Poster · 2025 · AACR

Silicon Membrane Technology Enables Novel Live-Cell Assays Through Delivery of Diverse Cargos and Screening of Impermeable Compounds Prior to Medchem Optimization

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

Publication text

Silicon Membrane Technology Enables Novel Live-Cell Assays Through Delivery of Diverse Cargos and Screening of Impermeable Compounds Prior to Medchem Optimization Zhihui Song1,Sophia Hirsch1, Darby Kreienberg1, Andrew Larocque1, Eleni Rogers1, Xi Ai2, James Vasta3, Matthew Robers3, Alec Barclay1, Armon Sharei1 1 Portal Biotechnologies, 134 Coolidge Ave, Watertown, MA 02472, USA. www.portal.bio 2Merck Research Laboratories, 2015 Galloping Hill Rd, Kennilworth, NJ, 07033 3Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711 USA Research-Scale System Gateway™ Compact bench-top unit that controls delivery parameters MicroBooster™ delivery cartridge Defined pore size for deformation 18mm 35mm Cell type-specific consumable for optimal mechanoporation Introduction to Portal’s Approach: Implementing a Universal Cell Engineering Solution Mechanical Disruption via Boosting Intracellular DeliveryCells and Cargo Cells Cargo 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

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

Galaxy™ Galaxy system for use with liquid handlers In-line integrations with existing robotics Consistent Delivery of Cargo Across Pre-loaded Plates by Automated In line dispensing 24h post-boost10 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 In-line Integrations Boost with no cargo Dispense into 96 well plates pre-loaded with dextran Concentration Kinase Tracer Intracellular Protein Detection with Promega’s LgBiT Reagents ● 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 Enabling High Throughput Screening by Dispensing into Pre-loaded Multiwell Cargo Plates Instrument and Consumable Independent Instrument 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.

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

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. Mechanoporation Can be Used to Deliver Membrane Impermeable Cargo to Cells Improved delivery using mechanoporation Mixed PBMCs Cell impermeable small molecules A cell impermeable JAK-STAT pathway inhibitor was identified in a cell-free screen. This small molecule was delivered to mixed PBMCs using either electroporation (orange) or mechanoporation (blue). Flow cytometry was used to assess viability and delivery of the JAK compound into the cells (left). Activity of the inhibitor was confirmed by assessing knockdown of STAT in the mixed PBMCs (right). Li J, et al. 2017. ACS Chemical Biology. Efficient JAK Compound Delivery Corresponding STAT Knockdown Plate 2 Plate 7 Dextran Delivery to HeLa Cells by Automated Dispensing 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. Efficient Delivery of Labeled Macrocycle to HEK293 Cells HEK293 cells FITC-labeled macrocycle UntreatedBoost FITC Transmitted light HEK293 cells were mechanoporated with a FITC-labeled macrocycle. Viability and macrocycle delivery to the untreated and boosted samples were measured by flow cytometry (left) and fluorescence microscopy (right). Scale bar = 300 μm. Macrocycle Delivery to HeLa cells Shows Robust Dose Response Boost with no cargo Pipette into plate pre-loaded with labeled macrocycle Macrocycle 0 μM 12 μM 24 μM 48 μM 96 μM Concentration Kinase Inhibitor (Top) HeLa cells were transfected with SRC-NanoLuc Fusion vector then mechanoporated with varying amounts of impermeable kinase tracer. NanoGlo substrate was added and BRET (Bioluminescence resonance energy transfer) was detected on a plate reader. (Bottom) HeLa cells transfected with SRC-NanoLuc Fusion vector were incubated for 2 hours with Src kinase inhibitors (Ponatinib or Bosutinib) then mechanoporated with an impermeable kinase tracer. NanoGlo substrate was added and BRET was detected on a plate reader.

Summary

● Portal’s mechanoporation technology can be used for drug discovery and cell engineering ● Compatible across broad cell types including: NK cells HSCs B cellsPBMCs T cells iPSCs ● Portal’s Research Scale Gateway™ and High Throughput Galaxy™ systems enable complex cell engineering and delivery of cell impermeable molecules with a simple workflow ○ Small molecules, peptides, and proteins can be delivered into cells at scale for use in downstream screening Connect with Portal! Contact us for beta program eligibility www.portal.bio Robust Multiplex Delivery in Unstim. T Cells Mixed dextran, mRNA, & RNP Naive T cells Activated T cells Engineered with Simultaneous circRNA Delivery for IL-12 and CD19 CAR Human Pan-T cells were isolated and activated for 6 days. Activated T cells were mechanoporated with CD19 CAR circRNA and membrane bound IL-12 (mbIL-12) circRNA. Boosted cells were co-cultured 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). Activated T cells

(d6)

CD19 CAR circRNA mblL-12 circRNA

Cd19 Car

mbIL-12 HeLa cells were boosted then manually added into wells of a 96-well plate pre-loaded with FITC-labeled macrocycle at 0-0.2 mg/ml. Flow cytometry was conducted post-boost to assess viability and efficiency of macrocycle delivery into boosted cells. DELs Peptides Macrocycle Antibodies Small molecules ● Can be used to deliver single or multiplexed cargoes including: GFP expression B2M expression Unstimulated T cells isolated from PBMCs were mechanoporated with multiplexed GFP mRNA, fluorescently-labeled dextran polymers, and β2 microglobulin (B2M) CRISPR RNPs. Flow cytometry was performed after 40hr. Concentration Kinase Inhibitor Mechanoporated mRNA Yields Similar BRET Profile Compared to Digitonin-permeabilized Control NanoLuc mRNA was introduced by either mechanoporation (top) or permeabilization with digitonin (bottom) and BRET signal was measured using a plate reader. Intracellular Target Engagement Assays with Impermeable Molecules

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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.

# Silicon Membrane Technology Enables Novel Live-Cell Assays Through Delivery of Diverse Cargos and Screening of Impermeable Compounds Prior to Medchem Optimization 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: Silicon Membrane Technology Enables Novel Live-Cell Assays Through Delivery of Diverse Cargos and Screening of Impermeable Compounds Prior to Medchem Optimization - Document type: Poster - Year: 2025 - Outlet: AACR - Cells: T cells, PBMCs, Cancer Cell Lines - Cargos: mRNA, CRISPR RNPs, Antibody, Oligo, Peptide, Protein, small molecules, Polymers, circRNA - Page URL: https://portal.bio/publications/2025-silicon-membrane-technology-enables-novel-live-cell-assays-through-delivery-of-divers--ajkGEhEAAC4Ac90r - Markdown URL: https://portal.bio/publications/2025-silicon-membrane-technology-enables-novel-live-cell-assays-through-delivery-of-divers--ajkGEhEAAC4Ac90r.md - Original file: https://portal-bio.cdn.prismic.io/portal-bio/aFoqeHfc4bHWipk9_AACR_DrugDiscovery_2025Poster.pdf ## Publication text Silicon Membrane Technology Enables Novel Live-Cell Assays Through Delivery of Diverse Cargos and Screening of Impermeable Compounds Prior to Medchem Optimization Zhihui Song1,Sophia Hirsch1, Darby Kreienberg1, Andrew Larocque1, Eleni Rogers1, Xi Ai2, James Vasta3, Matthew Robers3, Alec Barclay1, Armon Sharei1 1 Portal Biotechnologies, 134 Coolidge Ave, Watertown, MA 02472, USA. www.portal.bio 2Merck Research Laboratories, 2015 Galloping Hill Rd, Kennilworth, NJ, 07033 3Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711 USA Research-Scale System Gateway™ Compact bench-top unit that controls delivery parameters MicroBooster™ delivery cartridge Defined pore size for deformation 18mm 35mm Cell type-specific consumable for optimal mechanoporation Introduction to Portal’s Approach: Implementing a Universal Cell Engineering Solution Mechanical Disruption via Boosting Intracellular DeliveryCells and Cargo Cells Cargo 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. Williams AR, Bao S, Miller DL. Biotechnol Bioeng. 1999 Galaxy™ Galaxy system for use with liquid handlers In-line integrations with existing robotics Consistent Delivery of Cargo Across Pre-loaded Plates by Automated In line dispensing 24h post-boost10 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 In-line Integrations Boost with no cargo Dispense into 96 well plates pre-loaded with dextran Concentration Kinase Tracer Intracellular Protein Detection with Promega’s LgBiT Reagents ● 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 Enabling High Throughput Screening by Dispensing into Pre-loaded Multiwell Cargo Plates Instrument and Consumable Independent Instrument 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. 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 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. Mechanoporation Can be Used to Deliver Membrane Impermeable Cargo to Cells Improved delivery using mechanoporation Mixed PBMCs Cell impermeable small molecules A cell impermeable JAK-STAT pathway inhibitor was identified in a cell-free screen. This small molecule was delivered to mixed PBMCs using either electroporation (orange) or mechanoporation (blue). Flow cytometry was used to assess viability and delivery of the JAK compound into the cells (left). Activity of the inhibitor was confirmed by assessing knockdown of STAT in the mixed PBMCs (right). Li J, et al. 2017. ACS Chemical Biology. Efficient JAK Compound Delivery Corresponding STAT Knockdown Plate 2 Plate 7 Dextran Delivery to HeLa Cells by Automated Dispensing 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. Efficient Delivery of Labeled Macrocycle to HEK293 Cells HEK293 cells FITC-labeled macrocycle UntreatedBoost FITC Transmitted light HEK293 cells were mechanoporated with a FITC-labeled macrocycle. Viability and macrocycle delivery to the untreated and boosted samples were measured by flow cytometry (left) and fluorescence microscopy (right). Scale bar = 300 μm. Macrocycle Delivery to HeLa cells Shows Robust Dose Response Boost with no cargo Pipette into plate pre-loaded with labeled macrocycle Macrocycle 0 μM 12 μM 24 μM 48 μM 96 μM Concentration Kinase Inhibitor (Top) HeLa cells were transfected with SRC-NanoLuc Fusion vector then mechanoporated with varying amounts of impermeable kinase tracer. NanoGlo substrate was added and BRET (Bioluminescence resonance energy transfer) was detected on a plate reader. (Bottom) HeLa cells transfected with SRC-NanoLuc Fusion vector were incubated for 2 hours with Src kinase inhibitors (Ponatinib or Bosutinib) then mechanoporated with an impermeable kinase tracer. NanoGlo substrate was added and BRET was detected on a plate reader. ### Summary ● Portal’s mechanoporation technology can be used for drug discovery and cell engineering ● Compatible across broad cell types including: NK cells HSCs B cellsPBMCs T cells iPSCs ● Portal’s Research Scale Gateway™ and High Throughput Galaxy™ systems enable complex cell engineering and delivery of cell impermeable molecules with a simple workflow ○ Small molecules, peptides, and proteins can be delivered into cells at scale for use in downstream screening Connect with Portal! Contact us for beta program eligibility www.portal.bio Robust Multiplex Delivery in Unstim. T Cells Mixed dextran, mRNA, & RNP Naive T cells Activated T cells Engineered with Simultaneous circRNA Delivery for IL-12 and CD19 CAR Human Pan-T cells were isolated and activated for 6 days. Activated T cells were mechanoporated with CD19 CAR circRNA and membrane bound IL-12 (mbIL-12) circRNA. Boosted cells were co-cultured 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). Activated T cells ### (d6) CD19 CAR circRNA mblL-12 circRNA ### Cd19 Car mbIL-12 HeLa cells were boosted then manually added into wells of a 96-well plate pre-loaded with FITC-labeled macrocycle at 0-0.2 mg/ml. Flow cytometry was conducted post-boost to assess viability and efficiency of macrocycle delivery into boosted cells. DELs Peptides Macrocycle Antibodies Small molecules ● Can be used to deliver single or multiplexed cargoes including: GFP expression B2M expression Unstimulated T cells isolated from PBMCs were mechanoporated with multiplexed GFP mRNA, fluorescently-labeled dextran polymers, and β2 microglobulin (B2M) CRISPR RNPs. Flow cytometry was performed after 40hr. Concentration Kinase Inhibitor Mechanoporated mRNA Yields Similar BRET Profile Compared to Digitonin-permeabilized Control NanoLuc mRNA was introduced by either mechanoporation (top) or permeabilization with digitonin (bottom) and BRET signal was measured using a plate reader. Intracellular Target Engagement Assays with Impermeable Molecules ## 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.