the peptide sequence of anastomosis photocaged 1 ( apc1 ) contains seven lysine residues that are protonated at neutral ph , keeping the peptide soluble and in its monomeric unfolded state . as will be shown , a sol - gel phase transition can be initiated by triggering the folding of the peptide into an amphiphilic -hairpin . once folded , apc1 is designed to rapidly self - assemble into a fibrillar hydrogel network , where each fibril is composed of a bilayer of -hairpins that are intermolecularly hydrogen - bonded along the long - axis of a given fibril , figure 1b ( transition i ) and 2b . earlier studies in our lab support this proposed mechanism where the formation of the hydrophobic interface that defines the fibril bilayer provides most of the thermodynamic driving force for self - assembly . we , and the pochan laboratory , have shown that hydrogels formed from hairpin peptides display shear - thin / recovery rheological behavior . when these gels experience a shear stress , such as that delivered by a syringe plunger , some of the interactions that stabilize their fibril network can be disrupted allowing the material to flow . when the application of shear stress ceases , the network heals and the gel recovers ( figure 1b , transition ii ) . as will be shown , apc1 exhibits similar behavior , allowing its syringe - based delivery . to induce the final gel - sol transition that allows the material within the sutured vessel to dissolve , we sought to disrupt the hydrogel network by destabilizing the hydrophobic interior of its fibrils , figure 2b . apc1 incorporates a photocaged glutamic acid , namely 4-methoxy-7-nitroindolinyl glutamic acid [ e(mni ) ] on its valine - rich face , which would reside in the hydrophobic bilayer of the peptide fibril . when the gel is irradiated with 365 nm light , or alternatively through 2-photon irradiation at 720 nm , the cage is released introducing a negatively charged glutamate side chain into the hydrophobic bilayer , which is energetically unfavorable . this locally disruptive interaction is additive and should result in the global destabilization of the fibril network defining the gel state , thus initiating the final gel - sol transition ( figure 1b , transition iii and figure 2a ) . the mni cage , chosen for its efficient aqueous photolysis and cytocompatibility , is incorporated at position 14 of apc1 's primary sequence . this is a central position on the hairpin 's hydrophobic face that corresponds to a fully buried site within the fibril bilayer . incorporating the cage at this position necessitates the placement of a glycine residue on the opposing strand of the hairpin at position 7 . in the self - assembled state , the small glycine provides a hole on the hydrophobic face of one folded hairpin into which the caged side chain from a neighboring hairpin can reside . this lock and key side chain packing arrangement accommodates the large photocage within the tight steric constraints of the bilayer interior . for example , aryl residues can be found in sheet interiors with their aromatic side chains laying over a glycine residue in a neighboring strand . the formation of favorable - interactions between the aromatic side chain and the glyl - amide backbone drives this fold . although the extended length and flexibility of the caged side chain could allow for multiple modes of packing , molecular modeling shows that in the context of the apc1 hairpin , the proposed lock and key arrangement results in a well - packed hydrophobic face that is conducive to bilayer formation ( figure 2b ) . figure 2c shows a cut - away view of one monolayer formed from three hairpins . here , the caged side chain from one hairpin lays over the glycine of a neighboring hairpin below it , with the indoline group making hydrophobic contacts with proximal valine side chains . the top - most hairpin in the assembly shown in panel c highlights the glycine hole into which a photocaged side chain could be placed if an additional hairpin were to join the fibril assembly . in addition to apc1 , a second peptide was designed to study the positional effect of cage placement on gel performance . peptide apc2 contains the [ e(mni ) ] residue at position 16 , which is slightly closer to the hairpin 's c - terminus . two control peptides were also prepared , namely capc1 and capc2 , which contain uncaged glutamate residues at positions 14 and 16 , respectively . the ability of apc1 and apc2 to fold and associate into -sheet rich assemblies was monitored using circular dichroism ( cd ) spectroscopy . in water at 5 c , the cd spectra of 1 wt% solutions of apc1 and apc2 indicate that the peptides are unfolded ( figure 3a ) . however , figure 3b shows that folding and self - assembly can be triggered by adding ph 7.4 buffer that contains nacl to the aqueous peptide solution and increasing the temperature to 25 c for apc1 and to 37 c for apc2 . the nacl screens the lysine point charges and increasing the temperature drives the hydrophobic effect , both of which favor folding and assembly . the exact temperatures needed to induce gelation was determined by monitoring the mean ellipticity at 216 nm as a function of temperature , figure s3 . in these cd experiments , self - supporting hydrogels of both peptides ( ph 7.4 , 150 mm nacl ) form directly in the cuvette . spectra of both peptides in the gelled state display distinct minima at 216 nm , which is indicative of  sheet secondary structure . also evident in the spectra are absorptions due to the indoline cage , which occur in the near uv - cd ( 240 - 380 nm ) . this indicates that in the unfolded state of each peptide , the achiral aromatic indoline ring resides in an achiral environment , that is , it is solvent exposed . however , when the peptides fold and assemble , the ring is placed into a chiral environment , such as that provided by the fibril 's hydrophobic interior . importantly , under the same solution conditions that support apc1 and apc2 assembly , the control peptides capc1 and capc2 remain unfolded , figure s4 . this suggests that the control peptides ' negatively charged glutamate side chains are not accommodated within the hydrophobic interior of the fibril bilayer and thus , peptide folding and assembly are disfavored . importantly , it also supports the assertion that if the glutamate 's negative point - charge were to be unmasked when the peptides are in the folded and assembled fibrillar state , that this event should be disruptive . oscillatory rheology was employed to study the rheological behavior of each gel under experimental conditions that mimic its use during the anastomosis procedure . here , the storage modulus ( g ) , a measure of the gels ' mechanical rigidity , is monitored in the rheometer under environmental conditions that mimic : ( 1 ) the initial ( sol - gel ) formation of the hydrogel in the syringe ; ( 2 ) shear thinning of the material during injection with subsequent hydrogel recovery in the vessel lumen ; and ( 3 ) the disruption of the gel network ( gel - sol ) by uv photolysis after suturing . monitoring gel behavior in a rheometer , although not exactly capturing the gel 's response during the actual anastomosis procedure , allows quantitative and reproducible measurement of gel mechanical properties under highly controlled conditions . i , the rate of hydrogel formation ( sol - gel ) is assessed by monitoring the evolution of g after peptide folding and assembly is triggered . apc1 forms a semi - rigid gel within the first few minutes that further rigidifies with time ( g2,500 pa after 30 minutes ) . after completion of the initial time sweep , 1000% strain is applied to the material for 30 seconds in regime ii to mimic syringe delivery of the gel . this application of strain results in an immediate decrease in g indicative of shear thinning that results in a viscous gel capable of flow . after the 30 seconds , the applied strain is decreased allowing the apc1 gel network to recover to about 75% ( 1900 pa ) of its original rigidity . lastly , using an optically clear parallel plate in the rheometer , the recovered hydrogel is subjected to irradiation by uv light ( 365 nm ) for the first 10 minutes of regime iii . the data clearly show that the gel network is rapidly degraded with an almost immediate decrease of g to 180 pa . taken together , the rheological data suggests that apc1 is capable of triggered gelation , shear - thin delivery via syringe and rapid post - delivery recovery . importantly , irradiation by uv rapidly disrupts the gel network affording a viscous material , which should be capable of dissolution when exposed to the shear of blood flow . figure 3d shows the same experiment for the apc2 gel , where a significant lag phase before the onset of gelation is observed . however , with time , apc2 forms a gel that is significantly more stiff ( 20,000 pa ) then that formed from apc1 . in regime ii , the apc2 gel shear thins but is unable to self - heal effectively and recovers only about 5% of its original mechanical rigidity . irradiation in regime iii although apc2 initially forms a rigid gel , its inability to recover after being shear thinned precludes it use in the anastomosis procedure . overall , this rheological data indicates that the positional placement of the photocage within the peptides ' primary sequence is important . here , incorporation of the cage near the hairpin 's terminus hampers the ability of its network to recover . the rheological data shows that irradiation of the recovered apc1 gel leads to a significant decrease in g presumably as a result of releasing the glutamate cage . this was confirmed by following the fate of caged apc1 during photolysis by uv spectroscopy and lcms , which showed nearly complete release of the mni cage and evolution of corresponding carboxylate - containing peptide within 90 seconds of irradiation , figure s8 . in figure 4 , transmission electron microscopy ( tem ) shows the morphology of fibrils isolated from a 1 wt% apc1 hydrogel before ( a ) and after ( b ) irradiation . before irradiation , the hydrogel network is composed of long fibrils whose lengths are distributed over a range of 150 nm to over 1000 nm , figure 4c . irradiation results in the formation of small fibril segments as shown in panels ( b ) and ( d ) . the average length of these fibrils is on the order of  150 nm demonstrating that the majority of longer fibrils have been converted to smaller segments as a result of photolysis . further , cd spectroscopy of the irradiated gel shows an attenuation of -sheet signal at 216 nm , which is consistent with the disruption of the fibril network , figure s9 . the fact that some small fibrils persist may be due to either incomplete release of the cage , or that a population of the uncaged peptide remains in the fibrillar state . at any rate , the rheological data demonstrates , and as will be confirmed in the in vivo studies , this level of fibril disruption is sufficient to ensure the necessary gel - sol phase transition . the ability of the apc1 hydrogel to serve as a temporary aid in the suturing process was assessed in a mouse femoral artery end - to - end anastomosis model . this challenging microsurgical setting tests the gel with regards to ease , safety , precision , and speed of arterial anastomosis . the mouse femoral artery is approximately 200 microns in diameter and , conventionally , is anastomosed via a technically challenging and time - consuming  no - touch  under - water suturing technique . in contrast , the apc1 hydrogel is designed for facile administration directly to the in situ vessel to distend the lumen and add stability to the vessel wall . this should enable more precise and quick placement of stitches , resulting in increased vessel patency . in this model , an incision is made in the groin crease of mice to expose the femoral artery , which is dissected and subsequently clamped , figure 5a . optical coherence tomography ( oct )   of the vessel cross - section shows that the lumen is collapsed , figure 5b . without the aid of a stent or luminal filler an intraluminal injection of a 2 wt% apc1 hydrogel was administered by syringe to both severed ends of the vessel , distending the lumen ( figure 5c ) and mechanically supporting the vessel wall after injection , figure 5d . a longitudinal cross - section oct image of the proximal ( left ) and distal ( right ) ends of the vessels after injection with the apc1 hydrogel is shown in figure 5e . vascular distention caused by the gel helped maintain a cylindrical vessel shape , facilitating identification of a single vessel wall leading to more uniform suture spacing and closure . further , hydrogel can be applied between the vessels , aiding their approximation ( see video s1 ) . vessel ends can be inserted into the gel , where local thinning occurs proximal to the vessels during their movement within the gel . the needle can be passed directly through the optically clear gel to place the sutures . upon placement of the final suture , the external gel is washed away , and the vessel is irradiated at the suture site for 2 minutes using a hand - held 365 nm led uv light to remove the interior gel . resumption of blood flow after removing the clamps clears the disrupted gel as evidenced by volume doppler oct ( figure 5f ) and blood flow speed measurements , figure s10 . the efficacy of the final gel - sol phase transition is critical since remaining solid material could lead to thrombus formation , diminished perfusion and tissue ischemia . thus , vessel patency was assessed in a series of experiments that monitor vascular perfusion . first , high - resolution micro - ct was used to follow the perfusion of a polymeric contrast agent ( microfil ) one hour after gel - based end - to - end anastomosis of the femoral artery and resumption of blood flow . figure 6a - c and video s2 show that polymer completely fills the distal tibial and fibular vessels as well as the plantar arch on the footpad and the digital branches to the toes . separate experiments in which animals were dissected to directly observe polymer distribution support the ct data ( figure 6d - f ) , confirming occlusion free vascular patency throughout the entire extremity that is similar to the contralateral control limb . lastly , perfusion of a near - infrared dye was followed throughout an explanted hind limb after irradiating a gel that had been implanted into the femoral artery , figure 6g - i . the leading edge of the dye ( green arrow ) immediately penetrates the limb from its initial injection site ( yellow arrow ) , quickly penetrating another major vessel ( white arrow , h ) and distant vascular regions and surrounding tissue ( i ) . taken together , these experiments indicate that the peripheral vascular and capillary bed is well perfused without any evidence of luminal narrowing or occlusion due to gel remnants . we also investigated gel biocompatibility , where histology shows a similar peri - vascular inflammatory response in vessels anastomosed with or without gel , a result of surgical tissue traumatization and post - operative healing , figure s11 . further , subcutaneously injected gel produced no gross local inflammation and a typical foreign - body response that resolved , figure s12 . designing soft materials from self - assembling peptides allows their bulk properties to be engineered at the molecular level for specific applications . physical attributes endeared to the peptide monomer are translated to the properties of the self - assembled gel network . the multiple phase transitions of apc1 , which enable its use in facilitating the anastomosis of ultra - small vessels , are due to the exact placement of natural and non - natural amino acids within its sequence that render the peptide , and its corresponding gel , responsive to environmental change . thus , de novo peptide design can afford a self - assembled material that represents a promising alternative to currently available non - injectable stents . general methods describing the synthesis and analytical characterization of the photocaged glutamic acid and all peptides , as well as detailed procedures for all of the experiments employed in this study ( including the number of animals used and replicates ) can be found in the supplementary information in the online version of the paper . the supplementary information also contains afm , additional rheological studies , histological analysis , and videos showing the gel - aided anastomosis and three - dimensional reconstruction of hind - limb vasculature .
many surgeries are complicated by the need to anastomose , or reconnect , micron - scale vessels . 
 although suturing remains the gold standard for anastomosing vessels , it is difficult to place sutures correctly through collapsed lumen , making the procedure prone to failure . here , we report a multi - phase transitioning peptide hydrogel that can be injected into the lumen of vessels to facilitate suturing . 
 the peptide , which contains a photocaged glutamic acid , forms a solid - like gel in a syringe and can be shear - thin delivered to the lumen of collapsed vessels ( where it distends the vessel ) , and the space between two vessels ( where it is used to approximate the vessel ends ) . 
 suturing is performed directly through the gel . 
 light is used to initiate the final gel - sol phase transition that disrupts the hydrogel network , allowing the gel to be removed and blood flow to resume . 
 this gel adds a new tool to the armamentarium for micro- and supermicrosurgical procedures .