Multifunctional lipid-coated polymer nanogels crosslinked by photo-triggered Michael-type addition †

Department of Materials Science and En DuPont Hall, Newark, DE 19716, USA. E-ma Biomedical Engineering, University of Dela Delaware Biotechnology Institute, 15 Innov † Electronic supplementary information four-arm nitrobenzyl protected PEGhydrogels and PEG-Mal control; storage different concentrations; DLS of lipidtemperatures; full uorescence spectra microscopy image of the COOH-NG contr Cite this: Polym. Chem., 2014, 5, 1728


Introduction
Nanotechnology has had a signicant impact on the development of nanoscale vehicles and devices in the eld of drug delivery and tissue engineering, [1][2][3][4][5] and many types of nanoparticles have been developed over the past few decades, including inorganic nanoparticles, lipid-based carriers, and polymeric nanostructures (e.g., polymer-drug conjugates and block copolymer micelles). 6,7lthough metallic nanoparticles provide opportunities for magnetic resonance imaging (MRI) 8 or photothermal therapy, 9 they are not biodegradable and many are not small enough to be cleared from circulation easily, which may lead to long-term toxicity due to potential accumulation in the body.Polymeric nanoparticulate systems have attracted much attention as potential drug carriers due to enhanced therapeutic efficacy that is imparted by improvements in the solubility of hydrophobic drugs, extension of circulation half-life, reduction of immunogenicity, and sustained drug release. 10,11In addition, nanoparticles that are responsive to external stimuli such as pH, 12 light, 13,14 temperature, 15,16 magnetic elds, 17 and ultrasound 18 have also been developed to achieve on-demand/triggered drug release. 198][29][30][31][32] Composed of highly crosslinked hydrophilic polymer chains, nanogels maintain structural integrity even under dilute conditions.Due to their gel-like structures, nanogels have a high degree of porosity that allows efficient encapsulation of therapeutic molecules. 335][36] Nanogels have thus emerged as promising materials for the delivery of a wide range of therapeutic molecules including chemotherapeutics, 29,31 proteins, 30,32 and siRNAs. 33 major obstacle in the preparation of nanogels is macrogelation, which ultimately promotes bulk gelation (to yield either a microgel or hydrogel). 37Many procedures have been employed to avoid macrogelation during crosslinking, including microemulsion/inverse microemulsion polymerization methods. 38n these approaches, each droplet of polymer solution serves as a nanoreactor, permitting isolated crosslinking of each droplet and the facile incorporation of bioactive molecules. 39However, the necessary use of organic solvents in the formation of the emulsions can be detrimental to the therapeutic cargo. 40Alternatively, preparation of nanogels from nanotemplates such as liposomes largely avoids the use of organic solvents during chemical crosslinking, 41 as hydrogels are formed in the aqueous lumen of the liposome.In addition, the use of liposomes generally permits better control over the size of the resulting nanogels, given the uniformity of liposome populations and the simplicity of the manipulation of their size via extrusion methods. 42lthough the preparation of nanogels using liposometemplated methods is a common approach, [43][44][45][46][47][48][49] most reported strategies are based on radical polymerization of vinyl monomers/ methacrylated macromers or ionic crosslinking of alginate, which exerts less control of network development and may result in heterogeneous network structures.While Michael-type addition reactions have been widely used as a crosslinking strategy for hydrogel preparation under mild, physiologically relevant conditions, 50,51 they have not been commonly employed in the eld of nanogel synthesis, 52 owing to their spontaneous reaction kinetics. 53][66][67] Recently, photolabile chemistry has also been utilized as a strategy for light-triggered crosslinking of alginate hydrogels, which displayed improved mechanical properties and homogeneity. 68The application of these strategies to yield photolabile, protected precursors for Michael-type additions would permit the lightdependent production of homogeneous network structures with more precise control of the reaction, which would thus expand the utility of Michael-type additions in the synthesis of nanogels.
We report here the production of novel lipid-coated polymer nanogels, which are crosslinked by photo-triggered Michael-type additions of thiols and maleimides, via liposome-templated methods.Specically, photolabile o-nitrobenzyl protecting groups have been employed to protect PEG-thiols and thus to permit triggering of nanogel formation upon photo-irradiation; the o-nitrobenzyl groups undergo photocleavage into o-nitrosobenzaldehyde upon irradiation with UV light, releasing free thiols for subsequent crosslinking reactions with maleimides. 54,69he photo-triggered chemistry provides temporal control of the reaction and avoids macroscale gelation during nanogel synthesis, while the lipid coating offers additional functional features to the nanogels by preventing burst release of therapeutic molecules and providing surface versatility.The photo-triggered gelation of bulk hydrogels was monitored via oscillatory rheology and the size distribution and colloidal stability of the nanogels were measured via dynamic light scattering and electrophoretic light scattering, respectively.The morphology and size of the nanogels were conrmed via transmission electron microscopy (TEM).The chemical reactivity of the nanogels was probed via their modication with a uorescent dye.Owing to the ability to control degradation of the resulting polymeric networks, these nanogels have potential application as targeted drug-delivery vehicles and imaging probes.

Synthesis of photolabile, protected PEG-SH
Four-arm PEG-SH, protected with o-nitrobenzyl groups, was synthesized via previously published methods, with some modications. 62,70Four-arm, thiol-terminated PEG (150 mg or 0.015 mmol) was dissolved in 5 mL of DMF, and then neutralized with TEA (100 mL or 0.72 mmol).The solution was cooled down to 0 C and a solution of 2-nitrobenzyl bromide (50 mg or 0.23 mmol in 5 mL acetone) was added dropwise over 15 min.The reaction mixture was stirred vigorously at 0 C for 1 h and then at room temperature for 24 h.The resulting solution was dialyzed against DI water for 24 h (MWCO 1000) and then lyophilized to afford a white powder product.The functionality of the resulting polymers was determined via 1 H NMR (in CDCl 3 ), with an average value of 71 AE 5%. 1

Bulk gel preparation via photo-triggered Michael-type addition and subsequent rheological analysis
The PEG hydrogel was prepared by the photo-triggered Michaeltype addition of protected PEG-SH and PEG-Mal (at a nal concentration of 5 wt% or 15 wt%).The solution was well mixed by vortexing and then was exposed for 2 hours to UV light (365 nm; Black-Ray® mercury lamp) at an intensity of 10 mW cm À2 .Oscillatory rheology experiments conducted on a stresscontrolled AR-G2 rheometer (TA Instruments, New Castle, DE, USA) were used to characterize the photo-triggered cross-linking and subsequent mechanical properties of the bulk PEG hydrogels.Experiments were conducted at room temperature using an 8 mm diameter, steel plate geometry with 100 mm gap distance, with oscillatory time, frequency and strain sweeps performed.Strain sweeps were performed on samples from 0.1% to a maximum strain of 1000% to determine the limit of the linear viscoelastic region.Dynamic oscillatory time sweeps were collected at angular frequencies of 6 rad s À1 and 1% strain chosen from the linear viscoelastic region.Rheological properties were examined by frequency sweep experiments (u ¼ 0.1 to 100 rad s À1 ) at a xed strain amplitude of 1%.The protected PEG-SH and PEG-Mal precursors were dissolved in water separately with a concentration of 5 wt% or 15 wt%.The precursors were then combined, vortexed, and loaded onto the rheometer stage.Hydrogels were formed by exposing the solution to UV light continuously for 2 hours (365 nm at 10 mW cm À2 ; OmniCure® S2000, 200 W UV lamp with bandpass lters and high power ber light guide) or periodically by shuttering the light (365 nm at 10 mW cm À2 ; light on for 5 min, light off for 20 min); the G 0 and G 00 values were monitored throughout the crosslinking experiments.

Synthesis of lipid-coated nanogels (NGs)
A chloroform solution (1 mL) with 5 mg egg-PC and 0.5 mg of cholesterol was placed in a round-bottom ask, with evaporation of the chloroform under nitrogen ushing to yield a thin lm of the lipid that was then dried under vacuum overnight at room temperature to remove the organic solvent.A 15 wt% PEG solution was prepared by dissolving 75 mg PEG-Mal and 75 mg o-nitrobenzyl-protected PEG-SH in 1 mL DI water; this solution was used to hydrate to the lipid lm, with ten alternating cycles of 30 s vortexing and 5 min quiescent incubation at room temperature. 48The solution was then sonicated for 30 min to completely solubilize the lipid.The resulting multilamellar liposomes were extruded 20 times through a 0.8 mm polycarbonate membrane (Whatman, Piscataway, NJ, USA), 20 times through a 0.2 mm membrane, and nally 21 times through a 0.1 mm membrane using an Avanti® Mini-Extruder (Avanti Polar Lipids Inc., Alabaster, AL, USA).The resulting unilamellar liposomes were then diluted 5 times (to avoid macroscale gelation upon UV irradiation).The solutions were irradiated under UV light (365 nm) for 2 hours with a light intensity of 10 mW cm À2 .The nanogels were stored at 4 C until further use.

Surface functionalization of lipid-coated nanogels
Chloroform solution (1 mL) with 4 mg egg-PC, 1 mg DSPE-PEG 2000 -COOH and 0.5 mg cholesterol was placed in a roundbottom ask, with nitrogen ushing and drying to yield a thin lm.Carboxylic acid-functionalized liposomes, and then lipidcoated nanogels (COOH-NGs), were produced as described above and stored at 4 C until further use.A solution of COOH-NG (1 mL, 1.2 mg mL À1 ) was then added to EDC-sulfo-NHS solution (50 mM EDC and 25 mM sulfo-NHS dissolved in phosphate buffer pH ¼ 7.2) to yield a total nal volume of 2.5 mL.The solution was stirred for 45 min at 4 C to activate the carboxylic acid groups and then 0.5 mg 1-aminomethylpyrene (dissolved in 50 mL DMSO) was added dropwise to achieve a nal dye concentration of 0.2 mg mL À1 .The solution was subsequently stirred for 4 hours at room temperature.The resulting dye-conjugated nanogels (dye-conjugated NGs, 2.5 mL) were then dialyzed against 2 L water overnight and washed 3 times with water at 4000 rpm for 20 min using Amicon Ultra-15 Centrifugal Filter Units (MWCO: 30 kDa, Millipore, Billerica, MA) to remove unconjugated dye molecules.The emission uorescence spectrum of the dye-conjugated nanogels was measured from 350-600 nm with a slit width of 1.5 nm (aer excitation at 333 nm with a slit width of 3 nm) using a HORIBA Jobin Yvon SPEX FluoroMax-4 spectrouorometer.Microscopy images of the uorescently labeled NGs were obtained via confocal laser scanning microscopy (CLSM, Zeiss, 510 NLO, Germany).A COOH-NG control was prepared under identical reaction conditions and procedures, without the coupling reagents, to conrm that non-reacted dye was removed under the reported conditions.

Dynamic light scattering (DLS) and zeta potential analysis
The average size and size distribution of the nanogels were analyzed via dynamic light scattering (DLS) and the zeta potential determined via analysis of light scattering on a Malvern Zetasizer Nano ZS apparatus with Malvern Instruments DTS soware (v.6.01)(Malvern Instruments, UK).The analyses were conducted on solutions of the nanogels in DI water and at 25 C.For the stability measurement, analyses were conducted in PBS buffer at different temperatures.The mean hydrodynamic diameter of the nanogels (d h ) was computed from the intensity of the scattered light using the Malvern soware package based on the theory of Brownian motion and the Stokes-Einstein equation.The zeta potential of the nanogels was analyzed using the electrophoretic light scattering spectrophotometer of the instrument.

Transmission electron microscopy (TEM) characterization of nanogel morphology
Transmission electron microscopy (TEM) images were acquired using a FEI Tecnai-12 microscope operating at an accelerating voltage of 120 kV.Images were collected on a Gatan CCD camera.TEM samples were prepared by applying a drop of nanogel solution ($3 mL) onto a carbon-coated copper TEM grid (300 mesh size).The excess solution was wicked away aer 1 min using a piece of lter paper.A droplet of 1% phosphotungstic acid aqueous solution ($3 mL) was then added onto the grid and wicked away aer 1 min using a piece of lter paper.The samples were dried at room temperature for 3 hours before imaging.

Photodeprotection and photo-triggered gelation
The photolabile, protected PEG-SH was synthesized as shown in Scheme 1.The thiols from the PEG chains were protected by the photolabile o-nitrobenzyl groups to yield o-nitrobenzyl thioether moieties. 1H NMR spectroscopy was used to conrm the functionality of the polymer protection by o-nitrobenzyl groups.0][71] Based on the integration of the aromatic and ethylene group protons, a functionality of 71 AE 5% was obtained; this functionality was consistently obtained across multiple syntheses.Upon UV irradiation, the o-nitrobenzyl groups undergo photocleavage into o-nitrosobenzaldehyde, releasing free thiols for subsequent in situ Michael-type additions.
The photo-triggered gelation experiments were carried out at ambient temperature by irradiation of the mixed solution of hydrogel precursors at a wavelength of 365 nm and an intensity of 10 mW cm À2 (Scheme 2).The hydrogel turned yellow aer gelation, which may be attributed to the o-nitrosobenzaldehyde produced by the photocleavage. 62Hydrogel formation was conrmed via oscillatory rheology in situ during network development (Fig. 1).The data in Fig. 1 show that homogeneous solutions of protected PEG-SH and PEG-Mal in water did not exhibit any increase in the elastic modulus (G 0 ) before UV irradiation, while the Michael-type addition between thiol and maleimide usually occurs within a few seconds under similar conditions. 50,72Aer 5 min irradiation, a rapid increase in G 0 was observed, conrming gelation and supporting the expected photo-triggered crosslinking mechanism.The rheology results are also plotted in log scale as shown in Fig. S2, † to better show that the viscous modulus (G 00 ) remained essentially unchanged before UV irradiation and ultimately increased to approximately 100 Pa when irradiation was completed.The slow increase of G 0 at later time points during the measurement is likely a result of some continued polymerization (owing to the slow deprotection kinetics) and in part due to the radical polymerization of the remaining PEG-Mal functional groups that have not yet reacted with thiol. 73Data presented in Fig. S3 † illustrate that the G 0 of a PEG-Mal control (lacking PEG-SH) began to increase aer approximately 30 min, indicating the polymerization of PEG-Mal and suggesting that the increase in G 0 of the PEG-Mal/PEG-SH bulk gel aer 30 min irradiation may arise in part from this polymerization.In order to demonstrate that the crosslinking reaction between thiol and maleimide groups occurs mainly as a result of UV exposure, the polymerization was interrupted at approximately 15, 40 and 65 min, for a period of 20 min each time, simply by shuttering the UV light source.The data in Fig. 1b illustrate that little or no increase in elastic modulus was observed during the periods when there is no UV irradiation, indicating the temporal control of this light-dependent crosslinking strategy.Similar to other light-dependent thiol-ene polymerization strategies as those previously reported by Bowman, Anseth and co-workers, 74 the photo-triggered Michaeltype addition might also have signicant potential in the preparation of biomimetic hydrogels.The temporal control achieved by this strategy would allow premixing and appropriate positioning (e.g., in a tissue defect) of hydrogel precursors prior to crosslinking.The spatial control enabled by this chemistry, while not investigated here, might extend the capability of Michael-type additions to create patterned devices, materials and structures.
It should be noted that the mechanical properties of these hydrogels can be readily tuned not only by varying the UV irradiation time, but also by changing the polymer concentration.As shown in Fig. S4, † an expected increase in elastic modulus was observed upon an increase in polymer concentration.Specically, the measured G 0 of the 5 wt% gels is 2 kPa, while that of the 15 wt% gels increased to approximately 12 kPa.Such changes in hydrogel properties with variation in crosslinking density have been regularly employed to modify the delivery properties of various hydrogels, which could be potentially applied to these nanogel systems. 50,75nthesis and characterization of lipid-coated nanogels (NGs) The lipid-coated PEG nanogels were prepared via a liposometemplated method as shown in Scheme 3. Upon hydration of a lipid thin lm with the polymer solutions, multilamellar liposomes form with hydrogel precursors encapsulated in the aqueous lumen.By extrusion of the multilamellar liposome suspension through polycarbonate membranes of specic pore size, small unilamellar liposomes with well-dened size distribution can be prepared. 44The solutions of the unilamellar liposomes, prepared via these accepted strategies, were then diluted to prevent the potential macrogelation of any unencapsulated precursors outside the liposomes.Aer UV irradiation, precursors were crosslinked by photo-triggered Michaeltype addition inside the lipid vesicles, forming lipid-coated nanogels.
The hydrodynamic diameter and surface charge of the lipidcoated nanogels in aqueous solution were determined via dynamic light scattering (DLS) and zeta potential measurements, respectively.As shown in Fig. 2, the lipid-coated nanogels have an average diameter of 160 nm with a polydispersity index (PDI) of 0.25, which is very close to that of the liposomes prepared under the same conditions (154 nm, PDI 0.14).The similarity in size between the lipid-coated nanogels and liposomes conrmed the successful use of the template to form nanogels of prescribed size as expected based on the use of other liposome-templated nanogel systems. 42,45For example, work reported by An et al. 42 demonstrated the size control and monodispersity of nanogels prepared from liposome templates by varying the polycarbonate membrane with specic pore sizes.The lipid-coated nanogels exhibited slightly negative zeta potentials (À7.1 mV), which may be attributed to the lipid layer since the surface charge of bare nanogels became neutral when the lipid layer was removed by detergents such as Triton X-100 (data not shown).Work reported by Wang et al. 76 also indicated that conventional liposomes composed of egg-PC and cholesterol (2 : 1 molar ratio) demonstrate a zeta potential value of À2.7 mV (average diameter: 110 nm), which is in good agreement with our results.Retention of the lipid layer offers advantages for the subsequent use of these hydrogel nanoparticles; the slight negative charge introduced by the lipid layer should improve the stability of the nanogels in solution (and in circulation) by preventing self-aggregation and non-specic cellular uptake, based on electrostatic repulsion. 77,78he morphology of the lipid-coated nanogels dried from aqueous solution was conrmed via transmission electron microscopy (TEM); representative data from these experiments are shown in Fig. 3.As illustrated in the images, the lipid-coated nanogels showed a reasonably well-dened spherical morphology.The average diameter of the nanogels (calculated from analysis via Image J of over 100 nanoparticles on the TEM images) is 110 AE 35 nm, which is in good agreement with results obtained from the DLS measurements presented in Fig. 2. The relatively smaller size observed from the TEM images compared   with DLS results is likely due to the dehydration of the nanogels during the TEM sample preparation, which has been commonly observed. 30,31,79he stability of these nanogels under physiologically relevant buffer conditions was evaluated via DLS at different temperatures.The data shown in Fig. S5 † indicate that changes in hydrodynamic size of the nanogels upon temperature changes are almost negligible between 25 C to 50 C, suggesting their stability against aggregation at physiological temperatures.The nanogel solutions were also stable and showed no signicant aggregation for several months when stored at 4 C.

Surface functionalization of lipid-coated nanogels
Owing to their high surface area-to-volume ratio, the therapeutic effect of nanoparticles can be enhanced by surface modication with ligands that allow targeting of specic tissues and/or uorescence imaging. 7,80To examine the potential suitability of these lipid-coated nanogels for such applications, carboxylic lipid-coated nanogels (COOH-NGs) composed of PEG based nanogels and a mixture of egg-PC and DSPE-PEG 2000 -COOH in a 4/1 ratio (w/w) were formulated.Poly(ethylene glycol) (PEG) on nanogel surfaces has been demonstrated to prolong circulation and improve evasion of immune clearance. 81A reactive uorescent dye, 1-aminomethylpyrene, was then introduced onto the surface of the COOH-NGs via an EDCsulfo-NHS coupling reaction to yield dye-conjugated nanogels (dye-conjugated NGs) (Scheme 4).A control experiment was performed on COOH-NGs under identical conditions and procedures, without the coupling reagents, to demonstrate that unconjugated pyrene can be removed under such conditions.3][84] The uorescence spectra of various nanogels were normalized to the o-nitrosobenzaldehyde peak at 360 nm (Fig. S6 †), at which both the COOH-NGs and dye-conjugated NGs revealed strong uorescence emission due to the o-nitrosobenzaldehyde residues encapsulated in the nanogel networks; the concentration of the o-nitrosobenzaldehyde is expected to be similar in the various nanogels. 69The emission from the dye-conjugated NGs closely matches the spectrum of pyrene dissolved in DMSO, suggesting that the chromophore remains intact aer conjugation.These results illustrate that pyrene has been covalently conjugated to the surface of nanogels via EDC-sulfo-NHS coupling; the fact that no signicant signal was observed in the same region from the COOH-NG control demonstrates that unbound pyrene molecules were removed under these experimental conditions.
The hydrodynamic diameter and surface charge of the COOH-NGs and dye-conjugated NGs in aqueous solution were also measured by dynamic light scattering (DLS) and electrophoretic light scattering, respectively, as shown in Fig. 5.The COOH-NGs have an average diameter of 156 nm with a PDI of 0.17, which closely matches that of the COOH-functionalized liposome (141 nm, PDI: 0.12), as expected.The size analysis of the dye-conjugated NGs revealed that they have an average diameter of 181 nm with a PDI of 0.23, illustrating the lack of signicant change in hydrodynamic diameter aer conjugation of the dye.The slight increase in size could be explained by slight swelling of the nanogels due to the retention of a small amount of DMSO from the reaction (Fig. 5a). 31As shown in Fig. 5b, the COOH-NGs exhibit a slightly greater negative surface charge (À16.3 mV) than the NGs, as expected owing to the presence of the carboxylic acid group.The negative zeta potential of dye-conjugated NGs (À15.8 mV) became only slightly less negative aer the conjugation of pyrene, which is likely due to the relatively low coupling efficiency of EDCmediated reactions at pH ¼ 7.2. 85 The surface modication was also conrmed via uorescence microscopy of the pyrene-modied NGs; a representative image from confocal laser scanning microscopy is shown in Fig. 6.As shown in the gure, the dye-conjugated nanogels appear as individual green dots on the dark background, while no green particles were observed in the uorescence microscopy image of the COOH-NG control (Fig. S7 †).Work recently reported by Singh et al. 32 also conrmed the conjugation of carboxyourescein-labeled model peptide to nanogels and the removal of unbound peptide via similar methods.Consistent with this report, these confocal microscopy data are consistent with the spectroscopy data and conrm the successful conjugation of pyrene.These results also suggest that the nanogels likely have the capacity to be functionalized with a broad range of biomolecules to enhance therapeutic efficacy.
In contrast to traditional radical-chain-growth photopolymerization of polymers modied with acrylate or methacrylate moieties, formation of hydrogels via the step-growth mechanism of Michael-type addition may afford networks of greater homogeneity and improved mechanical integrity. 86Also, compared to step-growth thiol-ene chemistry 74 and the recently developed photocaged amine-catalyzed thiol-Michael addition, 56 the photolabile-protection strategy of thiol groups permits triggering of subsequent crosslinking reactions without the addition of any initiators or catalysts, and might thus positively affect the integrity and bioactivity of encapsulated therapeutic agents.In addition to the o-nitrobenzyl moiety employed here, similar photolabile-protection of thiols or amines could be achieved by coumarin and p-methoxyphenacyl moieties. 66,67,71Therefore, this photo-triggered Michael-type addition crosslinking strategy provides an initiator/catalystfree photo-crosslinking method for polymer network formation with the associated advantages of homogeneity and spatiotemporal control, potentially diversifying the uses for the photo-mediated click reaction toolbox.
Compared with other existing nanogel systems, the lipidcoated nanogels synthesized from photo-triggered Michaeltype addition in this report provide mild reaction conditions and control of the network development, offering new strategies in the design of nanogel materials.The surface charge and functionality introduced by the lipid coating may increase circulation stability and offer versatility of the nanogels in multiple applications, making them promising candidates as nanoparticle therapeutics.
5,79 In particular, our group has exploited the reversibility of Michael-type reactions in the presence of glutathione (GSH), a thiol-containing tripeptide localized in cellular compartments or tumor microenvironments. 87Hydrogels prepared with arylthiol-modied PEGs based on thiol-maleimide Michaeltype addition showed signicantly more rapid degradation under conditions similar to those in intracellular compartments and tumor microenvironments (10 mM GSH) than under reducing conditions in blood circulation (10 mM GSH). 72herefore, the incorporation of these GSH-responsive arylthiolmaleimide linkages in nanogel synthesis suggests potential opportunities in the development of nanoparticles for intracellular or targeted delivery of therapeutics with increased stability in blood circulation.Considering the hydrophilic nature of these nanogels, hydrophilic drugs and proteins could be incorporated into these systems via physical encapsulation or chemical conjugation.Our ongoing studies have also preliminarily shown that these nanogels have higher drug encapsulation efficiency compared to liposome carriers due to their crosslinked network structure.Investigations of such nanogel systems with stimuli responsiveness are underway and will be the subject of future reports.

Conclusions
Novel, well-dened lipid-coated polymer nanogels were successfully synthesized via photo-triggered Michael-type addition using liposome templates.The nanotemplate method offers facile control of the size of the nanogels and mild synthetic conditions.The photo-triggered crosslinking and  temporal control achieved by these chemical strategies, conrmed by oscillatory rheology experiments, permit the synthesis of these nanogel templates and suggest opportunities for expanding the tool-box for photo-triggered Michael-type additions and patterning bulk gels via these methods.Through surface modication with functional ligands such as targeting moieties and cell penetration peptides, these nanogels provide a promising platform for nanoparticle therapeutics.

Fig. 1
Fig. 1 Oscillatory rheology time sweeps of 15 wt% PEG hydrogels formed by photo-triggered Michael-type addition with (a) continuous and (b) periodic irradiation (365 nm and 10 mW cm À2 ).

Fig. 2
Fig. 2 Dynamic light scattering analysis of the hydrodynamic size of lipid-coated nanogels and liposomes in water at room temperature.

Fig. 3
Fig.3TEM images of the lipid-coated nanogels dried from aqueous solution.

Fig. 4
Fig.4Fluorescence spectra of carboxylic acid-functionalized, lipidcoated nanogels and dye-conjugated nanogels in aqueous solution; l exc ¼ 333 nm, 20 C; spectra of nanogels were normalized to the onitrosobenzaldehyde peak at 360 nm, which should be of similar intensity for the various nanogels.

Fig. 5
Fig. 5 Dynamic light scattering and zeta potential analysis of functional lipid-coated nanogels.Size distribution (a) and surface charge density (b) of carboxylic acid-functionalized liposomes, carboxylic acid-functionalized lipid-coated nanogels and dye-conjugated nanogels in water at room temperature.