Dissolving microneedles enabled delivery of Oxaliplatin- sodium butyrate loaded outer membrane vesicles against rectal cancer

Abstract

Background

Oxaliplatin (OXA) is a commonly used drug for the treatment of rectal cancer (RC). However, traditional administration methods are plagued by low efficiency, high systemic side effects, and poor patient tolerance. Therefore, there is an urgent need to develop a targeted drug delivery system to enhance efficacy and reduce toxicity.

Methods

A strain of Clostridium butyricum with anti-cancer properties was screened, and outer membrane vesicles (OMVs) rich in sodium butyrate (NaB) were prepared. These OMVs (NaB-OMVs, NOMVs) were used to address the challenge of combined administration caused by differences in the administration methods and physicochemical properties of OXA and NaB. OXA was encapsulated into NOMVs via sonication to obtain OXA-loaded NaB-OMVs (OXA@NOMVs, ONOMVs). To improve stability and enable targeted delivery, a dissolving microneedle (MNs) system (ONOMVs@MNs) was developed using PVP K90 and sodium hyaluronate via the mold method. The system was characterized for morphology, size, zeta potential, mechanical strength, and rectal mucosa permeability. Additionally, targeted therapy advantages were evaluated using methods including anal microneedle administration in mice and fluorescence labeling of vesicles.

Results

Characterization showed that ONOMVs exhibit a saucer-like morphology with a diameter of 100 nm and a zeta potential of 20 mV. The ONOMVs@MNs were conically shaped and possessed sufficient mechanical strength to penetrate the anal mucosa. In rectal mucosa permeability experiments, the 2-hour permeability of the MNs group was 1.78 times higher than that of the liquid (liquor) group. Anal microneedle administration in mice and fluorescence labeling confirmed the targeted therapy advantages of NOMVs@MNs.

Conclusions

ONOMVs@MNs is an efficient local drug delivery platform that combines NaB and OXA, providing a potential therapeutic approach for RC.

Graphical abstract

Introduction

Rectal cancer (RC) constitutes over 60% of colorectal cancer, a disease that ranks third in global incidence and mortality rate, imposing a significant disease and economic burden. The management of RC encompasses a variety of clinical approaches, including comprehensive treatments that integrate surgery, radiotherapy, chemotherapy, molecular targeted therapy, immunotherapy, and biotherapy [1]. Additionally, Puzzo M et al. explored the complex landscape of multi-level resistance mechanisms in the epidermal growth factor receptor (EGFR) pathway of colorectal cancer, which are constituted by mutations including KRAS, NRAS, and BRAF V600E, and proposed that nanotechnology-assisted therapy could potentially be harnessed in the future to bypass these multi-level resistance mechanisms [2]. More notably, oxaliplatin (OXA) stands out as a pivotal chemotherapy agent in the treatment of RC. For instance, OXA, when combined with 5-fluorouracil or capecitabine, serves as the standard adjuvant chemotherapy post-surgery for stage III RC, and is the preferred first-line treatment for advanced RC [3].

Despite its efficacy, OXA is notorious for its significant toxicity profile, with cumulative dose-related toxicities (such as neurotoxicity, hepatorenal toxicity, and myelosuppression) escalating markedly when cumulative doses exceed 800 mg/m² [4, 5]. Additionally, drug resistance further restricts its therapeutic benefits, making the exploration of novel adjuvant drugs to mitigate chemotherapy resistance and cumulative toxicity a critical avenue for overcoming current RC treatment limitations. Earlier studies found that sodium butyrate (NaB) combined with OXA exerts a synergistic anti-RC effect (combination index 0.123) [6]. As the sodium salt of butyric acid, NaB inhibits RC cell proliferation, invasion, and metastasis while inducing apoptosis by regulating complex molecular networks [7]. It also helps overcome OXA-related chemotherapy resistance and cumulative toxicity, and beyond its anti-cancer effects, suppresses inflammation, strengthens gut barrier function, and enhances mucosal immunity [8, 9], thereby preventing inflammation-related colorectal cancer through improved inflammatory processes [10].

However, OXA (a cytotoxic drug requiring intravenous administration) and NaB (a biological agent for oral use) have different administration modes and physicochemical properties, making conventional combined administration unable to ensure synergistic drug concentration at tumor sites. Thus, modern preparation technology is urgently needed to construct new high-efficiency, low-toxicity compound formulations for optimal combination effects. Bacterial outer membrane vesicles (OMVs)—20–300 nm spherical nanoscale phospholipid bilayer particles released by Gram-negative bacteria that retain parental membrane characteristics [11]—contain bioactive proteins, lipids, nucleic acids, and metabolites [12]. With high biocompatibility, low immunogenicity, easy editability, and targeting, OMVs have been widely used in anti-tumor therapy [13]. For example, Qin et al. [14] engineered EcN-derived OMVs with neurobinding peptide NP41 to deliver L-arginine (Lar@NP-OMVs), suppressing pancreatic cancer cell proliferation/migration and improving chemotherapy outcomes. Kim et al. [15] showed OMVs target tumors via the EPR effect, accumulating passively through leaky tumor vasculature. Overall, OMVs as drug carriers are expected to solve the difficult problem of co-administration caused by different administration methods and physicochemical properties of OXA and NaB.

However, OMVs have demanding preservation conditions, are susceptible to environmental factors, and exhibit poor long-term stability. Additionally, aggregating at RC tumor sites to ensure dose concentration is difficult, necessitating integration with other formulation techniques to maintain biological vesicle stability [16]. Emerging microneedle (MN) drug delivery technology offers a new direction: MNs precisely target sites, enable local drug delivery, and enhance efficient absorption (especially of macromolecules), overcoming OMV delivery challenges [17]. Dissolving MNs—micron-sized needle tip arrays on a base—are a physical penetration advancement, enabling accurate targeting, local delivery, and efficient drug absorption [18]. Using MNs for bacterial vesicles offers advantages: slow/controlled drug release, needle body dissolution post-poration, and room-temperature storage [17]. Their safety as carriers for inflammatory bowel disease (IBD) validates feasibility for RC anti-tumor therapy [19]. In conclusion, bacterial OMVs provide a platform for the combined administration of OXA and NaB, and MNs as drug carriers can improve local administration and drug concentration in tumors.

Combination and co-administration of drugs face challenges such as pharmacokinetic mismatch, differences in physicochemical properties, and additive toxicity, while nanotechnology (such as bacterial outer membrane vesicles and degradable polymeric nanoparticles) provides innovative solutions for addressing such issues through precise delivery, controlled release, and microenvironment-responsive properties [20–23]. In this study, bacterial OMVs were applied to solve the problem of co-administration caused by different administration methods and physicochemical properties of OXA and NaB, and MNs were used to deliver OMVs to improve OMVs’ stability (Graphic figure). The application of MNs to carry OMVs for drug delivery was proposed innovatively, providing a new idea of MN drug delivery technology in the field of microbial application and anti-tumor therapy, which has important scientific value and clinical significance.

Methods and materials

Materials

Cell experiments

The HCT116 human RC cell line was acquired from the Type Culture Collection Cell Bank of the Chinese Academy of Sciences, located in Shanghai, China. For cell culture, RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was employed, supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Inc.). The cells were maintained in a humidified incubator (Sanyo XD-101; Sanyo Electric Co., Ltd., Osaka, Japan) at 37 °C under a 5% CO₂ atmosphere. OMVs were applied to treat the RC cell lines.

Animal experiments

Male C57BL/6J Nifdc mice, aged six weeks, were procured from Beijing Visonglihua Experimental Animal Technology Co., Ltd. After a one-week acclimatization period, the mice were anesthetized using isoflurane inhalation. A surgical procedure was performed to open the abdominal cavity, during which MC38/GFP cells (Fuheng, FH0991, 1 × 10⁷) were injected into the rectal wall. The abdominal cavity was then sutured using a 4 − 0 suture to close both the muscle and skin layers. Postoperatively, the mice received an anti-inflammatory injection of penicillin at a dose of 80,000 units.

Methods

Inclusion of volunteers

This study recruited 10 healthy participants who attended routine health check-ups at the health examination center of Huzhou Central Hospital from January 2023 to April 2023. The research protocols and consent procedures were reviewed and authorized by the Ethics Committee of Huzhou Central Hospital (Approval No. 202312005-01. Written informed consent was obtained from all participants in accordance with the ethical standards set by the Ethics Committee of Huzhou Central Hospital. Participant demographics and characteristics are summarized in Table 1. Inclusion criteria: (1)Individuals who underwent a full medical assessment. (2)Individuals who signed an informed consent form. Exclusion criteria: (1)Individuals with significant chronic or systemic health conditions. (2)Individuals with a history of psychiatric disorders or impairments affecting cognitive or communicative abilities. (3)Individuals who had consumed antibiotics or gut microbiota-modulating drugs within the three months prior to enrollment.

Table 1.

Clinical information of the volunteers

Sample	Sex	Age	BMI	State
1	Female	48	24.14	Normal
2	Male	67	19.03	Normal
3	Female	57	20.20	Normal
4	Female	57	26.56	Normal
5	Female	66	21.76	Normal
6	Female	68	24.03	Normal
7	Female	58	19.53	Normal
8	Female	54	20.70	Normal
9.	Male	58	20.03	Normal
10.	Female	53	23.59	Normal

Collection and quality control of fecal samples

Collection of fecal samples

Fecal samples weighing approximately 5–10 g were collected from participants who had not used laxatives or lubricants. The samples were obtained using a fecal collector within 30 min of defecation, which occurred before breakfast in the morning. The fecal samples were clearly identified and stored in an -80 °C ultra-low temperature refrigerator less than 1 month.

Quality control of fecal samples

To ensure the quality of the fecal samples, a pre-test involving PCR amplification was conducted. The primers used for this process were 27 F (AGRGTTYGATYMTGGCTCAG) and 1492R (RGYTACCTTGTTACGACTT). Samples yielding faint bands after PCR purification were excluded from further analysis, and replacement samples were collected to proceed with the study.

Isolating and culturing Clostridium Butyricum

The fecal samples were dissolved, diluted and coated on TSN medium (Formula: tryptone 17 g/L, soy peptone 3 g/L, sodium chloride 5 g/L, potassium dihydrogen phosphate 2.5 g/L and glucose 2.5 g/L) and anaerobic in a 37℃ incubator for 48 h to observe colony morphological characteristics. Colonies of interest were subjected to Gram staining, and their structural features and staining properties were examined using an oil immersion microscope. Colonies meeting the morphological characteristics of Clostridium butyricum were underlined to inoculate RCM medium (Formula: yeast dipped powder 3 g/L, beef extract 10 g/L, peptone 10 g/L, soluble starch 1 g/L, glucose 5 g/L, cysteine hydrochloride 0.5 g/L, sodium chloride 3 g/L, and sodium acetate 3 g/L). These colonies were further cultured to obtain pure isolates. Subsequently, physiological and biochemical tests were performed, along with 16 S rDNA sequencing for identification (reference sequence: NCBI-KC196777.1).

Detection of NaB

Ten strains of Clostridium butyricum were cultured in RCM medium under anaerobic conditions at 37 °C. Samples were taken every 2 h and centrifuged at 4 °C at 3000 g for 10 min. The concentration of NaB in the supernatant was measured by high-performance liquid chromatography (HPLC) [24].

Isolation of OMVs rich in NaB (NOMVs)

The Clostridium butyricum culture in RCM medium was subjected to centrifugation at 4 °C and 8000 g for 30 min. The resulting pellet was discarded, and the supernatant was further centrifuged at 4 °C and 20,000 g for 70 min. The supernatant from this step was carefully collected, filtered through a 0.22 μm membrane, and then centrifuged again at 4 °C and 120,000 g for 120 min. After discarding the supernatant, the pellet was resuspended in a small volume of sterile phosphate-buffered saline (PBS). The suspension was mixed thoroughly with PBS and centrifuged once more under the same conditions (4 °C, 120,000 g, 120 min) to wash the pellet. This washing step was repeated twice. Finally, the pellet was resuspended in 1 mL of PBS to achieve a concentrated solution of OMVs, which was stored at -20 °C for future use.

Preparation of the OXA-NOMVs (ONOMVs)

A 5% (w/v) glucose solution was prepared, containing OXA at a concentration of 5 mg/mL and OMVs at varying concentrations of 1, 3, and 5 mg/mL. The mixtures were sonicated using a Tekmar sonic disruptor equipped with a 0.25-inch tip, operating at 20% amplitude. The sonication process consisted of six cycles, each involving 30 s of active sonication followed by 30 s of rest, totaling 2 min, with a 1-minute cooling interval between cycles. This procedure yielded a homogeneous OXA-OMVs suspension. Subsequently, the concentrations of OXA and OMVs within the vesicles were quantified, and the encapsulation efficiency was calculated. The optimal formulation was determined based on the synergy ratio, with the best performance indicated by a combination index (CI) of 0.123.

Characterization of ONOMVs

The OMVs were diluted to the desired concentration using phosphate-buffered saline (PBS). A small volume of the diluted OMVs was applied to copper grids coated with a standard carbon film and then stained with 2% phosphotungstic acid. After allowing the samples to adhere to the grids, they were examined using a transmission electron microscope (TEM) for detailed imaging.

For particle size and concentration analysis, the OMVs were similarly diluted with PBS to the appropriate concentration. The samples were loaded into the sample chamber of a Malvern nanoparticle tracking analyzer using a loading needle. Data analysis was performed using NanoSight NTA software to determine the size distribution and concentration of the OMVs. The particle concentration was calculated using the formula: OMVs particle concentration = detected concentration×dilution factor of the sample.

Inhibitory effect of ONOMVs on cancer cells

The CCK-8 assay was used to measure the proliferation inhibition rate, flow cytometry was applied to analyze apoptosis, the scratch-wound assay and Transwell invasion assay were carried out to evaluate migration and invasion abilities, and cell cycle analysis was also performed. Controls were set up in each experiment, and two interventions were carried out: ①5 mg/ml OXA (5 µl); ②5 × 10⁹ Particles/mL ONOMVs from ONOMVs-loaded MNs (ONOMVs@MNs) (10 µl).

CCK-8 assay

RC cells in the exponential growth phase were seeded into 96-well plates at a density of 5 × 10³ cells per 100 µL and allowed to adhere for 24 h. Subsequently, 5 µL of OXA solution (5 mg/mL) was introduced into each well, and the cells were cultured for 8, 16, and 24 h, respectively. After the designated incubation periods, 10 µL of CCK-8 reagent (KGA317, Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) was added to each well, followed by an additional 3-hour incubation. The optical density (OD) at 450 nm was quantified using a microplate reader (ELx800; BioTek Instruments, Inc., Winooski, VT, USA). Physiological saline was used as the blank reference, while untreated RC cells served as the control group. The cell inhibition rate (IR) was determined using the formula: IR = [1 - (OD450 of experimental group - OD450 of blank group) / (OD450 of control group - OD450 of blank group)]×100%.

Analysis of cell apoptosis by flow cytometry

HCT116 cells were plated in 6-well plates at a density of 5 × 10⁵ cells per well and exposed to 10 µL of OXA at concentrations of 2.5, 5, 10, and 15 mmol/L for 24 h at 37 °C. Following treatment, the cells were rinsed twice with 0.1 mmol/L PBS. The cells were then stained with 5 µL of annexin V fluorescein isothiocyanate (FITC; KGA105, KeyGen Biotech Co., Ltd.) and propidium iodide (PI; KGA511, KeyGen Biotech Co., Ltd.), and incubated in the dark at room temperature for 15 min. The stained cells were analyzed using a FACS flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) equipped with a 488 nm argon laser. The cell populations in each quadrant were quantified using the instrument’s integrated software system.

Scratch-wound assay

RC cells were grown in 12-well plates until they formed a confluent monolayer. A straight scratch was then made in the monolayer using a sterile 20 µL pipette tip. The cells were gently washed three times to remove debris, followed by treatment with OXA-OMVs at a concentration of 5 × 10⁹ particles/mL (10 µL) for 24 h. The width of the scratch area was measured at different time points to assess the migration capacity of the cells.

Transwell invasion assay

HCT116 cells were collected and suspended in 200 µL of serum-free medium, followed by seeding into the upper compartments of Transwell plates pre-coated with Matrigel at a density of 2 × 10⁴ cells per well, utilizing membranes with 8 μm pores. The lower compartments were supplemented with complete culture medium. After a 48-hour incubation period at 37 °C in a 5% CO₂ environment, the cells were fixed with 4% formaldehyde for 30 min and then stained with 0.4% crystal violet for 10 min at room temperature. Cells that had traversed the membrane to the lower side were randomly imaged using a microscope. The invasive capacity of untreated HCT116 cells and those exposed to OXA-OMVs (5 × 10⁹ particles/mL, 10 µL) was assessed by quantifying the number of cells in five randomly chosen microscopic fields at 200× magnification.

Cell cycle analysis

HCT116 cells were plated in 6-well plates at a density of 3.0 × 10⁹ cells per well and incubated in culture medium for 24 h. The cells were subsequently exposed to OMVs at a concentration of 5 ×  10⁹ particles/mL (10 µL) for 48 h. After treatment, the cells were rinsed, collected, and fixed in 70% ethanol, followed by storage at 4 °C overnight. The fixed cells were then treated with Tris-HCl buffer (pH 7.4) containing 1% RNase A (catalog number KGA511; KeyGen Biotech Co., Ltd.). Staining was performed using propidium iodide (PI) at a concentration of 5 mg/mL. The stained cells were analyzed using a flow cytometer (FACSCalibur; Becton-Dickinson) and cell cycle analysis software (FlowJo, version 7.6.5; KeyGen Biotech Co., Ltd.). All experiments were conducted in triplicate to ensure consistent and reproducible results.

Preparation of ONOMVs@MNs

A PDMS-based conical MN mold, featuring a 15 × 15 array of conical openings with a depth of 800 micrometers and a base width of 300 micrometers, was obtained from Xiamen Qipu Medical Technology Co., Ltd., China. An aqueous solution containing 6% (w/v) hyaluronic acid (HA) and 0.1% (w/v) ONOMVs was prepared and poured into the mold. The mold was then carefully transferred to an incubation dish, ensuring an airtight seal to limit oxygen exposure. To fill the microneedle cavities, the mold was centrifuged at 2500 g for 3 min, after which any excess solution was removed. The filled mold was dried in a vacuum desiccator, allowing the formation of the needle tips. Following the initial filling, a 15% (w/v) polyvinylpyrrolidone (PVP) solution was applied to the mold, which was then centrifuged again at 2500 g for 3 min. The mold was dried under the same conditions for 3 days, ultimately yielding the ONOMVs@MNs.

Mechanical characterization of ONOMVs@MNs

Appearance and dimension observation of MNs

The surface morphology, arrangement, and distribution of the MNs, including the needle tips and backing layer, were examined using both optical microscopy and scanning electron microscopy (SEM). Key dimensional parameters, such as the height of the MNs, bottom diameter, tip diameter, and the spacing between individual MNs, were measured and recorded.

Mechanical strength measurement of MNs

A tensile test was executed on an Instron 68SC-05 apparatus to evaluate the mechanical properties of the ONOMVs@MNs patch. The MN bases were firmly affixed to a planar surface via double-sided adhesive tape, and the needles were submitted to a vertical compression test using a force sensor. Pressure was continuously applied to the MN and the pressure changes during the MN compression were recorded continuously. In addition, the fragility of MN can also be assessed from the gradient of the force-displacement slope.

In vitro drug release from tlevs@mn patches

The in vitro drug release behavior of MNs was measured using the Franz diffusion pool and the content of OXA and NaB at the sites.

Assessment of puncturability and safety of MNs in vivo

MNs containing OXA-loading vesicle were applied to the rectal mucosa of 18 male Wistar rats. The rats were euthanized at three time points: before administration, 2 h post-administration, and 24 h post-administration. The extent of mucosal damage was evaluated through visual inspection, and rectal mucosal tissue was collected for sectioning. The puncturability and safety of the MNs were assessed by analyzing hematoxylin-eosin (HE) stained sections of the rectal mucosa.

Animals groups

The mice were divided into control group, blank MN group (DMNs-Control), OXA intravenous injection group (OXA-I.V.), OXA-loaded blank vesicles@MNs group (OXA-OMVs@MNs), and ONOMVs@MNs group. The NC group had no other intervention. The OXA-I.V. group was given OXA 130mg/m² tail intravenously once every 3 weeks. The DMNs-Control group, OXA-OMVs@MNs and ONOMVs@MNs group were applied DMNs to the anal site once a week, respectively. Tumor area at the rectal site of mice was examined 2 weeks later by the animal fluorescence imaging apparatus.

OMVs-targeting assay

ONOMVs were labeled with Cy5 fluorescein (Solaibao, S1053). The MNs were prepared with Cy5-ONOMVs and the MNs were visualized under a fluorescence microscope. The MC38-GFP tumor-bearing mice (2 weeks after tumor-bearing) were treated with tail vein injection of Cy5-ONOMVs a and MNs at the anal site, respectively. OMVs targeting was assessed 1 h later by photographing on an animal fluorescence imaging device.

Measurement of tumor size

The MNs-Control group, OXA-OMVs@MNs and ONOMVs@MNs group were applied MNs to the anal site once a week, respectively. Tumor area at the rectal site of mice was examined 2 weeks later by the animal fluorescence imaging apparatus.

Results

Clostridium butyricum’s OMVs

Based on the butyrate-producing capacity, one out of 10 Clostridium butyricum strains was selected and isolated to obtain its bacterial OMVs containing butyrate (Fig. 1A). Ten Clostridium butyricum strains were tested for butyrate acid production capacity. The highest producing strain was selected and NaB in the medium of the highest producing strains reached a concentration of 8.33 mmol/L after 24 h (Fig. 1B). Meanwhile, its microscopic structure under TEM is displayed (Fig. 1C). Clostridium butyricum has greater anticancer ability compared with standardized Clostridium butyricum (ATCC 19398) (Fig. 1D). The OMVs isolated from Clostridium butyricum also have a stronger anticancer capacity (Fig. 1E). Moreover, the anticancer ability of 1000 g/ml Clostridium butyricum OMVs was better than that of 500 g/ml and 200 g/ml (Fig. 1F). Clostridium butyricum OMVs is named NaB-OMV because it is rich in NaB.

Fig. 1.

Screening of Clostridium butyricum and isolation of OMVs. A Flow chart of screening Clostridium butyricum and isolated OMVs. B The amount of sodium butyrate secreted in 10 Clostridium butyricum strains (every 2 h within 24 h). C The ultrastructure of bacterial vesicles was observed under electron microscope. D The effect of Clostridium butyricum and standard Clostridium butyricum on RC cell proliferation was determined by CCK-8. E The effects of bacterial vesicles secreted by Clostridium butyricum and standard Clostridium butyricum on the proliferation of RC cells were detected by CCK-8 method. F The effect of bacterial vesicles at different concentrations of Clostridium butyricum on RC cell proliferation was examined by CCK-8 assay

OXA-loading capacity of ONOMVs

ONOMVs were characterized by TEM and NTA (Fig. 2A). The NTA particle size analysis showed that ONOMVs’ size ranged from 20 to 500 nm and the concentration of ONOMVs was 2.7 × 10¹¹ Particles / mL (Fig. 2B–C). Compared to NOMVs before loading OXA, the particles size of ONOMVs was increased (Fig. 2D), and the zeta potential of ONOMVs was decreased (Fig. 2E). Preliminary in vitro stability test found that the particle size distribution of ONOMVs changed with time, with the particle size exceeding 200 nm at all detection time points (Fig. 2F). The detection of ONOMVs by acoustic degradation method showed that the Zeta potential was the highest (~ 155.67mV) and the encapsulation rate was the highest (~ 93.11%) when the particle size was about 265 nm (Fig. 2G–H).

Fig. 2.

Preparation and characterization of ONOMVs. A Flow chart of characterization and functional detection for ONOMVs. B TEM was used to observe the morphology of OMVs and ONOMVs. C Evaluate the concentration of ONOMVs with different particle sizes. D Compare the particle size of OMVs and ONOMVs. E Compare the zeta potential of OMVs and ONOMVs. F On days 0, 3, 6, 9 and 12, the particle size distribution of drug-loangding vesicles was detected by MicrotracNanotrac Wave II. G Zeta potential of vesicles with different particle size distributions. H Encapsulation rate of vesicles with different particle size distributions

Fabrication and characterization of the ONOMVs@MNs

ONOMVs@MNs were fabricated and characterized (Fig. 3A). The ONOMVs@MNs were observed under SEM and stereoscope and the MNs were conical array (15*15) (Fig. 3B–C). The ONOMVs were mainly concentrated at the tip of the needle in fluorescence stereoscope (Fig. 3D). The pressure tension test to determine the mechanical strength of MNs showed that the maximum pressure of MNs could withstand was 0.2 N/Needle when the deformation was 0.5 mm (Fig. 3E). Since the force for successful insertion into the skin barrier is approximately 0.098 N/Needle, the fabricated ONOMVs@MNs exhibit sufficient strength for transdermal drug delivery [25]. Comparing the contents of ONOMVs on the tip and the base of MNs suggested that the ONOMVs in these fabricated MNs were basically gathered at the tip (Fig. 3F). Furthermore, Cy5-ONOMVs and Cy5-ONOMVs@MNs were prepared (Fig. 3G). Fluorescence of the drug-loaded vesicles in the MNs was observed by fluorescence microscopy (Fig. 3H).

Fig. 3.

Characterization of ONOMVs-loaded MNs. A Flow chart of characterization for MNs. B Scanning electron microscope image of MNs. C Confocal microscope image of MNs. D Images of MNs under fluorescence. E Pressure-displacement curve. F The contents of ONOMVs on the tip and the base of MNs. G The MNs were visualized by fluorescence microscopy. H The fluorescence microscope image of Cy5-ONOMVs@MNs

Stability, safety, and drug delivery capability of ONOMVs@MNs

Stability examination found that there was no significant change in the encapsulation rate of MNs within 30 days, but the encapsulation rate of vesicle solution group decreased significantly, indicating MNs well maintaining the stability of ONOMVs preparations at room temperature (Fig. 4B). In the rectal mucosa permeability experiment, the 2 h permeability of the MNs group was 181.9 µg/kg and the liquor group was 101.9 µg/kg, and the penetration efficiency of MNs was 1.78 times higher than that of the liquor. In this way, the feasibility of drug delivery by MNs through the rectum was demonstrated (Fig. 4C). Moreover, in vivo experiments found that MNs did not cause significant damage to rectal tissue (Fig. 4D–F) and other organs such as heart, liver, spleen, lung and kidney (Fig. 4G), suggesting that MNs are relatively safe. In addition, MNs ultimately increased local tumor site concentrations of NaB and OXA compared to other drug delivery routes (Fig. 4H–I). In conclusion, the involvement of MNs improved the stability of ONOMVs, showed high biosafety, and increased the concentration of drugs at the rectal tumor site.

Fig. 4.

Stability and safety assessment of the ONOMVs@MNs. A Flow chart of stability and safety assessment of the ONOMVs@MNs. B Stability of ONOMVs@MNs. C In vitro transrectal penetration curve. D Rectum administration of blank MNs and drug-MNs. E Rectal tissue after microneedle rectal administration. F Histopathological images of the anus and rectum of rats before and after microneedle rectal administration, and the degree of rectal injury was evaluated after HE staining. G Pathological images of heart, liver, spleen, lung and kidney. H After sodium butyrate (NaB) oral administration, enema and microneedle intervention, the concentration of NaB at tumor site in rats was detected every 6 h from 0 h to 24 h. I OXA intravenous, enema and microneedle intervention were performed on rats, and OXA concentration was detected every 6 h at tumor site in rats from 0 h to 24 h

The precise delivery of ONOMVs to the RC site by ONOMVs@MNs

To clarify the rectal cancer targeting of ONOMVs@MNs, Cy5-ONOMVs injection with tail vein and Cy5-ONOMVs@MNs intervention were used for the MC38-GFP tumor-bearing mice, respectively. One hour later, mice were observed with animal fluorescence imaging equipment and the Cy5-ONOMVs of MNs group was concentrated and rectal sites (Fig. 5A–B).

Fig. 5.

Rectal cancer targeting of ONOMVs@MNs. A Vesicle targeting experiments after tail vein injection for intervention in MC38 mice. B Vesicle targeting experiments of MNs after intervention in MC38 mice

Antitumor effect of ONOMVs@MNs

As shown in Fig. 6A, the experimental setup with human RC cell lines HCT116 was used to evaluate the impact of ONOMVs@MNs. The survival rate of RC cells treated with ONOMVs from dissolved MNs was found to be the lowest, and the apoptosis rate was the highest (Fig. 6B-C). This clearly indicates the potent tumor cytotoxicity of ONOMVs@MNs. When compared with OXA, ONOMVs from dissolved MNs demonstrated superior performance in inhibiting the proliferation, migration, and invasion of RC cells (Fig. 6D-F). Additionally, a relatively larger proportion of RC cells were arrested in the G1 phase, suggesting that ONOMVs@MNs possess a stronger ability to induce G1 phase arrest of tumor cells (Fig. 6G).

Fig. 6.

Anticancer effect evaluation of the ONOMVs@MNs in vitro. A Flow chart of anticancer effect evaluation of the ONOMVs@MNs in vitro. B HCT116 was intervened with free oxaliplatin (OXA) and ONOMVs from dissolved MNs, respectively, and the survival rate of cancer cells with different treatment was observed. C Group a used OXA intervention, group b used the ONOMVs from dissolved MNs. Apoptosis of cancer cells was detected by Annexin V-FITC/PI double staining. D Caner cell proliferation was detected by CCK-8 method. E Caner cell migration was detected via scratch test. F Transwell method was used to detect the invasion ability of cancer cells. G PI single staining method was used to analyze the cycle of cancer cells

Based on the in vivo experiments with MC38-GFP tumor-bearing mice as shown in Fig. 7A, live imaging was conducted. The relative fluorescence area of MC38-GFP tumor-bearing mice in the control group, blank MN group (MNs-Control), OXA intravenous injection group (OXA-I.V.), OXA-loaded blank vesicles@MNs group (OXA-OMVs@MNs), and ONOMVs@MNs group was measured to be 707.7 × 10⁻² cm², 703.0 × 10⁻² cm², 654.7 × 10⁻² cm², 631.3 × 10⁻² m², and 564.3 × 10⁻² cm², respectively (Fig. 7B). These results strongly suggest that ONOMVs@MNs are the most effective in treating RC.

Fig. 7.

Anticancer effect evaluation of the ONOMVs@MNs in vivo. A. Intervention methods and flow chart of anti-rectal cancer efficacy of OMVs was assessed by MC38-GFP tumor-bearing mice. B: Display and comparison of fluorescence intensity in live animal imaging. A total of 5 groups were divided into control group, blank microneedle group (MNs-Control), OXA intravenous injection group (OXA-I.V.), OXA-loaded blank vesicles@MNs group (OXA-OMVs@MNs), and ONOMVs@MNs group

Discussion

An innovative ONOMVs@MNs was prepared in this study. This MN can be used as a drug delivery platform for the combination of NaB and OXA. The dose cumulative toxicity of OXA was also avoided while enhancing the drug effect against rectal cancer. Furthermore, the safety of the MN was demonstrated in this study.

Due to the inherent endogenous capabilities of OMVs, including low toxicity, minimal immunogenicity of cargo transport, and high biocompatibility and stability, the application of OMVs in the field of cancer therapy has been improving over the last decade [26]. OMVs can be used as a drug delivery system for various types of cancer. Doxorubicin, a commonly [27] drug used for the treatment of solid tumors, has been loaded into exosomes. OMVs have been used in colon cancer, osteosarcoma [28], ovarian cancer [29], breast cancer [30] and pancreatic cancer [31] et al. This study innovatively applied OMVs loading NaB and OXA to treat rectal cancer, fully exploiting the characteristics of OMVs. However, engineered OMVs are difficult to remain stable in a complex gut environment and a long-term in vitro environment. Moreover, it is still difficult to guarantee the concentration of drugs in RC tumor sites by oral loaded OMVs alone.

Emerging MN techniques can precisely locate tumor sites, achieve local administration, improve local drug concentration, and reduce cumulative drug dose, providing a new direction to overcome the problem of combined administration of NaB and OXA [32]. MNs is a novel local delivery device that combines the advantages of local injection and transdermal administration to increase the local drug concentration within the tumor. The array structure of the MNs allows for a more uniform drug distribution, while overcoming the problem of diminished drug efficacy caused by insufficient local tumor penetration [33]. MN matrix material can effectively wrap the drug and protect the drug from degradation by the external environment, thus improving the stability of the drug and enabling effective drug release into the local tumor [34]. In conclusion, MNs may become a important route of tumor administration with the advantages of less invasive, painless, strong targeting and uniform drug administration. In this study, OXA-OMVs-loaded MNs were prepared based on the multiple characteristics of MNs and an innovative combination of exosome loading techniques. This MNs can reduce the dose-induced cumulative toxicity of OXA, providing a new weapon for the treatment of rectal cancer.

In the past, rectal administration has usually been employed for antipyretic drugs [35] in cases where oral administration is limited. In this study, a rectal-administered anti-tumor formulation was developed, and this formulation has a great advantage in terms of antitumor effect. (1) Firstly, MNs can form tiny channels on the rectal mucosa, breaking through the limitation of the mucosal barrier in traditional rectal administration modes. These channels can significantly increase drug permeability and enable drugs to enter the blood circulation more rapidly and efficiently. (2) Secondly, rectal administration can avoid the first-pass effect of oral drugs through the liver, reducing the metabolism and degradation of the drug in the liver and thus improving the bioavailability of the drug. Rectal administration of MNs further enhances this advantage, enabling more drugs to directly enter the blood circulation and reducing the burden of drugs on organs such as the liver. (3) Thirdly, MNs can enable the concentrated release of the drug at the rectal site, improve the local concentration of the drug, enhance the therapeutic effect, and reduce the side effects of the drug on other parts of the body. (4) Finally, MNs can be designed into easy-to-use dosage forms such as patches and suppositories. Patients can receive treatment in outpatient clinics or administer the medication by themselves. It is convenient and fast, improving the convenience and compliance of treatment.

Nonetheless, this study does have certain limitations. Firstly, owing to the mode of mucosal administration, the existing MNs cannot be readily utilized for the treatment of RC. In the future, a potential approach could be to incorporate endoscopic technology to develop MNs capable of treating RC. Secondly, the drug-loading capacity of MNs remains an issue. Shortening the medication interval is one possible solution to consider. Moreover, seeking methods to increase the loading of OMVs also represents a promising avenue. Finally, it may be challenging for MNs to substitute postoperative adjuvant therapy. The reason is that there is currently no conclusive evidence indicating that the effect of drugs on the local rectal mucosa is comparable to that of intravenous administration. Nevertheless, we are fully confident that the application of MNs in the treatment of RC holds great potential for a bright future. It is noteworthy that materials such as liposomes, exosomes, polymeric nanoparticles, and metal nanoparticles have also demonstrated unique potential in combination drug delivery. The exploration of these materials provides multi-dimensional references for optimizing the ONOMVs@MNs system—future efforts could integrate targeted modification, stimulus-responsive release, or multimodal therapeutic strategies to further overcome limitations in drug loading and mucosal administration, thereby promoting the clinical translation of combined drug delivery systems for RC.

Conclusion

In summary, NaB-OMVs loaded with OXA were developed, and MNs were prepared based on these OMVs. The MNs-enhanced stability of OMVs was fully utilized while preserving their drug-combination properties. The ONOMVs@MNs feature uniform size, good dispersion, and high stability. Furthermore, the prepared MNs possess adequate mechanical properties, negligible mucosal irritation, and the ability to target rectal cancer. Thus, ONOMVs@MNs serves as an efficient local drug-delivery platform that combines NaB and OXA. This platform enables the avoidance of the dose-cumulative toxicity of OXA, achieves good therapeutic effects, and presents potential treatment options for RC.

Abbreviations

RC

Rectal cancer

OXA

Oxaliplatin

MNs

Microneedles

NaB

Sodium butyrate

Na+

Sodium ions

OMVs

Outer membrane vesicles

NOMVs

OMVs rich in NaB/NaB-OMVs

ONOMVs

OXA-loaded NaB-OMVs

ONOMVs@MNs

ONOMVs-loaded MNs

PBS

Phosphate-buffered saline

TEM

Transmission electron microscope

SEM

Scanning electron microscope

HE

Hematoxylin-eosin

NTA

Nanoparticle tracking analysis

Author contributions

Chu Jian and Qu Zhanbo drafted the manuscript. Wu Yinhang and Xu Yating revised the manuscript. Huang Bo and Wang Yingchen performed the experiment. Wu Zheng carried out the data analysis. Zhuang Jing and Wang Zefeng designed and drew figures. Yu Xiang and Han Shuwen conceived and reviewed the manuscript.

Funding

This research was supported by Zhejiang Provincial Natural Science Foundation (No.LTGY24H160001), Public Welfare Technology Application Research Program of Huzhou (No.2024GY22) and Medical and Health Research Project of Zhejiang Province (No.2024KY410).

Data availability

All data generated or analysed during this study are included in this published article.

Declarations

Ethics approval and consent to participate

The clinical protocols involving the patients and the informed consent form were approved by the Ethics Committee of Huzhou Central Hospital (No.202312005-01). The animal experiments were approved by the Experimental Animal Management and Ethics Committee of Huzhou Central Hospital (No.202401001).

Consent for publication

All authors read and approved the final manuscript.

Competing interests

The authors declare that no potential conflicts of interest exist.