RNA Nanotechnology to Solubilize Hydrophobic Antitumor Drug for Targeted Delivery

Abstract Small‐molecule drugs are used extensively in clinics for cancer treatment; however, many antitumor chemical drugs dissolve poorly in aqueous solution. Their poor solubility and nonselective delivery in vivo often cause severe side effects. Here, the application of RNA nanotechnology to enhance the solubility of hydrophobic drugs, using camptothecin (CPT) for proof‐of‐concept in targeted delivery for cancer treatment is reported. Multiple CPT prodrug molecules are conjugated to RNA oligos via a click reaction, and the resulting CPT‐RNA conjugates efficiently self‐assemble into thermodynamically stable RNA three‐way junction (3WJ) nanoparticles. The RNA 3WJ is covalently linked with seven hydrophobic CPT prodrug molecules through cleavable ester bonds and a folic acid ligand for specific tumor targeting while remaining soluble in aqueous solutions without detectable aggregation at therapeutic dose. This CPT‐RNA nanoparticle exhibits efficient and specific cell binding and internalization, leading to cell apoptosis. Tumor growth is effectively inhibited by CPT‐RNA nanoparticles; the targeted delivery, strengthened by tumor ligand, further enhances tumor suppression. Compared with the traditional formulation, solubilization of CPT in aqueous buffer using RNA nanoparticles as a carrier is found to be safe and efficacious, demonstrating that RNA nanoparticles are a promising platform for the solubilization and the delivery of hydrophobic antitumor drugs.

with Agilent PLRP-S 8µM 300Å column. MALDI-TOF mass spectrometry was carried out on Bruker MicroFlex. UV/Vis absorbance was measured on NanoDrop 2000 by Thermo Scientific. Gel electrophoresis system was from Bio-Rad, and all gels were scanned on GE Typhoon FLA 7000. The temperature gradient gel electrophoresis (TGGE) system was from Biometra. Dynamic light scattering (DLS) was done on Malvern zetasizer. Confocal microscopy was done on the Olympus FV 3000 Spectra Confocal system and flow cytometry was done on Becton Dickinson FACSCalibur Flow Cytometer in OSUCCC Analytical Cytometry Shared Resource (ACSR). Olympus IX71 Inverted microscope was used for cell morphology observation.
The reaction was monitored by TLC to ensure the complete consumption of CPT. After the reaction was completed, it was washed with 1 M HCl three times and the organic layer was dried over anhydrous MgSO 4 . Upon the removal of dichloromethane by rotavaporation, the crude product was purified by recrystallization in methanol: dichloromethane (95: 5) followed by washing the product with a small amount of cold methanol. The synthesis yield was 65%. 1

Synthesis of chemically-modified RNA oligos
Standard procedures were followed for solid-phase oligonucleotide synthesis at 1 µmol scale. All pyrimidines were chemically modified by 2'-fluoro (2'-F) or 2'-O-propargyl and all purine were 2'-OH. The sequences are listed, and underlined letters indicate the position of 2'-O-propargyl nucleotides if they are incorporated in the sequences.

Conjugation of CPT prodrug to alkyne-displaying RNA oligos
CPT prodrug was conjugated to alkyne-displaying RNA oligos via Copper-catalyzed azide-alkyne cycloaddition (CuAAC). 100 mM CuBr solution (tBuOH: DMSO= 1:3) was mixed with 100 mM TBTA (tBuOH: DMSO= 1:3) in a ratio of 1:2 to yield Cu-TBTA complex. 10 µL of such freshly prepared Cu-TBTA solution was added to 60 µL CPT prodrug solution (DMSO, 20 mM) followed by adding 50 µL alkyne-displaying RNA oligos (3WJ a -3alkyne or 3WJ b -4alkyne) solution (water, 1-2.5 mM measured by RNA absorbance at 260 nm). Additional 130 µL DMSO was added to keep CPT prodrug soluble. The reaction was done overnight at room temperature. Water was added to reach 400 µL (total volume) followed by adding 40 µL 3 M sodium acetate (pH 5.2) and 1100 µL ethanol (200 proof). The tube was stored at -20 ºC for at least two hours to precipitate CPT-RNA conjugate. After centrifugation at maximal speed (13100 RPM) at 4 ºC for 30 minutes and washing the pellet with 70% cold ethanol, the pellet was dried by spin vacuum. 100 µL water was used to dissolve CPT-RNA conjugate and the partially precipitated CPT prodrug was removed by filtration. CPT-RNA conjugate was further purified by reverse phase HPLC (Solvent A: 0.1 M TEAA (triethylamine acetate) in water and solvent B: 75% acetonitrile and 25% water with 0.1 M TEAA).

Confirmation of CPT-RNA conjugation
Successful CPT-RNA conjugation was resolved on 16% 8 M urea denaturing PAGE or reverse-phase HPLC with an increasing gradient of solvent B (75% Acetonitrile with 0.1 M TEAA) from 5% to 90 % in one hour. Maldi-Tof Mass Spectrometry was performed on Bruker MicroFlex using 3-hydroxypicolinic acid (3-HPA) as the matrix.

Evaluation of improved water solubility by CPT-RNA conjugates
The molarity of CPT-RNA conjugate was measured according to the absorbance at 260 nm which was mainly resulted from RNA. For 3WJ a -3CPT, the concentration of CPT was readily obtained by multiplying CPT-RNA molarity by three. For CPT free drug, the calculated amount of CPT was weighed on analytical balance and mixed with expected amount of water or DMSO. For CPT free drug in water, a homogeneous suspension was made by sonication for 5 minutes and UV absorbance of these saturated aqueous solutions were measured after the high-speed centrifugation to remove undissolved CPT free drug.
Absorbance at 354 nm was recorded for all three solutions and plotted against CPT molarity (theoretical molarity was used for CPT free drug in water as its solubility in water was poor).

Release of CPT in serum
10 µM CPT-RNA conjugate (3WJ a -CPT) was incubated in 50% FBS. 3 µL samples were taken at different time points (up to 12 hours) and fast frozen on dry ice and stored at -80 ºC until analyzed on 16% 8 M urea denaturing PAGE. CPT release yield against incubation time was then quantified by ImageJ.

Assembly of RNA 3WJ samples
All RNA 3WJ samples were prepared by mixing three separate strands (3WJ a , 3WJ b , and 3WJ c ) at equimolar concentration in PBS buffer, annealed at 85 ºC for 5 minutes, and allowed to cool to room temperature over 40 minutes. The assembly was characterized on 12% native PAGE using TBE buffer (120V, 4 ºC, 90 minutes). All assembled RNA 3WJ samples studied in cell and animal experiments were sterilized by 0.22 µm filter. Filtered 3WJ samples were also used in the measurements of dynamic light scattering.

Cell culture
Human KB cells (American Type Culture Collection, ATCC) cultured in RPMI-1640folate deficient (Thermo Scientific) medium containing 10% FBS and 1% Penicillin-Streptomycin in a 37 ºC incubator under 5% CO 2 and a humidified atmosphere.

In vitro cell binding and internalization assay
Concentration is based on assembled RNA nanoparticles. For flow cytometry assay, 25 nM, 100 nM and 400 nM Alexa647 labeled FA-7CPT-3WJ and 7CPT-3WJ nanoparticles were incubated with KB cells at 37 ºC for 1 hour. Besides, 100 nM Alexa647 labeled FA-3WJ and 3WJ without CPT were also used for incubation as control groups. 100 nM Alexa647 labeled FA-7CPT-3WJ and 7CPT-3WJ nanoparticles were also incubated with HepG2 cells at 37 ºC for 1 hour. After washing with PBS twice, the cells were resuspended in PBS for flow cytometry analysis. It was performed by OSUCCC Analytical Cytometry Shared Resource (ACSR). The data was analyzed by FlowJo software.
For confocal microscope imaging, KB cells were seeded on glass slides at 70% confluence one day before treatment. 100 nM FA-7CPT-3WJ-Alexa647 and 7CPT-3WJ-Alexa647 were incubated with cells at 37 ºC for 1 hour. For specificity study, 100 µM folic acid were added to FA-7CPT-3WJ-Alexa647 for co-incubation with cells. After incubation, the cells were washed with PBS and fixed by 4% paraformaldehyde (PFA). The cytoskeleton of cells was stained by Alexa Fluor 488 Phalloidin after pre-treated by 0.1% Triton-X 100 for 5 minutes. The slides with cells were finally mounted with DAPI for nucleus staining. The internalization assay was analyzed by Olympus FV-3000 Spectra Confocal Microscope.

In vitro cytotoxicity effects study
All concentrations are calculated based on CPT. The plate was gently shaken by shaker to get a uniformly colored solution. The absorbance at 570 nm was read by Synergy 4 microplate reader (Bio-Tek).

In vitro apoptosis study
For Caspase-3 assay, KB cells were seeded to 24-well plate overnight. The cells were treated with FA-7CPT-3WJ, 7CPT-3WJ, FA-3WJ, 3WJ as well as CPT alone as a control.
The Caspase-3 activity was measured by Caspase-3 Assay Kit (BD Pharmingen) following the manufacture's instruction. Briefly, cell lysate was collected after 6h, 24h and 48h treatment using Cell Lysis Buffer in the kit. For each group, 40 µL of cell lysate was incubated with 2 μL reconstituted Ac-DEVD-AMC substrate in 80 μL of 1× HEPES buffer and incubated at 37 ºC for 1 hour. The AMC released from Ac-DEVD-AMC was measured by a Fluorolog spectroflurometer (Horiba Jobin Yvon) with excitation wavelength of 380 nm over an emission wavelength of 400-500 nm.
For PI& FITC Annexin V staining assay, KB cells were seeded into 24-well plate overnight. The cells were treated with samples described above. After 48-hour incubation, the assay was conducted using FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen).
The cells were trypsinized, washed by PBS twice and re-suspended in the 1× Binding Buffer (1×10 6 cells/mL). 100 µL of solution was transferred to flow tube. 5 µL FITC Annexin V and 5 µL propidium iodide (PI) were added for 15-minute incubation at room temperature. Finally, 400 µL 1× Binding Buffer was added to each tube for analysis by flow cytometry within 1 hour. When the tumor size reached about 50 mm 3 , KB tumor xenograft bearing mice were randomly divided into 4 groups (n=5). PBS, FA-7CPT-3WJ, 7CPT-3WJ and CPT alone (Formulated in 10% DMSO and 5% Tween 80) were injected to the mice for a total of four doses at 4.3 mg/kg (CPT/mice weight) every other day. PBS treated mice were served as a control group. The tumor volume was measured every day and calculated as (length × width 2 )/2. The mice weight was also recorded every day to monitor whether the treatment caused toxicity and weight lost. At day 10 post-treatment, the mice were sacrificed, and tumors were harvested. The tumor weight of each mice was measure and recorded. Figure S1. Comparison of reverse-phase HPLC spectra (Absorbance at 260nm) of 3WJ b -4alkyne (Black) and 3WJ b -4CPT after purification (Red).