Microfluidic-enabled intracellular delivery of membrane impermeable

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Letter

Microfluidic-enabled intracellular delivery of membrane impermeable inhibitors to study target engagement in human primary cells Jing Li, Bu Wang, Brian M Juba, Michael Vazquez, Steven W. Kortum, Betsy S. Pierce, Michael Pacheco, Lee Roberts, Joseph W Strohbach, Lyn H. Jones, Erik Hett, Atli Thorarensen, Jean-Baptiste Telliez, Armon Sharei, Mark Bunnage, and Jonathan Brian Gilbert ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00683 • Publication Date (Web): 31 Oct 2017 Downloaded from http://pubs.acs.org on November 2, 2017

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ACS Chemical Biology

Microfluidic-enabled intracellular delivery of membrane impermeable inhibitors to study target engagement in human primary cells Jing Li 1§, Bu Wang2●, Brian M Juba3, Michael Vazquez1, Steve W. Kortum4, Betsy S. Pierce 4○ , Michael Pacheco4, Lee Roberts1◊, Joseph W Strohbach1, Lyn H. Jones1∆, Erik Hett1◊, Atli Thorarensen1, Jean-Baptiste Telliez4, Armon Sharei 2, Mark Bunnage 1₸, Jonathan Brian Gilbert2§ 1

Medicine Design, Pfizer Inc, 1 Portland Street, Cambridge, MA, 02139, USA SQZ Biotechnologies, 134 Coolidge Avenue, Watertown, MA, 02472, USA 3 Inflammation and Immunology Research Unit, Pfizer Inc, 1 Portland Street, Cambridge, MA 02139, USA 4 Medicine Design, Pfizer Inc, Eastern Point Road, Groton, CT 06340, USA 2

§

Correspondence: [email protected]; [email protected] [email protected]

● Present address: Reinen, LLC 90 State Street STE 700, Office 40, Albany, NY 12207 ○ Present address: Kalexsyn, 4502 Campus Drive, Kalamazoo, MI 49008, USA ◊ Present address: MRL Exploratory Science Center, 320 Bent Street, Cambridge, MA, 02141, USA ∆ Present address: Jnana Therapeutics, 50 Northern Avenue, Boston, MA, 02210, USA ₸ Present address: Vertex Pharmaceuticals, 50 Northern Avenue, Boston, MA, 02210, USA

1 ACS Paragon Plus Environment


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Abstract Biochemical screening is a major source of lead generation for novel targets. However, during the process of small molecule lead optimization, compounds with excellent biochemical activity may show poor cellular potency, making structure-activity relationships difficult to decipher. This may be due to low membrane permeability of the molecule, resulting in insufficient intracellular drug concentration. The Cell Squeeze® platform increases permeability regardless of compound structure by mechanically disrupting the membrane, which can overcome permeability limitations and bridge the gap between biochemical and cellular studies. In this study, we show that poorly permeable Janus kinase (JAK) inhibitors are delivered into primary cells using Cell Squeeze®, inhibiting up to 90% of the JAK pathway, while incubation of JAK inhibitors with or without electroporation had no significant effect. We believe this robust intracellular delivery approach could enable more effective lead optimization and deepen our understanding of target engagement by small molecules and functional probes.


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Introduction A common challenge faced during target-based drug discovery programs is that lead optimization can be hampered due to target engagement issues, making structure -activity relationships difficult to interpret. For example, “hits” identified from target-based screening may not always reach their intracellular protein target to show activity in cellular assays. The poor translation from biochemical to cellular assays is often attributed to lack of cellular permeability, the presence of efflux pumps, or native ligand competition.1-3 However, it is difficult to delineate these aspects from one another and ascertain how each contributes to the lack of translation. A simple and robust method that can overcome at least one of these challenges, such as cell permeability, could more specifically illuminate the discrepancies between biochemical and cellular assays. 4 Ideally, this technology would satisfy the four-pillars concept,5 which requires that the drug is exposed to the site of action (pillar 1) so it can engage the target (pillar 2) and lead to a functional effect (pillars 3 and 4). Poorly permeable small molecules, chemical probes, and other uncharged materials, such as proteins, have been historically challenging to introduce into the intracellular space by traditional means such as electroporation (EP) or transfection reagents.6 To tackle this challenge, we employed a recently discovered vector-free microfluidic platform that temporarily disrupts the cell membrane to enhance intracellular delivery, enabling the assessment of cellular activity by various functional assays. Specifically, with this delivery system (dubbed Cell Squeeze®) cells undergo rapid mechanical deformation as they pass through constriction points in microfluidic channels. As a result, the formation of transient pores in the membrane facilitates the passive diffusion of materials-of-interest into the cytosol (Figure 1A).7-9 Using this technology, we performed a proof-of-concept study showing that a panel of poorly permeable JAK inhibitors greatly reduced the phosphorylation of STAT5 (pSTAT5) of human primary peripheral blood mononuclear cells (PBMCs). Furthermore, we demonstrated that an impermeable Alexa 647-labeled JAK kinase chemical probe was successfully delivered into PBMCs and inhibited the level of p-STAT5. In comparison, the microfluidic device produced significant improvements relative to electroporation (EP), such as



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enhanced delivery efficiency (% delivery into live cells), and in particular, enabling the delivery of uncharged molecules. Overall, our study reveals an attractive strategy that has the potential to not only allow better understanding of why some biochemically potent small molecules fail to elicit cellular responses, but also provides a platform for delivering membrane impermeable chemical probes into cells to facilitate further target engagement and mechanism-of-action studies. Results and discussion To explore the possibility of intracellular delivery of small molecules into PBMCs, we first applied microfluidic cell squeezing in the presence of cascade blue-labeled 3kDa dextran and monitored its delivery by flow cytometry, as 3kDa dextran delivery has been shown to mimic the delivery of small molecules and proteins.7 Interestingly, with squeezing we observed a right shift with >2 log in fluorescence compared to “endocytosis”, a control condition mimicking uptake by inherent cellular endocytosis where the cells were treated with dextran without squeezing (Figure 1B). The delivery and viability (measured by propidium iodide assay) can be modified using different conditions, but for this study we chose a harsher condition to ensure higher delivery. In addition to dextran, we tested a different fluorescent molecule, Alexa 488, with a size similar to the molecules of interest (vide infra) and examined its intracellular delivery by fluorescent imaging (Figure 1C). In line with previous results,10 the fluorescent signal was found to be diffuse in the cytoplasm and not in endocytic vesicles as previously seen with electroporation.11 Similar to dextran, no fluorescence was detected in the controls and we observed an increase in fluorescence signal when the cells were squeezed at a higher pressure of 60 psi in the presence of Alexa 488. As expected, the higher pressure leads to more rapid cell deformation and induces higher delivery. In order to identify an optimal working condition for the intracellular delivery of small molecules into PBMCs, we evaluated various parameters including different chip designs (S1A), pressures (S1B), temperatures (S1C), and buffers (S1D). Based on the FACS results, we found that using a microfluidic design 30 m in length and 4 m in width with the cells suspended in PBS at 4oC at 60 psi achieves efficient intracellular delivery of molecules while still maintaining reasonable cell viability.


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To assess the potential of the microfluidic platform in supporting drug design and chemical biology research we performed a case study on a well-understood biological system. Janus kinases (JAK) belong to a family of intracellular, non-receptor tyrosine kinases that are required for signaling through type I/II cytokine receptors.12 Upon cytokine binding to gamma-common (c or CD132)-containing receptors, JAK3 and JAK1 are activated leading to the phosphorylation of STAT5, amongst other STATs, to control adaptive immune functions. However, dysregulation of the JAK signaling can lead to inflammatory responses; therefore, targeting JAK1/3 is considered a promising therapeutic strategy for the treatment of inflammatory diseases.13 In an effort to identify potent JAK1 inhibitors, we discovered that a series of small molecules exhibited excellent biochemical potency but low cell (RRCK) permeability (Table 1, Synthesis is described in the SI). Therefore, we suspected that these molecules may not be able to show cellular potency, thus providing an excellent model to test the Cell Squeeze® technology. First, we determined that the JAK-STAT signaling pathway remains intact after squeezing in PBMCs as shown by the western blot analysis of p-STAT5 (Figure 2A). We then examined a poorly cell permeable JAK inhibitor, compound 1. As shown by FACS analysis, 1 weakly inhibits p-STAT5 in PBMCs (Figure 2B). In contrast, after squeezing PBMCs in the presence of the compound, we observed an approximately 90% reduction in p-STAT5 as displayed by a reduction in the high intensity peak, which represents phosphorylated STAT5, and an increase in the low intensity peak, which represents non-phosphorylated STAT5. Interestingly, attempted delivery of 1 by electroporation (EP) was ineffective in inhibiting pSTAT5 (Figure 2B - Left). Additionally, we tested a second JAK inhibitor 2, and likewise, we observed the same phenomenon, inhibition via squeezing and lack of inhibition via EP (Figure 2B - Right). Finally, for further validation we examined a panel of JAK inhibitors with low membrane permeability. As expected, all of these molecules (compounds 3-7) showed decreased levels of p-STAT5 upon cell squeezing, highlighting the advantage of this microfluidic approach (Figure 2C). We carried out a proof-of-principle study using 8 (Synthesis is described in the SI), a poorly permeable JAK chemical probe tagged with Alexa 647 (Figure 3A), which allowed us to evaluate



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its intracellular delivery efficiency via flow cytometry as well as its functional effects simultaneously for direct comparison. After the cells were passed through the microfluidic chip with the chemical probe, we monitored the delivery of 8 by flow cytometry and saw that cell squeezing was more efficient in delivery of 8 compared to non-squeeze endocytosis control (Figure 3B). When we analyzed the delivery of 8 as well as fluorescently labeled model materials via EP, there was an increase in fluorescence for the treated samples (Figure 3B & S2). However, in Figure 3C, as seen previously in Figure 2B, we did not see effective inhibition. Furthermore, in the absence of the compound, Cell Squeeze® alone does not cause effective inhibition (Figure 3D). This indicates that the intracellular concentration of compounds after delivery via EP is too low to show functional effects which could be due to the lower delivery intracellular delivery efficiency with electroporation and/or that the fluorescence measurements for EP are not sufficiently representative of intracellular delivery to the cytosol. Although electroporation can access the cytoplasm in certain cases, there is evidence that electroporated materials can primarily associate with the cell membrane and/or endosomes and thus reduce the impact on an intracellular pathway. Furthermore, for electroporation, physicochemical properties of the materials, such as charge and size, directly influence delivery efficiency.6, 11, 14-16 These data thus highlight the potential of Cell Squeeze®'s diffusive delivery mechanism to enable robust delivery of a wide range of compounds to the cytosol without corruption of normal cell function while minimizing concerns related to material physiochemical properties or sub-cellular localization. As the Cell Squeeze® delivery mechanism depends on diffusion, Cell Squeeze® supports the ability to deliver a wide range of compounds into the intracellular space independent of their structure and charge. Herein, we report the use of the vector-free Cell Squeeze® platform to promote intracellular delivery of poorly permeable JAK inhibitors and chemical probe. The successful intracellular delivery of these small molecules was sufficient to exert their functional effect, as indicated by marked attenuation of downstream STAT5 phosphorylation. By confirming the relationship between biochemical and cell based assays, researchers can trigger further structure-activity relationship studies with increased confidence in the hit of interest.


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In general, intracellular delivery of poorly membrane permeable molecules into human primary cells is a challenge. Existing technologies such as electroporation or chemical/lipid methods have several drawbacks, including their reliance on exogenous materials or electrical fields, which may lead to toxicity or off-target effects.7 In particular, we found that electroporation seemingly leads to delivery as measured by fluorescence; however, when cells were treated with compounds via EP they did not show functional effects. Thus, it is unclear if the compounds are available in the cytosol of if they were embedded in the cytoplasmic membrane or present in endosomes. Nevertheless, cell squeezing overcame these concerns, demonstrating efficient uptake, minimal toxicity as assessed by propidium iodide viability and on-target effect (e.g. siRNA mediated knockdown, antigen presentation) in primary immune cells.6, 9, 17, 18 Importantly, in our study, cell squeezing showed the unique capability of delivering molecules with low membrane permeability without disrupting key cellular functions. However, other signaling mechanisms will require further investigation. Previously, the microfluidic method has shown its ability to deliver a wide range of macromolecules including carbon nanotubes, proteins, antigens and siRNA into stem cells and immune cells.7, 9, 18 Herein, we feature a new application of the method through intracellular delivery of poorly membrane permeable small molecules to address a common issue faced in early drug discovery. Additionally, many activity-based probes exhibit low membrane permeability and therefore can only be used on cell lysates, which potentially do not reflect the physiological environment where proteins are intricately regulated.19, 20 To address this concern, we showed that we were able to deliver a poorly permeable JAK chemical probe 8 into primary cells by microfluidic squeezing and quickly assess its functional effect. Moreover, during the process of conversion of a hit in a phenotypic screen into a functional chemical probe for target identification studies, molecular weight is often added, thus leading to lower cellular permeability. Therefore, the Cell Squeeze® technique would also be potentially useful for enabling chemical biology target identification experiments. In summary, we believe the technology also has the potential to deliver poorly permeable small molecules and functional probes to relevant cell systems to rapidly validate or invalidate targets of interest or to determine in-cell selectivity. By overcoming the challenge of intracellular delivery of low 7 ACS Paragon Plus Environment


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membrane permeability compounds, independent of structure or charge, this approach could enable more effective lead optimization and further enhance our understanding of intracellular target engagement by small molecules. ACKNOWLEDGEMENTS We would like to thank Brittany Stokes and Scott Sauer for their helpful comments and edits in writing this manuscript.

Supporting information, including methods and procedures, is available free of charge via the internet at http://pubs.acs.org. Experimental Procedures Methods and procedures are provided in the Supporting Information. Figure and table legends Figure 1: Delivery of Materials into cells using the Cell Squeeze® technique A) Schematic of the Cell Squeeze® intracellular delivery process whereby cells are deformed to cause transient pores in the cell membrane. B) Histogram of intracellular delivery of fluorescent dextran into PBMCs using Cell Squeeze®. Endocytosis (Endo) was exposed to the same dye, but not squeezed. C) Fluorescent microscopy (40x) of PBMCs with free Alexa488 delivered via the Cell Squeeze® process. Endocytosis was exposed to the same dye, but not squeezed. Figure 2: Intracellular delivery and functional assessment of poorly permeable JAK inhibitors A) Western blot to measure the phosphorylation of the JAK/STAT5 pathway act in the absence or presence the Cell Squeeze® treatment; there are two duplicates of each condition. B) Comparison of low membrane permeable kinase inhibitor activity (compound 1 & 2) when delivered via electroporation or Cell Squeeze®. C) Further validation of the Cell Squeeze® capability to deliver multiple low membrane permeable kinase inhibitors (The mean is shown; error bars, SD). Figure 3: Intracellular delivery and functional assessment of a JAK chemical probe using Cell Squeeze® A) Structure of compound 8 B) Comparison of intracellular delivery of compound 8 when delivered via electroporation or Cell Squeeze®. Endocytosis (Endo) was exposed to the same dye, but not squeezed. 8 ACS Paragon Plus Environment

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C) Demonstration of intracellular activity using model compound 8 at 10 M. D) In the presence or absence of Cell Squeeze®, DMSO carrier alone does not disrupt the pathway of interest. Table 1: A list of poorly cell permeable JAK inhibitors References 1. Strelow, J., Dewe, W., Iversen, P. W., Brooks, H. B., Radding, J. A., McGee, J., and Weidner, J. (2004) Mechanism of Action Assays for Enzymes, In Assay Guidance Manual (Sittampalam, G. S., Coussens, N. P., Nelson, H., Arkin, M., Auld, D., Austin, C., Bejcek, B., Glicksman, M., Inglese, J., Iversen, P. W., Li, Z., McGee, J., McManus, O., Minor, L., Napper, A., Peltier, J. M., Riss, T., Trask, O. J., Jr., and Weidner, J., Eds.), Bethesda (MD). 2. Bunnage, M. E., Gilbert, A. M., Jones, L. H., and Hett, E. C. (2015) Know your target, know your molecule, Nat Chem Biol 11, 368-372. 3. Hughes, J. P., Rees, S., Kalindjian, S. B., and Philpott, K. L. (2011) Principles of early drug discovery, Br J Pharmacol 162, 1239-1249. 4. Hann, M. M., and Simpson, G. L. (2014) Intracellular drug concentration and disposition--the missing link?, Methods 68, 283-285. 5. B., G., and L.H., J. (2014) Target validation using in-cell small molecule clickable imaging probes, Medicinal Chemical Communications 5, 247-254. 6. Stewart, M. P., Sharei, A., Ding, X., Sahay, G., Langer, R., and Jensen, K. F. (2016) In vitro and ex vivo strategies for intracellular delivery, Nature 538, 183-192. 7. Sharei, A., Zoldan, J., Adamo, A., Sim, W. Y., Cho, N., Jackson, E., Mao, S., Schneider, S., Han, M. J., Lytton-Jean, A., Basto, P. A., Jhunjhunwala, S., Lee, J., Heller, D. A., Kang, J. W., Hartoularos, G. C., Kim, K. S., Anderson, D. G., Langer, R., and Jensen, K. F. (2013) A vector-free microfluidic platform for intracellular delivery, Proc Natl Acad Sci U S A 110, 2082-2087. 8. Kollmannsperger, A., Sharei, A., Raulf, A., Heilemann, M., Langer, R., Jensen, K. F., Wieneke, R., and Tampe, R. (2016) Live-cell protein labelling with nanometre precision by cell squeezing, Nat Commun 7, 10372. 9. Sharei, A., Trifonova, R., Jhunjhunwala, S., Hartoularos, G. C., Eyerman, A. T., Lytton-Jean, A., Angin, M., Sharma, S., Poceviciute, R., Mao, S., Heimann, M., Liu, S., Talkar, T., Khan, O. F., Addo, M., von Andrian, U. H., Anderson, D. G., Langer, R., Lieberman, J., and Jensen, K. F. (2015) Ex vivo cytosolic delivery of functional macromolecules to immune cells, PLoS One 10, e0118803. 10. Lee, J., Sharei, A., Sim, W. Y., Adamo, A., Langer, R., Jensen, K. F., and Bawendi, M. G. (2012) Nonendocytic delivery of functional engineered nanoparticles into the cytoplasm of live cells using a novel, high-throughput microfluidic device, Nano Lett 12, 6322-6327. 11. Paganin-Gioanni, A., Bellard, E., Escoffre, J. M., Rols, M. P., Teissie, J., and Golzio, M. (2011) Direct visualization at the single-cell level of siRNA electrotransfer into cancer cells, Proc Natl Acad Sci U S A 108, 10443-10447. 12. Laurence, A., Pesu, M., Silvennoinen, O., and O'Shea, J. (2012) JAK Kinases in Health and Disease: An Update, Open Rheumatol J 6, 232-244. 13. Hofmann, S. R., Ettinger, R., Zhou, Y. J., Gadina, M., Lipsky, P., Siegel, R., Candotti, F., and O'Shea, J. J. (2002) Cytokines and their role in lymphoid development, differentiation and homeostasis, Curr Opin Allergy Clin Immunol 2, 495-506. 14. Escoffre, J. M., Portet, T., Favard, C., Teissie, J., Dean, D. S., and Rols, M. P. (2011) Electromediated formation of DNA complexes with cell membranes and its consequences for gene delivery, Biochim Biophys Acta 1808, 1538-1543. 9 ACS Paragon Plus Environment


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15. Golzio, M., Teissie, J., and Rols, M. P. (2002) Direct visualization at the single-cell level of electrically mediated gene delivery, Proc Natl Acad Sci U S A 99, 1292-1297. 16. Sun, C., Cao, Z., Wu, M., and Lu, C. (2014) Intracellular tracking of single native molecules with electroporation-delivered quantum dots, Anal Chem 86, 11403-11409. 17. Griesbeck, M., Ziegler, S., Laffont, S., Smith, N., Chauveau, L., Tomezsko, P., Sharei, A., Kourjian, G., Porichis, F., Hart, M., Palmer, C. D., Sirignano, M., Beisel, C., Hildebrandt, H., Cenac, C., Villani, A. C., Diefenbach, T. J., Le Gall, S., Schwartz, O., Herbeuval, J. P., Autran, B., Guery, J. C., Chang, J. J., and Altfeld, M. (2015) Sex Differences in Plasmacytoid Dendritic Cell Levels of IRF5 Drive Higher IFN-alpha Production in Women, J Immunol 195, 5327-5336. 18. Szeto, G. L., Van Egeren, D., Worku, H., Sharei, A., Alejandro, B., Park, C., Frew, K., Brefo, M., Mao, S., Heimann, M., Langer, R., Jensen, K., and Irvine, D. J. (2015) Microfluidic squeezing for intracellular antigen loading in polyclonal B-cells as cellular vaccines, Sci Rep 5, 10276. 19. Bunnage, M. E., Chekler, E. L., and Jones, L. H. (2013) Target validation using chemical probes, Nat Chem Biol 9, 195-199. 20. Jones, L. H. (2015) Cell permeable affinity- and activity-based probes, Future Med Chem 7, 21312141.

Notes: Research was conducted in accordance with all acceptable Pfizer policies including IRB/IEC approval.


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A

B 1

Cells and target material in suspension

2

High-speed cell deformation (squeezing)

3

Temporary disruption of the cell membrane

4

Target material enters the cell

5

Membrane reseals

150mg/mL fluorescent dextran Endo|PBS 88% viable

SQZ|PBS 48% viable

fluorescence intensity

C

ACS Paragon Plus Environment

Figure 1


A

Figure 2

No SQZ

B

SQZ

Compound 1

Compound 2 Electroporation | RPMI Inefficient inhibition

Endocytosis | RPMI

SQZ | PBS Efficient inhibition

Endocytosis | PBS

C

P-STAT5 inhibition assay 1.2

1

P-STAT5 level

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0.8 No SQZ

0.6

SQZ 0.4

0.2

0 PF-04939821

3

PF-00599855

4

ACS Paragon Plus Environment PF-00924546 PF-04989163 PF-00537353

5

6

7

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A

B

1mM compound 8

EP|RPMI 79% viable Alexafluor647

SQZ|PBS 48% viable

Endo|RPMI 88% viable

Compound 8

Endo|PBS 88% viable Alexa Fluor 647 fluorescence intensity (Compound 8)

Normalized to mode

C

Compound 8 10mM

D

0.2% DMSO (negative control)

SQZ

SQZ

Endo

Endo

Alexa Fluor 647 fluorescence intensity (P-STAT5 antibody)

Normalized to mode

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Figure 3

Alexa Fluor 647 fluorescence intensity (P-STAT5 antibody) ACS Paragon Plus Environment

Table 1


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JAK1 IC50 (mM)

RRCK (10-6 cm/sec)

1

0.016

0.241

2

0.001

0.419

3

0.086

2.509

4

0.058

0.484

5

0.002

0.722

6

0.001

0.729

7

0.005

0.382

compound ID

Structure


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A

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C


B

CHIP DESIGN

PRESSURE

90 psi 65% viable

30-4 68% viable

60 psi 76% viable

10-4 62% viable

30 psi 76% viable

Endocytosis 88% viable


Pacific Blue fluorescence intensity


D

TEMPERATURE


SI Figure 1

BUFFER

Ice 56% viable

PBS 58% viable

Room Temperature 67% viable

OptiMEM 67% viable

Endocytosis 87%



Figure S1: Effect of key delivery parameters on delivery efficiency and viability. A) microfluidic chip design: ACS Paragon Plus Environment 30-4/10-4 = length-width B) Applied pressure to fluid C) Temperature and D) Delivery Buffer were altered to identify the optimal conditions using PBMCs.


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A

Dextran Endo|RPMI

EP|RPMI

Pacific blue fluorescence intensity

Figure S2: Delivery of model molecule dextran through electroporation as measured by flow cytometry.


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SI Figure 2

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Impermeable Inhibitor/chemical probe

Intracellular Delivery

Live-cell Inhibition


SQZ Biotechnologies | Confidential | 1

Microfluidic-enabled intracellular delivery of membrane impermeable

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