle's medium (DMEM) was obtained from Corning Cellgro (Manassas, Va.). Absolute ethanol was obtained from Pharmco-Aaper (Brookfield, Conn.; 200 proof). Milli-Q water with a resistivity of 18.2 MSΩ cm was obtained from an inline Millipore RiOs/Origin water purification system.
Characterization methods. Scanning electron microscopy (SEM) analyses and energy-dispersive X-ray spectroscopy (EDS) elemental mappings were performed on a Hitachi SU-8010 field-emission scanning electron microscope at 15.0 kV. Transmission electron microscopy (TEM) and high-resolution TEM (HRTEM) imaging were carried out using a Hitachi model H-7650 transmission electron microscope at 200 kV. Wide-angle powder X-ray diffraction (PXRD) patterns were acquired on a Rigaku D/MAX-RB (12 kW) diffractometer with monochromatized Cu Kα radiation (λ=0.15418 nm), operating at 40 KV and 120 mA. The UV-Vis absorption spectra were recorded using a Perkin-Elmer UV/vis Lambda 35 spectrometer. Fluorescence emission measurements were carried out using a fluorescence spectrometer (Perkin-Elmer LS55). Three-color images were acquired using a Zeiss LSM510 META (Carl Zeiss MicroImaging, Inc.; Thornwood, N.Y., USA) operated in channel mode of the LSM510 software.
UiO66-NH2 NPs synthesis. UiO-66-NH2 NPs were synthesized following previously reported methods (Yoo et al., 2011) with no modification. Briefly, 25.78 mg ZrCl4 (0.11 mmol) and 14.49 mg 2-aminoterephthalic acid (0.08 mmol) were dissolved in 10 mL of DMF solution. Then 1.441 g acetic acid (0.024 M) was added into the above solution. The mixed solution was placed in an oven (120° C.) for 24 h. After cooling down the reaction mixture to room temperature, obtained NPs were subsequently washed with DMF and methanol via centrifugation redispersion cycles. The synthesized UiO-66-NH2 NPs were stored in EtOH before use.
MIL-100(Fe) NPs synthesis. MIL-100(Fe) NPs was synthesized following previously reported methods with no modification (Yan et al., 2017). Briefly, 2.43 g iron(III) chloride hexahydrate (9.0 mmol) and 0.84 g trimesic acid (4.0 mmol) in 30 ml H2O were mixed in a Teflon tube, sealed, and placed in the microwave reactor (Microwave, Synthos, Anton Paar). The temperature of the mixed solution was increased from room temperature to 130° C. under solvothermal conditions (P=2.5 bar) within 30 seconds, and then kept at 130° C. for 4 minutes and 30 seconds, and finally cooled down again to room temperature. The synthesized NPs were centrifuged down and then washed twice with EtOH. The dispersed NPs were allowed to sediment overnight. The remaining supernatant of the sedimented suspension was filtrated (filter discs grade: 391, Sartorius Stedim Biotech) three times to finally yield the MIL-100(Fe) NPs. The synthesized MIL-100(Fe) NPs were stored in EtOH before use.
Mesoporous silica NPs (MSN) synthesis. MSN NPs was synthesized following previously reported methods in our group with no modification (Villa et al., 2016). Briefly, 0.29 g of CTAB (0.79 mmol) was dissolved in 150 mL of 0.51 M ammonium hydroxide solution in a 250 mL beaker, sealed with parafilm (Neenah, Wis.), and placed in a mineral oil bath at 50° C. After continuously stirring for 1 h, 3 mL of 0.88 M TEOS solution in EtOH and 1.5 μL APTES were combined and added immediately to the mixed solution. After another 1 h of continuous stirring, the particle solution was stored at 50° C. for another 18 h under static conditions. Next, the solution was passed through a 1.0 μm Acrodisc 25 mm syringe filter (PALL Life Sciences, Ann Arbor, Mich.) followed by a hydrothermal treatment at 70° C. for 24 h. To remove the CTAB, the synthesized MSN NPs were transferred to 75 mM ammonium nitrate solution in ethanol, and placed in an oil bath at 60° C. for 1 h with reflux and stirring. The MSN NPs were then washed in 95% ethanol and transferred to 12 mM HCl ethanolic solution and heated at 60° C. for 2 h with reflux and stirring. Finally, MSN NPs were washed in 95% ethanol, then 99.5% ethanol, and stored in 99.5% ethanol before use.
Fe3O4 NPs synthesis. Bare Fe3O4 NPs was synthesized following the reported methods with no modification (Villa et al., 2015). Briefly, 0.687 g of Fe(acac)3 (1.94 mmol) was dissolved in 9 mL of benzyl alcohol. The mixed solution was heated to 170° C. with reflux and stirring at 1500 rpm for 24 h. After the reaction was cooled down to room temperature, 35 mL EtOH was added into the mixed, and then centrifuged at 20000 rpm for 10 min. The supernatant was discarded, and the resulted precipitate was washed with EtOH twice to yield to the Fe3O4 NPs. The synthesized Fe3O4 NPs were stored in EtOH before use.
MSN@ZIF-8 NPs synthesis. 2.5 mg MSNs were suspended in 2.5 mL water. Next, 250 μL of Zn(NO3)2 (0.134 M) and 1 mL of 2-MIM (0.219 M) were subsequently added into the solution. The mixed solution was stirred for 0.5 h, and then centrifuged at 20000 rpm for 10 min. The supernatant was discarded, and the resulted precipitate was washed with EtOH twice to yield to the NPs. The synthesized MSN@ZIF-8 NPs were stored in EtOH before use.
Fe3O4@ZIF-8 NP synthesis. 2.5 mg Fe3O4 were suspended in 2.5 mL water and then 250 μL of Zn(NO3)2 (0.134 M) and 1 mL of 2-MIM (0.219 M) was subsequently added into the solution. The mixed solution was stirred for 0.5 h, and then centrifuged at 20000 rpm for 10 min. The supernatant was discarded, and the resulted precipitate was washed with EtOH twice to yield to the NPs. The synthesized Fe3O4@ZIF-8 NPs were stored in EtOH before use.
All the animal procedures complied with the guidelines of the University of New Mexico Institutional Animal Care and Use Committee and were conducted following institutional approval (Protocol 11-100652-T-HSC and 17-200658-HSC). Human RBCs were acquired from healthy donors with their informed consent. All blood samples were collected and stored in BD Vacutainer® blood collection tubes (Becton Dickinson, N.J., USA) containing 1.5 mg of EDTA per mL of blood for anticoagulation purposes. The purification of whole blood was carried out using Ficoll® density gradient centrifugation procedure (Magnani & Rossi, 2014).
Synthesis of Armored RBC-UiO-66-NH2. 5 million RBCs were suspended in 500 μL of 1× PBS (pH 5) solution containing 400 μg/mL UiO-66-NH2 NPs . After 10 s vortexing and 30 s of incubation, 500 μL of 40 μg/mL tannic acid in 1× PBS (pH 5) solution was added with 30 s vigorous mixing. The formed Armored RBC-UiO-66-NH2 was then rinsed with 1× PBS (pH 7.4), and stored in 1× PBS (pH 7.4).
Synthesis of Armored RBC with MIL-100(Fe) NPs coating. 5 million RBCs were suspended in 500 μL 1× PBS (pH 5) solution containing 200 μg/mL MIL-100 NPs. After 10 s vortexing and 20 s of incubation, 500 μL of 32 μg/mL tannic acid in 1× PBS (pH 5) solution were added with 30 s vigorous mixing. The formed Armored RBC-MIL-100(Fe) was then rinsed with 1× PBS (pH 7.4), and stored in 1× PBS (pH 7.4). This process represents a typical procedure for single MIL-100(Fe) NP shell formation and could be repeated one or two times to achieve multi-layered coating.
Synthesis of Armored RBC-MSN@ZIF-8. 5 million RBCs were suspended in 500 μL 1× PBS (pH 5) solution containing 400 μg/mL MSN@ZIF-8 NPs. After 10 s vortexing and 20 s incubation, 500 μL of 32 μg/mL tannic acid in 1× PBS (pH 7.4) solution were added with 30 s vigorous mixing. The formed Armored RBC-MSN@ZIF-8 was then rinsed with 1× PBS (pH 7.4), and stored in 1× PBS (pH 7.4).
Synthesis of Armored RBC-Fe3O4@ZIF-8. 5 million RBCs were suspended in 500 μL 1× PBS (pH 5) solution containing 250 μg/mL Fe3O4@ZIF-8 NPs. After 10 s vortexing and 20 s incubation, 500 μL of 40 μg/mL tannic acid in 1× PBS (pH 7.4) solution were added with 20 s vigorous mixing. The formed Armored RBC-Fe3O4@ZIF-8 was then rinsed with 1× PBS (pH 7.4), and stored in 1× PBS (pH 7.4).
Native and Armored RBCs were rinsed with 1× PBS (pH 7.4) solution and then suspended in 1× PBS (pH 7.4) solution at room temperature for 7 days. After centrifugation (300 g, 5 min), the absorbance of hemoglobin in the supernatant was measured by a BioTek microplate reader (Winooski, Vt.) at 540 nm to calculate the hemolysis percentage. Double distilled (D.I.) water and 1× PBS (pH 7.4) solution containing native RBCs were used as positive control (100% hemolysis) and negative control (0% hemolysis), respectively. The hemolysis percentage of each sample was determined using the reported equation (Villa et al., 2016). Percent hemolysis (%)=100×(Sample Abs540nm−Negative control Abs540nm)/(Positive control Abs540nm−Negative control Abs540nm)
The Antigenic protective capability of Armored RBC was assessed by investigating the attenuation of antibody-mediated agglutination of RBCs. Briefly, 1 million native RBC or Armored RBC-MIL-100(Fe) samples were suspended in 450 μL 1× PBS (pH 7.4) solution, and then 50 μL of anti-type sera that included A, B, and Rh were added. After 15 min, the bright field images were taken by Leica DMI3000 B inverted microscope to evaluate the agglutination.
Native and Armored RBC-MIL-100(Fe) were rinsed with 1× PBS (pH 7.4) solution and then suspended in different concentration of NaCl solution (0 to 0.8%, w/v). After centrifugation (300 g, 5 min), the absorbance of hemoglobin in the supernatant was measured by a BioTek microplate reader (Winooski, Vt.) at 540 nm to calculate the hemolysis percentage.
Native RBCs and Armored RBC-MIL-100(Fe) were rinsed with 1× PBS (pH 7.4) solution and then incubated in 1× PBS (pH 7.4) with different concentrations of Triton X-100. After centrifugation (300 g, 5 min), the absorbance of hemoglobin in the supernatant was measured by a BioTek microplate reader (Winooski, Vt.) at 540 nm to calculate the hemolysis percentage.
Native RBCs and Armored RBC-MIL-100(Fe) were rinsed with 1× PBS (pH 7.4) solution and then incubated in 1× PBS (pH 7.4) with different concentrations of Stöber sphere silica NPs at room temperature for 3 h in continuous rotating state. After centrifugation (300 g, 5 min), the absorbance of hemoglobin in the supernatant was measured by a BioTek microplate reader (Winooski, Vt.) at 540 nm to calculate the hemolysis percentage.
The cryopreservation was tested by referring to the reported paper with slight modifications (Wang et al., 2014). Hydroxyethyl starch (HES) were dispersed in 1× PBS (pH 7.4) solution with the concentration of 175.0 and 215.0 mg/mL. 50 million/mL native RBCs and Armored RBC-MIL-100(Fe) were rinsed with 1× PBS (pH 7.4) solution and then suspended in 1× PBS (pH 7.4) solution or HES solution. Each sample was frozen by immersion in liquid nitrogen (−196° C.) for 2 h prior to thawing. Thawing of samples was undertaken by transferring samples to 4° C. in the fridge for a minimum of 2.5 h. Slow thawing process promoted extensively ice recrystallization while ensuring samples were fully thawed. Next, the samples were centrifuged (300 g, 5 min) and the absorbance of hemoglobin in the supernatant was measured by a BioTek microplate reader (Winooski, Vt.) at 540 nm to calculate the cell recovery. Double distilled (D.I.) water and 1× PBS (pH 7.4) solution containing native RBCs were used as the positive and negative controls, respectively. The cell recovery percentage of each sample was determined using the same equation above.
Luminol-Based chemiluminescence was used to evaluate oxygen carrying capacity of RBCs (Mukthavaram et al., 2014). Briefly, 70 mg sodium perborate, 500 mg sodium carbonate, and 200 mg luminol were added to 5 mL water and dissolved by sonication. The luminol solution was left undisturbed for 5 min in a dark room. For imaging purposes, 1 mL of luminol solution was added to 4 mL samples (20 million native RBC or Armored RBCs) in 1× PBS (pH 7.4) solution. The optical image was taken by a Sony ILCE-5100 Camera (ISO-100 and exposure time of 1/15 s). The chemiluminescence optical image was taken in a dark room by a Sony ILCE-5100 Camera (ISO-6400 and exposure time of 30 s). For luminescence assay, 100 μL of samples in 1× PBS (pH 7.4) solution were added into white 96-well plates at a density of 5 million cells/mL. After that, 20 μL of luminol solution was added to each well and the contents were mixed for 2 min on shaker in the dark. Luminescence was measured using a BioTek microplate reader. The luminescence was expressed as a relative.
Capability of reversibly binding oxygen was detected by analyzing changes of UV-Vis absorption spectra (300-700 nm) in oxygenated and deoxygenated solutions. For complete deoxygenation, nitrogen gas was in flown into the sample solution to deplete most of the oxygen. After 2 h, sodium dithionite (Na2S2O4) was added, and UV-Vis absorption spectrum was scanned by a BioTek microplate reader. For oxygenation, sample solutions were exposed to atmospheric oxygen for more than 2 h, and UV-Vis absorption spectrum was recorded. This process represents the typical procedure for reversibly binding oxygen capability. Technical replica amount to 3.
The vascular flow characteristics of Armored RBCs were tested using Ex ovo Chick embryo model as described previously (Villa et al., 2016) and was conducted following institutional approval (Protocol 11-100652-T-HSC). Briefly, eggs were acquired from East Mountain Hatchery (Edgewood, N. Mex.) and placed in a GQF 1500 Digital Professional incubator (Savannah, Ga.) for 3 days. Embryos were then removed from shells by cracking into 100 ml polystyrene weigh boats. Ex ovo chick embryos were covered and incubated at 37° C., 100% humidity. 20 million cells/mL of native RBCs and Alexa Fluor 647-labeled-Armored RBC-MSN@ZIF-8 were incubated in 1× PBS (pH 7.4) solution with 10 mg/mL bovine serum albumin (BSA) for 20 min and then rinsed and stored in 1× PBS (pH 7.4) solution. 100 μL of samples in 1× PBS (pH 7.4) solution were injected into secondary or tertiary veins via pulled glass capillary needles. Embryo chorioallantoic membrane (CAM) vasculature was imaged using a customized avian embryo chamber and a Zeiss Axio Examiner upright microscope with heating stage.
All the animal procedures complied with the guidelines of the University of New Mexico Institutional Animal Care and Use Committee and were conducted following institutional approval (Protocol 17-200658-HSC). The experiments were performed on female Albino C57BL/6 mice (6 weeks). To evaluate the circulation half-life of NPs and Armored RBCs, DyLight 800-labeled MSN@ZIF-8 hybrid NPs and the related Armored RBCs were used. Briefly, both samples were incubated in 1× PBS (pH 7.4) solution with 10 mg/mL bovine serum albumin (BSA) for 30 min and then rinsed and stored in 1× PBS (pH 7.4) solution. 150 μL of NPs (1 mg/mL) and the related Armored RBCs (1 mg/mL NPs on Armored RBCs) were injected into the eye of the mice. The blood was collected at 0.5, 1, 2, 6, 12, and 24 h following the injection. Each time point group contained three mice. The collected blood samples were diluted with the same amount of 1× PBS before fluorescence measurement. Particle retention in circulation at these time points was determined by measuring the fluorescence on a BioTek microplate reader (Winooski, Vt.). Pharmacokinetics parameters were calculated to fit a two-compartment model.
To study the biodistribution of the NPs and Armored RBCs in various tissues, 150 μL of NPs (1 mg/mL) and the related Armored RBCs (1 mg/mL NPs on Armored RBCs) were retro-orbital injected to mice. At 12 and 24 h time points following the particle injection, three mice were randomly selected and euthanized. Their liver, spleen, kidneys, heart, lung, and blood were collected. The collected organs were examined with an IVIS fluorescence imaging system (Xenogen, Alameda, Calif.), and the fluorescence intensity of the NPs and Armored RBCs in different organs was further semi-quantified by the IVIS imaging software.
The Armored RBCs were rinsed with 1× PBS and then suspended in 20 mM EDTA in 1× PBS (pH 7.4) solution for different times (maximum time: 15 min) to allow the controlled destruction of MOF NPs. Then the RBCs were then rinsed with 1× PBS (pH 7.4), and stored in 1× PBS (pH 7.4).
Preparation of NO solution followed a reported protocol in NO sensor studies (Godfrin et al., 2012). 10 mM NaOH and 1× PBS (pH 7.4) solutions were pre-bubbled with nitrogen for 2 h to deplete the dissolved oxygen. NO precursor Diethylamine NONOate sodium salt was added to a 10 mM NaOH solution to make the 500 μM stock solution. The stock solution was diluted with 1× PBS (pH 7.4) solutions to generate various concentrations of NO solutions. The NO-containing PBS solutions were set for at least 15 min to allow the NO concentrations to saturate before NO sensor studying. For in vitro NO study, 2.5 million DAR-1-loaded Armored RBCs were suspended in 1 mL of NO-containing PBS solution. For fresh blood NO study, the collected flash blood samples were first diluted with the same amount of 1× PBS, and then incubated with 1 mL solution containing 2.5 million sensing Armored RBCs. After 5 min incubation, the fluorescence emission spectrum was obtained.
The magnetic Armored RBC-Fe3O4@ZIF-8 were oriented in the direction of an external magnetic field produced by a neodymium magnet. The bright field images were taken by Leica DMI3000 B inverted microscope to evaluate the magnetic guidance.
Three different fluorescently labelled MSN@ZIF-8 NPs were used for modular nanoparticles superassembly. For Armored RBC construction, 5 million RBCs were suspended in 500 μL of 400 μg/mL mixed MSN@ZIF-8 nanoparticles (˜1:1:1 ratio) in 1× PBS (pH 5) solution. After 10 s vortex and 20 s incubation, 500 μL of 32 μg/mL tannic acid in 1× PBS (pH 7.4) solution were added with 30 s vigorous mixing. The formed multi-fluorescent Armored RBCs were then rinsed with 1× PBS (pH 7.4), and stored in 1× PBS (pH 7.4).
Armored RBCs has a striking advantage for the design of multifunctional, hierarchical nano-assemblies employed for drug delivery and molecular imaging. It can revert to the full range of metal organic framework nanoparticles, as functional, robust and modular building blocks. When constructing a multifunctional, protective shell around single RBCs, these MOF-based NP form a fast exoskeleton based on particle-particle super-assembly and interlocking at the proximal RBC membrane surface. The exoskeleton assembly occurs in two steps: at first, MOF NPs attach and concentrate onto the native RBC surface. Since RBC membranes are rich in carbohydrates and proteins, they have a highly negatively charged surface (Bondar et al., 2012). Due to the frangibility and sensitivity of RBCs, a strong interaction between NPs and RBC membranes always causes RBC rupture and hemolysis. MOF NP surfaces comprise well-defined, long periodic arrangements of metal nodes and organic ligands, which allow precise tuning of the coordination and interactions with organic moieties on the RBC membrane surface. Based on zeta potential (ζ) measurements, MOF NPs used within this study (UiO-66-NH2, MIL-100(Fe), and ZIF-8) have a negative charge ranging from −3.0 to −29.1 mV (
As a demonstration of the armored cell concept, individual purified RBCs (
The first impressive property of Armored RBCs is their enhanced cytoprotection against external stressors. To benchmark the protective effect, we exposed armored RBC-MIL-100(Fe) to various harsh environmental conditions including antibody-mediated agglutination, osmotic pressure, detergents, toxic NPs, and freezing conditions (
An important feature of RBCs is their oxygen carrier capability and long-circulation times in blood. To facilitate the in vivo bioapplication of the designed Armored RBCs, Armored RBCs must exhibit the very same behaviors as native RBCs. At first, luminol-based chemiluminescence was employed to reveal the presence of hemoglobin in Armored RBCs (
RBCs are well known to easily traverse the microvasculature with dimensions that are smaller than their size and display long circulation times in vivo. To investigate the circulation behavior of Armored RBCs, real-time fluorescence wide-field imaging was carried out on a chick embryo ex ovo (chorioallantoic CAM model;
To further characterize circulation properties of the Armored RBCs in vivo, albino C57BL/6 mice were used to examine their pharmacokinetic and biodistribution behaviors. Mice were injected with control DyLight 800-labeled MSN@ZIF-8 hybrid NPs and the corresponding MSN@ZIF-8 Armored RBCs by retro-orbital injection, respectively, at a dose of 150 μg NPs/mouse. Syngeneic RBCs were used to create Armored RBCs, negating blood cell type complications. To study the circulation half-life, at various time points following the injection (
Furthermore, to analyze the related biodistribution, at 12 h and 24 h post injection, mice were euthanized and their liver, spleen, kidneys, heart, lungs, and blood were harvested for fluorescence analysis (
In summary, Armored RBCs exhibit enhanced in vivo circulation/residence times compared to many other classes of nanoparticles (Brenner et al., 2018; Anselmo et al., 2013; Pan et al., 2018; Anselmo et al., 2015) and could serve as sources of MOFs (or conceivably other nanoparticles) that could be released and targeted to various organs over time. This could extend the in vivo applications of MOFs and enable targeting and delivery to difficult-to-reach sites in the body.
Given the chemical diversities of MOF nanobuilding blocks, the armored RBC concept can be generally extended based on a plethora of MOF types and combinations. It is basically unlimited in generating diverse functionalities and hence serves as a promising technology to satisfy the growing need of multifunctional nanoparticles in biomedical applications, which we demonstrate in the following section. Depending on the MOF building block, armored MOF shells can (i) not only be created but also be biocompatibly disassembled if necessary. Depending on the underlying MOF NPs, armored RBCs offer (ii) unique physiochemical properties such as optical, magnetic and sensing properties. But most importantly: (iii) the armored RBC concept profits enormously from multiplexing: MOF NP associated properties can be linearly combined within the Armored RBC shell during a mixed super-assembly synthesis.
Armored RBCs hold great promise for drug delivery applications. In order to function as a Trojan horse, the encapsulation shell of Armored RBCs plays a pivotal role. Not only its synthesis, but also its triggered disassembly enables Armored RBCs to serve as a nanoparticle reservoir and source fulfilling the Trojan Horse concept. The designed armored RBCs, based on UiO-66-NH2 MOF NPs exhibit this behavior. Their shell can be disassembled in ethylenediamine-tetraacetic acid (EDTA) solution in a programmed fashion due to the responsive nature of metal-phenolic complexation. The armored MOF shell can be progressively disassembled over the time course of 15 min (as shown by fluorescence microscopy for UiO-66-NH2-covered RBCs labeled with fluorescein isothiocyanate (
To demonstrate the sensor capabilities of Armored RBCs, the modular properties of MOF NPs were used to design Armored RBCs that detect nitric oxide (NO) in blood. NO is a key signaling molecule acting as a potent vasodilator that relaxes the arteries (
Armed RBCs inherit the collective properties of their MOF or other NP building blocks imparting non-native desirable properties. To demonstrate this idea, magnetic Armored RBCs were created that can be externally controlled. Based on metal-phenolic linker chemistry, magnetic Fe3O4 (˜8.0 nm) embedded in ZIF-8 MOFs (
Finally, modular super-assembly of Armored RBCs provides far-reaching possibilities for extensions via co-assembly of different functional MOF- (or NP-) based nano-objects into multimodal nano-structures. These could integrate various, simultaneous functionalities, such as contrast for different imaging modalities, thermal therapies and drug delivery. To introduce Armored RBCs as multimodal super-architecture, multi-fluorescent RBCs were created by incubating native RBCs simultaneously with almost equal concentrations of three different fluorescently labelled MSN@ ZIF-8 NPs in a one-pot process for less than 2 minutes (
A general, simple, and modular approach is described for a class of hybrid biomaterials termed “armored cells” with diverse possible functionalities. Using metal-phenolic chemistry native RBCs were encapsulated with MOF NP-based exoskeletons in seconds without RBC lysis. The modularity and simplicity of this method arises from fast MOF NPs super-assembly at the RBC membrane surface and enables the transformation of different MOF building blocks and RBC vehicles into diverse functional hierarchitectures. Armored RBCs preserve the original properties of native RBCs, show enhanced resistances against external stressors, and exhibit extraordinary new properties that are foreign to native RBCs based on the highly modular nature of MOF nanobuilding blocks integrated into the RBC exoskeletons. The presented approach profits from the wide range of variable MOF NPs and opens the door to design of multimodal nano-superstructures for multimodal imaging, image-guided therapies and theranostics. The strategy of Armored RBCs, however, is not restricted to red blood cells alone, but can be further extended to any other cell types like leucocytes playing a pivotal role in immunity and inflammatory processes. We believe our findings will open new avenues for artificially designed cell-inspired functional materials for wide ranging biomedical applications.
A modified viable (functional) vertebrate cell encased in (comprising a coat of) reversibly interlinked metal-organic framework (MOF) nanoparticles is provided. In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is a human cell. In one embodiment, the cell is a red blood cell. In one embodiment, the cell is a hematopoietic cell. In one embodiment, the cell is a B cell, T cell or macrophage cell. In one embodiment, the MOF nanoparticles comprise ZIF-8, MIL-100(Fe), UiO-66, UiO-66-NH2, magnetic iron oxide (Fe3O4) NPs@ZIF-8, or mesoporous silica NP@ZIF-8, or a combination thereof. In one embodiment, the nanoparticles have a diameter of about 100 nm to about 500 nm. In one embodiment, the nanoparticles have a diameter of about 100 nm to about 200 nm. In one embodiment, the nanoparticles have a diameter of about 400 nm to about 500 nm. In one embodiment, the nanoparticles have a diameter of about 5 nm to about 500 nm, about 10 nm to about 300 nm or about 15 nm to about 250 nm. In one embodiment, the nanoparticles comprise Zn, Fe, Zr, or Co. In one embodiment, the nanoparticles comprise iron oxide. In one embodiment, the nanoparticles comprise Au, Ni, Mn, Ti, W, Mg, Al, Cu or Cr. In one embodiment, the nanoparticles are linked via a metal-phenolic interaction. In one embodiment, the nanoparticles are linked via a boronic acid-phenolic acid interaction. In one embodiment, the nanoparticles comprise silica. In one embodiment, the nanoparticles further comprise covalently attached moieties. In one embodiment, the mesopores in the nanoparticles further comprise a moiety. In one embodiment, the moiety comprises an optically detectable molecule, a diagnostic molecule or a therapeutic molecule. In one embodiment, the diagnostic molecule comprises a contrast agent or magnetic molecule. Also provided is a composition comprising a population of the encased cells. In one embodiment, the composition further comprises a buffer, e.g., the buffer has a pH less than 7.4.
In one embodiment, a method of making vertebrate cells encased in reversibly interlinked metal-organic framework (MOF) nanoparticles is provided. The method includes combining a population of vertebrate cells and a population of MOF nanoparticles under conditions that allow for attachment of the MOF nanoparticles to the surface of the cells, thereby providing a mixture; and adding an amount of a ligand to the mixture under conditions that allow for interconnecting the MOF nanoparticles.
In one embodiment, a method of making vertebrate cells encased in reversibly interlinked metal-organic framework nanoparticles is provided. The method includes providing a population of vertebrate cells, a population of MOF nanoparticles and a ligand; and adding the MOF nanoparticles and ligand to the cells under conditions that allow for interconnecting the MOF nanoparticles.
In one embodiment, the MOF nanoparticles have a negative charge ranging from −3.0 to −30 mV in 0.2× PBS. In one embodiment, the ligand comprises tannic acid, epigallocatechin gallate, epicatechin gallate myricetin, quercetin, quercetagetin, eupafolin, luteolin, scutellarein, dicaffeoylquinic, theaflavin, heaflavin-3′-gallate (TF1). theaflavin-3, 3′-digallate (TF2) or a combination thereof. In one embodiment, the MOF nanoparticles are in an acidic isotonic buffer that alters the zeta potential of the MOF nanoparticles, e.g., the buffer ranges from pH 5 to pH 7. In one embodiment, the cells are red blood cells. In one embodiment, the MOF nanoparticles comprise ZIF-8, MIL-100(Fe), UiO-66, UiO-66-NH2, magnetic iron oxide (Fe3O4) NPs@ZIF-8, or mesoporous silica NP@ZIF-8, or a combination thereof. In one embodiment, the nanoparticles have a diameter of about 100 nm to about 500 nm. In one embodiment, the nanoparticles have a diameter of about 100 nm to about 200 nm. In one embodiment, the nanoparticles have a diameter of about 400 nm to about 500 nm. In one embodiment, the nanoparticles further comprise a covalently attached moiety, e.g., the moiety is a cell targeting moiety, for instance, an antibody or an antigen binding fragment thereof. In one embodiment, mesopores in the nanoparticles further comprise a molecule. In one embodiment, the moiety or molecule comprises an optically detectable molecule, a diagnostic molecule or a therapeutic molecule. In one embodiment, the diagnostic molecule comprises a contrast agent or a magnetic molecule.
In one embodiment, a method to disrupt linkages in vertebrate cells encased in reversibly interlinked metal-organic framework (MOF) nanoparticles, which includes contacting a population of vertebrate cells encased in reversibly \interlinked metal-organic framework nanoparticles with an agent that disrupts the linkages, e.g., a metal chelator.
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All publications, patents and patent applications are incorporated herein by reference. While in the foregoing specification this invention has been described in relation to certain preferred embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein may be varied considerably without departing from the basic principles of the invention.
This application claims the benefit of the filing date of U.S. application No. 62/808,674, filed on Feb. 21, 2019, and U.S. application No. 62/735,585, filed on Sep. 24, 2018, the disclosures of which are incorporated by reference herein.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/052669 | 9/24/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62735585 | Sep 2018 | US | |
| 62808674 | Feb 2019 | US |
