From Macro to Micro to Nano the Development of a Novel Lysine Based Hydrogel Platform and Enzyme...

Journal of Materials Chemistry BMaterials for biology and medicinewww.rsc.org/MaterialsB

ISSN 2050-750X

PAPERXiao-Hong Qin, Chih-Chang Chu et al.From macro to micro to nano: the development of a novel lysine based hydrogel platform and enzyme triggered self-assembly of macro hydrogel into nanogel

Volume 3 Number 11 21 March 2015 Pages 2233–2412

Journal ofMaterials Chemistry B

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From macro to m

aKey Laboratory of Textile Science & Techn

Textiles, Donghua University, No. 2999 No

201620, China. E-mail: [email protected] of Biomedical Engineering, Co

E-mail: [email protected] of Fiber Science and Apparel

14853, USA

Cite this: J. Mater. Chem. B, 2015, 3,2286

Received 17th November 2014Accepted 8th January 2015

DOI: 10.1039/c4tb01902d

www.rsc.org/MaterialsB

2286 | J. Mater. Chem. B, 2015, 3, 228

icro to nano: the development ofa novel lysine based hydrogel platform and enzymetriggered self-assembly of macro hydrogel intonanogel

De-Qun Wu,ac Jun Wu,b Xiao-Hong Qin*ac and Chih-Chang Chu*bc

A novel L-lysine basedmultifunctional crosslinker family was developed and utilized to fabricate a 100% pure

poly(ester amide) (PEA) hydrogel with unique structure via a novel gelation strategy in a single and rapid step.

Enzyme triggered biodegradation was utilized to turn the resultant macro hydrogel into abundantmicrogels

with a 3 micron size in the early stage. Nanogels of 50 nm in diameter were then formed after 8 days of

biodegradation in the enzyme solution. The enzyme degradation of doxorubicin (DOX) loaded macro-

gel indicated that the micro and nano gels containing DOX could be obtained using the same strategy

while retaining their sustained drug release performance. This study reports a new biocompatible and

biodegradable crosslinker and hydrogel system, and illustrates a new nanotechnology capable of

controllably producing nanogels using the enzymatic degradation of macro hydrogels.

1. Introduction

Biodegradable polymeric nanoparticles have been widelyutilized for drug delivery or theranostic applications.1–4 Whencompared with nanoparticles, biodegradable nano size poly-meric hydrogels are more attractive for certain types of drugdelivery applications because of their high water content and 3-D porous network structure.5–10 This has been widely demon-strated by macro size hydrogels.11–15 However, preparing a nanosize hydrogel in a reproducible and facile manner is still chal-lenging.16–19 Herein, we propose a novel, but simple andstraightforward strategy to fabricate nano size hydrogels: thebiodegradation of macro size hydrogel into nano size hydrogel,which will maximally retain the original unique hydrogelproperty and structure.

To utilize this strategy, a specially designed macro hydrogelplatform is required with the following properties/structure: (1)high permeability and porosity allowing the enzyme to reach themost interior sites of the hydrogel network; (2) selectivelydistributed structures/sites for the enzyme to attack and cut themacrogel into the desired shape and size; and (3) suitablechemical bonds for enzyme to attack. Based on the aboverequirements, a lysine based poly(ester amide) (PEA) hydrogel

ology Ministry of Education, College of

rth Renmin Road, Songjiang, Shanghai,

rnell University, Ithaca, NY, 14853, USA.

Design, Cornell University, Ithaca, NY,

6–2294

platform associated with a novel gelation strategy was developedfor this study. PEAs were selected here because they have beenshown to exhibit excellent biocompatibility and controllableenzyme biodegradation behavior.14,20–23 However, the reportedPEA hydrogel systems are very limited and they are all fabricatedvia photocrosslinking with the assistance of polyethylene dia-crylate or other polymers containing diacrylate groups. Thisresults in a very slow biodegradation rate and contributes to anuncontrollable and complicated biodegradationmechanism.24–26

Herein, a new pure PEA hydrogel fabrication strategywas developed, utilizing the theory of gelation in polymer chem-istry.27–29 For this purpose, multifunctional PEA monomers (f > 2)are required in the polymerization to form gels under certainconditions.30 However, to the best of our knowledge, there havenot been any reports on multifunctional PEA monomers suitablefor this goal. Therefore, a new L-lysine based PEAmonomer familywas developed for the rst time in this report. Monomers Lys-x (x¼ 4, 6, 8) were synthesized according to a newlymodied protocolby reacting L-lysine with a fatty diol in the presence of toluene-sulfonic acid. This water soluble monomer family has 4 func-tional primary amine groups (f ¼ 4) and is non-toxic to cells evenat high concentrations. The 4-arm functional monomer familyhas shown robust crosslinking capability via its rst time appli-cation for fabricating hydrogels in this report.

2. Experimental2.1 Materials

L-Lysine monohydrochloride (Lys), p-toluenesulfonic acidmonohydrate (TosOH$H2O), adipoyl chloride, sebacoyl

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Fig. 1 (a) Chemical structure of the di-p-nitrophenyl monomers. (b)Chemical structure of the Lys-x monomer. (c) Illustration of thechemical structure for the hydrogel. The Lys-x part in the structureindicates the monomer shown in (b), p-toluenesulfonic acid and thehydrochloride salt of lysine diester. The wavey lines represent the AA-PEA chains.

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chloride, succinyl chloride, 1,4-butanediol, (Alfa Aesar, WardHill, MA) and p-nitrophenol (J. T. Baker, Phillipsburg, NJ) wereused without further purication. Triethylamine (TEA) fromFisher Scientic (Fairlawn, NJ) was dried via reuxing withcalcium hydride followed by distillation. Other solvents, such asbenzene, toluene, ethyl acetate, acetone, N,N-dimethylaceta-mide (DMAc) and dimethyl sulfoxide (DMSO) were purchasedfrom VWR Scientic (West Chester, PA) and were puried usingstandard methods before use. Trypsin (Type IX-S, from bovinepancreas, lyophilized power, 13 000–20 000 BAEE units per mgprotein), hydralazine, 1-[3-(dimethylamino)propyl]-3-ethyl-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) werepurchased from VWR Scientic (West Chester, PA). 5-(and 6)-Carboxyuorescein succinimidyl ester (NHS-uorescein, exci-tation maximum 491 nm and emission maximum 518 nm) waspurchased from Pierce (Rockford, IL).

2.2 Synthesis of the Lys-x monomers

As shown in Fig. 1, the di-p-toluenesulfonic acid salt of bis-L-lysine ester monomer (Lys-4) was prepared as one example toillustrate the Lys-x monomers library. Typically, L-lysine(0.132 mol), p-toluenesulfonic acid monohydrate (0.132 mol),and 1,4-butanediol (0.06 mol) in 250 mL of toluene were placedin a ask equipped with a Dean–Stark apparatus, a CaCl2 dryingtube and a magnetic stirrer. The solid–liquid reaction mixturewas heated at 90 �C and reux for 16 hours until 4.3 mL(0.24 mol) of water was collected.

The reaction mixture was then cooled to room temperature,ltered and dried, and nally puried by dissolving in isopropylalcohol at 40 �C and cooling the solution to�20 �C overnight forthe precipitation of the di-p-toluenesulfonic acid salt of bis-L-lysine ester from isopropyl alcohol. This process was repeatedthree times. Isopropyl alcohol was changed every time aerprecipitation, the white sticky mass was dried in vacuo at 40 �Cfor 24 hours. The nal product was a white powder and the yieldwas 70–80%. This new Lys-4 monomer differs from the previ-ously published Lys-based monomers in two ways: protectionand deprotection during the synthesis are not required, and theavailability of four amine groups in each monomer instead ofone functional carboxylic acid or amine group.31,32

2.3 Synthesis of the monomers of di-p-nitrophenyl esters ofthe dicarboxylic acids

The synthesis of the di-p-nitrophenyl succinate (NSu), di-p-nitrophenyl adipate (NA) and di-p-nitrophenyl sebacate (NS)monomers are described in the literature31 and are illustrated inFig. 1a.

2.4 Fabrication of lysine based PEA polymers and theirmacrogels

The Lys-based biodegradable and pure AA-PEA polymers andtheir macrogels were prepared by a direct polycondensationreaction of Lys-4 monomer and p-nitrophenyl diester mono-mers at 80 �C in DMAc. In brief, NS (or NA, NSu) and Lys-4monomers at a feed molar ratio of 1.5 (or 2) were added to a vialand dissolved in DMAc at 50 �C, and then triethylamine (TEA)


(4.5 eq. to Lys-4) was added. The above mixed solution wasplaced in an 80 �C oil bath for a specic time and macrogelformation occurred, i.e., polymerization and crosslinking reac-tions occurred simultaneously. The resultant macrogels werecarefully removed from the vials and washed several times withDMSO and acetone, and then with distilled water to remove anyresidual chemicals. The distilled water was replaced periodi-cally. Aer this purication process, the macrogels were soakedin distilled water until swelling equilibrium, and then removedand dried in vacuo at room temperature for 48 hours forcharacterization.

2.5 Fourier transform infrared (FTIR) spectroscopy

The FTIR spectra of the monomers and macro hydrogels wererecorded on a spectrophotometer (Perkin-Elmer Magna-IR560Spectrometer) to characterize the chemical structures of the Lys-4 monomer and its hydrogels. The samples were ground intopowders, compressed into KBr discs and the FTIR spectrarecorded over the wavenumber range of 550–4000 cm�1. FTIRspectra were then obtained with a PerkinElmer (Madison, WI)Nicolet Magana 560 FTIR spectrometer with Omnic soware fordata acquisition and analysis.

2.6 Interior morphology of macrogels

Scanning electron microscopy (SEM) was employed to analyzethe interior morphological structure of the Lys-4 macrogels as afunction of the precursor feed ratio before and aer biodegra-dation. Typically, aer being incubated in distilled water atroom temperature for 3 days to reach its equilibrium swelling,

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the macrogel was then gently removed and immediately trans-ferred into liquid nitrogen to freeze and retain the swollenstructure. The samples were subsequently freeze-dried for threedays in a Virtis (Gardiner, NY) freeze drier in vacuo at �50 �C.

For the preparation of the particles generated from the bio-degraded macrogels for SEM observation, the solution mediumin which the Lys-based PEA macrogel was immersed wasremoved at pre-determined periods of immersion and placedonto a glass coverslip, dried overnight in a fume hood, andnally xed on aluminum stubs and coated with gold for30 seconds prior to SEM observation with a Hitachi (MountainView, CA) S4500 SEM instrument. Image analyses of SEM datawere performed using the public domain NIH image program.

2.7 Swelling kinetics of Lys-based PEA macrogel

The swelling kinetics of the Lys-basedmacrogels weremeasuredat room temperature (25 �C). The dried Lys-based macrogelwere weighed and immersed in 10 mL of distilled water at roomtemperature for pre-determined intervals; they were thenremoved and the water on the macrogel surface gently wipedusing wet lter paper and weighed until there was no furtherweight change. The swelling ratio (Q) was calculated as follows:

Q ¼ Ws �Wd

Wd

� 100% (1)

whereWs is the weight of the swollen macrogel at time t andWd

is the weight of the dry macrogel at t ¼ 0.

2.8 Mechanical testing

The mechanical testing of the Lys-based macrogels was per-formed on a DMA Q800 Dynamic Mechanical Analyzer (TAInstruments Inc., New Castle, DE) in “controlled force” mode.The swollen macrogels samples (disc of 1.2 cm diameter) weresubmerged in distilled water and mounted between a movablecompression probe (diameter 15 mm) and the uid cup. Acompression force from 0.01 to 0.05 or 0.30 N (depending on thegel strength) at a rate of 0.02 or 0.05 N min�1 was applied atroom temperature. The compression elastic modulus (E) of theswollen macrogel was calculated from the plot the compres-sional stress versus strain.

2.9 In vitro enzymatic biodegradation of the Lys-based PEAmacrogel

The biodegradation of Lys-based macrogels was carried out in asmall vial containing a small piece of known weight dry mac-rogels sample (ca. 50 mg) and 10 mL of PBS buffer (pH 7.4,0.1 M) with trypsin at a concentration of 0.1 mg mL�1. A purePBS buffer was used as the control. The vial was then incubatedat 37 �C. The incubation media were refreshed daily in order tomaintain the enzymatic activity. At predetermined immersiondurations, the macrogel samples were removed from the incu-bation medium, and then washed three times with PBS, andthen lyophilized in vacuo with a FreeZone Benchtop andConsole Freeze Dry System (Model 7750000, LABCONCO Co.,Kansas City, MO) at �48 �C for 72 hours to a constant weight.

2288 | J. Mater. Chem. B, 2015, 3, 2286–2294

The degree of biodegradation was estimated from the weightloss of the macrogels based on the following equation:

Wlð%Þ ¼ W0 �Wt

W0

(2)

where Wl is the percentage weight loss, W0 was the originalweight of the dried lysine based macrogel sample beforeimmersion, andWt was the dried lysine based macrogel sampleweight aer an incubation time t. The weight loss data from theaverage of three specimens was recorded.

To observe the nanogel generated from the macrogelbiodegradation in the enzyme solution at a predetermined time,a few drops of the biodegradation solution were collected andplaced on silicon slides for SEM characterization.

2.10 Fluorescein microscopy

The nanogels generated from the biodegraded Lys-basedhydrogels were modied by coupling a uorescence dye forvisual purposes in the subsequent biological studies. In brief,aer the macro hydrogel was biodegraded for 5 days in 0.1 mgmL�1 trypsin medium, the biodegradation solution wascollected and dialyzed through a dialysis tube (MW cutoff100 000) to remove the trypsin enzyme. Aer dialysis, pre-determined amounts of NHS-uorescein were added into thedialyzed solution containing the nanogel and the solution pHadjusted to 5.0. NHS-uorescein was dissolved in DMSO, andthen added to the above nanogel solution, the reaction lastedfor 12 hours with stirring. The solution was collected and dia-lyzed against di-water using a dialysis tube (MW cutoff 12 000)to remove the unreacted NHS-uorescein and solvent DMSO.Finally, the resulting solution was free dried using a freeze drierto collect the dye-tagged Lys-based PEA nanogel. To obtain theimage of the NHS-uorescein attached nanogel, the freeze-driednanogel sample was dispersed in a PBS solution, sonicated for10 min, and then observed by uorescence microscopy. Fluo-rescence microscopy was performed using a confocal uores-cence microscope (C1-si, Nikon, Japan) tted with standardFITC and Texas Red lter sets.

2.11 Nanogel size and zeta potential measurement and sizedistribution

The size, size distribution and zeta potential of the Lys-basedPEA nanogel were determined using a Zeta sizer (Nano ZS,Malvern Instruments). The nanogel contained aqueous solution(200 mg L�1) was passed through a 0.45 mm pore size lter priorto measurement.

2.12 Drug loading in Lys-based PEA nanogel and in vitrorelease

To investigate the DOX loading in the Lys-based PEA nanogel,Lys-4, NA monomers and DOX were dissolved in DMAc and theDOX-impregnated Lys-based PEA hydrogels were fabricatedusing the procedure described towards the fabrication of lysinebased PEA polymers and their macrogels. In a typical case,10 mg of DOX, 1.0 g of Lys-4 and 0.85 g of NA were dissolved inDMAc and heated to 80 �C for 3min to form a DOX-impregnated




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Lys-based PEA macrogel. The resultant macrogel was immersedin acetone, DMSO, and then di-water to remove any unreactedregents. The macrogel biodegradation procedure was the sameas that described previously in the in vitro enzymatic biodeg-radation of lysine based macrogel with biodegradation durationof 6 days in 0.1 mg mL�1 trypsin solution. The obtained Lys-based PEA nanogel containing DOX were dialyzed in a dialysistube (MW cutoff at 100 000 g mol�1) for one day. Aer dialysis,the dialysis tube was directly immersed into 400 mL PBS solu-tion (pH ¼ 7.4, 0.1 M). Aliquots of 3 mL were subsequentlywithdrawn from the PBS periodically at 37 �C for predeterminedtime. Aer each sampling, 3mL of fresh PBS solution was addedback to the dialysis tube to maintain a constant volume ofsolution. The amounts of DOX released from the nanogel weremeasured using UV absorbance at 497 nm of the 3 mL aliquotremoved at predetermined periods (the calibration curve ofDOX is not shown).

To determine the drug entrapment efficiency and drugloading efficiency of the Lys-based nanogel, the amounts ofDOX released from the preloaded Lys-4 based macrogel duringthe swelling was tested (M1). Additionally, the amounts of DOXreleased in the course of biodegradation and dialysis of thenanogel were calculated (M2). The amount of DOX wasmeasured using the UV-vis absorbance at 497 nm according tothe calibration curve. The nal amounts of DOX loaded into thenanogel were calculated using:

ML ¼ M0 � M1 � M2 (3)

where M0 is the initial amounts of DOX preloaded into themacrogel during macrogel formation.

The drug entrapment efficiency (DEE) and drug loadingefficiency (DLE) in the biodegraded nanogel were dened asfollows.

DEE ¼ Mass of loaded drug in nanogel

Mass of drug fed initially� 100% (4)

DLE ¼ Mass of loaded drug in nanogel

Mass of drug loaded nanogel� 100% (5)

2.13 In vitro cytotoxicity of the Lys-based PEA nanogels

200 mL of Hela cells in DMEMmedium with a cell concentrationof 1.00 � 104 cells per mL were added to each well in a 96-wellplate. The number of Hela cells in each well is 2.0 � 103. Aerincubation for 24 h in an incubator (37 �C, 5% CO2), the culturemedium was replaced with 200 mL of DMEM containing theFITC-labeled 4-Lys-4 nanogel [4-Lys-4 (1.5 : 1)-FITC] or DOX-loaded 4-Lys-4 (1.5 : 1)-FITC nanogel at predetermined nanogelconcentrations and the mixture was further incubated foranother 48 hours. Then, the DMEM medium containing either4-Lys-4 (1.5 : 1)-FITC nanogel or DOX-loaded 4-Lys-4 (1.5 : 1)-FITC nanogel were replaced with fresh DMEM and 20 mL of MTTsolution (5 mg mL�1) was added to the Hela cells in each well inthe 96-well plate. Aer incubation for 4 h, 200 mL of DMSO wasadded and the samples shaken at room temperature. The


optical density (OD) was measured at 570 nm with a MicroplateReader Model 550 (BIO-RAD, USA). The cell viability wascalculated using the following equation. Cell viability ¼ODtreated/ODcontrol, where ODcontrol was obtained in the absenceof nanogels (DMEM medium only) and ODtreated was obtainedin the presence of nanoparticles with or without DOX. All cellviability data was normalized against the blank controls (nonanogels and no DOX).

2.14 Cell internalization of 4-Lys-4-FITC biodegradationnanogel

Hela cells were used for the cell internalization study of FITC-tagged Lys-based PEA [4-Lys-4 (1.5 : 1)] nanogel with or withoutDOX [4-Lys-4 (1.5 : 1)-FITC]. Hela cells were cultured on LabTekchamber slide dishes (2.0 � 104 cells per plate) to grow to about70% conuence. The culture medium was then removed and1 mL of fresh DMEM containing 4-Lys-4 (1.5 : 1)-FITC nanogel(1 mgmL�1) with or without DOX was added to each well. All thecells were incubated in the plates containing DMEM and poly-mers for 6 h, 12 h and 24 h at 37 �C, respectively. Then, themedium was removed and the cells were washed three timeswith PBS solution. Then, 1 mL of PBS solution was added intoeach well. The uorescence images of the cells were observedunder excitation at 488 nm using confocal laser scanningmicroscopy (CLSM) (Leica TCS SP2AOBS, Germany).

3. Results and discussion3.1 Synthesis of the Lys-x monomers and hydrogels

Polymerization occurred between lysine monomers and diacidbased monomers (NA, NS) (Fig. 1a and b) via a single steppolycondensation reaction (Fig. 1c). The 1H NMR spectrum ofthe lysine monomer is illustrated in Fig. 2a. The chemical shid at 8.34 ppm was attributed to amine group attached on-NH2TosOH (d), d at 8.02 ppm (hydrogen chloride attached toamine group, (l)) d at 7.75 and 7.14 ppm (the phenyl groupof TosOH attached to amine group, (b and c)), d at 4.15 ppm ((e)–C]O–O–CH2), d at 3.81 ppm ((g) –C]O–O–CH–NH2), d at 2.75ppm ((k) –CH2–NH2), d at 2.21 ppm ((a) phenyl-CH3), d at 1.78ppm ((j) –CH2–CH2–CH2–), d at 1.68 ppm ((h) –CH–CH2–CH2–),d at 1.59 ppm ((f) –CH2–CH2–CH2–) and d at 1.51 ppm ((i) –CH2–

CH2–CH2–). FTIR: 3640 cm�1 (–NH2, wide) and 1745 cm�1

(–C]O–O–). The nal products aer polymerization could bepolymers or crosslinked polymers (macro gels), mainlydepending on the monomer ratio (Fig. 1c). The formation of thehydrogels was demonstrated by the FTIR spectra shown inFig. 2c, the characteristic bands at 1742 cm�1 attributed to theC]O stretch; 1640 cm�1 attributed to amide I; and 1560 cm�1

attributed to amide II. These corresponding characteristicbands at 1742 cm�1, 1640 cm�1 and 1560 cm�1 were observed inthe 4-Lys-4 (1.5 : 1), 4-Lys-4 (2 : 1), 8-Lys-4 (1.5 : 1) and 8-Lys-4(2 : 1) hydrogels. It can be veried that hydrogels were formedvia the condensation reaction of the amino groups in Lys-4 andthe carbonyl groups of NA or NS. As demonstrated in Fig. 2b, thecharacteristic bands of 3300 cm�1 were attributed to N–Hstretch with the corresponding strength of 4-Lys-4 (1.5 : 1) being

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larger than that of 4-Lys-4 (2 : 1) due to the different feed ratio ofNA to Lys-4. As for the former, the feed ratio of NA to Lys-4 is1.5 to 1, that indicates there are more free amino groups in thehydrogels, the same case was observed in the 8-Lys-4 (1.5 : 1)and 8-Lys-4 (2 : 1) hydrogels. Below the gelling point ratio,soluble polymers will be formed and above that point, the gelwill form. Aer optimization, it was found that the gel point ismainly affected by the reaction temperature, monomer ratio,and concentration of the monomer (Table 1). When comparedto the reported PEA hydrogel systems, this new Lysine strategyhas a few unique advantages, including: (1) one step hydrogelfabrication by simply mixing and heating the monomer solu-tions together within minutes, (2) eliminating the reportedneed for tedious protection and deprotection procedures of theamino acids that is costly, time consuming and results in lowyields,31 (3) double bond free gelation and (4) a hydrogel having100% pure PEA component. Subsequently, a 100% pure lysinebased PEA hydrogel library was developed with the detailed gelsynthesis protocol reported in the study. Here is one simpleexample for 4-Lys-4 (x-Lys-y, where x and y represent themethylene in the diacid monomer and diol monomer, respec-tively) gel: 4-Lys-4 gel was prepared by mixing NA and Lys-4 witha molar ratio of 2 : 1 for 2.0 min at 80 �C.

The polymerization/gelation rate of Lys-4 with NS is fasterthan with NA in DMAc at 80 �C, i.e., 2 min vs. 4 min. Thepolymerization/gelation of Lys-4 monomers with Nsu monomer(x¼ 1 in Fig. 2b) under the same reaction conditions as with NA

Fig. 2 (a) 1H NMR spectrum of the Lys-4 monomer (300 MHz, DMSO-d6). (b) Swelling kinetics of the Lys-4 basedmacrogels in distilled waterat 25 �C. (c) FTIR spectra of the lysine based hydrogel. (A) 4-Lys-4(2 : 1); (B) 4-Lys-4 (1.5 : 1); (C) 8-Lys-4 (2 : 1); (D) 8-Lys-4 (1.5 : 1). 1742cm�1 attributed to the C]O stretch; 1640 cm�1 attributed to amide I;and 1560 cm�1 attributed to amide II. (d) Optical images of thehydrogels. (A) 8-Lys-4 (1.5 : 1); and (B) 4-Lys-4 (1.5 : 1). The molar ratioof 1.5 : 1 indicates NA or NS to the Lys-4 monomer. (e) SEM images ofthe Lys-4 based hydrogels before degradation. (A) 8-Lys-4 (2 : 1); (B)8-Lys-4 (1.5 : 1); (C) 4-Lys-4 (2 : 1); and (D) 4-Lys-4 (1.5 : 1).

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or NS, however, was unsuccessful. This was due to the presenceof less methylene or activity in the Nsu monomer that form ashort spacer when polycondensation occurred, which made itdifficult to fabricate a hydrogel.

3.2 Swelling kinetics of the Lys-based PEA macrogel andmechanical properties

Fig. 1c is an illustration of the hydrogel crosslinking formedupon reaction of the monomer A and monomer B used to formthe hydrogel. Interestingly, it was found that the polymeriza-tion/gelation rate and gel structure were determined by themonomer structure under xed reaction conditions. Forexample, Lys-4 shows a faster gelation rate when reacting withNS than with NA in DMAc at 80 �C, i.e., 2.0 min vs. 4.0 min. Thepolymerization/gelation of Lys-4 monomers with the Nsumonomer (x¼ 1 in Fig. 1b) under the same reaction conditions,however, was only able to form a very so hydrogel or viscousmixture. This may be due to the weaker reactivity or presence ofless methylene in the Nsu monomer, which made it difficult tofabricate a hydrogel. The modulus of the resultant hydrogelswas also affected by the monomer structure: higher x or y wouldcause a higher modulus (Table 2). For example, the 8-Lys-4hydrogel had a signicantly higher compression modulus(more than 25 times) than the 4-Lys-4 hydrogel with the samemonomers feed ratio, i.e., 670 � 53 kPa vs. 25 � 4.5 kPa,respectively (Table 2).

To successfully obtain nanogels via an enzyme biodegrada-tion strategy, one of the key factors is the three dimensionalstructure of the macrogel, which is directly determined orrelated to the intrinsic chemical structure of the macro hydro-gel. Before the biodegradation study, the interior morphology ofthe freeze dried Lys-x (x ¼ 4, 6, 8) based macro hydrogel librarywas systematically investigated by scanning electron micros-copy (SEM). As shown in Fig. 2e, before biodegradation, all thehydrogels exhibited well-dened, three-dimensional porousand completely connected network structures with an averagewall thickness of around 1.0–4.0 mm and average pore sizeranging from 4.0 to 10.0 mm. It was found that the x, y andm to nratio affects the pore size and wall thickness. For example, asshown in Fig. 2e, the average pore size of the hydrogel were

Table 1 Preparation of Lys-4 based macrogels

Sample code NA NS Lys-4 Lys-6 Lys-8 DMAc Et3N

4-Lys-4 (1.5 : 1) 0.76 g — 1.0 g — — 2.0 g 0.65 g4-Lys-4 (2 : 1) 1.02 g — 1.0 g — — 2.0 g 0.65 g8-Lys-4 (1.5 : 1) — 0.87 g 1.0 g — — 2.0 g 0.65 g8-Lys-4 (2 : 1) — 1.16 g 1.0 g — — 2.0 g 0.65 g4-Lys-6 (1.5 : 1) 0.67 g — — 1.0 g — 2.0 g 0.65 g4-Lys-6 (2 : 1) 0.95 g — — 1.0 g — 2.0 g 0.65 g4-Lys-8 (1.5 : 1) 0.65 g — — — 1.0 g 2.0 g 0.65 g4-Lys-8 (2 : 1) 0.89 g — — — 1.0 g 2.0 g 0.65 g8-Lys-6 (1.5 : 1) — 0.81 g — 1.0 g — 2.0 g 0.65 g8-Lys-6 (2 : 1) — 1.08 g — 1.0 g — 2.0 g 0.65 g8-Lys-8 (1.5 : 1) — 0.76 g — — 1.0 g 2.0 g 0.65 g8-Lys-8 (2 : 1) — 1.01 g — — 1.0 g 2.0 g 0.65 g




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6.0 mm and 4.0 mm for 4-Lys-4 (1.5 : 1) (1.5 : 1 is the molar ratioof monomer NA to Lys-4) and 4-Lys-4 (2 : 1), and 10.0 mm and8.0 mm for 8-Lys-4 (1.5 : 1) and 8-Lys-4 (2 : 1), respectively. The4-Lys-4 hydrogels had thinner cell walls than the 8-Lys-4hydrogels, which indicates 8-Lys-x hydrogels had a morecompact structure. In addition, the average pore size decreasedwith an increase in x. With an increase in amide group densityformed in the hydrogel, i.e., an increase in cross-linking density,the average pore size decreased, which is consistent with theswelling ratio and mechanical data of the macro hydrogels(Fig. 2b).

Fig. 3 (a) SEM images of the lysine based macrogels with an enzymetrypsin concentration of 0.1 mgmL�1 in PBS buffer (pH 7.4, 0.1 M) after1 day of biodegradation. (A) 4-Lys-4 (1.5 : 1); (B) 4-Lys-4 (2 : 1); (C) 8-Lys-4 (1.5 : 1); and (D) 8-Lys-4 (2 : 1). (b) SEM images of the lysinebased hydrogel with an enzyme trypsin concentration of 0.1 mg mL�1

3.3 In vitro enzymatic biodegradation of Lys-based PEAmacrogel

The enzyme biodegradation mechanism of the cross-linkedlysine based hydrogels was subsequently investigated using ahydrolytic enzyme with a series of concentrations in PBS buffersolutions. The weight loss, size distribution and SEM were usedto characterize and monitor the biodegradation progress of thehydrogels. The hydrolytic enzyme, trypsin, is utilized here as amodel enzyme because of its general degradation capability. Inaddition, we have focused on the material related properties inthis study. The results indicated that the degradation behavioris affected by a variety of factors, including crosslinking density,concentration of enzyme, biodegradation time, the density ofthe hydrophobic moiety and the molecular weight of the back-bone chain. For example, as shown in Fig. 4a, the degradationbehavior of the lysine based hydrogel depended on the cross-linking density of the amide groups and the hydrophobicity ofthe hydrogels. For the 4-Lys-4 (1.5 : 1) hydrogel immersed inPBS solution without trypsin, its weight loss was only 5.0 wt%aer 10 days, which indicates the contribution from thehydrolytic degradation of the buffer was very small or negligible.Therefore, the degradation contribution is mainly due toenzyme attack. The biodegradation rate of the hydrogel couldbe regulated by tuning the crosslinking density. For example,4-Lys-4 (1.5 : 1) is faster than 4-Lys-4 (2 : 1), which was ascribedto the lower crosslinking density of the amide groups in thehydrogels. With an increase in the ratio of the monomer NA toLys-4, the hydrogel has a higher crosslinking density of theamide groups. A similar trend was observed in the 8-Lys-4hydrogels. In general, the degradation rate of the hydrogel isrelated to several network parameters such as the number ofcrosslinkers per backbone chain, molecular weight of thebackbone unit and the proportion of biodegradable groups inthe main and side chains.33–35 Therefore, the biodegradation

Table 2 Compressive modulus of the Lys-4 based macrogels

Sample code Monomer molar feed ratioCompressivemodulus (kPa)

4-Lys-4 (1.5 : 1) NA : Lys-4 ¼ 1.5 : 1 25 � 4.54-Lys-4 (2 : 1) NA : Lys-4 ¼ 2 : 1 46 � 6.18-Lys-4 (1.5 : 1) NS : Lys-4 ¼ 1.5 : 1 670 � 538-Lys-4 (2 : 1) NS : Lys-4 ¼ 2 : 1 1350 � 126


rate decreased with an increase in crosslinking density. It wasfound that the hydrogel with a higher swelling ratio had a fasterbiodegradation rate in our study, which is consistent with theabove rules. Aer screening, 8-Lys-4 and 4-Lys-4 gels werechosen for the following study.

SEM was used to monitor the morphology change of theswollen 8-Lys-4 and 4-Lys-4 hydrogels during enzymaticbiodegradation. On the second day of biodegradation, there area lot of regular microspheres on the hydrogel surface and in thehydrogel networks, and the average size ranges from 0.5 mm to4.0 mm (Fig. 3a). This is the rst time that the synthesizedhydrogel was reported to be able to produce microspheres(microgels) aer being treated with an enzyme. With the pro-longed biodegradation time, the enzymatic biodegradation ofthe hydrogel network continues and the average size of themicrosphere was decreased to below 3.0 mm aer 2 days. Themorphology of the microspheres became less regular and someof the microspheres linked like a string of beads, indicating the1st stage biodegradation was incomplete (Fig. 3b). Moreover,the average pore size of the hydrogels increased during thebiodegradation process. As shown in Fig. 3c, the average poresize of the 8-Lys-4 (1.5 : 1) and 8-Lys-4 (2 : 1) was around10.0 mm and 6.0 mm, respectively, and the size for 4-Lys-4

in PBS buffer (pH 7.4, 0.1 M) after 3 days of biodegradation. (A) 4-Lys-4(1.5 : 1); (B) 4-Lys-4 (2 : 1); (C) 8-Lys-4 (1.5 : 1); and (D) 8-Lys-4 (2 : 1).(c) SEM images of the lysine based macrogels with an enzyme trypsinconcentration of 0.1 mg mL�1 in PBS buffer (pH 7.4, 0.1 M) after 4 daysbiodegradation. (A) 4-Lys-4 (1.5 : 1); (B) 4-Lys-4 (2 : 1); (C) 8-Lys-4(1.5 : 1); and (D) 8-Lys-4 (2 : 1). Microgels and nanogels are invisiblebecausemost of themhave beenwashed out with PBS. (d) SEM imagesof the lysine basedmacrogels with an enzyme trypsin concentration of0.1 mg mL�1 in PBS buffer (pH 7.4, 0.1 M) after 5 days of biodegra-dation. (A) 4-Lys-4 (1.5 : 1); (B) 4-Lys-4 (2 : 1); (C) 8-Lys-4 (1.5 : 1); and(D) 8-Lys-4 (2 : 1). Microgels and nanogels are invisible because mostof them have been washed out with PBS.

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(1.5 : 1) and 4-Lys-4 (2 : 1) was around 12.0 mm and 10.0 mm,respectively. Aer 4 days, the average pore size of the 8-Lys-4(1.5 : 1), 8-Lys-4 (2 : 1) was around 20.0 mm and 15.0 mm,respectively; the average size for 4-Lys-4 (1.5 : 1) and 4-Lys-4(2 : 1) was 30.0 mm and 40.0 mm, respectively, and the regular 3-D structure of the hydrogels was almost broken (Fig. 3d). Thezeta potential of the 4-Lys-4 (1.5 : 1) and 4-Lys-4 (2 : 1) nanogelwas 14.5 mV and 9.6 mV, respectively, indicating net freeprimary amine groups on the microsphere surface.

At 5 days, the microspheres on the hydrogel surface werepartially biodegraded and some of the broken microsphereswere observed (Fig. 3d). The hydrogel network almost collapsedand the average pore size enlarged. We also investigated thestatus of the biodegradation solution during the enzymebiodegradation treatment. On the sixth day, a large number ofmicrogels were obtained by degradation and their sizedecreased to about 200 nm (Fig. 4b). Aer 8 days biodegrada-tion, the size of the obtained nanogels was about 50 nm with anarrow size distribution (Fig. 4c). The size of the micro andnano gels in PBS solution were characterized by DLS. Theenzyme trypsin was removed by dialysis (cutoff weight 100 000).

3.4 Drug loading in the Lys-based PEA nanogels and in vitrorelease

The biodegradation behavior of the drug loaded Lys basedmacro hydrogels were further examined using the DOX as amodel drug. The hydrogels were biodegraded using 0.1 mg

Fig. 4 (a) The weight loss of the lysine based hydrogels duringdegradation. The hydrogels were immersed in PBS buffer (pH 7.4,0.1 M) with trypsin at a concentration of 0.1 mg mL�1 and pure PBSused as a control. (b) SEM images of the lysine based 4-Lys-4 (1.5 : 1)macrogels with an enzyme trypsin concentration of 0.1 mg mL�1 inPBS buffer (pH 7.4, 0.1 M) after (A) 6 days of biodegradation; and (B)7 days of biodegradation. (C) DLS of the 6 day biodegradation nanogeland (D) DLS of the 7 day biodegradation nanogel. (c) SEM images of thelysine based 4-Lys-4 (1.5 : 1) hydrogels with an enzyme trypsinconcentration of 0.1 mg mL�1 in PBS buffer (pH 7.4, 0.1 M) after (A)8 days of biodegradation and (B) 9 days of biodegradation. (C) DLS ofthe 8 day biodegradation nanogels and (D) DLS of the 9 day biodeg-radation nanogels.

2292 | J. Mater. Chem. B, 2015, 3, 2286–2294

mL�1 trypsin PBS solution, as described in the previous section.The DOX was preloaded into the hydrogels during the macrohydrogel fabrication. In this study, for a 5.0 wt% drug added,the drug entrapment efficiency (DEE) and drug loading effi-ciency (DLE) was 51.6% and 2.6%, respectively. The DOX loadednanogels were obtained and chosen for a release study aer5 days of degradation. The resultant nanogels were washed withPBS before testing the DOX release. The yield of the above 4-Lys-4 (1.5 : 1) nanogel was 18.6%. As demonstrated in Fig. 5b, at atime of 5 hours, the cumulative DOX release from the nanogelsin both PBS solution (pH 7.4, 0.1 M) and enzyme trypsin solu-tion (0.1 mg mL�1) was about 10.0%. However, the release ofDOX became a virtually constant rst order rate aer 15 hoursover a 10 day period for nanogels in PBS solution (pH 7.4, 0.1 M,0.1 mg mL�1 trypsin).

With the degradation of the ester and amide bonds of thehydrogel, the hydrophilic lysine segments of the nanogel dis-solved and diffused into the buffer solution and nanogelbecame more and more loose. When the degradation rate of thelysine based nanogel came to a moderate stage, the releasedDOX amounts increased in a linear relationship as a function oftime (1st order). Aer degrading the 4-Lys-4 (1.5 : 1) nanogel,the release behavior came to an end. This indicates that therelease of DOX was mainly controlled by the dissolution anddissociation of the 4-Lys-4 (1.5 : 1) nanogels rather than by adiffusion mechanism.

3.5 Cell internalization of the 4-Lys-4-FITC biodegradationnanogel

Monomer Lys-4 has 4 free amine groups, when the molar ratioof NA to the lysine based monomer Lys-4 is 1.5 : 1, the resultant4-Lys-4 (1.5 : 1) hydrogel has free amine groups aer hydrogelformation compared to the 4-Lys-4 (2 : 1) hydrogel. The freeamine groups endow the nanogel a positive charge for in vitro

Fig. 5 (a) CLSM images of the biodegradation particles: (A) em 595 nmfor DOX; (B) em 515 nm for FITC and (C) a physically overlaid image ofpanels A and B. (b) Cumulative release of DOX from the 4-Lys-4(1.5 : 1) hydrogel biodegraded nanogels at 37 �C in PBS solution (pH7.4, 0.1 M) at a trypsin concentration of 0.1 mgmL�1. (c) Cytotoxicity ofthe 4-Lys-4 (1.5 : 1)-FITC nanogels, DOX loaded 4-Lys-4 (1.5 : 1)-FITCnanogels and free DOX at different concentrations. Control for freeDOX indicates no DOX for cell culture.



Fig. 6 Confocal laser scanning microscopy images of Hela cells after6 h (A1, B1, C1, D1); 12 h (A2, B2, C2, D2); 24 h (A3, B3, C3, D3); and freeDOX (A0, C0, D0) incubated with DOX loaded 4-Lys-4 (1.5 : 1)-FITCnanogels and free DOX (20 mg mL�1) as the control in the DMEMmedium at 37 �C. (A1, A2, A3, A0): bright field images, B1, B2, B3, B4:confocal green fluorescence images; C1, C2, C3, C4: confocal redfluorescence images; D1, D2, D3, D4: merge.


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cellular uptake and chemical conjugation, such as uorescencedye attachment. CLSM was used to investigate the 4-Lys-4(1.5 : 1)-FITC biodegradation nanogels and DOX loaded 4-Lys-4(1.5 : 1)-FITC nanogel's uptake by HeLa cells and their subse-quent accumulation. The uoresence dye, FITC, was chemicallyconjugated to the nanogel upon reaction with its amine groupsand was used to track the nanogel in the cell for imaging. Aer6 h of incubation with the DOX loaded 4-Lys-4 (1.5 : 1)-FITCnanogels suspension (1 mg mL�1) at 37 �C, uorescence wasobserved in HeLa cells (Fig. 6). With the incubation timeincreasing, a higher uorescence intensity was observed, asshown in Fig. 6B2. When the HeLa cells grew to 24 h with theDOX loaded 4-Lys-4 (1.5 : 1)-FITC nanogels, a much higherintensity of the red uorescence was seen inside the Hela cells.This is consistent with the MTT results of the DOX loaded 4-Lys-4 (1.5 : 1)-FITC nanogels, as shown in Fig. 5c. The cytotoxicityincreased with the increased concentration of DOX loaded4-Lys-4 (1.5 : 1)-FITC nanogels. When the concentration of DOXloaded 4-Lys-4 (1.5 : 1)-FITC nanogels is 0.25 mg mL�1, the cellviability is less than 20%. When the concentration is 0.5 mgmL�1, the cell viability decreased to 10%. Finally, at a concen-tration of 1.0 mg mL�1, the cell viability decreased to 8%.

4. Conclusions

In conclusion, a novel strategy was utilized to develop func-tional microgels and nanogels from macrogels using thecontrolled enzyme biodegradation of the screened macrogels.To achieve the goal of nanogel fabrication, a new


multifunctional L-lysine based diester monomer was speciallydeveloped and a corresponding crosslinking method used tochemically fabricate a new family of pure amino acid-basedpoly(ester amide) hydrogels with desired chemical structures.For some screened macrogels, it was found that sphericalnanogels with controllable size were formed and the nal sizecould be below 50 nm aer being biodegraded for 8 days. Withthe abundance of free amine groups, the degradedmicrogel andnanogels could easily conjugate with biologically active reagentsor dyes and demonstrate a controllable and sustained release,therefore, expanding the biomedical applications of this gelplatform.

Acknowledgements

This research is supported by a grant from the Rebecca Q.Morgan Foundation. The research is also supported by theFundamental Research Funds for the Central Universities andsponsored by the Shanghai Pujiang Program 14PJ1400300. Theauthors are grateful for the Nanobiotechnology Center (NBTC)for using the research facilities in Cornell University for cellrelated work.

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From Macro to Micro to Nano the Development of a Novel Lysine Based Hydrogel Platform and Enzyme...

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