Worm-like micelles facilitate the intestinal mucus diffusion and drug accumulation for enhancing colorectal cancer therapy
Yihan Zhou , Duo Gao , Yaying Wang , Li Liang , Qingyu Zhang , Wenwen Han , Jie Wang , Chunliu Zhu , Xinxin Zhang , Yong Gan
Colorectal cancer (CRC) is a prevalent gastrointestinal malignancy worldwide, with a mortality rate ranking fourth globally [1,2]. Gastrointestinal administration, especially rectal administration, can increase the drug concentration at the disease site to avoid systemic absorption [2,3], ensuring a higher level of safety and efficacy [4], which plays an important role in the treatment of CRC. To avoid a cascade of mucosal physiological obstacles in the gastrointestinal tract [5,6] after rectal administration of drugs, such as pH and enzymes [7,8], nanoparticle-based drug delivery strategies are employed to enhance the stability of encapsulated drugs and facilitate their transport to the vicinity of the mucosal layer [9–11]. Colonic mucus consists of a thick inner layer and a fast-flowing outer layer, forming a dense three-dimensional network [12,13]. The thickness of mucus on the surface of the colon is approximately 110–160 µm [14–16]. Foreign nanoparticles entering the mucus layer may be easily trapped and captured by the network structure through hydrophobic interactions [17,18]. To enable drugs to reach cells and exert pharmacological activities effectively, it is necessary to overcome the mucosal barrier, in which the mucus layer serves as the main gated barrier.
Both of the neutral surface charge and polyethylene glycol-modification of nanoparticles could prolong the retention and release time of nanoparticles within the mucus layer [19,20] to effectively overcome mucus barrier [21]. However, these strategies are not conducive to cellular uptake of the carriers [22]. In addition, the complexity of designing surface-modified nanoparticles, coupled with the high preparation costs, significantly hampers their practical application [22–24]. Anisotropic particles, such as rod-shaped [25], disk-shaped [12,26], and needle-shaped particles [27], have shown promising properties, including enhanced mucus diffusion and tumor permeability [27,28]. Compared with the above shapes, filamentous nanoparticles exhibit higher flexibility and can effectively resist the harsh physical and chemical environmental pressures in the body [29]. Of late years, research on utilizing existing filamentous particles as a strategy to enhance the absorption barriers of drugs in the colon remains relatively unexplored.
Recently, segmented filamentous bacteria (SFB) found in the large intestine are filamentous in shape, with diameters ranging from 0.7 µm to 1.8 µm and lengths ranging from 40 µm to 60 µm, demonstrating an aspect ratio of approximately 30 to 50. SFB selectively colonize the end of the ileum or adhere to the absorbent villi of the epithelial cells in the cecum mucosa [30], moving swiftly through the mucosal barrier and possessing excellent mucus permeability [31]. Based on this information, we speculate that designing filamentous nanoparticles based on SFB could facilitate polymer micelles in overcoming the mucosal barrier and effectively delivering the drug nanoparticles to epithelial cells [29,32,33]. However, research on the morphology design of filamentous particles remains limited.
Herein, we constructed four different shapes of poly(1,4-butadiene)-b-poly(ethylene oxide) (PB-PEO) block copolymeric micelles (linear micelles (LMs), worm-like micelles (WLMs), large spherical micelles (LSMs), and small spherical micelles (SSMs)) to investigate their ability to overcome drug delivery barriers and improve the therapeutic effect of colorectal cancer. The WLMs displayed prolonged residence within the mucus layer, rapid permeation through colonic mucus, and effective infiltration into tumor tissue, ultimately resulting in potent anti-tumor effects. This study provided valuable insights for the design of antitumor drug delivery systems, particularly in terms of carrier morphology, offering potential advancements in cancer treatment strategies.
Polymer micelles of varying morphologies were prepared using the thin film hydration method, with PB-PEO copolymer as the raw material. The hydrophobic core of micelles could non-covalently encapsulate hydrophobic drugs, improving drug solubility and stability [34,35]. Firstly, we investigate the physicochemical properties of micelles. Fig. 1A showed the molecular structural formula of the main polymer material, and Fig. 1B displayed the morphological diagrams and transmission electron microscopy (TEM) of the four polymeric micelles. LMs had a linear structure, with a width of 15–30 nm and length of 1–3 µm. WLMs were vermicular in shape, with diameters of 15–30 nm and lengths of 300–500 nm. The aspect ratio of WLMs was about 15–30, which was similar to that of SFB. The particle size of the micelles was measured using a dynamic light scattering method (Fig. 1C). LSMs had diameters of about 80–120 nm, while SSMs were fully spherical with a uniform particle size of about 15–30 nm. The hydration particle size of WLMs and LSMs was similar, making them appropriate control groups. The zeta potential (Fig. 1D) of all the groups was about −22.2 ± 2.0 mV. The findings from Fig. 1E demonstrated that the micelles exhibited stability in vitro within 48 h.
As depicted in Fig. S1 (Supporting information), hydroxycamptothecin (HCPT) loading yield was up to 4.63%. Controlling the drug dosage of the four groups of micelles allowed for consistent dosages and ensured the reliability of the following drug research. HCPT cumulative release from micelles was less than 40% at pH 6.8, but accelerated after lowering the pH to 5.5 (Fig. 1F and Fig. S2 in Supporting information). This indicated that HCPT-loaded micelles were relatively stable during colonic transport, without premature leakage. These micelles could be quickly released and accelerated drug accumulation in tumor cells. We confirmed that PB-PEO material in the concentration range of 5–1000 µg/mL had no significant cytotoxicity to HCT-116 cells (Fig. S3 in Supporting information), making it suitable for subsequent cell experiments. Consequently, we successfully synthesized four micelles of varying shapes that demonstrated excellent stability and biocompatibility, while maintaining comparable physical and chemical properties. This allowed us to accentuate the distinct differences in their shapes.
To assess the diffusivity of polymeric micelles in mucus, we used multi-particle tracking techniques to collect 100 particles and calculate their total motion within the mucus. Trajectories of LMs, WLMs, LSMs, and SSMs in fresh colonic mucus were described in Fig. 2A. It was reported that the reduction of particle size would increase the diffusion coefficient [28]. The results indicated that SSMs could diffuse more quickly in mucus due to their small size. Conversely, LMs were trapped within the mucus network owing to their large size and limited pore size of the mucus. The mean square displacement (MSD) of WLMs was approximately 2.13- and 5.10-fold higher than LSMs and LMs, respectively (Fig. 2B). Additionally, the data presented in Fig. 2C revealed that WLMs displayed the highest distribution of effective diffusivities (Deff), indicating their exceptional mucus diffusion capacity. These findings suggested that beyond the size of the carrier, the shape of polymeric micelles also played an important role in their movement. Different shapes were trapped in the mucus to varying degrees and exhibited different mobility due to the limited pore size of the mucus, which was consistent with previous reports of Bao et al. [36]. Importantly, molecular dynamics simulations revealed that particles with high aspect ratio owned excellent mucus permeability. WLMs were found to diffuse faster than spherical micelles with similar size, indicating potential for efficient drug delivery.
To further investigate the mucus permeability of micelles, we used confocal laser scanning microscopy (CLSM) to capture three-dimensional images of the mucus after micelle incubation. As depicted in Fig. 2D, WLMs displayed the deepest penetration depth along the z-direction. The mean fluorescence intensity (MFI) of two-dimensional coverage image of WLMs was approximately 1.41- and 2.64-fold higher than LSMs and LMs, respectively (Fig. S4 in Supporting information). Conversely, SSMs were promptly cleared due to their small size and limited adhesion to the mucus layer. LMs had long and winding crosslinking form, leading to mostly wrap around the mucus surface and be easily captured by reticular mucus. On the other hand, the shape and aspect ratio of WLMs resembled filamentous bacteria, facilitating their greater diffusion and permeability in mucus than their counterparts. These results demonstrated that WLMs hold potential as candidates for improved mucus penetration and drug delivery.
To simulate the clearance of colonic mucus and assess the permeation ability of micelles with different shapes in flowing mucus environments, we developed a transwell model in vitro [18,37]. As depicted in Fig. 2E, the transwell system consisted of colonic mucus at the bottom of the chamber, micelles in the apical chamber, and HCT-116 cells in the basolateral medium. WLMs exhibited the highest MFI in the lowest medium, being 1.60- and 2.57-fold higher than LSMs and LMs, respectively (Fig. 2F), indicating greater penetration capacity after mucosal clearance. These findings were consistent with the results of mucus penetration in vitro. Regarding cellular uptake capacity, WLMs demonstrated the highest uptake efficiency, which was 1.43-fold higher than LSMs (Figs. 2G and H). Despite the trapping effect of the network structure and the self-cleaning effect of mucus, WLMs could effectively penetrate colonic mucus and exhibit superior cellular uptake efficiency in comparison to other micelles, owing to their unique shape characteristics.
Cellular viability of HCT-116 cells was evaluated after incubation with free HCPT or HCPT-loaded micelles. As illustrated in Fig. 2I, HCPT-loaded micelles exhibited significant reductions in cellular viability compared to free HCPT group, indicating their potential as anticancer carriers. WLMs exhibited the lowest half maximal inhibitory concentration (IC50) value of 0.172 µg/mL (Fig. 2J), underscoring the efficacy in releasing anticancer drugs and reducing cell proliferation. Furthermore, the in vitro antitumor activity was found to be related to micellar shape. We utilized the mucus-clearing transwell system to explore the impact of HCPT-loaded micelles on inducing tumor cell apoptosis. These results demonstrated that the apoptosis rate of the WLMs was 2.25-fold higher than LMs (Figs. 2K and L). The enhanced inhibitory effect of WLMs was primarily attributed to the high concentrations of WLMs, which promoted rapid intracellular drug aggregation and inhibited cell proliferation. The shape of the micelles played a crucial role in residing within the mucus layer. The micellar shape influenced both drug accumulation and release within colonic epithelial cells, ultimately enhancing the anti-tumor effects.
We then assess whether the advantageous properties of WLMs could enhance in vivo delivery efficiency. The animal welfare and experimental procedures underwent a thorough review and received approval from the Animal Ethics Committee of the SIMM. To visualize the intestinal retention behavior, we employed the IVIS spectrometer system. Upon local rectal administration in BALB/c nude mice, DiR-labeled micelles with distinct geometries exhibited varying retention rates when passing through the intestinal tract (Figs. 3A and B). As revealed by fluorescence intensity analysis, WLMs had the higher fluorescence intensity in the intestinal tract compared to the LSMs at 2 h post-administration. Remarkably, we observed intestinal clearance in all preparation groups at 10 h, with LMs exhibiting the fastest clearance rate, followed by SSMs. This could be attributed to the unfavorable shape of both LMs and SSMs, which hindered their ability to stick to the intestine. WLMs, owing to their optimal length, could moderately reside within the mucus mesh. This residence helped reduce the clearance caused by mucus peristalsis, facilitating their prolonged retention in the intestinal mucus. Moreover, we collected the section of colonic villus, and investigated the absorption of DiI-labeled LMs, WLMs, LSMs, and SSMs by CLSM (Figs. 3C and D). As expected, WLMs exhibited high fluorescence intensity, demonstrating uniform and efficient absorption in the epithelial cells due to the strong mucus penetration capability. Notably, MFI of WLMs was 1.41-, 1.61-, and 2.64-fold higher than LSMs, SSMs, and LMs, respectively. Thus, owing to the worm-like appearance and shorter length, WLMs could swiftly penetrate through the mucosal barrier and be effectively absorbed by the colorectal villi. To further explore the accumulation of micelles in tumor tissue, we developed an orthotopic colorectal cancer model in BALB/c nude mice. Colorectal tumor tissues were sliced after incubating with four distinct DiI-labeled formulations for 4 h, and CLSM imaging was captured. As shown in Figs. 3E and F, a significant area of fluorescence was observed in WLMs-treated tumor tissue, indicating that WLMs had high tumor tissue infiltration capacity. The MFI of WLMs in the tumor was 3.18- and 5.47-fold higher than LSMs and LMs, respectively. The fibrous structure of the tumor interstitial space was similar to the mucus, suggesting that the mechanism of rapid penetration of WLMs into the tumor tissue could be analogous to that of mucus [12]. These results demonstrated that WLMs enable to enhance mucus retention and accumulate in the colorectal tumor tissue, making them promising candidates for the treatment of colorectal cancer.
To investigate the biological distribution of micelles with different geometries in vivo, the pharmacokinetic characteristics and penetration capability were explored. The pharmacokinetic profiles of DiI-labeled micelles were examined in Sprague-Dawley rats, following rectal administration, and the resulting plasma concentration was assessed. As shown in Fig. 3G, WLMs exhibited the highest plasma concentrations among all time points. The area under the curve (AUC) of WLMs was 1.91- and 3.07-fold higher than LSMs and LMs, respectively (Fig. 3H). WLMs also had a longer elimination half-time following rectal administration compared to other groups, approximately 4.26 h (Fig. 3I). The drug within the micelles were stable and released slowly. Thus, the peak time in vivo was also longer than the free DiI group. In addition, LMs exhibited a more elongated shape in comparison to WLMs and demonstrated the lowest Cmax, whereas spherical micelles possessed average pharmacokinetic parameters owing to their symmetrical nature, uniform sizes, and drug release kinetics. These results identified significant differences in the in vivo distribution of micelles based on their shape, with varying rates of absorption and removal. As evidenced by the pharmacokinetic parameters, different preparation groups demonstrated distinct reabsorption processes once they entered the bloodstream. WLMs had higher bioavailability and could accumulate around tumor tissues during reabsorption, leading to better therapeutic outcomes against colorectal cancer. This systemic absorption and treatment effect may even impact the metastasis of colorectal cancer in rats, rather than just locally absorbing the drug. Our findings may serve as a research basis for subsequent overall pharmacodynamic evaluation.
Based on the favorable outcomes in vitro, we evaluated the in vivo anti-tumor efficacy of micelles loaded with HCPT against CRC. The mice were randomly divided into six groups and administered various formulations rectally every 2 days for eight total repetitions (on days 0, 2, 4, 6, 8, 10, 12, and 14). We monitored tumor growth in nude mice using the IVIS spectrometer system to gauge the efficacy of HCPT-loaded micelles in vivo. After 14-day treatment, each drug-loaded preparation group showed significant tumor growth inhibition (Figs. 4A and B). Notably, the lowest total flux of HCT-Luc tumors cells was observed in the group treated with WLMs, indicating strong suppression of tumor growth. We extracted colonic segments from mice to weigh tumors and found that WLMs exhibited a markedly superior effect in inhibiting tumor growth during treatments. The tumor weight of LSMs was approximately 1.96-fold higher than WLMs (Figs. 4C and D). H&E staining of tumor tissues confirmed the successful destruction of tumor cells by WLMs (Fig. 4E). Time-body weight curves showed no significant reduction in the four formulation groups, but the free HCPT group had slight reductions, indicating that direct HCPT administration can lead to certain toxicity (Fig. S5 in Supporting information). Importantly, we observed no obvious tissue necrosis or morphological changes in major organs, indicating good safety of micelles in vivo (Fig. S6 in Supporting information). These results suggested that HCPT-loaded micelles were biocompatible and held great potential as safe candidates for CRC therapy. To be specific, WLMs showed the best outcome in terms of antitumor treatment compared to other formulations. However, the lengthy structure of LMs impeded its ability to pass through the mucus grid, making it susceptible to getting trapped in the mucus layer. Consequently, LMs displayed the worst tumor inhibition among all formulations. With the small spherical shape, SSMs could easily pass through mucus pores, but they were prone to be cleared by mucus, resulting in the loss of more particles and poor penetration ability, and leading to suboptimal tumor inhibition effects. In comparison to WLMs, LSMs had a smaller surface area, which reduced contact with mucus. However, their capacity of resistance to mucus clearance was lower than that of WLMs, indicating slightly lower drug delivery capability.
In summary, we developed four distinct shapes of polymeric micelles that exhibited various differences in overcoming drug delivery barriers, ultimately enhancing cancer therapy. LMs were easily captured by mucus, whereas not easily cleared by mucus and had a lower efficiency in overcoming the mucosal barrier compared to the two spherical micelles. WLMs, with an aspect ratio similar to SFB, had worm-like single-chain forms that require minimal energy consumption to enter mucosal pores, resulting in higher retention time and movement rate in mucus. Furthermore, WLMs accumulated rapidly in tumor through morphological adhesion, enabling drug release and improving tumor penetration, and thereby exhibiting excellent antitumor effects [26,27]. The morphological strategy that we had developed showed promise for clinical colorectal cancer treatment. Overall, this research provided invaluable insights into how different shapes of polymeric micelles could overcome colonic mucosal absorption barriers, broadening the understanding in the physical pharmaceutics field and providing valuable strategies for developing efficient delivery systems of insoluble drugs in polymer materials.
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
The authors would sincerely acknowledge the financial support from the National Natural Science Foundation of China (Nos. 82003678, 82222066, 82025032), Chinese Pharmacopoeia Commission (No. 2021Y30). We are also grateful for Lihui Xin and Yao Li from the National Facility for Protein Science in Shanghai for the use of TEM and Integrated Laser Microscopy System.
Supplementary material associated with this article can be found, in the online version, at doi:
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Figure 1 Characterization of polymeric micelles. (A) The molecular structural formula of main polymeric materials. (B) The TEM images of micelles. (C) The number sizes and (D) zeta potentials of LMs, WLMs, LSMs, and SSMs (n = 3). Scale bar: 50 nm. (E) The stability of micelles in phosphate buffered saline (PBS) (n = 3). (F) The cumulative release of HCPT in pH 5.5 (n = 3). The data are presented as the mean ± standard deviation (SD).
Figure 2 The polymer micelles overcome the mucosal and cellular barriers in vitro. (A) Representative trajectories of multiple-particle tracking for LMs, WLMs, LSMs, and SSMs in the colonic mucus of mice on a time scale of 1 s. (B) Ensemble-averaged geometric MSD as a function of time scale. The data represent experiments that tracked 100 particles. (C) Distributions of the logarithms of individual particle effective diffusivities (Deff) for particles at a time scale of 1 s (n = 100). (D) Three-dimensional images of the mucus penetration. Green: mucus stained with Alexa Fluor 488-labeled wheat germ agglutinin (WGA). Red: DiI-labeled micelles. Scale bar: 20 µm. (E) Schematic diagram of HCT-116 uptake after simulated colonic mucus clearance using a transwell system. (F) The MFI of the micelles reaching the bottom of the chamber after passing through the mucus layer (n = 3). P < 0.05. (G) CLSM images represent the shape effects of the micelles on mucinous penetration and cellular uptake. Blue: nuclei stained with DAPI. Red: DiI-labeled micelles. Scale bar: 10 µm. (H) Quantitative results of cellular uptake of micelles after mucus penetration determined by a BCA kit (n = 3). **P < 0.01. (I) Cell viability after incubation with different formulations (n = 3). (J) IC50 values of various formulations in (I) (n = 3). P < 0.05. (K) Flow cytometric examination and (L) the quantitative analysis of HCT-116 cell apoptosis after the different treatments (n = 3). **P < 0.01. The data are presented as the mean ± SD.
Figure 3 The distribution of polymeric micelles in the intestinal tracts, tumor tissues and in vivo blood circulation. (A) Representative retention pictures of intestinal segments from nude mice after ex vivo imaging. Scale bar: 4 cm. (B) Radiant efficiency (RE) of micelles in the intestinal segments (n = 3). P < 0.05. (C) Representative CLSM images of frozen sections of the colon after bowel loops. Blue: nuclei stained with DAPI. Red: DiI-labeled micelles. Scale bar: 50 µm. (D) Quantification of the coverage of micelles in the colon sections (n = 3). The data are presented as the mean ± SD. P < 0.05. (E) Representative CLSM images of micelles distribution in the colorectal tumor sections. Blue: nuclei stained with DAPI. Red: DiI-labeled micelles. Scale bar: 400 µm. (F) MFI of tumor section (n = 3). ****P < 0.0001. (G) The plasma concentration, (H) the AUC and (I) elimination half-time of micelles (n = 3). **P < 0.01. The data are presented as the mean ± SD.
Figure 4 In vivo antitumor efficacy of HCPT-loading micelles against CRC. (A) Representative in vivo bioluminescence imaging of the orthotopic colorectal tumor at the indicated time. The HCT-116 cells were labeled with luciferase (n = 3). Scale bar: 2 cm. (B) Tumor growth profiles obtained through quantifying the bioluminescence in panel A. The data are presented as mean ± SD (n = 3). P < 0.05. (C) Representative photos of the tumors harvested at the end of the experiment (Day 14). Scale bar: 1 cm. (D) Tumor weights of mice receiving different formulations (n = 3). P < 0.05. (E) Hematoxylin-eosin (H&E) staining of tumor tissue. Scale bar: 100 µm.
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