Cornea‐SELEX for aptamers targeting the surface of eyes and liposomal drug delivery

Abstract Cornea is the major barrier to drug delivery to the eye, which results in low bioavailability and poor efficacy of topical eye treatment. In this work, we first select cornea‐binding aptamers using tissue‐SELEX on pig cornea. The top two abundant aptamers, Cornea‐S1 and Cornea‐S2, could bind to pig cornea, and their K d values to human corneal epithelial cells (HCECs) were 361 and 174 nм, respectively. Aptamer‐functionalized liposomes loaded with cyclosporine A (CsA) were developed as a treatment for dry eye diseases. The K d of Cornea‐S1‐ or Cornea‐S2‐functionalized liposomes reduces to 1.2 and 15.1 nм, respectively, due to polyvalent binding. In HCECs, Cornea‐S1 or Cornea‐S2 enhanced liposome uptake within 15 min and extended retention to 24 h. Aptamer CsA liposomes achieved similar anti‐inflammatory and tight junction modulation effects with ten times less CsA than a free drug. In a rabbit dry eye disease model, Cornea‐S1 CsA liposomes demonstrated equivalence in sustaining corneal integrity and tear break‐up time when compared to commercial CsA eye drops while utilizing a lower dosage of CsA. The aptamers obtained from cornea‐SELEX can serve as a general ligand for ocular drug delivery, suggesting a promising avenue for the treatment of various eye diseases and even other diseases.


P7-704
CAAGCAGAAGACGGCATACGAGATGCTCAGGA GTGACTGGAGTTCAGACGTGTGCTCT TCCGA TCTGGAGGCTCTCGGGACGAC The enriched pool from round 12 was used as the DNA template and was subjected to another PCR reaction using forward primer (P5-503) and reverse primer (P7-704) containing unique index sequences were used.Agarose gel was used to purify the PCR products subjected to gel extraction using a small DNA fragment extraction kit (IBI Scientific).The concentration of the purified DNA was quantified.The samples were submitted to McMaster University Genomics Facility for Illumina sequencing.

Additional experimental methods Chemicals
Porcine eyeballs were obtained from a local market (Highland Packers, Hamilton).Phospholipids were purchased from Avanti Polar Lipids.All aptamers and primers were purchased from Integrated DNA Technologies, and their sequences are listed in Table S1.Cyclosporine, Fluoromount™ Aqueous Mounting Medium, proteinase K, and deoxyribonucleic acid from salmon sperm were purchased from Sigma-Aldrich.3K (3K MWCO and 10K MWCO Amicon Ultra-0.5 mL Centrifugal Filters were purchased from Millipore Sigma.Bovine serum albumin was purchased from HyClone.Streptavidin agarose resin and cell culture-related chemicals including medium, serum, and antibiotics were purchased from Fisher Scientific Inc. Culture flasks and Hoechst 33342 solution were purchased from Thermo Fisher.Sodium chloride, Isol-RNA Lysis Reagent, and MTT were purchased from VWR. PBS, iScript™ cDNA Synthesis Kit, Micro bio-spin chromatography columns, and SsoFast EvaGreen supermixes were purchased from Bio-Rad.Glass bottom dishes were purchased from Greiner Bio-One.Milli-Q water was used to prepare all buffers, solutions, and suspensions.All buffers and solutions were prepared with Milli-Q water.

Dry eye cell model
HPV-immortalized HCECs were gifted from Dr. Maud Gorbet at the University of Waterloo.The culture method was based on Dr. Gorbet's method with a few modifications. [2]The cells (passages 6-10) were maintained in DMEM/F12 medium supplemented with 1% FBS and 1% penicillin/streptomycin at 37 o C, 95% humidity, and 5% CO2.The cell medium was changed every 2 days.To establish an in vitro DED model, sodium chloride was added to the culture medium to increase the osmolarity of the medium by 200 mOsM.

Cell viability
HCECs were seeded in 96-well plates at 12,000 cells/well and incubated for 24 h.Afterward, HCECs were treated with vehicle, CsA (0.001% in medium), aptamer or non-aptamer CsA liposomes under serum-free hyperosmolarity medium for 24 h.HCECs cultured in the isosmotic medium with a vehicle were used as a control.Cell viability was measured using MTT assay, following the manufacturer's instructions.

Figure S1 .
Figure S1.The 8 most abundant sequences in the sequencing results.The sequences have more than 0.1% counts in the sequencing results were analysed and grouped in 6 families after sequence alignment.

Figure S2 .
Figure S2.Assessment of binding properties of FAM-labelled S2.2 aptamers to HCECs by flow cytometry.(A) Flow cytometry data for FAM-labelled Cornea-S1 and Cornea-S2 aptamer in HCECs.(B) The Kd value of S2.2 aptamers to HCECs was calculated based on the FAM fluorescence intensity determined by flow cytometry.(C) Binding of S2.2 aptamer in HCECs.The binding ability of FAM-labelled Cornea aptamers in HCECs.The cell nucleus was stained with DAPI to give blue fluorescence and the green fluorescence was from FAMlabelled aptamers.Scale bar: 20 μm.

Figure S3 .
Figure S3.Assessment of binding properties of FAM-labelled aptamer to surface proteindisrupted HCECs by flow cytometry.HCECs were detached from the culture flask by 0.25% trypsin and 0.1 mg mL -1 proteinase K followed by incubation with FAM-labelled cornea aptamers.The mean fluorescence intensities of (A) Conrea-S1 and (B) Cornea-S2 in intact cells (black curve) and disrupted cells (red curve) were measured by flow cytometry.

Figure S4 .
Figure S4.Absorbance of free aptamer at 260 nm after aptamer inserted to the liposome.

Figure S5 .
Figure S5.Time-dependent cellular uptake of liposomes.HECEs were treated with nonaptamer, Corena-S1 or Corean-S2 CsA liposomes for 15 min, 30 min, 1 h, 2 h, or 4 h in a hyperosmolarity medium.The cellular uptake of liposomes was studied using fluorescence microscopy.The cell nucleus was stained with DAPI to give blue fluorescence and the red fluorescence was from Rhod PE.Scale bar: 20 μm.Non-apt: non-aptamer.The results indicated the presence of Conrea-S1 and Cornea-S2 aptamers allowed cellular uptake of liposome in 15 min and accumulation of liposome in 4 h in HCECs, while the non-aptamer group required longer incubation time (30 min) to have observable liposome uptake.

Figure S6 .
Figure S6.Evaluation of fluorescein punctate staining and tear film.(A) The cornea was divided into five regions for fluorescein punctate assessment.The images of (B) a stable tear film and (C) unstable tear films with tear film break-up (arrow) were observed under tearscope with a grid pattern.

Figure S7 .
Figure S7.SELEX condition and progress.(A) List of library concentrations in each round.(B) SELEX progress monitoring by RT-PCR results.The y-axes is the number of RT-PCR cycles it took to reach 50% of the maximum fluorescence signal or the midpoint cycle.

Table S1 .
Oligo sequences used in this work.

Table S2 .
Sample preparation for DNA sequencing