This work describes how a small-molecule chemical trigger, reacting through the mediatory of an inverse electron demand Diels–Alder reaction, results in enhanced cellular uptake and selective nanoparticle disintegration and cargo liberation, via gross polymeric morphological alterations. The power of these responsive nanoparticles is demonstrated through encapsulation of the anti-cancer agent doxorubicin and its triggered release, allowing controlled cell death in response to a small-molecule chemical trigger

Allen

Blanazs

Cabral

Denard

Discher

Elsabahy

Esser-Kahn

Fleige

Jain

Jin Geng

Karver

Kataoka

Kevin Neumann

Kurra

Mark Bradley

Meng

Mura

Napoli

Pasut

Pattni

Sarthak Jain

Savić

Schild

Schoch

Shapira

Stuart

Venkataraman

Wang

Wijnen

Yang

Zhang

Šečkutė

English

PubMed

Crossref

Chemical Communications

Nanoparticle “switch-on” by tetrazine triggering

Neumann, Kevin

Jain, Sarthak

Geng, Jin

Bradley, Mark

Edinburgh Research Explorer

     Edinburgh Research Explorer                                      Nanoparticle “switch-on” by tetrazine triggeringCitation for published version:Neumann, K, Jain, S, Geng, J & Bradley, M 2016, 'Nanoparticle “switch-on” by tetrazine triggering',Chemical Communications, vol. 52, no. 75, pp. 11223-11226. https://doi.org/10.1039/C6CC05118ADigital Object Identifier (DOI):10.1039/C6CC05118ALink:Link to publication record in Edinburgh Research ExplorerDocument Version:Peer reviewed versionPublished In:Chemical CommunicationsGeneral rightsCopyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s)and / or other copyright owners and it is a condition of accessing these publications that users recognise andabide by the legal requirements associated with these rights.Take down policyThe University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorercontent complies with UK legislation. If you believe that the public display of this file breaches copyright pleasecontact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately andinvestigate your claim.Download date: 11. May. 2020Journal	Name	 	COMMUNICATION	This	journal	is	©	The	Royal	Society	of	Chemistry	20xx	 J.	Name.,	2013,	00,	1-3	|	1 		Please	do	not	adjust	margins	Please	do	not	adjust	margins	Received	00th	January	20xx,	Accepted	00th	January	20xx	DOI:	10.1039/x0xx00000x	www.rsc.org/	Nanoparticle	“Switch-on”	by	Tetrazine	Triggering		Kevin	Neumann,‡	Sarthak	Jain,‡	Jin	Geng*	and	Mark	Bradley*	We	show	how	a	small-molecule	chemical	trigger,	reacting	through	the	mediatory	of	an	inverse	electron	demand	Diels-Alder	reaction,	results	 in	 enhanced	 cellular	 uptake	 and	 selective	 nanoparticle	disintegration	 and	 cargo	 liberation,	 via	 gross	 polymeric	morphological	 alterations.	 The	 power	 of	 these	 responsive	nanoparticles	 is	demonstrated	through	encapsulation	of	the	anti-cancer	 agent	 doxorubicin	 and	 its	 triggered	 release,	 allowing	controlled	 cell	 death	 in	 response	 to	 a	 small-molecule	 chemical	trigger.	Nanotechnology-based	 systems	 for	 drug	 delivery	 have	received	 tremendous	 attention	 and	 delivered	 impressive	 and	symbiotic	 progress	 in	 materials	 science	 and	 pharmaceutical	development.1-3	 Nanoparticles	 (NP),	 have	 been	 used	 to	improve	 drug	 solubility	 and	 enhance	 therapeutic	effectiveness4,5,	 owing	 to	 targeting	 to	 tumor	 tissues	 with	improved	pharmacokinetics	and	pharmacodynamics	and	active	intracellular	 delivery.	 In	 this	 context	 nanoparticle-based	polymersomes,	 generated	 from	 polymeric	 amphophiles,	 are	popular	allowing	the	rapid	generation	of	stable	vesicles,6-8	and	micelles9-11	 in	 water.	 Typically,	 the	 membranes	 of	polymersomes	 are	 thicker,	 stronger,	 and	 inherently	 more	stable	 than	 those	 found	 in	 conventional	 liposomes,	while	 the	scope	 of	 polymeric	 building	 blocks	 available	 for	 membrane	formation	 allows	 for	 much	 greater	 chemical	 control	 and	tunability	then	conventional	lipid	nano-structures.12,13	Typically	micelles	 encapsulate	 hydrophobic	 compounds,	 while	 vesicles	can	 encapsulate	 hydrophilic	 molecules	 within	 their	 aqueous	interior	 as	 well	 as	 trapping	 hydrophobic	 moieties	 within	 the	“membrane”	 with	 drugs	 such	 as	 doxorubicin	 well	 retained.14	Polyethylene	 glycol	 (PEG)	 has	 been	 widely	 used	 as	 the	hydrophilic	 block	 in	 polymersomes,	 conferring	 properties	 to	block	 immunological	 recognition,	 and	 enhance	biocompatibility.15		Triggered-release	nanoparticles	offer	a	sophisticated	approach	to	 drug-delivery	 enabling	 control	 over	 when	 and	 where	 the	drug	 is	 released,	 enhancing	 therapeutic	 efficacy	 and	minimising	 side-effects.16,17	 In	 general,	 triggering	 causes	changes	 in	 the	 hydrophilic-to-hydrophobic	 balance	 of	 the	polymer,	thereby	resulting	in	morphological	disturbance	of	the	self-assembled	 polymersome	 structure,	 and	 nanoparticle	conformational	instability.18	Controlled	release	from	nanoparticles	through	the	application	of	an	external	stimulus	can	be	broadly	divided	into	remote	and	local	 triggers.	 Remote	 triggers	 use	 an	 external	 physical	stimulus	such	as	temperature	(e.g.	polymers	exhibiting	a	lower	critical	 solution	 temperature,	 for	 example,	 poly(N-isopropylacrylamide))19,	 ultrasound	 or	 light	 (which	 can	 alter	the	 properties	 of	 the	 nanoparticles	 with	 the	 help	 of	 a	molecular	switch	such	as	an	azobenzene	unit20).	Local	triggers	utilise	the	environment	of	the	target	site,	for	example,	an	up-regulation	 in	 a	 specific	 enzyme	 (e.g.	 a	 protease),	 a	 change	 in	pH,	or	reactive	oxygen	species	(often	associated	with	tumors).	As	 such	 reactive	 oxygen	 species,	 such	 as	 hydrogen	 peroxide	and	 hydroxyl	 radicals,	 have	 been	 employed	 as	 triggers	 to	create	 oxidation	 sensitive	 drug	 delivery	 systems	 based	 on	thioether21	 or	 selenium	 oxidation22.	 In	 addition,	 drugs	 have	been	 released	 from	 polymers	 through	 cleavage	 of	 disulphide	linkages	by	glutathione	(GSH)23,24	or	dithiothreitol	 (DTT)25,26	 in	the	form	of	degradable	polymer	aggregates.	A	powerful	reaction	that	has	recently	been	widely	exploited	in	bioconjugation27,28	 strategies	 is	 the	 additive	 free,	 inverse	electron	demand	Diels-Alder	reaction,	between	tetrazines	and	electron	rich	dienophiles,29	while,	the	inverse	electron	demand	Diels-Alder	 reaction	 in	 an	 aqueous	 environment	 displays	 an	acceleration	 behaviour	 as	 previous	 reported	 by	 both	 us	 and	others.30,31	 Here	 we	 report	 the	 development	 of	 a	 fully	bioorthogonal,	small	molecule	activated	nanoparticle,	with	on-demand	drug	release.	Using	a	small	molecule,	in	the	form	of	a	tetrazine	as	an	external	 trigger,	polymer	 chains	 that	make	up	the	nanoparticles	undergo	a	series	of	inverse	electron	demand	Diels-Alder	 reactions,	 disrupting	 the	 nanoparticle,	 with	COMMUNICATION	 Journal	Name	2 	|	J.	Name.,	2012,	00,	1-3	 This	journal	is	©	The	Royal	Society	of	Chemistry	20xx	Please	do	not	adjust	margins	Please	do	not	adjust	margins	concomitant	release	of	an	encapsulated	drug	–	 in	essence	we	demonstrate	 tetrazine	 responsive	 nanoparticles	with	 “switch-on”	 release	of	 the	 anti-cancer	 agent	doxorubicin.	 PC3	human	prostate	 cancer	 cell	 lines	 were	 chosen	 for	 investigating	 their	response	to	doxorubicin	loaded	nanoparticles.	Poly(allyl	 glycidyl	 ethers)	 (PAGE)	 have	 been	 explored	 as	 a	chemically	 flexible	 alternative	 to	 PEG,	 stemming	 from	 the	pendant	 allyl	 groups	 which	 are	 amenable	 to	 a	 range	 of	modifications.	In	this	study,	a	diblock	copolymer,	poly(ethylene	glycol)-b-(allyl	 glycidyl	 ether)	 (PEG-b-PAGE),	 was	 synthesised	via	 anionic	 ring-opening	 polymerisation	 using	 potassium	alkoxide/naphthalenide	 as	 the	 initiator32	 to	 give	 a	 block	copolymer	 that	 contained	 a	 hydrophilic	 PEG	 block	 with	 a	weight	fraction	of	30	%	and	a	hydrophobic	block	with	terminal	allyl	 pendant	 units.	 This	 was	 formulated	 to	 form	compartmental	 self-assembled	 nanoparticles	 (Fig.	 1),	 with	transmission	electron	microscopy	(TEM)	analysis	revealing	the	formation	 of	 characteristic	 hollow	 nanoparticles	 with	 a	 well-defined	 spherical	 morphology,	 exhibiting	 a	 mono-modal	distribution	 of	 particles	 of	 ca	 150	 ±	 32	 nm,	 with	 a	 wall	thickness	estimated	to	be	4	±	1	nm	(Fig.	1c).		As	 a	 reactive	 specie,	 it	 is	 important	 to	 keep	 the	 balance	 of	solution	stability	and	fast	reaction	kinetics.33	We	have	previous	reported	that	the	tetanize	used	 in	this	study	undergoes	a	fast	reaction	 with	 poly(allyl	 glycidyl	 ethers)	 in	 water.30 Upon	treatment	 of	 the	 nanoparticles	 with	 a	 water-soluble	 and	hydrophilic	 tetrazine,	 the	 polymer	 backbone	 was	 rapidly	modified,	 with	 dynamic	 light	 scattering	 studies	 revealing	 a	dramatic	 reduction	 in	 diameter	 from	 150	 nm	 to	 30	 nm	 after	four	hours	 incubation	 (interestingly	 this	displayed	a	 lag	phase	of	2	hours,	see	Fig.	2a).	The	reaction	will	be	slow	initially	since	that	the	hydrophilic	tetrazine	has	to	reach	to	the	hydrophobic	layers,	but	following	the	modification,	the	rate	will	increase,	as	the	 membrane	 becomes	 more	 and	 more	 hydrophilic.	 TEM	measurement	 confirmed	 that	 a	 population	 of	 micelles	 was	generated	with	a	uniform	diameter	of	30	±	5	nm	(Fig.	1d,	and	e).	 These	 changes	 take	 place	 due	 to	 the	 modification	 of	 the	hydrophobic	 moieties,	 arising	 from	 the	 conversing	 of	 the	hydrophobic	moieties	 into	hydrophilic	 side	chains	 (due	 to	 the	nature	 of	 the	 tetrazine	 used	 and	 the	 high	 level	 of	 polymer	modification).	 In	 support	 of	 these	 observation,	 the	 zeta-potential	 values	 of	 the	 NP	 exhibited	 a	 dramatic	 change	 from	0.12	mV	to	24.10	mV	following	tetrazine	modification.  					Journal	Name	 	COMMUNICATION	This	journal	is	©	The	Royal	Society	of	Chemistry	20xx	 J.	Name.,	2013,	00,	1-3	|	3 	Please	do	not	adjust	margins	Please	do	not	adjust	margins	The	 IC50	 values	of	 the	NPs	and	 tetrazine	against	 the	PC3	cells	were	 determined	 as	 5	 mg	 ml-1	 and	 100	 µM	 respectively,	indicating	 relatively	 low	 cytotoxicity. To investigate cell internalisation, FITC labelled PEG-b-PAGE	 (FITC-PEG-b-PAGE)	was	 prepared.	 The	 self-assembled	 NPs	 showed	 a	 time-dependent	 increase	 in	 fluorescence	 intensity	 in	 cells	 with	 an	enhanced	internalisation	capacity	in	the	present	of	tetrazine	as	evidenced	by	flow	cytometry	and	cell	imaging	(Fig	3). To	explore	the	cargo	release	profile	of	the	nanoparticles	upon	the	 addition	 of	 tetrazine,	 nanoparticles	 loaded	 with	Doxorubicin	 (DOX)	were	prepared	 (the	DOX	 loading	efficiency	and	dimensions	of	the	DOX-loaded	NPs	are	shown	in	table	1).	For	measurement	 of	 release	 profiles,	 DOX	 loaded	 NPs	 (3	mg	ml-1	 in	PBS)	were	dialysed	 (MWCO	20	KDa)	against	PBS	 (pH	=	7.4,	 10	mM)	 at	 37	 °C	 and	 tetrazine	 (50	 µM,	 4	 equiv.	 to	 allyl	ether	units)	was	introduced	and	the	release	of	doxorubicin	was	monitored	 spectrophotometrically	 at	 485	 nm.	 The	 triggered	nanoparticles	 exhibited	 full	 cargo	 release,	 with	 the	 release	profile	 mirroring	 the	 reduction	 in	 particle	 diameter	 (see	 Fig.	2b.	 In	 the	absence	of	 tetrazine,	 the	amount	of	 liberated	DOX	was	 negligible	 over	 48	 h,	 while	 in	 the	 in	 the	 presence	 of	tetrazine,	 the	 release	 was	 rapid,	 with	 almost	 quantitative	release	 over	 10	 hours.	 The	 trigger	 release	 of	 DOX	 was	attributed	to	the	gross	morphological	changes	occurring	to	the	polymer.	Hydrophobic	DOX	 is	 encapsulated	 in	 the	membrane	of	 the	 vesicles.	 The	 reaction	 of	 tetrazine	with	 the	 allyl	 group	will	 cause	 vesicles	 collapse	 rapidly	 as	 the	 global	 properties	change	 quickly,	 but	 small	 hydrophobic	 pockets	 were	 still	binding	DOX	release.		Cell	 viability	 studies	were	undertaken	 in	 the	presence	of	DOX	loaded	NPs,	with	 and	without	 the	 tetrazine	 trigger	 using	 PC3	cells	stained	with	the	vital	cell	stain	CellTrackerTM	green.	Fig.	4	shows	 the	 images	of	cells	 incubated	with	empty	NPs	and	NPs	loaded	with	DOX	with	and	without	the	tetrazine	(videos	were	created	through	continuous	image	captures	under	microscope,	see	 supporting	 information).	 After	 72	 hours	 cell	 viability	 was	quantified	 by	 flow	 cytometry	 using	 propidium	 iodide	 as	 a	live/dead	cell	discriminator.	The	proportions	of	viable	and	non-viable	cells	were	also	evaluated	as	shown	in	Figure	5	with	DOX	loaded	NPs	by	flow	cytometry.	The	1	and	6	µM	DOX-NP’s	were	non-cytotoxic	over	72	hours	in	the	absence	of	tetrazine,	while	the	 18	 µM	 DOX-NPs	 exhibited	 some	 cytotoxicity	 (87%	 cell	viability	compared	to	95%	for	 the	control).	These	DOX	 loaded	nanoparticles	 demonstrated	 significant	 effects	 on	 the	 cells	following	 the	 introduction	 of	 the	 tetrazine,	 which	 triggered	nanoparticle	 collapse	and	DOX	 release,	 resulting	 in	 cell	 death	in	 a	 dose	 dependent	 manner.	 As	 shown	 in	 Fig.	 4,	 upon	 the	addition	of	a	trigger	tetrazine,	a	cytotoxic	effect	was	observed	for	 all	 concentrations	 with	 the	 18	 µM	 DOX	 loaded	 NP’s	resulting	 in	 98%	of	 cells	 taking	up	 the	 stain	 propidium	 iodide	(which	is	excluded	by	the	plasma	membrane	in	healthy	cells)	–	an	 indication	 of	 cell	 death.	 In	 addition	 to	 triggering	 cargo		Table	1	Characterisation	of	the	prepared	DOX-loaded	nanoparticle.	DOX	loading	Conc.	(µM)	d	(nm)	 PDI	 Encapsulation	Efficiency	(%)	5	 150	±	24	 0.112	 22	±	4.1	20	 145	±	21	 0.107	 32	±	3.6	50	 160	±	36	 0.098	 35	±	6.2	100	 178	±	39	 0.126	 30	±	4.1	d	=	hydrodynamic	diameter,	PDI	=	polydispersity	index.	n=	3.	COMMUNICATION	 Journal	Name	4 	|	J.	Name.,	2012,	00,	1-3	 This	journal	is	©	The	Royal	Society	of	Chemistry	20xx	Please	do	not	adjust	margins	Please	do	not	adjust	margins	release	 the	 tetrazine	 reaction	 also	 promoted	 cellular	 uptake,	resulting	 in	 DOX-loaded	 nanoparticles	 increasingly	 entering	cells	as	the	reaction	progressed.34,35	Conclusions	In	 summary,	 a	 novel	 approach	 to	 allow	 the	 selective	 control	and	manipulation	 and	 subsequent	 triggering	 of	 nanoparticles	by	 the	 application	 of	 an	 external	 bio-orthogonal	 chemical	stimulus	 in	the	form	of	a	tetrazine	 is	presented.	The	tetrazine	mediated	 inverse	electron	demand	Diels	Alder	 reaction	alters	the	morphology	 of	 the	 nanoparticles,	 triggering	 drug	 release.	With	a	 tetrazine	 trigger,	 the	NPs	also	exhibited	enhanced	cell	uptake	 due	 to	 the	 switching	 of	 the	 surface	 charge	 and	 a	reduction	in	size.	This	new	class	of	responsive	material	offers	a	new	control	 strategy	 for	 triggered	release	through	a	chemical	stimulus	 using	 a	 tetrazine,	 with	 potential	 in	 dosage	 control.	There	 are	 a	 number	 of	 strategies	 for	 controlled	 drug	 release	that	 have	 been	 designed	 to	 increase	 intracellular	 drug	concentrations,	 but	 efficient	 delivery	 of	 therapeutics	 into	tumour	cells	still	remains	a	major	challenge	for	cancer	therapy.	Recently,	 tailor-made	 dual-responsive	 drug	 delivery	 devices	have	 been	 designed	 to	 overcome	 drug	 resistance	 and	inefficient	 cellular	 uptake.36,37	 Our	 approach	 offers	 a	 new	strategy	 in	 the	 manipulation	 of	 polymeric	 nanoparticles;	gaining	 control	 over	 both	 size	 and	 morphology	 of	 the	 self-assembled	 structures.	With	 chemical	 handles,	 our	 design	 can	be	 simply	 employed	 in	 the	 creation	 of	 robust	 multiple-responsive	delivery	devices	by	 the	combination	of	a	 tetrazine	trigger	with,	for	example,	pH	sensitivity,	potentially	providing	a	novel	and	versatile	approach	for	efficient	cancer	therapy.	This	 work	 was	 supported	 by	 the	 European	 Research	 Council	(Advanced	 Grant	 ADREEM	 ERC-2013-340469)	 and	Biotechnology	 and	 Biological	 Sciences	 Research	 Council	(BB/L00609X/1).	We	thank	Dr.	M.	Waterfall	for	flow	cytometry	analysis.	Notes	and	references	1	 S.	Mura,	J.	Nicolas	and	P.	Couvreur,	Nat	Mater,	2013,	12,	991–1003.	2	 M.	De,	P.	Ghosh	and	V.	Rotello,	Adv	Mater,	2008.	3	 H.	Cabral,	N.	Nishiyama	and	K.	Kataoka,	Accounts	Chem	Res,	2011,	44,	999–1008.	4	 A.	Shapira,	Y.	D.	Livney,	H.	J.	Broxterman	and	Y.	G.	Assaraf,	Drug	Resistance	Updates,	2011,	14,	150–163.	5	 S.	Venkataraman,	J.	L.	Hedrick,	Z.	Y.	Ong,	C.	Yang,	P.	L.	R.	Ee,	P.	T.	Hammond	and	Y.	Y.	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Nanoparticle “switch-on” by tetrazine triggering

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