mRNA Sonotransfection of Tumors with Polymeric Microbubbles: Co‐Formulation versus Co‐Administration

Abstract Despite its high potential, non‐viral gene therapy of cancer remains challenging due to inefficient nucleic acid delivery. Ultrasound (US) with microbubbles (MB) can open biological barriers and thus improve DNA and mRNA passage. Polymeric MB are an interesting alternative to clinically used lipid‐coated MB because of their high stability, narrow size distribution, and easy functionalization. However, besides choosing the ideal MB, it remains unclear whether nanocarrier‐encapsulated mRNA should be administered separately (co‐administration) or conjugated to MB (co‐formulation). Therefore, the impact of poly(n‐butyl cyanoacrylate) MB co‐administration with mRNA‐DOTAP/DOPE lipoplexes or their co‐formulation on the transfection of cancer cells in vitro and in vivo is analyzed. Sonotransfection improved mRNA delivery into 4T1 breast cancer cells in vitro with co‐administration being more efficient than co‐formulation. In vivo, the co‐administration sonotransfection approach also resulted in higher transfection efficiency and reached deeper into the tumor tissue. On the contrary, co‐formulation mainly promoted transfection of endothelial and perivascular cells. Furthermore, the co‐formulation approach is much more dependent on the US trigger, resulting in significantly lower off‐site transfection. Thus, the findings indicate that the choice of co‐administration or co‐formulation in sonotransfection should depend on the targeted cell population, tolerable off‐site transfection, and the therapeutic purpose.

mRNA Sonotransfection of Tumors with Polymeric Microbubbles:

Co-formulation vs. Co-administration
Junlin Chen 1 , Bi Wang 1 , Yuchen Wang 1 , Harald Radermacher 1 , Jinwei Qi 1 , Jeffrey Momoh 1 , Twan Lammers 1 , Yang Shi 1 , Anne Rix 1# , Fabian Kiessling 1# * In vivo inflammatory response to streptavidin-coated MB: Eight female Balb/cAnNrJ mice aged 10-12 weeks (Janvier) were randomly assigned using Excel random numbers to receive either uncoated PBCA-MB or Streptavidin-coated PBCA MB (Strep MB).Housing conditions were like those described in the main text.After the acclimatization phase of one week, approximately 100 µl of blood was taken retrobulbar from the animals under inhalation anesthesia to obtain an individual baseline blood count for each animal.All blood analyses were performed from a person blinded to the treatment groups.One week after the first blood draw, the animals were administered the respective type of MB in a concentration of 2 × 10 9 MB/ml in a volume of 50 μl of 0.9% NaCl via a tail vein catheter.Subsequently, blood was taken retrobulbar from the animals to detect acute changes in the blood.A final blood sample was taken two days after MB injection to determine various blood parameters.The animals were euthanized by cervical dislocation, and gross necropsy was performed to assess changes in the liver, kidneys, and spleen weights.

Quantification of Streptavidin on MB:
Streptavidin-conjugated MB were prepared as described in the Experimental section (page 24, last paragraph): 10 9 /mL MB were destroyed by applying an ultrasonic cleaner for 1 minute at 60 W. The Pierce TM BCA Protein Assay was performed according to the manufacturer's instructions (Thermo Fisher Scientific, Schwerte, Germany).Briefly, bovine serum albumin (BSA) standards were prepared with concentrations ranging from 0 to 2 mg/mL.Aliquots of the protein samples (25 μL) or the standards were mixed with the BCA working reagent (200 μL) in a 96well plate.The plate was incubated at 37 °C for 30 min to allow the formation of the purple-colored complex.After the incubation, the absorbance of the samples was measured at 562 nm using a microplate reader (Tecan, Maennedorf, Switzerland).A standard curve was generated using the BSA standards, and the protein concentrations of the samples were calculated based on the standard curve.Liposomes and biotinylated liposomes are stable for more than 3 days at 4 °C and 37 °C as indicated by a stable size.At 4°C, streptavidin-MB are stable for more than 24 hours.At 37 °C, their size starts to decrease after 12 hours.Liposome-MB complexes remain stable for 10 hours at 4 °C.When exposed to 37 °C, their size starts to decrease after 3 hours, indicating that liposomes detach from the MB.

Figure S4 .
Figure S4.The quantification of streptavidin on MB.The addition of EDC significantly increases streptavidin binding.***p < 0.001.

Figure S5 .
Figure S5.Quantification of mRNA contents in suspensions of plain MB, plain liposomes, mRNAlipoplexes, and lipoplex-MB, as determined by the Ribogreen assay.

Figure S6 .
Figure S6.Sizes of liposomes, MB, and liposome-MB complexes at 4 °C and 37 °C over time.Liposomes and biotinylated liposomes are stable for more than 3 days at 4 °C and 37 °C as indicated by a stable size.At 4°C, streptavidin-MB are stable for more than 24 hours.At 37 °C, their size starts to decrease after 12 hours.Liposome-MB complexes remain stable for 10 hours at 4 °C.When exposed to 37 °C, their size starts to decrease after 3 hours, indicating that liposomes detach from the MB.

Figure S7 .
Figure S7.Sonotransfection of breast cancer-bearing mice.Mice were anesthetized with isoflurane and placed in prone position on a 37 °C heating pad.The transducer was kept 2.5 cm away from the center of the tumor using a caliper.US gel was placed on the tumor to fill the gap between the skin and the transducer.

Figure S8 .
Figure S8.Colocalization of the mCherry and anti-mCherry antibody signals in mouse tumor tissue.A)-C) show representative histological images of 4T1 tumors with the expression of mCherry (A), the immunostaining of the mCherry protein (B), and the co-localization of both signals (C).D)-F) 2.5D topological representation of confocal laser microscopy images of mouse tumors after administration of mCherry mRNA and DOTAP/DOPE liposomes.D) The direct observation of mCherry protein fluorescence (yellow) only gains a low signal.E) Immunofluorescence staining with the additional mCherry antibody DyLight 680 (red) significantly improves the protein detection.F) The anti-mCherry antibody DyLight 680 signal co-localizes with the mCherry protein signal, turning it orange.Scale bar = 50 μm.

Figure S9 .Figure S10 .
Figure S9.Representative example for measuring the distances between transfected cells and the closest vessel performed using ImageJ.

Figure S11 .
Figure S11.Quantification of the percentage of A) transfected endothelial cells and B) smooth muscle cells (SMA) based on the co-staining of CD31, SMA, and mCherry antibodies.The transfected endothelial cells (%) were calculated by dividing the number of CD31-positive and mCherry-positive cells by the total mCherry-positive cells.Co-formulation (Co-F) with US resulted in more transfected endothelial cells than lipoplexes alone or co-administration (Co-A).The transfected SMA cells (%) were also calculated by dividing the number of SMA-positive and mCherry-positive cells by the total mCherry-positive cells.*p< 0.05.