Cancer Treatment Strategies and Challenges Research Paper

Abstract

Exosomes are cell secretory vesicles with a bilayer membrane structure of 30-150 nm in diameter. Their unique structural and intrinsic biological functions such as long blood circulation and immune escaping capability and exceptional biocompatibility, showing compelling potential for use as natural nanocarriers for the delivery of various cargos such as chemicals, proteins, nucleic acids, and gene therapeutic agents. Various advanced design/synthetic strategies have been invested in tailoring exosome-based nanoplatforms for the optimization of diagnostic and/or therapeutic use and mitigation of undesirable side effects in recent years. Nevertheless, making use of both the distinguished inherent benefits of exosomes as natural carriers with cutting-edge nanotechnology with the excellent theranostic performance that can simultaneously achieve the diagnostic and therapeutic with a single “combo” nanoplatform has been rarely reported. Moreover, under or over treatment is hard to prevent because of the lack of imaging method for therapy response real-time monitoring. Herein, for the first time, exosomes biohybrid-based cocktail strategy is designed to achieve targeted robust theranostic with the capability of highly sensitive and molecular specific diagnostic imaging as well as local triple-combination (gene/chemo/photothermal) synergistic tumor therapy. Practically, the intracellular microRNA-2I (miR-2I), a widely-studied novel tumor diagnostic marker, can specifically lead to the conformational change of the conjugated molecular beacon to produce fluorescent signal, allowing miR-2I targeted responsive molecular imaging. Subsequently, according to this diagnosis information, the designer exosome-based nanoplatform was guided to the target tumor site by an external magnetic field. Exosome nanoplatform that accumulated in tumor can generate the localized hyperthermia under near-infrared irradiation, which not only can programmable minimally invasive ablate tumor without affecting surrounding tissue, but also can impact the stability of biological exosomes membrane to promote the release of the encapsulated chemical drug from exosomes on demand. Meanwhile, upon endocytosis, the molecular beacon can hybridize with the cellular miR-2t to form duplex structure and inhibit miR-21 function to promote cancer cell apoptosis. The miR-2I responsive fluorescence can also serve as real-time Eil feedback for the tumor treatment monitoring and evaluation. Experiments conducted both in living organisms and artificial environments demonstrate that our designer exosomes-based cocktail strategy can robustly inhibit tumor growth with high efficiency, and the exosomes cocktails strategy for cancer CI theranostic is expected with promising potential in accelerating the advancement of exosome-based next-generation individualized precision nanomedicines.

Introduction

Cancer is one of the most deadly health conditions, taking millions of lives worldwide. Current cancer treatment strategies such as chemotherapy[refl, immunotherapy[ref], radiotherapy[ref], gene therapy[ref], and thermal therapy[retl, still have many unmet challenges, including relatively low efficacy, dose-limiting toxicities, and systemic side effects. The advent and subsequent refinement of the combination therapy, which integrates two or more of these cancer treatment modalities, significantly improved therapeutic potency to cancerous cells while reducing the side effects. The design of such nanomedicine platforms mainly relies on the integration of different components into one single nano-system. Although these therapeutic effects are promising, more efforts need to be dedicated to exploring a high-performance natural nanoplatform capable of co-delivering various cargos once in all into desired tumor sites, and with optimized biocompatible properties.

Exosomes, endogenous nanosized particles (50-150 nor), secreted by various cells and absorbed by recipient cells, are efficient natural carriers in the biomedical field for therapeutic agents delivery due to their unique structural compositional characteristics and fascinating physicochemical/biochemical properties such as manometer size, low cytotoxicity, satisfactory stability with the double membrane in circulation without opsonization and the capability to overcome the biological barriers and escape immune surveillance. Despite the successful exploration of exosomes for cancer diagnosis and therapy, in recent years, combining cancer diagnosis with therapy simultaneously of exosomes still remains a great challenge.

Inspired by the merits of the natural nanovesicles and the state of the art of the advanced engineering versatility of synthetic nanomaterials, we previously presented the synthetic and biological hybrid exosomes for targeted synergistic chemo and photothermal tumor therapy based on chemical edit membrane strategy. Taking into consideration the more extraordinary virtue of non-invasive early cancer diagnosis, here, for the first time, a new version of multifunctional integrated cocktail biological and the synthetic hybrid designer exosome-based cancer-targeted theranostic platform is described. The motivation for its use is early diagnosis, a higher precision of molecular imaging, accounting for better treatment as well as continuing evaluation of treatment efficacy (Figure 1).

Briefly, we cultured donor cells in normal cell culture medium added with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl(polyethyleneglycol)-biotin (DSPE-PEG-Biotin). DSPE-PEG-Biotin can be inserted into the donor cells membrane themselves and this modification can be naturally inherited by their released exosomes through cellular metabolic pathways. The DSPE-PEG functionalization not only extends the circulation half-life of exosomes encapsulated drugs but also reduces non-specific protein binding or cell adhesion.Later, chemical drugs (DOX) were encapsulated into the isolated exosomes by electroporation. Meanwhile, we coated the magnetic Fe3O4 nanoparticles with polydopamine (PDA) and modified surface with avidin and molecular beacon (MB) by self-polymerization under basic conditions. Practically, Fe3O4@PDA can prompt exosomes to massively accumulate at the tumor site under the magnetic field. Meanwhile, the PDA coated on Fe304 can strongly quench the fluorescence of the MB Upon entering tumor cells, she fluorescent of the MB is switched on due to the specific recognition of the intracellular microRNA-2I (miR-2I), a well-known tumor diagnostic and prognostic marker resulted in simultaneous miR-21 responsive fluorescence imaging and antitumor therapy by forming duplex structure and inhibiting miR-21 function. Therefore, our designer MB combined exosome platform famish the feasibility as a theranostic probe of cancer, and the therapeutic function of miR-21 provide the possibility of real-time monitoring via the molecular imaging without using other large and extensive imaging apparatus, such as magnetic resonance imaging (MRI), single-photon emission computed tomography (SPECT), positron emission tomography (PET), and computed tomography (CT). More importantly, PDA polymerized on the surface of the magnetic beads can realize efficient absorption of near-infrared light and rapid conversion into heat energy. Since the laser spot size is controllable, this photothermal can on-demand selectively destroy cancer cells without affecting the surrounding normal cells, and cause instability of exosomes and promotes the release of encapsulated chemical drugs (DOX). Therefore, our exosome cocktail strategy can simultaneously achieve the local triple chemo/gene/photothermal combination synergistic cancer therapy through on demand-controlled drug release and inhibit tumor relapse in a programmable manner.

To verify the properties of our designed platform, experiments in vitro and in vivo were detailed conducted. We illustrated that our comprehensive theranostic exosomes-based platform possesses the following advantages: (I) This synthetic and biological hybrid designer exosome theragnostic system can be facilely constructed by a donor cell-assisted chemical insert membrane functionalization strategy. (2) This theragnostic system possesses a robust targeting ability under external magnetic control. (3) A specific cancer diagnosis can be accomplished both in laboratory settings as well as in living organisms with high sensitivity tumor-related real-time miR-2 I imaging. (4) The remarkable photothermal properties of this system can destabilize exosomes membranes and on-demand release the encapsulated drugs. (5) It provides programmed synergistic chemo/gene/photothermal therapy and significantly improves the anticancer effect both in artificial and natural settings. This system may potentially make a breakthrough for designing exosomes with a prospective comprehensive nanocarrier system for cancer precise theranostic treatment.

Experimental Section

Reagents and instruments: All the cell-culture medium, FBS, and phosphate-buffered saline (PBS) were bought from Gibco life technologies manufacturer (Grand Island, NY, USA). All the culture consumables needed for cell culture were supplied by Coming (Acton, MA). DAPI, Calcein acetoxymethyl ester (Calcein-AM), propidium iodide (Pt), DOX, phorbol-12-myristate-12-acetate (PMA), avidin and dopamine hydrochloride came primarily from Sigma-Aldrich (St. Louis, MO, USA). Biotin functionalized 1,2-dioleoyl-m-glycerol-3-phosphoethanolamine-poly (ethylene glycol)-2000 (DSPE-PEG-Biotin) was bought from Ponsure Biotech (Shanghai, China). DNA and RNA sequences were synthesized and purified by Sangon biotechnology company (Shanghai, China). All the chemicals purchased for the present research met the reagent grade standards. A Millipore Milli-Q system was used for the creation of ultrapure water (18 M.Q.cm). Testing the hydrodynamic diameters and concentrations of suspended purified exosomes, Fe3O4@PDA, and Exo-Fe304@PDA was made possible thanks to transmission electron microscope (8H-7000FA, Hitachi, Japan) and Nanosight (Malvern Instruments, UK). Lastly, the evaluation of the UV—Vis absorption spectra was made with the spectrophotometer (UV2550, Shimadzu, Japan). The records of Fe3O4@PDA, Fe3O4@PDA-MB and Exo-Fe3O4@PDA-MB were logged with the use of an FTIR spectrometer. FTIR analyses were conducted in terms of pure exosomes at the speed of 2 mm/s by and in the range of 400-4000/cm (VERTEX 70, Broker, Germany).

Cell culture: The American Type Culture Collection (ATCC, Manassas, VA) was the source of the human THP-1 cells. With the addition of 10% (v/v) FBS 1% (v/v) and antibiotic, the RPMI 1640 medium was chosen for the maintenance of the THP-1 cells. The requested circumstances for their incubation included 37 °C in a 95% air/ 5% CO2 atmosphere.

Synthesis of engineered exosomes: The induction of THP-I cells into macrophages was enabled through the incubation with 320 x M PMA for 12 h. With the purpose of macrophages’ modification with biotin, the former were cultured in RPMI 1640 medium containing 10% exosome-depleted FBS, DSPE-PEG-Biotin (50 ug/mL). Its maintenance under normal conditions took three days. The supernatants of starved macrophages cells were removed from the biotin-labeled exosomes after being centrifuged at 120000 g for 2 hours. Later, we created a mixture of the engineered exosomes (2 mg/mL, 50 pL) and DOX in 250 AL electroporation buffers in 0.4 cm cuvette (Bio-Rad). This helped us to load DOX into our engineered exosomes. Bio-Rad Gene Pulser Xcell Electroporation System was used for electroporation at 250 V and 350 pF on a B. Lastly, to restore the membrane of the electroporated exosomes, we proceeded with the incubation of the mixture at 37 T for 30 min.

Synthesis of Fe3O4@PDA: We synthesized of Fe3O4i@PDA by resuspending Fe3O4 nanoparticles (diameter, 5 am) with 05 mL of 10 rnM Trio buffer solution (pH 8.5), then we added 2 mg/mL of dopamine and shaking at room temperature for 3 hours. These actions resulted in a change of color from brown to black. After the reaction, we placed it in an ultrasonic cleaner for three minutes to remove the unreacted dopamine and then centrifuged it at 2000 rpm for 20 minutes, washed it with water three times and resuspend it in PBS. Synthesis of molecular beacon: We dissolved the molecular beacon with a DEPC buffer solution. 5’Cy5GCGCGTCAACATCAGTCTGATAAGCTACGCGC-AA-3′ is our ID molecular beacon (MB) sequence, S’-TAG CTT ATC AGA CTG ATG TTG A -3′ is the DNA sequence of miR-21. Preparation of Exo-Fe3O4@PDA-MB: We incubated Fe3O4@PDA (1 mL) with 100 gL of avidin (1 mg/mL) and MB (100 mM) for 8 hours and then centrifuged this mixed solution at 8000 rpm for 10 mins and washed with water for three times. Finally, we incubated this engineered Fe3O4@PDA with the biotin-labeled exosomes at 4 °C shaking overnight and removed the redundant exosomes by the magnet to obtain our engineered Exo-Fe3O4@PDA.

Characterization of Exo-Fe3O4@PDA-MB: We dispensed the Exo-Fe3O4@PDA-MB ID solution onto a copper mesh covered with carbon film and stained with 2% phosphotungstic 0 acid. The sample was observed by a transmission electron microscope (TEM) of 75 kV (8H-7000 FA, Hitachi, Japan).

Evaluation of the amount of MB attached to Exo-Fm0c@PDA-MB. The attached amount of MB on the Exo-Fe3O4@PDA-MB was measured by the fluorescence of the labeled dye. In particular, we converted the intensity of the fluorescence of the supernatant with free MB to the concentration of the respective MB with a standard linear calibration curve. Known concentrations of MB were used to calculate the standard curve. Lastly, we divided the subtracted concentration of MB by the Exo-FmOrdPDA-MB concentration, which yielded us the average number of MB per particle.

Cytotoxicity of Exo-Fea(X@PDA-MB: HeLa cells (4010C cells/mL, 100 pL per well) in the logarithmic growth phase were seeded in a 96-well plate, which was cultured in 5% CO/ incubator at 37 °C for 12 h. Then, 100 pL PBS with varying levels of concentrations of sterile 0.45 on filter membrane filtered Exo-DOX, Exo-EmOr@PDA, and Exo-DOX-Fm0o@PDA-MB (100 pg/mL) were added respectively. What followed after the four-hour period of incubation was the exposure to MR light (808 nm, IA W/cm2) condition for 1 min and non-stop incubation for 20 h. Conversely, the rest of the groups underwent direct incubation with the samples for 24 h. The treatment above was concluded by the calculation of cell viability. Namely, we added CCK-8 solution (10 9L) and determined its absorbance at 450 mu.

Establishment of Xenografts in nude mice: The establishment of cervical cancer-bearing mouse model started of with the subcutaneous inoculation of HeLa cells (110s cells in 100 pL medium) into the flanks of the male nude mice (18-20 g, four weeks old). We evaluated the size of the xenografts to track the tumor growth every two days. The formula used for the calculation of the tumor volumes goes as follows: Volume = (Length x Width)/2.

Characterization of the targeted ability effect of Exo-Fe3CLI@PDA-MB in vivo: Here, we used the un-fluorescent MB sequence 5′-CFCGCGTCAACATCAGTCTGATAAGCTACGCGC-AA-3′ is our molecular beacon (MB) sequence to modify the exosomes. We used a ph 85 buffer solution for labeling the engineered exosomes by the NHS-Cy55 (mass ratio of 100:1); the procedure took four hours. Once the tumor grew as large as 50 mmt, the tumor-bearing mice were weighed and put into two groups. All of them underwent intravenous injection with un-fluorescent engineered exosomes (5 mg/mL, 200 pL). The experimental group was prescribed magnetic field treatment whereas the control group was not. Injections at 3, 6, 9, 18, 24, and 48 h were followed by imaging using the CRI Maestro in vivo fluorescence imaging system. 48 hours after the start of the treatment, the mice were put down, and their major organs were collected. In administering the aforementioned procedures, we adhered strictly to Huazhong University of Science and Technology’s guidelines for animal research. All protocols were previously approved by the Institutional Animal Care and Use Committee.

We used a 4.0 % (v/v) paraformaldehyde solution for keeping tumors intact overnight and rinsed them twice with PBS to make sure that all excess formaldehyde was removed. Tissue sections were encased in paraffin and dissected into sections (7pm). In preparation for further observations, we applied hematoxylin and eosin (H&E) to them and used an optical microscope (Olympus IX51, Japan) for analysis. Another selected staining method was the TUNEL apoptosis staining, for which the fixed tumor sections were stained by 50 pL TUNEL reaction mixture (Roche) for 60 min at 37 °C as per the manufacturer’s guidelines. The visualization of the cell nuclei was possible thanks to staining with DAPI. We took images of the fixed tumor sections with an inverted fluorescence microscope (IX71, Olympus, Japan).

Characterization of the targeted photothermal effect of Exo-Fe304@PDA-MB: The photothermal conversion effect of Exo-Fe304@PDA-MB was evaluated both in vitro and in vivo. We added 200 gL of the Exo-Fe304@PDA-MB into the well of a detachable 96-well plate and monitored its thermal imaging and recorded its temperature under 808 nm MR laser irradiation to test its photothermal effect in vitro. Furthermore, we injected saline (200 pL) or Exo-Fe304@PDA-MB (200 pl., 5 mg/mL) into mice bearing loLa tumors via tail vein. As mentioned before, the experimental and control groups differed in their treatment with a magnetic field. The distinction was made to show its targeted photothermal effect in living organisms. After two days, the tumors were exposed to radiation by an 808 nm MR laser (ID W/cm2, 6 min). A photothermal imaging system (P1400, Optris, Germany) helped with monitoring the thermal imaging and temperature surge.

Characterization of the MR imaging ability of Exo-Fe3O4@PDA-MB: 3T MRI scanner (MR Solutions Ltd., Guildford, UK) was able to confirm the MRI contrast enhancement effect of Exo-Fe3O4@PDA-MB that was observed both in vitro and in vivo. When it came to experimentation in an artificial setting, we prepared MR1 samples of HeLa cells with various formulations (controls and experimental samples) in plastic dishes. A mouse body coil was key to performing standard TI- and T2-weighted MRI scans (MR Solutions Ltd.). Conversely, experimentation on living organisms required a systematic administration of Exo-Fe3O4@PDA-MB, and the mice were imaged at day 12. Similarly, we acquired standard TI-and T2-weighted MM scans, which were later forwarded to a local workstation. For their analysis, we used a commercial 313 analysis software titled lnveon Research Workplace (IRW) (Siemens HealthCare, Knoxville, TN).

Characterization of the targeted miR-21 imaging of Exo-Fe3O4@PDA-MB: The first step was to test the laboratory detection of miR-21 ability. To achieve this, we incubated twenty microliters (20 oL) of Exo-Fe3O4@PDA-MB with varying volumes of miRNA-21 dissolved in diethyl pyrocarbonate (DEPC) water (100 ttL). The procedure was administered at 37 °C for the designed times in the DNA hybridization buffer (10 mM Tris-HC1, pH=75, and 50 mM NaC1) with the purpose of determining fluorescence intensity. What followed was the collection of the fluorescence spectra from 500 to 700 nm under excitation at 490 nm.

The next step was to confirm the targeted imaging of Exo-Fe30o@PDA-MB ability in living organisms. To do this, the tumor-bearing mice were injected with Exo-Fe3O4@PDA-MB (200 pL, 5 mg/mL) through the tail vein; later, they were treated with a magnetic field. At 3, 6, 12, 24, and 48 h post-injection, HeLa tumor-bearing nude mice were examined using the CRI Maestro in vivo fluorescence imaging system. Two days later, the mice were put down to sleep for organ harvesting.

In ▪ vivo antitumor effects: We created six groups (five mice in each group) and assigned our subjects to them at random. This was done to assess the efficacy of DOX-loaded Exo-Fe3O4@PDA-MB in vivo in fighting cancer cells. The mice were injected with PBS (G1), Exo-DOX (G2), Fe3O4@PDA (G3), Exo-DOX-Fe3O4@PDA (G4), Exo-DOX-Fe3O4@PDA-MB (G5), and Exo-DOX.Fe3O4@PDA-MB plus magnetic field (G6) at a DOX dose of 5 mg/kg. As seen from above, G6 was the only group where mice also underwent a magnetic treatment. The tumors were treated by placing permanent magnets (100 x 50 x 20 mm, 0.6 T) for four hours after the injection. The injections took place for the next three days and were administered up to four times a day. A day post-injection, the tumors were exposed to radiation by the NIR laser (808 non, 1.0 W/cm’) for 6 min every 12 hours. We logged the changes in tumor growth and body weight. The tumors were extracted and documented on camera after the last measurement. A microbalance was used for weighing tumors that were later fixed with 4% paraformaldehyde.

Results and Discussion

Characterization of Exo-Fe₃O₄@PDA-MB

Exosomes can transport molecules that are bioactive, for instance, nucleic acids, as well as proteins between cells and, can cross physiological barriers including blood-brain barrier and extravasate from tumor blood vessels and penetrate into deep tumor tissues without being clearing by the immune phagocytosis system. By virtue of its non-immunogenic characteristic due to similar composition as the body’s other cells and stable lipid bilayer membrane structure, they can maintain the encapsulated drug stability, especially for nucleic acid or protein drugs, and improve the solubility of hydrophobic drugs. Thus, it is not impossible to make use of exosomes of natural systems enabling drug delivery in living organisms. Yet, normal cell-derived exosomes are typically not capable of targeting cancer cells directly, which may compromise their therapeutic efficacy, especially when it comes to complicated procedures. Conversely, tumor-derived exosomes do not share this drawback with cell-derived exosomes. In fact, they are capable of selectively delivering drugs, targeting not only the tumor itself but also metastasis and even the premetastatic niche. Apparently, this phenomenon can be attributed to their tumor-homing properties.

For all their advantages, tumor-derived exosomes should be used with caution. They are involved in unwanted biological processes such as tumor progression, metastasis formation, and drug resistance. For this research, we selected THP-1 macrophages cell as our donor cell (immune cell) for deriving exosomes. It has been found that macrophage-derived exosomes do possess the valuable umor-targeting property. At the same time, they retain topology of plasma membrane proteins and avoid entrapment in mononuclear phagocytes as shown in recent research.

Figure S1 depicts the process of testing the proteins of exosomes using western blot. In the process, we discovered the existence of two typical exosome marker proteins (CD81 and CD63) and Actin protein (control). On the other hand, we were not able to identify the negative exosome marker protein (Calnexin). That allowed us to conclude that the vesicles were indeed macrophage-derived exosomes. We were able to introduce a reproducible and bio-friendly approach to integrate the synthetic nanomaterials with engineered exosomes via a donor cell-assisted membrane modification strategy in alignment with our previously developed method. Through the same approach, the exosomes can be equipped with biotin, which can form the strongest known non-covalent interaction with avidin. In order to collect biotin functionalized donor cells, we incubated the macrophages with DSPE-PEG-biotin (50 µg/mL) for 48 hours. We demonstrated this method feasibility by incubating the biotin modified THP-1 cells and its secreted exosomes with FITC-avidin (50 µg/mL) for 2 hours and removed the excess fluorescent dye by centrifugation for 3 times. The feasibility of this donor cell assisted exosome membrane modification method was analyzed using flow cytometry (Figure S2). The fluorescent of DSPE-PEG-biotin treated cells and exosomes were significantly higher than unmodified cells and exosomes, illustrated that biotin were effective attached on cells and exosomes membrane. Polydopamine (PDA), a melanin-like biopolymer, has a robust binding property with excellent biocompatibility and low toxicity, and easy degradation ability. It can be easily and stably polymerized on the surface of nanoparticles under alkaline conditions. Because it has a large number of catechol groups, it can form a stable π-π bond with DNA molecules and adsorb DNA molecules. Besides, it also can adsorb avidin through Schiffjian base reaction and Michael addition reaction. The mean size of exosomes was 141.2 nm, and the concentration was 1.03 × 10⁹ particles/mL (Figure S3). We watered it down one thousand times for the electron microscope analyses. The transmission electron microscopy (TEM) image of purified exosomes, synthesized Fe₃O₄ nanoparticles, and the Fe₃O₄-functionalized exosomes are demonstrated in Figure 2A. It proved that it was indeed possible to combine Fe₃O₄ nanoparticles and the engineered exosomes. To ensure that the synthesized complex has been functionalized on exosome, we further processed the X-ray photoelectron spectroscopy (XPS) to measure the surface elemental composition (Figure 2B), and Fourier transforms infrared (FTIR) spectroscopy analysis (Figure 2C) to compare pure exosomes, Fe₃O₄@PDA, and Exo-Fe₃O₄@PDA-MB-avidin to investigate the interactions between the Fe₃O₄, PDA, MB and avidin. The characteristic adsorption band at 1545.48 cm^-1 from exosomes was allegedly caused by the Amide vibration. The characteristic adsorption band at 591.03 cm^-1 was attributed to the Fe-O stretching vibration. The complex peaks at 1514.36 cm^-1, 1403.28 cm^-1 and 1302.81 cm^-1 from the Fe₃O₄@PDA are explained by the skeletal stretching vibrations of the phenyl ring in PDA, supported the successful modification of the Fe₃O₄ with polydopamine. The C–O antisymmetric deformation vibration of epoxy group at 878.08 cm^-1 indicating a successful fabrication of avidin and MB onto the Fe₃O₄@PDA. The Exo-Fe₃O₄@PDA-MB-avidin complex both have characteristic adsorption band at 591.03 cm^-1 from exosomes and have characteristic adsorption band at 2923.32 cm^-1 from Fe, clearly proved that the Fe₃O₄@PDA-MB-avidin were put on top of the engineered exosomes.

In recent years, magnetic nanoparticles coated with PDA have been successfully demonstrated to have significant quenching ability for molecular beacons with hairpin structures due to the fluorescence resonance energy transfer (FRET) phenomenon. When the target DNA sequence present, the hairpin structure of the molecular beacon opens and restores the fluorescent signal because the distance between the fluorescent group and the magnetic nanoparticles coated with dopamine is far. MiR-21, one of the most investigated miRNAs to date, is overexpressed in numerous types of cancer and associated with cancer initiation, proliferation, apoptosis, and metastasis.Moreover, the distribution and expression level of miR-21 in cells can be adjusted by responding to their interior genetic program systems or external stimulus.^[9][10][11] Therefore, miR-21 can be used as a tumor diagnostic and prognostic marker, and the inhibition of the over-expressed miR-21 function is envisaged to be a robust strategy for the treatment of cancer.

To demonstrate the specificity of miR-21 detection by our designer exosome hybrid, we first tested its quench ability, as shown in Figure 2D. We added purified exosomes, synthesized Fe₃O₄ nanoparticles, Fe₃O₄@PDA, and exosomes-Fe₃O₄@PDA into Cy5 labeled hpDNA solution. We found that PDA coating could significantly improve the fluorescent quenching ability since the fluorescent of exosomes-Fe₃O₄@PDA and Fe₃O₄@PDA was much weaker than purified Fe₃O₄. Further, we added Exo-Fe₃O₄@PDA with different concentrations (0, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5 mg/mL) to 50 nM Cy5 labeled hpDNA solution and reacted for one hour in the dark environment. Finally, we tested the fluorescence of supernatant after centrifugation. As shown in Figure 2E, the fluorescence value of the supernatant gradually decreased with the increase of the concentration of Exo-Fe₃O₄@PDA. When the Exo-Fe₃O₄@PDA concentration reached to 0.5 mg/mL, the fluorescence almost disappeared, similar to the control PBS group (Figure 2F). After successfully confirming the quenching ability of Exo-Fe₃O₄@PDA, we further investigated the ability of our designed functional Exo-Fe₃O₄@PDA-MB to respond to miR-21. We added different concentrations of miR-21 to Exo-Fe₃O₄@PDA-MB solution (0.5 mg/mL) and reacted for 1 hour in the dark environment, and the fluorescence value of the supernatant was measured after centrifugation (Figure 2G). As shown in Figure 2H, as the concentration of miR-21 increased from 0 nM to 50 nM, the fluorescence value of supernatant increased from 0 AU to 130 AU, which successfully confirmed that our designed Exo-Fe₃O₄@PDA-MB can response to miR-21 efficiently. The hairpin structure of the molecular beacon can be opened and restore the fluorescence of the initially quenched element when miR-21 binds to the recognition site of Exo-Fe₃O₄@PDA-MB. Next, we further tested its ability to suppress miR-21 expression in living cells. The RT-PCR amplification curve of miR-21 (Figure 2I) shows that the Exo-Fe₃O₄@PDA-MB could bind to miR-21 and suppress its expression. Moreover, the miR-21 expression level of HeLa cells treated by 0.5 mg/mL Exo-Fe₃O₄@PDA-MB was significantly lower than treated by 0.2 mg/mL Exo-Fe₃O₄@PDA-MB (Figure 2J).

MiRNA Imaging and Photothermal Ability of Exo-Fe₃O₄@PDA-MB

We first transfected HeLa cells with different concentrations of miR-21 (5 pM, 10 pM, and 20 pM). Then we cultured these cells with the same amount of Exo-Fe₃O₄@PDA-MB (100 pM) for six hours and observed the cell fluorescence using a confocal microscope with an excitation light of 647 nm. The result showed that the fluorescence intensity of Cy 5 increased with the miR-21 concentration surged from 5 pM to 20 pM (Figure 3A). Based on this result, we additionally incubated different amounts of Exo-Fe₃O₄@PDA-MB with the same concentration of HeLa cells for 6 hours, observed the fluorescence of Cy 5 in the cells, and calculated the average fluorescence intensity by flow cytometry. Figure S4 demonstrates that the fluorescence intensity of Cy 5 in the cells increased with the concentration of Exo-Fe₃O₄@PDA-MB climbed from 1 pM to 100 pM, the fluorescence reached saturation when the amount of Exo-Fe₃O₄@PDA-MB was 50 pM. Next, we added our designer Exo-Fe₃O₄@PDA-MB to different types of cells and observed their fluorescence by confocal microscope. As shown in Figure 3B, the expression of miR-21 of tumor cell lines (HeLa cells) is significantly higher than that of normal cells (NIH 3T3 cells), which is also consistent with the quantitative results by flow cytometry (Figure S5). It showed that the Exo-Fe₃O₄@PDA-MB is suitable for different cells and specific to cancer cells.

We have successfully demonstrated the thermo sensitivity of the exosomal membrane is beneficial to realize controlled release of the encapsulated drug in our previous studies. Therefore, we put forward the idea about the possibility of NIR light energy to heat energy conversion using the Fe₃O₄@PDA joined exosomes. This process could make possible the destabilization of the exosome membrane and facilitate drug release (Figure 3C). As shown in Figure 3D, the exosomes membrane could be ruptured by the NIR irradiation. The next point of interest for us was the photothermal activity of Exo-Fe₃O₄@PDA-MB. We observed any alterations in local temperature at different times of laser irradiation. After laser irradiation (808 nm, 2.0 W/cm²) for 3 min, the PBS solution only showed a slight temperature change, slowly increased from the initial 20.1 ℃ to 32.8 ℃. In contrast, the temperature of the Exo-Fe₃O₄@PDA-MB solution dramatically increased from 20.5 ℃ to 69.1 ℃ (Figure 3E). Cancer cells have lower heat resistance than normal cells; high temperatures around 45 ℃ are able to target and destroy cancer cells without doing harm to healthy tissues. The temperature of Exo-Fe₃O₄@PDA-MB can be increased to 40 ℃ within 30 seconds, thereby destroying the exosome membrane and allowing the rapid and massive release of the encapsulated drugs. On the other hand, the local heat energy will increase the permeability of the cancer cell membrane, so that cancer cells can take up drugs more efficiently. Next, we investigated the cytotoxic effects of the Exo-Fe₃O₄@PDA-MB with NIR laser irradiation for 30 seconds on HeLa cells (Figure 3F). It showed that the cell apoptosis rate increased from 8.2 % to 50.4 %, with the concentration of Exo-Fe₃O₄@PDA-MB increased from 1 pM to 200 pM. We further compared the cytotoxic effects of PBS, Exo-DOX, Exo- Exo-DOX-Fe₃O₄@PDA, and Exo-DOX-Fe₃O₄@PDA-MB with and without NIR laser treatment of HeLa cells. Figure 4A contains fluorescent images that show that unlike Exo-DOX, cells treated with Exo-DOX-Fe₃O₄@PDA-MB displayed significantly stronger PI fluorescence signals, although both of them were irradiated with the laser. Besides, we analyzed the in vitro cytotoxicity of Exo-DOX-Fe3O4@PDA-MB. It was done by assessing cell viability through CCK8 assay, which helped us understand the chemo/gene/photothermal therapeutic efficiency of the proposed treatment. As seen in Figure 4B, the treatment yielded better results when it was combined with NIR irradiation. Furthermore, Exo-DOX-Fe₃O₄@PDA-MB plus NIR laser irradiation treatment had the most significant impact on cell viability. Therefore, one may infer that the release of DOX from Exo-DOX-Fe3O4@PDA-MB can be further enhanced by NIR irradiation. Meanwhile, the molecular beacon on Exo-DOX-Fe₃O₄@PDA-MB could bind with the miR-21 and inhibit its function. Another useful property of the NIR irradiation is its ability to boost cytotoxicity against cancerous tissues.

Tumor Targeting of Exo-DOX-Fe₃O₄@PDA-MB In Vivo

The in-vivo performance of the Exo-DOX-Fe3O4@PDA-MB was assessed through a series of experiments on tumor-bearing mice. We administered Cy5.5-labeled Exo-DOX-Fe3O4@PDA-MB (5 mg/mL) injections through the tail veins of the mice. Further, the animals were observed with the help of a non-invasive near-infrared optical imaging method. The method entailed calibrating the excitation and emission wavelengths at 675 and 720 nm, respectively. In the mice that were not exposed to the magnetic field (MF), we did not observe intense fluorescence in the tumor site (Figure 4C). However, when we applied an MF for 48 hours, we were able to detect a robust Exo-DOX-Fe3O4@PDA-MB fluorescence signal. This finding hinted at the outstanding magnetic-targeting properties of Exo-DOX-Fe3O4@PDA-MB. The mice were euthanized 48 hours post-injection, after which their the excised tumor tissues and other major organs were taken for ex-vivo fluorescence imaging. In alignment with the in vivo imaging conclusion, the mice that underwent the Exo-DOX-Fe₃O₄@PDA-MB therapy demonstrated the significantly higher tumor fluorescence signal with the application of the MF, compared with the tumor-bearing mice from the control group that did not undergo MF treatment (Figure 4D). Quantitative methods allowed us to discover that the fluorescence intensity of the tumor with the MF application was approximately 3.5-fold than the cancer without the MF application (Figure 4E). These results verified Exo-DOX-Fe₃O₄@PDA-MB as suitable nanomaterial for active magnetic showing a good success rate killing cancer cells.

Photothermal Efficiency of Exo-DOX-Fe₃O₄@PDA-MB In Living Organisms

Since the Exo-DOX-Fe₃O₄@PDA-MB proved to be well-suited for targeting tumors in vivo, we were compelled to study its tumor-targeted photothermal therapeutic effects on tumor xenograft mice. We used the intravital fluorescence imaging system to identify and log the time‐dependent biodistribution of NIR signals. At 48 hours after injection, the tumors received NIR laser irradiation treatment (Figure 4F). We tested the temperature at tumor after the mice treated with PBS, Exo-DOX-Fe₃O₄@PDA-MB without the application of MF, and Exo-DOX-Fe₃O₄@PDA-MB with the implementation of MF. All these mice were irradiated with NIR laser (808 nm, 1.0 W/cm², 3 min) and being observed by an IR thermal camera. The result illustrated that the tumor temperature of mice that underwent Exo-DOX-Fe₃O₄@PDA-MB with MF application treatment could be increased by 21.33 ± 3.17 °C within 3 min laser irradiation. Conversely, the identical laser irradiation treatment of tumors after an Exo-DOX-Fe₃O₄@PDA-MB injection without the application of MF yielded 16.73 ± 2.88 °C of temperature increment. As the control group, the temperature of the same laser irradiation of tumors administrated with PBS only increased by 9.83 ± 1.15 °C (Figure 4G). All these results showed that the exceptional targeting ability of Exo-DOX-Fe₃O₄@PDA-MB could result in an outstanding photothermal efficacy of tumor in vivo.

Biocompatibility of Exo-DOX-Fe₃O₄@PDA-MB In Vivo

BALB/c mice received Exo-Fe₃O₄@PDA-MB (5 mg/mL, 200 µL) injections into the tail vein to evaluate the biosafety of Exo-Fe₃O₄@PDA-MB in vivo. It was critical to understand how toxic Exo-Fe3O4@PDA-MB could be to mice’s major organs. Therefore, we conducted H&E staining, hematological analysis, and liver/kidney function indices (liver functions indices: alanine aminotransferase (ALT) and aspartate aminotransferase (AST); kidney function indices: creatinine (CRE) and blood urea nitrogen (BUN)), which was revealed in a blood biochemistry test. Figure 5A demonstrates that mice in the Exo-Fe₃O₄@PDA-MB group did not suffer apparent organ injury when compared with those in Exo-Fe₃O₄@PDA, Exo-Fe₃O₄ and PBS (control) group. The results suggested that histological toxicity was truly negligible, proving Exo-Fe₃O₄@PDA-MB treatment to be safe and biocompatible. At the three-week mark, there were no major changes in blood cells, hemoglobin, and platelets, as determined by hematological analysis (Figure S6). Nor did we see any changes in the ALT/AST/CRE/BUN concentration in comparison with the control group (Figure S7). On the basis of these findings, we were able to conclude the biocompatibility of Exo-Fe₃O₄@PDA-MB as a nanocarrier and the absence of grave side effects in living organisms.

MiRNA Imaging of Exo-DOX-Fe₃O₄@PDA-MB In Vivo

MiR-21 is considered to be one of the markers of cancer development, which is why it is crucial to develop non-invasive screening methods of miR-21. This is especially true given that at present, conventional molecular biology techniques have been found to be not exactly effective or applicable for imaging purposes. Here, we tested the targeted miR-21 imaging ability of Exo-Fe₃O₄@PDA-MB in vivo without any fluorescent dye modification. Different xenograft tumor models (mice that underwent MCF-7and HeLa cells treatment) were employed to determine the practical aspects of Exo-Fe3O4@PDA-MB in miRNA-21 imaging. We first investigated the microRNA imaging ability in mice model subcutaneously implanted with HeLa cells.The intravital fluorescence imaging system was utilized to capture the time-dependent biodistribution of Cy5 signals generated by the molecular beacon on the Exo-Fe₃O₄@PDA-MB. As shown in Figure 5B, the overall fluorescence result illustrated that the Exo-DOX-Fe₃O₄@PDA-MB distributed throughout the whole body of the mouse after being injected from the tail vein and was enriched in the tumor within 48 h. The mice put to sleep 48 h after the injection in vivo imaging, and their tissues were excised for ex vivo fluorescence images (Figure 5C). The quantitative result demonstrated that the fluorescence observed in the tumor was more pronounced than in other major organs (Figure 5D). Next, we applied the Exo-DOX-Fe₃O₄@PDA-MB to the mice model subcutaneously implanted with MCF-7 cells (Figure 5E and Figure S8). Intense fluorescence was discovered at the tumor sites compared with the other organs after 48 h tail vein injection. This finding validated the capacity of Exo-DOX-Fe₃O₄@PDA-MB to reliably visualize the miRNA-21 in various cancerous tissues under external magnetic fields. The liver fluorescence was explained by the presence of the phagocytic cells of the reticuloendothelial system engulfed Exo-DOX-Fe₃O₄@PDA-MB.

The Synergistic Combination of Chemo/Gene/Photothermal Effects of Exo-DOX-Fe₃O₄@PDA-MB

We inquired whether Exo-DOX-Fe₃O₄@PDA-MB was capable of fighting cancer in nude mice suffering from a HeLa subcutaneous cancer. At the 10, 13, 16, and 19-day mark, we administered tail intravenous injections with (G1) PBS, (G2) Exo-DOX, (G3) Fe₃O₄@PDA, (G4) Exo-DOX-Fe₃O₄@PDA, (G5) Exo-DOX-Fe₃O₄@PDA-MB and (G6) Exo-DOX-Fe₃O₄@PDA-MB plus magnetic field at a DOX dose of 5 mg/kg. In the mice assigned to G6, the tumor site had the permanent magnets (100 × 50 × 20 mm, 0.6 T) installed for four hours post-injection. One day (24 h) after the injections, all the tumors of mice were exposed to the NIR laser (808 nm, 1.0 W/cm²) irradiation for 5 min every 12 hours. At the end of the treatment, we took pictures of common tumors, which is demonstrated in Figure 5F. The images helped us improve our understanding of the efficacy of various treatments in attacking tumors. When comparing all the treatment groups, we discovered that the Exo-DOX-Fe3O4@PDA-MB plus magnetic field with the NIR irradiation resulted in the smallest tumor size. Thus, it was safe to conclude that Exo-DOX-Fe₃O₄@PDA-MB could be used for chemo/gene/photothermal antitumor treatment. The quantitative analysis of tumor volume and weight were illustrated in Figure 5G and Figure 5H, the targeted triple chemo/gene/photothermal therapy (G6) is far more efficient than other non-targeted single or dual treatments. The mice did not vary much in terms of weight, which could potentially explain their good tolerance for the experimental procedures (Figure 5I). The next step was to evaluate apoptosis in the tumor tissues, which was accomplished by using TUNEL. It denoted the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. As shown in Figure 5J, tumor cells following Exo-DOX-Fe₃O₄@PDA-MB treatment with the magnetic field showed a more significantly higher apoptosis rate and substantial cell remission than tumors treated without a magnetic field, further confirming the Fe₃O₄ nanoparticles mediated targeted therapy on suppressing tumor growth. These results provide convincing evidence that the Exo-DOX-Fe₃O₄@PDA-MB is instrumental to the achievement of superior therapeutic performance.

Conclusion

In summary, we designed for the first time an original theragnostic exosome-based platform that could integrate targeted cancer therapy and miRNA responsive real-time therapeutic efficacy monitoring to prevent under/over-treatment of tumors. This system can be facilely constructed by a biocompatible and safe donor membrane modification technique. The experimental testing of the biological and synthetic hybrid designer exosome theranostic system in laboratory settings and living organisms yielded a number of positive results. Namely, we discovered its bio-friendliness, robust tumor-targeting capability, in situ miRNA-responsive cancer cell visualization, controllable drug release and superior chemo/gene/photothermal synergistic cancer therapy efficiency with reduced side effects. The molecular beacon functionalized on our platform not only proved its suitability for hybrid cancer treatment with diagnosis by identifying endogenous miR-21, impeding miR-21 function, but also can be instrumental to evaluate the therapeutic efficacy. We believe this strategy could bestow exosomes with other cancer specific microRNA-responsive characteristics by changing the sequences of the molecular beacon recognize element. This approach paves a new path to tailor exosome-based theranostic nanoplatforms for precise cancer treatment.