Oral Administration of PLGA Nanoparticles to Deliver Antisense Oligonucleotides to Inflammatory Lesions in the Gastrointestinal Tract

Yuta Yagi; Yiwei Liu; Jinting Li; Shunsuke Shimada; Munetaka Ohkouchi; Yasushi Taguchi; Teruki Nii; Takeshi Mori; Yoshiki Katayama

doi:10.1248/bpb.b23-00769

Abstract

In this study, we prepared antisense oligonucleotide (ASO)-encapsulated nanoparticles (NPs) with a suitable profile for oral administration for the treatment of inflammatory bowel disease (IBD). We chose a water-in-oil-in-water (w/o/w) method to prepare the NPs using poly(lactide-co-glycolide) as a matrix and Pluronic as a stabilizer. The obtained NPs had a suitable diameter (158 nm) for the penetration of the mucus layer, endocytic uptake by enterocytes, and accumulation in inflammatory lesions in the intestine. The amount of ASOs in the NPs was relatively large (6.41% (w/w)). When the NPs were stably dispersed in solutions that mimicked gastrointestinal (GI) juice, minimal leakage of ASOs was demonstrated over the required period. The NPs were administered orally to mice with colitis induced by dextran sodium sulfate, which reduced target gene expression in the colons and rectums of the mice, whereas naked ASO administration caused no reduction in gene expression. Thus, the NPs have the potential of promising oral carriers of ASOs for the treatment of IBD that specifically target inflammatory lesions in the GI tract, thereby reducing the non-specific toxic effects of ASOs.

INTRODUCTION

Inflammatory bowel disease (IBD) severely reduces the QOL of patients through the effects of chronic inflammation and this may ultimately necessitate bowel resection. Recently, biologic agents such as anti-tumor necrosis factor α and anti-interleukin-12/23 antibodies that target inflammatory processes have been approved for the treatment of IBD. However, these agents show reductions in efficacy following long-term administration because of the generation of anti-drug antibodies (ADAs) and are associated with a risk of lymphoma development, owing to their immunosuppressive effects.^1,2)

Antisense oligonucleotides (ASOs) are another potential means of IBD treatment that are under development,³⁾ and include Alicaforsen (ISIS 2302) and Mongersen.^4–6) Although ADAs are generated against such ASOs, their efficacy and safety are not compromised.⁷⁾ Furthermore, ASOs target disease-specific genes, which minimizes their oncogenicity. Alicaforsen is an ASO that is administered via enema to target intercellular adhesion molecule-1, which is upregulated in many types of cells, including the colonic epithelial cells of patients with IBD. This ASO has been shown to successfully improve the disease activity indices of patients with ulcerative colitis in a clinical trial.⁴⁾ Mongersen is an orally administered ASO that targets Smad7 in epithelial and lamina propria cells. It has been shown to have anti-inflammatory effects in clinical trials, but its development was discontinued following a phase III trial, owing to its poor therapeutic effect.^5,6) One possible explanation for this may be its instability in the harsh conditions of the gastrointestinal (GI) tract, including the low pH of the stomach contents,⁸⁾ and another may be the poor delivery of the ASO to intestinal cells, because of the low permeability of the mucus layer.^9,10) The oral administration of ASO may represent a promising means of avoiding the side effects associated with systemic administration, such as off-target effects and hepatotoxicity owing to less absorption from the GI tract into the bloodstream,^11,12) and thereby facilitate the safe and efficient treatment of IBD.

Recently, nanoparticle (NP) formulations have attracted attention as a medium for oral drug delivery, because they are protected against harsh GI conditions, can penetrate the mucus layer when their surface characteristics are optimized, and spontaneously target inflammatory GI lesions.^13–16) These features of NP formulations should facilitate the oral delivery of ASO for the treatment of colitis. Previously, NP formulations based on poly(D, L-lactide-co-glycolide) (PLGA) have been used for the oral delivery of nucleic acids, such as plasmids¹⁷⁾ and decoy DNA.⁸⁾ Although they were shown to ameliorate colitis in mice, these formulations were not optimal for mucus penetration or specifically delivered for inflammatory lesions.

In this study, we applied recent knowledge of the most suitable NP formulation for mucus penetration and the targeting of inflammatory lesions to the design of ASOs suitable for oral delivery. As a cargo model, we used an ASO targeting non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (Malat1), which is ubiquitously expressed in vivo.^18,19) The purpose of this study is to examine the knockdown effect of Malat1 in gastrointestinal organs after oral application of ASO-containing NP. To the best of our knowledge, this is the first report of an NP formulation of ASOs for oral delivery. We created an NP formulation that has the potential for effective knockdown in inflammatory GI lesions.

MATERIALS AND METHODS

Materials

Resomer^® RG 752 H, Poly(D, L-lactide-co-glycolide) (lactide:glycolide 75 : 25, MW 4000–15000), and Pluronic F-127 were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Polyethylene glycol (PEG)(5000)-b-PLA(5000) and Diblock Polymer were purchased from Polysciences (Warrington, PA, U.S.). An oligonucleotide (ON) (5′-GCA AAG TCT GAC TAA CTC CCC-3′, 21-mer, all DNA) and an ASO (5′-GGG TcA GCT GCC AAT GcT AG-3′ (20-mer, the underlined part encodes 2′-O-methoxyethyl, the non-underlined part is DNA, the lower case letters indicate 5-methylated bases, and it was fully phosphorothioated) were custom synthesized by Nitto Denko (Osaka, Japan). The ASO targets the long non-coding RNA Malat1.²⁰⁾

Preparation of PLGA NPs

ON or ASO-loaded PLGA NPs were prepared using the solvent diffusion method described by Tsujimoto et al.²¹⁾ and the water-in-oil-in-water (w/o/w) method described by Takami and Murakami.²²⁾

The NPs were prepared by the solvent diffusion method as follows: PLGA (50 mg) was dissolved in 10 mL of an acetone/ethanol mixture (3 : 1), then the ON solution (1 mg in 750 µL of distilled water) was added to the PLGA solution. The resulting organic solution was added dropwise to 9.1 mL of aqueous phase (75 mg of polyvinyl alcohol (PVA) solution, 4 mg of chitosan, and 12 mg of citric acid in Milli-Q water) and stirred (500 rpm) at room temperature overnight.

NPs were prepared by the w/o/w method as follows: the ON or ASO solution in Milli-Q water (3.75 mg/150 µL or 6.00 mg/300 µL, respectively) was added dropwise to 3 mL of 2% (w/v) PLGA and 1% (w/v) PEG-b-PLA solution in a toluene and dichloromethane mixture (density adjusted to 1 g/cm³), then homogenized [15 min, 20000 rpm] or sonicated [40 s–5 min, 20–60 W, pulse output (on 0.4 s, off 0.6 s)] on ice. The primary emulsion obtained was poured into 15 mL of aqueous Pluronic F-127 solution (8% (w/v)), homogenized [5 min, 10000 rpm] and/or sonicated [2–5 min, 20 W, pulse output (on 0.4 s, off 0.6 s)] on ice. The secondary emulsion obtained was evaporated by stirring (200 rpm) at room temperature overnight, then ultracentrifuged (100000 × g, 20 min, 4 °C) to collect the NPs. Finally, the suspension buffer was replaced with 10% (w/v) trehalose solution or Milli-Q water, and the mixture was lyophilized. The lyophilized NPs were stored at −80 °C overnight, and they were reconstituted by adding the same amount of water.

Evaluation of the Physicochemical Properties of the NPs

The particle distribution and ζ-potential were determined using a Zetasizer Pro (Malvern Panalytical, Malvern, U.K.). The amount of ON encapsulated in NPs was determined by quantifying the ON remaining in the supernatant of the final emulsion using HPLC with diode-array detection (HPLC-DAD) (Chromaster; Hitachi, Tokyo, Japan). The ONs or ASOs were separated and eluted using the following gradient: 10% B (0–0.5 min), 10–80% B (0.5–5.0 min), increased to 80–90% B (5.0–5.1 min), maintained at 90% B (5.1–6.0 min), reduced from 90 to 10% B (6.0–6.1 min), and then maintained at 10% B (6.1–10 min) at a flow rate of 1.0 mL/min. Chromatographic separation was performed on an InertSustain AQ-C18 column (5 µm, 4.6 I.D. × 50 mm; GL Sciences, Tokyo, Japan). The mobile phases A and B consisted of 100 mM triethylamine acetate (TEAA) in Milli-Q water and 100 mM TEAA in acetonitrile, respectively. The sample injection volume was 20 µL, and UV detection was performed at 260 nm. A calibration curve was created using the ON and the ASO standard solutions. The calibration curve showed good linearity (r² > 0.99) using 1, 2, 5, 10, 20, 50, 100, 200, 500, and 1000 µg/mL dilutions. The encapsulation efficiency (%) or the content (% (w/w)) was calculated using the amount of encapsulation, derived from the concentration in the supernatant and the amount used for the NP preparation, or the mass of lyophilized NP in the absence of a cryoprotectant, respectively.

In Vitro NP Stability Test

The lyophilized NPs in 10% w/v trehalose solution were dispersed in simulated gastric fluid (34.2 mM NaCl, 227 mM HCl, pH approx. 1.2) and simulated intestinal fluid (50 mM KH₂PO₄, 23.6 mM NaOH, pH approx. 6.8). After incubation for 0, 2, and 4 h at 37 °C, the particle size distributions of the NPs were analyzed using a Zetasizer Pro. The remaining NP suspension was ultracentrifuged (100000 × g, 20 min, 4 °C), the supernatant was collected, and the concentration of free ASOs in the supernatant was determined using HPLC-DAD.

Evaluation of the Effects of ASO on Colitis in Mice

Animal experiments were performed according to the guidelines of the Animal Care and Use Committee of Mochida Pharmaceutical Co., Ltd. (Tokyo, Japan) (Approval Number: PK22146003; August 22nd, 2022). Male 7-week-old C57BL/6J mice were purchased from The Jackson Laboratory Japan, Inc. (Kanagawa, Japan). They were maintained under a 12-h light/dark cycle in a temperature-controlled environment and provided with a CE-2 diet and tap water ad libitum. After 5 d of acclimation, the mice were provided with free access to tap water or tap water supplemented with 2.5% (w/v) dextran sulfate sodium (DSS) (MW 36000–50000; MP Biomedicals, Santa Ana, CA, U.S.A.) for 5 d, then switched to tap water on day 6. The success or failure of the induction of enterocolitis by DSS was identified based on the presence or absence of diarrhea, hematochezia, stains around the anus, and body mass. The test substance was administered 9 d after the start of DSS administration at the doses shown in Table 1. The NP dose of 29 mg ASO/kg was the maximum dose that could be dispersed in the volume used (400 µL). Twenty-four hours after administration, the mice were euthanized by blood release under anesthesia, and duodenal, jejunal, ileal, colonic, and rectal samples were collected. The Malat1 expression in each was measured using real time-quantitative PCR (RT-qPCR) and the ASO concentration in each was measured using LC-tandem mass spectrometry (LC-MS/MS).

Table 1. Doses Used for the in Vivo Study of Mice with DSS-Induced Colitis

Sample	Route	Dose as NP (mg/kg)	Dose as ASO (mg/kg)	Dose volume (mL/kg)	NPs concentration (mg/mL)	ASO concentration (μg/mL)
Vehicle (Healthy)	P.O.	—	—	20	—	—
Vehicle	P.O.	—	—	20	—	—
NP	P.O.	492	29	20	24.6	1450
ASO	P.O.	—	29	20	—	1450
ASO	S.C.	—	50	5.0	—	10000

P.O. and S.C. represent oral administration and subcutaneous administration, respectively.

RNA Isolation and RT-qPCR Analysis

Tissue samples were homogenized twice in 1 mL of Trizol Reagent (Life Technologies, Carlsbad, CA, U.S.A.) using zirconia beads using a Micro Smash (3000 rpm, 4 °C, 2 min), then left to stand at room temperature for 5 min. The samples were then vortexed in 200 µL chloroform, then left to stand at room temperature for 3 min. After centrifugation (12000 × g, 4 °C, 15 min), 250 µL of each supernatant was added to equal volumes of 70% ethanol, and the entire volumes were applied to RNeasy columns (Qiagen, Hilden, Germany). The RNA obtained was purified and eluted in distilled water, according to the manufacturer’s protocol. The RNA concentrations were determined using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, U.S.A.), and a minimum 260/280 nm absorbance ratio of 1.8 was required. The RNA samples were reverse-transcribed using SuperScript IV VILO Master Mix and an ezDNase Enzyme kit (Thermo Fisher Scientific), according to the user’s guide. RT-qPCR analysis was then performed in duplicate using QuantStudio 3 (Thermo Fisher Scientific) and a FAM-based TaqMan gene expression assay kit (Thermo Fisher Scientific), according to the manufacturer’s recommendations. Malat1 expression was normalized to that of Gapdh.

Statistical Analysis

The data of characteristics of NPs and the qualification data by LC-MS/MS were described using the mean ± standard deviation (S.D.) or single value. Malat1 RNA expression level by RT-qPCR was described as the mean ± standard error of the mean (S.E.M.), and statistical analysis was performed with Student’s t-test and one-way ANOVA with Dunnett’s post-hoc test using statistical analysis JMP Statistics software (version, 11; SAS, NC, U.S.A.).

RESULTS

Preparation of NPs Using a w/o/w Method

We selected a w/o/w method for the preparation of ON-containing NPs. This is suitable for the encapsulation of hydrophilic materials such as ASOs in a hydrophobic matrix. Here, we used PLGA as a matrix, because of its biocompatibility and its ability to biodegrade. The preparation procedure is depicted in Fig. 1. The conditions for the preparation of NPs using the w/o/w method were optimized to achieve a high ON content by assessing the use of homogenization or sonication for each w/o and w/o/w process. The density of the DCM and toluene mixture was adjusted to 1 g/cm³ to stabilize the w/o and w/o/w emulsions, and PEG-b-PLA was added to stabilize the w/o emulsion.²¹⁾ The properties and size distributions of the NPs prepared using the three different conditions are summarized in Table 2 and Fig. 2. The diameters of the NPs ranged from 115 to 158 nm, with narrow size distributions. Condition No. 3, in which sonication was used for both the w/o and w/o/w processes, was associated with the highest encapsulation efficiency (73.6%).

Fig. 1. Scheme for the Preparation of NPs Using a w/o/w Method

Table 2. Characteristics of NPs Prepared Using the w/o/w Method (Nos. 1–4) or the Solvent Diffusion Method (No. 5)

No.	Content	W/o formation	W/o/w formation	Size (nm)	Polydispersity Index	Encapsulation efficiency (%)	ON or ASO content (% (w/w))
1	ON	H (20k rpm, 15 min)	H (10k rpm, 15 min)+ S (20 w, 5 min)	115 ± 1	0.296 ± 0.001	56.1 ± 0.5	3.39
2	ON	H (20k rpm, 15 min)	S (20 w, 2 min)	131 ± 1	0.251 ± 0.001	51.5 ± 0.5	—
3	ON	S (60 w, 40 s)	S (20 w, 2 min)	142 ± 1	0.179 ± 0.002	73.6 ± 0.7	3.82
4	ASO	S (20 w, 5 min)	S (20 w, 2 min)	158 ± 2	0.189 ± 0.013	63.6 ± 1.4	6.41
5	ON	Solvent diffusion method		312	0.0708	109 ± 28	2.05

Data are the mean ± S.D. (n = 3). Some values are based on a single experiment. H and S indicate homogenization and sonication, respectively.

Fig. 2. Size Distributions of NPs Obtained Using Conditions No. 1 (A), 2 (B), 3 (C), 4 (D), and 5 (E)

In panel D, the solid and dashed lines represent the original and reconstituted NPs, respectively.

We also evaluated a solvent diffusion method for the preparation of NPs (No. 5 in Table 2, Supplementary Fig. S1) that has been reported to incorporate ONs with high efficiency.²⁰⁾ Here, PLGA was used as a matrix, and cationic chitosan was used for complex formation with ONs, which rendered the ONs hydrophobic, to facilitate their efficient incorporation into PLGA NPs.⁸⁾ We found that the encapsulation efficacy of ONs was almost quantitative using this method, but the mass of ONs in the NPs (2.05% (w/w)) was lower than that of those prepared using the w/o/w method (3.82% (w/w)). The diameter of the NPs prepared in this way was very large (312 nm). Therefore, we concluded that the w/o/w method was suitable for our purpose. Based on condition No. 3, ASO-encapsulated NPs were prepared using condition No. 4, in which the sonication power used for the w/o process was reduced to avoid undesirable damage to the ASOs. The ASO used here targeted Malat1, which is a long non-coding RNA transcript that is highly expressed in various tissues.^17,18) The NPs prepared were of a comparable size and had similar encapsulation efficacy to those of the ON-encapsulated NPs prepared under condition No. 3 (Table 2). The ζ-potential of the ASO-encapsulated NPs was −2.6 mV, which is almost neutral. Notably, the ASO-encapsulated NPs can be stored in their lyophilized form in the presence of trehalose as a cryoprotectant. The size distribution of the reconstituted NPs was almost identical to that of the original NPs (Fig. 2).

In Vitro Stability of ASO-Encapsulated NPs

The ASO-encapsulated NPs should not be degraded or aggregate under the conditions encountered in the GI tract, for the protection of the ASOs and their efficient uptake by enterocytes. To evaluate NP aggregation and the release of the encapsulated ASOs in the GI tract, the lyophilized NPs were reconstituted in simulated gastric fluid (pH 1.2) and simulated intestinal fluid (pH 6.8), and the changes in the NP size distribution and the amount of ASOs released over 4 h were monitored. This is the time taken for GI contents to reach the colon after oral administration to mice.²³⁾ As shown in Figs. 3A, B and Supplementary Table S2, the size distribution of the NPs showed no difference when at pH 1.2 or pH 6.8, indicating a lack of aggregation. The released ASOs accounted for <10% of the total for up to 4 h, indicating that the degradation of the NPs was very slow under these conditions (Fig. 3C). The diameters of the ON-encapsulated NPs prepared using the solvent diffusion method were also measured in these two solutions. The NPs were stable in the pH 1.2 solution but aggregation occurred at pH 6.8 (Supplementary Fig. S2). For this reason, NPs prepared using the solvent diffusion method are not suitable for the oral administration of NPs.

Fig. 3. Size Distributions of the ASO-Encapsulated NPs in pH 1.2 Buffer (A) and pH 6.8 Buffer (B)

The colored line represents the particle size distributions at each time point (0, 2, and 4 h) when incubated at 37 °C. (C) ASO release ratio from the NPs in the pH 1.2 and pH 6.8 solutions at each time point.

Gene-Silencing Effects in Mice with DSS-Induced Colitis

To evaluate the efficacy of the oral delivery of ASOs to inflamed intestinal tissues by the NPs, we assessed the knockdown of gene expression in mice with DSS-induced colitis. First, we measured the basal expression of Malat1 before and after the induction of colitis. As shown in Fig. 4A, the Malat1 expression in the colon and the rectum of mice with colitis was low, which can be ascribed to the inflammation in these components of the GI tract.²⁴⁾ As a control, naked ASOs were administered via the oral or subcutaneous routes. As shown in Fig. 4B, the subcutaneous administration of naked ASOs caused significant knockdown in all of the tissues evaluated, whereas the oral administration of naked ASOs did not. The oral administration of the NPs reduced the rectal Malat1 expression vs. that associated with the oral administration of naked ASOs. Oral NP administration also reduced Malat1 expression in the colons of two of four mice, but this effect was not statistically significant.

Fig. 4. Malat1 RNA Variation in Each GI Tissue after Administration of NP and ASO

(A) Comparison of the Malat1 expression of healthy mice and mice with DSS-induced colitis 24 h after vehicle administration. Data are mean ± S.E.M. (n = 4). Student’s t test was used to compare Malat1 RNA level differences between Healthy and DSS-induced colitis in each tissue, statistical significance was set at p < 0.05. (B) Results of mMALAT1-ASO-mediated gene silencing in mice with DSS-induced colitis 24 h after oral or subcutaneous administration of each formulation. Data are mean ± S.E.M. (n = 4). The plots indicate individual values. One-way ANOVA and Dunnett’s test were used to compare Malat1 RNA level differences between Vehicle P.O. and other administration groups in each tissue, statistical significance was set at p < 0.05. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant.

We also measured the amount of ASO that reached each tissue using LC-MS/MS (Table 3). A large amount of ASO was identified following the subcutaneous administration of naked ASOs. Almost no accumulation of ASO was identified following the oral administration of the NP, but the oral administration of naked ASOs was associated with the low level of accumulation in the ileum. This implies that the NPs reduce the intestinal accumulation of ASOs, although the NPs may protect the ASOs from harsh GI conditions.

Table 3. Concentrations of mMALAT1-ASO in Each Component of the Gut of Mice with DSS-Induced Colitis 24 h after the Oral or Subcutaneous Administration of Each Test Substance

Sample	Route	ASO dose (mg/kg)	ASO amount (µg/g tissue)
Sample	Route	ASO dose (mg/kg)	Duodenum	Jejunum	Ileum	Colon	Rectum
NP	P.O.	29	—^a⁾	—^a⁾	—^a⁾	—^a⁾	—^a⁾
ASO	P.O.	29	—^a⁾	—^a⁾	0.143 ± 0.091	—^a⁾	—^a⁾
ASO	S.C.	50	6.74 ± 1.00	6.11 ± 0.66	7.53 ± 1.00	16.4 ± 1.5	16.1 ± 2.7

a) Below the limit for quantification (0.05 µg/g tissue). The amount of ASO is shown as mean ± S.D.

DISCUSSION

In the present study, we aimed to prepare NPs with suitable properties for the penetration of the mucus layer in the intestine and for endocytotic uptake into enterocytes. For the penetration of the mucus layer, NPs with a neutral polymer modification have been reported to show superior performance.²⁵⁾ For the endocytotic uptake by enterocytes, a diameter of 100–200 nm was reported to be suitable.^26,27) We used a combination of homogenization and sonication to prepare the w/o/w emulsions that become NPs after the evaporation of the organic solvent. Pluronic F-127 (triblock copolymer of PEG₁₀₀-PPG₆₅-PEG₁₀₀) was used as a surfactant for the preparation of the w/o/w emulsion, which resulted in the coating of the NP surfaces with neutral PEG chains. We screened various preparation conditions and identified four conditions, shown in Table 2, that generated NPs with optimum diameters (between 100 and 200 nm). The coating with the neutral PEG chains and the diameters of the NPs are suitable for the penetration of the mucus layer and endocytotic uptake by enterocytes. Using sonication for both steps of the w/o and w/o/w preparation (in methods Nos. 3 and 4) resulted in the highest encapsulation efficacy of ONs and ASOs in the resulting NPs. For the encapsulation of the content in the w/o/w emulsions, w/o emulsions of smaller particle size than those of the w/o/w emulsions are suitable, which enables straightforward incorporation of w/o emulsions into the w/o/w emulsions without disrupting the w/o emulsions. Because sonication usually generates finer w/o emulsions than homogenization, the leakage of the contents of w/o emulsions should be minimized.^22,28)

We also evaluated the preparation of NPs using the solvent diffusion method reported as ONs-encapsulated NPs. It was the most efficient method to encapsulate ONs, but larger particle diameters (approx. 300 nm) and the aggregation that was observed at pH 6.8 (Supplementary Fig. S2) mean that this approach is not suitable for oral delivery. The aggregation of the NPs at pH 6.8 may be due to the weakened charge repulsion among the chitosan on the NP surface at pH.^29,30)

The NPs encapsulating mMALAT1-ASO showed a relatively high content of ASO (6.4% (w/w)), which is critical for efficient gene knockdown in cells that take up the NPs. The stability of the NPs and the leakage of ASOs from the NPs were also evaluated under the gastric and intestinal pH conditions for up to 4 h (Fig. 3), and no aggregation and a negligible amount of leakage were identified, implying that these NPs should be suitable for ASO delivery to target enterocytes in the absence of aggregation or ASO leakage. Notably, the NPs can be lyophilized for storage without a change in size or content leakage from the reconstituted NP.

The in vivo study showed that NP administration tended to reduce the expression of Malat1 in the colons and rectums of mice with DSS-induced colitis (Fig. 4B). This can be explained by the accumulation of the NPs in sites of inflammation. Inflammation is known to be more marked in the large intestine than in the small intestine of such mice,³¹⁾ and the lower expression of Malat1 in the colon and rectum (Fig. 4A) is the result of DSS-induced inflammation in these components of the gut.²⁴⁾ The uptake of NPs into the inflamed tissue is facilitated by the greater permeability and retention (EPR) induced by the breakdown of the intestinal barrier.³²⁾ Thus, the NPs may be a unique ASO carrier that selectively knock down gene expression at sites of inflammation in the GI tract, which should minimize the risk of side effects associated with accumulation in non-target healthy tissues. On the other hand, subcutaneous administration of ASO strongly decreased Malat1 expression in whole GI tissues in a nonspecific manner (Fig. 4B). According to the quantification results (Table 3), ASO was not detected in most of the tissues after the administration of naked ASO and the NPs. This may be explained by the lower IC₅₀ of a Malat1 ASO than the lower limit of quantification.³³⁾

The possible reasons for the weak knockdown effect of the NPs will be as follows. First, the uptake efficiency of the NPs by enterocytes is low because of the PEGylation on the surface of the NPs. To facilitate this process, ligand modification on the surface of the NPs will work. Fructose, as a ligand for glucose transporter 2, and dipeptides, as ligands for peptide transporter 1, may be suitable because of their relatively high expression in enterocytes.^34,35) Second, after the endocytotic uptake, the release of ASO from the NPs may not be efficient due to the tolerance of the NPs toward endosomal acid conditions. To facilitate the release of ASO, raising the glycolic acid content of PLGA which increases the hydrolysis rate will work.³⁶⁾ Finally, the efficiency of endosomal escape of ASO may be poor. To improve this, the co-incorporation of substances such as cationic lipids into the NPs which promote endosomal escape may be helpful.^37,38)

In conclusion, we have prepared ASO-encapsulated NPs that are suitable for oral administration and target inflamed GI tissue. The NPs may protect ASOs against the pH conditions of the stomach and intestine. The oral administration of NPs tended to induce specific gene knockdown in inflamed colon and rectum but did not affect healthy tissues. Although the effect of the NPs alone may not be sufficient for clinical use, their use in combination with mucolytic agents or the modification of ligands on the NP surface may improve their knockdown effects. Thus, these NPs have the potential of promising oral carriers of ASOs for the treatment of IBD, with specificity for inflammatory lesions in the GI tract, and should therefore minimize the non-specific toxic effects of ASOs.

Acknowledgments

We thank colleagues of Mochida Pharmaceutical Ltd. for their technical assistance. We also thank Professor Yoshihiko Murakami (Tokyo University of Agriculture and Technology) for the discussion. We thank Mark Cleasby, PhD for editing a draft of this manuscript. YY, SS, MO and YT are the employees of Mochida Pharmaceutical Co., Ltd. whose company funded this study.

Conflict of Interest

Yuta Yagi, Shunsuke Shimada, Munetaka Ohkouchi and Yasushi Taguchi are employees of Mochida Pharmaceutical Co., Ltd. Yiwei Liu, Jinting Li, Teruki Nii, Takeshi Mori, and Yoshiki Katayama declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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