Preparation and characterization of the PLGA-NP drug delivery system
Compared with other nano-drug delivery systems, PLGA, as FDA-approved polymers, have excellent biodegradability, strong ability to improve the solubility of hydrophobic drugs, and high encapsulation efficiency that effectively deliver more drugs to the site of disease and slow release of the encapsulated drug. Further, surface decoration of PLGA-PEG-Mal NPs is simple and can improve targeting efficiency; also, the PEG-Mal linker may increase circulation time in vivo and bioavailability of loaded drugs while protecting them from the immune response during circulation [52, 53].
To establish the PLGA-NPs and loaded them with BA and Res, we first optimized the composition for synthesizing BA-loaded NPs. Then we adjusted the ratio of Res to BA for incorporation. We fabricated five different formulations with a varied weight proportion of BA and Res. The formulation with the BA/Res proportion of 1:2 exhibited the highest BA/Res loading and encapsulation efficiencies. HPLC analysis revealed that the entrapment efficiencies of BA and Res in BA/Res@NP were 74.54% and 52.33%, and the loading capacities were 8.71% and 1.69%, respectively (Additional file 1: Table S2). TEM images showed that the morphology of both BA/Res@NP and BA/Res@NP-PBP were spherical, and the size distribution was narrow (Fig. 2A, B). Surface modification of PBP had no significant change in morphology. The particle sizes and zeta potentials were measured using the dynamic light scattering (DLS) method. The average size of BA/Res@NP was about 164.18 ± 0.8 nm, and zeta potential of − 25.46 ± 2.87 mV, while the respective measurements of BA/Res@NP-PBP were 184.3 ± 7.1 nm and − 28.02 ± 1.58 mV (Fig. 2C, D), indicating that after the surface decoration of the PBP, the physical characterization only slightly changed. In addition, BA/Res@NP-PBP had a narrow polydispersity index (PDI) of 0.051, suggesting that they are homogeneous in size, consistent with our TEM observations. Fourier transform infrared (FTIR) spectroscopy analysis also confirmed the successful coupling of PLGA-PEG-Mal and PBP (Fig. 2E).
We next investigated the cumulative release of BA and Res from the BA@NP, Res@NP, and BA/Res@NP in vitro. At different pH solutions, NPs showed similar drug release profiles (Fig. 2F–H), with ~ 60% of the encapsulated BA and Res releasing from the NPs at 24 h, and after 48 h the cumulative release reached ~ 80%. The drug release profiles indicated that BA and Res could be liberated from the hydrophobic core to yield a sustained drug release, avoiding sudden release.
Evaluation of the NPs’ BRET-FRET effect
To build the FRET system, we chose two lipophilic dyes for an efficient energy transfer (Fig. 3A). DiL and DiD were selected to be incorporated in the hydrophobic inner cavity of PLGA-NPs (Fig. 1A). We first optimized the concentration of DiL in NPs to obtain the most robust fluorescence. We detected the fluorescence intensity of DiL at different concentrations. The results showed that DiL reached the highest fluorescence intensity at 80 µM, and the fluorescence intensity decreased with increasing concentration (Fig. 3B). After setting the concentration of DiL at 80 µM, we then mixed DiD to obtain different proportions of DiL/DiD mixture and detected their fluorescence intensity. The fluorescence intensity was the highest when the ratio of DiL to DiD was 9:1 (Fig. 3C). To get the highest energy transfer efficiency, we set the ratio at 9:1 and then adjusted the overall concentration of the DiL and DiD. The results showed that the fluorescence intensity increased with the concentration from 60 to 80 µM, and a strong fluorescence signal was observed at 670 nm (Fig. 3D). However, as the concentration increased, the fluorescence intensity decreased. Therefore, in the follow-up experiments, the concentration of DiL in the DiL/DiD-loaded BA/Res@NP-PBP was set as 80 µM and the ratio of DiL to DiD was 9:1. The final concentration was 100 µM to obtain the highest fluorescence intensity at 670 nm. Under a confocal microscope, we found that the DiL and DiD in NPs were overlapped together (Fig. 3E), indicating that DiL and DiD were encapsulated in NP and were very close to each other, which was a necessary condition between the donor and acceptor molecules for FRET (≤10 nm) [54, 55].
To further determine the BRET-FRET effect in a nonconjugated state between DiL/DiD-loaded BA/Res@NP-PBP and luminol, we established an in vitro MPO reaction with or without NPs. IVIS imaging system was used to detect luminescence. Large amounts of reactive oxygen species (ROS) and MPO were induced by adding phorbol myristate acetate (PMA) to the cell culture medium to generate photoemission of luminol. We detected a significantly stronger luminescence signal in the presence of luminol and NPs than luminol alone (Fig. 3F). Meanwhile, the signal was strong at 550 nm in the presence of luminol alone and much stronger at 670 nm in the presence of both luminol and NPs, indicating that the BRET-FRET effect between the luminol and DiL/DiD-loaded BA/Res@NP-PBP could extend the wavelength of the light emitted by luminol and MPO and red-shift to NIR region.
The MPO imaging capability of luminol and DiL/DiD-loaded BA/Res@NP-PBP NPs was further determined in the DSS-induced UC model, in which increased MPO activity presents in the inflammatory site of the colon. We improved the parameters of luminol and NPs bioluminescence imaging. The strongest luminescence signal was detected with the doses of luminol and NPs at 4 mg/mouse and 100 µL/mouse (Fig. 3G, H), respectively. Bioluminescence imaging was performed every 2 min after co-delivery of luminol (4 mg) and NPs (100 µL). And the strongest signal was obtained at 4 min after injection (Fig. 3I). Subsequently, the luminescence signal gradually decreased but could be detected until 30 min, providing sufficient time for MPO-dependent imaging. Also, injection of DiL/DiD-loaded BA/Res@NP-PBP and luminol together provided the peak bioluminescence signal with a nearly 24-fold increase (0.747 × 105 versus 0.031 × 105 photons/s) compared with luminol alone (Fig. 3J, K), suggesting an effective BRET-FRET effect between luminol and DiL/DiD-loaded BA/Res@NP-PBP in an MPO-dependent manner. Further, luminol and DiL or DiD loaded NPs resulted in only a 12-fold (0.389 × 105 or 0.426 × 105 versus 0.031 × 105 photons/s, respectively) increase in luminescence emission (Fig. 3J, K). Therefore, the optimal BRET-FRET luminescence images were obtained after co-delivery of 4 mg luminol and 100 µL DiL/DiD-loaded BA/Res@NP-PBP NPs per mouse at 4 min, which can be used for the subsequent diagnosis and treatment of the UC model.
Targeting ability of BA/Res@NP-PBP
To assess whether P-selectin could be used as a target for drug delivery in the inflamed area of the colon, we investigated P-selectin expressions in clinical samples from chronic colitis patients and associated the results with the progression of cancer using immunohistochemistry (IHC). As shown in Additional file 1: Fig. S1, the expression of P-selectin in healthy tissues was deficient but was much high in inflammatory colon sites and tumors at different stages. In addition, the expression level of P-selectin was positively correlated with the clinical stages of cancer (Additional file 1: Fig. S1), indicating that P-selectin could serve as a specific delivery target in UC therapy.
The key for effective treatment of inflammation was to ensure that the drugs reach the inflammation site and are effectively internalized by the targeted cells. Raw 264.7 macrophage cells and Colon-26 epithelial-like are two major cells in colon-targeting drug delivery, which were chosen to evaluate the uptake efficiency of BA/Res@NP-PBP in vivo. DiL was encapsulated in BA/Res@NP-PBP as a fluorescent dye. After the DiL-labeled BA/Res@NP-PBP were incubated with Raw 264.7 and Colon-26 cells for 6 h, the localization of DiL-labeled BA/Res@NP-PBP in cells was observed by confocal microscope. Confocal imaging showed that after 6 h of incubation, a high DiL signal could be detected in the cells (Fig. 4A), indicating that the BA/Res@NP-PBP were successfully uptaken by Raw 264.7 cells and Colon-26 cells. However, compared with PBP modified BA/Res@NP (BA/Res@NP-PBP), the fluorescence signal of non-surface-functionalized NP (BA/Res@NP) in cells was weak (Additional file 1: Fig. S2), suggesting the limited target ability of BA/Res@NP and PBP modification can improve cell uptake efficiency. Furthermore, field-emission scanning electron microscopy (FE-SEM) was utilized to visualize the real-time binding of BA/Res@NP-PBP with Raw 264.7 cells and Colon-26 cells. The versatility of FE-SEM allowed us to obtain a detailed characterization of NPs’ processes of adsorption and endocytosis. FE-SEM images revealed that large amounts of BA/Res@NP-PBP (marked by red arrow) were present on the surface of Raw 264.7 cells and Colon-26 cells (Fig. 4B), indicating PBP-medicated active-targeting could significantly improve the binding ability between NPs and cells.
Next, we next studied the enhanced targeting effect of BA/Res@NP-PBP to the inflamed colon compared to BA/Res@NP in mice. DSS-induced colitis mice were intravenously injected with DiR-loaded BA/Res@NP-PBP or DiR-loaded BA/Res@NP. As a lipophilic NIR fluorescent dye, DiR is the best dye for in vivo imaging because of its deep penetration depth, which makes deep tissue imaging possible. After 12 h administration, collected the major organs (including the colon) in each group, obtained the in vivo imaging by IVIS. In fact, mice with colitis received the DiR loaded BA/Res@NP-PBP and showed significantly stronger fluorescent signals in the colon than the DiR loaded BA/Res@NP treated groups (Fig. 4C, D) validating a significantly improved colon-targeting effect. Additionally, fluorescence was also observed in the main organs from both groups (Fig. 4C, D). In contrast, in the DiR-loaded BA/Res@NP-PBP group, we found significantly reduced accumulation of NPs in the liver and kidney, suggesting PBP modification avoids potential NP systemic biodistribution and toxicity. Quantitative DiR fluorescent intensity from the colon and other organs was analyzed by region of interest (ROI) and represented by a histogram, as shown in Fig. 4E. Both BA/Res@NP-PBP and BA/Res@NP target the inflamed colon in the colitis mice, with the former showing slightly higher targeting ability.
Synergistic anti-inflammatory effects of BA/Res@NP in vitro
The anti-inflammatory effect of BA/Res@NP was demonstrated in a model of LPS-induced inflammation in macrophages. After LPS induction, the level of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α, and IL-12) in macrophages increased rapidly, which was significantly higher than the expression of the negative control group (Fig. 5A–D). Strikingly, pretreatment of BA@NP or Res@NP could effectively reduce the expression levels of pro-inflammatory factors, suggesting that BA and Res had significant anti-inflammatory activity, which was consistent with the previous reports. Moreover, BA/Res@NP down-regulated the expression levels of pro-inflammatory cytokines most, compared to BA@NP alone and Res@NP alone groups, suggesting a potential synergistic effect between BA and Res.
LPS-induced inflammation of Raw 264.7 cells inevitably led to cell death. The percentage of apoptotic was revealed by Annexin V-FITC/PI assays. The results showed the proportion of apoptotic cells in LPS group was the highest (36.89 ± 7.4%), BA@NP and Res@NP group could reduce the number of apoptotic cells (31.48 ± 9.2%, 24.53 ± 2.1%), but the proportion of apoptotic cells in the synergistic administration group was the lowest (23.12 ± 2.1%) (Fig. 5E), demonstrating anti-inflammatory effects of BA and Res, and the combination of the two drugs was better. The results were presented by flow cytometry.
To confirm the synergistic effect of BA and Res, we also compared the effects of BA@NP alone, Res@NP alone, and BA/Res@NP combination treatments on cell viability using MTT assays. At a certain concentration, both BA and Res could reduce the cell viability in a dose-dependent manner (Fig. 5F, G). The Chou-Talalay method was a widely accepted method for quantitative analysis of drug synergies. Finally, the combination index (CI) could describe the additive, synergistic and antagonistic effects of drugs. The results were denoted by the fraction affected (Fa) plot and the isobologram, respectively. So the cell viability with the CI values of 0.60, 0.43, 0.52, and 0.51 for the BA/Res@NP concentration decreasing 10% of cell viability IC10, IC20, IC30, and IC40, respectively, reflecting a significant synergistic effect between BA and Res. In addition, the CI values versus Fa plot (Fig. 5H) and the isobologram (Fig. 5I) confirmed that BA and Res have a synergistic therapeutic effect indeed.
Biocompatibility of NP in vitro and in vivo
Biocompatibility is a key factor to consider when evaluating a novel nano delivery platform. The biocompatibility of Blank@NP on Colon-26 cells in vitro was investigated by MTT assay. The Colon-26 cells were treated with Blank@NP at the different tested concentrations (up to 400 µg/mL). After 24 h of co-culture, the cell viability was not significantly affected (Additional file 1: Fig. S3A). When the incubation time was extended to 48 h, other conditions remained unchanged, the cell viability was not significantly affected either (Additional file 1: Fig. S3B), indicating this nano delivery system had no toxicity to cells.
Then we also evaluated the biocompatibility of Blank@NP in vivo. Blank@NP was injected intravenously for a week and measured the bodyweight change daily. The results showed that there was no significant change in weight loss compared with the control group (Additional file 1: Fig. S4). Tissue sections stained with H&E showed that there was no damage to the main organs in the Blank@NP group, which was consistent with the control group (Additional file 1: Fig. S5). The structure of the endocardium, myocardial membrane, and epicardium of heart tissue are apparent in both the Blank@NP group and the control group. Hepatocytes and hepatic lobules were intact, and no fibrosis was found in lung specimens. No abnormality was found in blood analysis and biochemistry examination (Additional file 1: Fig. S6). The liver and kidney injury indicators were also within the normal range in both the Blank@NP and control groups. The in vitro and in vivo results indicated that Blank@NP were with good biocompatibility and non-toxic, which could be served as a safe nanocarrier for drug delivery.
Administration of BA/Res@NP attenuates acute colitis
To evaluate the anti-inflammatory effect of BA and Res in vivo, we first investigated whether BA@NP, Res@NP, and BA/Res@NP could relieve DSS-induced acute colitis. DSS-induced acute colitis in mice is a model with high similarity to human colitis, and the specific implementation method is shown in Fig. 6A. During the treatment, we observed that BA@NP, Res@NP, and BA/Res@NP significantly reduced the body weight loss and lowered the DAI within 7 days compared to the DSS group (Fig. 6B, C), indicating reduced inflammation DAI with NPs treatment. On the 8th day of the experiment, the mice were sacrificed, and the colon tissues were obtained. By measuring the length of the colons, the DSS group was the shortest, while BA@NP, Res@NP, and BA/Res@NP groups were longer than the DSS group (Fig. 6D, Additional file 1: Fig. S7). In addition, we measured colonic MPO activities, a factor that reflected inflammation. DSS treatment significantly boosted the MPO activity but was notably decreased by BA@NP, Res@NP, and BA/Res@NP, with the BA/Res@NP combinational therapy, had the best effect on reducing the MPO activity (Fig. 6E).
We then examined the expression level of pro-inflammatory factors in colon tissues of different groups of mice. The results showed that BA@NP, Res@NP, and BA/Res@NP significantly decreased the mRNA level of TNF-α, IL-1β, IL-12, and IL-6 (Fig. 6F–I), suggesting that BA@NP, Res@NP, and BA/Res@NP could reduce the transcription of pro-inflammatory factors and effectively alleviated DSS-induced acute colitis.
Statistical analysis of histological scores of BA@NP, Res@NP, and BA/Res@NP on acute UC was performed by H&E-stained sections. DSS-induced but untreated mice exhibited significant signs of inflammation, including ulceration, goblet cell decreased, crypt disappeared, mucosal thickening, and lymph node formation (Fig. 6J). In contrast, treatment with BA@NP, Res@NP, or BA/Res@NP attenuated these inflammatory manifestations, particularly in the context of local lymphocytic infiltration. BA/Res@NP group presented the best efficacy reflected by the histological score levels (Additional file 1: Fig. S8). These findings indicate that BA and Res exert excellent anti-inflammatory effects and combinational treatment results in the best attenuation for colonic inflammation in the DSS-induced UC.
Impact of BA/Res@NP on gut microbiota
Studies have shown a strong correlation between intestinal diseases and its gut microbiota. Gut microbiota was an important part of the intestinal environment, and the normal gut microbiota maintained a relatively stable state. Gut microbiota could promote the operation of nutrients in the intestinal tract, preserve the healthy homeostasis of the intestine, and promote the development of the immune system. A large number of studies have confirmed that the microbiota of UC patients is significantly different from that of healthy people, which due to the changes in the occurrence and development of UC [56,57,58]. To determine whether generated BA/Res@NPs could maintain gut microbiota homeostasis, 16S rRNA sequencing analysis was used to investigate the gut microbiota from collected fecal samples. The method validation study showed that with the increasing sample size, the curves representing species diversity and richness gradually flattened (Fig. 7A, B), indicating that the sample size was enough for sequencing. The rank abundance curve also represented the richness and evenness of samples (Fig. 7C). These results showed that the results analyzed by 16S rRNA sequencing were reliable.
The beta diversity (number of species) in the healthy control mice was higher than other groups. NP-treated groups showed significantly increased beta diversity (Shannon index of operational taxonomic units [OTUs]) that was reduced by DSS-only treatment (Fig. 7D). A similar result was reflected by the Chao index of OTUs (Fig. 7E). Moreover, there was a positive correlation between the diversity of gut microbiota and the effect of NPs on UC treatment, indicating that the more microbiota, the better the therapeutic effect of UC. As shown in the Venn diagram (Fig. 7F, H), certain bacterial species appeared in all three groups, indicating these microbiotas might contribute to maintaining the homeostasis of the gut environment. Data presented in Fig. 7G showed that the majority of the dominant bacteria in the feces of the healthy control group were symbiotic bacteria, such as Firmicutes, Proteobacteria, and Bacteroidota. On the contrary, the DSS group showed a significant reduction in the diversity of symbiotic bacteria. While, the variety of symbiotic bacteria in NP treatment groups increased within the dominant species, in which BA/Res@NP had the most increased diversity. These change trends of predominant bacteria were consistent with the treatment results of UC. Moreover, heatmap of the gut microbiota composition in different treatment groups represented that the more similar the composition was to that of the healthy control group, the better the therapeutic effect was generated of UC (Fig. 7I). These results demonstrated that the imbalance of intestinal microbiota is closely related to the inflammation in the gastrointestinal tract. Moreover, BA/Res@NP could modulate the imbalanced gut microbiota ratio towards a near-healthy ratio, similar to the effect of BA/Res@NP in treating acute UC.
Administration of BA/Res@NP attenuates chronic colitis
We also established a chronic UC model induced by DSS, which could well reflect the relapsing and long-lasting of IBDs in humans so as to evaluate further the anti-inflammatory effect of BA@NP, Res@NP, and BA/Res@NP. The modeling method is shown in Fig. 8A. After three rounds of DSS feeding, the weight of mice decreased significantly in the DSS group. Mice treated with each NP showed slight bodyweight loss, DAI, and increased colon length (Fig. 8B–D). Results further showed that treatment with BA@NP, Res@NP, or BA/Res@NP significantly decreased the expression of colonic pro-inflammatory cytokines, such as TNF-α, IL-1β, IL-6, and IL-12 (Fig. 8E–H). Anti-inflammatory effects of BA@NP and Res@NP were further verified by histological analysis of H&E-stained sections, including reduced neutrophil infiltration and disappeared ulcers (Fig. 8I). Overall, the performance of BA@NP and Res@NP in the chronic colitis model was consistent with that in acute UC, and the BA/Res@NP combined with two drugs had a synergistic effect.