Poster · 2025 · SLAS

Enabling novel live cell assays with a silicon membrane-based platform for mechanical delivery of impermeable small molecules, peptides, and probes to accelerate early drug discovery

Cells: T cells, iPSCs, PBMCs, Cancer Cell Lines
Cargos: mRNA, CRISPR RNPs, Antibody, Oligo, Peptide, Protein, small molecules, Polymers, circRNA

Publication text

Enabling novel live cell assays with a silicon membrane-based platform for mechanical delivery of impermeable small molecules, peptides, and probes to accelerate early drug discovery Sophia Hirsch, Andrew Larocque, Eleni Rogers, Darby Kreienberg, Zhihui Song, Crystal Tsui, Jacquelyn Hanson, Alec Barclay, Armon Sharei Portal Biotechnologies, 134 Coolidge Ave, Watertown, MA 02472, USA. www.portal.bio Delivering Promega’s LgBiT Reagents for Intracellular Protein Detection ● MiaPaCa-2 cells endogenously tagged with HiBiT at the KRAS(G12C) locus were treated with the PROTAC LC-2 for 24 hours (0-10 μM) and mechanoporated with LgBiT protein (Promega) ● Nano-Glo® Endurazine™ was added and luminescence was detected on a GloMAX plate reader to detect degradation of KRAS(G12C) ● Potential utility in other intracellular labeling assays to determine PPIs, degradation and other live cell kinetics Concentration LC-2 Endogenous protein with HiBiT LgBiT delivery for luminescence Degradation removes signal Macrocycle Delivery to HeLa cells Shows Robust Dose Response Macrocycle Boost with no cargoHeLa cells Pipette into plate pre-loaded with labeled macrocycle HeLa cells were boosted then manually added into wells of a 96-well plate pre-loaded with FITC-labeled macrocycle at 0 - 96 μM. Flow cytometry was conducted post-boost to assess viability and efficiency of macrocycle delivery into boosted cells. 0 μM 12 μM 24 μM 48 μM 96 μM Research-Scale System Gateway™ Compact bench-top unit that controls delivery parameters and easily fits in hood MicroBooster™ consumable delivery cartridge Defined pore size for deformation 18mm 35mm Cell type-specific consumable for optimal mechanoporationmediated cargo delivery Introduction to Portal’s Approach: Implementing a Universal Cell Engineering Solution Mechanical Disruption via Boosting Intracellular DeliveryCells and Cargo Cells Cargo ● Low pressure requirements enable integration into existing robotics ● Mechanoporation parameters including pore size can be optimized using the research-scale system ● Ready for early adopters 2025 High-Throughput Capabilities “Powered by Portal” Consumables ● Portal is creating a transformative technology for cellular engineering compatible across cell types and cargos ○ Small molecules, oligos, peptides, antibodies, DELs, mRNA, siRNA, DNA, RNPs, proteins, and other cargos ○ Primary and patient-derived cell lines including immune and stem cells ● Cells are boosted through a membrane at low operating pressure (3-15 PSI) ○ Membrane format1 enables high-throughput cellular engineering

Sharei, et al., PNAS, 2013
Szeto, et al., Scientific Reports, 2015
Sharei, et al., PLOS One, 2015
Lee and Sharei, et al. Nano Letters 2012
Sharei et al., Integrative Biology 2014
DiTommaso, et al., PNAS, 2018

Background on Mechanoporation ● Previous work has shown that mechanical deformation of cells through a microfluidic channel enables cytosolic intracellular delivery1 ○ Delivers macromolecules2,3,4 to both primary cells and cell lines ● Cells are squeezed through a narrow channel at high pressure. The cell membrane is temporarily disrupted, allowing the target cargo to enter the cytosol before the membrane seals5. ○ Cells can be mixed with cargo or added before membrane re-sealing ● Mechanoporation has minimal impact on cell function and gene expression, in contrast to electroporation and viral delivery methods6

Williams AR, Bao S, Miller DL. Biotechnol Bioeng. 1999

Enabling High Throughput Screening with by Dispensing into Pre-loaded Multiwell Cargo Plates

Summary

● Portal’s mechanoporation technology can be used for drug discovery and cell engineering ● Efficient delivery of diverse impermeable molecules for use in downstream screening including: ● Compatible across broad cell types including: NK cells HSCs B cellsPBMCs T cells iPSCs Connect with Portal! Contact us for beta program eligibility www.portal.bio ● Simple workflow enabling high throughput capabilities ● Can be used to deliver single or multiplexed cargoes ● Little impact on normal cellular function or morphology HEK293 cells + Other cell lines DELs Peptides Macrocycle Antibodies Small molecules Consistent Delivery of Cargo Across Pre-loaded Plates by Automated In line dispensing 24h post-boost Dextran Delivery to HeLa Cells by Automated Dispensing Well # (Plate 2) Well # (Plate 7) 10 min post-boost A cartridge was designed for in-line integration of mechanoporation with a robotic single-head dispenser. HeLa cells were automatically dispensed into 96-well plates pre-loaded with fluorescent dextran. Flow cytometry was used to assess dextran delivery. Data for delivery across the 2nd (wells 97-192) and 7th (wells 577-672) 96-well plates are shown here. Phase contrast images (right) show morphology of HeLa cells 10 minutes (left) and 24 hours (right) post-boost. Scale bar (white) = 300 μm. Consumable only Instrument and Consumable In-line Integrations Independent Instrument Robust Multiplex Delivery in Unstimulated T Cells GFP expression B2M expression Mixed dextran, mRNA, & RNP Naive T cells Naive T cells were isolated from PBMCs and mechanoporated with a multiplexed cargo containing GFP mRNA, fluorescently-labeled dextran polymers, and β2 microglobulin (B2M) CRISPR RNPs. After 40h, expression of mRNA and B2M protein were assessed by flow cytometry. Galaxy™ Galaxy device mechanoporates cells and outputs into a trough ready for liquid handler or manual multi-channel pipette FITC-labeled oligo, and Cascade blue-labeled 3 kDa dextran were mixed and dispensed into 96-well plates. The Galaxy system was used to mechanoporate HeLa cells into a trough before dispensing into wells of 5 96-well plates with a multichannel. Flow cytometry was conducted post-boost to assess delivery of the cargos to the cells. Data from plates 1 and 5 shown here. Activated T cells Engineered with Simultaneous circRNA Delivery for IL-12 and CD19 CAR Human Pan-T cells were isolated and activated with CD3/CD28 Dynabeads for 48h and T cells were cultured an additional 4 days. Activated T cells were mechanoporated with CD19 CAR circRNA and membrane bound IL-12 (mbIL-12) circRNA. Boosted cells were rested for 2 h before co-culturing with Raji cells at different ratios of effector cells (E) to target cells (T). Flow cytometry was conducted 48h post-boost to assess expression of CD19 CAR and mbIL-12 (left) and cytotoxicity (right). Cytotoxicity was measured by quantifying re;/duction in CD19+ Raji cells relative to counting beads. Activated T cells

(d6)

CD19 CAR circRNA mblL-12 circRNA

Cd19 Car

mbIL-12 Robust DNA Encoded Library Delivery in iPSCs and HeLa Cells iPSCs Fluorescent-tagged OR iPSC Boost HeLa Boost Induced pluripotent stem cells (iPSCs) or HeLa cells were mechanoporated with a fluorescently-tagged DNA encoded library (DEL) probe. Delivery efficiency was measured using flow cytometry to detect the fluorescent tag immediately after boosting. Portal cartridge integrated in-line with the fluidics of an existing robot in custom integrations. Boost with no cargo Dispense into 96 well plates pre-loaded with dextran Efficient Delivery of Labeled Macrocycle to HEK293 Cells HEK293 cells FITC-labeled macrocycle Untreated FITC Transmitted light HEK293 cells were mechanoporated with a FITC-labeled macrocycle. Flow cytometry was used to assess viability and macrocycle delivery to the untreated and boosted samples. Delivery was also assessed by fluorescence microscopy (right). Scale bar = 300 μm.

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Some publications reference SQZ Biotechnologies, a company Dr. Armon Sharei and colleagues previously founded. Portal is the next generation company leveraging Dr. Sharei's platform technology.

# Enabling novel live cell assays with a silicon membrane-based platform for mechanical delivery of impermeable small molecules, peptides, and probes to accelerate early drug discovery This page is a text version of a Portal Biotechnologies publication. The bibliographic record is labeled in the text so it can be read without the surrounding website. ## Bibliographic record - Title: Enabling novel live cell assays with a silicon membrane-based platform for mechanical delivery of impermeable small molecules, peptides, and probes to accelerate early drug discovery - Document type: Poster - Year: 2025 - Outlet: SLAS - Cells: T cells, iPSCs, PBMCs, Cancer Cell Lines - Cargos: mRNA, CRISPR RNPs, Antibody, Oligo, Peptide, Protein, small molecules, Polymers, circRNA - Page URL: https://portal.bio/publications/2025-enabling-novel-live-cell-assays-with-a-silicon-membrane-based-platform-for-mechanical--ajkGNhEAAC4Ac92M - Markdown URL: https://portal.bio/publications/2025-enabling-novel-live-cell-assays-with-a-silicon-membrane-based-platform-for-mechanical--ajkGNhEAAC4Ac92M.md - Original file: https://portal-bio.cdn.prismic.io/portal-bio/aFosjHfc4bHWiplN_PortalSLASposter2025.pdf ## Publication text Enabling novel live cell assays with a silicon membrane-based platform for mechanical delivery of impermeable small molecules, peptides, and probes to accelerate early drug discovery Sophia Hirsch, Andrew Larocque, Eleni Rogers, Darby Kreienberg, Zhihui Song, Crystal Tsui, Jacquelyn Hanson, Alec Barclay, Armon Sharei Portal Biotechnologies, 134 Coolidge Ave, Watertown, MA 02472, USA. www.portal.bio Delivering Promega’s LgBiT Reagents for Intracellular Protein Detection ● MiaPaCa-2 cells endogenously tagged with HiBiT at the KRAS(G12C) locus were treated with the PROTAC LC-2 for 24 hours (0-10 μM) and mechanoporated with LgBiT protein (Promega) ● Nano-Glo® Endurazine™ was added and luminescence was detected on a GloMAX plate reader to detect degradation of KRAS(G12C) ● Potential utility in other intracellular labeling assays to determine PPIs, degradation and other live cell kinetics Concentration LC-2 Endogenous protein with HiBiT LgBiT delivery for luminescence Degradation removes signal Macrocycle Delivery to HeLa cells Shows Robust Dose Response Macrocycle Boost with no cargoHeLa cells Pipette into plate pre-loaded with labeled macrocycle HeLa cells were boosted then manually added into wells of a 96-well plate pre-loaded with FITC-labeled macrocycle at 0 - 96 μM. Flow cytometry was conducted post-boost to assess viability and efficiency of macrocycle delivery into boosted cells. 0 μM 12 μM 24 μM 48 μM 96 μM Research-Scale System Gateway™ Compact bench-top unit that controls delivery parameters and easily fits in hood MicroBooster™ consumable delivery cartridge Defined pore size for deformation 18mm 35mm Cell type-specific consumable for optimal mechanoporationmediated cargo delivery Introduction to Portal’s Approach: Implementing a Universal Cell Engineering Solution Mechanical Disruption via Boosting Intracellular DeliveryCells and Cargo Cells Cargo ● Low pressure requirements enable integration into existing robotics ● Mechanoporation parameters including pore size can be optimized using the research-scale system ● Ready for early adopters 2025 High-Throughput Capabilities “Powered by Portal” Consumables ● Portal is creating a transformative technology for cellular engineering compatible across cell types and cargos ○ Small molecules, oligos, peptides, antibodies, DELs, mRNA, siRNA, DNA, RNPs, proteins, and other cargos ○ Primary and patient-derived cell lines including immune and stem cells ● Cells are boosted through a membrane at low operating pressure (3-15 PSI) ○ Membrane format1 enables high-throughput cellular engineering 1. Sharei, et al., PNAS, 2013 2. Szeto, et al., Scientific Reports, 2015 3. Sharei, et al., PLOS One, 2015 4. Lee and Sharei, et al. Nano Letters 2012 5. Sharei et al., Integrative Biology 2014 6. DiTommaso, et al., PNAS, 2018 Background on Mechanoporation ● Previous work has shown that mechanical deformation of cells through a microfluidic channel enables cytosolic intracellular delivery1 ○ Delivers macromolecules2,3,4 to both primary cells and cell lines ● Cells are squeezed through a narrow channel at high pressure. The cell membrane is temporarily disrupted, allowing the target cargo to enter the cytosol before the membrane seals5. ○ Cells can be mixed with cargo or added before membrane re-sealing ● Mechanoporation has minimal impact on cell function and gene expression, in contrast to electroporation and viral delivery methods6 1. Williams AR, Bao S, Miller DL. Biotechnol Bioeng. 1999 Enabling High Throughput Screening with by Dispensing into Pre-loaded Multiwell Cargo Plates ### Summary ● Portal’s mechanoporation technology can be used for drug discovery and cell engineering ● Efficient delivery of diverse impermeable molecules for use in downstream screening including: ● Compatible across broad cell types including: NK cells HSCs B cellsPBMCs T cells iPSCs Connect with Portal! Contact us for beta program eligibility www.portal.bio ● Simple workflow enabling high throughput capabilities ● Can be used to deliver single or multiplexed cargoes ● Little impact on normal cellular function or morphology HEK293 cells + Other cell lines DELs Peptides Macrocycle Antibodies Small molecules Consistent Delivery of Cargo Across Pre-loaded Plates by Automated In line dispensing 24h post-boost Dextran Delivery to HeLa Cells by Automated Dispensing Well # (Plate 2) Well # (Plate 7) 10 min post-boost A cartridge was designed for in-line integration of mechanoporation with a robotic single-head dispenser. HeLa cells were automatically dispensed into 96-well plates pre-loaded with fluorescent dextran. Flow cytometry was used to assess dextran delivery. Data for delivery across the 2nd (wells 97-192) and 7th (wells 577-672) 96-well plates are shown here. Phase contrast images (right) show morphology of HeLa cells 10 minutes (left) and 24 hours (right) post-boost. Scale bar (white) = 300 μm. Consumable only Instrument and Consumable In-line Integrations Independent Instrument Robust Multiplex Delivery in Unstimulated T Cells GFP expression B2M expression Mixed dextran, mRNA, & RNP Naive T cells Naive T cells were isolated from PBMCs and mechanoporated with a multiplexed cargo containing GFP mRNA, fluorescently-labeled dextran polymers, and β2 microglobulin (B2M) CRISPR RNPs. After 40h, expression of mRNA and B2M protein were assessed by flow cytometry. Galaxy™ Galaxy device mechanoporates cells and outputs into a trough ready for liquid handler or manual multi-channel pipette FITC-labeled oligo, and Cascade blue-labeled 3 kDa dextran were mixed and dispensed into 96-well plates. The Galaxy system was used to mechanoporate HeLa cells into a trough before dispensing into wells of 5 96-well plates with a multichannel. Flow cytometry was conducted post-boost to assess delivery of the cargos to the cells. Data from plates 1 and 5 shown here. Activated T cells Engineered with Simultaneous circRNA Delivery for IL-12 and CD19 CAR Human Pan-T cells were isolated and activated with CD3/CD28 Dynabeads for 48h and T cells were cultured an additional 4 days. Activated T cells were mechanoporated with CD19 CAR circRNA and membrane bound IL-12 (mbIL-12) circRNA. Boosted cells were rested for 2 h before co-culturing with Raji cells at different ratios of effector cells (E) to target cells (T). Flow cytometry was conducted 48h post-boost to assess expression of CD19 CAR and mbIL-12 (left) and cytotoxicity (right). Cytotoxicity was measured by quantifying re;/duction in CD19+ Raji cells relative to counting beads. Activated T cells ### (d6) CD19 CAR circRNA mblL-12 circRNA ### Cd19 Car mbIL-12 Robust DNA Encoded Library Delivery in iPSCs and HeLa Cells iPSCs Fluorescent-tagged OR iPSC Boost HeLa Boost Induced pluripotent stem cells (iPSCs) or HeLa cells were mechanoporated with a fluorescently-tagged DNA encoded library (DEL) probe. Delivery efficiency was measured using flow cytometry to detect the fluorescent tag immediately after boosting. Portal cartridge integrated in-line with the fluidics of an existing robot in custom integrations. Boost with no cargo Dispense into 96 well plates pre-loaded with dextran Efficient Delivery of Labeled Macrocycle to HEK293 Cells HEK293 cells FITC-labeled macrocycle Untreated FITC Transmitted light HEK293 cells were mechanoporated with a FITC-labeled macrocycle. Flow cytometry was used to assess viability and macrocycle delivery to the untreated and boosted samples. Delivery was also assessed by fluorescence microscopy (right). Scale bar = 300 μm. ## Provenance Some publications reference SQZ Biotechnologies, a company Dr. Armon Sharei and colleagues previously founded. Portal is the next generation company leveraging Dr. Sharei's platform technology.