We have previously demonstrated that intraperitoneal injections of 2′-O-methyl-phosphorothioate (2′OMePS) antisense oligoribonucleotides adsorbed onto a cationic core-shell nanoparticles (NPs), termed ZM2, provoke dystrophin restoration in the muscles of
The X-linked recessive Duchenne muscular dystrophy (DMD) affects 1 in 3500 newborn boys [
Two AON chemistries, 2′OMePS and phosphorodiamidate morpholino oligomer (PMO), have already been the subject of clinical trials in humans [
In order to increase stability and half-life in biological fluids, thereby improving AON efficacy, different polymeric nanoparticles have been developed; their subcellular and submicron size allows them to penetrate deeply into tissues through the fine capillaries, crossing the fenestration present in the epithelial lining. We have previously demonstrated the effectiveness of polymethylmethacrylate (PMMA) T1 (Figure
Nanoparticles characteristics. Representation of the interactions between antisense oligoribonucleotide (M23D AON) and quaternary ammonium groups on the surface of T1 (a) and ZM2 (b) nanoparticles.
The
Indeed, treatment of
The objective of this work was to study the biodistribution, elimination, and efficacy of orally administered NPs-AON in
In order to protect molecules and facilitate drug permeation, different kinds of delivery systems are under study in order to inhibit hydrolysis, prolong intestinal retention, and thereby enhance drug bioavailability [
Alginate is one of the most common and suitable biopolymers. It is nontoxic, biodegradable, and employed to make acidic pH-resistant hydrogel [
Swelling of alginate is minimal in the stomach, while it increases moving toward the intestine, due to the pH increase [
These results encourage further research into the oral administration route for antisense molecules.
To assess the biodistribution and elimination pathways, a novel NP sample was prepared, nominated ZM4. ZM4 features the same size, size distribution, and surface hydrophilicity as ZM2, but it also exposes at the surface, or in the swelled shell, primary amino groups. These groups are necessary to interact with the isothiocyanate groups of the NIR-797 fluorescent dye (Sigma-Aldrich), giving rise to ZM4-IR nanoparticles in which the dye is covalently incorporated into the shell (see Table
Nanoparticles characteristics.
Sample | Diameter (SEM) nm (±standard error of the mean) | Diameter (PCS) nm (±polydispersity index) | Quaternary ammonium groups |
Primary amino groups |
NIPAM | Studies |
---|---|---|---|---|---|---|
ZM2 |
|
|
202 | — | yes | Functional analysis (dystrophin rescue) with ZM2-AON and ZM2-AON alginate |
| ||||||
ZM4 |
|
|
107 | 10.2 | yes | Biodistribution and elimination using dye NIR-797 |
Schedule of IR-dye conjugate nanoparticle treatments for biodistribution and elimination analysis.
No. ofmice | Oral formulations | Frequency | No. of administration | Time of analysis |
---|---|---|---|---|
|
ZM4-IR (2.5 mg) | 1/week | 1 | Elimination (metabolic cages): 12 hours after administration |
| ||||
|
ZM4-IR (2.5 mg) | 2/week | 15 | Time of collection of Odyssey images: before the new administration |
The Odyssey
Biodistribution of ZM4-IR-dye. Three mice (1, 2, 3) were analyzed at 12 (a), 24 (b), 48 (c), and 72 (d) hours post ZM4-IR single administration. Fluorescence was visualized using the Odyssey Infrared Imaging System. Green fluorescence represents the IR signal (~800 nm) associated to the nanoparticle. The signal appears localized to the abdominal region (intestine) of the mouse. (e) Mice located on the mouse pod of the Odyssey scanner. (f) The image shows the absence of fluorescence signal in
In the multiple administration schedule, ZM4-IR treated
Odyssey analysis of organ/muscle cryosections (20
In the nanoparticles clearance studies, feces and urine samples were collected and analyzed with Odyssey. The feces of 3 untreated
Table
Schedule of
Oral formulations | Frequency | No. of administrations | Total dose | Time of analysis | |
---|---|---|---|---|---|
Group 1 |
ZM2 (2.5 mg)-AON (225 |
2/week | 32 | 240 mg/Kg | 1 week after last administration |
ZM2 (2.5 mg) |
2/week | 32 | — | 1 week after last administration | |
| |||||
Group II |
ZM2 (2.5 mg)-AON (200 |
3/week | 36 | 240 mg/Kg | 1 week after last administration |
ZM2 (2.5 mg) |
3/week | 36 | — | 1 week after last administration | |
| |||||
Group III |
— | — | — | — | 1 week after last administration |
The amount of AON loaded onto alginate-coated ZM2 nanoparticles was lower with respect to the one used for ZM2 uncoated nanoparticles, 200
The only side effect we observed was a mild laxative effect after the treatment with alginate formulations.
In order to test the effect of ZM2-AON treatments on the dystrophin transcript, we assessed exon 23 skipping levels using a Real-Time PCR exon-specific assay (ESRA), as previously reported [
To evaluate the presence and amount of AON in tissues, an AON sequence-specific hybridization ligation assay was performed. We analyzed the diaphragm from treated and untreated mice given that the diaphragm from alginate-ZM2-AON treated mice resulted positive in ESRA analysis (8% of skipping). To compare the data with a muscle that was negative in the ESRA analysis, we also analyzed the quadriceps from untreated and treated mice. The AON hybridization assay revealed the absence of AON both in diaphragm and quadriceps from all the treatments, probably due to the low amount of AON administered in our experiments (240 mg/kg) and M23D could be too low to be detectable by this assay (data not shown).
In order to evaluate the presence and the correct localization of dystrophin, wild type (WT), treated and untreated
After 12 weeks of treatment with alginate ZM2-AON, immunofluorescence analysis revealed a slight rescue of dystrophin only in intestinal smooth muscle (Figure
Immunofluorescence analysis of intestinal smooth muscle of wild type (WT), untreated (
Immunofluorescence analysis of dystrophin protein in the diaphragm of wild type (WT), untreated (
In mice treated with ZM2-AON alginate-free complexes, no dystrophin was detected in any of the muscles analyzed.
Western blot analysis, performed as previously described [
Immunoblotting of dystrophin in the intestine of untreated
For WT samples, the total protein loaded was 1/10 (15
In this work, we demonstrate that the oral route is very appealing as administration for AON and, more in general, drugs. The promising fact is the very low, but measurable, dystrophin rescue on the diaphragm that needs to be further studied to improve efficiency, reaching a remarkable dystrophin restoration. We believed that our data, although preliminary, might represent the first step for further studies aiming at delivering nanoparticle-AON orally.
Here, we reveal the biodistribution and elimination of orally administered NIR-dye marked NPs as well as the exon skipping efficacy of NPs combined with AON molecules in
Preclinical studies and clinical trials [
Here, we use alginate as an encapsulating agent for the oral formulation in order to protect the AON further, in addition to its combination with nanoparticles, against the gastric pH and to increase the intestinal adhesiveness of ZM2-AON complexes. Sodium alginate is biocompatible, biodegradable, nontoxic and approved by the US Food and Drug Administration for oral use [
Alginate has never been used as a protective agent for exon-skipping inducing RNA molecules systemic delivery.
After a single oral administration, ZM4 remains in the intestine for about 72 hours, before being adsorbed and/or eliminated. The imaging in live animals before each dose of the multiple treatment shows the absence of NP accumulation. Adsorption through the intestinal wall is demonstrated by Odyssey cryosection analysis showing ZM4 in abdominal lymph nodes one week after treatment; no ZM4 was detected in the organs of mice sacrificed 1 month after the last administration. ZM4 clearance studies reveal that they are eliminated almost exclusively through feces and mainly in the first 48 hours after administration. This result suggests that the positivity observed in the intestine 72 hours after administration is represented by a low percentage (10%) of residual ZM4 and that the biodistribution evaluation could be affected by this early elimination of the majority of the administered NPs. Furthermore, in this context, the potential functional effect of NP-AON complexes in terms of dystrophin protein rescue could be reduced by the low amount of systemically available therapeutic molecules.
Following the verification of the intestinal absorption of the ZM4 we continued to demonstrate that oral treatment with NP-AON complexes is able to restore dystrophin synthesis, though at very low level, in
Our data demonstrate that only in
Summary of results of dystrophin restoration studies.
Oral formulations | Tissues | Exon 23 skipping |
IHC | WB | |
---|---|---|---|---|---|
Group I |
ZM2 (2.5 mg)-AON (225 |
Diaphragm | 0 | − | − |
Intestine | 0 | − | − | ||
Gastrocnemius | 0 | − | − | ||
Quadriceps | 0 | − | − | ||
Heart | 0 | − | − | ||
ZM2 (2.5 mg) |
Diaphragm | 0 | − | − | |
Intestine | 0 | − | − | ||
Gastrocnemius | 0 | − | − | ||
Quadriceps | 0 | − | − | ||
Heart | 0 | − | − | ||
| |||||
Group II +alginate |
ZM2 (2.5 mg)-AON (200 |
Diaphragm | 8 | + | − |
Intestine | 0 | + | + | ||
Gastrocnemius | 0 | − | − | ||
Quadriceps | 0 | − | − | ||
Heart | 0 | − | − | ||
ZM2 (2.5 mg) |
Diaphragm | 0 | − | − | |
Intestine | 0 | − | − | ||
Gastrocnemius | 0 | − | − | ||
Quadriceps | 0 | − | − | ||
Heart | 0 | − | − | ||
| |||||
Group III |
— | Diaphragm | 0 | − | − |
Intestine | 0 | − | − | ||
Gastrocnemius | 0 | − | − | ||
Quadriceps | 0 | − | − | ||
Heart | 0 | − | − |
It is known that dystrophin is present in several cell types in the gastrointestinal wall, in smooth muscle cells (SMCs) of the muscularis externa and muscularis mucosae, in the myoid cells located in the mucosa, in the perivascular SMCs, and all the submucous and myenteric neurons [
Notably, our results give the first evidence that the oral delivery of an antisense oligoribonucleotide co-formulated with ZM2 and alginate can produce a functional effect on RNA splicing. We observe that the protective action of the alginate is essential for preventing damage to the AON molecules by gastric acid since only with alginate-coated ZM2-AON we observe dystrophin synthesis, showing that ZM2 alone are not sufficient for AON protection. The low efficiency on the restoration of dystrophin protein might be due to the inability of ZM2 to efficiently overcome the intestinal barrier, as documented by the biodistribution studies by Odyssey. Concomitantly, the observed laxative effect of alginate may result in a reduced amount of available ZM2-AON complexes and a consequent decrease in the contact time between particles and the intestinal epithelium, leading to a lower uptake of the molecules. AON specific ELISA concordantly demonstrated the absence of AON in mice tissues. Nonetheless, some AONs are protected enough to pass the body barriers and reach at least the diaphragm with measurable functional effect.
Smaller size or/and modified hydrophilicity of NPs and AON chemical modifications may also improve the
All experiments were performed on male
ZM4 nanoparticle sample were prepared by emulsion polymerization of methyl methacrylate employing as emulsion stabilizers, different functional comonomers (N-isopropyl acrylamide (NIPAM), (N,N-dimethyl N-octyl ammonium) ethyl methacrylate bromide (MOAEMA) and 2-aminoethyl methacrylate hydrochloride (AEMA)).
In particular, 30.0 mL of methyl methacrylate were introduced in a flask containing 500 mL of an aqueous solution of 2.34 g (6.7 mmol) of MOAEMA, 0.55 g (3.3 mmol) of AEMA and 1.13 g (10 mmol) NIPAM. The flask was fluxed with nitrogen under constant stirring for 30 minutes, then 85 mg (0.313 mmol) of the cationic free radical initiator AIBA, dissolved in water, were added. The polymerization was performed at
Particle sizes and size distributions were measured by dynamic light scattering and scanning electron microscopy (SEM) analysis. Dynamic light scattering analysis was performed at 25°C, with a Malvern Zetasizer Nano ZS system at a fixed scattering angle of 90°, using a He-Ne laser and a PCS software (Malvern, U.K., version 6.11). Five individual measurements were performed for each sample. The values that we report are the average of these 5 measurements. SEM analysis were performed with a Field Emission Gun Inspect F microscope from FEI Company. The SEM micrographs were elaborated by the Scion Image processing (NIH, public domain) program. From 200 to 250 individual nanospheres were measured for each sample. The characteristics of the obtained nanoparticles are reported in Table
1,2 mL of a solution 3 mg/mL of dye NIR-797 Isothiocyanate (Sigma-Aldrich,
2.5 mg of ZM4-IR NPs dissolved in 200
Prior to the acquisition of
At the end of treatments, mice were sacrificed, their tissues and organs excised, frozen in liquid N2-cooled isopentane, and stored at −80°C. Twenty-micrometer-thick frozen transverse sections were cut from different specimens, analyzed by Odyssey and stained with hematoxylin and eosin (H&E) (Sigma-Aldrich, Milan, Italy).
M23D(+07-18) (5′-GGCCAAACCUCGGCUUACCUGAAAU-3′) AON against the boundary sequences of the exon and intron 23 of mouse
ZM2 nanoparticles were prepared by emulsion polymerization of methyl methacrylate employing, as emulsion stabilizers, two functional comonomers (N-isopropyl acrylamide (NIPAM) and (N,N-dimethyl N-octyl ammonium) ethyl methacrylate bromide) as previously reported [
Suspension of naked ZM2 or ZM2-AON complexes in water were mixed with a solution of sodium alginate (0.52 mg alginate/mg of ZM2) under magnetic stirring for 10 minutes. CaCl2 (3 mM) was added to the solution and kept under agitation for 10 additional minutes. After centrifugation at 11000 g for 15 minutes the supernatant was removed and collected in new tubes, while alginate-ZM2 or alginate ZM2-AON (200
Two groups of
Table
Total RNA was extracted from frozen sections of muscle biopsies using TRIzol (Invitrogen, Milano, Italy), and reverse-transcribed into cDNA using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Frankfurt, Germany). We performed Exon-Specific Real Time Assays (ESRAs) using RNA pool for each treatment group. ESRAs were used on exons 8, 23, and 25 to quantify the percentage of exon-23 skipping in treated, with respect to untreated mice (ΔCt method), using
The assay for measuring the concentration of 2OMePS oligoribonucleotide 23AON in tissue samples is based on a hybridization ligation assay [
One week after the last injection,
Seven-micrometer-thick frozen transverse sections were cut from at least two-thirds of the length of heart, diaphragm, gastrocnemius, quadriceps muscles and intestine; for each muscle at least 5 slices were cut at 150
Western blot analysis was performed as previously described [
To quantify the restoration of dystrophin protein in treated versus wild type mice, a densitometric analysis of autoradiographic bands was performed with a Bio-Rad Densitometer GS 700 (Bio-Rad, Milan, Italy), followed by normalization with the quantity of total protein loaded onto the gels.
The authors declared no conflict of interests.
The Telethon Italy Grant GGP09093 (to A. Ferlini, Department of Medical Science, Section of Medical Genetics, University of Ferrara, Ferrara, Italy) is acknowledged. The authors are grateful to Judith CT van Deutekom (Prosensa Therapeutics B.V., Leiden) for her help and suggestions with AON hybridization ligation assay.