Cellular AND Gates: Synergistic Recognition to Boost Selective Uptake of Polymeric Nanoassemblies

Article abstract of DOI:10.1002/anie.202002748

The development of nanoparticle-based biomedical applications has been hampered due to undesired off-target effects. Herein, we outline a cellular AND gate to enhance uptake selectivity, in which a nanoassembly–cell interaction is turned on, only in the concurrent presence of two different protein functions, an enzymatic reaction (alkaline phosphatase, ALP) and a ligand–protein (carbonic anhydrase IX, CA IX) binding event. Selective uptake of nanoassemblies was observed in cells that overexpress both of these proteins (unicellular AND gate). Interestingly, selective uptake can also be achieved in CA IX overexpressed cells, when cocultured with ALP overexpressed cells, where the nanoassembly presumably acts as a mediator for cell–cell communication (bicellular AND gate). This logic-gated cellular uptake could find use in applications such as tumor imaging or theranostics.

Full text of DOI:10.1002/anie.202002748

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: Cellular AND Gates: Synergistic Recognition to Boost Selective
Uptake of Polymeric Nanoassemblies
Authors: Jingjing Gao, Peidong Wu, Ann Fernandez, Jiaming Zhuang,
and Sankaran Thayumanavan
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202002748
Angew. Chem. 10.1002/ange.202002748
Link to VoR: http://dx.doi.org/10.1002/anie.202002748
http://dx.doi.org/10.1002/ange.202002748

10.1002/anie.202002748
Angewandte Chemie International Edition
COMMUNICATION
Cellular AND Gates: Synergistic Recognition to Boost Selective
Uptake of Polymeric Nanoassemblies
Jingjing Gao[a], Peidong Wu[a], Ann Fernandez[a], Jiaming Zhuang*[a], S. Thayumanavan*[a][b][c]
[a]
J. Gao, P. Wu, A. Fernandez, Dr. J. Zhuang, Prof. Dr. S. Thayumanavan,
Department of Chemistry
[b]
[c]
Molecular and Cellular Biology Program
Center for Bioactive Delivery
University of Massachusetts Amherst, Amherst, MA 01003 (USA)
Email: jzhuang@chem.umass.edu; thai@chem.umass.edu
Supporting information for this article is given via a link at the end of the document.
Abstract: Development of nanoparticle based biomedical
applications has been hampered due to undesired off-target effects.
Here we outline a set of cellular AND gate concepts to enhance
selectivity, where a nanoassembly-cell interaction is turned on, only in
the concurrent presence of a two different protein functions, an
enzymatic reaction (ALP) and a ligand-protein (CA IX) binding event.
Selective uptake of nanoassemblies was observed in cells that
overexpress both of these proteins (unicellular AND gate).
Interestingly, selective uptake can also be achieved in CA IX
overexpressed cells, when cocultured with ALP overexpressed cells,
where the nanoassembly presumably acts as a mediator for cell-cell
communication (bicellular AND gate). Demonstration of these logic-
gated cellular uptake will find use in applications such as tumor
imaging or theranostics.
incorporated onto a polymeric nanoassembly, but it is masked
with another functionality, making it unavailable for binding to the
cell surface protein. We envisaged designing the masking
functionality such that it will be removed upon reaction with ALP.
With this design, the nanoassembly would require the concurrent
presence of both CA IX and ALP for activated uptake by cells
(Figure 1).
Biological systems rely on cellular communications to generate
precise responses, where cell surface receptors and enzymes are
major conduits.[1] Similarly, cell surface proteins are the primary
handles in targeting therapeutic molecules and imaging
modalities in many diseases, such as in immunotherapy and in
classifying cancer types.[2] Despite identifying cell surface target
proteins, efforts towards specific on-target accumulation have
resulted in minimal success.[3] Because, although active targeting
relies on the incorporation of ligands on nanoparticles to
recognize receptors overexpressed on tumor cell surfaces,[4]
presence of low level of receptors in off-target locations can still
hamper the specificity of these ligand-decorated nanocarriers.
Nature often overcomes these specificity issues through Boolean
logic, where the need for the concurrent presence of two different
signals are imposed for a process to occur, an AND gate.[5]
Inspired by this capability, we sought out to designing a cellular
AND gate, where two different signals must be present in a single
cell for the cell to efficiently take up a polymer nanoassembly –
unicellular AND gate.
Additionally, biological systems also use cell-cell communications
to execute many functions.[6] With logic-based polymeric
assemblies as the tool, we were interested in also designing
systems, where the processing of the nanoassembly by a specific
protein in one cell activates it for a different cell with a
complementary surface signature – a bicellular AND gate.
To test this possibility, we chose alkaline phosphatase (ALP) and
carbonic anhydrase IX (CA IX) as the two cell surface proteins, as
both of these are known to be overexpressed on certain
pathological cells.[7] Our design strategy for this purpose is shown
in Figure 1. Here, the ligand moiety for CA IX protein is
FIgure 1. Schematic representation of cellular AND gated nanoparticle uptake.
Path a: ligand on the surface of the nanoassembly is unmasked by the enzyme,
but no active cellular uptake due to the absence of the complementary protein.
Path b: the ligand is not unmasked due to the absence of the unmasking
enzyme and is not available for active cellular uptake despite the presence of
complementary protein; Path c: the presence of both the unmasking enzyme
and the complementary protein causes receptor-mediated cellular uptake.
The molecular design strategy for the polymer assembly is shown
in Scheme 1. We designed an amphiphilic diblock copolymer
(Mn= 8.8 K Da, Đ= 1.02.), containing polyethylene glycol (Mn = 5
K Da) as the hydrophilic block and a coumarin-bearing
alkylmethacrylate as the hydrophobic block. The critical cell-
surface recognizing functionality was incorporated at the
hydrophilic terminus, as this part of the polymer would be solvent-
exposed and available for binding, when assembled in aqueous
phase. We chose aryl sulfonamide as the ligand for binding CA
IX. The key ligand-protein binding interaction here is based on
the sulfonamide moiety binding to the zinc ion in the active site of
the CA IX protein.[8] Therefore, we use the sulfonamide moiety to
introduce a masking group, as this would both functionally and
sterically make the aryl sulfonamide moiety unavailable for the
protein. Thus the sulfonamide moiety was protected with p-
substituted benzyl carbamate, as shown in Scheme 1. The
substitution at the para-position is based on a phosphate moiety.
Here, the ALP-induced cleavage of the phosphate moiety would
l i b e r a t e t h e p - p h e n o l i c f u n c t i o n a l i t y
1
This article is protected by copyright. All rights reserved.

10.1002/anie.202002748
Angewandte Chemie International Edition
COMMUNICATION
Scheme 1. Polymer synthesis route and ALP induced exposure of sulfonamide ligands.
which would undergo
sulfonamide moiety. The synthetic approach to this polymer is
shown in Scheme 1.
a
cascade reaction to reveal the
Our design hypothesis is that the polymeric nanoassembly would
not be able to bind to the target enzyme CA IX, until the ALP
enzyme covalently cleaves the phosphate mask to reveal the
sulfonamide ligands. To test this possibility, we used a
competitive displacement assay, where 5-(dimethylamino)-1-
naphthalenesulfonamide (DNSA) is used as the probe. The
fluorescence signal of DNSA is higher when it is complexed to
carbonic anhydrase and is much lower when is released into the
aqueous phase.[9] Here, a CA-DNSA pre-complex would be
fluorescent, but if the complexed DNSA is replaced by the surface
sulfonamide ligand on the nanoassembly, its fluorescence at 460
nm would decrease significantly (Figure 3a).Since CA IX is a
membrane protein, we used bovine carbonic anhydrase (bCA) as
the surrogate protein for making the pre-complex. Our studies
showed that when the ligands on the Nanoassemblies were
The amphiphilic nature of diblock copolymer P2 resulted in the
formation of a nanoassembly in aqueous phase, with an apparent
hydrodynamic diameter of ~120 nm (Figure 2b). To further
stabilize the polymer assembly, light-induced dimerization of the
coumarin moieties within the hydrophobic interior was used to
crosslink the assembly’s core. This dimerization-based
crosslinking of the coumarin moieties was conveniently monitored
by the decrease in the corresponding coumarin absorption peak
(Figure 2c). The time-dependent nature of the decrease shows
that the crosslink density of the assembly can be tuned, if
necessary. The fact that the crosslinking process did not alter the
size suggests that the morphology of the assembly is retained,
which is further supported by transmission electron microscopy
studies (Figure 2d,e).
(a)
Self-assemble
UV irradiation
Polymer
Crosslinked assembly
Micelle
(b)
(c)
1.2
30
25
20
15
10
5
Series1
0 min
3 min
Series3
6 min
micelle
Micelle
1
0.8
0.6
0.4
0.2
0
Series2
nXanliongkele-5dmainssembly
UV
9 min
Series4
12 min
Series5
0
280
300
320
340
360
380
1
10
100
1000
Wavelength (nm)
Size (Dh, nm)
(d)
(e)
Figure 2. a) Preparation of crosslinked nanoassembly; b) DLS profile of micelle
and crosslinked assembly; c) UV induced crosslinking of micelles; TEM images
of (d) micelles and (e) crosslinked assembly.
Figure 3. a) Scheme of DNSA-bCA competitive displacement assay, (b)
Emission spectrum of DNSA-bCA complex when treated with Nanoassembly
and ALP.
2
This article is protected by copyright. All rights reserved.

10.1002/anie.202002748
Angewandte Chemie International Edition
COMMUNICATION
masked by the aryl phosphate moiety, it did not competitively
remove DNSA, suggested by the little change of fluorescence
or a CA IX inhibitor or the combination of the two, during the
cellular uptake experiments. Indeed, the Saos-2 cells treated with
intensity before and after the nanoassembly was added. However, either one or both of the inhibitors exhibited substantially lower
when ALP was added to this system, significant fluorescent signal
decrease was observed over 30 minutes, indicating that the
unmasked ligand on the polymer nanoassembly surface was able
to displace DNSA from the active site of bCA (Figure 3b). TEM
images and DLS profile showed that morphology of
nanoassemblies didn’t change after ALP treatment (Figure S4,
S5), zeta potential of particles increased from -23 mV to +2 mV,
suggesting the cleavage of the phosphate mask (S5).
cellular uptake, compared to the untreated cells (Figure 4b).
These findings support our unicellular AND gate hypothesis that
ALP and CA IX need to be concurrently present on the cell surface
for the nanoassemblies to be taken up efficiently. These findings
also confirm that the dye molecules are not passively diffusing into
the cells.
To further investigate the unicellular AND gate possibility, we
evaluated the selectivity of the nanoassemblies with four different
cell lines. These cell lines were chosen for their variations in the
levels of expression of ALP and CA IX on their surfaces, viz.
Saos-2 (ALP+, CA IX+), MDA-MB-231 (ALP+, CA IX-), HT-1080
(ALP-, CA IX+) and MCF-7 (ALP-, CA IX-) cell lines. If the
proposed unicellular AND gate pathway is operational, the
accumulation of nanoassemblies will be only observed in the
Saos-2 cells, where ALP and CA IX are concurrently
overexpressed. The absence of either protein expression in other
cell lines will suppress the accumulation of nanoassemblies.
Indeed, DiI-loaded nanoassemblies are readily taken up by Saos-
2 cells, but not by MDA-MB-231, HT-1080 or MCF-7 cells (Figure
4c,d).
Figure 4. a) Schematic representation of ‘Unicellular AND gate’ b) Quantitative
analysis of the fluorescence intensity of Cy3-labeled nanoparticles in SAOS-2
cells, c) Histograms of cellular uptake of DiI loaded nanogels in MCF-7, HT1080,
MDA-MB-231 and SAOS-2 cells, d) Mean intensity of four cell lines showed the
DiI loaded nanogel accumulation in four cell lines. Statistical significance was
calculated via one-way ANOVA with a Tukey post-hoc test. *P < 0.05; **P <
0.01.
Following this confirmation, we were interested in testing the
implications of this logic gate in cellular uptake (Figure 4a). SAOS-
2 is a human osteosarcoma cell line that overexpresses both ALP
and CA IX.35 Because of the presence of both these proteins on
the same cell surface, this cell should serve to test the unicellular
AND gate possibility, where the ALP processes the
nanoassembly to reveal the sulfonamide ligand, which when
recognized by the cell surface CA IX, will undergo an activated
uptake inside the cells. To test this hypothesis, we loaded the
polymeric assembly with a hydrophobic dye, 1,1'-dioctadecyl-
3,3,3'3'-tetramethyl-indocarbocyanine perchlorate (DiI), to track
and quantify the cellular uptake using flow cytometry and confocal
laser scanning microscopy. We were excited to find that the
accumulation of the assembly in Saos-2 cells is high (Figure S6).
When treated Saos-2 cells with ALP pretreated Nanoassemblies,
we observed a similar cellular uptake, suggesting ALP on the
Saos-2 cells are sufficient to remove the phosphate mask and
reveal the sulfonamide ligands (Figure S9). However, it is the
comparison with appropriate controls that would clearly test if
there is indeed an activated uptake based on the designed AND
gate. To this end, we treated the Saos-2 cells with an ALP inhibitor
Figure 5. a) Schematic representation of ‘Bicellular AND gate’, b) Flow
cytometry dual fluorescence density plot of HT1080 and MDA-MB-231 coculture
(nanogel was loaded with DiI dye, HT1080 was stained with membrite dye), c)
Histograms of nanogel uptake in HT1080 and MDA-MB-231 cells.
Unicellular AND gate operates when two different proteins are
present on the same cell. Inspired by the cell-cell communication
modalities seen in nature using two distinct proteins in two
different cells, we were interested in investigating the possibility
of bicellular AND gates in cellular uptake (Figure 5a). Here, the
ALP on the surface of one cell would unmask the ligand moieties
in the nanoassembly, while the CA IX on the surface of the second
cell would cause active uptake. To test this idea, we cocultured
two different cell lines, HT-1080 and MDA-MB-231, which
overexpress CA IX and ALP on their surfaces respectively. The
key here is the ability to differentiate the two cells in the
microscope. For this purpose, HT1080 cells were stained with
membrite dye (408 nm). DiI-loaded nanoassemblies were then
added to the coculture and incubated for 1 hour. Although these
3
This article is protected by copyright. All rights reserved.

10.1002/anie.202002748
Angewandte Chemie International Edition
COMMUNICATION
Szabó, E.; Sarkadi, B. Biomark. Med. 2013, 7, 803–819. g) Shy, A. N.;
Kim, B. J.; Xu, B. Matter 2019, 1 , 1127–1147.
assemblies do not readily enter HT-1080 (CA IX+) cells or MDA-
MB-231 (ALP+) cells when they were cultured separately, we
observed a significant accumulation in HT-1080 (CA IX+) cells
when they are cocultured with MDA-MB-231 (ALP+) (Figure 5b,c).
These experiments show that the cell surface ALP in the latter cell
line processes the nanogels to be taken up by the former cells
after binding to the carbonic anhydrase.
[3]. a) Maeda, H. Adv. Drug Deliv. Rev. 2015, 91, 3–6. b) Battistella, C.;
Callmann, C. E.; Thompson, M. P.; Yao, S.; Yeldandi, A. V.; Hayashi, T.;
Carson, D. A.; Gianneschi, N. C. Adv. Healthc. Mater. 2019, 1901105. c)
Byrne, J. D.; Betancourt, T.; Brannon-Peppas, L. Adv. Drug Deliv. Rev.
2008, 60, 1615–1626. d) Bae, Y. H.; Park, K. J. Control. release 2011,
153, 198. e) Long, M. J. C.; Poganik, J. R.; Aye, Y. J. Am. Chem. Soc.
2016, 138, 3610–3622. f) A. B. Chinen, C. M. Guan, J. R. Ferrer, S. N.
Barnaby, T. J. Merkel, C. A. Mirkin, Chem. Rev. 2015, 115, 10530–10574.
g) M. Elsabahy, G. S. Heo, S. Lim, G. Sun, K. L. Wooley, Chem. Rev.
2015, 115, 10967−11011. h) A. R. Maity, D. Stepensky, Mol. Pharm. 2015,
13, 1–7. i) M. W. Tibbitt, J. E. Dahlman, R. Langer, J. Am. Chem. Soc.
2016, 138, 704−717.
[4]. a) Shi, J.; Xiao, Z.; Kamaly, N. Farokhzad, O.C. Acc. Chem. Res. 2011,
44, 1123-1134. b) Cheng, Y.; Morshed, R.A.; Auffinger B.; Tobias, A.L.
Lesniak, M.S. Adv. Drug Deliv. Rev. 2014, 66, 42-57. c) Byrne, J.D.;
Betancourt, T.; Brannon-Peppas, L. Adv. Drug Deliv. Rev. 2008, 60,
1615-1626. d) Low, P.S. Henne W.A.; Doorneweerd, D.D., Acc. Chem.
Res. 2008, 41, 120-129. e) Feng, Z. Q. Q.; Wang, H. M.; Wang, S. Y.;
Zhang, Q.; Zhang, X. X.; Rodal, A.; Xu, B. J. Am. Chem. Soc. 2018, 140,
9566-9573
[5]. a) de Silva, A. P.; Uchiyama, S. Nat. Nanotechnol. 2007, 399–410. b)
Margulies, D.; Melman, G.; Felder, C. E.; Arad-Yellin, R.; Shanzer, A. J.
Am. Chem. Soc. 2004, 126, 15400–15401. c) Erbas-Cakmak, S.; Akkaya,
E. U. Angew. Chemie Int. Ed. 2013, 52, 11364–11368. d) De Silva, P. A.;
Gunaratne, N. H. Q.; McCoy, C. P. Nature 1993, 364, 42–44. e) Gao, J.;
Liu, X.; Secinti, H.; Jiang, Z.; Munkhbat, O.; Xu, Y.; Guo, X.;
Thayumanavan, S. Chem. - A Eur. J. 2018, 24, 1789–1794.
[6]. a) Ellison, D.; Mugler, A.; Brennan, M. D.; Lee, S. H.; Huebner, R. J.;
Shamir, E. R.; Woo, L. A.; Kim, J.; Amar, P.; Nemenman, I.; et al. PNAS,
2016, 113, E679–E688. b) Lawrence, T. S.; Beers, W. H.; Gilula, N. B.
Nature 1978, 272, 501–506. c) Perbal, B. Cell Commun Signal, 2003, 1,
3.
In summary, we have demonstrated a set of cellular logic gates
that exhibit efficient uptake of polymeric nanoassemblies in
specific cells. The polymeric system contains a functional
hydrophilic terminus based on a caged carbonic anhydrase-
specific ligand, which can be unmasked with an ALP-catalyzed
cleavage of an aryl phosphate group and a subsequent self-
immolation reaction. Such a design offers to exhibit cellular AND
gates in unicellular and bicellular settings. We show that these
nanoassemblies are taken up efficiently and selectively by Saos-
2 cells in single cell cultures, because this is the only cell line
overexpresses both the unmasking enzyme ALP and the receptor
protein CA IX, among the four cells tested. The same concept was
then tested in a two-cell co-culture, where one cell overexpresses
ALP (MDA-MB-231) and the other overexpresses CA IX (HT-
1080). Here, only in the co-culture does the latter cell exhibits
higher degree of uptake of the nanoassembly, because the former
cell was able to unmask the ligand from the nanoassembly. The
design insights and the concept of cellular AND gates provided
here will find use in many applications where specificity in
targeting is critical, because an AND-gate requires the concurrent
presence of two different cell surface markers. Since proteins are
arguably the most prominent pathological biomarkers, these dual
protein-based cellular AND gate uptake of polymeric
nanoassemblies would open up new possibilities for tumor
imaging, diagnostics and targeted delivery.
[7]. a) Rao, S. R.; Snaith, A. E.; Marino, D.; Cheng, X.; Lwin, S. T.; Orriss, I.
R.; Hamdy, F. C.; Edwards, C. M. Br. J. Cancer 2017, 116, 227–236. b)
Fishman, W. H.; Inglis, N. R.; Green, S.; Anstiss, C. L.; Gosh, N. K.; Reif,
A. E.; Rustigian, R.; Krant, M. J.; Stolbach, L. L. Nature 1968, 219, 697,
[8]. Krishnamurthy, V. M.; Kaufman, G. K.; Urbach, A. R.; Gitlin, I.; Gudiksen,
K. L.; Weibel, D. B.; Whitesides, G. M. Chem. Rev. 2008, 108, 946–1051.
[9]. H. N. Li, Y. X. Ci, Anal. Chim. Acta 1995, 317, 353–357.
Acknowledgements
We thank the National Science Foundation (CHE-1307118) for
supporting this work. The microscopy data was gathered in the
Light Microscopy Facility and Nikon Center of Excellence at the
Institute for Applied Life Sciences, UMass Amherst with support
from the Massachusetts Life Sciences Center. We thank Dr. Amy
S. Burnside for help with Flow Cytometry data analysis.
Conflict of interest
The authors declare no conflict of interest.
Keywords: AND gate • Targeting • Cellular uptake • Enzyme-
responsive polymeric nanoassembly
[1]. Mittelbrunn, M.; Sánchez-Madrid, F. Nat. Rev. Mol. Cell Biol.
2012, 13, 328.
[2]. a) Seo, J.; Kim, S.; Park, H. H.; Choi, D. Y.; Nam, J. M. Nano- Sci. Adv.
2019, 5, 1–14. b) Callmann, C. E.; Barback, C. V.; Thompson, M. P.; Hall,
D. J.; Mattrey, R. F.; Gianneschi, N. C. Adv. Mater. 2015, 27, 4611–4615.
c) Long, M. J. C.; Poganik, J. R.; Aye, Y. J. Am. Chem. Soc. 2016, 138 ,
3610–3622. d) Wu, L.; Qu, X. Chem. Soc. Rev. 2015, 44, 2963–2997. e)
Liu, H. W.; Chen, L.; Xu, C.; Li, Z.; Zhang, H.; Zhang, X. B.; Tan, W. Chem.
Soc. Rev. 2018, 21, 7140–7180. f) Várady, G.; Cserepes, J.; Németh, A.;
4
This article is protected by copyright. All rights reserved.

10.1002/anie.202002748
Angewandte Chemie International Edition
COMMUNICATION
Entry for the Table of Contents
Insert graphic for Table of Contents here.
Insert text for Table of Contents here.
We showed here a set of cellular logic gates that exhibit efficient uptake of polymeric nanoassemblies in specific cell type, which
were achieved by synergized effects of two different enzymes that are overexpressed on cell surface.
5
This article is protected by copyright. All rights reserved.