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.

Keywords: Microneedles; Outer membrane vesicles; Oxaliplatin; Rectal cancer; Sodium butyrate.

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

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

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

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

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

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

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