1. Introduction
Exosomes are natural nano-sized cell-secreted vesicles that vary in size from 30 nm to 150 nm. They can transport various biomolecules and play important roles in the interactions with the immune system, making them highly suitable for precise drug delivery [
1]. In recent years, advancements in exosome engineering technology have generated substantial interest in using targeted exosomes as a promising avenue for delivering therapeutics focusing on cancer treatment [
2,
3,
4]. These attributes include biocompatibility, low immunogenicity, and an intrinsic capacity for transporting biomolecules, including proteins, lipids, and nucleic acids, between cells [
5,
6,
7]. Furthermore, exosomes can traverse biological barriers, thereby establishing their suitability for the targeted delivery of diverse therapeutic agents, such as small molecules, siRNAs, and miRNAs, to specific cellular targets within an organism [
8,
9].
17-Dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) is a potent heat shock protein 90 (HSP90) inhibitor used to reshape cancer treatment [
10,
11,
12]. HSP90 is a chaperone protein that plays a pivotal role in stabilizing and properly folding various client proteins, many of which are integral to cell growth, proliferation, and survival pathways [
11]. By inhibiting HSP90, 17-DMAG disrupts the G2/M cell proliferation cycle, indicating profound implications in cancer therapy [
13,
14]. Its ability to orchestrate cell cycle arrest, induce apoptosis, and effectively curtail tumor proliferation underscores its potential for enhancing anticancer efficacy. The antitumor effects of 17-DMAG on gastric cancer cell lines have previously been explored [
10]. However, a targeted delivery system might help increase the efficacy of anticancer treatments and reduce their toxicity.
Gastric cancer is a global burden, being the fifth most common cancer and the third most common cause of cancer-related death globally [
15]. Although many cytotoxic chemotherapy agents and targeted and immune-modulatory agents have shown strong performance in cancer treatment, drug resistance issues remain. Therefore, novel approaches to cancer treatment are essential. To obtain optimally targeted exosomes, we needed to identify a specific target expressed on the membrane of the gastric cancer cell line. MKN45 is one of the most common gastric cancer cell lines and expresses many biomarkers related to tumor progression [
16,
17,
18]. Among the studies of the biomarkers within MKN45, a study by Sahin et al. [
19] employed a phage display peptide library to screen for proteins with a specific binding affinity for MKN45 gastric cancer cells. Their research established that the DE532 peptide (VETSQYFRGTLS) exhibited specific binding capabilities in MKN45 gastric cancer cells.
In this study, we proposed that 17-DMAG-loaded MKN45-targeting exosomes effectively deliver 17-DMAG to gastric cancer cells, resulting in enhanced antitumor effects. This innovative approach leverages the advantages of exosome-based delivery systems, such as biocompatibility and the ability to cross biological barriers, while exploiting MKN45-specific targeting of tumor cells. Additionally, we conducted the same experiments using the adenocarcinoma gastric (AGS) cell line to explore the efficacy of 17-DMAG-loaded DE532 exosomes in a gastric cancer cell line other than MKN45. Our research aims to provide insights into the design of targeted exosome-based therapies for gastric cancer in terms of potential clinical applications.
3. Discussion
The current research involves the encapsulation of conventional therapeutic drugs within nanoparticles designed to target gastric cancer specifically. This approach is expected to enhance the therapeutic efficacy against gastric cancer and significantly reduce adverse effects. This study investigated the anticancer effects of genetically engineered exosomes loaded with 17-DMAG for treating gastric cancer. The targetability and efficacy of the intravenously administered 17-DMAG-loaded DE532 exosomes [
10] were investigated in vitro and in vivo using mouse gastric cancer xenograft models. tEx(D) demonstrated higher targetability than that of other materials, such as tEx, Ex(D), and Ex, and showed promising antitumor effects, as evidenced by the smallest tumor size, lowest tumor growth rate, and lowest weight of excised tumors. Moreover, tEx(D) administration resulted in the highest upregulation of the pro-apoptotic marker BIM and the lowest downregulation of the anti-apoptotic marker Bcl-xL, with the lowest expression of antioxidant enzymes, such as SOD2 and catalase, within tumor tissues derived from mouse gastric cancer xenograft models. Consequently, 17-DMAG-loaded DE532 exosomes are believed to exhibit the highest anticancer efficacy by achieving targetability in gastric cancer.
The need for new anticancer treatments has arisen from the limitations of existing therapies. Cytotoxic chemotherapeutic agents, such as 5-fluorouracil, cisplatin, and oxaliplatin, are widely used to treat stomach cancer because of their effectiveness in attacking cancer cells. However, these treatments also affect normal cells, leading to various side effects [
20]. Targeted treatments have been developed to alleviate some of these side effects; however, problems remain. For example, trastuzumab for human epidermal growth factor receptor 2-positive gastric cancer and ramucirumab for advanced gastric cancer have shown improved results compared to those of traditional chemotherapy. However, they are not applicable to all patients, are expensive, and have considerable resistance issues, similar to those of other cytotoxic anticancer drugs [
21].
17-DMAG belongs to the category of drugs known as HSP90 inhibitors [
10,
11,
12]. HSP90 is a chaperone protein that plays a crucial role in stabilizing and assisting in the correct folding of various client proteins, many of which are involved in cell growth, proliferation, and survival pathways [
11]. The inhibition of HSP90 disrupts these essential cellular processes, making it a promising target for cancer therapies. The general anticancer effect of 17-DMAG lies in its ability to inhibit HSP90, which leads to the degradation of client proteins, ultimately leading to cell cycle arrest, apoptosis (programmed cell death), and tumor growth inhibition. In the context of gastric cancer, 17-DMAG has been observed to inhibit the proliferation of gastric cancer cells, induce apoptosis (programmed cell death) in these cells, reduce tumor growth in preclinical gastric cancer models, and enhance the sensitivity of gastric cancer cells to various chemotherapeutic agents [
10,
11,
12,
22].
17-DMAG does not exclusively target tumor cells, leading to the occurrence of several adverse effects when used as an anticancer therapy. The well-known adverse effects of 17-DMAG include gastrointestinal disturbances, fatigue, hepatotoxicity, hematological effects such as leukopenia and thrombocytopenia, electrocardiographic changes indicative of cardiotoxicity, neurological effects such as peripheral neuropathy or dizziness, and skin rash [
10,
11,
12]. Some of the more severe side effects of 17-DMAG, such as liver toxicity and myelosuppression, may be life-threatening [
11,
23]. Consequently, vigilant monitoring of patients during 17-DMAG treatment is imperative. Therefore, the development of targeted therapies that selectively deliver 17-DMAG to tumor cells holds promise for enhancing its anticancer efficacy while mitigating its adverse effects. Our findings revealed that loading 17-DMAG into exosomes expressing proteins targeting MKN45 cells resulted in the highest degree of targeted delivery with the most pronounced antitumor effects, as evidenced by both in vitro and in vivo experiments.
The experimental results demonstrate that the drug-loaded targeted exosomes exhibited the most substantial therapeutic efficacy in both in vitro and in vivo experiments. Although this study did not assess adverse effects, it is anticipated that targeted delivery will augment the anticancer effect of the drug while concurrently diminishing adverse effects. Subsequent experiments will be conducted to elucidate this. The anticancer effects of 17-DMAG-loaded DE532 exosomes were comprehensively confirmed, including their effect on cell proliferation and alterations in apoptosis-related markers and antioxidant enzymes in a xenograft model, which resulted in reduced tumor size and weight. DE532 exosomes demonstrated a certain degree of anticancer effect, suggesting the involvement of MKN45 protein expressed on the surface of gastric cancer cells in the oncogenesis of gastric cancer [
18]. These experimental results collectively highlight the application of drug delivery within genetically engineered exosomes as an efficacious anticancer approach. Given that various chemotherapeutic agents currently lack tumor-specific targetability, the use of targeted exosomes as vehicles has the potential to confer targetability to nonspecific chemotherapeutic agents.
Currently, this study has not been able to present an alternative for the development of exosome mass production and purification technology, and there is a lack of information on methods to improve the targeting for each type of cancer cell. Additionally, in the current study, experiments were conducted with a known target of a cancer cell line called DE532; however, its applicability to actual patient cancer cells may be difficult, acknowledging a need for the continued development of appropriate targets. Strategies to prevent autoimmune reactions caused by the excessive activation of the immune system by exosomes are also needed.
4. Material and Methods
4.1. Cell Culture
Human ASCs were obtained from Hurim BioCell Co. (Seoul, Republic of Korea) and cultured in DMEM/low glucose (GibcoBRL, Waltham, MA, USA) supplemented with antibiotics (penicillin–streptomycin; GibcoBRL) at 37 °C in a humidified atmosphere with 5% CO2 in an incubator. AGS and MKN45 cells were procured from the Korea Cell Line Bank (KCLB, Seoul, Republic of Korea). AGS and MKN45 gastric cancer cells were maintained in RPMI1640 (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Hyclone) and 1% penicillin/streptomycin (Gibco BRL, Waltham, CA, USA) at 37 °C in a humidified atmosphere with 5% CO2 in an incubator.
4.2. Generation of Genetically Engineered Exosomes
To generate tEx, the ASC cultures were grown to 90% confluence and subjected to starvation by removing fetal bovine serum for 24 h. After transfecting 4 µg of pDisplay-DE532 (MKN45 cell-targeted peptide: VETSQYFRGTLS(Val-Glu-Thr-Ser-Gln-Tyr-Phe-Arg-Gly-Thr-Leu-Ser)) into ASCs for 24 h, the conditioned medium was collected and centrifuged at 2500× g for 15 min at 4 °C to remove cell debris. Exosome isolation reagent (Thermo Fisher, Waltham, MA, USA) was then added to the conditioned medium, and the solution was incubated overnight at 4 °C. Exosomes were isolated from the conditioned medium using differential centrifugation at 10,000× g for 60 min at 4 °C. The precipitated exosomes were recovered through standard centrifugation at 10,000× g for 60 min, and the pellet was resuspended in phosphate-buffered saline (PBS).
4.3. Cargo Loading into the Exosomes
To investigate whether electroporation could effectively load cargo (17-DMAG) into exosomes, ASC exosomes were isolated using either total exosome isolation reagent or ultracentrifugation and then resuspended in an electroporation buffer. Electroporation was conducted using a NEPA 21 SUPER Electroporator with 0.2-cm cuvettes and aluminum electrodes. The samples, which had a volume of 100 µL (containing a maximum of 100 µg of total exosome protein), were electroporated at 200 V for 5 ms. Finally, the electroporated samples were analyzed using NTA and TEM, as previously described.
4.4. Flow Cytometry Analyses
Exosomes were suspended in ice-cold PBS (108 particles/mL) and incubated for 2 h at 4 °C with 20 μL of monoclonal antibodies(1:50): CD63-PE (BD Biosciences, Dubai, UAE, catalog number 556020), CD81-FITC (BD Bioscience, catalog number 555445), c-Myc-Alexa Fluor 488 (R&D systems, Minneapolis, MN, USA, catalog number IC3696G), and c-Myc-PE (R&D systems, catalog number IC3696P). Analysis was performed using the NxT Attune Flow Cytometer cell analyzer. All control samples were run along with the experimental samples, and all experiments were repeated three times.
4.5. NTA
NTA was performed using the ZetaView instrument (Particle Metrix GmbH, Ammersee, Bavaria, Germany). Each measurement involved the capture of 11 videos in one cycle. All measurements met the quality criteria of 50–150 particles per frame, 107 particles/mL concentration, and valid tracks greater than 20%. The built-in ZetaView software (Version 8.05.16 SP3) was used to analyze the videos immediately after capture.
4.6. TEM
For TEM analysis, Ex and tEx suspended in PBS were placed on Formvar carbon-coated grids and left for 10 min. Subsequently, excess liquid was removed using filter paper. Negative staining was performed using a 1% uranyl acetate solution for 10 min, and any excess liquid was removed using filter paper. The grids were then left to dry at room temperature(25 °C). The adsorbed Ex and tEx were observed using a Hitachi transmission electron microscope (HT7800) operating at 80–100 kV.
4.7. Cell Viability Assay
In a 96-well plate, 1 × 104 AGS and MKN45 gastric cancer cells were seeded and incubated for 24 h before treatment. The Ez-Cytox cell viability assay kit (Daeil Lab Service Co., Ltd., Seoul, Republic of Korea) was used to measure the cell viability after 48 h. Absorbance was measured at 450 nm using a Synergy HTX Multi-Mode Microplate Reader (BioTek Instruments, Inc., Winooski, VT, USA).
4.8. Detection of Mitochondrial Superoxide
AGS and MKN45 gastric cancer cells were stained with MitoSOX (Molecular Probes, Waltham, MA, USA) to detect the mitochondrial superoxide levels. The cells were kept in the dark at 37 °C for 10 min and then analyzed using a laser-scanning microscope (Eclipse TE300: Nikon, Tokyo, Japan).
4.9. Real-Time PCR
The total RNA was extracted from AGS, MKN45, and mouse tumor tissues using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Reverse transcription was performed with 1 µg of RNA using the RT-premix kit (TOYOBO, Osaka, Japan), following the manufacturer’s instructions. SYBR Green real-time quantitative PCR was conducted using the following primers: mouse Bcl-xL forward 5′-AAC ATC CCA GCT TCA CAT AAC CCC-3′ and reverse 5′-GCG ACC CCA GTT TAC TCC ATC C-3′; mouse Bax forward 5′-CTGCAGAGGATGATTGCCG-3′ and reverse 5′-TGCCACTCGGAAAAAGACCT-3′; mouse GAPDH forward 5′-CGACTTCAACAGCAACTCCCACTCTTCC-3′ and reverse 5′-TGGGTGGTCCAGGGTTTCTTACTCCTT-3′. Reactions were performed using the Applied Biosystems StepOnePlus Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA).
4.10. Western Blot Analysis
AGS and MKN45 gastric cancer cells and mouse tissues were lysed using the EzRIPA Lysis kit (ATTO Corporation; Tokyo, Japan) and quantified using the Bradford reagent (Bio-Rad, Hercules, CA, USA). Proteins were visualized using Western blot analysis using primary antibodies (1:1000 dilution) from Cell Signaling Technology (Beverly, MA, USA) followed by horseradish peroxidase-conjugated secondary antibodies (1:2000 dilution) from Vector Laboratories (Burlingame, CA, USA). Specific immune complexes were detected using the Western Blotting Plus Chemiluminescence Reagent (Millipore, Bedford, MA, USA). Primary antibodies against Bcl-xL, BAX, and β-actin and horseradish peroxidase-conjugated secondary antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). The original Western blot images are available in the ‘Original Western blots’.
4.11. Immunohistochemical Analysis
For immunohistochemical analysis, formalin-fixed paraffin-embedded tissue sections were deparaffinized, rehydrated in an ethanol series, and subjected to epitope retrieval using standard procedures. Antibodies against Bcl-xL, BIM, SOD, and catalase were used for immunohistochemical staining (Cell Signaling Technology, Danvers, MA, USA). The samples were examined under a laser-scanning microscope (Eclipse TE300; Nikon, Tokyo, Japan) to analyze the expression of these antibodies.
4.12. Animals and Study Design
Five-week-old male BALB/c nude mice (Orient Bio, Seongnam, Republic of Korea) were used for the comparative modeling of subcutaneous tumor growth. AGS and MKN45 gastric cancer cells (5 × 10
6) were injected subcutaneously into each mouse. All animal studies were conducted in compliance with the guidelines of the Institute for Laboratory Animal Research, Korea (IRB No: CUMC-2022-0141-01). The mice were weighed twice weekly. Detailed weights are provided in a
Supplementary file. Seven days after tumor cell injection, all the mice had measurable tumors. The in vivo efficacy was evaluated by randomly grouping the mice (n = 5 per group) and treating them intravenously with PBS (control), Ex (10
10 Ex particles in 100 µL of PBS, twice a week), tEx (10
10 Ex particles in 100 µL of PBS, twice a week), Ex (17-DMAG, 2.5 mg/mL; 10
10 Ex particles in 100 µL of PBS, twice a week), or tEx (17-DMAG, 2.5 mg/mL; 10
10 Ex particles in 100 µL of PBS, twice a week) for 30 days. Ex and tEx were labeled using the ExoGlowTM Ex Labeling Kit (SBI Biosciences, Palo Alto, CA, USA). ExoGlow-Vivo-labeled Ex and tEx showed robust signals in vivo. The tumor size was measured twice a week using a caliper, and the tumor volume (V) was calculated using the following formula: length × width
2 × 0.5236. After treatment completion, all mice were euthanized. The mice were anesthetized for 5 min using a gas mixture of inhaled oxygen (3 mmHg) and nitrous oxide (7 mmHg), which was administered through a specially designed chamber. After the experiment was completed, the mice were euthanized with 100% carbon dioxide gas for 5 min to obtain tissue samples.
4.13. Statistical Analysis
All data were analyzed using SPSS software (version 11.0; SPSS Inc., Chicago, IL, USA) and presented as the mean ± standard deviation. Statistical comparisons among the groups were performed using the Kruskal–Wallis test. Probability values of p < 0.05 were considered statistically significant.